The Mediterranean Diet An Evidence-Based Approach

The Mediterranean Diet An Evidence-Based Approach The Mediterranean Diet An Evidence-Based Approach Edited by Victor R. Preedy King’s College London, London, UK Ronald Ross Watson University of Arizona, Tucson, AZ, USA AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright © 2015 Elsevier Inc. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. 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Antonio Agudo, MD, MSc, PhD (47), Catalan Institute of Oncology (ICO), Barcelona, Spain Americo Bonanni (589), IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli, Italy Marisa Alarcón (601), Institut Botànic de Barcelona (IBBCSIC-ICUB), Barcelona, Spain Gerardo Bosco, MD, PhD (271), University of Padova, Padova, Italy Juan José Aldasoro (601), Institut Botànic de Barcelona (IBB-CSIC-ICUB), Barcelona, Spain Genevieve Buckland, MSc, PhD (47, 417), Catalan Institute of Oncology (ICO), Barcelona, Spain Smaragdi Antonopoulou, PhD (379), Harokopio University, Athens, Greece Yardena Arnoni, MSc (3), Hebrew University-Hadassah Medical School, Jerusalem, Israel Sara Arranz, PhD (153), Department of Internal Medicine, Hospital Clı́nic, Institut d’Investigacions Biomédiques August Pi i Sinyer (IDIBAPS), University of Barcelona, Barcelona, Spain; CIBER CB06/03 Fisiopatologı́a de la Obesidad y la Nutrición, (CIBERobn), Girona, Spain Hebatallah Husseini Atteia (441), Faculty of Pharmacy, Zagazig University, Zagazig, Sharkia Gov., Egypt Elena Azzini (249), National Institute for Food and Nutrition Research, Rome, Italy Lina Badimon (367), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; IIB-Santpau, Barcelona, Spain; CIBEROBN Instituto de Salud Carlos III, Madrid, Spain; Cátedra de Investigación Cardiovascular, (UABHSCSP-Fundación Jesús Serra), Barcelona, Spain Sara Bastida, PhD (217, 491), Universidad Complutense de Madrid, Madrid, Spain Giovanni Beccari, PhD (563), Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy Elliot M. Berry, MD, FRCP (3), Hebrew UniversityHadassah Medical School, Jerusalem, Israel Francesca Biandolino (165), CNR—Institute of Coastal Marine Environment (IAMC), Taranto, Italy Marialaura Bonaccio (589), IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli, Italy Santiago Bonachela, PhD (23), University of Almerı́a ceiA3, Almerı́a, Spain Nadia Calabriso, PhD (135, 291), C.N.R. Institute of Clinical Physiology, Lecce, Italy; C.N.R. Institute of Clinical Physiology, Pisa, Italy Oguzhan Caliskan (621, 629), Department of Horticulture, Mustafa Kemal University, Antakya-Hatay, Turkey Maria Annunziata Carluccio, PhD (135, 291), C.N.R. Institute of Clinical Physiology, Lecce, Italy; C.N.R. Institute of Clinical Physiology, Pisa, Italy J. Jesús Casas, PhD (23), University of Almerı́a ceiA3, Almerı́a, Spain Rosa Casas, Predoctoral Student (153), Department of Internal Medicine, Hospital Clı́nic, Institut d’Investigacions Biomédiques August Pi i Sinyer (IDIBAPS), University of Barcelona, Barcelona, Spain; CIBER CB06/03 Fisiopatologı́a de la Obesidad y la Nutrición, (CIBERobn), Girona, Spain Itandehui Castro-Quezada, MSc (13), University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain; CIBER Fisiopatologia Obesidad y Nutrición, Instituto de Salud Carlos III, Lourdes, Spain Gemma Chiva-Blanch, PhD (153), Department of Internal Medicine, Hospital Clı́nic, Institut d’Investigacions Biomédiques August Pi i Sinyer (IDIBAPS), University of Barcelona, Barcelona, Spain; CIBER CB06/03 Fisiopatologı́a de la Obesidad y la Nutrición, (CIBERobn), Girona, Spain Gea Oliveri Conti, MSc, PhD (547), Department of Hygiene and Public Health “G.F. Ingrassia,” Laboratory of Environmental Hygiene and Food (LIAA), University of Catania, Catania, Italy xvii xviii Contributors Chiara Copat, MSc, PhD (547), Department of Hygiene and Public Health “G.F. Ingrassia,” Laboratory of Environmental Hygiene and Food (LIAA), University of Catania, Catania, Italy Roberto Fallico, MD (547), Department of Hygiene and Public Health “G.F. Ingrassia,” Laboratory of Environmental Hygiene and Food (LIAA), University of Catania, Catania, Italy Patrick Couture, MD, PhD (325), Institute of Nutrition and Functional Foods, Laval University, Québec QC, Canada Paul Farajian, PhD (69), Agricultural University of Athens, Athens, Greece Lorenzo Covarelli, PhD (563), Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy Encarnación Fenoy, MD (23), University of Almerı́a ceiA3, Almerı́a, Spain Vincenzo De Feo (649), Università degli Studi di Salerno, Fisciano (Salerno), Italy J.M. Fernandez, PhD (513), Lipids and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital/ University of Córdoba and CIBER Fisiopatologı́a Obesidad y Nutrición (CIBEROBW), Health Institute Carlos III, Madrid, Spain Giovanni de Gaetano, MD, PhD (589), IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli, Italy Maria Luz Fernandez, PhD (357), University of Connecticut, Mansfield, CT, USA Michel de Lorgeril, MD (337), Faculté de Médecine, Grenoble, France José Manuel Fernández-Real, PhD (505), Hospital Dr. Josep Trueta, Girona, Spain Laura De Martino (649), Università degli Studi di Salerno, Fisciano (Salerno), Italy Margherita Ferrante, MD (547), Department of Hygiene and Public Health “G.F. Ingrassia,” Laboratory of Environmental Hygiene and Food (LIAA), University of Catania, Catania, Italy Raffaele De Caterina, MD, PhD (291), “G. d’Annunzio” University, Chieti, Italy Christiana A. Demetriou, PhD (407), Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Department of Epidemiology and Biostatistics, School of Public Health, London, UK Elizabeth Fragopoulou, PhD (379), Harokopio University, Athens, Greece Emmanuel J. Diamantopoulos, MD, PhD (313), Evangelismos State General Hospital, Athens, Greece F. Fuentes-Jimenez, MD, PhD (513), Lipids and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital/ University of Córdoba and CIBER Fisiopatologı́a Obesidad y Nutrición (CIBEROBW), Health Institute Carlos III, Madrid, Spain Zora Djuric (451), University of Michigan, Ann Arbor, MI 48118, USA José J. Gaforio, MD, PhD (205, 281), University of Jaén, Jaén, Spain Maria Benedetta Donati, MD, PhD (589), IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli, Italy Marta Garaulet, PhD (237), University of Murcia, Murcia, Spain Jorge Doreste-Alonso (61), University of Las Palmas de Gran Canaria, Madrid, Spain Vanessa Garcia-Larsen, PhD (123), Respiratory Epidemiology, Occupational Medicine, and Public Health, National Heart and Lung Institute, Imperial College London, London, UK Paraskevi Detopoulou, PhD (379), General Hospital Korgialenio-Benakio, Athens, Greece Karima El Rhazi, MD, PhD (123), Department of Epidemiology and Public Health, Faculty of Medicine of Fes, University Sidi Mohamed Ben Abdillah, Fes, Morocco Sahar Elsayed El-Swefy (441), Faculty of Pharmacy, Zagazig University, Zagazig, Sharkia Gov., Egypt Manuel Espárrago Rodilla, PhD (491), Hospital de Mérida, Mérida, Badajoz, Spain Ramon Estruch, MD, PhD (153), Department of Internal Medicine, Hospital Clı́nic, Institut d’Investigacions Biomédiques August Pi i Sinyer (IDIBAPS), University of Barcelona, Barcelona, Spain; CIBER CB06/03 Fisiopatologı́a de la Obesidad y la Nutrición, (CIBERobn), Girona, Spain Hannah Gardener, ScD (345), University of Miami Miller School of Medicine, Miami, FL, USA Eva Gesteiro (491), Hospital de Mérida, Mérida, Badajoz, Spain Mark R. Goldstein, MD, FACP (259), NCH Physician Group, Naples, FL, USA Carlos A. González, MD, PhD (417), Catalan Institute of Oncology (ICO-Idibell), Barcelona, Spain Evanthia Gouveri, MD (313), Evangelismos State General Hospital, Athens, Greece Contributors xix Sergio Granados, PhD (205), The Methodist Hospital Research Institute, Houston, TX, USA Luca Mascitelli, MD (259), Comando Brigata alpina “Julia”, Multinational Land Force, Udine, Italy Keith Grimaldi, PhD (105), National Technical University of Athens, Athens, Greece Marika Massaro, PhD (291), C.N.R. Institute of Clinical Physiology, Pisa, Italy Andreas Hadjisavvas, PhD (407), Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus F. Xavier Medina (37), Universitat Oberta de Catalunya (UOC), Barcelona, Spain Patricia Henrı́quez-Sánchez (61), University of Las Palmas de Gran Canaria, Madrid, Spain; Instituto de Salud Carlos III, Madrid, Spain Joaquı́n Hernández, PhD (23), University of Almerı́a ceiA3, Almerı́a, Spain Licia Iacoviello, MD, PhD (589), IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli, Italy Marcello Iriti, PhD (143, 199), Milan State University, Milan, Italy Dimitra Karageorgou, MSc (91), Agricultural University of Athens, Athens, Greece Niki Kontou, RD, PhD (393), Saint Savvas Cancer Hospital, Athens, Greece; Harokopio University, Athens, Greece Kyriacos Kyriacou, PhD (407), Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus Guiomar Mendieta (367), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; IIB-Santpau, Barcelona, Spain Marta Mesı́as, PhD (185), Consejo Superior de Investigaciones Ciencı́ficas, (CSIC), Spain Renata Micha, PhD (91), Agricultural University of Athens, Athens, Greece; Harvard School of Public Health, Boston, MA, USA José Marı́a Moreno-Navarrete, PhD (505), Hospital Dr. Josep Trueta, Girona, Spain Francisco J. Moyano, PhD (23), University of Almerı́a ceiA3, Almerı́a, Spain M. Pilar Navarro, PhD (185), Consejo Superior de Investigaciones Ciencı́ficas, (CSIC), Spain Filomena Nazzaro (649), Istituto di Scienze dell’Alimentazione, Avellino, Italy Giuseppe La Torre, MD, MSc, DSc (533), Sapienza University of Rome, Rome, Italy Chakib Nejjari, MD, PhD (123), Department of Epidemiology and Public Health, Faculty of Medicine of Fes, University Sidi Mohamed Ben Abdillah, Fes, Morocco Denis Lairon, PhD (303), Aix-Marseille University, Marseille, France Lena Maria Nilsson, PhD (579), Umeå University, Umeå, Sweden Benoı̂t Lamarche, PhD (325), Institute of Nutrition and Functional Foods, Laval University, Québec QC, Canada Tzortzis Nomikos, PhD (379), Harokopio University, Athens, Greece Jaime Lee (81), Swinburne University, Hawthorn, VIC, Australia Francesca Oliviero, PhD (461), Department of Medicine— DIMED, University of Padova, Padova, Italy Maria A. Loizidou, PhD (407), Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus Ilkay Erdogan Orhan (639), Gazi University, Ankara, Turkey; Eastern Mediterranean University, Famagusta, Turkey Alicia López-Biedma, MSc (281), University of Jaén, Jaén, Spain Antonio Paoli, MD, BSc (105, 271), University of Padova, Padova, Italy Helen Macpherson, PhD (81), Swinburne University, Hawthorn, VIC, Australia Christopher Papandreou, PhD (429), Harokopio University of Athens, Athens, Greece Giuseppe Maiani (249), National Institute for Food and Nutrition Research, Rome, Italy Matthew Pase, PhD (81), Swinburne University, Hawthorn, VIC, Australia Emilia Mancini (649), Università degli Studi di Salerno, Fisciano (Salerno), Italy Mariangela Pellegrino, PhD (135), C.N.R. Institute of Clinical Physiology, Lecce, Italy Colin R. Martin (519), Faculty of Health, Uxbridge, UK Andrew Pipingas, PhD (81), Swinburne University, Hawthorn, VIC, Australia Miguel Ángel Martı́nez-González (61), Instituto de Salud Carlos III, Madrid, Spain; University of Navarra, Madrid, Spain Concetta Potenza, MD, MSc (533), Sapienza University of Rome, Rome, Italy xx Contributors Ermelinda Prato (165), CNR—Institute of Coastal Marine Environment (IAMC), Taranto, Italy Cristina Sánchez-Quesada, MSc (281), University of Jaén, Jaén, Spain Victor R. Preedy (519), King’s College London, London, UK Mario Pulido-Moran, MSc (205), Granada University, Granada, Spain Almudena Sánchez-Villegas (61), University of Las Palmas de Gran Canaria, Madrid, Spain; Instituto de Salud Carlos III, Madrid, Spain Leonardo Punzi, MD, PhD (461), Department of Medicine—DIMED, University of Padova, Padova, Italy Rosella Saulle, MD, MSc (533), Sapienza University of Rome, Rome, Italy José L. Quiles, PhD (205), Granada University, Granada, Spain Andrew Scholey, PhD (81), Swinburne University, Hawthorn, VIC, Australia Ma del Carmen Ramı́rez-Tortose, PharmD, PhD (205, 281), University of Granada, Granada, Spain Salvatore Sciacca, MD (547), Department of Hygiene and Public Health “G.F. Ingrassia,” Laboratory of Environmental Hygiene and Food (LIAA), University of Catania, Catania, Italy Caroline Richard, RD, PhD (325), Institute of Nutrition and Functional Foods, Laval University, Québec QC, Canada Beatriz Rodrı́guez Bernal (491), Universidad Complutense de Madrid, Madrid, Spain Blanca Román-Viñas, MD, PhD (13), CIBER Fisiopatologia Obesidad y Nutrición, Instituto de Salud Carlos III, Lourdes, Spain; Fundación para la Investigación Nutricional, University of Barcelona Science Park, Barcelona, Spain Emilio Ros, MD, PhD (175), Lipid Clinic, Endocrinology & Nutrition Service, Institut d’Invesigacions Biomèdiques, August Pi Sunyer, Hospital Clinic, Barcelona, Spain; CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III (ISCIII), Barcelona, Spain D. Rosado-Alvarez, MD, PhD (513), Lipids and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital/ University of Córdoba and CIBER Fisiopatologı́a Obesidad y Nutrición (CIBEROBW), Health Institute Carlos III, Madrid, Spain José-Luis Rı́os (611), Universitat de València, Burjassot, València, Spain Cristina Ruano (61), University of Las Palmas de Gran Canaria, Madrid, Spain; Instituto de Salud Carlos III, Madrid, Spain Semra Akar Sahingoz, PhD (115), University of Gazi, Ankara, Turkey Patricia Salen, BSc (337), Faculté de Médecine, Grenoble, France José L. Sánchez Benito, MD (519), Vocalia de Alimentación y Nutrición del Colegio de Farmacéuticos de COFM, Madrid, Spain Eva Sánchez Soriano, MD (519), Nurse Hospital Puerta de Hierro, Madrid, Spain Francisco J. Sánchez-Muniz, PhD (217, 491), Universidad Complutense de Madrid, Madrid, Spain Egeria Scoditti, PhD (135, 291), C.N.R. Institute of Clinical Physiology, Lecce, Italy; C.N.R. Institute of Clinical Physiology, Pisa, Italy Isabel Seiquer, PhD (185), Consejo Superior de Investigaciones Ciencı́ficas, (CSIC), Spain Leda Semyonov, MD (533), Sapienza University of Rome, Rome, Italy Lluı́s Serra-Majem, MD, PhD (13, 37, 61), University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain; CIBER Fisiopatologia Obesidad y Nutrición, Instituto de Salud Carlos III, Lourdes, Spain; Fundación para la Investigación Nutricional, University of Barcelona Science Park, Barcelona, Spain; International Foundation of Mediterranean Diet, London, UK; University of Las Palmas de Gran Canaria, Madrid, Spain; Instituto de Salud Carlos III, Madrid, Spain Dean A. Sewell, BA, PhD (473), Heriot-Watt University, Edinburgh, United Kingdom Paolo Sfriso (461), Department of Medicine—DIMED, University of Padova, Padova, Italy Aziz Sheikh, BSc, MSc, MD (473), The University of Edinburgh, Edinburgh, United Kingdom Nevena Skroza, MD, MSc (533), Sapienza University of Rome, Rome, Italy Paolo Spinella, MD (461), Department of Medicine— DIMED, University of Padova, Padova, Italy Laura Tosi, PhD (563), Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy Ibrahim Tumen (639), Bartin University, Bartin, Turkey Palmira Valderas-Martinez, Predoctoral Student (153), Department of Internal Medicine, Hospital Clı́nic, Institut d’Investigacions Biomédiques August Pi i Sinyer (IDIBAPS), University of Barcelona, Barcelona, Contributors xxi Spain; CIBER CB06/03 Fisiopatologı́a de la Obesidad y la Nutrición, (CIBERobn), Girona, Spain Paolo Vineis, MD, PhD (407), Department of Epidemiology and Biostatistics, School of Public Health, London, UK Elena M. Varoni, DD, PhD (143, 199), Milan State University, Milan, Italy; McGill University, Montreal, QC, Canada; University of Eastern Piedmont ‘A. Avogadro’, Novara, Italy Fernando Warleta, BSc (281), University of Jaén, Jaén, Spain Gemma Vilahur (367), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; IIB-Santpau, Barcelona, Spain; CIBEROBN Instituto de Salud Carlos III, Madrid, Spain Clinton B. Wright, MD, MSc (345), University of Miami Miller School of Medicine, Miami, FL, USA Leo R. Zacharski, MD (259), Veterans Affairs Hospital, White River Junction, VT, USA; Geisel School of Medicine at Dartmouth College, Hanover, NH, USA Lorena Villalon (81), Swinburne University, Hawthorn, VIC, Australia Antonis Zampelas, PhD (69, 91), Agricultural University of Athens, Athens, Greece Gemma Xifra Villarroya (505), Hospital Dr. Josep Trueta, Girona, Spain Sara Zuber, MD (533), Sapienza University of Rome, Rome, Italy Preface The Mediterranean diet is frequently considered a nutritional “elixir” that reduces risk factors associated with disease. Hitherto, there has been a great deal of ambiguity about the evidence supporting the concept of the Mediterranean diet, and there have been many unfounded myths associated with its composition and the therapeutic effectiveness of adopting such a dietary regimen. However, the past few decades have seen major advances in the understanding of the Mediterranean diet and its application. Unfortunately, details of the Mediterranean diet are fragmentary or unfocused with little attention to its composition, ethos, and potential applications. These limitations have been succinctly addressed in The Mediterranean Diet: An Evidence-Based Approach. This book is an authoritative synopsis of many of the complex features of the Mediterranean diet, ranging from its historical basis, supportive evidence and epidemiological studies, to the antioxidant, anti-inflammatory, hypolipidemic and other properties of individual components. This book embraces a holistic approach and effectively investigates the Mediterranean diet from the cell level to the nutritional well-being of geographical populations. The book is divided into 4 sections: Section Section Section Section 1: The Mediterranean Diet: Concepts and General Aspects 2: Components of the Mediterranean Diet 3: Health and Nutritional Aspects of the Mediterranean Diet 4: Novel Nutraceuticals and Edible Plants Used in the Mediterranean Region Section 1 begins with material on the Mediterranean diet with respect to origins and evolution, nutritional adequacy, agricultural practices, sustainability, mortality, quality of life, children and adolescents, cognitive health, cardiovascular diseases, genomics, diet quality, nutritional knowledge and socio-economic features. There follows in Section 2 coverage of olive oil, wine, beer, fish, nuts, minerals, melatonin, hydroxytyrosol and frying. Section 3 has material on metabolism, metabolic syndrome, obesity and diabetes, cardiovascular disease, cancer, brain and behavior, immunology, life stages, organs and applications, adverse aspects, and methods for interventions with, or adherence to, the Mediterranean diet. Finally, Section 4 deals with genetic diversity of plants, apoptotic activities of plant species, hawthorn fruit, figs, Cupressus sempervirens and essential oils. Contributors are authors of international and national standing, leaders in the field and trendsetters. Emerging fields of the Mediterranean Diet science and important discoveries relating to diet and nutrition are included here. This book represents essential reading for nutritionists, dieticians, health care professionals, research scientists, biochemists, physicians, general practitioners, and public health practitioners, as well as those interested in health in general. Victor R. Preedy Ronald Ross Watson xxiii Chapter 1 On the Origins and Evolution of the Mediterranean Diet Yardena Arnoni, MSc and Elliot M. Berry, MD, FRCP Hebrew University-Hadassah Medical School, Jerusalem, Israel. INTRODUCTION Ample empirical evidence exists for the health benefits of the Mediterranean diet. The nutritional benefits of the foods incorporated in the modern Mediterranean diet have been well elucidated, and a plethora of literature has been published to support the claims of health and disease prevention [1]. Yet the historical and biblical origins of the Mediterranean diet remain to be emphasized. While the name itself attests to the geographical location, the composition of the original Mediterranean diet needs to be considered in its original environment. The word diet is derived from the Greek diaeta, meaning not just food but “way of life.” The Mediterranean diet is an interwoven, multicultural labyrinth that has developed over time, absorbing the richness of the diverse cultures of different occupiers/conquerors while maintaining original local traditions. The term Mediterranean diet was coined in 1960 by the American physiologist Ancel Keys and his wife Margaret in their book How to Eat Well and Stay Well the Mediterranean Way [2]. They identified the eating behaviors of countries such as Greece—Crete in particular—and southern Italy, with extension to other geographical areas around the Mediterranean basin. Based on their pioneer studies, the dietary patterns of these countries were associated with longevity and reduced rates of coronary heart disease morbidity and mortality, cancers, and other chronic diseases related to diet in the 1960s [3,4]. The Mediterranean diet, however, did not begin in the 1960s but extends further back to biblical times. GEOGRAPHIC AND EVOLUTIONARY HISTORY The world of the biblical Mediterranean diet covers regions of land connected to a sea and extending to an ocean. The geographic and evolutionary origins of the diet are of interest because they encompass the history of Western civilization. The rise of agriculture (domestication of crops) and animal husbandry took place from ca. 10,000 to 4000 BCE, whereas the wheel, metallurgy, writing, and city-states arose from ca. 4000 to 1000 BCE. These essential developments in the evolution of civilization originated in the Fertile Crescent: from Mesopotamia, the ancient Near East, Canaan, and Egypt. The biblical period from the time of the patriarchs until King David in Jerusalem was from ca.1950 to 1000 BCE; the fall of Troy and the Greek colonization of Ionia occurred in the twelfth century BCE and David’s capture of Jerusalem in ca. 1000 BCE. Evidence of dietary patterns has been obtained from archaeo-botany and written records. The origins and spread of domesticated grains has been traced to the Fertile Crescent, spreading from Mesopotamia (the cradle of civilization) [5–7] (Figures 1 and 2). It seems that such food use reached the Middle East before the Greek islands. While there are many similarities between the traditional Greek (Cretan) Mediterranean diet and that described in the Pentateuch, the differences are of interest because they have special nutritional benefits. The Bible speaks of the seven species—wheat, barley, grapes, figs, pomegranates, olives, and honey—as well as a land “flowing with milk and (date) honey” (Deuteronomy 8:8), which are, coincidently, the basic staples of Mediterranean cuisine. In Roman times, historians recorded that the produce of the land of Israel was of particularly high quality and was served at the best tables. Such cross-cultural influences may be seen during the Last Supper, which possibly was a Passover seder and was itself adapted from the Greek symposium brought to the region after the conquests of Alexander the Great in the fourth century BCE. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 3 4 SECTION 1 The Mediterranean Diet: Concepts and General Aspects U.S.S.R. FIGURE 1 Distribution of wild emmer (Triticum dicoccoides) in the Near East. Solid circles represent known sites in which wild emmer is common [7]. Reprinted with permission from Science. TURKEY SYRIA LEBANON IRAQ JO R DA N ISRAEL FIGURE 2 Distribution of known and reasonably certain sites of wild barley [7]. Reprinted with permission from Science. ORIGINS OF THE MEDITERRANEAN DIET AND THE BIBLICAL DIET The strength of the Mediterranean diet is its connection to ancient biblical culture; it is not a transitional trend [8]. The origin of the Mediterranean diet encompasses the history of Western civilization. In its traditional form, the food in the region was eaten according to the season, dictated by climate and agriculture and over and above commemoration of landmarks such as annual festivals. Origins of the Mediterranean Diet Chapter 1 5 Countries were influenced by factors of religion and tradition, economics and foreign governing powers. Eating in the Mediterranean culture surpasses the indispensable consumption of calories for energy. The social element of eating elevated meals to become more than just a means of fulfilling biological (animal) instincts: “We do not sit at the table only to eat, but to eat together” (quote attributed to Plutarch, 46–120 CE). On some occasions meals had a religious status, as seen in the Jewish Sabbath, New Moon, and festival meals. These feasts were of extended duration, with special ritual foods to mark the occasions. The dietary laws of the Bible forbade mixing milk with meat (Exod. 23:19 and 34:26; Deut. 14:21) and proscribed eating pig and shellfish, allowing only animals with a cloven hoof and that “chewed the cud” (Lev. 11:3), while fish had to have both fins and scales (Lev. 11:9). The reasons for this are not clear but might relate to eating only those animals that were preyed upon and not predators. Meat is neither common nor outlawed in the ancient Mediterranean diet. In biblical times, meat was generally eaten only on special occasions, and in later Christian tradition lamb was part of the Easter meal. In the Jewish religion the law institutes certain obligations and duties regarding the consumption of animals. The animal is killed without suffering by a trained authority. Blood cannot be consumed; only ruminant mammals and certain birds are deemed kosher. In the Christian tradition, the New Testament put an end to the Jewish prohibitions on food but maintained the ritual of avoiding gluttony and greed. In Islam certain prohibitions related to meat (including not eating pork) are enforced for it to be ordained Halal and suitable for consumption, whereas wine is forbidden. In the biblical land “flowing with milk and (date) honey” the seven species (Exod. 3:8) were given high priority. Archeological excavations have uncovered these products eaten during the Minoan period and dating back to the Bronze Age civilization that arose on the island of Crete (2700–1450 BCE). Before this, in the Copper Age, plants and food from the sea were almost the sole sources of nutrients. MEDITERRANEAN LIFESTYLE Human genetic profiles have not changed significantly over the past 10,000 years, whereas lifestyle has been revolutionized. Modern industrialized populations are characterized by reduced energy expenditure and increased energy intake. Fat intake in the form of trans and saturated fats has increased, and there is a decreased intake of fiber, complex carbohydrates, fruits and vegetables (vitamins and antioxidants), protein, and calcium [9]. In the United States there were three times more deaths due to cancer and coronary heart disease than in Crete [10], and this differential has only been increasing. The Lyon Heart Study adapted the Cretan diet for the French population and showed cardioprotective and anticancer effects, thereby demonstrating that the Mediterranean diet can be modified to suit other populations [11]. Current trends of eating while watching television promote unhealthy, quick meals and exclude social/family communication. Other factors that may contribute to the Mediterranean lifestyle include a relaxing psychosocial environment, mild climate, preservation of the extended family structure, and even a siesta [12], as well as regular activity, mainly through walking [13]. Sleep and exercise deficiencies have been correlated with chronic illnesses such as diabetes and heart disease [14]. COMPONENTS OF THE ORIGINAL MEDITERRANEAN DIET A homogeneous Mediterranean diet is hard to deduce; each Mediterranean country brings its unique culture, history, and tradition to its food and eating. Diversity notwithstanding, Mediterranean regions share certain common dietary practices that remain a firm foundation for the food consumed that is responsible for health benefits. The Keys’ research highlighted the three primary components of the Mediterranean diet: olive oil, wine, and bread. The generic Mediterranean diet is characterized by a high monounsaturated fat-to-saturated fat ratio, low total fat (<30%), low saturated fat (<10%), moderate alcohol intake (essentially wine), and high intake of vegetables, fruits, legumes, and grains (complex carbohydrates and dietary fiber) [15]. Contemporary adjustments in the Mediterranean cuisine have occurred as a result of new products and influences stretching from Asia to America with the introduction of tomatoes, potatoes, cornbeans, and cane and sugar, which, while widespread in modern cuisine, are not native to the region. The following sections describe the health benefits of different components of the biblical diet in addition to the conventional Mediterranean diet recommended today. THE BIBLICAL SEVEN SPECIES The biblical seven species—wheat, barley, grapes, figs, pomegranates, olives, and date honey—together with some indigenous foods from the Middle East, are now scientifically recognized as healthy foods, and the addition of some of these could further enhance the benefits of the Mediterranean diet. Tables 1 and 2 detail some of the biblical and historical references to the seven species. 6 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 1 Some Selected Biblical References to Fruits and Vegetables in the Mediterranean Diet Food Location Ancient Use Olives Hills of Galilee and Samaria Grown in the summer dry season (Deuteronomy 11:4) Olive oil was used in religious ceremonies: as an offering to God in the Tabernacle (Exodus 29:40), in anointing of priests and kings (Exodus 29:7, Exodus 30:30, Leviticus 8:12, I Samuel 16:13, I Kings. 1:39, Psalms 23:5) Symbol of joy “oil of gladness” (Psalms 45:7) Symbol of peace “And the dove came back to him in the evening, and behold, in her mouth was a freshly plucked olive leaf” (Genesis 8:11) Cosmetic use: “Let not oil be lacking on your head” (Ecclesiastes 9:8) Medicinal benefit (Isaiah 1:6, Mark 6:13, Luke 10:34) “a green olive tree, beautiful with good fruit” (Jeremiah11:16) The trees once went out to anoint a king over them, and they said to the olive tree, ‘Reign over us.’ But the olive tree said to them, ‘Shall I leave my abundance, by which gods and men are honored, and go hold sway over the trees?’ (Judges 9:8–9) Grapes Southern plains and Judean hills Grown in the summer dry season (Deuteronomy 11:4) Wine: social, cultural and nutritional value “makes life merry” (Ecclesiastes 10:19) and gladdens the heart (Psalms 104:15) “Grapes. . .with some of the pomegranates and the figs” (Numbers 3:23) “wisdom has prepared her meat and mixed her wine” (Proverbs 9:1–6) Importance of wine shown by Noah “Noah began to be a man of the soil, and he planted a vineyard” (Genesis 9:20) Wine as a symbol of God’s care of His People “Song of the Vineyard” (Isaiah 5) Vineyards as a symbol of peace as in the reign of Solomon “And Judah and Israel lived in safety, from Dan even to Beersheba, every man under his vine and under his fig tree, all the days of Solomon” (I Kings 4:25) and “land of grain and vineyard” (II Kings 18:32) Wine was red deduced from reference connecting wine to blood “drank foaming wine made from the blood of the grape” (Deut. 32:14) Jesus at the Last Supper “this is my blood” (Matthew 26:27–28) Medicinal use: “bound up his wounds, pouring on oil and wine” (Luke 10:34) “use a little wine for the sake of your stomach and your frequent ailments” (I Tim. 5:23) Moderation emphasized in Bible against intoxication “And do not get drunk with wine, for that is debauchery” (Ephesians 5:18) and (I Peter 4:3) Figs Hills of Samaria Grown in the summer dry season (Deuteronomy 11:4) High sugar content as a sweetener “good and sweet” (Judg. 9:11) and source of energy “cake of pressed figs and two cakes of raisins. He ate and was revived” (I Samuel 30:12) Medicinal use “Prepare a poultice of figs and apply it to the boil”(Isaiah 28:21) “. . .a hundred cakes of figs and a skin of wine” (2 Samuel 16:1) Destruction of fig trees prophetic symbol of destruction of the land “they shall eat up your vines and your fig trees” (Jer. 5:17, 8:13, Hos. 2:12) Dates Jericho High sugar content used as honey “a land flowing with milk and honey” (Exodus 3:8, Deuteronomy 26:15) “a little honey, some spices and myrrh, some pistachio nuts, and almonds” (Gen. 43:11) Medicinal use “use as a laxative and strengthen the body” (Babylonian Talmud Tractate Ketuboth) Spiritual symbol on the Temple “. . .carved engraved figures of cherubim and palm trees. . .” (I Kings 6:29) Symbol of righteous characteristics “The righteous flourish like the palm tree” (Psalms 92:12) and as one of the Four Species in the Lulav (palm branch) Roman conquerors of Palestine embossed the date palm on coins minted in celebration of the victory over the Jews and destruction of the Temple in 70 CE Symbol of life in the desert where it was a sign that water was available “they took branches of palm trees and went out to meet him” (John 12:13) Pomegranates Hills of Galilee and Samaria Religious symbolism High priest’s robe (Exodús 28. 33–34) Solomon’s Temple ornaments (I Kings 7:18) Symbol of beauty “Your cheeks are like halves of a pomegranate behind your veil” (Song of Songs 6:7) Origins of the Mediterranean Diet Chapter 1 7 TABLE 2 Some Selected Biblical References to Grains in the Mediterranean Diet Food Location Ancient Use Barley Near East, sown along the shores of the Mediterranean and Judean hills “They came to Bethlehem at the beginning of the barley harvest” (Ruth1:22) Poor people’s food and was the last remaining grain in times of famine. (Judges 7:13; 2 Kings 4:42) Wheat Southern plains Passover “they baked cakes of unleavened bread” (Exodus 12:39) Fundamental basic food “. . . all support of bread” (Isaiah 3:1) and Ezekiel 4:16 As well as the food of kings (II Kings 25:29) Spiritual symbol: “priest gave him the holy bread, for there was no bread there but the bread of the Presence” (I Samuel 21:6) The Showbread (12 loaves) were placed on a gold table in the Temple (Leviticus 24:5) Blessing over bread was given by Jesus at the Last Super “Jesus took bread, and after blessing it broke it and gave it to the disciples” (Matthew 26:26) Grains The pivotal role of bread is further emphasized by its important status and use during festivals. In the tenth century BCE, carvings on limestone describe the harvest seasons in the land of Israel according to the Gezer calendar, which is a rhythmic enumeration of the agricultural seasons. In the dry summer months vines were pruned; figs, dates, pomegranates, and grapes ripened; and the wheat was harvested, whereas in the spring season barley was harvested. Wheat flour and grain have provided the staple (breads, pitas, etc.) for different types of meals throughout the Mediterranean basin. Fresco wall paintings at Knossos show the prime role of bread in the Cretan diet. It is the basic food, par excellence, and is at the center of food culture in addition to its great religious meaning and social value. Cereals include wheat, barley, rye, spelt, and oats. Barley, as opposed to wheat, contains little gluten, is coarse, and is tough to chew. The method of preparation of grains was inventive, including kneading, fermenting, drying, baking, and cooking. Means of consumption were diverse: leavened or unleavened bread, cornmeal (Italian polenta), and porridge (Spanish gachas), semolina, and pasta [16]. The process of bread production in ancient times was an intense, complex process that differentiated between domesticated man and the nomad. Olives The olive in the form of fruit and its derivative, olive oil, is arguably the primary element of the Mediterranean diet, maintaining its position from biblical to modern times. Definitions of the Mediterranean basin have even been ascribed to regions where olive trees grow. When saturated fats are replaced with monounsaturated fats in the human diet, plasma cholesterol concentrations improve [17]. In murine models of atherogenesis, the most impressive beneficial effect is shown for extra virgin oil enriched with green tea polyphenols [18]. In ancient times olives were consumed by farmers and carried by travelers and nomads; furthermore, olives were a popular appetizer. Romans served olives as starters and desserts in their rich banquets. The majority of fat in olive oil contains monounsaturated fatty acids. Fat from edible olives and olive oil consists of oleic acid [c18:1, n 9] (75%), saturated fat (15%), and polyunsaturated fat (10%). Olive oil is extracted from deeply pigmented olives that are rich in phytonutrients, including the phenolics hydroxytyrosol and oleorupein. In biblical times, olive oil was cold pressed and stored in dark, opaque glass containers as a means to protect the taste from what is now known as the powerful oxidative action of sunlight. Unprocessed olive oil has the greatest antioxidant effect. Comparison of extracted phenolic compounds from extra virgin olive oil and processed olive oil showed that extra virgin olive oil has significantly greater antioxidant effects than processed olive oil [19]. The unprocessed extra virgin olive oil of the biblical diet had a higher concentration of antioxidants, which may prevent low-density lipoprotein (LDL) oxidation. In addition to the advantageous effects on blood cholesterol, olive oil also has anticarcinogenic actions [20]. It seems, therefore, that some of the phenolic content and health benefits are reduced during the modern process of olive oil refinement. Dates In the Persian Gulf, going back to biblical days, dates have remained a prominent source of sweetness in the Mediterranean cuisine. The date was a convenient source of nutrients for nomads and travelers. Fruits of the date palm (Phoenix dactylifera 8 SECTION 1 The Mediterranean Diet: Concepts and General Aspects L. arecaceae) are an important dietary component in the Middle East and north Africa. Dates are an ideal high-energy food because they contain high sugar content. They are also a good source of fiber and minerals such as calcium, iron, magnesium, potassium, and zinc [21]. Date fruit is used in folk medicine for the treatment of various infectious diseases and cancer [22] because of the apparent immunomodulatory activity [22], antibacterial capacity [23], and antifungal properties that have become evident with use [24]. Furthermore, aqueous extracts of dates have been shown to have potent antioxidant activity [25], since they inhibit in vitro lipid and protein oxidation, and possess substantial free radical scavenging capacity. Pomegranates The pomegranate has been cultivated in the Mediterranean region since ancient times and was introduced into Egypt from Syria and from Israel around 1600 BCE. In the Bible, the coat of the high priest was adorned with pomegranates (Exod. 39:34–26). There are many quotations concerning this luscious fruit, especially in the Song of Songs: “As a piece of pomegranate are thy temples” (6:7); “I would cause thee to drink of spiced wine and the juice of my pomegranate” (Song of Sol. 8:2). In Greek mythology, the pomegranate was a symbol of life and rejuvenation. It has potent antioxidants, including ellagitannin polyphenolic compounds such as punicalagins and punicalins, as well as ellagic acid and gallic acid. Pomegranates have been shown to reduce LDL oxidation [26] and to decrease the progression of prostate cancer [27]. Furthermore, drinking just 50 mL of pomegranate juice daily for 3 months can significantly lower blood pressure by 5%. Pomegranate is a major source of most potent antioxidants (tannins, anthocyanins), and leaf extracts from pomegranate may also be effective in weight loss since pomegranate consumption reduces fat absorption from the intestine without affecting plasma triglycerides concentrations and is a potent nutraceutical agent against cardiovascular disease [28,29]. Figs The fig is the fruit of lust and is believed to be an omen for fertility. The large amount of fiber in figs stimulates bowel movement. Excavations at Gezer uncovered remains of dried figs from the Neolithic Age, and an old seed was germinated recently from a deposit near the Dead Sea [30]. Figs are native to the Mediterranean, growing on the ficus tree (Ficus carica), and were one of the first fruits to be cultivated. The fruit is rich in sucrose and simple sugars, minerals, and fibers and is a good source of potassium, calcium, magnesium, iron, copper, and manganese. Dried figs are popular because they last for a long time and have a high calcium content (250 mg calcium/100 g dried fruit weight). Grapes Vine cultivation and wine production originated in Mesopotamia. The culture of wine consumption, however, belongs to the Mediterranean. An Egyptian inscription from 2375 BCE records how a military governor, Uni, sent troops to put down a revolt in Israel during the reign of Pharaoh Pepi I and how they “destroyed the fortresses. . . and felled the fig trees and vines.” A mural from the reign of Amenopsis II (fifteenth century BCE) shows the preparation of wine in Egypt by the Apirou, considered by some to be a reference to the Hebrews [31]. From early Egyptian civilization through the times of the Roman Empire, wine has been of importance, especially to the civilized elite. Consumption of wine in the Mediterranean diet is subject to different cultural norms, especially in Muslim countries that prohibit wine intake. Hence, during the period of Arab rule, vineyards in occupied regions deteriorated. Red wine is rich in antioxidants from the flavonoid phenolics family, including cathechin, querchitin, anthocyanins, and resveratrol. Resveratrol is a trihydroxystilbene phenolic compound found in grape seeds and skins, and it was shown to increase blood high-density lipoprotein cholesterol, to protect against LDL oxidation, and to attenuate blood clotting [32]. Resveratrol has been reported to have antiaging effects and be protective against carcinogenesis [33]. In the Mediterranean culture, wine is consumed as part of a meal, whereas Western cultures may consume wine independent of meals. Alcohol consumption unaccompanied by food leads to rapid alcohol absorption and increases the risk of intoxication. It is of interest that the Rambam (Maimonides) noted the health benefits of wine more than 800 years ago [34]. The lower antioxidant activity of white wines compared to red wines lies in the reduced amount of polyphenols extracted from the grape skin; red wine is prepared after spending a long time (1 month) in contact with the grape skin. The potent antioxidant activity of an Israeli red wine also was demonstrated in a study of a UK wine, where the antioxidant capability of the UK wine was lower than that of the Israeli wine. Flavonols are potent polyphenolic antioxidants, and this may explain the above results. There is wide variation in the flavonol content of different red wines Origins of the Mediterranean Diet Chapter 1 9 throughout the world, and the amount of sunlight to which the grapes are exposed during cultivation, when flavonols are synthesized, is a major determinant of production of flavonols in grapes. Thus, the climatic conditions under which grapes are grown could explain the fivefold increased content of flavonols in and the higher antioxidant potency of the specific Israeli red wine studied compared to the UK wine [35]. ADDITIONAL BIBLICAL FOODS The Mediterranean diet, particularly that from Crete, has a balanced intake of polyunsaturated essential fatty acids—omega 6 and omega 3—in a ratio of 2:1, in contrast to the much higher ratios observed in the diets of western and northern Europe and the United States [9]. In the traditional Mediterranean diet a sweet tooth was satisfied by intake of carobs and figs stuffed with walnuts as a snack. Carob During the Roman persecution, Rabbi Shimon Bar Yochai and his son survived, according to tradition, for 13 years in a cave by eating carobs and dates and drinking water [36]. Carob is a legume native to the Mediterranean. The word carob is derived from the Arab kharrub and means “pod”; it also gave the name carat to the measure of gold. Carob beans can be dried, ground, and roasted to produce carob flour or powder. Carob is caffeine free and naturally sweet; it is a rich source of calcium and potassium and smaller amounts of iron and some B vitamins. Insoluble fiber extracted from carob pulp has been shown to have beneficial effects, lowering LDL cholesterol and triglycerides [37]. Nuts Nut consumption has an inverse relationship with risk for cardiovascular diseases [38]. Nuts are rich in protein, fiber, phytonutrients, and polyphenolic antioxidants, as well as monounsaturated fatty acids and polyunsaturated fatty acids. Walnuts in particular have a high content of omega 3 fatty acids, in addition to high amounts of fiber, B vitamins, magnesium, and several types of antioxidants. Walnuts are reported to be active in improving blood vessel elasticity and in reducing atherosclerotic plaque accumulation, LDL cholesterol concentration in blood, and the inflammatory C-reactive protein biomarker [39]. Peanuts were shown to contain the flavonoid resveratrol (about 70 mg per ounce of peanuts), whereas almonds contain the flavonoids quercetin and kaempferol. Nuts are a rich source of fiber, vitamin E, and phytochemicals such as ellagic acid, flavonoids, phenolics, luteolin, isoflavones, and tocotrienols. They are also an excellent source of magnesium, zinc, selenium, copper, iron, riboflavin, niacin, and folic acid. CONCLUSION Western lifestyle has penetrated Mediterranean regions, whether by a welcome invitation or via indirect diffusion through economic globalization and modern industrialization [40]. An obesogenic environment, characterized by easily available concentrated sugar and high-fat foods along with activity-reducing habits, has infiltrated the territory of the Mediterranean basin and broken the traditions of family-centered, season-orientated meals. Adoption of a food pyramid with an emphasis on traditional biblical food would be beneficial [8] (Figures 1–3). From biblical times through the centuries, the Mediterranean diet has been able to adapt and incorporate the beneficial food and customs of different foreign rules. In modern times and in the future, the role of the Mediterranean diet may be to add to its proven health benefits by incorporating biblical foods, with their respective health benefits, within the diet and lifestyle. SUMMARY POINTS l l l l The Mediterranean diet is reviewed in the context of history and culture. The ancient Mediterranean diet focuses on the seven biblical species—wheat, barley, grapes, figs, pomegranates, olives, and date honey—together with other indigenous foods from the Middle East. These have been shown to have beneficial health properties. The original, traditional Mediterranean lifestyle involves physical activity and communal meals. Adopting such habits and including biblical foods in the Mediterranean diet will help combat the obesogenic environment and counter the risks of the noncommunicable diseases of modern life. 10 SECTION 1 The Mediterranean Diet: Concepts and General Aspects FIGURE 3 Proposed additions of biblical components to the Mediterranean diet pyramid. ACKNOWLEDGMENT The authors thank Dr. Sig Geller for editorial revision. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] Trichopoulou A, Vasilopoulou E. Mediterranean diet and longevity. Br J Nutr 2000;84(2):S205–9. Keys AB, Keys M. How to eat well and stay well the Mediterranean way. New York: Doubleday; 1975. Trichopoulou A, Costacou T, Bamia CDT. Adherence to a Mediterranean diet and survival in a Greek population. N Engl J Med 2003;348:2599–608. Sofi F, Cesari F, Abbate R, Gensini GF, Casini A. Adherence to Mediterranean diet and health status: meta-analysis. BMJ 2008;337:1344. 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Curr Aging Sci 2008;1:145–51. [33] Alarcón de la Lastra C, Villegas I. Resveratrol as an anti-inflammatory and anti-aging agent: mechanisms and clinical implications. Mol Nutr Food Res 2005;49(5):405–30. [34] Ben-Ami R, Berry EM. Maimonides on nutrition and lifestyle: is his advice still applicable today? In: Collins K, Kottek S, Rosner S, editors. Moses Maimonides and his practice of medicine. Haifa, New York: Maimonides Research Institute; 2013. p. 85–107. [35] Howard A, Chopra M, Thurnham D, Strain J, Fuhrman B, Aviram M. Red wine consumption and inhibition of LDL oxidation: what are the important components? Med Hypotheses 2002;59:101–4. [36] Kohelet Raba 10:8. [37] Zunft HJF, Lüder W, Harde A, Haber B, Graubaum HJ, Koebnick C, et al. Carob pulp preparation rich in insoluble fibre lowers total and LDL cholesterol in hypercholesterolemic patients. Eur J Clin Nutr 2003;42(5):235–42. [38] Sabate J. Nut consumption, vegetarian diets, ischemic heart disease risk, and all-cause mortality: evidence from epidemiologic studies. Am J Clin Nutr 1999;70:500S–3S. [39] Vinson JA, Cai Y. Nuts, especially walnuts, have both antioxidant quantity and efficacy and exhibit significant potential health benefits. Food Funct 2012;2:134–40. [40] Palma G, Padilla M. The Mediterraneisation of food fashions in the World. In: Mediterra. Paris: Presses de Sciences Po; 2012, ISBN: 9782724612486 [chapter 6]. Chapter 2 Nutritional Adequacy of the Mediterranean Diet Itandehui Castro-Quezada, MSc1,2, Blanca Román-Viñas, MD, PhD2,3 and Lluı́s Serra-Majem, MD, PhD1,2,3 1 University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain. 2 CIBER Fisiopatologia Obesidad y Nutrición, Instituto de Salud Carlos III, Lourdes, Spain. 3 Fundación para la Investigación Nutricional, University of Barcelona Science Park, Barcelona, Spain. ABBREVIATIONS AI ANR EAR INL IOM KIDMED MDP MUFA NAR NIVs SFA UNL WDP adequate intake average nutrient requirement estimated average requirement individual nutrient level Institute of Medicine Mediterranean diet score in children Mediterranean dietary pattern monounsaturated fatty acids nutrient adequacy ratio nutrient intake values saturated fatty acids upper nutrient level Western dietary pattern INTRODUCTION In recent years there has been an increased interest in finding a dietary pattern that fulfills the nutritional requirements of a population. Assessing the quality of a diet to establish nutritional recommendations for individuals or populations is a priority [1]. Nutrient requirements are traditionally based on the minimum amount of a nutrient needed by an individual to avoid deficiency and are defined by the physiological needs of the body. Alternatively, the requirement can be defined as the intake at which health is optimal, including the prevention of chronic diet-related diseases [2]. The Mediterranean diet is recognized as a healthy dietary pattern [3]. It is characterized by a beneficial fatty acid profile with large amounts of monounsaturated fatty acids (MUFAs) and a higher MUFA-to-saturated fatty acid (SFA) ratio than Western-type dietary patterns [4,5]. In addition, consumption of a small amount of carbohydrates [5], combined with elevated intake of dietary fiber [6] and antioxidants [7,8], may work together to produce favorable effects on health status. Greater adherence to a Mediterranean diet has been related to a reduced risk of all causes of mortality, as well as lower incidence of or mortality from cardiovascular diseases, certain types of cancer, neurodegenerative diseases (Parkinson’s and Alzheimer’s disease), and type 2 diabetes [9,10]. Nutritional adequacy can be estimated by the comparison between the nutrient requirement and a certain individual’s or population’s intake. Because neither the real intake nor the real requirement for one individual is known, the assessment of the adequacy of an individual’s or population’s nutrient intake can be calculated as the probability of adequacy [1,11]. NUTRITIONAL ADEQUACY AND PUBLIC HEALTH Rapid changes in diets and lifestyles resulting from industrialization, urbanization, economic development, and market globalization are having a significant effect on the health and nutritional status of populations [12]. Countries such as Spain and Italy have demonstrated a downward trend in adherence to the Mediterranean dietary pattern in recent decades [13,14]. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 13 14 SECTION 1 The Mediterranean Diet: Concepts and General Aspects The Mediterranean diet by the consumption of virgin olive oil, fruits, vegetables, whole-meal cereals, and fish, used to be rich in vitamins and minerals, making the risk of deficient micronutrient intakes quite infrequent. This could explain why inadequate intake of the vitamins such as B1, B2, niacin, B6, folates, or B12 were rare and intake of antioxidant vitamins (vitamins E and C) and carotenes were high in the Mediterranean basin [15,16]. The changes in the traditional Mediterranean diet in recent decades, however, include the incorporation of low nutrient-dense foods (such as soft drinks, sweets, bakery products) and variations in food processing methods (for example the refinement of flour). This transition has contributed to an increased risk of deficient intakes of some vitamins, especially folates and vitamins A and D, in particular among certain vulnerable population groups or collectives [15,16]. In 2011, Román-Viñas et al. [17] estimated the prevalence of inadequate nutrient intake in Europe using nutrient intake published data. The analysis of a number of micronutrients among adult and elderly European populations showed a mean prevalence of inadequate intake for zinc, iron, and vitamin B12 among 11% or less of the elderly population and a prevalence between 11% and 20% for inadequate copper intake in the adult and elderly populations, inadequate vitamin B12 in the adult population, and inadequate vitamin C in elderly Europeans. Finally, vitamin D, folic acid, calcium, selenium, and iodine were micronutrients with a prevalence of inadequacy in more than 21% of the adults and elderly; vitamin C had this prevalence of inadequacy only in the adult population [17]. Therefore surveillance of nutrient adequacy in Europe is needed to identify population groups at nutritional risk [11]. Nutritional adequacy can be used to determine the risk of deficiency of a nutrient in terms of low intake or high intake; for example, the adverse effects of high levels of sodium intake may be applicable to reducing the risk of certain chronic diseases or conditions such as hypertension [11,18]. The complexity of the relationships between dietary intake and pathology, however, cannot be attributed to a single nutrient but rather to multiple nutrients and foods. Thus the correct exposure should be measured to understand such a relationship. Furthermore, not only nutrients but also foods—and the interaction between them—are of concern for this kind of evaluation. Food pattern analysis is a key issue when investigating the relationship between nutrition and disease [11,19]. METHODS OF ASSESSING NUTRITIONAL ADEQUACY 1.0 1 1.0 2 3 0.5 0.5 0. 0 Increasing intake of nutrients Risk of excess Risk of inadequacy The quality of a diet can be assessed in terms of nutrient intake and the level of compliance with nutrient requirements or in terms of food or food group intake and diet patterns [20,21]. Traditionally, as a consequence of the necessity of meeting the body’s needs for certain nutrients and avoiding deficiencies, nutrient intake assessment in populations has compared intake against the requirement for the nutrient. Different types of analyses are used to evaluate the nutrient adequacy of a diet. The method used depends on the purpose of the analysis (to evaluate individuals or a population), the nutrient being studied, and the type of distribution of the intakes [21]. Obviously, the recommendations used for the comparison are country specific and evidence based [11]. Recommended nutrient intake values (NIVs) vary between countries in the amount of nutrients and the terms used to describe the requirement [22]. Using the NIVs as a common set of terms and definitions, as a standardized terminology has been proposed by a group of experts from United Nations University (UNU), in collaboration with the Food and Agriculture Organization (FAO), the World Health Organization (WHO), and the United Nations Children’s Fund (UNICEF). The NIVs include three terms: the average nutrient requirement (ANR), the individual nutrient level (INLx), and the upper nutrient level (UNL) (Figure 1) [23]. FIGURE 1 Nutrient intake values and the risk of nutrient inadequacy or excess, showing the average nutrient requirement (1), the individual nutrient level (2), and the upper nutrient level (3). Adapted from the Institute of Medicine [18]. Nutritional Adequacy and Mediterranean Diet Chapter 2 15 The ANR is defined as the average or median usual intake value that is estimated to meet the requirement for a specific criterion in a group at a certain life stage or of a particular sex. The ANR is equivalent to the estimated average requirement (EAR) used by the Institute of Medicine (IOM). The INLx is the recommended nutrient level for all healthy individuals in a specific subpopulation. The committees frequently add two standard deviations to the ANR, which covers the needs of most of the population (i.e., 98%), assuming that the distribution is symmetrical. The INL98 is equivalent to the recommended dietary allowance used by the IOM. Most nutrients have an UNL that is the highest daily intake that can be tolerated without risk of adverse health effects [11,23,24]. The term used by the IOM is tolerable upper intake level. Finally, an adequate intake (AI) is estimated if there is not enough scientific evidence to establish values for an ANR or INLx. The AI has been included in IOM recommendations but not in the standardized terminology proposed by the UNU [11,24]. These nutritional requirements are applied to both the nutritional assessment and the planning of dietary interventions on an individual and population-based level [1]. Although the first dietary recommended intakes were published in 1940 [25], little or no guidance on how to use them was given until the IOM published guidelines in 2000 describing how requirements where derived and their application. Even with these guidelines, not all researchers follow such advice; they apply the nutritional requirements in different ways to assess nutritional intake adequacy [1,11]. According to the IOM, the prevalence of inadequate intakes for groups can be estimated using two methods: the probability approach and the EAR (ANR) cut-point method [11,26]. The probability approach requires estimating the probability of inadequate intakes for each individual in a population subgroup, averaging the probabilities, and then using this average as an estimate of the prevalence of inadequacy [21]. The EAR cut-point method measures the prevalence of inadequate intakes as the proportion of the population with usual intakes below the ANR (or EAR). However, the EAR cut-point method requires the following conditions to be fulfilled: intakes and requirements for the nutrient being studied must be independent, nutrient requirements must be distributed symmetrically, and the variance of the distribution of requirements should be smaller than the variance of the usual intake distribution [27]. However, the relationship between dietary intake and disease is complex and cannot be reduced to the study of the effect that certain nutrients have on health. Not only nutrients but also foods and the interactions between them are of concern for such an evaluation. The first methods used to assess nutritional adequacy was the association between a combination of nutrients or foods and health [19]. The nutrient adequacy ratio (NAR) is an index of adequacy that compares an individual’s daily intake of a nutrient with the INL98 for that nutrient [28]. Mean adequacy ratio calculates the average for the NARs for the selected nutrients for a certain subject [28]. Diet indexes are known as a priori defined because they are based on previous knowledge of nutrition (dietary guidelines or recommendations). The so-called a posteriori approach consists of defining food patterns once the dietary data are collected and using statistical analyses to identify the current relevant food patterns within the study population [11,19]. The main statistical procedures used to analyze dietary data and identify dietary patterns are known as factor analysis or cluster analysis [29]. Both a priori hypothesis-oriented diet indices and a posteriori-defined patterns have been related to the incidence of health outcomes (hard clinical end points) and biomarkers in epidemiological or clinical studies. Some of these dietary patterns have been related to nutrient adequacy [11]. This approach parallels a validation study based on the rationale that if the classification of participants according to their adherence to the dietary pattern is able to determine whether they fail to reach the optimal nutrient intake, the use of the dietary pattern is sufficiently valid [19]. Some a priori-defined diet indices have been correlated with the adequacy of certain nutrients, for example, the revised Diet Quality Index [30], Healthy Eating Index [31], Dietary Diversity Score [32], and the Food Variety Score [33]. Similarly, the Mediterranean diet has been quantified in several diet indices established a priori as an attempt to globally evaluate the quality of the diet based on a traditional Mediterranean reference pattern [34]. For example, the Mediterranean Adequacy Index (MAI) is obtained by dividing the sum of the percentage of total energy from typical Mediterranean food groups by the sum of the percentage of total energy from atypical Mediterranean food groups. It was developed to assess how close a diet is to the Healthy Reference National Mediterranean Diet. Alberti et al. [35] found that MAI values of diets consumed by elderly participants from 10 European countries followed for 10 years were inversely associated with total mortality (HR: 0.83; 95% CI: 0.75–0.92). Serra-Majem et al. [36] developed the KIDMED (Mediterranean diet score in children) index to assess the adequacy of Mediterranean dietary patterns (MDPs) in children and young people; the results are discussed later in the section “Mediterranean Diet in Children and Nutritional Adequacy.” Referring to the a posteriori-defined analysis, the studies evaluating the adequacy of nutrient intake associated with dietary patterns showed that the prudent pattern (defined by factor analysis as a diet rich in vegetables, fruits, legumes, whole grains, and fish) was valid for assessing the intake adequacy of a-carotene, lycopene, and lutein for men [37] 16 SECTION 1 The Mediterranean Diet: Concepts and General Aspects and b-carotene, vitamin C, vitamin B6, and folic acid for women [38]. Despite the method used to identify dietary patterns, vitamin B12 and vitamin E were found to be the micronutrients with lower probabilities of being effectively assessed. Nevertheless, scientific evidence shows that diet indices are tools with fair to moderate validity for assessing micronutrient intake adequacy [11,19]. MEDITERRANEAN DIET, WESTERN DIET, AND NUTRITIONAL ADEQUACY The MDP and the Western dietary pattern (WDP) differ not only in terms of food intake characteristics but also when nutrient adequacy is the comparative point. The relationship between nutrient adequacy and these two dietary patterns was recently assessed in a Mediterranean country by the Seguimiento de la Universidad de Navarra cohort study. In this study, the probability of adequate intake of nutrients was estimated using a probability approach and NIVs [5]. In Table 1 are shown the correlation coefficients between food consumption and factors representing both patterns. The WDP was positively correlated with the intake of red and processed meat, eggs, sauces, precooked food, fast food, energy drinks, sweets, full-fat dairy products, and potatoes and negatively correlated with the consumption of low-fat dairy products. Higher adherence to the WDP showed the highest percentages of individuals with noncompliance to recommendations for iodine, vitamin E, magnesium, iron, vitamin A, selenium, vitamin C, and folic acid. Furthermore, when adherence to the WDP was higher, the number of unmet nutrient intakes increased (Figure 2). Subjects in the highest quintile of the WDP had a 2.5fold increased risk for 10 unmet NIVs when compared to the WDP group with the lowest adherence [15]. The food groups identified in the MDP were olive oil, poultry, fish, low-fat dairy products, legumes, fruits, and vegetables [5]. Higher adherence to the MDP was associated with a lower percentage of energy coming from total fat and SFA. The ratio of MUFA to SFA increased with increased adherence to the MDP (P for trend <0.001). Protein intake (as a percentage of energy) increased across categories of adherence to the MDP. Carbohydrate intake was low (43–44%), showing TABLE 1 Correlation Between Baseline Food Consumption and Factors Representing Mediterranean and Western Dietary Patterns in the Seguimiento de la Universidad de Navarra Cohort Study (n ¼ 17,197) Dietary Patterns Food Groupsa Factor 1 (Western)b Factor 2 (Mediterranean)b Olive oil - 0.32 Poultry - 0.38 Red meat 0.54 - Processed meat 0.5 - Eggs 0.37 - Fish - 0.59 Sauces 0.42 - Precooked food 0.41 - Fast food 0.57 - Caloric soft drinks 0.35 - Commercial sweets 0.4 - Whole-fat dairy 0.43 - Low-fat dairy -0.31 0.37 Legumes - 0.3 Vegetables - 0.68 Fruits - 0.54 Potatoes 0.45 a Values are presented in grams per day. bCorrelation coefficients <0.3 were omitted for simplicity. Adapted from Serra-Majem et al. [5] - Nutritional Adequacy and Mediterranean Diet Chapter 2 FIGURE 2 Mean number of nutrients with intakes not meeting recommended levels across quintiles of Western dietary pattern score. Adapted from Serra-Majem et al. [5]. 17 Mean* NIVs not met 4.5 4 3.5 3 2.5 2 Q1 Q2 Q3 Q4 Q5 Quintiles of adherence to the Western dietary pattern *Adjusted for age and sex FIGURE 3 Mean number of nutrients with intakes not meeting recommended levels across quintiles of Mediterranean dietary pattern score. Adapted from Serra-Majem et al. [5]. Mean* NIVs not met 4.5 4 3.5 3 2.5 2 Q1 Q2 Q3 Q4 Q5 Quintiles of adherence to the Mediterranean dietary pattern *Adjusted for age and sex a similar value across all the quintiles; on the other hand, consumption of dietary fiber increased according to the levels of adherence to the MDP. All the nutrients studied (except sodium) showed increasing values with increasing quintiles of adherence to the MDP. Therefore, subjects with a higher score for the MDP had a better nutrient profile, and a lower proportion of individuals showed inadequate intakes of micronutrients (Figure 3). Subjects in the highest quintile of the MDP had a lower risk of failing to meet 10 NIVs [OR: 0.02 (95% CI: 0.00–0.16, P for trend <0.001] when compared to the lowest category of adherence to MDP [5]. The MDP had similarities with the healthiest patterns (prudent [37,39], healthy [38], and health-conscious [40] patterns) defined in non-Mediterranean countries: a positive correlation with intakes of fruits, green leafy vegetables, poultry, and fish 18 SECTION 1 The Mediterranean Diet: Concepts and General Aspects and certain lifestyle habits such as not smoking and being more physically active [41]. When the association of the dietary patterns with their nutrient intake profiles was analyzed, however, differences arose, especially in relation to fat intake. The prudent and healthy patterns had lower intakes of total and saturated fat [37,42,43], and some studies found even lower intakes of MUFAs [42,43]. Healthy patterns showed higher percentages of energy coming from proteins and carbohydrates and lower percentages of energy coming from fat [38,44] when comparing the highest quintile with the lowest. In 2011, Maillot et al. [45] conducted a study that applied individual diet modeling in a representative sample of French adults to evaluate the smallest dietary changes needed to fulfill a whole set of nutrient recommendations for each individual. They found that including foods typically found in the Mediterranean diet was strictly necessary to achieve French nutrient recommendations. Therefore, MDP was directly associated with the MUFA-to-SFA ratio, showing a healthier profile for the quality of fat intake when compared to other studies conducted in non-Mediterranean countries. Furthermore, as adherence to the Mediterranean diet increases, the probability of nonfulfillment of nutrient recommendations decreases [5]. MEDITERRANEAN DIET IN CHILDREN AND NUTRITIONAL ADEQUACY The Mediterranean dietary pattern has also been associated with nutritional adequacy in children. The enKid study was a cross-sectional study conducted in Spain that assessed 3166 individuals aged from 6 to 24 years [46]. A short questionnaire was used to evaluate the quality of the Mediterranean diet (KIDMED Index) (Table 2). This tool allowed us to classify the food intake of subjects into three categories of the Mediterranean diet quality (poor, medium, or high) [47]. The nutrient intake adequacy was estimated as the percentage of population with intakes less than two-thirds of the recommended nutrient intakes (<2/3 INL). TABLE 2 KIDMED Test to Assess Adherence to the Mediterranean Diet KIDMED Test Scoring Eats a fruit or drinks fruit juice every day +1 Eats a second fruit every day +1 Eats fresh or cooked vegetables regularly once a day +1 Eats fresh or cooked vegetables more than once a day +1 Consumes fish regularly (at least 2–3/week) +1 Goes to a fast food restaurant (hamburgers) >1 time/week 1 Likes legumes and eats them >1 time/week +1 Consumes pasta or rice almost every day (5 servings/week) +1 Eats cereals or grains (bread, etc.) for breakfast +1 Consumes nuts regularly (at least 2–3 times/week) +1 Uses olive oil at home +1 Skips breakfast 1 Eats a dairy product for breakfast (yogurt, milk, etc.) +1 Has commercially baked goods or pastries for breakfast 1 Eats two yogurts and/or some cheese (40 g) daily +1 Eats sweets and candy several times every day 1 KIDMED Index Score Adherence to Mediterranean Diet 3 points Poor 4–7 points Medium 8 points High Adapted from Serra-Majem et al. [36]. Nutritional Adequacy and Mediterranean Diet Chapter 2 19 Serra-Majem et al. found in the enKid study that total energy intake tend to increase according to the KIDMED Index in male adolescents aged 15 to 24 years. In children and adolescents the consumption of fiber, calcium, iron, magnesium, potassium, phosphorus, and vitamins, with the exception of vitamin E, increased according to the KIDMED Index. The proportion of children with inadequate intake of calcium, iron, and vitamins A and C (in girls), magnesium, and vitamin B6 (excluding boys aged 6–14 years), and decreased when the quality of the Mediterranean diet increased (Table 3) [36]. The high nutritional quality of the Mediterranean diet contributes to the health benefits that have been ascribed to this dietary model. For this reason, in addition to better dietary fat quality and the increased quantity of antioxidants [7,48,49], we may consider nutritional adequacy as another favorable component of the Mediterranean diet. The enKid study demonstrated that when the level of adhesion to a Mediterranean diet model is optimal, there is a reduced risk of inadequate intakes, thus making fortification and supplementation of almost all vitamins and minerals unnecessary. Therefore, health promotion strategies should be prioritized to promote the Mediterranean diet instead of alternatives such as fortification or supplementation [27,50]. SUMMARY POINTS l l l l l In recent decades changes in the adherence to the MDP have contributed to the risk of inadequate intake of nutrients in the population. Nutritional adequacy should be assessed by comparing usual nutrient intake estimates with recommendations. However, dietary patterns may also be used because they correlate quite well with nutritional adequate intakes. The Mediterranean diet has similarities with other healthy dietary patterns; however, individuals with higher adherence to an MDP show a healthier profile of fat intake. When adherence to an MDP is high, the probability of fulfilling the nutrient recommendations increases. Health promotion strategies should prioritize the promotion of the Mediterranean diet instead of alternatives such as fortification or supplementation. TABLE 3 Percentage of Inadequate Intakes (<2/3 INL) in School Children According to Adherence to the Mediterranean Diet KIDMED Index for Children Aged 6-14 Years (Male/Female) Poor quality: score 3 (%) Medium quality: score 4–7 (%) High quality: score 8 (%) P for trend Energy 4.8/13.3 1.9/6.9 1.3/6.3 0.303/0.467 Protein 0.0/0.0 0.0/0.0 0.0/0.0 - Calcium 4.8/26.7 2.6/10.4 0.4/4.2 0.027/<0.001 Iron 0.0/33.3 0.8/23.8 0.0/15.4 0.300/0.008 Magnesium 19.0/0.0 9.8/4.2 4.6/2.9 0.004/0.707 Thiamin 0.0/0.0 0.4/0.4 0.0/0.4 0.464/0.871 Riboflavin 0.0/0.0 1.1/1.2 0.4/0.8 0.559/0.881 Niacin 0.0/0.0 0.4/0.4 0.0/0.4 0.464/0.871 Vitamin B6 0.0/33.3 3.0/10.8 2.9/5.0 0.724/<0.001 Folate 14.3/46.7 9.8/32.3 5.0/23.8 0.021/0.010 Vitamin B12 0.0/0.0 0.0/0.0 0.0/0.0 - Vitamin C 47.6/13.3 18/15.4 5.4/4.6 <0.001/<0.001 Vitamin A 57.1/80.0 63.9/61.5 59.6/54.2 0.523/0.024 Vitamin D 100.0/100.0 95.9/99.6 95.8/97.1 0.618/0.024 Vitamin E 28.6/66.7 43.2/60.8 36.3/57.1 0.394/0.310 Adapted from Serra-Majem et al. 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[42] Zhang C, Schulze MB, Solomon CG, Hu FB. A prospective study of dietary patterns, meat intake and the risk of gestational diabetes mellitus. Diabetologia 2007;49:2604–13. [43] Fung TT, Willett WC, Stampfer MJ, Manson JE, Hu FB. Dietary patterns and the risk of coronary heart disease in women. Arch Intern Med 2001;161:1857–62. [44] Esmaillzadeh A, Kimiagar M, Mehrabi Y, Azadbakht L, Hu FB, Willett WC. Dietary patterns and markers of systemic inflammation among Iranian women. J Nutr 2007;137:992–8. [45] Maillot M, Issa C, Vieux F, Lairon D, Darmon N. The shortest way to reach nutritional goals is to adopt Mediterranean food choices: evidence from computer-generated personalized diets. Am J Clin Nutr 2011;94:1127–37. [46] Serra-Majem L, Garcı́a-Closas R, Ribas L, Pérez-Rodrigo C, Aranceta J. Food patterns of Spanish school children and adolescents: the enKid Study. Public Health Nutr 2001;4:1433–8. [47] Serra-Majem L, Ribas L, Ngo J, Ortega RM, Garcı́a A, Pérez-Rodrigo C, et al. Food, youth and the Mediterranean diet in Spain. Development of KIDMED, Mediterranean Diet Quality Index in children and adolescents. Public Health Nutr 2004;7:931–5. [48] Saura-Calixto F, Goñi I. Definition of the Mediterranean diet based on bioactive compounds. Crit Rev Food Sci Nutr 2009;49:145–52. [49] Su Q, Rowley KG, Itsiopoulos C, O’Dea K. Identification and quantification of major carotenoids in selected components of the Mediterranean diet: green leafy vegetables, figs and olive oil. Eur J Clin Nutr 2002;56:1149–54. [50] Bemelmans WJ, Broer J, de Vries JH, Hulshof KF, May JF, Meyboom-De Jong B. Impact of Mediterranean diet education versus posted leaflet on dietary habits and serum cholesterol in a high risk population for cardiovascular disease. Public Health Nutr 2000;3:273–83. Chapter 3 Agricultural Practices in the Mediterranean: A Case Study in Southern Spain J. Jesús Casas, PhD, Santiago Bonachela, PhD, Francisco J. Moyano, PhD, Encarnación Fenoy, MD and Joaquı́n Hernández, PhD University of Almerıá ceiA3, Almerıá, Spain. ABBREVIATIONS BP CAP EC EU IWUE RIS before present Common Agrarian Policy electrical conductivity (dS m 1) European Union marketable crop production over total crop irrigation water supply (kg m 3) ratio of irrigation water supply to crop water requirements INTRODUCTION The current patterns and processes in environmental and sociocultural issues of the Mediterranean Region cannot be understood without taking into account the deep transformation produced by human activities across the Mediterranean since the Neolithic agricultural revolution [1,2]. Various authors—artists, historians, and ecologists—mainly from north-central Europe, have thought of this region as a “ruined landscape” or “lost Eden” [1]. This idea mainly stems from the perception that a pristine nature, rich in exuberant forests, was destroyed over 10 millennia of unsustainable use of resources by humans [3,4]. Despite much evidence supporting this view, it seems to be somewhat fragmentary. Indeed, a great body of knowledge also indicates that the nature of the Mediterranean region was not always such an “Eden” because of pure natural environmental constraints [1]. Other authors have adopted an approach midway between these two perceptions. For instance, Blondel and Aronson [5] view human populations as “sculptures of Mediterranean landscapes”: to them, the conspicuous anthropogenic transformations of the environment have often determined complex socio-ecosystems characterized by high levels of both wild and domesticated biodiversity and equipped with an outstanding resilience after millennia of interaction between human and natural processes. Other authors went beyond claiming, either explicitly [6] or implicitly [3,7], that a complex “co-evolution” shaped these interactions through long-lasting and constantly evolving land use practices. Reciprocal evolutionary changes between interacting parts summarize the common essence of the various concepts of co-evolution. There is much evidence of evolutionary changes in domesticated and wild Mediterranean species, either through direct selection or, indirectly, by means of habitat alterations by humans [5]; the putative effects on humans, however, remain a matter difficult to disentangle. The “gen-cultural co-evolution” hypothesis might help enlighten this co-evolutionary view. This is a part of the more general “niche construction” theory, which focuses on the capacity of some species, particularly humans, to modify natural selection by changing their environment and thereby acting as co-directors of their own evolution and that of other species [8], which may confer stability to the entire system [9]. Accordingly, the profound cultural changes (including technology, diet, and social organization) that occurred in the Mediterranean region since the Neolithic agricultural revolution are likely to have acted as self-selective filters for humans, leading to highly resilient traditional socio-agro ecosystems. During the past 100 years, however, the intensification of agricultural activities has induced profound alterations in traditional socio-agro ecosystems. Under the production paradigm that aimed at the “green revolution” worldwide, new science-driven technologies—that is, new cultivars; massive use of mineral fertilizers, pesticides, and hormones; and mechanization—have dominated agriculture [10], resulting in conspicuous increases in productivity but producing The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 23 24 SECTION 1 The Mediterranean Diet: Concepts and General Aspects numerous environmental drawbacks. These are likely to disrupt the presumed resilience mechanisms of the traditional Mediterranean socio-agro ecosystems and their provision of tangible and intangible goods and services to humans. It was not until the mid-1980s that a general concern about environmental and health problems pushed scientists and policymakers to formulate the current paradigm of agricultural sustainability, that is, maintaining or increasing food productivity with minimal adverse effects on the environment to promote the persistence of the whole system [11]. Despite the progress in food output originated by the production paradigm, its main goal of eradicating hunger was not globally fulfilled [10]. In industrialized as well as in many developing countries, a parallel transition toward calorie-rich diets has been associated with increasing incidence of obesity-related diseases [11]. This undesirable dietary transition also has been consistently detected in Mediterranean countries during the past 30 years, particularly in the European ones [12]. This chapter gives a region-wide perspective of agricultural practices and their consequences on productivity and environmental and health issues. However, for most detailed information we focus on Andalusia (southern Spain), a region well known by the authors. Thus, this review embraces the following topics: (i) a brief description of the environmental and cultural determinants of agriculture in this region; (ii) a description of the recent changes in agricultural practices associated with intensification/extensification trends; and (iii) a description of recent trends in practices that apply specifically to three distinct farming systems producing dietetically valued foods typically related to the Mediterranean diet—the extensive Iberian pig production system, the olive production system, and the greenhouse vegetable agro industry. ENVIRONMENTAL AND CULTURAL DRIVERS SHAPING TRADITIONAL AGRICULTURAL LANDSCAPES Mediterranean agriculture is characterized by its remarkably high diversity of crop species and varieties. This could be explained as the result of two interacting factors: (i) the position of this region as the crossroads of successive indigenous and nearby civilizations, leading to an intense exchange of species; and (ii) its outstanding environmental heterogeneity promoted by its transitional climate, which is affected by interactions between mid-latitude and tropical processes, as well as its complex topography, coastline, and lithology, all of which have fostered the successful establishment of many crops. Thus, many species, including citrus, apples, apricots, cherries, hazelnuts, walnuts, sugar cane, bananas, rice, spinach, aubergines, and carrots, seem to have been imported from the Middle East, Asia, or India by the armies of Alexander the Great, the Romans, and later by the medieval travels of Christians and Muslims [13,14]. At the onset of the Modern Age, the exploration of the Americas initiated by Christopher Columbus prompted the intense trans-Atlantic commerce that greatly improved the wealth of Mediterranean agriculture with valuable crops such as maize, potatoes, squash, beans, peppers, and tomatoes. Since the dawn of agriculture—ca.11,500 years BP (a conservative estimation)—civilizations that flourished around the Mediterranean Sea have struggled with the scarcity and irregular availability of water and their consequences: fire and severe floods and droughts. Thus, agricultural practices have fundamentally evolved in this region to cope with fluctuating environmental conditions, eventually leading to management packages adapted to confer stability to the socio-agro systems. This does not mean that traditional Mediterranean agro systems were exempt from disequilibrium crises over their lengthy trajectory, but it is also true that Mediterranean productivity has been sustained across eight millennia of agricultural land use as a consequence of human and environmental resilience [3]. Fire seems to be a foundational farming practice in the Mediterranean, as well as in other agricultural centers worldwide. The onset of the Holocene was characterized by fire expansion, possibly favored by increasing seasonality. This might have promoted the spread of most early domesticated cereals and legumes, as well as animals dependent on herbaceous plants, by preserving open grasslands and limiting the encroachment of trees, as is currently practised in some Mediterranean agro-pastoral systems [1]. If true, Neolithic communities might have taken advantage of this setting to manage their local resources using wildfires and/or prescribed burns [15]. Fire became more frequent and clearly associated to human activities from the onset of the Bronze Age until present, and it seems to be synchronous with the development of slash-and-burn agriculture, animal husbandry, and mining activities [16]. Irrigation revolutionized farming productivity, promoted food security, and gave rise to the exuberant Mesopotamian and Egyptian civilizations. Although initially dependent on natural irrigation (river floods), irrigation schemes (similar to the present standards) were soon developed and used in these two regions as far back as 7000 years BP. In the Nile floodplain, water-lifting devices or the construction of high-waterhead canal systems fed a highly resilient and complex system of perennial irrigation independent of river floods [17]. The methods invented were refined with time and gradually spread throughout the region, primarily by Phoenicians, Greeks, and Romans, who incorporated their own contributions Agricultural Practices in the Mediterranean Chapter 3 25 [18]. Under Roman rule, hydraulic structures spread throughout the region—dams, reservoirs, cisterns, canals, and aqueducts—for domestic and irrigation purposes, and many of them are still in use. During the Middle Age Islamic period, many of these infrastructures were maintained or improved to expand irrigated land, particularly after the introduction of crops with high water demand (e.g., rice, sugar cane, citrus) [1]. Traditional farming systems were essentially consolidated until the Renaissance. Butzer [7] highlights the remarkably sophisticated risk-reducing strategies of these traditional systems to cope with environmental fluctuations and fit into the local environmental mosaic. As mentioned by Butzer, this traditional farming was based in four components: (a) Rain-fed, less frequently irrigated, outfield cultivation of a selection of cereals and legumes suited for local soils and climates in biennial or triennial cycles of crop rotation, including fallow and/or intercropping to maintain or restore soil fertility, to improve water-use efficiency, and to reduce disease and pest risks (b) Irrigated cultivation of various green vegetables and condiments in kitchen or market gardens, which diversified and buffered the diet during the prolonged summer droughts (c) Vineyards, olive groves, and fruit tree orchards: Intercropping fruit trees and herbaceous crops was the norm, particularly in areas with gentle slopes, where this practice and terracing confer additional advantages for preventing soil erosion (d) Animal husbandry mainly exploited household animals, hens, pigs, and, more rarely, cattle, together with pack animals (donkeys, mules, oxen) and goats and sheep herds, all ensuring diverse and flexibly available sources of proteins (eggs, dairy products, meat), wool, leather, and manure. Herding of sheep and goats and sometimes pigs was practised in mountainous communes and/or by grazing on stubble after harvest or in fallow areas to provide manure. Livestock often were moved seasonally in ascending/descending transhumant cycles, a buffer remedy to summer droughts in lowlands and winter frost on mountains. RECENT TRENDS IN AGRICULTURAL PRACTICES: INTENSIFICATION VERSUS EXTENSIFICATION In a broad sense, the Mediterranean region is characterized by cyclic alternative periods of agricultural intensification and deintensification [7]. During periods ruled by the hegemony of Phoenicians, Greeks, and, in particular, Romans, the sociopolitical integration of the region and economic stability propelled commercial success, which in turn stimulated an intensification of the rural economy (i.e., wheat, olive oil, and wine) and population growth. But these surges alternated with periods of sociopolitical disintegration and a slowdown in trade and agricultural activities. The technological advances during the twentieth century, primarily in mechanization, crop varieties, chemical fertilization, and pest, weed, and disease control, have determined an unprecedented growth in agricultural production worldwide. However, the intensification of Mediterranean agriculture during the last century was relatively low compared to that of northern Europe (Table 1) because of idiosyncrasies that stem from the relatively high abundance of areas with unfavorable soils, precipitation, and topography, in addition to sociopolitical constraints. Thus, at the regional scale a geographical polarization in farming intensification has taken place in most Mediterranean countries in the European Union (EU), driven by the concentration of intensive farming in the most easily accessible fertile irrigated lowlands, whereas the traditional/extensive systems were gradually abandoned in the highly inaccessible mountainous areas because of their low economic competitiveness [19]. Abandoning farmland in upland zones has resulted in a reduction of soil erosion due to vegetation recolonization but increasing incidence of fire, whereas erosion and general landscape degradation affected abandoned fields in semi-arid environments [20]. However, during the 1990s the CAP (EU Common Agrarian Policy), together with the strengthening of national and international markets, with increasing demand for Mediterranean products, encouraged the expansion and intensification (i.e., high tree density and irrigation) of almond and olive orchards and vineyards into marginal slopping lands, which leads to increased soil erosion [21]. Irrigation practices are pivotal for agricultural intensification in Mediterranean regions. Thus, irrigation represents between 70% and 80% of the total water withdrawal in Mediterranean countries, with the overall irrigated area accounting for more than 16 Mha, and during the 1980s and 1990s, irrigation increased by 3 Mha [22]. This expansion has caused conspicuous overexploitation problems of surface and groundwater: massive river regulation and pollution; wetland loss and degradation; and groundwater depletion, nitrate contamination, and saline water intrusion in coastal aquifers [23]. The effects of agricultural intensification on the environment and human welfare have created growing public concern that has been reflected in the latest EU CAP reforms, starting with the Agenda-2000 package [19]. CAP reforms envisage an increased emphasis toward targeted agro-environmental measures to promote low-input systems, which seem to have 26 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 1 Indicators of Agricultural Intensification in Selected Northern and Mediterranean EU Countries Total Consumption of Mineral N Fertilizers (kg ha 1)a Sales of Pesticides (kg Active Ingredient ha 1)a,b 1985 2010 80–89 00–09 Northern countries Denmark 143 71 2.6 1.2 France 87 82 3.3 3.0 Germany 134 105 1.8 1.8 Netherlands 250 117 10.1 4.5 UK 98 64 2.6 1.4 Mediterranean countries Greece 117 51 6.7 2.8 Italy 81 45 7.1 6.5 Portugal 36 20 0.1 4.4 Spain 38 37 1.6 1.4 a Surface area corresponds to total utilized agricultural area. bData correspond to means of at least 3 years within each period of 10 years, depending on data availability (own calculations). Source: EUROSTAT Agri-environmental statistics 2010. succeed in reversing or slowing down overall European agricultural intensification during the past two decades (see Table 1). Policy changes also have created a favorable framework for the development of organic farming, which was regulated and granted subsidies in EU Mediterranean countries during the 1990s. There are currently more than 5 Mha of land dedicated to organic farming in the Mediterranean region [24]. THE EXTENSIVE IBERIAN PIG PRODUCTION IN DEHESA AGRO-SYLVO-PASTORAL SYSTEMS The dehesa agroforestry ecosystem is characterized by a combination of woodland and natural pastures that occupy more than 3 Mha in the southwest of the Iberian Peninsula, of which 2.7 Mha are located in Spain (1 Mha in Andalusia; Figure 1). The dehesa ecosystem is the result of human action in the Mediterranean forest over thousands of years to obtain large grazing surfaces and also is characterized by the presence of arboreal species of Quercus (Q. rotundifolia and Q. suber) at a density of 30–50 trees ha 1, with an understory composed of croplands, grasslands, and shrublands [25]. The shrub species and other less profitable herbaceous species have been eliminated by human actions to make better use of the resources. The dehesa is characterized by a Mediterranean semiarid climate, with average precipitation ranging from 300 to 800 mm year 1 and with dry summers and cold winters. Such conditions determine the level of grass productivity, which has two peak production periods in spring and autumn. The presence of hard and acid substrates with silica, granite, or slate rocks results in shallow soils containing a small amount of organic matter and phosphorus, nitrogen, and potassium deficiencies. Under these conditions, arable farming is not possible or profitable, and the only possible form of rational, productive, and sustainable production is based in the extensive production of ruminants (beef cattle, sheep, goat) and of the renowned Iberian pig [26]. The Iberian pig is a native breed present in the Iberian Peninsula from ancient times; it has adapted to an extensive production system characterized by the use of the natural meadowland resources of pastures and acorns. The products obtained from the Iberian pig have healthy properties. When raised under the traditional system, characterized by a finishing growth stage called montanera (in Andalusia), which begins in October and ends in December or January, a pig consumes a large amount of grass (at least 3 kg) and acorns (6–10 kg) each day and reaches a live weight of nearly 150 kg, the size required for slaughtering (Figure 2). The montanera is conditioned by numerous factors including the abundance and maturity of acorns and the quality and quantity of fresh grass as protein and a vitamin supplement. A pig needs to consume 10–15 kg of acorns for each 1-kg gain in live weight [27]. Agricultural Practices in the Mediterranean Chapter 3 27 FIGURE 1 Map of Andalusia (southern Spain) showing the surface area occupied by three main types of farmland use in this region characterized in this chapter. FIGURE 2 Herd of Iberian pigs feeding under an oak in a dehesa from Sierra Morena (northwest Andalusia), as the final fattening stage before the slaughter. Photo courtesy of Enrique López-Carrique. As occurs in other monogastrics, the Iberian pig incorporates fats into body tissue in the same form in which they are ingested; hence the lipid profile of its meat shows high levels (nearly 55%) of oleic fatty acid, the monounsaturated fatty acid in olive oil, and relatively low concentrations of linoleic (8%) and palmitic acids (20%) [25]. Scientific reports [28] have shown that these fats exercise a beneficial effect on health by increasing the amount of good cholesterol (high-density lipoproteins) and reducing bad cholesterol (low-density lipoproteins). Cured Iberian hams obtained from pigs fed a diet of acorns contain a total proportion of unsaturated fatty acids of >75%, making the fat from Iberian hams the most “cardio healthy” of all animal fats—even healthier than some fats of plant origin. Although the increased insaturation would cause an increase in meat oxidation, this is limited by the effect of tocopherols present both in acorn (g-tocopherol) and grass (a-tocopherol). In addition, it contains a smaller amount of cholesterol (60 mg/100 g) than beef. The traditional Iberian pig production system, which has little in common with industrial systems of pig production, contributes to the maintenance of the dehesa agro-sylvo-pastoral ecosystem. In addition, the Iberian pig sector accounts for more than 14% of total agricultural production in the regions where it is located, and farms and producers play an important role in the conservation and development of these less-favored areas. Nevertheless, in the past 20 years the increased demand for Iberian pig products has determined the appearance of production systems that lie well apart from 28 SECTION 1 The Mediterranean Diet: Concepts and General Aspects the traditional one. This has resulted in the increased presence in the markets of products not showing the abovementioned valuable nutritional properties. The quality of Iberian pig products has recently been normalized and regulated as a response to the demands of traditional producers who perceive that all the positive features of this ancient production system may be at risk. THE OLIVE GROVE SYSTEM Andalusia (southern Spain) is the region with the highest olive production in the world. The total surface area of olive groves in this region (1.55 Mha; Table 2 and Figure 1) represents about 24% of the olive groves worldwide: 27% of the groves in the EU, 62% of the groves in Spain, and 30% of the agricultural land in Andalusia. Most olive orchards are destined to produce olive oil (94%), although a small surface area (6%) is dedicated to producing table olives. The importance of the olive economy in Andalusia dates back to the Roman Empire, when olive oil from this region enjoyed a deserved prestige and was exported throughout the western Roman world. Olive groves currently can be viewed as a key multifunctional agricultural system in Andalusia. This crop represents the second agricultural subsector, after TABLE 2 Selected Technical Characteristics of Olive Farming in Andalusia, Differentiating Conventional and Organic Farming Conventional Farming Organic Farming Variable 1990–95 2005–10 1990–95 2005–10 Total surface area 1.24 Mha 1.50 Mha 9000 ha 45,000 ha Rainfed (% surface area) 90 62 - 94 Irrigated (% surface area) 10 38 - 6 Yield 1 Rainfed 1500–2200 kg ha Irrigated 3200–10,000 kg ha Price paid to growers 0.5–2.5 € kg 1 13% to +20% a 1 +16% to +52% a Density of plantation (trees ha 1) <100–200 Rainfed Irrigated 100–200 200–2500 b Soil management (% farms) Highly frequent Nonrecommended (slope > 10%) or shallow tillage 66 0 18 65 Mineral fertilizers 90 66 Manure (compost) 5 70 0 15 Rainfed 30 20 Irrigated 50 40 Tillage Herbicides Vegetal covers b Fertilization (% farms) Green manure (legumes) Nonrenewable energy used (MJ l 1 b oil) a Range of percentages relative to nearby farms under low–medium intensity conventional farming. Source: Alonso-Mielgo A. La economıá del olivar ecológico. In: Guzmán-Casado G, Coord., editor. El olivar ecológico. Madrid: Consejerıá de Agricultura y Pesca, Servicio de Publicaciones y Divulgación, Mundi-Prensa; 2011. p. 391–417. bSource: Guzmán GI, Alonso A. A comparison of energy use in conventional and organic olive oil production in Spain. Agri Syst 2008. http://dx.doi.org/10.1016/j.agsy.2008.06.004. Main sources: Spanish Ministry of Agriculture, Food and Environment; Department of Agriculture, Fisheries and Environment, Government of Andalusia. Agricultural Practices in the Mediterranean Chapter 3 29 horticulture, in economic importance, generating 37% of the agricultural gross margin and 30% of the agricultural employment in this region. Given its wide-ranging territorial dimension, farming practices in olive groves have critical environmental transcendence in Andalusia. Olive groves also provide a habitat for many species, particularly in low-intensity olive farming areas with limited use of agrochemicals and old trees with herbaceous covers that can harbor high levels of biodiversity. Olive groves in Andalusia, as in many other Mediterranean regions, have experienced periods of expansion and contractions related to demographic, economic, and political challenges. For instance, by the middle of the last century rural depopulation caused the scarcity and increasing costs of labor, which, coupled with increasing market competition from seed-oils cheaper than olive oil, triggered a severe crisis in this agro industry [29]. However, after the entry of Spain into the EU (in 1986), subsidies for olive oil production, in addition to a renewed market appreciation of this product, fostered the sector, promoting the expansion and intensification of olive groves (Table 2). Olive trees (Olea europaea L.) have adapted to tolerate drought and poor, stony soils. Thus it is no wonder that traditional olive agriculture was based on rain-fed farming predominantly confined to slopes or rugged land—often transformed using terraces—leaving more fertile plains for crops with higher requirements. Traditional olive groves (Figure 3) are characterized by the use of locally adapted varieties using a relatively low plantation density (50–150 trees ha 1) [30] and dominated by old trees (often >50 years old, with several trunks per tree) and relatively low chemical inputs. Despite the high environmental value attributed to these low-input systems, tillage for weed control is relatively frequent in conventional olive farming (Table 2), causing severe soil loss and surface water contamination with sediments and agrichemicals, particularly in high-slopping lands [31]. Traditional systems often render small yields (e.g., between 800 and 1500 kg ha 1) [30], but need large amounts of labor for harvesting, which is normally carried out by beating the fruit off trees with poles onto nets. Low productivity coupled with a tendency of olive trees toward alternate bearing and aggravated by interannual fluctuating weather conditions (droughts and frosts) and a cyclic crisis of low market prices determine high socioeconomic instability in traditional monoculture olive zones, complicating the viability of the system. Consequently, a redesign of olive orchards toward intensification started in the 1980s, regulated and funded by the Spanish agricultural policies and EU CAP subsidies based on farm production levels that ruled during the last two decades of the twentieth century. Olive grove intensification fundamentally entails high plantation densities, with only one trunk per tree to facilitate mechanical harvesting and reduce costs and fertigation mostly applied using drip systems following deficit irrigation principles. Both practices have resulted in increased yields—up to 10 times higher than yields obtained in rain-fed, low-density olive groves (Table 2). Intensification has spread primarily by new olive plantations in plain or low-slopping areas, where mechanization is feasible. Systems with super-high-density plantations (>1500 trees ha 1) (Figure 4) are now frequent in these areas, where Arbequina is the most common cultivar. These systems require high establishment costs (>6000 € ha 1) but produce the earliest crop (>1000 kg oil ha 1) the third year after planting, with yields of >1800 kg oil ha 1 from the 5th to 10th years, using between 150 and 250 mm of irrigation water annually [29]. The new technology for olive oil extraction greatly improves oil quality. This consists of a continuous, two-phase centrifugation process that generates a liquid phase (olive oil) and an organic slurry (olive mill pomace). This by-product FIGURE 3 Traditional olive groves under organic production located in Jaén (Andalusia), showing an herbaceous cover of spontaneous flora. Photo courtesy of Miguel Pastor Muñoz-Cobo. 30 SECTION 1 The Mediterranean Diet: Concepts and General Aspects FIGURE 4 A superintensive olive grove located in Campo de Nijar (Almerı́a, Andalusia). Photo courtesy of Julian Cuevas. has traditionally represented a major environmental problem, causing conspicuous surface water contamination. Nowadays, the use of olive mill pomace, preferably composted, for organic amendment of soil is a common practice in many Mediterranean countries, where soils often have low organic matter contents, thus contributing to the sustainability of the system [32]. Intensification of conventional olive groves can be considered relatively low compared to other crop systems. Given the extensive area occupied by this crop in Andalusia, however, the increasing use of practices such as tillage, irrigation, herbicides, mineral fertilizers (Table 2), and pesticides has raised awareness about human welfare and environmental conservation. For instance, the inadequate use of pesticides can impair the safety of olive products [33], and overfertilization with nitrogen can reduce the dietary quality of olive oil [34]. Moreover, in Jaén, the province with the highest extension of olive groves, mean herbicide concentrations above the limit for drinking water have been detected in surface waters and ground waters [35]. But the same study also highlights an acceptable evolution during the past decade of herbicide concentrations in surface and ground waters (decrements between 20% and 70% for several substances), which was associated with the current regulations and actions on this matter. In fact, during the past decade considerable progress has been made to implement agro-environmental measures in intensified olive groves. The recent rural development plan of Andalusia (2007–2013), according to a European Commission regulation (no. 1698/2005), established and funded specific agro-environmental measures to practise integrated production in olive groves, particularly in those situated in basins that supply surface or ground water to populations and in Natura 2000 areas. More than 155,000 ha of olive groves in this region are currently managed by integrated production methods. In his report on the environmental impacts of olive groves in the EU, Beaufoy [31] concluded that “. . .the negative environmental effects of intensification could be reduced considerably by means of appropriate farming practices; and that, with appropriate support, traditional low-input plantations could continue to maintain important natural and social values in marginal areas.” The last seems particularly true for organic olive groves (Figure 3), which effectively contribute to maintaining high standards of food safety and quality and reducing hazards to the environment (Table 2). Organic olive groves are located in Andalusia, mainly in mountainous areas, where intensification is difficult and olive yield is low. In these areas organic olive grove production offer an added value by means of agro-environmental subsidies and through market opportunities for organic products. Therefore, as long as these opportunities remain in force these low-input systems will continue to be economically advantageous and sustainable. THE GREENHOUSE VEGETABLE AGRO INDUSTRY Greenhouse cultivation is a steadily growing agricultural sector worldwide, especially in areas with warm climates, such as Mediterranean coastal areas. This system is able to satisfy increasing consumer demand for a wide range of high-quality products throughout the year using highly efficient resources. Vegetable crops are grown all over the Mediterranean basin under different production systems and with varying degrees of intensification. They have traditionally been grown in open fields, mostly during the summer period. However, over the past 40 years intensive horticultural crops, especially greenhouse horticulture, have become widespread in coastal areas of the northern and southern Mediterranean Basin, where the Agricultural Practices in the Mediterranean Chapter 3 31 mild winter climate enables vegetable production in simple greenhouse structures. The Mediterranean coast of southeast Spain represents the largest greenhouse area in Europe, [36] with about 37,000 ha, mostly concentrated on the Almerı́a coast and dedicated to producing fruit vegetables (Figure 1). Greenhouse horticulture in Almerı́a has undergone spectacular growth—from barely 3000 ha in 1970 to about 27,000 ha in 2010—transforming the area into a prosperous and competitive farming system. However, since the early 2000s the surface area dedicated to greenhouse production has hardly increased, principally because of the increase in input costs and the stabilization of vegetable sale prices [37]. Tomatoes, sweet peppers, cucumbers, melons, watermelons, zucchini (courgettes), eggplants (aubergines), and beans are grown in greenhouses mostly during the winter period, when these products have the strongest potential and competitiveness in European markets. Most greenhouse farms are small (1–2 ha), family-run concerns based on the use of enarenado soils and low-cost plastic greenhouses [37]. The introduction of the so-called enarenado technique [38], which consists of artificially layered soils with topsoils of gravel-sand mulches, spread very rapidly in the region from the early 1960s because it offered several advantages to greenhouse production: (i) improving water use efficiency by decreasing evaporation from the soil; (ii) mitigating soil salinity by avoiding superficial salt accumulation when irrigating with water of moderate salinity (2–4 dS m 1); (iii) improving the soil temperature in winter; (iv) reducing weed infestation; and (v) preventing soil compaction by farm workers and machinery. The greenhouse type known as Parral (Figure 5a), which is used by about 98% of local farmers [39], is an adaptation of the former structure used to support grape vines (Figure 5b) in the region [40], consisting of vertical rigid pillars on which a double grid of iron wire is placed and to which is attached a flexible plastic film. Although this structure has been largely improved by progressively increasing the volume-to-surface ratio, the ventilation rate (sidewall and roof vents), and the slope of the roof, microclimate control is still rather limited and crop growth conditions are usually outside the optimum range. Moreover, in these greenhouses the microclimate objectives change drastically depending on the time of year. During the cold period (late autumn and winter) the main objective is to increase the temperature, whereas the opposite occurs during late spring, summer, and early autumn. FIGURE 5 (a) Parral greenhouses in Vicar, Almerı́a, in eastern Andalusia. (b) Structure used to support grape vines (parral in Spanish), which was locally adapted to develop the Parral-type greenhouse in Almerı́a in eastern Andalusia. (a) Photo courtesy of Joaquín Hernández. (b) Photo courtesy of Oscar Lescure. 32 SECTION 1 The Mediterranean Diet: Concepts and General Aspects FIGURE 6 Cucumber crop in a greenhouse with an internal plastic cover (Almerı́a, eastern Andalusia). Photo courtesy of Santiago Bonachela. Mediterranean greenhouses are mostly low-cost structures covered with plastic film and have no active climate control systems, cultivating soil-grown crops [39]. In these greenhouses, passive techniques are usually used to improve the microclimate during both the cold and hot growth periods; active climate control systems consuming fossil energy are seldom used. In winter, internal plastic covers (Figure 6), plastic and gravel/sand mulches, or reduced natural ventilation are the most frequently used techniques, whereas natural ventilation and whitewashing the external surface covering are the main cooling methods used in spring and autumn. Consequently, the main microclimate variables (air temperature, humidity, and carbon dioxide [CO2] concentration and radiation) are usually below or above the optimum for fruit/ vegetable production during most of the crop cycles [41] and have a negative effect on yield and fruit quality [42]. As a result, crop productivity in these greenhouses is usually much lower than in the high-technology greenhouses of central and northern Europe and northern America, where microclimate conditions are usually maintained close to the optimum by active climate control systems (heating, dehumidification, supplementary lighting, etc.), which involve high energy consumption and, consequently, environmental effects. Baille [43] estimated annual greenhouse heating requirements of about 3000 MJ m 2 year 1 in northern European countries; more recently, an annual greenhouse heating requirement of 1180 MJ m 2 year 1 has been estimated in The Netherlands [44]. Moreover, Mediterranean greenhouses are not usually enriched with CO2 and they can therefore be considered CO2 sinks. The concentrated greenhouse areas on the Almerı́a coast are extremely vulnerable to the propagation of pests and diseases because of their high density, proximity, and low airtightness, as well as the overlapping of different crop cycles. In the past, most farmers applied intensive chemical programs to control pests and diseases, but this strategy resulted in the main pest populations developing resistance to the applied active ingredients [45], in particular Western flower thrips (Frankliniella occidentalis Pergande), the sweetpotato whitefly (Bemisia tabaci Gennadius), and beet armyworm (Spodoptera exigua Hübner). This, in turn, led to the intensification of chemical pesticide use, which became totally unsustainable in several crops, particularly sweet peppers, and therefore led to severe problems of chemical residues on fruits [45]. This problem was first alleviated by the introduction of bumblebee pollination in tomato crops, and since 2007 it has been practically solved by the large-scale introduction of biological control agents as the main component of integrated pest management and production systems [45]. During the 2012/2013 crop season, biological control was routinely applied in approximately 75–80% of the total greenhouse area of Almerı́a (Jan van der Bloom, personal communication). Key beneficial species in the biological control systems are Orius laevigatus Fieber, Amblyseius swirskii Athias-Henriot, Eretmocerus mundus Mercet, and Nesidiocoris tenuis Reuter [45]. The extended use of biological control within integrated production systems has drastically reduced the use of chemical pesticides and almost totally eliminated chemical residues on fruits [45]. The fast development of the greenhouse sector in Almerı́a also brought other challenges and negative environmental effects. In its initial stage in the 1970s and 1980s, the sector developed without any type of territorial planning and organization. This led to the overexploitation and contamination of aquifers and to the uncontrolled dumping of waste (organic, plastic, packaging, etc.). Different rural hygiene plans have been implemented since the early 2000s to improve the collection of many types of waste materials and their treatment, which have reduced this problem [37]. We should also highlight the growing awareness of farmers regarding the necessity to maintain a clean environment, particularly as a result of the extended incorporation of biological control techniques in farming. As a result, most residues are now adequately collected and treated, although new techniques are required to treat or recycle the huge amount of crop residues produced in the region. Agricultural Practices in the Mediterranean Chapter 3 33 Water is an increasingly scarce resource in Mediterranean regions, such as the Mediterranean coast of southeastern Spain, because of increased consumption and salinization. Greenhouse crops on the Almerı́a coast are drip irrigated, and the irrigation head usually consists of a pump, water filters, a fertilizer injector, and control mechanisms. Water is distributed by polyethylene drip lines with emitters placed every 0.5 m with nominal discharge rates of about 3 l h 1 [46]. Most farmers have small ponds close to their greenhouses to ensure the availability of water for irrigation [47]. Over 8000 ponds, most used for greenhouse irrigation, have recently been inventoried in Almerı́a [48], and this relatively high farm–pond density might have important implications for biodiversity conservation in an area with scarce wetlands [49]. Mean annual irrigation water supply for the main greenhouse crop rotations ranges between 444 [46] and 495 mm [50]. These values are lower than those found when these crops are grown outdoors [46,51] because of lower potential evapotranspiration (less solar radiation and wind and higher air humidity; use of plastic and gravel/sand mulches and cycles around the winter period) and the application of advanced irrigation technologies (drip irrigation, reuse of drainage water, etc.). Greenhouse cultivation also is characterized by greater water use efficiency due to a higher productivity (better control of microclimate, fertigation, and crop pests) and to the above-mentioned lower potential water use. Mean greenhouse values of irrigation water use efficiency ranged from 15.3 (autumn-winter green beans; Table 3) to 35.6 kg m 3 (spring watermelons) [46]. Moreover, the irrigation water use efficiency can be further improved by better greenhouse climate control, installation of automated fertigation systems, or the development of semiclosed greenhouses for Mediterranean climates. Finally, mean values of the relative irrigation supply (RIS, the ratio of irrigation water supply to crop water requirements) were close to 1 for most greenhouse vegetable crop cycles and rotations, indicating that, on average, the irrigation supply matched the maximum water requirements of these crops. However, the high variation coefficients observed for RIS and the analysis of the RIS dynamics throughout the cycles (Table 3) indicate that there are certain crops and cycles for which the irrigation water supply clearly does not match the crop water requirements [46]. Greenhouse irrigation water use in the Almerı́a coastal region can, therefore, be improved. Over the past 40 years, the province of Almerı́a (Spain’s driest province, with a mean annual rainfall of 250 mm) has endured a net water abstraction overdraft, which has led to serious groundwater imbalances mainly caused by increased demand for agricultural (e.g., greenhouse horticulture) and domestic needs (e.g., rapid urban growth and tourism development). Many of the underlying aquifers in the main greenhouse areas have been vastly overexploited, leading to significant water deficits and increasing salinization [52]. As a result, politicians and water planners have questioned long-term water sustainability in the region [53]. To solve this serious environmental and socioeconomic problem, the AGUA program [53] was developed by the national and regional governments in the early 2000s. This program promotes greater water efficiency resulting from higher water tariffs, increased water reuse, and water infrastructure improvements in conjunction with increasing use of desalinated water. However, it also points out that water savings alone will not be sufficient to meet future water demands in Spanish Mediterranean coastal areas and highlights desalination as the means to guarantee future availability and quality [53]. Consequently, during the past decade new, alternative water sources are TABLE 3 Values, Averaged for Six Cropping Seasons, and their Coefficients of Variation (in Parentheses) of Irrigation Water Supply (IWS), Relative Irrigation Supply (RIS), Irrigation Water Use Efficiency (IWUE) and Water Productivity (WP) of the Main Crop Cycles in Campo de Dalı́as (Almerı́a), Southeast Spain RIS Crop cycles IWS (l m 2) Cycle 1 2 3 IWUE (kg m 3) WP (€ m 3) Sweet pepper A-W 311 (32) 0.95 (36) 2.78 1.27 0.91 21.0(40) 13.1(50) Cucumber 270 (40) 1.62 (40) 3.53 1.48 1.20 33.2(52) 12.4(59) Green bean A-W 158 (33) 1.18 (24) 4.28 1.06 0.76 15.3(45) 15.9(45) Melon 177 (31) 1.00 (39) 3.52 1.19 0.52 22.8(34) 10.1(44) Watermelon 189 (38) 0.92 (33) 2.41 1.27 0.42 35.6(34) 7.8 (46) Green bean S 197 (24) 1.03 (28) 4.25 1.80 0.60 16.8(31) 15.4(53) Sweet pepper A-S 363 (30) 1.02 (27) 4.85 0.88 0.68 16.9(23) 8.7 (40) RIS values are presented for the whole cycle and during three crop periods along each cycle: crop establishment (1), crop development (2), mid to end of the season period (3). A, autumn; W, winter; S, spring. Source: Modified from Fernández et al. [46] (see list of references) 34 SECTION 1 The Mediterranean Diet: Concepts and General Aspects being used in Almerı́a’s greenhouse horticulture. Several desalination facilities have recently been installed in the region (the seawater desalination plant at Carboneras is currently Europe’s largest, with a net capacity of 42 hm3 year 1). About 6 hm3 of recycled wastewater, with high nutrient concentrations and low to medium conductivity values, is used in Almerı́a to irrigate intensive crops, particularly greenhouse tomatoes [53]. About 100 mm of the annual rainfall is already harvested on greenhouse covers by 50% of farmers [54]. In the near future, these additional water sources, together with the increasing water tariffs, may improve water use sustainability in the region. The greenhouse horticulture system of southeastern Spain presents appreciable nitrate contamination in the underlying aquifers [55]. Most of the areas where the greenhouses are concentrated have been declared nitrate vulnerable zones (Junta de Andalucı́a, Decreto 36/2008) in accordance with the EU Nitrate Directive (91/676/EEC). As a consequence, there is a legal requirement to implement practices that reduce nitrate leaching from greenhouse-grown crops. Most greenhouse farmers currently use fertigation systems to apply complete nutrient solutions by drip irrigation [39], but fertilizer and irrigation management is mostly based on accumulated local experience, with little consideration of environmental issues [56]. Fertilizers seem to represent the major environmental burden in Mediterranean greenhouses [57]. Most fertilizer leaching seems to be associated with huge manure applications [56] and excessive irrigation both before planting/sowing crops and while establishing crops [46,56]. Improvements should be focused on reducing fertilizer doses, adjusting the fertilizer–water balance, and implementing closed-loop irrigation systems [56]. For this purpose installing automated fertigation systems, together with using simple but robust systems of measuring water and nutrient drainage, seem to be essential. The salinity of greenhouse irrigation water in southeastern Spain varies appreciably. The electrical conductivity (EC) mostly varies between 0.5 and 4 dS m 1, but irrigation waters with moderate salinity predominate. Thus, the mean water EC from 97 irrigation ponds recently surveyed in the main greenhouse areas of Almerı́a and Granada was 1.9 dS m 1 [58]. Optimum production of greenhouse crops can be achieved under a range of EC values in the root environment, which depends mostly on crop characteristics and growth medium, fertigation management, and microclimatic conditions. Water salinity can negatively affect the yield of fruit vegetable crops, but it also offers the possibility of controlling crop growth and product quality. Increased salinity can improve various aspects of fruit quality in tomato crops, such as (i) the proportion of “extra” fruits (high visual quality); (ii) the soluble solids content; and (iii) the titratable acidity content [59]. However, the fruit size, a major determinant of price, is usually reduced. Increasing salinity may also increase the content of health-promoting attributes in tomatoes, such as lycopene content [60]. SUMMARY POINTS l l l l l Past and present farming practices in the Mediterranean are pivotal to interpreting current social and environmental patterns in this region. Farming practices in this region have evolved to cope with fluctuating environmental conditions such as water availability. Millennia of farmer–environment interactions have shaped highly resilient traditional agro systems. 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Effects of salinity on fruit yield and quality of tomato grown in soil-less culture in greenhouses in Mediterranean climatic conditions. Agric Water Manage 2008;95(9):1041–55. Kubota C, Kroggel M, Torabi M, Dietrich KA, Kim H, Fonseca J, et al. Changes in selected quality attributes of greenhouse tomato fruit as affected by pre- and postharvest environmental conditions in year-round production. HortScience 2012;47(12):1698–704. [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] Chapter 4 The Mediterranean Diet as an Intangible and Sustainable Food Culture Lluı́s Serra-Majem, MD, PhD1 and F. Xavier Medina2 1 University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain, and International Foundation of Mediterranean Diet, London, UK. 2 Universitat Oberta de Catalunya (UOC), Barcelona, Spain. ABBREVIATIONS CIHEAM CIISCAM CVD FDM GHG IFMED LCA MD MDP PREDIMED SCP SCPCS SCPFB UNESCO WDP International Centre for Advanced Mediterranean Agronomic Studies Interuniversity Research Centre on Mediterranean Food Cultures cardiovascular diseases Mediterranean Diet Foundation greenhouse gases International Foundation of the Mediterranean Diet life cycle assessment Mediterranean diet Mediterranean dietary pattern Mediterranean diet in the primary prevention of CVD Spanish current dietary pattern SCP estimated from the Household Consumption Surveys SCP estimated from food balance sheets United Nations Educational, Scientific and Cultural Organization Western dietary pattern INTRODUCTION The Mediterranean diet (MD) as a system is a cultural, historical, social, territorial, and environmental heritage transmitted from generation to generation for centuries, and it has been part, as a Mediterranean Food System, of the lifestyles of Mediterranean peoples throughout their history [1]. This legacy passed on and evolved in a constant temporal and spatial flow and acts as a living heritage in unique and outstanding cultural spaces; it is used to promote respect for cultural diversity and human creativity, an expression of sociability and communication between villages and individuals, a way to reinforce individuals’ identities in their places of origin, an integrative element of the nature and the history of communities, and a mechanism of defense for agriculture, sustainable rural development, and the landscape and environment of the Mediterranean area [2,3]. MD: AN INTANGIBLE CULTURAL HERITAGE Since November 16, 2010, the MD was inscribed on the United Nations Educational, Scientific and Cultural Organization’s (UNESCO) representative list of Intangible Cultural Heritage of Humanity [4]. The objective of this initiative was to safeguard the immense legacy representing the cultural value of the MD, as well as to share and disseminate internationally its values and benefits [5]. The culmination of this institutional process was fruit of the seed that had been sown in civil society in 2004—1 year after the approval of the UNESCO convention for the safeguarding of the intangible cultural heritage and 2 years before it came into force—as the Mediterranean Diet Foundation (FDM in its Spanish abbreviation), which hosted the first formal proposal to present this nomination, based on an original idea from L. Serra-Majem, the President of the FDM from April 1996 to November 2012. The proposal was decided after a difficult negotiation by the different The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 37 38 SECTION 1 The Mediterranean Diet: Concepts and General Aspects stakeholders involved in the decision process, who were very skeptical at the beginning of the process. The former Spanish Ministry of Agriculture, Fisheries and Food, patron of the FDM, played a fundamental role and was a vital link between the civil initiative and the governmental institutions. The Ministry made a firm commitment to this initiative, as did the Ministry of Italy, which showed the same support, favoring the transnational profile of the nomination right from the beginning. The implication of Greece and Morocco would firmly seal this joint endeavor. The presence and commitment of the Ministries of Culture confirmed another noteworthy aspect of this nomination: institutional transversality [1,2,6,7]. This process was conceived and had been germinating in civil society; had the privilege of counting on the involvement of national, regional, and local institutions; and received the unconditional support of the scientific community. Both governmental institutions and the scientific community were fundamental, and synchrony between all was decisive. It continued to enjoy the support and commitment of all the sectors that had worked in favor of this Mediterranean heritage for many years [1,8]. After publicly expressing the wish to nominate the MD as an Intangible Cultural Heritage of Humanity, there was a genuine explosion of enthusiasm and support from institutions and all types of associations, thus consolidating the transversal nature of the project. This elation demonstrated that a close bond and genuine identification persisted between Mediterranean societies and their cultural and food heritage [1,9]. The first event at which the proposal of the nomination was publicly presented took place on October 1, 2005, during the Year of the Mediterranean. The solemn setting of the University of La Sapienza de Roma provided an ideal context for the Third Euro-Mediterranean Forum on the “Dialogue through the civilisation and peoples of the Mediterranean: food cultures,” with the large participation of international scientists. It was there that the conference and the proposal of the nomination of the MD were delivered by Serra-Majem after convincing initially skeptical colleagues from other countries [10]. The proposal of the nomination received the unanimous support of the Third Forum, which included it in its final Declaration, The 2005 Rome Call: “To take into account that the Mediterranean Diet besides its health implications also has cultural and economic implications, therefore all Mediterranean countries need to agree and contribute to the process of preservation and promotion. To start the process of the recognition of the Mediterranean Diet Food Cultural Heritage behind the UNESCO, as an initial and shared common position to be coordinated from the Barcelona counterpart as an extension of the 1995 Barcelona Declaration, in collaboration with all the Mediterranean country representatives” [10]. In Barcelona in March 2006, during the VI International Congress on the MD, the international scientific community, following The 2005 Rome Call, renewed their unanimous support for the presentation of the nomination as well as the formalization and immediate commencement of the process and made an appeal to all Mediterranean institutions and organizations to follow suit and support the initiative [1]. In October 2007, on World Food Day, the FDM International Scientific Committee met in Barcelona and approved the “Declaration of Barcelona on the Mediterranean Diet as intangible cultural heritage” [1]. In December of that same year the former Spanish Ministry of Agriculture, Fisheries and Food held the first transmediterranean institutional meeting in Madrid, at which Spain, Greece, Italy, and Morocco agreed on the preparation of the nomination based on a strategic document prepared by the FDM and created their respective national teams. The following spring, in April 2008, at the Italian Ministry of Agricultural, Food and Forestry Policies in Rome, the four countries formalized the process through the Declaration of Rome and designated the FDM as technical transnational coordinator of the nomination [1]. The transversality of the MD and its environmental, economic, social, and cultural transcendence confer a unique dimension to the inscription. The derived duty to safeguard this heritage goes beyond the strict framework, whether institutional or otherwise, of “culture” and demands the same transversality in its management [6,9]. Indeed, the element inscribed is the MD and constitutes “a set of skills, knowledge, practices and traditions ranging from the landscape to the table, including crops, harvesting, fishing, conservation, processing, preparation and, particularly, consumption of food . . .” [4]. This unique lifestyle, determined by the Mediterranean climate and space, is also demonstrated through associated festivities and celebrations [1]. Through its social and cultural functions and their significance, the lifestyle embodies landscapes, natural resources, and associated occupations as well as the fields of health, welfare, creativity, and intercultural dialog and, at the same time, values such as hospitality or conviviality, sustainability and biodiversity [11]. All of these items compose this transversal cultural complex we call the MD and which is understood as a complete lifestyle. It is important to remark that the whole concept of the MD has been inscribed, not each of its components, whether tangible or intangible—neither olive oil from Jaen, nor the moussem from cherries, the market of Mistras, nor the Capponata. However, they are all individual examples of substantial components of the inscribed element; that is, they are a necessary constitutive part of the MD, the element recognized as an intangible cultural heritage of humanity. However, none of them, nor any other in the framework of this nomination and inscription, bears this recognition at an individual level [1]. The inscription of the MD has put numerous and important questions on the table. We cannot pretend to have solutions, responses, or strategies readily available for all, nor can we address them exhaustively. What is important is to confer to them the importance that they deserve and work without delay to answer them [1]. The first question is that of the Mediterranean Diet as Culture Chapter 4 39 geographical limits of the Mediterranean area, which we could call the “continent” of our heritage. In practical terms, or regarding the application of certain norms or regulations, certain limits can be agreed on and supported by the combination of various scientific and technical parameters [12]. Another question of utmost importance refers to the “content” of the MD, namely its tangible and intangible components. This can be conceptualized as the inventory, which is both an indispensible process and a tool that is constantly being updated. This is a complex task that does not start from scratch, because many sources are available today. It is demanding because the management of the tangible (utensils, artefacts, etc.), the intangible (festivities, knowledge, skills, etc.), and the landscapes and spaces are a constant concern. The task proposed is undoubtedly complex and should be addressed without delay through the pooling of concepts, selection criteria, processes, methodologies, and inventories, with their records and supports, to reach a consensus about all these items and more, which should allow us to become deeply acquainted with and define the content of the diet as to be able to value and decide what and how it is to be protected. A permanent surveillance system seems to be an indispensible tool in the medium term because this is live heritage, the evolution of which should be observed and analyzed carefully, without interruption, and, just as important if not more so, be appropriately accompanied by the coherence, consensus, and respect that this implies. This includes both the bearers of the MD heritage and the spatial-temporal contexts, or the tangible supports of the intangible manifestations of this heritage [13–18]. However, there is at present a deficit, especially at the government and public policies level, of acting practically in relation to safeguarding the MD. Real work of this nature is still urgent [19]. Responses concerning landscapes, techniques, elaborations, products, festivities, and so on will evidently be necessary, but the question is not simple. However, the difficulty and exigency of any project is always accompanied by the vector of progression and improvement [1]. The key to a respectful and sustainable future of the MD lies in the capacity for and commitment to investing resources, intelligence, and perseverance in sufficient quantities and applying them efficiently. The Convention points out the “the importance of intangible cultural heritage” as a “guarantee of sustainable development.” This criteria of sustainability is not exclusive of or inseparable from products, resources, spaces, or uses. We should also apply it to processes, decisions, and attitudes. Along these lines the questions that we have just put forward will find appropriate and sustainable responses [20]. The recognition of the MD as intangible cultural heritage of humanity by the UNESCO was never a final objective; it was not merely meeting any goal but the necessary impulse for a worthwhile outcome in the future, one that undoubtedly will require a great deal of clarity, composure, and perseverance. The nomination process was perfectly delimited and had clearly marked stages. The future that began on the day of the inscription is a succession of wide horizons in an unlimited time frame. The distance is huge and commands great respect. The Mediterranean is an area of great complexity because of both endogenous and exogenous vectors, and the MD is a cultural complex of capital importance that holds in its transversality and multifaceted nature some of its most defining and probably unique features. Throughout the whole process of the proposal, we always defended this complexity as a valued product of a history reaching back thousands of years; of processes of civilization, trade, and exchanges; of apprenticeships and transmissions; of tradition and innovations; of convergences and collisions. This complexity is one of the great strengths and potentialities of the MD as a vector of regional development, and, as such, it has contributed to the diet’s survival. It is also, however, an Achilles heel, discreet but vulnerable. The challenge of safeguarding the MD is enormous, but looking beyond foreseeable difficulties or the complexity of the objectives, the sum of efforts and commitments encourages and strengthens [7,12,18,19]. The Road Map for the future of the Mediterranean Diet development worldwide is currently under the auspices of a new Institution: The International Foundation of the Mediterranea Diet (IFMED). MD AND HEALTH Public health represents a major element in appraising the sustainability of the MD. In addition to its benefits in preventing cardiovascular disease, diabetes, and cancer, the MD has other numerous health benefits, related to immunity, allergic diseases, the psyche, or even quality of life, that are current fields of research [21–25]. The MD has an international projection and is thought to be the healthiest and the most sustainable eating pattern on the planet; it also is a key player in the public health nutrition field globally and especially in the Mediterranean area [19,24,26]. Relevant prospective epidemiological studies and some clinical or community trials, such as the PREDIMED study [23], have increased exponentially in the past five decades, as has the level and the quality of evidence around the MD. In the first systematic review of evidence from interventions using the MD conducted in 2006, the MD showed favorable effects on lipoprotein levels, endothelium vasodilatation, insulin resistance, metabolic syndrome, antioxidant capacity, myocardial and cardiovascular mortality, and cancer incidence in obese patients and in those with previous myocardial infarction [21]. 40 SECTION 1 The Mediterranean Diet: Concepts and General Aspects From the second published systematic review, a meta-analysis of the evidence of the relationship between the MD and health status by Sofi et al. [27], published in 2008 and revisited in 2010 [28] and 2013 [22], some interesting figures have been reported: a two-point increase in adherence score was significantly associated with a 9% reduction in overall mortality, a 10% reduction in cardiovascular disease mortality, a 6% reduction in neoplasm incidence or mortality, and a 13% reduction in incidence of Parkinson’s disease and Alzheimer’s disease among the general population. Furthermore, the PREDIMED trial [29] results pointed out that the MD, especially rich in virgin olive oil, is associated with higher levels of plasma antioxidant capacity. The plasma total antioxidant capacity is related to a reduction in body weight after 3 years of intervention using a Mediterranean-style diet rich in virgin olive oil in a population with high cardiovascular risk. Other PREDIMED results suggest there is no rationale to maintain the fear that Mediterranean foods rich in fats of vegetable origin (olive oil or tree nuts) may cause weight gain or be responsible for an increased risk of obesity, provided that the energy intake does not exceed energy expenditure [30]. The reviewed evidence from the elderly population is promising as well. In fact, in the PREDIMED study the MD was associated with a lower incidence of type 2 diabetes in both groups supplemented with nuts and supplemented with extra virgin olive oil [30]. Its uses in the primary prevention of CVD have recently been demonstrated [23]. The MD is the heritage of millennia of exchanges in the Mediterranean basin that have defined and characterized the eating habits of the countries in this region, but currently it is progressively being rapidly transformed [3] because of many factors of the Western economy and urban and technological society as well as the globalization of production and consumption. This food culture is now under three serious threats: (1) the American fast food culture based on meat, refined grains, potatoes, ice cream, candies, and beverages high in sugar; (2) the economic crisis affecting in particular the most disadvantaged groups and instigating decreased consumption of key MD food groups such as fruits, vegetables, virgin olive oil, nuts, and fish, accompanied by corresponding increases of refined grains, potatoes, and sugars; and (3) the promotion of high-protein diets, also prescribed by doctors and specialists, as a tool for weight loss or maintenance, with a major impact on health [31]. The erosion that can cause these threats, especially economic, must be countered with actions based on nutrition education, with the commitment that neither cost nor unfounded food choices cannot and should not be a barrier to the availability of basic foods of the MD: olive oil, fruits, vegetables, grains, dairy, nuts, or fish. Governments thus need to be committed to take appropriate actions that preserve this traditional and cultural knowledge and lead to a diversity of foods and diets, not only for the health benefits that they could provide in the short and long term [32]. MD AND SUSTAINABLE ENVIRONMENT Food systems’ environmental consequences are in public health agendas. Foods are produced, processed, distributed, and consumed, all of which have consequences on both human health and the environment [12,20,32]. In fact, food production is inevitably also a driver of environmental pressures, particularly in relation to climate change, water use, and toxic emissions [33]. For example, agriculture is one of the main contributors of emissions of greenhouse gases (GHGs), in particular methane and nitrous oxide, which are responsible for global warming. Other parts of the food system contribute to carbon dioxide emissions from the use of fossil fuels in processing, transportation, retailing, storage, and preparation. Food items differ substantially in their environmental footprint, which, among many other descriptors, can be measured as energy consumption, agriculture land use, water consumption, or GHG emissions [34]. Animal-based foods are by far more land and energy intensive compared to foods of vegetable origin [35]. Thus, dietary patterns can vary substantially in resource consumption and their impact on the environment as well as on the health of a given population [34]. Even though most of the literature regarding the environmental contribution of the different food groups is from different settings and types of analysis, their global statements generally converge. Plant-based foods contributed least to the selected environmental footprints. Although, as expected, in the Mediterranean dietary pattern (MDP), where meat and dairy represented a lower weight, in terms of water consumption and with a less extent energy consumption, vegetables had a much higher contribution compared to the other patterns [36]. Plant foods based on vegetables, cereals, and legumes are stated to have the lowest GHG emissions, even if processing and substantial transportation is involved [19,34]. In a recent study, legumes were included in the vegetable group as having similar low environmental impact values, as indicated in other studies; however, life cycle assessment data for legumes in and of themselves was not available. In fact, legumes are stated as alternatives to animal proteins because of their low environmental impact and long durability [34]. However, some foods of vegetable origin contribute substantially—together with dairy, in the case of the MDP and Spanish current dietary pattern (SCP)—to either water consumption (especially vegetable oils and to some extent nuts) or land use (cereals and vegetable oils) in their production. In both the SCP and the Western dietary pattern (WDP), vegetable oils were also important contributors to water consumption and energy consumption footprints, whereas animal-based foods were found to have a higher environmental cost in all dietary patterns (Table 1). In some studies, in the Spanish context meat and Mediterranean Diet as Culture Chapter 4 41 TABLE 1 Environmental Footprints for the Mediterranean Dietary Pattern (MDP), the Western Dietary Pattern (WDP), and the Two Estimates of Current Spanish Dietary Pattern (SCP) for the Total Spanish Population, as well as Estimated Current Real Pressure for Each Footprint 3 1 Agricultural land use (10 Ha year ) 1 Energy consumption (TJ year ) 3 1 Water consumption (km year ) Greenhouse gas emissions (Gg CO2 eq year) MDP SCPFB SCPCS WDP Current Real Pressure 8365 19,874 12,342 33,162 15,400 239,042 493,829 285,968 611,314 229,178 13.2 19.7 13.4 22.0 19.4 35,510 125,913 72,758 217,128 62,389 CS, consumption surveys; FB, food balance sheets. Source: Sáez-Almendros S, Obrador B, Bach-Faig A, Serra-Majam L. Environmental footprints of Mediterranean versus Western dietary patterns: beyond the health benefits of the Mediterranean diet. Environ Health 2013;12:118. dairy contributed most to environmental footprints [37,38], although in a lower absolute contribution than in the WDP. Regarding GHG emissions and land use, meat was undoubtedly the most contributing food item, with a large difference to other foods in both the WDP and SCP. It was observed that a reduction in meat consumption decreased GHG emissions [39] and land use and subsequently increased the availability of land for other uses [39]. Even though there is high production variability [34], 80% of global agriculture land use is related to livestock production and accounts for more than half of the GHG emissions from agriculture [40]. Meanwhile, dairy—as one of the main sources of animal protein in the MDP— was the largest contributor in terms of energy consumption in the three dietary patterns, and vegetable oils had a similar weight concerning water consumption in the WDP. In the MDP, dairy products were the food group that presented the highest footprint in all four analyzed footprints; in the MDP meat has the lowest weight in frequency and amount. Regarding GHG emissions, fish also made a remarkable environmental contribution in all the dietary patterns [38]. The most relevant dietary distinctions in terms of environmental cost are between animal-based versus plant-based diets but also seem to count the various ways foods are grown, processed, and transported. In general, the largest environmental impact of food production from the farm to consumers is associated with the primary production of agricultural products. In terms of energy consumption, differences in greenhouse production versus open-air cultivation of a certain crop, and canned or frozen-produce versus fresh-produce, are substantial [41,42]. In addition to the energy involved in agricultural production, the amount of energy used in household food storage, preparation, and waste is not negligible and in most cases is greater than in the intermediate processing [20,34]. Food policy and dietary guidelines need to go further from the classical approach which focuses only on nutrients to consider the environmental impact. Consumers tend to be more concerned each day about the environment and, even more, about their personal health, but food choices and cultural culinary traditions are not easily modified. Some studies state that even radical changes in food consumption patterns would induce quite small environmental benefits [43–45]. Thus, significantly reducing the environmental footprints by shifting from the SCP towards a Mediterranean-type diet would probably require substantial changes not only in consumers’ food choices but also especially in agrofood industry practices, public catering supply, and agricultural and trade policies [45–48]. An example, for instance, is the rationale of positively influencing prices of food subsidies. Spain is one of the major producers and exporters of typical Mediterranean products; thus it makes sense to stay with an MDP agricultural production model [38]. A shift from the SCP towards the MDP by complying with the food proportions and composition defined in the new MD pyramid [24] would be beneficial from both a health and environmental perspective. The MDP presented lower footprints than the SCP and to a much larger extent than the WDP. The MDP results in a lower environmental impact because of the consumption of more plant-derived products and fewer animal products [20,38]. The MDP is presented as not only a cultural model but as a healthy and environmentally friendly model, whose adherence in Spain would make a significant contribution to a greater sustainability of food production and consumption in addition to the well-known benefits on public health [20,38]. THE NEW MD PYRAMID The MD pyramid has evolved to adapt to the new way of life. By taking into consideration all scientific evidence for the health benefits of the MD and its protective effects against chronic diseases, as well as the present way of life and environmental constraints, the modern MD pyramid was developed: a lifestyle for today. As an initiative of the FDM together 42 SECTION 1 The Mediterranean Diet: Concepts and General Aspects with the Forum of the Mediterranean Food Cultures, and with the collaboration of numerous international entities, a wide range of experts in nutrition, anthropology, sociology, and agriculture reached a consensus about a new, richer educational tool (Figure 1) [24]. The pyramid addresses the healthy adult population and should be adapted for specific requirements in the case of children, pregnant women, and other health circumstances [29]. The new pyramid follows the previous pattern: At the base are food items that should sustain the diet and provide the highest energy intake, such as foods of plant origin (cereals, fruit, vegetables, legumes, tree nuts, seeds, and olives), with olive oil as the principal added fat source, which provide key nutrients and other protective substances that contribute to general well-being. At the upper levels are foods to be eaten in moderate amounts, such as those of animal origin and/or rich in sugars and fats which should be eaten in moderation; some of them should be left for special occasions. Moderate intake of alcohol, mainly in the form of wine during meals, is also stated. This plant-based diet is also characterized by high to moderate intake of fish and shellfish, moderate to low consumption of eggs, poultry, and dairy products (cheese and yogurt) [24]. This healthy MDP has been made popular through a pyramid representation, which graphically establishes guidelines about the foods to be consumed daily, weekly, and occasionally to follow a healthy and balanced diet. The first MD pyramid was presented in 1993 at the international conference on MDs held at the Harvard School of Public Health in Boston, MA [49]. Since then, together with the Oldways Preservation & Exchange Trust, various MD pyramids have been designed for several populations, particularly for North America [8]. Meals have an essential role in the MD, and thus the pyramid also reflects and introduces the concept of the composition of main meals. A balanced composition of main meals should include three basic elements: fruits, vegetables, and cereals, complemented by a lower contribution to daily energy intake from other plant foods, dairy products, and protein sources [31]. Cultural and Lifestyle Aspects Together with the proportion and frequency recommendations of consumption, the incorporation of lifestyle and cultural elements is one of the innovations of the pyramid. Thus, it is not just about prioritizing some food groups over others but also about paying attention to the methods of selecting, cooking, and eating them. Adopting a healthy lifestyle and FIGURE 1 The English version of the new Mediterranean diet pyramid, from an international consensus coordinated by the Mediterranean Diet Foundation. Adapted from Bach-Faig et al. [24]. Mediterranean Diet as Culture Chapter 4 43 preserving cultural elements should also be considered to acquire all the benefits from the MD. These elements are summarized though some key concepts [9,31]: Moderation. For public health reasons, and to fight against the obesity pandemic, serving sizes should be based on frugality and moderation to adapt urban and modern lifestyles to energy needs [50]. Conviviality. The aspect of conviviality is important for the social and cultural value of the meal, beyond nutritional aspects. Cooking, sitting around the table, and sharing food in the company of family and friends is a social support and gives a sense of community [24,31]. Culinary activities. Devoting enough time and space for culinary activities is stressed, accounting for their role in everyday meals, celebrations, and religious festivals in every culture [24,31]. Seasonality, biodiversity, eco-friendliness, traditional and local food products. These are presented at the bottom of the pyramid to highlight how the new revised modern MD is compatible with the development of a sustainable diet for present and future Mediterranean generations. The preference for seasonal, fresh, and minimally processed foods maximizes the content of protective nutrients and substances in the diet [24,31]. Physical activity. Regular moderate physical activity (at least 30 min throughout the day) is indicated as a basic complement to the diet for balancing energy intake, maintaining a healthy body weight, and achieving many other health benefits. Walking, taking the stairs instead of the elevator, doing housework, etc., are simple and easy ways of exercising. Practising leisure activities outdoors and preferably with others makes it more enjoyable and strengthens the sense of community [11,24,31]. The new pyramid is a result of international consensus and is based on the latest scientific evidence of health and nutrition published in hundreds of scientific articles in the past two decades, thus contributing to the harmonization of educational tools used in the promotion of the MD and responding to the need for a common framework among Mediterranean countries [24]. The use and promotion of this pyramid is recommended without any restrictions, and the 2010 edition has been adapted, translated, and edited in 10 different languages (English [Figure 1], Arabic [Figure 2], Catalan, Galician, Euskera, French, Spanish [Figure 3], Italian, Portuguese, and Greek) by the FDM in collaboration with some international organizations [24]. FIGURE 2 The Arabic version of the new Mediterranean diet pyramid, from an international consensus coordinated by the Mediterranean Diet Foundation. Adapted from Bach-Faig et al. [24]. 44 SECTION 1 The Mediterranean Diet: Concepts and General Aspects FIGURE 3 The Spanish version of the new Mediterranean diet pyramid: from an international consensus coordinated by the Mediterranean Diet Foundation. Adapted from Bach-Faig et al. [24]. FINAL CONSIDERATIONS Recognition from UNESCO, with the consequent increased visibility and acceptance of the MD around the world, along with better and more scientific evidence regarding its benefits and effectiveness on longevity, quality of life, and disease prevention, make this dietary pattern experience an unprecedented historical moment. This is a favorable situation that could possibly enable better knowledge about the MD worldwide, translating into a reduction of the environmental impact of production and transportation of food resources in the entire Mediterranean area [51]. To this end, the MD should be seen as what it is: an extremely healthy and environmentally sustainable food model, as well as an ancient cultural heritage that confers identity and belonging. SUMMARY POINTS l l l l l The MD as an ancient cultural heritage is a healthy and environmentally sustainable food model that confers identity and belonging. In 2010 the MD was inscribed into the UNESCO’s Representative List of Intangible Cultural Heritage of Humanity. Because of its transversality, the MD is a complex set of skills, knowledge, practices, and traditions ranging from the landscape to the table, including crops, harvesting, fishing, conservation, processing, preparation, and, in particular, consumption of food. This unique lifestyle, determined by the Mediterranean climate and space, is also shown through associated festivities and celebrations. The MD embodies landscapes, natural resources, and associated occupations as well as the fields of health, welfare, creativity, and intercultural dialog, and at the same time, values such as hospitality or conviviality, sustainability or biodiversity. Scientific evidence shows that the MD is a healthy and suitable pattern that relates to lower risk of mortality and other diseases such as cardiovascular disease, diabetes, and cancer, as well as having other numerous health benefits that are currently fields of research, such as immunity, allergic diseases, psychological factors, and quality of life. Mediterranean Diet as Culture Chapter 4 l l 45 The MDP results in a lower environmental impact because of the consumption of more plant-derived products and fewer animal products. 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Food consumption and civil society: Mediterranean diet as a sustainable resource for the Mediterranean area. Public Health Nutr 2012;14 (12A):2346–9. Chapter 5 The Mediterranean Diet and Mortality Genevieve Buckland, MSc, PhD and Antonio Agudo, MD, MSc, PhD Catalan Institute of Oncology (ICO), Barcelona, Spain. ABBREVIATIONS CBVD CHD CI CVD EPIC HR MD MDS MUFA PREDIMED PUFA rMED RTC SFA SUN cohort cerebrovascular disease death coronary heart disease confidence interval cardiovascular disease European Prospective Investigation into Cancer and Nutrition hazards ratio Mediterranean diet Mediterranean diet score monounsaturated fatty acids Prevención con Dieta Mediterránea polyunsaturated fatty acids relative Mediterranean diet score randomized control trial saturated fatty acids Seguimiento Universidad de Navarra cohort INTRODUCTION The relationship between the dietary patterns found in Mediterranean populations and different aspects of health has been extensively researched [1–5], and the epidemiological evidence suggests that the Mediterranean diet (MD) is an optimal diet for increasing longevity and preventing noncommunicable diseases. Studying mortality is a good general indicator of the overall health of a population, and assessing the relationship between adherence to an MD and overall mortality is of scientific interest because it reflects all the possible health benefits (known or unknown) that could be gained by following this dietary pattern, thereby giving an indication of its overall impact on health. The increased longevity in Mediterranean countries has been attributed in part to the MD’s protective role against major chronic diseases such as cardiovascular disease (CVD), certain cancers, type 2 diabetes, and some neurodegenerative diseases, as supported by findings from numerous observational studies [2,5,6]. These studies used dietary scores incorporating key elements that reflect the MD pattern to be able to evaluate the level of adherence to an MD. Randomized controlled dietary intervention trials also have demonstrated that following an MD reduces the incidence of type 2 diabetes [7], metabolic syndrome [8,9], and major CVD events and its risk factors [10–12]. The nutritional profile of the MD pattern that could be relevant due to its beneficial effects on mortality include its favorable fatty acid profile (due to regular consumption of olive oil, nuts and seeds, fish, and seafood and less frequent consumption of dairy products and meat), moderate ethanol intake (traditionally wine), high intake of plant proteins and low intake of animal proteins, high fiber content, and rich variety of vitamins and minerals from fresh fruit and vegetables [1,3,5]. A SYSTEMATIC REVIEW PROCESS TO UNCOVER THE LINK BETWEEN DIET AND MORTALITY No isolated study or study type can provide evidence that a single factor or exposure is protective or causative of a disease. For this reason systematic reviews of various epidemiological studies are an important way to objectively evaluate diet– disease relationships. In this chapter we provide a systematic review of the epidemiological studies (both observational and The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 47 48 SECTION 1 The Mediterranean Diet: Concepts and General Aspects experimental studies) to date that have researched the association between the MD and overall and cause-specific mortality in both Mediterranean and non-Mediterranean populations. The relative importance of the components within the MD scores (MDSs) to overall mortality, when available, also is reviewed. A MEDLINE search (until February 2013) including the terms Mediterranean diet or dietary pattern or diet in combination with other key words related to health status—mortality, survival, longevity, morbidity, CVD, coronary heart disease, stroke, cerebrovascular disease, neoplasm and cancer—was performed. The references from the extracted articles and reviews also were consulted to complete the search. We narrowed the search to include only articles examining the effect of the MD as a whole entity (i.e., the combined effect of the key MD components) on mortality. However, there was no restriction on the definition of the MD in terms of the inclusion of specific foods or nutrients. Studies were included if the main outcome was overall mortality or cause-specific mortality (i.e., death from CVD, cancer, or other causes). According to the criteria above, 21 articles from 18 independent studies were selected, of which 19 were cohort studies (one was a clinical trial analyzed as a cohort study) and 2 were randomized controlled trials (RCTs). The general characteristics of these studies and the main results are presented for the cohort studies and RCTs in Tables 1 and 2, respectively. ASSESSING LEVEL OF ADHERENCE TO THE MD The majority of the cohort studies in this area have used an MDS to assess each participant’s level of adherence to the MD (Table 1). The first a priori-defined MDS was developed over a decade ago by a Greek researcher, Dr. Trichopoulou, and her team [13]. It included nine key components of the MD, and a value of 0 or 1 was assigned to each participant depending on whether their intake of vegetables, fruit and nuts, legumes, cereals, fish and seafood, and monounsaturated fatty acid (MUFA)-to-saturated fatty acid (SFA) ratio was below or above the sex-specific medians. In contrast, 1 and 0 points were assigned for consumption below or above the sex-specific median intakes of meat and meat products and dairy products. Alcohol was scored dichotomously, assigning 1 point for moderate consumers (defined as 5-25 g/day for women and 10-50 g/day for men) and 0 points for intake above or below these sex-specific ranges [13]. Each participant’s points then were summed to give a range from 0 to 9, reflecting low to high adherence to the MD. There are now various modified versions of this MDS and other MDSs that are widely used to study the relation between the MD pattern and different health parameters [14]. For example, the relative Mediterranean diet (rMED) score is a modified version of the original MDS [13] developed for the EPIC-Spain cohort study [15]. It uses global tertiles as cutoffs instead, assigning 0, 1, or 2 points for intake within the first second and third tertiles of each component (calculated as a function of energy density) except alcohol. The scoring was reversed for the two components presumed not to fit the MD, and the total score ranged from 0 to 18. Instead of using the ratio of MUFA to SFA, the rMED includes olive oil intake, because this is one of the hallmarks of the MD. THE RELATIONSHIP BETWEEN THE MD AND MORTALITY The Magnitude of the Protective Effect Table 1 presents the main results from the 19 cohort studies [13,15–32] researching risk of mortality according to level of adherence to an MD (comparing participants with a high versus low MDS or a one- or two-unit increment in the MDS). In 2010 Sofi et al. [5] published a meta-analysis of eight of these cohort studies [17,19,22,26,28–30,32] published between 1995 and 2009 and reported that a two-unit increase in adherence to the MD was significantly associated with a reduced risk of mortality by 8% (relative risk, 0.92; 95% confidence interval [CI], 0.90-0.94). Two of the studies reported a negative but nonsignificant association [26,32]. Since this meta-analysis several large cohort studies [15,16,18,23,25] published results on the association between the MD and overall mortality. The SUN (Seguimiento Universidad de Navarra) cohort study [16] reported an impressive 62% reduced risk of dying (95% CI, 30-79) associated with high adherence to the MD, and a two-unit increment in their MDS was related to a 17% decrease in mortality (P for trend ¼ 0.006). The GISSI-Prevenzione trial [18] found a significant 15% decreased risk of mortality for each one-unit increase in their MDS. In the EPIC-Spain study [15] the MD was associated with a 21% decrease in mortality (95% CI, 9-31) for high versus low rMED score after adjusting for a large number of possible confounders. There was a 6% decreased risk of mortality (P for trend < 0.001) for each two-unit increment in the rMED score. TABLE 1 Study Characteristics and Main Findings for Cohort Studies Researching Adherence to the Mediterranean Diet and Mortality Cohort Country Reference Cohort N/ Follow-up Mortality Cause, n Age (years) Mediterranean Diet Score Definition SUN Cohort Spain Martinez-Gonzalez et al. [16] 9264/ 6.8 years All n ¼ 125 38 12 Oviedo Elderly Study Spain Lasheras et al. [17] 161/ 9.5 years All n ¼ 96 GISSI-Prevenzione trial Italy Barzi et al. [18] 11,323/ 6.5 years Greek cohort Greece Trichopoulou et al. [19] Main Findings HR (95% CI) MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Sex, age, education, BMI, smoking, physical activity, energy, egg, potato intake, h/d TV, hypertension, hypercholesterolemia, depression High versus low MDS: HR 0.38 (0.21, 0.70) +2p MDS: All HR 0.72 (0.58, 0.91) 65–95 MDS (no legume), eight items (+): cereals (including potatoes and bread), vegetables, fruit, fish, MUFA/SFA, (+m): alcohol**, (): meat and meat products, milk and dairy Sex, age, albumin concentration, self-assessment of health, physical activity, BMI, health related dieting +1p MDS (no legume): <80y: HR 0.69 (0.43, 0.93) 80y: HR 1.24 (0.60, 2.53) All n ¼ 1660 19–90 MD: (+) fish, fruit, raw vegetables, cook vegetables, olive oil Sex, age, smoking, hypertension, diabetes, cardiovascular risk factors and medications, HDL cholesterol +1p MDS: HR 0.85 (95% CI 0.82, 0.88) 182/ 6.8 years All n ¼ 53 >70 First MDS, eight items (+): cereals, vegetables, fruit, legumes, MUFA/ SFA, (+m): alcohol**, (): meat and meat products, dairy products Sex, age, smoking +1p MDS: HR 0.83 (0.69, 0.99) EPIC-Spain Buckland et al. [15] 40,622/ 13.4 years All n ¼ 1855 [CVD n ¼ 399 Cancer n ¼ 913 Other n ¼ 425] 29–69 rMED: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, and seafood, olive oil, (+m): alcohol**, (): meat and meat products, dairy products Sex, age, center, BMI, waist circumference, education, physical activity, smoking, energy intake High versus low rMED: All; HR 0.79 (0.69, 0.91) ♂ HR 0.76 (0.63, 0.90) ♀ HR 0.85 (0.68, 1.06) +2p rMED: HR 0.94 (0.90, 0.97) EPIC-Greece Trichopoulou et al. [13] 22,043/ 3.7 years All n ¼ 275 [CHD n ¼ 54 Cancer n ¼ 97] 20–86 MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Sex, age, BMI, smoking, education, energy intake, waist/hip circumference, egg, potato +2p MDS: All HR 0.75 (0.64, 0.87) ♂ HR 0.78 (0.65, 0.94) ♀ HR 0.69 (0.53, 0.90) EPIC-Greece Misirli et al. [20] 23,601/ 10.6 years CBVD n ¼ 196 20–86 MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Sex, age, BMI, smoking, education, physical activity, hypertension, diabetes, energy intake High versus low MDS: HR 0.88 (0.73, 1.06) +2p MDS: All HR 0.75 (0.64, 0.87) ♂ HR 0.94 (0.72, 1.23) ♀ HR 0.82 (0.64, 1.06) EPIC-Greece Dilis et al. [21] 23,929/ 10 years CHD n ¼ 240 20–86 MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Sex, age, height, BMI, physical activity, smoking, education, arterial blood pressure, alcohol, energy intake High versus low MD: HR 0.54 (0.37, 0.81). +2p MDS: All HR 0.78 (0.66, 0.92) ♂ HR 0.81 (0.66, 0.99) ♀ HR 0.75 (0.57, 0.98) 49 Continued The Mediterranean Diet and Mortality Chapter 5 Confounders Controlled For Cohort N/ Follow-up Mortality Cause, n Age (years) Mediterranean Diet Score Definition EPIC-Greece Trichopoulou et al. [22] 23,349/ 8.5 years All n ¼ 1075 20–86 British Diet and Nutrition Survey UK McNaughton et al. [23] 972/ 14 years All n ¼ 654 Danish branch of Euronut SENECA study Denmark Osler et al. [24] 202/6 years Vasterbotten Intervention Program N. Sweden Tognon et al. [25] Confounders Controlled For Main Findings HR (95% CI) MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk, and dairy Sex, age, education, smoking, physical activity, energy intake, waist/hip ratio, BMI +2p MDS: HR 0.86 (0.80, 0.93) 65 MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Sex, age, energy intake, social class, region, smoking, physical activity, BMI High versus low MDS: HR 0.78 (0.62, 0.98) All n ¼ 50 72 (mean) Reduced MDS, seven items (+): cereals, vegetables and legumes, fruit, MUFA/SFA, (+m): alcohol**, (): meat and meat products, milk and dairy products Sex, age, smoking +1p reduced MDS: HR 0.79 (0.64, 0.98) 77,151/ 10 years All n ¼ 2376 [Cancer n ¼ 974 CVD n ¼ 680 Ml n ¼ 305 Stroke n ¼ 144] 30–60 Refined mMDS, eight items: (+): whole-grain cereals, vegetables and potatoes, fruit and juices, fish and fish products, ratio of MUFA + PUFA to SFA (+m): alcohol, (): meat and meat products, dairy products Age, obesity, smoking, education, physical activity +1p refined mMDS ♂ HR 0.96 (0.93, 0.99) ♀HR 0.95 (0.91, 0.99) Scandinavian Women’s Lifestyle and Health Cohort (Swedish branch) Sweden Lagiou et al. [26] 42,237 (women)/ 12 years All n ¼ 572 [Cancer n ¼ 280] 30–49 MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Age, height, BMI, smoking, physical activity, education, energy, potato, egg intake PUFA, non-alcoholic beverages High versus low MDS: HR 0.89 (0.77, 1.04). +2p MDS: HR 0.93 (0.83, 1.03) <40y HR 1.09 (0.90, 1.32) 40y HR 0.87 (0.76, 0.98) Gothenburg Swedish Cohort Sweden Tognon et al. [27] 1037/ 8.5 years All n ¼ 630 70 Refined mMDS, eight items: (+): whole-grain cereals, vegetables and potatoes, legumes nuts and seeds, fruit and juices, fish and fish products, ratio of MUFA + PUFA to SFA (+m): alcohol, (): meat and meat products Sex, BMI, waist circumference, smoking, physical activity, marital status, education, birth cohort High versus low refined mMDS: HR 0.82 (0.67, 0.99) +1p refined mMDS HR 0.93 (0.89, 0.98) EPIC-Eldery 10 European countries Trichopoulou et al. [28] 74,607/ 7.4 years All n ¼ 4047 60 mMDS (modified Mediterranean diet score): cereals, vegetables, fruit and nuts, legumes, fish, MUFA + PUFA/SFA, (+m): alcohol**, (): meat and meat products, dairy products Sex, age, diabetes, education, smoking, physical activity, waist/ hip ratio, BMI, energy intake, eggs, potato, sugar and confectionary High versus low mMD: HR 0.91 (0.82, 1.01) +2p mMDS: HR 0.93 (89, 0.98) SECTION 1 The Mediterranean Diet: Concepts and General Aspects Cohort Country Reference 50 TABLE 1 Study Characteristics and Main Findings for Cohort Studies Researching Adherence to the Mediterranean Diet and Mortality—cont’d The Hale Project 11 European countries Knoops et al. [29] 2339/ 10 years All n ¼ 935 [CHD n ¼ 122 CVD n ¼ 371 Cancer n ¼ 233 Other n ¼ 145] 70–90 MDS no alcohol, eight items: (+): grains, vegetables and potatoes, fruit and fruit products, legumes and nuts and seeds, fish, MUFA/SFA, (): meat and meat products, dairy products Sex, age, BMI, study population, education, smoking, alcohol, physical activity +4p MDS no alcohol: HR 0.77 (0.68, 0.88) NIH-AARP USA Mitrou et al. [30] 380,296/ 10 years All* n ¼ 27,799 [CVD n ¼ 3451 Cancer n ¼ 5985 Other n ¼ 2669] 50–71 aMED, nine items (+): whole-grain cereals, vegetables (excluding potatoes), fruit, nuts, legumes, fish, MUFA/SFA, (+m): alcohol (5–25 g/ d) (): red and processed meat Age, energy intake, smoking variables, education, BMI, physical activity, race, marital status, menopausal hormone therapy (women) High versus low aMED: ♂ HR 0.79 (0.76, 0.83) ♀ HR 0.80 (0.75, 0.85) Nurses’ Health Study USA Fung et al. [31] 74,886 (women)/ 20 years CVD n ¼ 1077 CHD n ¼ 794 Stroke n ¼ 283 38–63 Adapted MDS, nine items (+): whole-grain cereals, vegetables (excluding potatoes), fruit, nuts, legumes, fish, MUFA/SFA, (+m): alcohol (5–15 g/d) (): red and processed meat Age, smoking, BMI, menopausal status, menopausal hormone therapy, energy intake, multivitamin intake, alcohol, physical activity, aspirin use Fifth versus first quintile adapted MDS HR:0.61 (0.49, 0.76) Anglo-Celtic, GreekAustralian cohort Australia KourisBlazos et al. [32] 330/N/S All n ¼ 38 70 MDS: nine items (+): cereals, vegetables, fruit and nuts, legumes, fish, MUFA/SFA, (+m): alcohol** (): meat and meat products, milk and dairy Sex, age, smoking, ethnic origin +1p MDS: All HR 0.83 (0.67, 1.02) Anglo-Celts HR 0.73 (0.45, 1.18) Greek-Australian HR 0.91 (0.69, 1.21) The Mediterranean Diet and Mortality Chapter 5 Abbreviations: hazard ratio (HR) and 95% confidence interval (CI); EPIC, European Prospective Investigation into Cancer and Nutrition; N/S, not specified; USA, United States of America; (+), positively scores high intake; +(m), positively scores moderate intake; (), positively scores low intake; SUN, Seguimiento Universidad de Navarra Project; NIH-AARP, National Institutes of Health and Diet Study; rMED, relative Mediterranean diet score, tertile cutoffs (g/2000 k cal/day); MDS, Mediterranean diet score, sex-specific median cutoffs; First MDS, original MD score by Trichopoulou et al. (1995), sex-specific median cutoffs; mMDS, modified Mediterraenan diet score; aMED, alternative Mediterraenan diet score; MUFA, monounsatured fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; *, All cases identified during 10 years of follow-up, cause-specific cases after 5 years of follow-up; **, alcohol range for women is 5–25 g/d and for men is 10–50 g/d. 51 52 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 2 Study Characteristics and Main Findings for Dietary Interventions Researching Adherence to the Mediterranean Diet and Mortality Study: Country Reference Cohort N/ Follow-up Mortality Cause Age (years) Mediterranean Diet Recommended Confounders Controlled For Main Findings HR/RR (95% CI) PREDIMED. Spain Estruch et al. [12] 7447/ 4.8 years All n ¼ 348 CVD n ¼ 87 67 (mean) MD (+): vegetables, fruit, butter, margarine or cream, sweet/ carbonated drinks, wine, legumes, fish or shellfish, chicken rabbit or white meat, sofrito, olive oil or nuts (): red meat and meat products, commercial sweets or pastries Sex, age, center, family history of CHD, smoking, BMI, waist/hip ratio, hypertension, dyslipidemia, diabetes MD with extravirgin olive oil or with nuts versus Control (low-fat): All HR 0.89 (0.71, 1.12) CVD HR 0.83 (0.54, 1.29) LYON Diet Heart Study France Lorgeril et al. [33] 605/ 3.8 years All n ¼ 38 Cardiac n ¼ 15 <70 Med-type diet (+): bread, root vegetables, green vegetables, fish, chicken, fruit, margarine (linoleic acid and a-linolenic acid enriched), rapeseed oil, olive oil, (+m) wine, () red meat, butter and cream Sex, age, smoking, cholesterol level, blood pressure, leukocyte count, aspirin use Med-type diet versus Control All RR 0.44 (0.21, 0.94) Cardiac RR 0.35 (0.15, 0.83) Abbreviations: CVD, cardiovascular disease; MD, Mediterranean diet; Med, Mediterranean; RR, risk ratio; HR, hazards ratio; (+), positively scores high intake; +(m), positively scores moderate intake; (), positively scores low intake. The magnitude of the protective effect was similar in a UK cohort study [23] that reported that high compared to low adherence to the MD was related to a 22% decreased risk in mortality (95% CI, 2-38). In two other studies outside the Mediterranean, a protective effect of the MD on mortality was also evident. For instance, a small cohort study [24] in Denmark found that a one-unit increment in MDS was related to an 11% decreased risk of mortality (95% CI, 2-36). A large cohort study set in Sweden [25] found that a one-unit increment in the MDS was related to a significant 4% (95% CI, 1-7) and 5% decreased risk of dying (95% CI, 1-9) in men and women, respectively. Two RCTs [12,33] investigated the effect of the MD on overall mortality (Table 2): the Spanish PREDIMED (Prevención con Dieta Mediterránea) intervention trial [12] and the Lyon Diet Heart Trial [33]. In PREDIMED, designed to assess the risk of major CVD events but including death from any cause, an MD supplemented with extra-virgin olive oil or nuts was associated with a negative but nonsignificant decreased risk of overall mortality (hazard ratio [HR], 0.89; 95% CI, 0.71-1.12) compared to a low-fat diet. The Lyon Diet Heart Trial [33] was also specifically designed to assess the effect of an MD on CVD death but also studied overall survival. The study reported that patients (survivors of an acute myocardial infarction) following an MD-type diet had an impressive 56% reduction in overall death (95% CI, 6-79). These more recent findings from both observational and experimental studies support the results from the 2010 meta-analysis [5], providing more solid evidence indicating that following an MD increases survival. In summary, epidemiological studies (mainly observational) exploring the relationship between the MD and mortality show that there is clear, consistent evidence that individuals who adhere to an MD are less likely to die. The magnitude of the protective effect varied depending on the study, but in general a high versus low MDS seemed to decrease risk of mortality by approximately 20%, and a two-unit increase in an MDS decreased mortality by 8%, according to a pooled meta-analysis of nine cohort studies [5]. The Mediterranean Diet and Mortality Chapter 5 53 Cause-Specific Mortality Eleven of the reviewed studies also researched the relationship between the MD and cause-specific mortality, providing more insight into how the MD actually is able to increase survival in terms of its specific health benefits. Table 3 presents results from the six cohort studies that provided sex-specific results of cause-specific mortality. CVD Mortality Adherence to the MD is associated with a reduction in mortality from CVD, in particular coronary heart disease (CHD), in all the studies researching this outcome. A high compared to low MDS significantly reduced the risk of CHD by 44% in the EPIC-Greece cohort [21], by 42% among women in the Nurses’ Health study [31], and by 39% in an elderly European cohort [29]. For a high versus low MDS, CVD mortality was significantly reduced by 34% in the EPIC-Spain cohort [15] (which was greater in women than in men), by 29% in the elderly European cohort [29], by 22% in men and 19% in women in the American Diet and Health Study [30], and by 39% in women in the Nurses’ Health study [31]. The two RCTs also reported on the effect of adhering to the MD on CVD mortality, and although there was a 17% decrease in CVD mortality in the PREDIMED trial [12], it was not statistically significant. In contrast, the Lyon Heart Study [33] found a significant 65% decreased risk of cardiac death related to following a Mediterranean-type diet, although there were only 15 cases in this analysis. An inverse but nonsignificant association between the MDS and CVD death was observed in the EPIC-Greece cohort [20]. The biological plausibility of the causal relation between the MD and CVD mortality is supported by strong evidence of the intermediate biological mechanisms and pathways involved [34], including the MD’s beneficial effects on blood lipids (decreasing triglycerides and total and low-density lipoprotein cholesterol and increasing high-density lipoprotein cholesterol), insulin sensitivity, markers of oxidation, inflammation, endothelial function, and CVD risk factors such as metabolic syndrome, diabetes, and overweight/obesity [1,2,10–12,35,36]. Cancer Mortality Results on the effect of the MD on death from cancer are mixed. Three studies found a significant protective effect; each two-point increment in the MDS was associated with a significant 24% reduced risk of cancer death in the EPIC-Greece cohort [13], high versus low MD adherence was related to a 17% reduced risk of cancer death in men and 12% in women in the American Diet and Health Study [30], and a one-point increase in the MDS was related to an 8% decreased risk of cancer death in the cohort from northern Sweden [25]. In contrast, three studies reported negative but nonsignificant associations between high adherence to the MD and cancer mortality: the Uppsala Health Care Cohort (HR, 0.80; 95% CI, 0.57-1.13 in women); EPIC-Spain (HR, 1.08; 95%CI, 0.81-1.15 in women and HR, 0.83; 95% CI, 0.68-1.08 in men) (Table 3), and the elderly European cohort (HR, 0.90; 95% CI, 0.70-1.17) (data not shown). In summary, the evidence for a relation between the MD and cancer mortality is much weaker than for CVD: only three of the six studies assessing this outcome reported that the MD significantly reduced death from cancer. Worth noting is that one of the studies reporting a significant reduction had by far the most number of cases (more than 3500 cancer deaths), indicating good study power to detect an association if one existed. In addition, there was a negative, albeit nonsignificant, association in the other studies. The MD’s potent antioxidant action and ability to reduce chronic inflammation, to offset the carcinogenic effects of N-nitroso compounds, and to downregulate cancer-related oncogenes could explain its anticarcinogenic role [6]. Mortality from Other Causes It is also worth noting is that several cohort studies [15,29,30] found that adhering to the MD was significantly associated with a decreased risk of dying from other causes, ranging from 23% (95% CI, 12-30) in men and 28% (95% CI, 13-41) in women in the American Diet and Health study to 29% (95% CI, 6-46) overall in the EPIC-Spain cohort and up to 39% (95% CI, 15-56) in the elderly European cohort [29]. In the study by Buckland et al. [15], this category included death from chronic diseases apart from cancer and CVD, death from respiratory disorders and other diseases and infections, as well as external causes. It is likely that the broad spectrum of nutritional attributes of the MD [1,3,37,38], such as its rich source of varied antioxidants, high fiber content, favorable lipid profile (with a high monounsaturated and polyunsaturated fat-to-saturated fat ratio and high intake of a-linolenic acid), and abundance of phytochemicals and other biologically active compounds and their synergistic interactions, could help prevent or improve the prognosis or response to treatment of a number of other diseases and illnesses [2,36], thereby reducing mortality. 54 Men Women Mortality Cause No. Cases Moderate vs Low MD Score High vs Low MD Score P-value trend No. Cases Moderate vs Low MD Score High vs Low MD Score P-value trend Misirli et al. [20] CBVD 104 0.63 (0.39, 1.02) 0.88 (0.50, 1.55) N/A 92 0.93 (0.61, 1.42) 0.60 (0.31, 1.16) N/A Dilis et al. [21] CHD 150 0.80 (0.55, 1.15) 0.62 (0.39, 0.98) N/A 210 0.89 (0.57, 1.39) 0.39 (0.17, 0.88) N/A Cancer 504 0.97 (0.77, 1.22) 0.83 (0.63, 1.08) 0.142 409 1.01 (0.80, 1.29) 1.08 (0.81, 1.45) 0.608 CVD 286 0.90 (0.66, 1.23) 0.71 (0.50, 1.01) 0.046 113 0.73 (0.47, 1.13) 0.54 (0.30, 0.96) 0.035 Other 277 0.73 (0.54, 0.99) 0.73 (0.51, 1.03) 0.098 148 0.71 (0.48, 1.04) 0.70 (0.43, 1.14) 0.129 Cancer 3717 0.86 (0.80, 0.93) 0.83 (0.76, 0.91) <0.001 2268 0.93 (0.85, 1.02) 0.88 (0.78, 1.00) 0.04 CVD 2425 0.95 (0.86, 1.04) 0.78 (0.69, 0.87) <0.001 1026 0.85 (0.74, 0.98) 0.81 (0.68, 0.97) 0.01 Other 1747 0.90 (0.81, 1.00) 0.77 (0.70, 0.88) <0.001 922 0.82 (0.71, 0.95) 0.72 (0.59, 0.87) <0.001 CVD N/A N/A N/A N/A 1077 0.89 (0.76, 1.08) 0.61 (0.49, 0.76) <0.0001 CHD N/A N/A N/A N/A 794 0.81 (0.65, 1.00) 0.58 (0.45, 0.75) <0.0001 Stroke N/A N/A N/A N/A 283 1.17 (0.81, 1.68) 0.69 (0.44, 1.07) 0.1 N/A N/A N/A N/A 280 0.91 (0.69, 1.18) 0.80 (0.57, 1.13) N/A Cohort—Country Author (year) EPIC—Greece EPIC—Spain Buckland et al.a [15] NIH-AARP Diet and Health Cohort—USA Mitrou et al. [30] b Nurses’ Health Study —USA Fung et al. [31] Uppsala Health Care—Sweden Lagiou et al. [26] Cancer Abbreviations: CVD, Cardiovascular disease; CHD, Coronary Heart Disease; CBVD, Cerebrovascular Disease N/A: Data Not Available; EPIC, European Prospective Investigation into Cancer and Nutrition; NIH-AARP, National Institutes of Health Assocation of Retired Persons Diet and Health Study; USA, United States of America a Additional sex specific data was provided from the author of this study, as was not published within this paper. bModerate MD score refers to third quartile and High MD Score refers to fifth quartile. SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 3 Results from Studies Reporting the Association Between the Mediterranean Dietary Pattern and Cause-Specific Mortality The Mediterranean Diet and Mortality Chapter 5 55 Overall, the results on cause-specific mortality reveal that the MD reduces the risk of mortality in part through its protective effect on CVD mortality, in particular CHD; in the reviewed studies a high versus low MDS decreased the risk of CVD mortality between 19% and 46% and the risk of CHD mortality from 42% to 61%. There was, however, no evidence that the MD reduced mortality from stroke or CBVD death. There is some evidence to suggest that the MD is associated with a reduction in mortality from cancer. Adhering to the MD is also linked to a reduction in mortality from a range of other diseases and illnesses not specified, grouped into “other causes.” The Benefits of the MD in Non-Mediterranean Populations Ten of the cohort studies researching the MD’s relation with mortality were carried out in non-Mediterranean countries (the United Kingdom, Denmark, Sweden, the United States, and Australia) or in European populations from both Mediterranean and non-Mediterranean countries. The findings consistently show that the MD was related to a decreased risk of overall mortality in non-Mediterranean populations but with a certain range in the magnitude of the negative association observed. In studies comparing the effect of a high versus low adherence to the MD on mortality, there was a reduced risk of 22% (95% CI, 2-38) in a British Cohort [23], 11% (95% CI, 4-23) in a Swedish cohort [26], 9% (95% CI, 1 to 18) in the EPIC-Elderly European cohort [28], 23% (95% CI, 12-32) in the Hale European cohort, and 21% (95% CI, 17-24) in men and 20% (95% CI, 15-25) in women in the American Diet and Health cohort [30]. There was also a reduced risk of mortality from CVD of 39% (95% CI, 24-51) in the Nurses’ Health Study [31]. A one-point increment in MDS was related to a 4% decrease in mortality (95% CI, 1-7) in men and a 5% decrease (95% CI, 1-9) in women in another Swedish cohort [25], a 21% decrease (95% CI, 2-36) in a Danish cohort [24], and a 17% decrease (95% CI, 2 to 33) in an Australian cohort. Overall, the findings from studies set outside the Mediterranean basin show that the benefits of the MD go beyond the geographic area where it was originally defined, and that even non-Mediterranean populations (ranging from Northern Europe, the United States, and even Australia) that do not traditionally have an MD-style eating pattern can and have adopted key features of Mediterranean cuisine and benefited in terms of increased longevity. The results are also of interest because the benefits of the MD could be partly attributed to the lifestyle habits with which it is intrinsically linked. Indeed, it is often noted that the MD is not just a dietary pattern but an expression of culture and history and a way of life [39]. The MD’s protective effect against mortality in non-Mediterranean populations indicates the benefits of the dietary pattern separate from the traditional Mediterranean lifestyle, although residual confounding by other healthy lifestyles associated with the MD in these populations cannot be ruled out. Relative Importance of Individual Components of the MD on Mortality Six cohort studies presented results on the association between the individual components of the MDS and overall mortality [15,16,18,20,22,25], as presented in Figure 1a–f. In general, the relationship between each separate component of the MDS and mortality was not very strong, and few components were significantly associated with overall mortality. However, alcohol was related to a reduced risk of mortality in three studies [15,22,25], fruit and nuts in another study [16] and olive oil in the EPIC-Spain studies [15]. In the GISSI-Prevenzione trial [18], however, all five food groups (fruit, raw vegetables, cooked vegetables, olive oil, and fish) were independently related with a reduced risk of mortality. Four cohort studies [15,22,25,27] also investigated the effect of the alternate exclusion of each component of the MDS (i.e., MDS without fruit or MDS without alcohol). This is useful to understand whether any one food group within the score would invalidate the effect of the entire score if it was excluded. However, because the association with mortality survived and was, in general, of a similar magnitude when each single component was alternately removed, the results demonstrate the robustness of the MDS (data not shown). The relative importance of the components of the MDS in reducing mortality was assessed in two EPIC studies [15,23] (presented as percentage change in HR). In the EPIC-Spain study [15], olive oil, alcohol, cereals, and fruit contributed most to the MDS, whereas in the EPIC-Greece study [23] alcohol, meat, vegetables, and fruit and nuts had a predominant effect on mortality. Overall these findings illustrate that the MDS is consistently a better indicator of survival than its individual food components. This could be partly due to interactions and synergisms between different foods and nutrients, and individual components may have health benefits that are undetectable alone but become more evident when integrated together within a dietary score. Dietary scores also have the advantage of overcoming problems of collinearity or confounding between food groups [40]. It should be noted, though, that in certain studies alcohol, fruit and nuts, cereals, olive oil, and meat were independently related to mortality. MD Component HR (95% CI) Fruit 1.03 (0.87, 1.21) Vegetables and potatoes 1.06 (0.90, 1.24) Monounsaturated to saturated lipids (ratio) 0.98 (0.84, 1.15) Cereals 1.01 (0.86, 1.19) Fish and seafood 0.96 (0.82, 1.13) Alcohol 0.77 (0.61, 0.97) Legumes, nuts, and seeds 0.98 (0.83, 1.16) Meat, meat products, and eggs 0.84 (0.71, 0.98) Dairy products 0.82 (0.70, 0.96) (a) 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 MD Component HR (95% CI) Fruits and nuts 0.93 (0.82, 1.06) Vegetables 1.02 (0.90, 1.16) Olive oil 0.85 (0.75, 0.96) Cereals 0.91 (0.81, 1.03) Fish and seafood 1.02 (0.90, 1.15) Alcohol 0.89 (0.80, 0.99) Legumes 1.00 (0.89, 1.12) Meat 1.08 (0.96, 1.22) Dairy products 1.08 (0.95, 1.24) (b) 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 MD Component HR (95% CI) Fruits and nuts 0.93 (0.82, 1.06) Vegetables 0.90 (0.76, 1.05) Monounsaturated to saturated lipids (ratio) 0.91 (0.79, 1.04) Cereals 0.99 (0.86, 1.13) Fish and seafood 1.08 (0.95, 1.22) Alcohol intake (low) 1.19 (1.03, 1.38) Alcohol intake (high) 1.47 (1.32, 1.91) Legumes 0.94 (0.83, 1.08) Meat 1.15 (0.99, 1.33) Dairy products 1.07 (0.93, 1.23) (c) 0.8 0.6 0.7 0.9 1 1.1 1.2 1.3 1.4 FIGURE 1 The association between the components of the Mediterranean diet and overall mortality in selected cohort studies. (a) The Gothenburg Swedish cohort, Tognon et al. [27] (b) The EPIC-Spain, Buckland et al. [15] (c) The EPIC-Greece, Trichopoulou et al. [22] The Mediterranean Diet and Mortality Chapter 5 MD Component HR (95% CI) Fruits and nuts 0.64 (0.43, 0.95) Vegetables 0.79 (0.53, 1.17) Monounsaturated to saturated lipids (ratio) 0.77 (0.51, 1.17) Cereals 0.83 (0.54, 1.27) Fish 0.95 (0.65, 1.38) Alcohol 0.79 (0.54, 1.17) Legumes 1.02 (0.67, 1.53) Meat/ meat products 0.97 (0.65, 1.44) Dairy products 1.04 (0.69, 1.58) (d) 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 MD Component HR (95% CI) Fruit 1.02 (0.97, 1.08) Vegetables 0.99 (0.95, 1.04) Monounsaturated to saturated lipids (ratio) 0.24 (0.07, 1.13) Cereals 1.10 (1.03, 1.17) Alcohol 0.81 (0.63, 1.05) Meat 0.99 (0.86, 1.13) Dairy products 1.00 (0.99, 1.01) (e) 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 MD Component HR (95% CI) Fruit 0.73 (0.54, 0.98) Raw vegetables 0.65 (0.51, 0.84) Cooked vegetables 0.70 (0.50, 0.99) Olive oil 0.76 (0.64, 0.91) Fish 0.76 (0.62, 0.94) (f) 0.5 0.6 0.7 0.8 0.9 1 57 1.1 1.2 1.3 1.4 FIGURE 1—CONT’D (d) The SUN cohort, Martı́nez-González et al. [16] (e) The Danish Euronut SENECA study, Osler et al. [24] (f) The GISSIPrevensione trial, Barzi et al. [18] 58 SECTION 1 The Mediterranean Diet: Concepts and General Aspects METHODOLOGICAL CONSIDERATIONS WHEN INVESTIGATING THE LINK BETWEEN THE MD AND MORTALITY The variability in magnitude or strength of the relationship between the MD and overall or cause-specific mortality may be related to a number of factors, including study design, geographic region of the study population, population characteristics, definition of MDS, methodology for collecting dietary data, and statistical analysis (Table 4). Because the MDS uses cutoffs based on medians or tertiles of the populations’ intake of each MD component (not quantitative amounts), the cutoffs will vary between studies depending on the dietary habits of the participants included. For example, the cutoffs defining low and high MD adherence may be uncharacteristically low in non-Mediterranean countries (especially because of the lower consumption of foods such as olive oil, legumes, or seafood in certain countries), and even Mediterranean countries may have this problem because of the documented disappearance of the “traditional” MD [41]. As a result, the MDS really reflects only “relative” adherence to an MD. In addition, the MDSs applied in the studies vary in the type and number of components included in their definition, and many do not include key characteristic components of a traditional MD, such as whole-grain cereals and moderate alcohol consumption or olive oil intake (the ratio of MUFA to SFA is normally used instead of olive oil). Therefore, caution should be taken when directly comparing mortality risk associated with high adherence to the MDS between different studies. Other methodological differences to consider when interpreting the results include the accuracy of the dietary data collected, which varies between studies and is related to the type of dietary questionnaire (most were validated); whether they were self-administered or completed during an interview; and the number of items included. Dietary measurement error consequently could influence (probably attenuate) the reported MD–mortality relationship. The population’s characteristics in terms of age, health status, and sex also are relevant. For instance, in the Swedish cohort study [26] adherence to the MD was only significantly related to overall and cancer mortality in women over 40 years old, which could be because of a stronger genetic influence for diseases such as cancer in younger women. The PREDIMED trial and Lyon Heart study also included selected health groups (individuals at high risk for CVD or with a previous myocardial infarction), and so the mortality risk estimates cannot be directly compared to the results from studies including healthy populations. Differences in the statistical analyses, such as the number and type of confounders controlled for in the statistical models, is also relevant because it is important to try and separate the positive effects of the healthy MD from those of a healthy lifestyle, as they often go hand in hand. Therefore, careful adjustment for socioeconomic and lifestyle factors related to mortality and the MD should help disentangle this, and although most studies adjusted for the main confounding variables, some residual confounding in observational studies is almost inevitable. FINAL COMMENTS There is substantial evidence showing that adhering to a Mediterranean-type diet is related to a reduction in risk of overall mortality, which is apparent among younger and older generations across Mediterranean and non-Mediterranean populations and is partly due to a reduction in the risk of dying from CVD and probably cancer. There are many nutritional attributes of the MD and related biological mechanisms that can explain the benefits of the MD on mortality. TABLE 4 The Methodological Factors to Consider when Interpreting Results of Epidemiological Studies on the Mediterranean Diet and Mortality Methodological Factors Variability Between Studies Reviewed Study design Observational (cohort) or experimental (RCT) Geographic origin of study population Mediterranean versus non-Mediterranean Study size and endpoints Total number of participants and endpoints Population characteristics Gender, age, socioeconomic level, health status Dietary data collection methodology Food frequency questionnaires, dietary history, multiple 24-h recalls (validated or not) Mediterranean diet definition Number and type of components included in Mediterranean diet score, cutoffs (sex-specific medians versus tertiles using energy-density method) Statistical analysis Confounding variables controlled for, type of sensitivity analyses The Mediterranean Diet and Mortality Chapter 5 59 SUMMARY POINTS l l l l l l l This chapter systematically reviews 21 epidemiological studies (19 observational and 2 experimental) researching the relationship between adherence to the MD and mortality. The results consistently show that individuals who adhere to the MD are less likely to die; in general a high versus low MDS was related to a decreased risk of mortality by 20%, and a two-unit increase in an MDS decreased mortality by 8%. The MD reduces risk of mortality partly due to its cardioprotective role, but it may also protect against mortality from cancer and other diseases or illnesses. Results from studies set in non-Mediterranean populations show that the health benefits of the MD, in terms of increased survival, are transferable outside the Mediterranean basin. The entire MDS is consistently a better indicator of survival than its individual food components; however, moderate alcohol intake and a high intake of fruit, vegetables, and olive oil were the components most heavily implicated in the score’s protective role against mortality. The benefits of the MD on mortality can be attributed to its rich source of varied antioxidants, favorable lipid profile, high fiber content, and abundance of phytochemicals and other biologically active compounds that have a cardioprotective and anticarcinogenic effect, amongst other health properties. Methodological differences between studies, including the geographic region where the study was set, population characteristics, definition of MDS, source of dietary data, and method of statistical analysis, could explain some of the differences in the magnitude and strength of results for overall and cause-specific mortality. REFERENCES [1] Perez-Lopez FR, Chedraui P, Haya J, Cuadros JL. Effects of the Mediterranean diet on longevity and age-related morbid conditions. Maturitas 2009;64:67–79. [2] Roman B, Carta L, Martinez-Gonzalez MA, Serra-Majem L. Effectiveness of the Mediterranean diet in the elderly. Clin Interv Aging 2008;3:97–109. [3] Trichopoulou A, Vasilopoulou E. 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Effect of a Mediterranean diet supplemented with nuts on metabolic syndrome status: one-year results of the PREDIMED randomized trial. Arch Intern Med 2008;168:2449–58. [9] Esposito K, Marfella R, Ciotola M, Di Palo C, Giugliano F, Giugliano G, et al. Effect of a Mediterranean-style diet on endothelial dysfunction and markers of vascular inflammation in the metabolic syndrome: a randomized trial. J Am Med Assoc 2004;292:1440–6. [10] Estruch R, Martinez-Gonzalez MA, Corella D, Salas-Salvado J, Ruiz-Gutierrez V, Covas MI, et al. Effects of a Mediterranean-style diet on cardiovascular risk factors: a randomized trial. Ann Intern Med 2006;145:1–11. [11] Estruch R. Anti-inflammatory effects of the Mediterranean diet: the experience of the PREDIMED study. Proc Nutr Soc 2010;69:333–40. [12] Estruch R, Ros E, Salas-Salvado J, Covas MI, Pharm D, Corella D, et al. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med 2013;368:1279–90. 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[32] Kouris-Blazos A, Gnardellis C, Wahlqvist ML, Trichopoulos D, Lukito W, Trichopoulou A. Are the advantages of the Mediterranean diet transferable to other populations? A cohort study in Melbourne, Australia. Br J Nutr 1999;82:57–61. [33] de Lorgeril M, Salen P, Martin JL, Monjaud I, Boucher P, Mamelle N. Mediterranean dietary pattern in a randomized trial: prolonged survival and possible reduced cancer rate. Arch Intern Med 1998;158:1181–7. [34] Panagiotakos DB, Pitsavos C, Polychronopoulos E, Chrysohoou C, Zampelas A, Trichopoulou A. Can a Mediterranean diet moderate the development and clinical progression of coronary heart disease? A systematic review. Med Sci Monit 2004;10:RA193–RA198. [35] Buckland G, Bach A, Serra-Majem L. Obesity and the Mediterranean diet: a systematic review of observational and intervention studies. Obes Rev 2008;9:582–93. [36] Serra-Majem L, Roman B, Estruch R. Scientific evidence of interventions using the Mediterranean diet: a systematic review. 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Chapter 6 Mediterranean Diet and Quality of Life Patricia Henrı́quez-Sánchez1,2, Jorge Doreste-Alonso1, Cristina Ruano1,2, Lluı́s Serra-Majem, MD, PhD1,2, Miguel Ángel Martı́nez-González2,3 and Almudena Sánchez-Villegas1,2 1 University of Las Palmas de Gran Canaria, Madrid, Spain. 2 Instituto de Salud Carlos III, Madrid, Spain. 3 University of Navarra, Madrid, Spain. INTRODUCTION Important changes in demographic structure have taken place throughout the last century. A decrease in the birth rate and an increase in longevity have determined an important process of aging worldwide. In 2010, 524 million people were over the age of 65, representing 8% of the world’s population, and this percentage may increase to 16% of the total population in 2050, surpassing the percentage of young people [1]. In addition to this increase in population with advanced ages, a spectacular increase in life expectancy also has been observed, leading to the appearance of chronic and degenerative diseases that determine the quality of life of the population. Nowadays, diseases such as cardiovascular disease, type 2 diabetes mellitus, cancer, and other chronic diseases represent more than half of all disease burden in developed countries [2]. For this reason, the interest in increasing the “quantity” of life must be accompanied by an increase in its quality. Although until only a few years ago health policies aimed to diminish the mortality and morbidity among populations, today there is a growing interest in understanding and improving individuals’ quality of life. QUALITY OF LIFE Nowadays, a person’s health status is evaluated beyond his or her physical capacity, taking into account the social context and mental health; therefore, the evaluation of quality of life has to be done in an integral way. The concept of quality of life was included in the area of the medicine in the 1950s. In 1994 the World Health Organization defined it as “the personal perception of an individual of his situation in life, inside the cultural context and values in which he lives, and in relation with his aims, expectations, values and interests” [3]. Quality of life is a multidimensional concept that includes physical, psychic, and social aspects, each of which has different components to be measured. They can represent both an objective (functioning and health status) and subjective (perceptions) dimension of health [4]. The measurement of quality of life is related to the subjective perception of health and well-being reported by an individual in relation to his or her social and cultural environment. Self-perceived health-related quality of life is a simple but effective indicator of the global health status and a useful tool to predict welfare needs and to organize prevention programs. It is obvious to think that the promotion of a healthy life is comparable to the concept of well-being and the concept of a good quality of life. The fact that one feels well determines the development of his daily life. A narrow relationship exists between how a person feels and what the person does. Questionnaires that measure quality of life have been frequently used in clinical populations and are of great interest in evaluating the quality of life of healthy populations, with the aim of determining the factors that could influence it. Several scientific studies show that self-perceived health-related quality of life is a good predictive factor of mortality [5–7]. From a public health perspective, it has been an increase in the evaluation of quality of life in population-based studies. QUALITY OF LIFE MEASUREMENT Because quality of life is not a biological measurement but one of perceived health, instruments ensuring the reliability and precision of the information collected must be used. In general, quality of life is determined by administering self-reported questionnaires. Multiple questionnaires exist to determine levels of quality of life, and they can be classified as generic or specific. Generic questionnaires have a purely descriptive use because they do not allow clinical changes produced by an intervention or treatment for a certain disease to be detected. They are applied to compare different populations and The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 61 62 SECTION 1 The Mediterranean Diet: Concepts and General Aspects pathological processes. Specific questionnaires are focused to study the quality of life of patients with a certain pathology, and they allow changes produced by the effects of a treatment to be evaluated. Specific questionnaires are the first choice to be applied in clinical trials. Five generic questionnaires are most often used. 1. 36-Item Short Form (SF-36) Health Survey. The SF-36 was developed over the past 10 years using questionnaires and data from the Medical Outcomes Study. It is a general health scale widely used and thoroughly validated [8]. This questionnaire contains 36 items, measuring 8 multi-item parameters of health status: physical functioning, role limitations because of physical health problems (role-physical), bodily pain (tolerance), general health perceptions, vitality, social functioning, role limitations because of emotional problems (role-emotional), and mental health (Table 1). The first four domains deal with physical aspects and the latter four reflect psychological features. Scores for each parameter are coded, summed, and converted into a scale from 0 (the worst possible condition) to 100 (the best possible condition). For example, for bodily pain a score of 100 means complete tolerance of pain. The SF-36 is appropriate for people aged 14 and older and is self-administered or administered via a computer or by a trained interviewer in person or over the telephone. It can be applied both in clinical populations and the general population and is characterized by easy administration—no more than 20 min are needed to complete it. There are two reduced versions of the questionnaire, the SF-12 and the SF-8, which also are validated and widely used, mainly in population-based studies. 2. EuroQol 5-Dimension (EQ-5D) Questionnaire. This questionnaire, developed by the EuroQol Group [9], is a descriptive tool designed to be self-administered and is characterized by very easy comprehension. This questionnaire measures health-related quality of life through five dimensions—mobility, self-care, daily activities, pain/discomfort, and anxiety/depression—instead of the eight dimensions measured in the SF-36 Health Survey. It also includes a visual analogical scale. The EQ-5D has been widely used as an instrument for measuring health status in clinical trials and observational and other public health studies and therefore has been using in both general populations and groups of people with diseases. The EQ-5D has been validated in different languages and is widely used in many countries including Europe, North America, Africa, and Asia. 3. Sickness Impact Profile (SIP). The SIP has 12 areas measured using numerical scales, and it is more complex than the SF-36: It takes more time to be administered, and it is difficult to apply it among outpatients and large populations. Therefore, although it has been used, it has not been as accepted as the SF-36. It has, however, been widely validated [10]. 4. Nottingham Health Profile (NHP). The NHP allows easy evaluation of personal perceptions of health. It consists of 38 questions with a yes/no response grouped in six dimensions: pain, physical mobility, sleep, energy, emotional reactions, and social isolation. The interpretation is based on the number of positive answers; higher marks represent a worse quality of life [11]. 5. General Health Questionnaire (GHQ). Aimed at detecting minor and functional psychiatric disorders, the GHQ is commonly used both in community and primary care settings. It originally included 60 items, but shorter versions, such as the GHQ-28 (assessing somatic symptoms, anxiety and insomnia, social dysfunction, and depression) and the GHQ-12, have been validated for screening purposes [12]. TABLE 1 SF-36 Domains Physical functioning Grade in which health problems can interfere with physical activities such as walking, self-care, or weight lifting Role physical Grade in which physical health problems can interfere with work activity or with other daily activities Bodily pain Pain intensity and its effect on work inside and outside home General health Personal belief regarding current health and future perspectives regarding health Vitality Vitality and energy feelings against tiredness or exhaustion Social functioning Grade in which mental health problems can interfere with habitual social life Role emotional Grade in which mental health problems can interfere with work activity or with other daily activities Mental health General mental health including depression, anxiety, emotional and behavior control and general positive effect Mediterranean Diet Quality of Life Chapter 6 63 On the other hand, there are a great number of specific questionnaires measuring health-related quality of life for diverse diseases: the Diabetes Quality of Life, the 39-item Quality of Life Parkinson Disease Questionnaire, the Chronic Respiratory Disease Questionnaire, and the Mild Hypertension Vital Signs Quality of Life Questionnaire. All the above-mentioned questionnaires are designed for the adult population, but there are also both generic and specific questionnaires to evaluate the quality of life of children and teenagers. Examples of generic instruments are the Functional Status Measure of Child Health and the Childhood Health Assessment Questionnaire Disability Index. Finally, it is worth mentioning that other types of measures combining quantity and quality of life are also used. Qualityadjusted life-years and disability-adjusted life-years, allowing us to consider the number of years lost because of premature death and incapacitation, are the most commonly used. MEDITERRANEAN DIET The positive health effects of the Mediterranean diet are strongly supported by evidence built on innumerable scientific achievements from the first study conducted by Keys in the 1950s. The Seven Countries Study was the first one recognizing the health effects of the diet followed by Mediterranean countries [13]. However, the Mediterranean diet pattern, recognized as an Intangible Cultural Heritage of Humanity by UNESCO, not only includes a set of “healthy” foods but also incorporates lifestyle and cultural elements other than those that are strictly dietetic. Daily physical activity and adequate rest also play a part in a balanced lifestyle, contributing to improved welfare and health [14]. The Mediterranean diet is a cultural model that leads to a higher level of health, better welfare, and better quality of life and is an efficacious primary and secondary prevention resource for those subjects adopting its principles. It also is able to reduce the burden of disease among developed and aged societies. The increasing interest in the relationships between food patterns and health is evidenced by the huge number of epidemiological studies published in the past decade [15]. The Mediterranean diet, along with a reduction in all-cause mortality [16–19], has been regarded as preventive for chronic pathologies such as cardiovascular disease [16], cancer [20], diabetes [21], depression [22], cognitive impairment [23], and Alzheimer’s disease [24]. MEDITERRANEAN DIET AND QUALITY OF LIFE The Mediterranean diet has also been associated with an improvement in quality of life; there are even several studies of this topic. Some clinic trials of both high-risk and diseased adult populations, have shown a better quality of life as a result of the implementation of a more healthy food pattern [25,26]. There exists supplementary evidence from cross-sectional studies of adult Mediterranean populations, namely those published in 2009 by Muñoz et al. [27] (n ¼ 8195) and Henrı́quezSánchez et al. [28] in 2012 (n ¼ 11,015), and it is noteworthy that the effect was found both in the physical and mental domains. The study by Henrı́quez-Sánchez et al. included participants in the SUN (Seguimiento Universidad de Navarra) Project, an ongoing, multipurpose study comprising a dynamic cohort of university graduates from Spain. Dietary intake was assessed through a validated 136-item semiquantitative food frequency questionnaire completed at baseline. Adherence to the Mediterranean diet was appraised according to the score created by Trichopoulou et al. [29]. This score includes nine components: intake of vegetables, legumes, fruits and nuts, cereals, fish, meat and meat products, and dairy products; moderate alcohol intake; and the ratio of monounsaturated fatty acids to saturated fatty acids (SFAs). Values of 0 or 1 were assigned to each component using the sex-specific medians in the studied population as cutoffs. Quality of life was assessed at the fourth year of follow-up using the validated Spanish version of the SF-36 Health Survey. Trichopoulou et al. reported that adjusted mean values for physical functioning, role-physical, bodily pain, general health, and vitality were significantly better in participants with higher Mediterranean diet scores (Figures 1 and 2). They also reported a significant multivariate-adjusted, direct linear association between the Mediterranean diet score and the physical and mental domains. As shown in Table 2, a one-point increment in the Mediterranean diet score was associated with a 0.50-point increment (95% CI 0.32-0.68) in the physical domain and 0.45-point increment (95% CI 0.26-0.62) in the mental health domain (Table 2). To our knowledge, regarding healthy pediatric populations, only a study of Greek adolescents has been published [30]. In that study adherence to the Mediterranean diet was positively correlated with health-related quality of life. It must be highlighted that overweight and obesity are increasing public health problems characterized not only by severe medical consequences occurring at a young age but also psychosocial consequences such as low self-esteem, insecurity, difficulties at school, impaired social interactions, and even depressive symptoms. Obese children and adolescents report significant impairments not only in total scores but also in all domains—physical, psychosocial, emotional, social, and school functioning—of health-related quality of life scales than healthy children and adolescents [31]. The likelihood of an obese 100 Mediterranean diet score SF-36 physical domains score 95 Low Low–moderate Moderate–high High P<0.001 90 P = 0.005 85 80 FIGURE 1 Adjusted means (95% confidence intervals) for SF-36 physical domains according to Mediterranean diet score categories. Means have been adjusted for age, sex, marital status, body mass index, smoking, leisure-time physical activity, total energy intake, and medical history of hypertension, diabetes, dyslipidemia, and coronary heart disease. PF ¼ physical functioning; RP ¼ role physical; BP ¼ bodily pain; GH ¼ general health. P = 0.005 75 70 P<0.001 65 60 55 50 PF RP GH BP 100 Mediterranean diet score SF-36 mental domains score 95 90 Low Low–moderate Moderate–high High score P =0.079 P = 0.061 85 80 FIGURE 2 Adjusted means (95% confidence intervals) for SF-36 mental domains according to Mediterranean diet score categories. Means are adjusted for age, sex, marital status, body mass index, consumption, leisure-time physical activity, total energy intake, and medical history of hypertension, diabetes, dyslipidemia, and coronary heart disease. SF ¼ social functioning; RE ¼ role emotional; MH ¼ mental health, VT ¼ vitality. P = 0.447 75 70 P<0.001 65 60 55 50 SF RE MH VT TABLE 2 Multivariate Regression Coefficients (95% Confidence Intervals)a for the Association Between Mediterranean Diet Score and Quality of Life Regression Coefficients 95% CI Vitality 0.50 0.32 to 0.68 Social functioning 0.18 0.02 to 0.34 Role emotional 0.34 0.03 to 0.66 Mental health 0.10 0.06 to 0.25 Physical functioning 0.33 0.22 to 0.43 Role physical 0.37 0.07 to 0.66 Bodily pain 0.27 0.04 to 0.50 General health 0.44 0.26 to 0.62 Mental component Physical component a Adjusted for age, sex, marital status, body mass index, smoking, leisure-time physical activity, total energy intake, and medical history of hypertension, diabetes, dyslipidemia, and coronary heart disease. Mediterranean Diet Quality of Life Chapter 6 65 child or adolescent having impaired health-related quality of life was 5.5 times greater than a healthy child or adolescent and similar to a child or adolescent diagnosed with cancer [32]. The main causes of obesity and overweight among children are a lack of physical activity and inappropriate food patterns. Adherence to a Mediterranean diet reduces the risk of obesity, and considering the relationship with quality of life, the advantages of promoting this healthy pattern at the earliest ages are fairly obvious. Several biological and physiological mechanisms could explain the beneficial effect of the Mediterranean diet on physical health. Adherence to the traditional Mediterranean diet has been associated with a reduction in low-grade inflammatory status, better endothelial function, an improved profile of coagulation markers [33,34], and less insulin resistance [35]. All these factors are thought to lead not only to a lower risk of chronic diseases but also to better metabolic control of already established diseases. Underlying mechanisms include, on one hand, effects of omega-3 fatty acids on neuronal membranes, enhancing synaptic membrane fluidity and serotonin transport [36]. On the other hand, B vitamins and folate are involved in the synthesis of serotonin and other monoamine neurotransmitters as well as other methylation reactions, for example, homocysteine catabolism [37]. A study conducted by Ruano et al. [38] showed a harmful association between the highest intake of trans unsaturated fatty acids compared with the lowest intake and several mental and physical SF-36 domains: vitality, social functioning, role-emotional, and bodily pain (Table 3). These associations could mean that the physiological changes that occur when this kind of fatty acid is consumed could mostly influence mental quality of life and therefore the self-perception of wellbeing. So, participants with the highest intake would perceive themselves as being more tired and worn out, having more social and role disability due to emotional problems and more severe limiting pain than the participants with the lowest intake of trans unsaturated fatty acids. An inverse association for SFA intake and some of the physical domains (physical functioning and general health) also was found, although this relationship was not statistically significant after adjusting for adherence to the Mediterranean diet score. Therefore, after adjusting for a dietary pattern such as the Mediterranean diet—rich in polyunsaturated fatty acids and other nutrients with beneficial effects—there is no harmful effect of SFAs on quality of life. Considering diverse Mediterranean diet components, there are more published studies regarding quality of life. In parallel to a higher consumption of fruit and vegetables, a reduction of perceived stress and depressive symptoms has been found [39,40], as well as a better self-reported physical function, which has less consistent effects on functional mental health [41]. These findings have been confirmed through randomized clinical trials in which such an intervention increased physical, but not mental, health status [42], although others have not obtained such clear results. It has been proposed that discrepancies can be explained by baseline self-perceived quality of life and differences in fruit and vegetable consumption. The same is observed for fish. Some authors report a positive relationship between fish consumption and the physical, but not mental, component of quality of life [43]. However, others found a positive association linking fish consumption TABLE 3 Multivariate Regression Coefficients (95% Confidence Intervals)a for the Association Between Extreme Quintile of Trans Unsaturated Fatty Acid Intake and Quality of Life Quintile 1 Quintile 5 Vitality 0 (ref.) 1.6 (3.0 to 0.2) Social functioning 0 (ref.) 1.7 (2.9 to 0.4) Role emotional 0 (ref.) 3.5 (6.0 to 1.0) Mental health 0 (ref.) 0.2 (1.4 to 0.7) Physical functioning 0 (ref.) 0.6 (1.4 to 0.2) Role physical 0 (ref.) 2.0 (4.4 to 0.3) Bodily pain 0 (ref.) 2.3 (4.1 to 0.5) General health 0 (ref.) 0.8 (2.2 to 0.6) Mental component Physical component a Adjusted for age, sex, marital status, body mass index, smoking, leisure-time physical activity, total energy intake, adherence to the Mediterranean diet score, and intake of polyunsaturated fatty acids, monounsaturated fatty acids, and saturated fatty acids. 66 SECTION 1 The Mediterranean Diet: Concepts and General Aspects Multivariate regression coefficients (95% confidence intervals) and self-reported mental, but not physical, health [44]. The consumption of fish could improve health-related quality of life because of its content of beneficial nutrients for health, such as omega-3 fatty acids, vitamins, and antioxidants. Concerning olive oil—the basic component of the Mediterranean diet—and in spite of its proven beneficial effects on a broad range of diseases and mental disorders, we found no studies of the eventual relationship between olive oil and quality of life. Interest in the ideas that people do not consume isolated foods or nutrients but include them in a varied overall dietary pattern, and that foods and nutrients can have synergistic or antagonistic effects when they are consumed together, have been growing in nutritional epidemiology. A cohort study that compared two dietary patterns [45]—one typified as a Western dietary pattern (characterized by high consumption of fast food, red and processed meats, high-fat dairy products, processed foods, refined cereals, eggs, commercial bakery goods, and sauces) and a second pattern labeled as a “Mediterranean dietary pattern” (characterized by high consumption of vegetables, fish and other seafood, fruit, poultry, olive oil, potatoes, low-fat dairy products, and legumes)—showed a significant inverse association between adherence to the Western dietary pattern and both physical and mental quality of life. On the contrary, a significant direct association was found between the Mediterranean dietary pattern and both dimensions of the SF-36, whereas baseline adherence to a Western dietary pattern was inversely associated with self-perceived quality of life after 4 years of follow-up, and baseline adherence to a Mediterranean dietary pattern was directly associated with better scores in quality of life 4 years later (Figure 3). From a public health perspective, implementing interventions promoting an increase in population health and a better quality of life, an objective to which the Mediterranean diet has to be a crucial contributor, is a priority. Bearing in mind that physical activity is a part of the Mediterranean dietary cultural pattern, it is appropriate to mention its beneficial effects on both physical and mental health through numerous broadly known mechanisms. Although there exists evidence of the positive effects of physical activity on individuals with disease, improving their prognosis and clinical course, knowledge of the relationship between physical activity and quality of life is scarce. A systematic revision published in 2007 [46] includes a limited number of studies of this topic. The majority are cross-sectional studies that found a positive association between physical activity and quality of life levels. Such a relationship is clearer for specific qualityof-life domains, namely vitality, physical functioning, and general health. A reverse causal association can be argued on the basis of the transversal nature of the studies; that is, it may be possible that the higher a subject’s welfare and health status, the higher their engagement in physical activity, rather than physical activity being the cause of a better quality of life. Besides this limitation, the consistency of results also is handicapped by the methods used to collect information about physical activity. Self-reported measures of physical activity lacking internal validity and yielding biased estimations have mainly been used. Beneficial effects of quality of life on physical activity have been found in studies using objective measurement methods such as accelerometers. However, those effects are 1 Mental component 0.8 0.6 Physical component 0.4 Physical component 0.2 0 Mental component -0.2 -0.4 -0.6 -0.8 -1 Mediterranean dietary pattern Western dietary pattern FIGURE 3 Multivariate regression coefficients (95% confidence intervals) for the association between empirically derived dietary patterns and quality of life. Values are adjusted for age, sex, marital status, body mass index, smoking, leisure-time physical activity, total energy intake, and medical history of hypertension, diabetes, dyslipidemia, and coronary heart disease. Mediterranean Diet Quality of Life Chapter 6 67 underestimated when subjective measurement methods are simultaneously used, given that individuals are prone to overreporting their physical activity, as published in a representative sample of the English general population [47]. Moreover, longitudinal studies carried out in healthy populations, regardless of whether they are observational or experimental in design, in general do not have enough quality to evaluate a causal relationship between physical activity and health-related quality of life. The results of a recent study regarding the physical activity and quality of life of a healthy Spanish cohort (n ¼ 11,938) have been published [48]. After the first 4 years of follow-up, those with higher baseline physical activity levels scored higher in all SF-36 domains, especially the mental ones. After 8 years of follow-up, mental domains scores improved, independent of previous scores, in those subjects who maintained or increased physical activity. Similar results have been found in other studies but referred to other domains [49]. Given the interactions between lifestyle determinants and quality of life, it is clear that prospective studies considering the Mediterranean diet and other related factors will improve our knowledge of its healthy effects. SUMMARY POINTS l l l l l Promotion of healthier lifestyles is a public health priority for developed countries. Welfare and quality of life have to be considered in the physical, mental, and social domains. Available data support withdrawal from a Western dietary pattern. The Mediterranean diet is a food pattern and a cultural model based on available natural foodstuffs served in moderate portions and shared among the family. 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Physical activity during leisure time and quality of life in a Spanish cohort: SUN (Seguimiento Universidad de Navarra) Project. Br J Sports Med 2012;46:443–8. [49] Van Oostrom SH, Smit HA, Wendel-Vos GC, Visser M, Verschuren M, Picavet SJ. Adopting an active lifestyle during adulthood and health-related quality of life: the Doetinchem Cohort Study. Am J Public Health 2012;102:e62–8. Chapter 7 Mediterranean Diet in Children and Adolescents Paul Farajian, PhD and Antonis Zampelas, PhD Agricultural University of Athens, Athens, Greece. ABBREVIATIONS BMI BP CVD GRECO study HRQOL KIDMED MAI MD PUFA SBP WC body mass index blood pressure cardiovascular diseases Greek Childhood Obesity study health-related quality of life Mediterranean Diet Quality Index for children and adolescents Mediterranean Adequacy Index Mediterranean diet polyunsaturated fatty acid systolic blood pressure waist circumference INTRODUCTION The term Mediterranean diet (MD) has been widely used to describe the traditional dietary habits followed by the populations in Greece, southern Italy, Spain, and other countries of the Mediterranean region during the 1960s. Although the definition of the MD is not consensual, mainly because this dietary pattern is rather heterogeneous among Mediterranean countries, the traditional MD, as defined by the various common types of the diets followed in Mediterranean countries, is characterized by high intake of vegetables, fruits, legumes, nuts, breads, and unrefined cereals (pasta, brown rice, bulgur, etc.); moderate to high intake of fish; moderate intake of dairy products (mostly cheese and yogurt); low intake of saturated fatty acids but high intake of monounsaturated fatty acids, coming mainly from the high consumption of olive oil; and low intake of red meat [1]. The large amount of vegetables, fresh fruits, and cereals and the abundant use of olive oil provide an adequate intake of dietary fiber, essential fatty acids, various vitamins and minerals, as well as several valuable bioactive compounds [2]. The MD has been associated with longevity [3] and has been promoted intensively during the past two decades for its numerous health benefits; there are many intervention and epidemiological studies suggesting that the MD is protective against several morbid conditions including atherosclerosis, coronary heart disease, diabetes, metabolic syndrome, and inflammation [4–6]. The majority of studies concerning the beneficial effects of the MD refer to adult populations. Nevertheless, there are studies of children conducted using modified versions of the MD score, an index introduced by Trichopoulou et al. [3], but the vast majority of the studies use the Mediterranean Diet Quality Index for children and adolescents (KIDMED) index, a tool recently developed to assess the degree of adherence to the MD, suggesting that MD may be an important health-promoting factor for children as well as adults [7]. MD INDICES AND ADHERENCE RATES IN CHILDREN AND ADOLESCENTS Although there are several reports demonstrating the beneficial effects of single foods or food groups that constitute the MD (mainly fruits, vegetables, dairy products, olive oil, and fiber-rich foods) on the risk of developing chronic diseases or in general on the relationship of diet and health outcomes, this approach does not consider the complexity of dietary behaviors, because foods and nutrients are not eaten in isolation. In addition, even if the importance of a single nutrient is well The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 69 70 SECTION 1 The Mediterranean Diet: Concepts and General Aspects established, it is widely accepted that individuals do not consume a single nutrient isolated from the rest but, rather, a whole variety of nutrients that may act synergistically or even antagonistically to each other, thus influencing their bioavailability and absorption [8]. Furthermore, although an individual can significantly increase the habitual intake of a food or a food group after being counseled to do so, one can also neglect to consume other foods to maintain a stable energy intake and not gain body weight. Therefore, it seems easier and more substantive to consider and evaluate the dietary intake of an individual or a population with a holistic approach and provide dietary counseling to promote adherence to a dietary scheme instead of particular foods or food groups [8]. To address this issue, investigators are now including indices of dietary patterns, quality, and variety in their research. Many dietary indices aiming to assess overall diet quality and to determine those dietary schemes and habits that promote human health are available for adults, as are indices evaluating the degree of adherence to the MD. Children’s adherence to a Mediterranean dietary pattern has mainly been evaluated using a recently developed tool, the (KIDMED score) [7]. The KIDMED index is based on the principles sustaining the Mediterranean dietary pattern as well as those that undermine it (such as frequent consumption of fast food and increased intake of sweets). The index includes 16 yes-or-no questions (Table 1). Questions denoting a negative connotation with respect to the MD are assigned a value of 1, and those with a positive aspect +1. The total score ranges from 4 to 12 and is classified into three levels: 8, optimal MD: 4–7, improvement needed to adjust intake to match the MD pattern; and <3, very low diet quality. More specifically, the index includes parameters that are based on the principles of the MD and assigns a value of +1 for the daily consumption of at least one serving of fruits and vegetables (although consumption of at least two servings of each is preferred), at least three daily servings of dairy products (one dairy product at breakfast and at least two servings of yogurt and/or cheese during the day), and daily consumption of grains and cereals for breakfast and consuming pasta or rice at least five times per week. In addition, weekly consumption of at least two to three servings of nuts and fish, and at least two servings of legumes, is suggested. Everyday use of olive oil is recommended. On the other hand, some of the dietary behaviors that are viewed as detrimental to the principles of the MD, and are assigned a value of 1, include the frequent intake of sweets and candies, the consumption of commercially baked goods and pastries for breakfast, eating meals in fast food restaurants, and skipping breakfast. Despite the fact that the MD prototype is used as an educational tool in Mediterranean as well as in non-Mediterranean countries, and despite the international promotion of the Mediterranean nutritional scheme that has taken place during the TABLE 1 KIDMED Test to Assess the Quality of the Mediterranean Diet +1 Eats a fruit or drinks fruit juice every day +1 Eats a second serving of fruit every day +1 Eats fresh or cooked vegetables regularly once a day +1 Eats fresh or cooked vegetables more than once a day +1 Consumes fish regularly (at least 2–3 times/week) 1 Goes more than once a week to a fast-food (hamburger) restaurant +1 Likes legumes and eats them more than once a week +1 Consumes pasta or rice almost every day (5 times/week) +1 Eats cereals or grains (bread, etc.) for breakfast +1 Consumes nuts regularly (at least 2–3 times/week) +1 Uses olive oil at home 1 Skips breakfast +1 Eats a dairy product for breakfast (yogurt, milk, etc.) 1 Eats commercially baked goods or pastries for breakfast +1 Eats two yogurts and/or some cheese (40 g) daily 1 Eats sweets and candy several times every day KIDMED, Mediterranean Diet Quality Index in children and adolescents. Adapted from Serra-Majem et al. [7], with permission from Cambridge University Press. Mediterranean Diet in Children and Adolescents Chapter 7 71 two decades, the MD has been gradually abandoned by the populations of the Mediterranean region, especially the younger generations. In a study aiming to analyze the worldwide trends of adherence to the MD, in 1961–1965 and 2000–2003 the Mediterranean Adequacy Index (MAI) was used [9]. This index is considered suitable for studying the adherence of a country or a population to the MD and is calculated by dividing the energy provided by the total sum of Mediterranean food groups by the energy from the non-Mediterranean food groups [9]. According to this study, although Mediterranean countries showed the highest MAI values in both periods compared to the rest of the countries, Mediterranean Europe and other Mediterranean countries experienced a significant decrease in their MAI values. According to the study, the Mediterranean European group (Albania, Cyprus, France, Greece, Italy, Malta, Portugal, Spain, Turkey, Yugoslavia SFR)—especially Greece, which had the greatest adherence in the 1960s—experienced the greatest decrease in MAI value (Table 2). TABLE 2 Ranking of Countries by Mediterranean Adequacy Index (MAI) Values in Both Time Periods 1961–1965 Countries Ranking 2000–2003 MAI Ranking MAI Greece 1 5.54 10 2.04 Albania 2 5.07 7 2.51 Turkey 3 5.03 5 2.80 Egypt 4 4.81 1 4.09 Tunisia 5 4.57 6 2.65 Japan 6 4.11 16 1.51 Romania 7 3.89 11 2.02 Libya 8 3.81 9 2.09 Algeria 9 3.61 4 2.81 Portugal 10 3.39 18 1.27 Morocco 11 3.37 11 3.25 Syria 12 3.35 8 2.25 Spain 13 3.35 21 1.19 Italy 14 3.30 15 1.62 Yugoslavia 15 3.13 22 1.15 Iran 16 2.87 2 3.65 Mauritania 17 2.87 13 1.77 Lebanon 18 2.70 14 1.72 Bulgaria 19 2.68 20 1.20 Cyprus 20 2.39 27 0.96 Chile 21 2.24 19 1.27 South Africa 22 1.87 12 1.78 Poland 23 1.84 23 1.12 Israel 24 1.62 24 1.09 Malta 25 1.56 17 1.42 Hungary 26 1.48 37 0.73 France 27 1.28 32 0.82 Continued 72 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 2 Ranking of Countries by Mediterranean Adequacy Index (MAI) Values in Both Time Periods—cont’d 1961–1965 2000–2003 Countries Ranking MAI Ranking MAI Argentina 28 1.13 25 0.97 Czechoslovakia 29 1.10 30 0.83 Finland 30 1.04 29 0.87 Austria 31 0.98 38 0.73 Ireland 32 0.97 33 0.80 Norway 33 0.88 26 0.97 Switzerland 34 0.88 39 0.72 Germany 35 0.82 34 0.76 Sweden 36 0.72 31 0.82 Canada 37 0.71 36 0.75 Australia 38 0.68 40 0.70 United Kingdom 39 0.68 28 0.87 Denmark 40 0.67 35 0.76 United States of America 41 0.63 41 0.64 Adapted from da Silva et al. [9], with permission from Cambridge University Press. Regarding the adherence rates of children and adolescents to the MD, relatively recent studies have shown low to average compliance. In a study of a large sample of Spanish schoolchildren aged 8–16 years, the KIDMED index classification among 8- to 10-year-olds was good in 48.6% of the population, average in 49.5%, and poor in 1.6%. Among the 10- to 16-year-olds, the KIDMED index classification was good in 46.9% of the population, average in 51.1%, and poor in 2.0% [10]. In another nationwide representative sample of Greek schoolchildren aged 10–12 years old, the rates of adherence to the MD pattern were much lower (Greek Childhood Obesity—GRECO study) [11]. Specifically, only 4.3% of children had an optimal score, whereas 46.8% were classified as low adherers to the MD. KIDMED score did not differ between boys and girls, but children from urban and semi-urban areas of the country had a higher KIDMED score compared with those form large urban areas. This study also provided evidence of the association between the level of adherence to the MD and the quality of the diet. In particular, it was shown that children with higher KIDMED score also had more frequent consumption of fruits, vegetables, legumes, dairy products, fish, bread, and nuts and had less frequent consumption of foods that, according to the MD scheme, should be consumed in moderation or rarely. Moreover, children with a higher KIDMED score reported having higher levels of physical activity, indicating that in addition to following a better-quality diet, they also adopt a healthier lifestyle (Table 3). Similarly, another study of Greek children and adolescents demonstrated low adherence to the dietary patterns of the MD, because only 11.3% of children and 8.3% of adolescents had an optimal KIDMED score [12]. Data from one more Mediterranean country further support these alarming findings. In a national study of Cypriot children by Lazarou et al. [13], only 6.7% of the sample was classified as high adherers of MD, whereas 37% had a poor KIDMED score; no differences were observed between boys and girls. The low proportion of children with high adherence to the MD could be explained by the nutrition transition theory and the westernization process of the Mediterranean countries [9,14]. It seems that enhanced socioeconomic conditions, increased income levels, increased commercial availability of food, the fact that certain foods are more affordable than before, and the high urbanization that has taken place in Mediterranean countries over the last decades are indicative of changes in food choices [15,16]. Dietary intakes have shifted toward increased consumption of animal protein, processed foods, restaurant-prepared foods, and, as a consequence, higher fat and sugar levels. Moreover, a stressful and demanding lifestyle, the long working hours of all family members, and less frequent family meals have been proposed as important determinants of the nutrition transition [15]. It is widely accepted that socioeconomic and demographic factors affect food choices and consequently health status. Children learn about eating not only through their own experiences but also by watching others—especially their parents, who act as role models. A growing body of research demonstrates similarities TABLE 3 Anthropometric, Lifestyle, and Dietary Characteristics According to KIDMED Score Categories in the Nationally Representative GRECO Study KIDMED Score (4 to 12) 3 4–7 8 N (%) 2240 (46.8) 2341 (48.9) 205 (4.3) Age (years) 10.94 0.76 10.91 0.74 10.90 0.73 0.46 48.4 49.3 51.7 0.59 Body mass index (kg/m ) 20.4 3.9 20.2 3.7 20.4 3.8 0.32 Waist circumference (cm) 68.9 9.7 68.4 9.6 68.4 9.8 0.13 Waist-to-hip ratio 0.83 0.08 0.83 0.07 0.83 0.08 0.55 Waist-to-height ratio 0.46 0.06 0.46 0.06 0.46 0.06 0.10 Body fat (%) 21.3 9.0 20.9 8.8 20.8 8.5 0.26 IPAQ score (1-5) 2.87 0.62 3.00 0.58 3.05 0.60 <0.001 Male sex (%) 2 Pa Starchy foods and cereals (%) <0.001 Breakfast ready-to-eat cereals 5 versus 4 times/week 29.5 46.8 74.6 <0.001 Pasta and rice 5 versus 4 times/week 51.0 68.9 85.9 Bread (%) 0.02 7 versus 6 times/week 26.1 29.5 32.3 <0.001 Legumes/Pulses (%) 2 versus 1 time/week 24.5 54.4 87.7 <0.001 Fruits (at least one fresh fruit) (%) 7 versus 6 times/week 85.1 96.8 98.5 31.2 71.7 98.0 <0.001 b Vegetables (at least one portion of vegetables) (%) 7 versus 6 times/week <0.001 Dairy products (at least 2 portions)c (%) 7 versus 6 times/week 48.1 77.4 92.2 Meat (%) <0.001 Red meat 2 versus 1 times/week 28.8 32.3 44.6 <0.001 Fish 2 versus 1 times/week 19.9 31.2 53.2 <0.001 d Fast foods (%) 2 versus 1 times/week 19.6 17.4 9.8 <0.001 Nuts (%) 3 versus 2 times/week 6.4 9.6 18.6 <0.001 Soft drinks (regular or diet) (%) 3 versus 2 times/week 17.7 12.8 9.9 22.8 16.0 9.6 e Sweets and foods high in sugar (%) 3 versus 2 times/week <0.001 Values are means standard deviations unless otherwise indicated. a P values between all groups as derived from analysis of variance or chi-square test. bCategory including vegetables and fresh legumes consumption. cCategory including milk, cheese, and yogurt consumption. dCategory including burgers and souvlaki (traditional food with meat) consumption. eCategory including chocolate, chocolate bars, and pastries. Adapted from Farajian et al. [11], with permission from Elsevier. 74 SECTION 1 The Mediterranean Diet: Concepts and General Aspects between parents’ and children’s food acceptance, preferences, and intake. In addition, the level of parents’ education is more likely to influence the beliefs, knowledge of nutrition, and health behaviors of the family, which in turn are involved in improved diet quality and nutritional habits of the children [17]. Results concerning the linkage between socioeconomic status and adherence to the MD pattern are contradictory. According to a study by Kontogianni et al. [12], parental education level was a significant predictor of the KIDMED score. In the study of Cypriot children by Lazarou et al. [13], however, no associations between socioeconomic level and KIDMED score classification were found. Although it has been demonstrated that healthier diets often are associated with higher education and income, there are studies of Mediterranean populations that have shown that adherence to the MD pattern is higher in lower social classes coming from rural populations [16]. Under the framework of the GRECO study, in an attempt to identify the social, demographic, family, and lifestyle parameters that constitute the profile of the adherent and nonadherent children to the MD in Greece, we found that both paternal and maternal education status were significant predictors of the KIDMED score, whereas family financial status did not seem to affect rates of adherence to the MD. Furthermore, the total hours of television viewing and playing video games during weekdays and the habit of watching television or using the personal computer while eating a meal increased the likelihood of children to be classified as low adherers to the MD. On the other hand, breakfast consumption, the habit of having family meals during the week, and higher adherence of parents to the MD increased the odds of a child have moderate to high adherence to the MD (unpublished data from the GRECO study). Prolonged time of television viewing has been associated with increased consumption of high-fat and high-sugar foods and reduced intake of fruits and vegetables, resulting in increased energy intake [18]. Furthermore, watching television during meals can lead to larger intake of food and poorer diet quality through less consumption of fruits and vegetables and higher intake of high-fat and high-sugar foods [19]. Finally, regular breakfast consumption has been shown to protect against excess body weight and adiposity in children and adolescents, whereas skipping breakfast has been associated with lower diet quality and consumption of high-energy snacks [20]. MD AND NUTRITIONAL ADEQUACY IN CHILDREN AND ADOLESCENTS Childhood and adolescence constitute a period of high nutritional demands resulting from increased dietary requirements for growth and the development of several tissues. Thus, nutritional inadequacies make children and adolescents vulnerable to impaired growth and current or future adverse health problems. The MD comprises foods that are naturally rich sources of vitamins, minerals, fiber and, because of the emphasis on minimal food processing, provides the maximum nutrient content without the losses that occur during food processing and manufacturing and as a result of few or no food additives. This explains why the risk of inadequate micronutrient intake was rather infrequent in populations that followed the traditional MD [21]. The dietary habits and nutritional status of Spanish children and adolescents with regard to compliance to the MD were evaluated by Serra-Majem et al. [21]. This study demonstrated that the consumption of fiber, calcium, iron, magnesium, potassium, phosphorus, and practically all vitamins (with the exception of vitamin E) increased according to the KIDMED index. Moreover, the percentage of inadequate intakes (less than two-thirds of the reference nutrient intake) declined with increasing KIDMED scores for calcium, magnesium, iron, and vitamins B6, C, and A in both sexes for most cases and almost all age groups. According to the investigators, in addition to the high nutritional quality, the MD provides enhanced nutritional adequacy; therefore, fortification of foods or the use of dietary supplements should not be considered necessary. In three unique studies, the bioavailability, status, and utilization of iron, zinc, and calcium—three micronutrients with crucial importance for the health and growth of adolescents and deficiencies of which are considered some of the most common—were investigated [22–24]. Impaired iron status in children and adolescents could have several important clinical implications for future physical and cognitive development and maturation. Iron deficiency is considered the most frequent single nutrient deficiency in the world and is most common in women [22]. However, iron deficiency is also frequent in children and adolescents; therefore, adequate iron must be provided by the diet on a daily basis within a pattern that promotes the bioavailability of dietary iron. Especially for adolescents, iron is necessary to support rapid physical growth, replace losses, and promote the increase in hemoglobin concentration [22]. Hence, two issues concerning the adequacy of iron, as well as the rest of the important nutrients, arise. First, how do the food habits of children and adolescents that have deviated from the MD pattern compromise the dietary intake and bioavailability of nutrients? Second, considering that one of the most characteristic features of the MD is the recommendation to consume red meat (main source of heme iron) a few times per week in moderate amounts, it is important to ensure that the adherence of children and adolescents to the MD pattern provides sufficient amounts of important nutrients, particularly iron, and promotes their bioavailability and utilization. Concerning the first issue, as previously described, the rates of adherence to the MD by children and adolescents are low, even among Mediterranean populations. Many studies have documented that a significant proportion of daily energy is Mediterranean Diet in Children and Adolescents Chapter 7 75 consumed from low-nutrient, energy-dense foods, snacking on high-fat and high-sugar foods, and eating in restaurants, which may cause low iron intake and bioavailability [25,26]. Regarding the second issue, a study by Mesias et al. [22], investigated the effects of a diet based on the Mediterranean pattern on iron bioavailability and status in adolescents. The study had a longitudinal design in which each subject was his own control. It was divided into two periods: a 3-day basal period, during which the subjects consumed their habitual diet, and a 28-day nutritional intervention period, during which an experimental diet and menu was designed according to the MD scheme. Dietary iron utilization was studied by means of iron intake and iron output in feces and urine, and iron status was analyzed in fasting blood samples collected at the end of each period. The experimental menu included more fish, legumes, cereals, fruits, and vegetables, less meat and meat products, and a higher monounsaturated fat-to-saturated fat ratio than the habitual intake. Olive oil was used as the main dietary fat. During the experimental period, adherence to the MD was significantly improved. Although meat consumption decreased, no significant differences in dietary iron intake (calculating both heme and nonheme) were observed, and iron absorption and retention significantly increased. According to the investigators, the characteristics of the MD increased iron absorption. It should be noted that nonheme iron is not absorbed as well as heme iron, but nonheme iron absorption is affected by enhancing or inhibiting factors. Therefore, although during the experimental period concentrations of phytate or iron-binding polyphenols were higher, the higher intake of ascorbic acid (a known enhancer of iron absorption) counteracted the inhibitory effect of phytic acid. Another explanation provided by the authors was that the intervention diet was high in legumes, which contain a form of iron that is bioavailable to the human intestine and whose absorption that is not influenced by phytic acid. Following the same methodology, in another study by Mesias et al. [23], zinc utilization and status in male adolescents was evaluated. Zinc is a mineral found in meat, poultry, fish, seafood, and dairy products and is required for the activity of over 200 enzymes (Zn dependent) involved in most major metabolic pathways. It participates in reactions involving either synthesis or degradation of major metabolites (carbohydrates, proteins, lipids, nucleic acids); participates in central nervous system development; and is an important factor for maturation and skeletal health [23,27]. Data analysis showed that zinc intake was close to the recommendations for adolescents and that, although a diet based on Mediterranean patterns is associated with factors that can negatively affect zinc absorption, such as high consumption of phytate, consumption in adequate amounts allows zinc status to be maintained during adolescence. Finally, in a study examining the effects of a dietary intervention based on a Mediterranean pattern on calcium utilization and metabolism, following the above-mentioned methodology [22,23], it was shown that dietary calcium utilization (calcium retention) during adolescence can be greatly improved by a Mediterranean-type diet [24]. This was mainly attributed to the reduced intake of sodium and the higher intake of magnesium, potassium, and phosphorus, which could contribute less urinary loss of calcium; to higher intake of vitamin D; higher intake of oligofructose and inulin, which may lead to significant increases in calcium absorption; as well as the high ratio of unsaturated-to-saturated fat intake, which may also favor calcium absorption [24,28,29]. This finding is of great importance because, in addition to the fact that calcium is the main nutrient for skeletal mineralization, promoting bone accretion during puberty, 45% of the adult skeleton is formed during the adolescent growth spurt, and achieving peak bone mass is considered the best strategy to avoid osteopenia and osteoporosis [30]. THE RELATIONSHIP OF THE MD WITH CHILDHOOD OBESITY Obesity among children and adolescents is a growing public health problem. Several studies have focused on the short- and long-term consequences of childhood obesity, and high body mass index (BMI) values have consistently been found to be associated with cardiovascular disease (CVD) risk factors such as insulin resistance, dyslipidemia, and increased blood pressure (BP) [31]. Recent findings from epidemiological studies support the increasing trend of obesity in Europe, showing, however, that southern European countries such as Spain, Italy, Portugal, and Greece report a higher prevalence of obesity compared with northern European countries [32]. The rapid increase in the prevalence of obesity in Mediterranean countries as it has been indicated in many studies published during the past decade, primarily suggests that behavioral factors, influenced by genetic, social, and economic environments, play a role. Although adherence to the MD has been associated with lower BMI and obesity risk in adults, [33] the effect in children and adolescents is not clear, because studies are limited and often inconsistent. There are studies showing that KIDMED score is negatively associated with BMI, [34,35] revealing a protective effect of the MD against the development of overweight and obesity, but these findings are not consistent and have not been confirmed by other studies. According to a study of a nationwide, representative sample of Greek schoolchildren aged 10–12 years old, Farajian et al. [11] found that the prevalence of overweight was 29.5% and the rate of obesity was 11.7%. Though children with higher KIDMED score reported higher levels of physical activity, no differences were found in the BMI of the groups with different KIDMED scores. Nevertheless, in a representative sample of young Spaniards among whom the association between 76 SECTION 1 The Mediterranean Diet: Concepts and General Aspects waist circumference (WC) and adherence to the MD was examined, age-adjusted linear regression revealed an inverse association between KIDMED category and both WC and waist-to-height ratio [36a]. Furthermore, after multivariate adjustment, a 5-point increase in KIDMED score was associated with a mean decrease of 1.54 cm in sex-, age-, and height-adjusted WC. In another report in the context of the Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants study, adherence to a Mediterranean-like dietary pattern was assessed using a modified version of the MD score based on the dietary data collected by a nonquantitative food frequency questionnaire [36b]. A large number of European children were measured at baseline and after 2 years. At baseline, high adherence to the MD was inversely associated with BMI as well as percentage of body fat, even after adjusting for several potential confounders. Longitudinally, a Mediterranean-like dietary pattern also was inversely associated with the highest change in BMI and central obesity markers (WC and waist-to-height ratio). Irrespective of the fact that the association between MD adherence and childhood obesity is not yet definitive, it has to be remarked that some of the components of the MD scheme, such as the high intake of fruits and vegetables and the high intake of fiber, have been linked with reduced risk for overweight and obesity and, in some cases, with lower likelihood of having increased WC [37]. In addition, some of the components of the MD that have been highlighted in the newly proposed graphical presentation of the MD, such as frequent family meals and adequate time dedicated to eating, have been shown to protect against obesity and promote healthier dietary habits, together with socialization [38,39]. Therefore, it could be claimed that promoting an MD pattern also helps tackle the childhood obesity problem. HEALTH EFFECTS OF THE MD IN CHILDREN AND ADOLESCENTS An increasing number of recent epidemiological studies of adult populations have established the health benefits associated with adherence to the MD pattern, mainly in relation to reducing the risk of developing type 2 diabetes, CVD, some neurodegenerative diseases, and cancers [40]. In addition, results of experimental studies have shown that the MD showed favorable effects on lipoprotein levels, endothelium vasodilatation, insulin resistance, metabolic syndrome, antioxidant capacity, myocardial and cardiovascular mortality, and cancer incidence in obese patients and in those with previous myocardial infarction [41]. Although there are fewer studies investigating the effects of the MD adherence to the risk of developing chronic diseases in children and adolescents, a growing number of studies reveal the beneficial effects of the MD on several health issues. Influence of the MD on Asthma in Children While the overall asthma prevalence has increased, with the highest incidence occurring in children, one of the potential environmental explanations that has been proposed for this increase relates to changes in diet [42]. According to researchers, a major change of the contemporary diet that increases the risk of developing asthma is the change in dietary fat intake characterized by the increment of n-6 polyunsaturated fatty acids (PUFAs) and reduction of n-3 PUFAs [42]. Others have proposed a mechanism that involves the decline of antioxidant defenses in the lungs, resulting in increased oxidant-induced airway damage caused by low intake of dietary antioxidants [43]. Many epidemiological studies have evaluated the role of individual nutrients or foods on asthma prevalence, suggesting a protective role of high fruit, vegetable, and fish intake [44]. Other studies have reported on the beneficial effects of single foods (such as citrus/kiwi fruit, apples, pears, tomatoes, nuts, and oily fish) rich in antioxidants, n-3 PUFAs, or flavonoids on asthma or wheeze [42]. In a systematic review and meta-analysis, Nurmatov et al. [45] concluded that although the available epidemiologic evidence regarding vitamins A, D, and E, zinc, fruits and vegetables, and the MD pattern is limited, data regarding these items and their role in the prevention of childhood wheeze and possibly asthma are encouraging. Analysis of dietary pattern accounting for the interaction and the intercorrelation among nutrients seems to have received much attention. Specifically, many studies have reported a protective effect of the MD on asthma in children and adolescents, although results do not always agree [42]. Adherence to the MD pattern provides high quantities of b-carotene and vitamin C (from fruits and vegetables), vitamin E (from olive oil and nuts), and various important antioxidant nutrients such as selenium, flavonoids, and polyphenols; therefore it seems reasonable to assume that the MD has protective effects against asthma development [44,46]. In a meta-analysis by Garcia-Marcos et al. [42] aiming to investigate the effects of MD adherence on asthma diagnosis and on current wheeze (or severe current wheeze) in children, adherence to the MD tended to be associated with lower occurrence of these three respiratory outcomes. This association was mainly driven by the results of studies of Mediterranean populations, although in non-Mediterranean studies there was an inverse association with asthma diagnosis but not with the other two conditions. When considering all centers together, however, MD was significantly associated with a lower prevalence of asthma (OR: 0.86; 95% CI 0.78–0.95; P ¼ 0.004). Mediterranean Diet in Children and Adolescents Chapter 7 77 Nevertheless, the protective effect of the MD on the prevalence of childhood asthma is not yet definitively established because of contradictory findings in the literature. These opposing findings might be due to the use of different Mediterranean indices, different asthma outcomes, dissimilar populations of children (e.g., preschoolers, school-age children, or adolescents), or even small sample sizes [42]. Therefore, better controlled trials using similar methodologies for different populations are necessary. The Association of the MD with BP Levels and Cardiovascular System Risk Factors High BP is detectable in children and adolescents and is increasing in prevalence. Estimates of the prevalence of high BP among children and adolescents are in the range of 3% to 5%, with much higher rates reported in certain populations and subgroups [47]. According to a position paper of the American and European Societies of Hypertension, therapeutic lifestyle changes are recommended as an initial treatment strategy for adults with abnormal BP values and children or adolescents with BP greater than the 90th percentile for age, sex, and height [48,49]. These lifestyle modifications include regular physical activity, maintaining normal weight, limiting dietary sodium intake, and increasing the consumption of fruits, vegetables, fiber, and low-fat dairy products. Sodium is the main mineral that has been linked to hypertension. The importance of sodium intake in determining BP levels in children and adolescents is shown in many observational studies and salt reduction trials. In a meta-analysis of 10 salt reduction trials with an average duration of 4 weeks, He and MacGregor [50] demonstrated that a modest reduction in salt intake had a significant effect on BP in children and adolescents. In modern diets, an estimated 75–80% of sodium intake comes from processed or restaurant-prepared foods, 10–12% occurs naturally in foods, and another 10–12% is from optional use of table salt. According to estimations, salt intake among children in developed countries has increased; the main contributors to this increase are starchy products (including bread, other baked goods, and breakfast cereals), meat products (and cold cuts), pastries, and processed foods in general. In a study by Magriplis et al. [51] in the context of the GRECO study, daily dietary sodium intake (excluding table salt and salt added during cooking) of 10- to 12-year-old Greek children was studied and associated with the MD pattern. The results of the study showed that 23% of children had high sodium intake (>2200 mg/day), without taking into account salt added at the table or while cooking. In addition, a further 20.9% of the children had a moderate sodium intake (between 1500 and 2200 mg/day), again only via foods. The major sources of sodium were pizza, hamburgers, fried potatoes, souvlaki (a traditional Greek meat product), bread, white and yellow cheese, and processed cereals (Figure 1). Interestingly, 34% of total sodium intake was found to be consumed by those foods regarded as “healthy” (i.e., bread, processed cereal, and white cheese). These foods, which are recommended to be consumed on a daily basis according to the MD scheme, are in fact everyday hidden sources of sodium because of the addition of sodium during manufacturing. Another important finding was that children adhering closer to the MD reported a higher dietary sodium intake. In particular, children who reported moderate and high adherence to the MD (i.e., KIDMED score >4) had higher sodium intake from the majority of the food groups. Moreover, a 1-unit increase in KIDMED score was associated with a 10% increase in likelihood of consuming total sodium >1500 mg/day (the upper level in the European Union). Although it would then be expected that children with high adherence to the MD would also have higher BP levels, this was not the case: There were no differences Junk foods Pizza Fried potatoes Greek souvlaki Hamburger Bread Healthy foods FIGURE 1 Sodium intake from healthy or “junk” foods as a percentage of total consumption other than table salt and salt added during cooking. Data are from the Greek Childhood Obesity study. Adapted from Magriplis et al. [51], with permission from Wolters Kluwer Health. Processed cereals Yellow cheese White cheese 0 5 10 15 % of total sodium intake 20 78 SECTION 1 The Mediterranean Diet: Concepts and General Aspects in the KIDMED score values between children sorted into different BP categories (unpublished data from the GRECO study). These data imply that although MD adherence can sometimes be accompanied by high intake of sodium, the MD still provides protection against increased BP levels, probably through the parallel high intake of potassium, mainly from increased consumption of fruits and vegetables. Concerning the association between the MD and BP levels, in addition the available studies of adults that demonstrate the inverse relation of MD adherence and arterial BP levels, there are few studies of childhood and/or adolescent populations. A study by Lazarou et al. [52] assessed the relationship between diet quality, determined using the Foods E-KINDEX, and BP levels. The Foods E-KINDEX is an index that assesses the consumption of foods recommended in the MD pyramid and cooking techniques. Compared with children with a low diet score, those with at least an average Foods E-KINDEX score were 57% less likely to have elevated systolic BP (SBP) levels, whereas children who scored below the mean on the Foods E-KINDEX (i.e., poor diet quality) were 2.3 times more likely to have elevated SBP. In a study of a healthy pediatric population (12-year-olds), the association of indices of arterial stiffness [augmentation index (AI) in the brachial artery, the ratio of peripheral pulse pressure to central pulse pressure, and the ratio of reflected wave transit time to height] with anthropometric and BP values and adherence to the MD was explored using the KIDMED index [53]. Measures of arterial stiffness are independent prognostic indicators of cardiovascular events and have been reported to be higher in obese compared with normal-weight children [53]. Among the main findings of this study was that children with higher BMI and WC had higher peripheral and central BPs and lower peripheral pulse pressure-to-central pulse pressure ratios. Furthermore, adherence to the MD was independently negatively correlated with AI. According to another interesting study by Mazaraki et al. [54], adolescents who adhered to the MD exhibited lower levels of albuminuria. In adult populations, albuminuria (apart from end organ damage in hypertension) constitutes a potent marker of atherosclerotic progression and increased cardiovascular risk [55]. In adolescents this marker is considered a predictor of pre-atherosclerosis, allowing assessment before the development of obvious CVD. In this study, urinary albumin excretion, expressed as the albumin-to-creatinine ratio, was higher in participants in the low KIDMED score category compared with those in the average and high categories. This difference remained significant even after controlling for age, sex, BMI, and systolic and diastolic BP, reflecting the protective effects of the MD on the vascular system. According to the authors, the pathophysiological explanation for the observed relation could be the protective effects of the MD on the endothelium and the antiatherosclerotic plus the anti-inflammatory properties of the dietary pattern. Health-Related Quality of Life and the Relationship with the MD Health-related quality of life (HRQOL) refers to an individual’s perception and subjective evaluation of their health and well-being within their unique cultural environment. Adolescence is a transitional stage of development, during which an individual experiences a variety of biological, social, and psychological changes that may positively or negatively affect the HRQOL. Although the self-perceived health of children and adolescents is recognized as an important outcome in public health studies, data from studies of pediatric or adolescent samples exploring adherence to the MD in relation to HRQOL are scarce. In adults, greater adherence to the MD has been shown to be associated with better HRQOL, whereas studies have shown that overweight and obesity in children and adolescents are associated with impaired HRQOL and that greater adherence to the MD pattern is associated with better academic performance [56]. In the sole study in this area examining the association between adherence to the MD and HRQOL among Greek adolescents, adherence to the MD was assessed using the KIDMED index and the perceived HRQOL using the KIDSCREEN-27 questionnaire, which assesses five dimensions of HRQOL: physical well-being, psychological well-being, parent relations and autonomy, social support and peers, and school environment [57]. Results of the study showed that adherence to the MD was positively correlated with all the components and the total score of HRQOL. Thus, it could be assumed that adolescents with better diet quality also have better physical and psychological health. It could also be postulated that the support of their family and the school environment is sufficient in a way that promotes healthy nutritional habits and behaviors. SUMMARY POINTS l l The MD has been promoted worldwide as one of the most healthy dietary patterns. In addition to the beneficial effects of the MD in adult populations, recent studies confirm that the MD may be an important health-promoting dietary scheme for children and adolescents. Children’s adherence to the Mediterranean dietary pattern has been assessed in several studies using a recently developed index. Like adults, children’s adherence to the MD in Mediterranean countries has been reported to be low to average and is affected by socioeconomic, parental, and modern lifestyle factors. Mediterranean Diet in Children and Adolescents Chapter 7 l l l 79 A growing number of studies reveal the beneficial effects of the MD on several health issues in children and adolescents. Greater adherence to the MD has been associated with the prevention of childhood wheeze and possibly asthma, better control of BP levels, lower levels of markers of atherosclerotic progression, and indices of arterial stiffness. In addition to its potential cardioprotective and anti-asthmatic effects, the MD provides enhanced nutritional adequacy during childhood and adolescence, when the nutritional demands for growth and maturation are increased. Although few in number, recent studies provide evidence that the intake, bioavailability, and utilization of iron, zinc, and calcium are sufficient. 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Mediterranean diet and waist circumference in a representative national sample of young Spaniards. Int J Pediatr Obes 2010;5:516–9. [36b]Tognon G, Hebestreit A, Lanfer A, Moreno LA, Pala V, Siani A, et al. Mediterranean diet, overweight and body composition in children from eight European countries: cross-sectional and prospective results from the IDEFICS study. Nutr Metab Cardiovasc Dis 2013;2(3):e000101. [37] Schr€ oder H. Protective mechanisms of the Mediterranean diet in obesity and type 2 diabetes. J Nutr Biochem 2007;18:149–60. [38] Bach-Faig A, Berry EM, Lairon D, Reguant J, Trichopoulou A, Dernini S. Mediterranean diet pyramid today. Science and cultural updates. Public Health Nutr 2011;14:2274–84. [39] Hammons AJ, Fiese BH. Is frequency of shared family meals related to the nutritional health of children and adolescents? Pediatrics 2011;2011(127): e1565–74. [40] Sofi F, Cesari F, Abbate R, Gensini GF, Casini A. Adherence to Mediterranean diet and health status: meta-analysis. BMJ 2008;337:a1344. [41] Serra-Majem L, Roman B, Estruch R. Scientific evidence of interventions using the Mediterranean diet: a systematic review. Nutr Rev 2006;64: S27–47. [42] Garcia-Marcos L, Castro-Rodriguez JA, Weinmayr G, Panagiotakos DB, Priftis KN, Nagel G. Influence of Mediterranean diet on asthma in children: a systematic review and meta-analysis. Pediatr Allergy Immunol 2013;24:330–8. [43] Seaton A, Godden DJ, Brown K. Increase in asthma: a more toxic environment or a more susceptible population? Thorax 1994;49:171–4. [44] Arvaniti F, Priftis KN, Papadimitriou A, Papadopoulos M, Roma E, Kapsokefalou M, et al. Adherence to the Mediterranean type of diet is associated with lower prevalence of asthma symptoms, among 10–12 years old children: the PANACEA study. Pediatr Allergy Immunol 2011;22:283–9. [45] Nurmatov U, Devereux G, Sheikh A. Nutrients and foods for the primary prevention of asthma and allergy: systematic review and meta-analysis. 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High sodium intake of children through ‘hidden’ food sources and its association with the Mediterranean diet: the GRECO study. J Hypertens 2011;29:1069–76. [52] Lazarou C, Panagiotakos DB, Matalas A. Foods E-KINDEX: a dietary index associated with reduced blood pressure levels among young children: the CYKIDS study. J Am Diet Assoc 2009;109:1070–5. [53] Lydakis C, Stefanaki E, Stefanaki S, Thalassinos E, Kavousanaki M, Lydaki D. Correlation of blood pressure, obesity, and adherence to the Mediterranean diet with indices of arterial stiffness in children. Eur J Pediatr 2012;171:1373–82. [54] Mazaraki A, Tsioufis C, Dimitriadis K, Tsiachris D, Stefanadi E, Zampelas A, et al. Adherence to the Mediterranean diet and albuminuria levels in Greek adolescents: data from the Leontio Lyceum ALbuminuria (3 L study). Eur J Clin Nutr 2011;65:219–25. [55] Tsioufis C, Dimitriadis K, Antoniadis D, Stefanadis C, Kallikazaros I. The inter-relationships of microalbuminuria with the other surrogates of the atherosclerotic cardiovascular disease in hypertensive subjects. Am J Hypertens 2004;17:470–6. [56] Costarelli V, Koretsi E, Georgitsogianni E. Health-related quality of life of Greek adolescents: the role of the Mediterranean diet. Qual Life Res 2013;22:951–6. [57] Robitail S, Ravens-Sieberer U, Simeoni MC, Rajmil L, Bruil J, Power M, et al. Testing the structural and cross-structural validity of KIDSCREEN-27 quality of life questionnaire. Qual Life Res 2007;16:1335–45. Chapter 8 The Influence of the Mediterranean Diet on Cognitive Health Helen Macpherson, PhD, Jaime Lee, Lorena Villalon, Matthew Pase, PhD, Andrew Pipingas, PhD and Andrew Scholey, PhD Swinburne University, Hawthorn, VIC, Australia. INTRODUCTION Almost 20 years has passed since the initial observation from the Seven Countries study that inhabitants of southern Europe, in the vicinity of the Mediterranean region, had lower rates of death due to coronary heart disease than those from northern Europe or the United States [1]. This was partially attributed to the consumption of olive oil, a key component of the diet followed by residents of the Mediterranean region. At a population level, adherence to this dietary pattern has since been linked repeatedly to lower risk of mortality [2,3], vascular events [4] and, importantly for this chapter, dementia [5]. Advancing age represents the major risk factor for dementia and, as a consequence of rapid population aging, the incidence of dementia in Western societies is increasing at an alarming rate. With an aging population and escalating rate of dementia, there is an increasing need to identify preventive strategies to delay the onset of dementia and reduce to the projected number of dementia cases in future years [6]. For this reason there is interest in modifying aspects of lifestyle, such as diet and exercise, to prevent or slow cognitive decline in older people. Alzheimer’s disease (AD) and vascular dementia are the most common forms of cognitive impairment in elderly people [7]. AD is a progressive degenerative disorder characterized by a loss of neurons that severely affects learning and memory [8]. Formation and accumulation of extracellular b-amyloid plaques and intraneuronal neurofibrillary tangles consisting of hyperphosphorylated tau protein are thought to be the key pathological events leading to neuronal damage [9]. Mechanisms such as neurovascular dysfunction, inflammatory processes, oxidative stress, and mitochondrial dysfunction also play critical roles in the neuropathology of AD [10]. In terms of cognitive function, AD is characterized as a substantial decline from previous functioning of short-term memory and a severe disruption in language, planning, or visual processing abilities [11]. Individuals with AD lose the ability to carry out activities of daily living and eventually the capacity to care for themselves. Individuals who have experienced a decline in cognition that exceeds their age and education level but does not impair activities of daily living are classified as having mild cognitive impairment (MCI) [12]. MCI often represents a preclinical stage of AD, and the annual conversion rate of MCI to AD is about 5–10% [13]. While age and apolipoprotein E status represent nonmodifiable risk factors for AD, a number of potentially modifiable risk factors include diabetes, midlife hypertension, obesity, depression, smoking, cognitive inactivity, and low education [14]. Cognitive deterioration occurs across the life span and is a feature of not only AD but also the normal aging process. Measures of fluid intelligence, which include a wide variety of abilities relying on memory, reasoning, and spatial abilities, are particularly vulnerable to the effects of aging [15]. A growing number of studies indicate that following a Mediterranean diet may help protect against dementia and the cognitive decline that occurs a part of the normal aging process [16]. CHARACTERISTICS AND ASSESSMENT OF THE MEDITERRANEAN DIET The Mediterranean diet refers to the dietary practices of populations from the Mediterranean region. The Mediterranean diet consists predominantly of fresh fruits, vegetables, breads, cereals, nuts, and legumes. Olive oil provides the main source of dietary fat. This dietary pattern features low to moderate consumption of dairy, fish, poultry, and alcohol served with meals. Red meat is consumed only in low quantities. The current consensus of the Mediterranean diet is shown in the food pyramid in Figure 1. The greatest difference between the Mediterranean diet and the Western diet is the sources and proportion of dietary fat. Western diets are high in saturated fats and refined carbohydrates, whereas the Mediterranean diet The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 81 82 SECTION 1 The Mediterranean Diet: Concepts and General Aspects We e kly Sweets ≤ 2 s Potatoes ≤ 3 s Red meat < 2 s Processed meat ≤ 1 s White meat 2 s Fish/seafood ≥ 2 s Eggs 2–4 s Legumes ≥ 2 s Olives/nuts/seeds 1–2 s herbs/spices Fruits 1–2 s Vegetables ≥ 2 s Olive oil Bread/pasta/rice/couscous Other cereals 1–2 s (preferably whole grain) Ev er ym ain me al Ev er yd ay Dairy 2 s (preferably low fat) Water and herbal infusions FIGURE 1 Current consensus of the Mediterranean diet. s, servings. Adapted from Bach-Faig et al. [17]. has a greater proportion of monounsaturated fats. The composition of the Western diet not only increases the risk of mortality from cardiovascular events and coronary heart disease but there also are growing concerns regarding the long-term effect of this dietary pattern on the brain [18]. An increasing number of health benefits are being attributed to the Mediterranean diet. This information has primarily been obtained from large population studies that examined information about dietary intake in relation to numerous health parameters and disease risk factors. To assess adherence to this dietary pattern, several indices have been proposed. The most commonly used measure is the MeDi score, a 10-point scale ranging from zero (low adherence) to nine (higher adherence) [3]. The MeDi score is calculated from sex-specific group medians of food intake obtained from a food frequency questionnaire. If consumption is above the median, one point is awarded for favorable components including fruit, vegetables, grains, nuts, legumes, and monounsaturated fatty acid-to-saturated fatty acid ratio. A value of zero is assigned if consumption of unfavorable components is above the median. An alternate scoring measure was developed by Panagiotakos et al. [19]. This measure also calculates a MedDiet score from a food frequency questionnaire. For items that are characteristic of the Mediterranean diet, such as fruits, vegetables, and olive, higher scores are assigned for more frequent intake. For foods not typical of this dietary pattern, such as meat and full-fat dairy products, lower scores are assigned for higher intake. For alcohol, both no consumption and low consumption receive higher scores. More recently, a briefer 14-item questionnaire was developed from the Prevencion con Dieta Mediterranea (PREDIMED) trial [20]. One point is assigned for each item that meets the criteria; higher scores indicate greater adherence to the Mediterranean diet. The advantage of this shorter measure is that it does not require any additional dietary information to be calculated. THE RELATIONSHIP BETWEEN DEMENTIA, COGNITIVE DECLINE, AND THE MEDITERRANEAN DIET There is growing evidence from large-scale, population-based studies that long-term adherence to the Mediterranean diet may help to protect against dementia and preserve brain and cognitive function in the later stages of the life span. For instance, several epidemiological studies identified a negative relationship between adherence to the Mediterranean diet and dementia or predementia syndromes in older adults. Findings from the longitudinal Washington Heights-Inwood Columbia Aging Project (WHICAP) revealed that after 4 years of greater adherence to the Mediterranean diet there Mediterranean Diet Cognitive Health Chapter 8 83 was a reduced risk for probable AD [21], development of MCI, and progression to AD [22], as well as reduced AD mortality [23]. Results from the Australian Imaging, Biomarkers, and Lifestyle Study of Aging indicated that the MeDi score was a significant predictor of MCI and AD over and above numerous AD risk factors [24] (Table 1). The Mediterranean diet has also been linked to a slower rate of cognitive decline in older individuals without dementia or MCI. These findings suggest there may be more general benefits of this dietary style on brain function. Recent results from the Reasons for Geographic and Racial Differences in Stroke study support this premise [25]. This study examined diet practices of US residents aged 45 years or older who were free from cognitive impairment at baseline. After an average follow-up period of 4 years, 7% of participants developed incident cognitive impairment. Among nondiabetic participants, higher adherence to the Mediterranean diet was associated with a lower likelihood of cognitive impairment. Findings from the Chicago Health and Aging Project indicated that among older people free of dementia (65 years old), following a Mediterranean diet was linked to a slower rate of global cognitive decline over 7 years [26]. In that study, cognition was assessed by a composite of four measures including immediate and delayed recall, digit symbol substitution, and the Mini-Mental State Examination (MMSE), a dementia screening tool that provides a global measure of cognitive function. Several other studies examined the relationship between adherence to the Mediterranean diet and score on the MMSE [32]. In the Three Cities trial, elderly residents of Bordeaux, France, who had greater adherence to a Mediterranean diet demonstrated less decline on the MMSE over a period of 5 years. This relationship was only observed among individuals who did not develop dementia and was not demonstrated for measures of specific cognitive domains [27]. Findings from the European Prospective Investigation into Cancer and Nutrition trial indicated that among residents of Greece older than 60 years of age, greater adherence to the Mediterranean diet and intake of olive oil and monounsaturated fats were weakly related to higher MMSE scores 6 to 13 years later [28]. Individual antioxidant-rich and flavonol-containing components of the Mediterranean diet, such as olive oil, nuts, and wine, also have been related to better memory scores in the Spanish PREDIMED study. Not all trials have identified a relationship between adherence to the Mediterranean diet and cognitive function. The French Supplementation with Vitamins and Mineral Antioxidants (SU.VI.MAX) examined the Mediterranean eating pattern among middle-aged adults in relation to cognitive performance 13 years later [29]. In that study, diet was not predictive of global cognitive performance, measured as a composite score of six neuropsychological tasks. The Australian PATH Through Life study indicated that following the Mediterranean diet was not related to the incidence of MCI 4 years later among individuals aged 60 to 64 years. Interestingly, high caloric and monounsaturated fat intake were stronger predictors [30]. However, in the study there were few cases of conversion to MCI over the 4-year period, potentially limiting the ability to detect a relationship between diet and MCI. Collectively, findings from these studies suggest that habitually following the Mediterranean diet may help protect against dementia. Heterogeneous findings in relation to populations without dementia may be due to differences in methodology or populations from which the participant sample was drawn. In the SU.VI.MAX trial, cognitive measures were not implemented at baseline [29], and only a subset of the overall sample underwent cognitive testing. This is an issue common to many large population studies that have collected cognitive information from only a smaller cohort of the overall study. Use of dietary supplements may also be an important consideration when examining the relationship between cognitive function and Mediterranean diet adherence. There is growing consensus that the use of dietary supplements may benefit aspects of health (including psychological well-being), especially in individuals with a suboptimal nutritional status. CARDIOVASCULAR MECHANISMS OF THE MEDITERRANEAN DIET Poor cardiovascular health is increasingly recognized as an important risk factor for developing dementia later [33]. A number of studies have largely attributed the health advantages of the Mediterranean diet to amelioration of oxidative stress, which in turn may alter the expression of inflammatory markers that are thought to play a role in the pathogenesis of vascular diseases and AD. For a review see Frisardi et al. [16]. Because cerebrovascular disease may often coexist in people diagnosed with AD [34], it has been suggested that, when maintained for years at a time, such a dietary pattern may have protective effects against cerebrovascular pathology, which in turn alters the onset and expression of cognitive changes [26]. Substantial cardiovascular benefits have been associated with the Mediterranean diet and may contribute to reduced prevalence of dementia. Panagiotakos and colleagues [19] examined the eating habits of more than 3000 healthy residents of Greece with no history of cardiovascular disease. They found greater adherence to the Mediterranean diet to be inversely associated with a number of clinical and biological markers of cardiovascular disease risk, including systolic blood pressure, total serum cholesterol, fibrinogen, C-reactive protein, and body mass index. There was also a positive association Participants Region Washington HeightsInwood Columbia Aging Project (WHICAP) [21] 2258 Adults aged 65 years at baseline United States Washington HeightsInwood Columbia Aging Project (WHICAP) [22] 1393 Adults aged 65 years at baseline Reasons for Geographic and Racial Differences in Stroke (REGARDS) [25] Cognitive Outcome Measures Results 4 Years Clinical Dementia Rating (CDR) measuring global cognition, DSM-III-R for all-cause dementia, NINCDS-ADRDA for Alzheimer’s disease Higher adherence to the MedDiet was associated with lower risk for Alzheimer’s disease United States 4.5 Years Neuropsychological battery containing tests of memory (short- and long-term verbal and nonverbal), orientation, abstract reasoning (verbal and nonverbal), language (naming, verbal fluency, comprehension, and repetition), and construction (copying and matching); CDR measuring global cognition, NINCDS-ADRDA for Alzheimer’s disease Higher MedDiet adherence was associated with a trend for reduced risk of developing mild cognitive impairment and reduced risk of mild cognitive impairment converting to Alzheimer’s disease 17,478 Adults aged >70 years at baseline United States 4 Years Six-item screener measuring global cognition Higher adherence to MedDiet was associated with a lower likelihood of incident cognitive impairment in nondiabetic participants only Chicago Health and Aging Project (CHAP) [26] 3790 Adults aged 65 years at baseline United States 7.6 Years MMSE and Symbol Digit Modalities Test scores were standardized and averaged for a global measure of cognitive function Higher MedDiet scores were associated with slower rates of cognitive decline Three City [27] 1410 Adults aged 65 years at baseline France 5 Years The MMSE, Isaacs Set Test (IST) of semantic verbal fluency, Benton Visual Retention Test (BVRT) of visual memory, Free and Cued Selective Reminding Test (FCSRT) of verbal episodic memory Higher MedDiet adherence was associated with slower decline on MMSE, but not other cognitive tests, or risk for incident dementia European Prospective Investigation into Cancer and Nutrition (EPIC) Greece cohort [28] 732 Participants 60 years at baseline Greece 6-13 Years MMSE Nonsignificant trend for a relationship between MedDiet adherence and cognition Supplementation with Vitamins and Mineral Antioxidants (SU.VI. MAX) [29] 3083 Adults aged 45 years France 13 Years Episodic memory, semantic fluency task, forward and backward digit span task of working memory, Delis-Kaplan trail-making test of mental flexibility. Scores were standardized to create a composite cognitive score; tests were administered only during follow-up MedDiet adherence was not related to cognitive function PATH Through Life [30] 1528 Adults aged Australia 4 Years Average z score of MMSE, California Verbal Learning Test, Symbol Digit Modalities Test, and Purdue pegboard test MedDiet was not protective of cognitive decline DSM-III-R, Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition. NINCDS-ADRDA, National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer Disease and Related Disorders Association [31]. SECTION 1 The Mediterranean Diet: Concepts and General Aspects Study Average FollowUp 84 TABLE 1 Longitudinal Studies Examining the Relationship Between Adherence to the Mediterranean Diet and Cognition Mediterranean Diet Cognitive Health Chapter 8 85 with total antioxidant capacity, which is considered to be an indicator of improved cardiovascular functioning. Moreover, more than 20% adherence to the diet was associated with a 37% decrease in the risk of having an acute coronary event. A large meta-analysis of 50 studies confirmed these results, indicating that adherence to the Mediterranean diet reduces the risk of a cluster of cardiovascular health conditions and is beneficial to waist circumference, high-density lipoprotein cholesterol levels, triglyceride levels, blood pressure levels, and glucose metabolism [35]. Because many of these cardiovascular parameters represent risk factors for dementia [33], it may be that beneficial cardiovascular effects of this diet maintain brain health into old age. OLIVE OIL Olive oil is rich in monounsaturated fatty acids and polyphenols and may be responsible for many of the cardiovascular benefits associated with this dietary profile. A recent study of more than 40,000 residents of Spain revealed that over 13 years there was a 26% reduction in all-cause mortality and a 44% decrease in cardiovascular disease mortality among those with the highest intake of olive oil [36]. The PREDIMED trial investigated the cardiovascular effects of the Mediterranean diet in 772 people 55 to 80 years old with multiple cardiovascular risk factors [37]. This randomized controlled trial found that following a Mediterranean diet supplemented with extra virgin olive oil or nuts for 3 months improved lipid profiles and reduced blood pressure, insulin resistance, and systemic markers of inflammation when compared with a low-fat diet. In a larger cohort of the PREDIMED study, followed for an average of 4.8 years, the Mediterranean diet supplemented with olive oil or nuts reduced the incidence of cardiovascular events relative to a low-fat diet [38]. Interestingly, when cognitive function was assessed in a sample of 285 participants from the PREMIMED trial after 6.5 years of adhering to the nutritional intervention, better fluency and memory tasks and a lower prevalence of MCI was observed for the Mediterranean diet supplemented with additional olive oil compared with controls [39]. Because these results were based on cognitive end points without reference to baseline cognitive assessments, there is a need to replicate this study. Nevertheless, findings from the PREDIMED study suggest that a Mediterranean diet rich in olive oil may contribute to cognitive health. DIETARY EFFECTS ON OXIDATIVE STRESS AND INFLAMMATION In addition to the advantages of a diet high in olive oil, it has been suggested that vascular processes and inflammatory pathways mediate the relationship between the Mediterranean diet and cognition [16]. Following a low-calorie Mediterranean diet for as little as 8 weeks has been demonstrated to be effective in reducing inflammatory markers in overweight individuals [40]. This may be a result of the combined effects of a range of components of the Mediterranean diet. Higher intake of fruit and vegetables has been associated with higher blood nutrient levels, lower oxidative stress, and better cognitive function [41]. Increasing fruit and vegetable intake over a period of 3 months has been demonstrated to improve nutrient levels in healthy individuals [42]. Consumption of fruit and vegetables or a diet that is full of antioxidants, serum carotenoids, vitamins, fiber, and magnesium are capable of reducing C-reactive protein [43], a marker of inflammation closely linked to cognition [44]. In addition, flavonoids found in fruit and vegetables and omega-3 polyunsaturated fatty acids found in high quantities in fish have demonstrated anti-inflammatory, cardioprotective, and neuroprotective properties [45,46]. Fish in particular contain omega-3 essential fatty acids, which have anti-inflammatory properties and increase cell membrane fluidity [47]. Despite the theoretical viewpoint that vascular mechanisms or reduced inflammatory processes are responsible for the relationship between the Mediterranean diet and dementia, this has not been confirmed in all trials [5,48]. Findings from the WHICAP study indicated that vascular variables including diabetes, stroke, hypertension, heart disease, and cholesterol levels did not account for the relationship between Mediterranean diet and AD [5]. In an analysis of data over 4 years from the WHICAP II study, levels of the inflammatory marker C-reactive protein and metabolic markers of fasting insulin and adiponectin did not mediate the relationship between Mediterranean diet and AD [48]. A potential issue with these cohorts was that participants were, on average, 75 years old when baseline measures were collected. Cardiovascular health at midlife is emerging as an important predictor of dementia [33], and following a cohort from early in middle age may be more informative regarding long-term mechanisms of the Mediterranean diet. ADDITIONAL BENEFITS OF EXERCISE Exercise is regarded as an important aspect of the Mediterranean diet. It is currently recommended that the diet be complemented by regular moderate physical activity equating to 30 min a day [17]. Combining a Mediterranean diet with 86 SECTION 1 The Mediterranean Diet: Concepts and General Aspects exercise is likely to maximize improvements to cardiovascular health. A study that examined the additive effects of a 3month Mediterranean diet and exercise intervention indicated that there was greater weight loss and blood pressure benefits when combined than when a Mediterranean diet intervention was used in isolation [49]. In an uncontrolled 4-month trial, a Mediterranean diet and exercise intervention reduced weight and body mass index from a classification of obese to overweight [50]. Exercise may also provide independent enhancements to brain function and cognition. A meta-analysis of 29 studies of older adults free from dementia demonstrated aerobic exercise interventions ranging from 8 to 72 weeks improved cognitive faculties including attention, processing speed, executive function, and memory [51]. Several mechanisms of action have been proposed to account for the cognitive benefits of exercise among older adults. Long-term effects of exercise may influence the brain, and consequently cognition, by reducing diabetes, hypertension, and cardiovascular disease [52]. Exercise may also increase the volume of the hippocampus, a brain region vital to memory function, as well as the rate of gray matter shrinkage in the brain [53]. MEDITERRANEAN DIET IN YOUNGER ADULTS Emerging research suggests that the favorable effect of the Mediterranean diet on cognitive functioning may not only apply to the elderly or those with cognitive impairment. A recent pilot study by McMillan et al. [54] investigated the effects of a 10-day Mediterranean diet intervention on mood and cognitive performance in healthy young women. Using a between-subjects design, the study found that, in comparison to participants who did not change their diet, participants following the Mediterranean diet had markedly improved reaction times for spatial working memory tasks. McMillan et al. suggested that the improvements in reaction time may be indicative of improvements in sustained attention. These findings are interesting in that they indicate another shorter-term mechanism for cognitive improvement than the previously suggested changes in cardiovascular health, oxidative stress, and inflammation, which would occur over a longer time frame [55]. Mood was another parameter improved in the study conducted by McMillan et al. [54]. Compared with the control (no change) group, participants who adhered to the Mediterranean diet for the 10 days had significant improvements in self-reported measures of vigor, contentment, and alertness, as well as decreased levels of depression, anxiety, and confusion. These findings were broadly consistent with previous epidemiological studies that found the prevalence of depression to be lower in Mediterranean countries, which has been attributed to the Mediterranean eating pattern [56]. The influence of the Mediterranean diet on cognition and mood over a period of days rather than years requires further investigation. The study of blood nutrients, oxidative stress, and inflammatory biomarkers in such study designs may be informative in terms of understanding the mechanisms that underlie any changes on behavioral indices. Components Mechanisms Polyphenols and antioxidants Monounsaturated fatty acids Reduce inflammation Cognitive health Reduce oxidative stress Reduce cardiovascular risk factors Vitamins and minerals Improve metabolic markers FIGURE 2 Components of the Mediterranean diet and potential mechanisms that influence cognitive health. Mediterranean Diet Cognitive Health Chapter 8 87 CONCLUSIONS AND FUTURE DIRECTIONS There is accumulating evidence that following a Mediterranean diet exerts positive effects on cardiovascular and brain health. Most studies have been conducted in older adults (>65 years old); therefore the evidence is strongest for the elderly. At a population level this may translate to a lower rate of cognitive impairment and dementia among individuals who follow this dietary pattern. In terms of the Western diet, it has been suggested that the high content of saturated fat may adversely affect the brain by damaging regions relevant to memory, increasing oxidative stress, promoting neuroinflammation, and compromising the integrity of the blood–brain barrier [18]. To a certain extent, positive findings regarding the Mediterranean diet may be equally reflective of the harmful effects of following a Western diet, which is high in processed foods and refined carbohydrates. This premise requires further investigation. Following from the PREDIMED intervention trial [39], there is now a need to extend research findings from epidemiological trials to randomized controlled trials to fully test the hypothesis that the Mediterranean diet can protect against dementia and cognitive decline. It may be necessary to conduct multiple randomized controlled trials among different geographic populations and age groups to gain a comprehensive picture of the cognitive benefits of the Mediterranean diet (Figure 2). SUMMARY POINTS l l l l l There is growing evidence that following a Mediterranean diet can benefit cardiovascular and brain health. At a population level, those who follow this dietary pattern seem to have a lower rate of cognitive impairment and dementia. A Mediterranean diet combined with exercise may exert maximal benefits on brain and cognitive health. Randomized controlled trials are required to determine the potential for the Mediterranean diet to protect against dementia. Future research should examine whether the Mediterranean diet can enhance cognition among both younger and older cohorts. REFERENCES [1] Keys A, Menotti A, Karvonen MJ, Aravanis C, Blackburn H, Buzina R, et al. The diet and 15-year death rate in the Seven Countries Study. 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Eur J Clin Nutr 2008;62(7):892–7. [51] Smith PJ, Blumenthal JA, Hoffman BM, Cooper H, Strauman TA, Welsh-Bohmer K, et al. Aerobic exercise and neurocognitive performance: a meta-analytic review of randomized controlled trials. Psychosom Med 2010;72(3):239–52. [52] Cotman CW, Berchtold NC, Christie LA. Exercise builds brain health: key roles of growth factor cascades and inflammation. Trends Neurosci 2007;30(9):464–72. [53] Ahlskog JE, Geda YE, Graff-Radford NR, Petersen RC. Physical exercise as a preventive or disease-modifying treatment of dementia and brain aging. Mayo Clin Proc 2011;86(9):876–84. [54] McMillan L, Owen L, Kras M, Scholey A. Behavioural effects of a 10-day Mediterranean diet. Results from a pilot study evaluating mood and cognitive performance. Appetite 2011;56(1):143–7. [55] Féart C, Samieri C, Allès B, Barberger-Gateau P. Potential benefits of adherence to the Mediterranean diet on cognitive health. Proc Nutr Soc 2013;72 (1):140–52. [56] Sánchez-Villegas A, Delgado-Rodrı́guez M, Alonso A, Schlatter J, Lahortiga F, Serra-Majem L, et al. Association of the Mediterranean dietary pattern with the incidence of depression: the Seguimiento Universidad de Navarra/University of Navarra follow-up (SUN) cohort. Arch Gen Psychiatry 2009;66(10):1090–8. Chapter 9 Mediterranean Diet and Cardiovascular Disease: An Overview of Recent Evidence Dimitra Karageorgou, MSc1, Renata Micha, PhD1,2 and Antonis Zampelas, PhD1 1 Agricultural University of Athens, Athens, Greece. 2 Harvard School of Public Health, Boston, MA, USA. ABBREVIATIONS CHD CI CVD EPIC HALE HDL HOMA-IR HR IL-1 IL-6 IL-7 IL-18 LDL MDP MDS Med-DQI MeDiet-PREDIMED MS MSDPS NCEP1 NOMAS PREDIMED RCT RR SUN coronary heart disease confidence interval cardiovascular disease European Prospective Investigation into Cancer and Nutrition The Healthy Ageing: a Longitudinal study in Europe high-density lipoprotein homeostatic model assessment insulin resistance hazard ratio interleukin 1 interleukin 6 interleukin 8 interleukin 18 low-density lipoprotein Mediterranean Dietary Pattern adherence index Mediterranean Diet Score Mediterranean Dietary Quality Index Mediterranean food pattern PREDIMED study Mediterranean Score Mediterranean Style Dietary Pattern Score National Cholesterol Education Program—step 1 Northern Manhattan Study PREvención con DIeta MEDiterránea randomized controlled trial relative risk Seguimiento Universidad de Navarra INTRODUCTION Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide, and, according to the World Health Organization, the annual number of deaths caused by CVD is expected to increase from 17 million in 2008 to 25 million in 2030. However, almost 80% of the CVD burden can be attributed to certain lifestyle habits, such as poor diet, physical inactivity, smoking, and alcohol consumption, the modification of which could lead to substantial reduction of premature heart disease and stroke [1]. These lifestyle habits cause certain intermediate metabolic and physiological changes in, for example, blood pressure, lipids, glucose, and body weight, which, in turn—and depending on the direction of these effects—can either cause or protect against the development of CVD [2]. Several foods have been found to be associated with CVD; increased consumption of fruits, vegetables, and olive oil and limited consumption of processed meat have been shown to be protective against CVD [3–5]. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 91 92 SECTION 1 The Mediterranean Diet: Concepts and General Aspects Although nutrient-based research is essential to elucidate potential underlying mechanisms, food-based research that takes into account the synergistic and antagonistic functions of nutrients could be more important in the modern era to understanding and reducing the disease burden associated with suboptimal dietary habits [6,7]. As a result, there has been increasing interest regarding the association between various diet patterns and individual nutrients and the effect they have on the prevention and development of chronic diseases and the associated mortality [8]. Among the different dietary patterns, such as the low-fat diet, the low-carbohydrate diet, the Dietary Approaches to Stop Hypertension diet, and the Mediterranean diet, there has been a growing interest over the past five decades in the latter as an optimal diet [9]. The global scientific interest regarding the beneficial effects of the Mediterranean diet on cardiovascular health was initially stimulated by the results of the Seven Countries Study, which showed that the incident of coronary heart disease (CHD) was significantly lower in the Greek population residing in Crete compared with other European populations [10]. Indeed, the Mediterranean diet has been shown to be associated with a decrease in all-cause mortality and higher longevity mainly because it has been found to have an inverse association with the development of obesity and chronic diseases such as diabetes, CVD, and cancer [2,4,9,11]. Thus, while Keys provided the hypothesis for the protective role of the Mediterranean diet in CVD, since then better tools, such as prospective cohorts and clinical trials, have been developed to examine this association, prove the causality of the relationship, and investigate the implicated mechanisms [5]. The Mediterranean diet has been repeatedly found to exert an atheroprotective role, whereas adherence to it has been associated with CVD risk reduction and with the improvement of attributing risk factors [2,9]. The purpose of this review is to investigate the effect of the Mediterranean diet on CVD, based on up-to-date evidence from prospective cohort studies and randomized controlled trials (RCTs), which, by design, provide results with higher validity when compared with other study designs (Figure 1). To evaluate and establish a causal relationship, the results of prospective cohort studies on primary prevention are compared with the results of RCTs. Because of the limited number of RCTs, though, RCTs of secondary prevention also are included. The end points of interest are both fatal and nonfatal disease outcomes (total CVD, CHD, stroke). Only results from RCTs were considered in the evaluation of intermediate metabolic factors. THE MEDITERRANEAN DIET The Mediterranean diet, first presented by Keys et al. [10], is based on the eating habits demonstrated in areas in the Mediterranean basin, such as Crete and, similarly, the rest of Greece, southern Italy, Spain, and France, during the early 1960s [10]. It is characterized by high consumption of fruits, vegetables, whole-grain cereals, legumes, nuts, and seeds; moderate consumption of fish, poultry, dairy products, and wine; and limited consumption of red meats and sweets [4]. Olive oil, which is high in monounsaturated fatty acids (MUFAs; i.e., oleic acid), is the main source of fat and a distinctive trait of the Mediterranean diet [13]. Originally, in addition to the dietary pattern, the lifestyle and the cultural characteristics of the areas in the Mediterranean basin, comprising physical activity and adequate rest throughout the day, contribute to the health benefits observed [14]. Almost 50 years later, however, the Mediterranean diet has not remained the same [15]. Strength of evidence Randomized trials of disease outcomes Prospective cohorts of disease outcomes Randomized trials of physiologic measures / risk factors Retrospective case-control studies of disease outcomes Animal studies Ecologic studies Case series/case reports Prevalence studies FIGURE 1 Study types and strength of evidence. The image shows the hierarchy of quality and strength of evidence of different study designs for evaluating causation of how dietary habits affect chronic disease. Randomized controlled trials (RCTs) along with prospective cohort studies of disease outcomes provide the best evidence. Adapted from Ref. [12]. Mediterranean Diet and Cardiovascular Disease Chapter 9 93 Westernization has a great impact on the food habits of Mediterranean countries, which has led to the increased consumption of refined carbohydrates, red meat, and saturated fat, whereas the consumption of whole-grain cereals and legumes has decreased [15]. Similar to all dietary patterns, the Mediterranean diet can be evaluated using two approaches: the a priori approach, which is the most commonly applied, and the a posteriori approach [15]. The a priori approach is a hypothesis-oriented approach based on the already known scientific evidence. This means that adherence to the Mediterranean diet is assessed using indices based on the eating habits already known to characterize this dietary pattern and not by taking into account the current food habits of the population [16]. This approach represents more efficiently the complexity of the diet but is also based on the assumption that all nutrients are of equal importance [15]. A variety of tools, indices, and scales have been developed and used as measures of adherence to the Mediterranean diet in large and smaller epidemiological studies focusing on the identification of the overall dietary pattern, but there is not yet a direct method to assess adherence [6]. The Mediterranean Diet Score (MDS) [17], Mediterranean Score (MS) [18], Mediterranean Dietary Quality Index (Med-DQI) [19], Mediterranean Dietary Pattern adherence index (MDP) [20], Mediterranean Style Dietary Pattern Score (MSDPS) [21], and the Mediterranean food pattern PREDIMED Study (MeDiet-PREDIMED) [22] are some of the indices used. However, there is an absence of common criteria defining the Mediterranean diet; hence each index is based on the available data and the study objectives [6]. One of the most widely used indices is the MDS, developed by Trichopoulou et al. [23] in 1995 and modified a few years later [24]. The score ranges from 0 to 9 and is based on the categorization of foods into food groups (beneficial effect groups: fruits, vegetables, legumes, cereals, fish; harmful groups: meat and dairy products). To determine the score for each food group, the sex-specific consumption median is used as the cutoff (scoring system: one point for each of the beneficial food groups and for the monounsaturated-to-saturated lipids ratio if the consumption is above the median; one point for each of the other two food groups if the consumption is below the median; and one point for ethanol daily consumption between 10 and 50 g for men and 5 and 25 g for women) [24]. Olive oil consumption, which is difficult to estimate in observational studies, is evaluated as part of the ratio of monounsaturated fat to saturated fat [24], which is a common way of evaluating it in most studies [8]. The MDS was modified in 2005 for non-Mediterranean populations to use it and the monounsaturated-to-saturated lipids ratio was replaced by the ratio of monounsaturated and polyunsaturated to saturated lipids [17]. Conversely, a second approach used to assess dietary patterns is the a posteriori approach, a result of factorial analysis to define the food patterns that reflect the existing habits of a population [15]. Therefore, when the Mediterranean diet is defined a priori, it responds to the food pattern described for the populations of Mediterranean countries in the 1960s, whereas the a posteriori approach reflects current habits. THE MEDITERRANEAN DIET AND CVD Total CVD Prospective Studies Several prospective cohort studies have investigated the potential positive association of the Mediterranean diet with CVD incidence and CVD mortality (Table 1). Sofi et al. [9] published a meta-analysis of prospective cohort studies in 2010, in which the a priori approach was used to assess adherence to the Mediterranean diet and CVD incidence. The meta-analysis included seven studies with 534,064 subjects and 8739 deaths or incidence cases (Figure 2). The outcomes included mortality from and/or incidence of CVDs. The results showed that a greater level of adherence to the Mediterranean diet was related to significantly higher protection against CVD; in particular, it was revealed that a two-point increase in the MDS [24] was associated with a 10% risk reduction (relative risk 0.90, 95% confidence interval [CI] 0.87–0.93) of death from and incidence of total CVD [9]. Following the meta-analysis by Sofi et al. [9], a few more researchers published their results on the beneficial association of the Mediterranean dietary pattern with CVD incidence. The Seguimiento Universidad de Navarra (SUN) study is a prospective cohort study with over 13,000 participants who were healthy at baseline; an inverse association between the Mediterranean diet and total CVD incidence was reported after almost 5 years of follow-up [15]. The end point of this study was a combined measure of incident cardiovascular death, acute coronary syndromes, revascularization procedures, or fatal or nonfatal stroke. They used the MDS to assess adherence to the traditional Mediterranean diet. The results showed that for those participants who exerted the highest adherence (total score 7) a 59% significantly lower CVD risk (hazard ratio [HR] 0.41, 95% CI 0.18–0.95) was reported compared with those with the lowest adherence score (total score 2). However, the continuous dose-response association was not reported to be significant; for every two-point increment in the MDS the adjusted HR for total CVD was estimated at 0.80 (95% CI 0.62–1.02) [15]. Results from 34,708 TABLE 1 Characteristics of the Prospective Cohort Studies and Randomized Controlled Trials Investigating Adherence to the Mediterranean Diet and Cardiovascular Disease Outcomes Study Sample Size Mediterranean Diet Adherence Index End Points Unit Hazard Ratio (95% CIa) Total CVD incidence (fatal, nonfatal) Two-point increment 0.90 (0.87–0.93)b Selection criterion: adjustment for potential confounders Studies with adjustment for confounders that include demographic, anthropometric, and traditional risk factors were considered of high quality Adjustment Prospective studies Meta-analysis of seven prospective cohort studies, Sofi [9] 534,064 The SUN Study, MartinezGonzalez [15] 13,609 MDS c Post hoc analysis “Post hoc Mediterranean diet”d Total CVD incidence (fatal, nonfatal) CHD incidence Total CVD incidence High 7 versus low 2 score two-point increment High 7 versus low 2 score two-point increment Upper versus lower quintile 0.41 (0.18–0.95) 0.80 (0.62–1.02) 0.42 (0.16–1.11) 0.74 (0.55–0.99) 1.14 (0.54–2.39) Age, sex, energy intake, CHD history, smoking, physical activity, body mass index (BMI) at baseline, history of hypertension or medication for hypertension, use of aspirin, diabetes and dyslipidemia at baseline The Dutch EPIC, Hoevenaar [25] 34,708 Modified MDSc Total CVD incidence (fatal, nonfatal) Total CVD incidence (fatal) Composite CVD incidence (fatal CVD, nonfatal myocardial infarction and nonfatal stroke) Myocardial infarction incidence Stroke incidence High 7 versus low 2 score Two-point increment High 7 versus low 2 score Two-point increment High 7 versus low 2 score Two-point increment High 7 versus low 2 score Two-point increment High 7 versus low 2 score Two-point increment 0.84 (0.75–0.96) 0.95 (0.91–0.98) 0.44 (0.30–0.66) 0.78 (0.69–0.88) 0.65 (0.53–0.80) 0.85 (0.80–0.91) 0.70 (0.54–0.92) 0.86 (0.79–0.93) 0.70 (0.47–1.05) 0.88 (0.78–1.00) Age, sex, smoking , physical activity, total energy intake, education level NOMAS, Gardener [26] 2568 MDSc Total CVD incidence (fatal, nonfatal) Myocardial infarction incidence Ischemic stroke incidence High 6 versus low 2 score One-point increment High 6 versus low 2 score One-point increment High 6 versus low 2 score One-point increment 0.75 (0.56–0.99) 0.94 (0.89–1.00) 0.61 (0.35–1.04) 0.93 (0.83–1.04) 0.98 (0.58–1.65) 0.98 (0.89–1.08) Education level, moderate-toheavy physical activity, average total daily kilocalorie consumption, and smoking Knoops [27] 2339 Based on MDS indexc CHD incidence (fatal) High 4 versus low <4 score 0.61 (0.43–0.88) Age, sex, education level, body mass index, dietary and lifestyle factors and study MDSc CHD incidence (fatal) CHD incidence (fatal, nonfatal) High 6 versus low 3 score two-point increment High 6 versus low 3 score Two-point increment 0.54 (0.37–0.81) 0.78 (0.66–0.92) 0.82 (0.66–1.02) 0.92 (0.84–1.02) Sex, age, BMI, height, physical activity, education level, energy intake, smoking, blood pressure The Greek EPIC, Dilis [28] 23,929 The Spanish EPIC, Buckland [29] 41,078 rMDSc CHD incidence (fatal, nonfatal) High 11 versus low 6 score one-point increment 0.60 (0.47–0.77) 0.94 (0.91–0.97) Sex, age, education level, physical activity, BMI, smoking, diabetes, hypertension, hyperlipidemia, energy intake, study center The Spanish EPIC, GuallarCastillon [16] 40,757 a posteriori analysis “Evolved Mediterranean diet”e CHD incidence (fatal, nonfatal) Upper versus lower quintile 0.73 (0.57–0.94) Age, sex, energy intake, BMI, waist circumference, education level, smoking, physical activity, diabetes, hypertension, hypercholesterolemia, cancer, oral contraceptives, menopausal status, hormone replacement therapy, study center Nurses Health Study, Fung [30] 74,886 alternate MDSc CHD incidence (fatal) CHD incidence (nonfatal) Stroke incidence (fatal, nonfatal) Stroke incidence (fatal) Stroke incidence (nonfatal) Upper versus lower Upper versus lower Upper versus lower Upper versus lower Upper versus lower 0.58 (0.45–0.75)b 0.78 (0.66–0.93)b 0.87 (0.73–1.02)b 0.69 (0.44–1.07)b 0.90 (0.75–1.08)b Age, smoking, BMI, menopausal status, postmenopausal hormone therapy, energy intake, multivitamin intake, alcohol, family history, physical activity, aspirin usage Me-Diet PREDIMED c Total CVD incidence (fatal, nonfatal) Total CVD incidence (fatal) Myocardial infarction incidence Stroke incidence Olive oil group versus Control group Nuts group versus Control Group Combined effect versus Control group Olive oil group versus Control group Nuts group versus Control Group Combined effect versus Control group Olive oil group versus Control group Nuts group versus Control Group Combined effect versus Control group Olive oil group versus Control group Nuts group versus Control Group 0.70 (0.54–0.92) 0.72 (0.54–0.96) 0.71 (0.56–0.90) 0.69 (0.41–1.16) 1.01 (0.61–1.66) 0.83 (0.54–1.29) 0.80 (0.51–1.26) 0.74 (0.46–1.19) 0.77 (0.52–1.15) 0.67 (0.46–0.98) 0.54 (0.35–0.84) 0.61 (0.44–0.86) Age, sex, smoking, BMI, waist/ height ratio, baseline hypertension, baseline dyslipidemia, baseline diabetes, family history of premature CHD, study center quintile quintile quintile quintile quintile Randomized controlled trials The PREDIMED Study, Estruch [3] 7447 High-risk population Continued TABLE 1 Characteristics of the Prospective Cohort Studies and Randomized Controlled Trials Investigating Adherence to the Mediterranean Diet and Cardiovascular Disease Outcomes—cont’d Study Sample Size Mediterranean Diet Adherence Index End Points Unit Hazard Ratio (95% CIa) Adjustment Combined effect versus Control group Lyon Diet Heart Study, De Lorgeril [31] IndoMediterranean Diet Heart Study, Singh [32] a 423 1000 Dietary survey/ participant visit Composite CVD prevalence 1 (cardiac death and nonfatal myocardial infarction) Composite CVD prevalence 2 (the preceding plus major secondary end points, like unstable angina, stroke, heart failure etc.) Composite CVD prevalence 3 (the preceding plus minor events requiring hospital admission) Mediterranean diet group versus Western diet group Mediterranean diet group versus Western diet group Mediterranean diet group versus Western diet group 0.28 (0.15–0.53)b 0.33 (0.21–0.52)b 0.53 (0.38–0.74)b Age, sex, smoking, cholesterol, systolic blood pressure, leucocyte count, aspirin use Food record Total CVD (fatal, nonfatal) Myocardial infarction (fatal) Myocardial infarction (nonfatal) Indo-Mediterranean diet group versus NCEP1 diet group Indo-Mediterranean diet group versus NCEP1 diet group Indo-Mediterranean diet group versus NCEP1 diet group 0.48 (0.33–0.71) f 0.67 (0.31–1.42) f 0.47 (0.28–0.79) f Age, gender, BMI, cholesterol and blood pressure CI, confidence interval. bRelative risk. cMDS ranges from 0 to 9: one point for each of the beneficial food groups (fruits, vegetables, legumes, cereals, fish) and for the monounsaturated-to-saturated lipids ratio if the consumption is above the sex-specific median; one point for each of the harmful food groups (meat and dairy products) if the consumption is below the sex-specific median; and one point for daily consumption of ethanol between 10 and 50 g for men and 5 and 25 g for women. The modified MDS replaces monounsaturated lipids with the sum of monounsaturated and polyunsaturated lipids in the numerator of the lipid ratio. The index Knoops et al. used ranges from 0 to 8, and it contains all MDS components except for alcohol. The alternate MDS (score range of 0–9) is based on the MDS; its scoring system is as follows: one point for above-median consumption of vegetables (excluding potatoes), fruits, nuts, whole grains, legumes, fish, and monounsaturated-to-saturated fat ratio; one point for below-median consumption of red and processed meats; one point for alcohol intake between 5 and 15 g/d. The Spanish EPIC study used a revised MDS, with scores ranging from 0 to 18, including 9 components, all of which (except alcohol) were expressed in energy per 1000 kcal. The Me-Diet PREDIMED is a 14-item screener evaluating the consumption frequency of olive oil, vegetables, fruits, red and processed meat, butter/margarine, sodas, legumes, fish/seafood, sweets, nuts, white meat instead of red, and sofrito. dThe “post hoc Mediterranean diet” includes vegetables, fruits, fish, poultry, whole-grain cereals, nuts and seeds, olive oil, and legumes. eThe “Evolved Mediterranean diet” does not include legumes, processed and unprocessed red meat, or wine. Furthermore, foods that score negatively in the MDS, such as fat-free dairy products and poultry, score positively in the evolved Mediterranean diet. fRate ratio. Mediterranean Diet and Cardiovascular Disease Chapter 9 Study Relative risk (95% CI) Weight (%) Relative risk (95% CI) Trichopoulou [23] 0.9 0.67 (0.47, 0.95) Knoops [27] 8.3 0.84 (0.76, 0.94) Mitrou (2007) (M) [40] 26.1 0.92 (0.89, 0.96) Mitrou (2007) (F) [40] 17.9 0.93 (0.88, 0.99) Fung (CHD) [30] 19.1 0.87 (0.82, 0.92) 1.8 0.80 (0.62, 1.03) Buckland [29] 15.6 0.95 (0.88, 1.01) Martinez-Gonzalez [15] 10.4 0.89 (0.81, 0.97) 100 0.90 (0.87, 0.93) Fung (Stroke) [30] Total (95% CI) 0.4 0.6 0.8 1 1.2 97 1.4 Reduced risk Increased risk FIGURE 2 The association between the Mediterranean diet and the risk of cardiovascular disease. This Forest plot shows the association between a twopoint increase in the score of adherence to the Mediterranean diet and the risk of mortality from or incidence of cardiovascular diseases. The center of each square indicates the relative risk of the study, and the horizontal lines indicate 95% CIs. The area of the square is proportional to the amount of information from the study. The diamond indicates pooled estimates. CHD, coronary heart disease. Adapted from Ref. [9]. participants in the Dutch European Prospective Investigation into Cancer and Nutrition (EPIC) study without CVD at baseline also suggest a protective role for the Mediterranean diet against CVD [25]. Using as end points fatal CVD, total CVD, and composite CVD (fatal CVD, nonfatal myocardial infarction, and nonfatal stroke) and the modified MDS to assess adherence to the dietary pattern, Trichopoulou et al. [17] showed that, after multivariate adjustment, greater adherence to the Mediterranean diet was associated with a 16% lower incidence of total CVD (HR 0.84, 95% CI 0.75–0.96), a 56% lower incidence of fatal CVD (HR 0.44, 95% CI 0.30–0.66), and a 35% lower incidence of composite CVD (HR 0.65, 95% CI 0.53–0.80) in comparison with nonadherence to very low adherence. They also showed the same association for dose-respondent assessment after adjustment, meaning that every two-point increase in MDS was associated with an HR of 0.78 (95% CI 0.69–0.88) for fatal CVD, 0.95 (95% CI 0.91–0.98) for total CVD, and 0.85 (95% CI 0.80–0.91) for composite CVD [25]. The Northern Manhattan Study (NOMAS), a prospective cohort study that investigated the relation between the Mediterranean diet and vascular events in a multiethnic population group with a 9-year follow-up, also showed an association between high adherence to the Mediterranean diet and a 25% reduction in risk of total CVD (adjusted HR 0.75, 95% CI 0.56–0.99), defined as a composite of incident ischemic stroke, myocardial infarction, and vascular death.[26] As expected, after further adjustment for potential mediators in the causal pathway for CVD development, in addition to sociodemographic and dietary factors, this association was no longer significant (HR 0.80, 95% CI 0.60–1.06) [26]. The above studies were based on an a priori approach to the Mediterranean diet, but to examine whether it manifests the same associations with cardiovascular health as it is currently followed, an a posteriori approach is necessary. The SUN study carried out a sensitivity analysis of the data, and in addition to investigating Mediterranean diet adherence with the help of MDS, they also performed a post hoc analysis [15]. This analysis revealed a “post hoc Mediterranean pattern” that showed no association between this dietary pattern and CVD incidence (HR 1.14, 95% CI 0.54–2.39) and a “Westernized pattern,” which was associated with a higher risk for CVD (HR 2.10, 95% CI 1.06–4.18). A limitation of this study, though, is that the sample included only university graduates; this could have interfered with some of the dietary habits observed in the post hoc Mediterranean pattern [15]. In conclusion, based on the results of the prospective cohort studies, there seems to be a protective association between the Mediterranean diet and CVD incidence and CVD mortality. Clinical Trials To our knowledge only two clinical trials have assessed the effect of the Mediterranean diet on total CVD as a disease outcome: one is about primary prevention, whereas the other is about secondary prevention. The Prevención con Dieta Mediterránea (PREDIMED) study assesses the effect of increased olive oil consumption within the frame of the Mediterranean diet on CVD [3]. The PREDIMED study is a large RCT that aims to assess the effect of the Mediterranean diet on the primary prevention of CVD based on three very distinctive cardiovascular end points: cardiovascular death, myocardial infarction, and stroke [3]. The sample in the trial consisted of men and women who had not developed CVD at baseline 98 SECTION 1 The Mediterranean Diet: Concepts and General Aspects but had either type 2 diabetes or at least three of the following cardiovascular risk factors: current smoking, hypertension, high low-density lipoprotein (LDL) cholesterol or lipid-lowering therapy, low high-density lipoprotein (HDL) cholesterol, overweight/obesity, and family history of premature CHD. According to the most recent publication from this research team, adherence to the Mediterranean diet, supplemented with either extra-virgin olive oil (mean consumption of extra-virgin olive oil at the end of the trial was 50 g/day) or nuts (mean consumption of nuts at the end of the trial was 30 g/day), and with no energy restriction compared to a low-fat control diet, resulted in the reduction of risk for the composite of cardiovascular death, myocardial infarction, and stroke (HR 0.70, 95% CI 0.54–0.92 for the olive oil group; HR 0.72, 95% CI 0.54–0.96 for the nuts group) [3]. There was no significant risk reduction regarding CVD mortality alone for Mediterranean diets combined (HR 0.83, 95% CI 0.54–1.29). A limitation of the PREDIMED trial, however, is that the control group was not intensively attended to and guided through the intervention for at least the first 3 years of the study, which might have resulted in a positive bias for the Mediterranean diet, but the differences between the initial period of the study and the one after the adjustment of the protocol were not significant regarding the aforementioned benefit [3]. Preceding PREDIMED, the Lyon Diet Heart Study was a randomized clinical trial that aimed to assess whether the Mediterranean Diet had a positive impact on recurrence rate after a first myocardial infarction [31]. In contrast with the PREDIMED study, the patients in the Lyon Diet Heart Study were assigned to two groups: one group was guided through following the Mediterranean diet, and the other group (control) received no dietary recommendations except for those commonly given by their doctors. The end points used were (1) cardiac death and nonfatal myocardial infarction, (2) cardiac death and nonfatal myocardial infarction plus major secondary end points (unstable angina, stroke, heart failure, etc.); and (3) cardiac death and nonfatal myocardial infarction, major secondary end points, and minor events requiring hospital admission. The Lyon Diet Heart Study also confirmed the cardioprotective effect of the Mediterranean diet, with adjusted HRs ranging from 0.28 to 0.53 between the two groups (HR 0.28, 95% CI 0.15–0.53 for the first end point; HR 0.33, 95% CI 0.21–0.52 for the second end point; and HR 0.53, 95% CI 0.38–0.74 for the third end point) [31]. Finally, an RCT that included both high-risk subjects and patients with a previous CHD incident assessed the effect of an Indo-Mediterranean diet on CHD development and progress and total CVD mortality compared with the dietary pattern recommended by the National Cholesterol Education Program—step 1 (NCEP1) [32]. The assessed components of the Indo-Mediterranean diet were fruits, vegetables, whole grains, walnuts or almonds, and mustard seed or soybean oil. The results showed that the risk of total CVD incidence (fatal and nonfatal) was reduced by 52% in the intervention group (rate ratio 0.48, 95% CI 0.33–0.71) [32]. Overall Prospective cohort studies conclusively show a protective association between the Mediterranean diet and total CVD incidence. Clinical trials, although limited in number, confirm this association, and their results show that the Mediterranean diet leads to the reduction of total CVD incidence. Taking these data together, the Mediterranean diet seems to have a beneficial association with total CVD, although there is no certain conclusion regarding causality (Figure 3). Coronary Heart Disease Prospective Studies Similar to total CVD, there are several cohort studies that have reported the beneficial effect of the Mediterranean dietary pattern on CHD [33] (Table 1). The SUN study revealed an inverse association between the Mediterranean diet and CHD incidents after almost 5 years of follow-up [15]. After multivariate adjustment, a higher MDS was associated with a lower risk of a CHD incident (HR 0.42, 95% CI 0.16–1.11) compared with those with the lowest score, whereas it was reported that a two-point increase in the MDS was associated with a 26% reduction of risk of a CHD incident (HR 0.74, 95% CI 0.55–0.99) [15]. Data from the The Healthy Ageing: A Longitudinal study in Europe (HALE) project showed that, among a sample of 2339 apparently healthy elderly participants from several European countries, higher adherence to the Mediterranean diet was associated with a 39% reduced risk of CHD mortality during a 10-year follow-up period (adjusted HR 0.61, 95% CI 0.43–0.88) [27]. The Dutch EPIC, which included over 34,000 participants without CVD at baseline, also showed that a greater modified MD adherence score was associated with a 30% lower incidence of myocardial infarction compared with nonadherence to very low adherence (adjusted HR 0.70, 95% CI 0.54–0.92). The protective association also was shown in the dose-response assessment: every two-point increase in the modified MDS was associated with 14% reduced risk of myocardial infarction incidence (adjusted HR 0.86, 95% CI 0.79–0.93) [25]. Moreover, data from a 10-year median follow-up of over 23,000 participants free of CHD, diabetes, and cancer at baseline in the Greek EPIC showed similar results [28]. The Greek EPIC investigated the association of the Mediterranean diet with CHD incidence (myocardial Mediterranean Diet and Cardiovascular Disease Chapter 9 99 FIGURE 3 The association between the Mediterranean diet and total cardiovascular disease incidence. The plot shows hazard ratios and confidence intervals (CIs) of prospective cohort studies and clinical trials (primary prevention) for total cardiovascular disease incidence. The center of each rhombus indicates the relative risk of the study, and the horizontal lines indicate 95% CIs (their values are given on the right side of the plot). *Relative risk. 1 Combined effect of the two intervention groups. 2Composite of fatal CVD, nonfatal myocardial infarction, and nonfatal stroke. infarction, angina, and other CHD) and CHD mortality. It revealed that, after multivariate adjustment, greater adherence to the Mediterranean diet was associated with 46% lower CHD mortality (HR 0.54 95% CI 0.37–0.81), whereas every two-point increase in the MDS was associated with 22% lower CHD mortality (HR 0.78, 95% CI 0.66–0.92), showing a continuous association. However, although an inverse association was found between high and low adherence to the Mediterranean diet and CHD incidence, this association seemed to be weaker than the one with CHD mortality (HR 0.82, 95% CI 0.66–1.02) [28]. A smaller multiethnic prospective cohort study of 2568 participants did not find significant association with incident myocardial infarction (HR 0.61, 95% CI 0.35–1.04), despite showing an association between adherence to the Mediterranean diet and CVD mortality [26]. The results from the Nurse’s Health Study and the 20-year follow-up of 74,886 women 38–63 years old revealed that a higher degree of adherence to the Mediterranean dietary pattern, measured using an alternate MDS, was associated with lower risk of combined fatal and nonfatal CHD (adjusted HR 0.71, 95% CI 0.62–0.82) [30]. This beneficial effect of the Mediterranean diet was observed despite the fact that the major source of monounsaturated fat in the US population came mainly from beef and other types of meat, whereas the contribution of olive oil, which is the primary source of MUFAs in Mediterranean countries, was limited to 10% of all MUFA intake [30]. Finally, the Spanish EPIC realized two different analyses of the data from over 40,000 participants: an a priori approach of the adherence to the Mediterranean diet [29] and an a posteriori investigation of the association of current dietary patterns and CHD [16]. According to the a priori assessment, in which a modified version of the MDS was used to estimate conformity to the Mediterranean diet, higher compared with lower adherence to the Mediterranean diet was significantly associated with 40% reduced risk of CHD after multivariate adjustment (HR 0.60, 95% CI 0.47–0.77) [29]. In the analysis by Guallar-Castillon et al. [16], the “evolved Mediterranean diet” that was revealed, characterized by consumption of plant-based foods and olive oil, is still associated with a lower risk of CHD incidents (HR 0.73, 95% CI 0.57–0.94) among the participants with the highest adherence compared with those with the lowest. Clinical Trials As mentioned earlier, the randomized clinical trial PREDIMED showed that a Mediterranean dietary pattern, supplemented with extra-virgin olive oil or nuts, leads to reduced risk for the composite of cardiovascular death, myocardial infarction, and stroke [3]. However, when the effect on myocardial infarction incidence was examined separately, the comparison 100 SECTION 1 The Mediterranean Diet: Concepts and General Aspects between the two Mediterranean groups and the control group did not reach statistical significance (HR 0.80, 95% CI 0.51– 1.26 for the olive oil group; HR 0.74, 95% CI 0.46–1.19 for the nuts group). On the contrary, Singh et al. [32] showed a significant reduction of stroke incidence in the intervention group following guidelines of an Indo-Mediterranean diet compared with the control group following NCEP1. The reduction was significant only for nonfatal myocardial infarction (rate ratio 0.47, 95% CI 0.28–0.79) [32]. Overall In conclusion, there seems to be a protective association between the Mediterranean diet and the development and progress of CHD (Figure 4). However, more clinical trials are necessary for this association to be established with greater validity. Stroke Prospective Cohort Studies Few studies regarding the Mediterranean diet and its separate association with stroke have been performed (Table 1). The Dutch EPIC study showed an inverse association between adherence to the Mediterranean diet and stroke incidence, although it did not reach statistical significance (HR 0.70, 95% CI 0.47–1.05) [25]. However, the dose-response association was marginally significant; the two-point increment in the modified MDS was associated with 12% reduced risk for incident stroke (adjusted HR 0.88, 95% CI 0.78–1.00). The results from the Nurse’s Health Study and the 20-year follow-up of 74,886 women revealed that a higher degree of adherence to the Mediterranean dietary pattern was associated with lower risk of incident stroke (adjusted HR 0.87, 95% CI 0.73–1.02) [30]. In particular, for the women with higher adherence to the Mediterranean diet, the relative risk for the development stroke was 0.87 compared with the women with lower adherence. The NOMAS, however, failed to find a significant association between adherence to the Mediterranean diet and incident ischemic stroke (HR 0.98, 95% CI 0.58–1.65) [26]. It should be noted, however, that the sample of the FIGURE 4 The association between the Mediterranean diet and coronary heart disease incidence. The plot shows hazard ratios and confidence intervals (CIs) of prospective cohort studies and clinical trials (primary prevention) for coronary heart disease incidence. The center of each rhombus indicates the relative risk of the study, and the horizontal lines indicate 95% CIs (their values are given on the right side of the plot). *Relative risk. 1Combined effect of the two intervention groups. Mediterranean Diet and Cardiovascular Disease Chapter 9 101 FIGURE 5 The association between the Mediterranean diet and stroke incidence. The plot shows hazard ratios and confidence intervals (CIs) of prospective cohort studies and clinical trials (primary prevention) for stroke incidence. The center of each rhombus indicates the relative risk of the study, and the horizontal lines indicate 95% CIs (their values are given on the right side of the plot). *Relative risk. 1Combined effect of the two intervention groups. NOMAS was significantly smaller than the sample of the Nurse’s Health Study, but even in this large cohort, when they divided the incident stroke into ischemic and hemorrhagic, they found no significant association between the Mediterranean diet and ischemic stroke, either [26]. Therefore, based on the current evidence, it is difficult to draw a definite conclusion regarding the association between the Mediterranean diet and stroke incidence. Clinical Trials The PREDIMED study showed a significant reduction in risk for stroke incidence between either of the two Mediterranean diet groups and the control group (HR 0.67, 95% CI 0.46–0.98 for the olive oil group; HR 0.54, 95% CI 0.35–0.84 for the nuts group) [3]. Overall The inconsistent results of the limited studies regarding the association of the Mediterranean diet with stroke do not allow a clear opinion of whether the Mediterranean diet has a protective association with stroke incidence and mortality to be formed (Figure 5). POTENTIAL UNDERLYING MECHANISMS The pathophysiology of CVD is linked to inflammation as a key process in its development and outcome. The process of inflammation is activated by stimuli such as hypercholesterolemia, hyperglycemia, and hypertension [34]. The protective effects of the Mediterranean diet against the development of CVD have been mainly attributed to the antioxidant and anti-inflammatory properties of its individual components [7]. Hence there is a great interest regarding the investigation of the effect of the Mediterranean diet on biomarkers characterizing the progression of inflammation progress [35]. As a plant-based diet, the foods that comprise the basis of the Mediterranean diet undergo minimal processing; thus their nutrients are more effectively preserved, which makes the overall diet richer in antioxidants [4]. Moreover, given the fact that olive oil is the main fat source and the consumption of meat and other animal products is low to moderate, the amount of saturated and trans fats that Mediterranean diet provides is very low [4,14]. A meta-analysis of seven RCTs comparing the effect of the Mediterranean diet and low-fat diets on cardiovascular risk factors showed that after 2 years of follow-up the Mediterranean diet was more beneficial regarding changes in systolic 102 SECTION 1 The Mediterranean Diet: Concepts and General Aspects blood pressure (weighted mean difference 1.7 mmHg, 95% CI 3.3 to 0.05), diastolic blood pressure (weighted mean difference 1.5 mmHg, 95% CI 2.1 to 0.8), total cholesterol (weighted mean difference 7.4 mg/day, 95% CI 10.3 to 4.4), and high-sensitivity C-reactive protein (weighted mean difference 1.0 mg/L, 95% CI 1.5 to 0.5) [2]. Another meta-analysis by Kastorini et al. [4] regarding the effect of the Mediterranean diet on the metabolic syndrome, which seems to be a strong predictor of cardiovascular events, showed that adherence to this dietary pattern is optimal not only for the metabolic syndrome but also for its distinct components (insulin resistance, HDL cholesterol, triglycerides, and blood pressure levels) [36], all of which are also potential risk factors for the development of CVD [4]. In particular, regarding insulin resistance, the combined results of 10 trials with 1742 participants showed that for the Mediterranean diet groups the value of homeostatic model assessment-insulin resistance (HOMA-IR) was significantly reduced (mean difference between intervention groups 0.45, 95% CI 0.74 to 0.16) [4]. According to 29 clinical trials investigating the effect of adherence to the Mediterranean diet on HDL-cholesterol, the participants who were assigned to the Mediterranean diet had significantly higher levels of HDL-cholesterol compared with the control group participants (mean difference between intervention groups 1.17, 95% CI 0.38-1.96). The combined effect of the same trials regarding triglycerides also showed that adherence to the Mediterranean results in significantly lower triglyceride levels (mean difference between intervention groups 6.14, 95% CI 10.35 to 1.93). With regard to blood pressure, the analysis of 14 trials with 3060 subjects showed a significant association between adherence to the Mediterranean diet and lower levels of systolic blood pressure (mean difference between intervention groups 2.35 mmHg, 95% CI 3.51 to 1.18 mmHg) and diastolic blood pressure (mean difference between intervention groups 1.58 mmHg, 95% CI 2.02 to 1.13 mmHg). Moreover, the PREDIMED study showed that adherence to the Mediterranean diet resulted in significant lowering of cellular lipid levels and LDL oxidation [37]. It also resulted in the reduction of apolipoprotein B and the increase of apolipoprotein A-I concentrations, whose increased ratio is a predictor of CHD [37]. Following this, using part of the PREDIMED study sample, Urpi-Sarda et al. [34] showed that the Mediterranean groups had lower plasma concentrations of interleukin (IL)-6 (change in olive oil group: 0.23 ng/L, 95% CI 0.4 to 0.003; change in nuts group: 0.33 ng/L, 95% CI 0.6 to 0.1) and of tumor necrosis factor (TNF) R60 and TNFR80, the receptors through which TNF-a expresses its activity (change in TNFR60 in olive oil group: 0.2 mg/L, 95% CI 0.4 to 0.1; change in TNFR60 in nuts group: 0.2 mg/L, 95% CI 0.3 to 0.1; change in TNFR60 in olive oil group: 0.6 mg/L, 95% CI 1.1 to 0.3; and change in TNFR60 in nuts group: 0.4 mg/L, 95% CI 0.9 to 0.1) after 1 year of intervention [34]. An earlier and smaller randomized clinical trial by Esposito et al. [38], had already proved the beneficial effects of the Mediterranean diet on endothelial function and vascular inflammatory markers. In this double-blind trial, 180 patients with the metabolic syndrome were divided in two groups: the intervention group received thorough guidelines on following the Mediterranean diet, and the control group followed a normal diet. The results after 2 years revealed that the patients assigned to the Mediterranean diet manifested a significant reduction in the concentration of the serum high-sensitivity C-reactive protein (change: 1.0 mg/L, 95% CI 1.7 to 0.3), IL-6 (change: 0.6 pg/mL, 95% CI 1.1 to 0.1), IL-7 (change: 0.5 pg/mL, 95% CI 0.9 to 0.1), and IL-18 (change: 15 pg/mL, 95% CI 28 to 2) compared with the control group. Furthermore, their insulin resistance decreased (HOMA-IR change: 1.1, 95% CI 1.9 to 0.3), whereas the endothelial function score was significantly improved (change: +1.7, 95% CI 1.0–2.4). More important, less than half of the patients of the intervention group (40 of 90) had characteristics of the metabolic syndrome after the 2 years of the trial, whereas this was observed in 78 of the 90 patients in the control group, suggesting that the Mediterranean diet can be effective in decreasing the prevalence of the metabolic syndrome and the cardiovascular risk associated with it [38]. Similar results were confirmed by a more recent trial by Rallidis et al. [39], who investigated the effect of the Mediterranean diet on the improvement of endothelial function in 90 healthy adults with abdominal obesity, a feature known to be associated with increased cardiovascular and type 2 diabetes risk. In this study, they also considered the degree of adherence to the diet; hence, all participants received counseling on the Mediterranean diet, but the counseling for the intervention group was more intensive and their daily and weekly food plan was very specific. The significant increase in fat consumption in the intervention group (P < 0.001) was attributed to virgin olive oil and almonds, as in the PREDIMED study. In addition to significantly improving the endothelial function of the intervention group subjects, the Mediterranean diet resulted in significant diastolic blood pressure reduction (P ¼ 0.041), and it tended to cause an increase in insulin sensitivity (P ¼ 0.072) [39]. Overall According to the results of clinical trials, adherence to the Mediterranean diet results in the improvement of plasma lipid profile and the reduction of insulin resistance and blood pressure, and it seems to have a beneficial effect on inflammation and oxidation biomarkers. Mediterranean Diet and Cardiovascular Disease Chapter 9 103 SUMMARY POINTS l l l l Prospective cohort studies show a protective association between the Mediterranean diet and total CVD incidence. Clinical trials, although limited in number, confirm this association. The Mediterranean diet seems to have a protective association with CHD incidence, but, again, more clinical trials with CHD as an end point are needed to establish significant causality. The evidence of stroke is contradictory; prospective studies report inconsistent results and the PREDIMED study shows a significant protective effect of the Mediterranean diet on stroke incidence. The Mediterranean diet has a protective effect on intermediate factors and contributes to the improvement of lipid profile, blood pressure, inflammation, and oxidation biomarkers. REFERENCES [1] Alwan A. Global status report on noncommunicable diseases 2010. Zurich: World Health Organisation; 2010. [2] Nordmann AJ, Suter-Zimmermann K, Bucher HC, Shai I, Tuttle KR, Estruch R, et al. Meta-analysis comparing Mediterranean to low-fat diets for modification of cardiovascular risk factors. Am J Med 2011;124(9):841–51, e842. [3] Estruch R, Ros E, Salas-Salvado J, Covas MI, Corella D, Aros F, et al. 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[17] Trichopoulou A, Orfanos P, Norat T, Bueno-de-Mesquita B, Ocke MC, Peeters PH, et al. Modified Mediterranean diet and survival: EPIC-elderly prospective cohort study. BMJ 2005;330(7498):991. [18] Goulet J, Lamarche B, Nadeau G, Lemieux S. Effect of a nutritional intervention promoting the Mediterranean food pattern on plasma lipids, lipoproteins and body weight in healthy French-Canadian women. Atherosclerosis 2003;170(1):115–24. [19] Gerber M. Qualitative methods to evaluate Mediterranean diet in adults. Public Health Nutr 2006;9(1A):147–51. [20] Sanchez-Villegas A, Martinez JA, De Irala J, Martinez-Gonzalez MA. Determinants of the adherence to an “a priori” defined Mediterranean dietary pattern. Eur J Nutr 2002;41(6):249–57. [21] Rumawas ME, Dwyer JT, McKeown NM, Meigs JB, Rogers G, Jacques PF. The development of the Mediterranean-style dietary pattern score and its application to the American diet in the Framingham Offspring Cohort. J Nutr 2009;139(6):1150–6. [22] Sanchez-Tainta A, Estruch R, Bullo M, Corella D, Gomez-Gracia E, Fiol M, et al. Adherence to a Mediterranean-type diet and reduced prevalence of clustered cardiovascular risk factors in a cohort of 3,204 high-risk patients. Eur J Cardiovasc Prev Rehabil 2008;15(5):589–93. [23] Trichopoulou A, Kouris-Blazos A, Wahlqvist ML, Gnardellis C, Lagiou P, Polychronopoulos E, et al. Diet and overall survival in elderly people. BMJ 1995;311(7018):1457–60. [24] Trichopoulou A, Costacou T, Bamia C, Trichopoulos D. Adherence to a Mediterranean diet and survival in a Greek population. N Engl J Med 2003;348(26):2599–608. 104 SECTION 1 The Mediterranean Diet: Concepts and General Aspects [25] Hoevenaar-Blom MP, Nooyens AC, Kromhout D, Spijkerman AM, Beulens JW, van der Schouw YT, et al. Mediterranean style diet and 12-year incidence of cardiovascular diseases: the EPIC-NL cohort study. PLoS One 2012;7(9):e45458. [26] Gardener H, Wright CB, Gu Y, Demmer RT, Boden-Albala B, Elkind MS, et al. Mediterranean-style diet and risk of ischemic stroke, myocardial infarction, and vascular death: the Northern Manhattan Study. Am J Clin Nutr 2011;94(6):1458–64. [27] Knoops KT, de Groot LC, Kromhout D, Perrin AE, Moreiras-Varela O, Menotti A, et al. Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women: the HALE project. JAMA 2004;292(12):1433–9. [28] Dilis V, Katsoulis M, Lagiou P, Trichopoulos D, Naska A, Trichopoulou A. Mediterranean diet and CHD: the Greek European Prospective Investigation into Cancer and Nutrition cohort. Br J Nutr 2012;108(4):699–709. [29] Buckland G, Gonzalez CA, Agudo A, Vilardell M, Berenguer A, Amiano P, et al. Adherence to the Mediterranean diet and risk of coronary heart disease in the Spanish EPIC Cohort Study. Am J Epidemiol 2009;170(12):1518–29. [30] Fung TT, Rexrode KM, Mantzoros CS, Manson JE, Willett WC, Hu FB. Mediterranean diet and incidence of and mortality from coronary heart disease and stroke in women. Circulation 2009;119(8):1093–100. [31] de Lorgeril M, Salen P, Martin JL, Monjaud I, Delaye J, Mamelle N. Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study. Circulation 1999;99(6):779–85. [32] Singh RB, Dubnov G, Niaz MA, Ghosh S, Singh R, Rastogi SS, et al. Effect of an Indo-Mediterranean diet on progression of coronary artery disease in high risk patients (Indo-Mediterranean Diet Heart Study): a randomised single-blind trial. Lancet 2002;360(9344):1455–61. [33] Mente A, de Koning L, Shannon HS, Anand SS. A systematic review of the evidence supporting a causal link between dietary factors and coronary heart disease. Arch Intern Med 2009;169(7):659–69. [34] Urpi-Sarda M, Casas R, Chiva-Blanch G, Romero-Mamani ES, Valderas-Martinez P, Salas-Salvado J, et al. The Mediterranean diet pattern and its main components are associated with lower plasma concentrations of tumor necrosis factor receptor 60 in patients at high risk for cardiovascular disease. J Nutr 2012;142(6):1019–25. [35] Camargo A, Delgado-Lista J, Garcia-Rios A, Cruz-Teno C, Yubero-Serrano EM, Perez-Martinez P, et al. Expression of proinflammatory, proatherogenic genes is reduced by the Mediterranean diet in elderly people. Br J Nutr 2012;108(3):500–8. [36] Santaniemi M, Ukkola O, Malo E, Bloigu R, Kesaniemi YA. Metabolic syndrome in the prediction of cardiovascular events: the potential additive role of hsCRP and adiponectin. Eur J Prev Cardiol 2013. [37] Sola R, Fito M, Estruch R, Salas-Salvado J, Corella D, de La Torre R, et al. Effect of a traditional Mediterranean diet on apolipoproteins B, A-I, and their ratio: a randomized, controlled trial. Atherosclerosis 2011;218(1):174–80. [38] Esposito K, Marfella R, Ciotola M, Di Palo C, Giugliano F, Giugliano G, et al. Effect of a Mediterranean-style diet on endothelial dysfunction and markers of vascular inflammation in the metabolic syndrome: a randomized trial. JAMA 2004;292(12):1440–6. [39] Rallidis LS, Lekakis J, Kolomvotsou A, Zampelas A, Vamvakou G, Efstathiou S, et al. Close adherence to a Mediterranean diet improves endothelial function in subjects with abdominal obesity. Am J Clin Nutr 2009;90(2):263–8. [40] Mitrou P, Kipnis V, Thiébaut A, Reedy J, Subar A, Wirfält E, et al. Mediterranean dietary pattern and prediction of all-cause mortality in a US population results from the NIH-AARP Diet and Health Study. Arch Intern Med 2007;167(22):2461–8. Chapter 10 Genomic Determinants of Mediterranean Diet Success Keith Grimaldi, PhD1 and Antonio Paoli, MD, BSc2 1 National Technical University of Athens, Athens, Greece. 2 University of Padova, Padova, Italy. INTRODUCTION The Mediterranean diet is recognized as one of the most healthy in the world, and variations of it seem to be the most successful nutritional regimens for reducing cardiovascular risk factors [1–5]. Why is the Mediterranean diet apparently so beneficial? In a recent review, Ordovas et al. [6] speculated that: the Mediterranean diet may be closer to the ancestral foods that were part of human development and our metabolism may have evolved to work optimally on such a diet rather than with the current diets richer in saturated fat and highly refined and processed foods. Therefore, it is possible that alleles that are associated with increased disease risk may be silenced in the presence of that more ancestral and traditional diet and lifestyle. This knowledge may provide the basis for successful public health as well individual approaches for disease prevention. Alleles refers to the presence of common genetic variants that can modify the way an individual responds to diets of differing composition. Two things are known for certain: genes affect response to diet, and diet affects gene expression (for example, personal genetic variation affects response to ultraviolet light, and ultraviolet light in turn affects gene expression). In general, the way genetic variation affects response to nutrients in the diet is termed nutrigenetics, whereas nutrigenomics is the term used for the effect of diet on gene expression. There are well-established gene–diet interactions for which specific dietary precautions are necessary, such as phenylketonuria, a genetic disorder requiring a lifetime of a phenylalanine-restricted diet, and so-called lactose intolerance, which is not a disease but a phenotype common to the majority of the world’s population (including in the Mediterranean region) who lack a polymorphism upstream of the lactase gene [7]. The effect of the polymorphism, common in central and northern Europe, is to ensure the continued production of the lactase enzyme, which would otherwise cease during childhood. The application of genetics and genomics to nutritional research is increasing detailed knowledge of metabolic pathways, but an added benefit is that it is bringing more consistency to nutritional research in general. Nutrition research as well as candidate gene-association studies have produced many inconsistent results over the decades, which translates into a level of confusion in the public health message. This is not related to the overall quality of the research; rather, it is because of the complexity of the effects of nutrition on long-term health and confounding due to individual variation. A good example is the association between coffee consumption and myocardial infarction (MI): many years of epidemiological studies yielded inconclusive results, which Cornelis et al. [8] hypothesized may be in part because of a lack of stratification according to caffeine metabolism genotype. Caffeine is metabolized by the enzyme CYP1A2, and a common genetic variation in this gene determines whether an individual metabolized caffeine quickly or slowly. In a study of over 4000 subjects, no significant cardiovascular risk was associated with coffee consumption in the entire study group; however, when stratified, the risk in fast metabolizers did not increase, whereas in the subset of slow metabolizers it increased significantly (odds ratio [OR] of 1.7 for 4 coffees per day) [8]. The point here is that in even a very large epidemiological study with a relatively simple-to-measure intake, the significant risk in a subsection of the population would not have been revealed, and in smaller studies the results would have been—and indeed were—inconsistent. The review by Ordovas et al. [6] mentioned above highlighted the status of early nutrigenomics studies in general as well as those with particular application to the Mediterranean diet (MD), and some significant reproducible gene–diet interactions were reported. This area of research has progressed significantly over the past ten years; there is more consistency, and increased numbers of gene–diet interactions, which are reviewed here, have been confirmed. The direct benefit of these studies is the possibility of using the knowledge for modifying nutritional advice for the individual. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 105 106 SECTION 1 The Mediterranean Diet: Concepts and General Aspects NUTRIGENETICS, NUTRIGENOMICS, AND DIET Nutrigenetics: Use of the MD to Neutralize Potentially Negative Effects of Some Common Genetic Variants Development of common complex diseases involves multiple genes plus multiple environmental factors, and actual individual risk is determined by gene–environment interactions [9]. In fact “genetic risk” associated single nucleotide polymorphisms (SNPs), such as skin color, have the potential to both increase and decrease risk, depending on the environment. A well-studied example of this interaction is the C677T SNP in the MTHFR gene and acid metabolism, which is important for thymidine synthesis and DNA methylation in pathways that have direct effects on genome stability and homocysteine levels, respectively [10,11]. The 677T allele causes an amino acid change that results in a much lower enzyme activity, having just 30% the activity of the 677C encoded enzyme [12]. In gene–disease association studies the T allele has been associated with increased risk of cardiovascular disease (CVD) [13] but may be protective against some cancers [10]; however, the effects are dependent on the availability of folic acid in the diet. At levels <400 mg/day, 677TT homozygotes are likely to have chronically high levels of homocysteine, which is an independent risk factor for CVD and osteoporosis [14]. With adequate folic acid in the diet, the potential increased risk for CVD and other diseases is neutralized and the protection against some cancers is maintained. Therefore the SNP itself does not increase or decrease any risks—its effects depends on diet. The geographic distribution of the SNP is interesting from this point of view. The variant T allele is very common in areas where traditional diets have high folic acid content (e.g., southern Europe; 20-25% of southern Italians are 677TT homozygous), but it is relatively rare in areas where folic acid is low (e.g., northern Europe; in Finland <4% are homozygous) [15–17]. The hypothesis is that the T allele was “allowed” by natural selection to become common in areas with high folic acid because it has protective effects when there is adequate folic acid and, vice versa, the prevalence of the SNP was reduced by migrations to northern Europe because of negative selection pressure of low folic acid. This dramatic effect of traditional diets and migration on the geographic prevalence of the C677T SNP is evidence that even single SNPs interacting with diet can have significant long-term health effects. Where nutrigenetic research identifies important gene–environment interactions, it becomes possible to intervene and reduce or neutralize the increased risks associated with particular genetic variants. Some recent studies have demonstrated this in the case of the MD and several potential risk alleles for common diseases. One of the first formal projects investigating the effects of genetic variation on the response to the MD was the Medi-RIVAGE study [18], which enrolled 212 male and female subjects with at least one cardiovascular risk factor between 1998 and 2002 and compared an MD to a standard low-fat diet. They eventually reported data from 169 subjects who were genotyped for 23 polymorphisms, showing interactions between some genetic variants and reduced cardiovascular risk [19]; although the numbers were small the project provided some early evidence of individual differences in response to the MD. Recent work in larger studies has reproducibly identified several gene-environment interactions relevant for components of the MD (Table 1). The FTO gene has been repeatedly associated with obesity in many different cohorts, and it is accepted as the gene most strongly associated with obesity (although of course it is still only one of many involved genes and its effect is small). Subsequent studies have suggested that the increased risk is dependent on diet and lifestyle, including levels of specific macronutrients such as saturated fats [21]. Moleres et al. [32] looked at the effects of fatty acids in 354 Spanish children and reported that the increased risk for obesity associated with the A allele (of the FTO rs9939609 polymorphism) was seen only when saturated fatty acid (SFA) consumption was high (>12.6% of total energy) and/or when the polyunsaturated fatty acid (PUFA) consumption was relatively low (PUFA-to-SFA ratio <0.43%); thus increasing PUFAs and reducing SFAs had the effect of neutralizing the risk associated with the allele and reducing odds of risk of obesity threefold. These and other data clearly show that the FTO genetic variant does not in itself increase the risk of obesity; it does so only under certain dietary conditions. The PPARg (peroxisome proliferator-activated receptor g) gene contains a polymorphism that changes the amino acid at position 12 in the protein from proline to alanine. PPARg is a transcription factor that is involved in glucose and lipid homeostasis. In association studies the Pro12 allele has been linked with a small increased risk in type 2 diabetes mellitus (T2DM) whereas the Ala12 is slightly protective [33]; on the other hand, depending on the type of diet, the Ala12 allele has been associated with increased weight gain [34,35]. Several studies of the MD have looked at the effects according to PPARg genotype. In 2009 Razquin et al. [20] published results of a 2-year intervention study of 774 patients at high risk of CVD in the PREDIMED randomized trial, which aimed at assessing the effect of the MD on CVD prevention. Genomic Determinants of Mediterranean Diet Success Chapter 10 107 TABLE 1 Examples of Gene—Mediterranean Diet (MD) Interactions (See Text for Details) Phenotype Gene–Diet Interaction Effect References MD versus Western-type diets Weight gain PPARg MD versus control diet Reduced increase in waist circumference among Ala allele carriers in the MD group ( >2-cm difference) [20] Obesity risk FTO Saturated fats PUFAs Increased obesity risk only when saturated fat (SFA) consumption was high (>12.6% total energy) and/or when PUFA consumption was relatively low [21] PPARg FTO Carbohydrates and physical activity Higher obesity risk in Ala allele (PPARg) and A allele (FTO) carriers with high intake of refined carbohydrates and sedentary lifestyle [22] ADIPOQ IL6 MD versus control diet Reduced risk for obesity in MD with high intake of olive oil and/or nuts [23] [24] FTO MC4R MD versus non-MD diets When adherence to the MD was low, carriers of the variant alleles had higher risk for T2DM [25] Various (10 SNPs, genetic risk score) “Prudent” (e.g., MD) versus Western diet With a high genetic risk score, those following the “prudent” diet had low odds of developing diabetes (OR 0.9–1.0), whereas those with the worst combination of high genetic risk score + Western diet were at an almost three times increased risk [26] Various (24 variants) Nutrigenetic versus normal diet Nutrigenetically enhanced traditional MD was more effective at improving risk biomarker profiles (glucose, LDL, homocysteine) and long-term weight loss [27] CLOCK Reduced-calorie MD Individuals with the G allele lost less weight than subjects with the more common AA genotype The minor allele carriers also had significantly higher ghrelin levels [28] [29] BMI APOA2 Saturated fats The CC genotype (15% of the populations) was associated with a 6.8% greater BMI in those consuming a diet high, but not a low, in saturated fat [30] Lipid profile APOA5 Saturated fats Carriers of the minor allele were somewhat protected against higher BMI and triglyceride levels associated with a higher-fat diet [31] T2DM Individual differences in the response to the MD Weight loss BMI, body mass index; LDL, low-density lipoprotein; OR, odds ratio; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SNP, single nucleotide polymorphism; T2DM, type 2 diabetes mellitus. Two groups followed an MD and the control group a standard low-fat diet. Over the course of the study, in the control group there was a higher change in waist circumference (>2 cm) in carriers of the Ala allele, but this was not seen in the MD groups, and there was a greater beneficial effect seen in patients with T2DM. This suggests that the Ala allele may be protective against T2DM but only under the right diet and lifestyle conditions (e.g., a Mediterranean-type diet); otherwise it can be negative. In another study of 978 elderly Spanish subjects, Galbete et al. [22] observed a higher risk of obesity in subjects with the Ala12 allele who were inactive or had a high carbohydrate intake (>246 g/day), and the risk was further increased in the presence of the FTO minor allele (rs9939609). The extensive data for these two genes may help to explain why, as shown by Elhayany et al. [36], a low-carbohydrate MD was more effective at reducing CVD and T2DM risk factors than the standard American Diabetic Association-recommended diet. Another gene associated with increased risk of obesity (and metabolic syndrome) is adiponectin. Raquin et al. [23] investigated the effect of a 3-year intervention with a Mediterranean-style diet in 737 overweight individuals with a high cardiovascular risk. The subjects were assigned to a low-fat diet or to one of two variant MDs: one with a high intake of virgin olive oil and another with a high intake of nuts. They reported that both MDs reversed the increased body weight gain associated with the higher risk adiponectin (ADIPOQ) risk alleles. In a related study the same group also observed that the 108 SECTION 1 The Mediterranean Diet: Concepts and General Aspects MD neutralized the effects of another variant in the interleukin-6 gene: the minor C allele was associated with increased adiposity, but carriers of this allele had the greatest reduction in body weight when following one of the MDs compared to the control diet [24]. Obesity is closely linked to T2DM, so it is not surprising that the same genes are associated with increased risk for both conditions. Both the FTO and melanocortin-4 receptor (MC4R) genes have been consistently associated with obesity risk and may also be linked to T2DM. In a very large case-control study of 7052 subjects with high cardiovascular risk (50% of whom were diabetics), Ortega-Azorin et al. [25] found that when adherence to MD was low, the carriers of the variant alleles (risk alleles for high body mass index [BMI]) had an increased risk of T2DM (OR 1.21) that disappeared when adherence was high (OR 0.97), and the gene-diet interactions were independent of BMI, suggesting a direct effect on T2DM risk. In the same study group the risk alleles also were found to be neutralized by increased physical activity [37]. As discussed above, common diseases have multiple components, including multiple genetic variants, that contribute to the development of the disease. The study of single gene-diet interactions will increase knowledge of the mechanisms involved but will address only one small aspect of the overall causation. A strategy for simultaneously assessing the overall effects of several genes is to calculate a “genetic predisposition score.” In a recent study Qi et al. [26] looked at 10 SNPs associated with T2DM and the effect of diet on the combined genetic score related risk. While they did not look specifically at the MD they showed that a “prudent” dietary pattern (high intake of vegetables, fruit, legumes, whole grains, fish, and poultry) abolished the significant T2DM risk seen in individuals who had a high genetic risk score and consumed a Western-type diet pattern (high intake of processed meat, red meat, butter, high-fat dairy products, eggs, and refined grains). Even with a high genetic risk score, those following the prudent diet had low odds of developing diabetes (OR 0.9–1.0), whereas the worst combination of high genetic risk score plus Western diet were at an almost a three times increased risk. In summary, there has been a great deal of consistency in studies analyzing genetic risk in the context of diet—in stark contrast to earlier genetic studies that looked at genetic association with disease without taking into consideration diet or lifestyle (in such studies inconsistency was the norm) [38]—and they clearly indicate that the effects of genetic risk alleles can be completely neutralized by specific dietary interventions; adherence to the MD seems to be particularly effective in counterbalancing genetic risk associated with the major complex diseases of the present age. Thus, even individuals with a high genetic risk for a common disease do not necessarily have a high actual risk; in fact, depending on diet and lifestyle, the overall risk can be reduced to significantly below average. Nutrigenetics: Individual Differences in Response to the MD The previous section looked at how the MD can be used to counteract the potentially negative effects of particular genetic variants. This section looks at further personalization of the MD itself. Just as one size does not fit all, so one MD does not fit all, and there is evidence that some aspects of the MD can be beneficially modified when certain genetic variants are present. The first evidence of this appeared in a study reporting the use of nutrigenetics in clinical practice that was published in 2007 [27], the subject of which was long-term weight loss; a total of 93 patients with a history of poor weight control were treated with a traditional MD; in the control group the MD was modified for each individual according to phenotypic and lifestyle parameters (e.g., food preferences, age, sex, BMI, activity levels), and the diets of the nutrigenetic group were further personalized according to genetic variation (among a panel of 24 genetic variants). The data reported showed that the nutrigenetically enhanced traditional MD was more effective at improving risk biomarker profiles (glucose, low-density lipoprotein, homocysteine) and long-term weight loss. After 12 months the majority of the control group had regained weight, whereas the nutrigenetic group did not, and the probability of maintaining weight loss over the long term was sixtimes higher in the nutrigenetic group (OR 6.1). Several more recent studies that demonstrate specific effects of individual genes on the response to Mediterranean-type diets have been published. The CLOCK gene is involved in control of the circadian rhythm and can have an influence on the response to weight-loss diets. Garaulet et al. [28] reported that the CLOCK rs1801260 SNP may predict the outcome of body weight reduction strategies based on reduced-calorie Mediterranean-type diets. They looked at 500 subjects who were prescribed a 28-week reduced-calorie MD and found that over this period individuals with the G allele lost less weight than subjects with the more common AA genotype (7.96 vs. 10.41 kg over 28 weeks). Another study by the same group showed similar results in a separate cohort of 1495 overweight subjects; the minor allele carriers were more resistant to weight loss, and the rate of loss slowed significantly, after 12 weeks. The minor allele carriers also had significantly higher ghrelin levels and they ate at different times (e.g., ate late breakfasts and more in the evening); there also was less compliance to the MD (with higher intake of processed foods and trans fatty acids). The minor allele carriers also slept less, which itself has been Genomic Determinants of Mediterranean Diet Success Chapter 10 109 associated with increased BMI [29]. A separate study by a different group suggested that more intense physical activity can be beneficial for weight loss in the presence of the minor allele [39]. Taken together, these interesting studies suggest that carriers of the CLOCK minor allele who are seeking to lose weight may not respond to a typical MD and require specific education about eating habits, exercise, and compliance if they are to achieve optimum results. The APOA2 gene codes for apolipoprotein 2, which is the second most abundant protein of the high-density lipoprotein particles, but its function remains largely unknown [40]. The interaction of this gene with diet, particularly saturated fats, has been well studied and results have been reproduced in several thousand subjects from six different populations. Corella et al. [30] analyzed gene–diet interactions between the APOA2-265T > C polymorphism and saturated fat intake (<22 or 22 g/day). In Mediterranean individuals, the CC genotype (15% of the population) was associated with a 6.8% greater BMI in those consuming a high-fat diet, but not a diet with low saturated fat. While the MD is naturally low in saturated fats, carriers of the CC allele apparently need to be particularly careful of saturated fat content to either lose weight or reduce the risk of weight gain. Genetic variability at the APOA5 locus also has been associated with increased CVD risk; however, the macronutrient content of the MD may affect this risk. An association was found between the APOA5-1131T > C SNP and fat intake; apparently carriers of the minor allele were somewhat protected against higher BMI and triglyceride levels associated with a higher fat diet [31]. These examples illustrate that genetic variation can influence the effects of the MD in different individuals. This line of research is growing rapidly and will no doubt yield further information that may be useful in making the MD even more effective at the individual level. Nutrigenomics: Investigating the Effect of the MD on Gene Expression Many studies have demonstrated that adherence to the MD leads to improvement in levels of biomarkers associated with inflammation and oxidative stress, which may explain, at least in part, the diet’s association with reduced risk for CVD [41]. The mechanisms for these actions of the MD have been the subject of recent nutrigenomics research. While genotype affects response to nutrients (metabolism, transport, excretion, etc.), as discussed in the previous sections, certain nutrients also have an effect on our genes, altering their expression in a specific and targeted manner. Diet also affects the level of many circulating metabolites, and recent technology developments allow the simultaneous detection of thousands of metabolic pathway intermediates; transcriptomics, proteomics, and metabolomics have been used to dissect the mechanisms responsible for the apparent benefits of the MD. The effects of certain critical ingredients of the MD on gene expression and on various metabolite levels have been investigated. Most work has looked at the effect of lipids, including monounsaturated fatty acids (MUFAs) present in olive oil and omega-3 PUFAs in fish and nuts. The first nutrigenomics studies of humans to be published appeared in 2009; they investigated the effects of PUFAs, MUFAs, and SFAs. These were single-blind crossover studies of 21 healthy men; peripheral blood mononuclear cells were isolated 6 hours after the intervention and the whole-genome expression was profiled. The study compared gene expression following a drink containing 55 g test fatty acids (65% PUFAs vs. 80% MUFAs vs. 70% SFAs). PUFAs led to upregulation of 225 genes and downregulation of 212 genes, and SFA upregulated and downregulated 196 and 101 genes, respectively. The studies showed that the main expression changes were associated with processes related to liver X receptor signaling, oxidative stress, inflammation, carbohydrate metabolism, and a variety of other processes, and the effects of PUFAs were the opposite of SFAs [42]. A separate double-blind clinical trial of elderly subjects using a 26-week intervention with fish oil containing a low or high dose of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (0.4 vs. 1.8 g, respectively) reported that high EPA + DHA changed the expression of 1040 genes compared with the control (high dose of oleic sunflower oil), which changed the expression of only 298 genes. Interestingly, EPA + DHA intake led to reduced expression of genes involved in inflammatory- and atherogenic-related pathways, such as nuclear transcription factor B signaling, eicosanoid synthesis, scavenger receptor activity, adipogenesis, and hypoxia signaling. The overall changes demonstrated that the 6-month intake of EPA + DHA led to a more anti-inflammatory and antiatherogenic gene expression profile [43]. Olive oil is a key ingredient of the MD and, when it has a high phenol content, has been shown to reduce inflammation, oxidative stress, and prothrombotic markers. The effects of the phenolic component of olive oil on the expression of over 30,000 genes was studied in blood mononuclear cells following a virgin olive oil-based breakfast with high (398 ppm) and low (70 ppm) content of phenolic compounds in 20 patients with metabolic syndrome. The analysis identified 98 genes whose expression was altered by the high phenolic content (79 underexpressed and 19 overexpressed), and many were linked to metabolic syndrome pathways; the reduced expression of several proinflammatory genes was observed [44]. 110 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 2 Nutrigenomics: The Effect of the Mediterranean Diet on Gene Expression Phenotype Dietary Components Effect References Inflammation Oxidative stress Saturated fats (SFAs) MUFAs PUFAs PUFAs led to upregulation of 225 genes and downregulation of 212 genes and SFA up- and downregulated 196 and 101 genes, respectively PUFAs had (beneficial) effects on expression opposite to those of SFAs [42] Inflammation Fish oil (EPA and DHA) EPA + DHA intake led to reduced expression of genes involved in inflammatory- and atherogenic-related pathways [43] Inflammation Olive oil (high vs. low phenols) Reduced expression of several proinflammatory genes was observed [44] Inflammation Oxidative stress Olive oil (high vs. low phenols) Decreased oxidative and inflammatory plasma markers Modified expression of genes involved in inflammation and oxidative stress, with enhanced effects seen in the high polyphenol group [45] EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid. In a separate controlled clinical trial of 90 healthy volunteers, a separate group measured the effects of a traditional MD with virgin olive oil or with washed olive oil (i.e., reduced phenolic content). Compared with a control diet, both of the MDs decreased oxidative and inflammatory plasma markers and affected the expression of genes involved in inflammation and oxidative stress; enhanced effects were seen in the high polyphenol group, and the authors concluded that the olive oil polyphenols had a significant role in the downregulation of proatherogenic genes in the context of a traditional Mediterranean diet [45] (Table 2). A recent review of the literature on the transcriptomic effects of the MD in humans confirmed the several lines of evidence that changes in gene expression in oxidative stress, inflammation, and atherogenic markers (such as tumor necrosis factor and interferon-g) is one mechanism of the MD effect [46]. These are interesting results from initial studies: the review concluded that “data in this field, although scarce, are promising”; progress is challenging because of the intensive nature of these types of investigations, which require highly controlled dietary interventions followed by detailed analysis of thousands of genes or metabolites. However, over the next few years researchers are likely to uncover many of the specific health-promoting pathways that are stimulated by components of the MD. CONCLUSIONS Over 50% of the adult population in the European Union is overweight or obese; levels have tripled over the past 20 years, and the trend shows no sign of slowing. The consequential serious social and economic problems are further compounded by the earlier onset of these diseases, causing, in addition to personal suffering, significant loss of work-years and a drain on resources [47]. Against this background, the MD is understandably receiving renewed attention by researchers and health care practitioners alike. During the twentieth century, great progress in improving health care was made. Notable successes include the eradication, at least in Europe, of most diseases of malnutrition; protection against infection through vaccination and antibiotics; and effective public health efforts against smoking. Over the past 50 years, however, rapid technological progress has been accompanied by (and has contributed to) negative effects on diet and lifestyle—the two most important factors in the cause of chronic disease. Our environment has changed enormously, leading to increased food choice and availability, along with the desire and, at least in part, the necessity for reduced costs accompanied by lower quality; we consume far more sugar and processed foods, and intensive agriculture has reduced the nutrient content of many vegetables [48–50]. In addition, developments in transport and technology have reduced the time we spend being physically active, resulting in an increased sedentary lifestyle. Also, in the face of this, the public health message has to combat ever more intense and creative commercial efforts to sell unhealthy foods and products. The resulting environment has become more “obesogenic” and “diseaseogenic.” The nutrigenetic and nutrigenomic studies presented in this chapter support the proposal by Ordovas and colleagues that the health benefits associated with the traditional MD may have genetic roots—that our genetic background evolved to work optimally with the MD and lifestyle. The answer to our health problems is “simple”: we should combine the healthier aspects of earlier decades while maintaining beneficial progress of the current era. Of course, the answer is not so simple: Genomic Determinants of Mediterranean Diet Success Chapter 10 111 (1) the public health message is either not heard, not understood, or is ignored, and (2) it does not meet the needs of the individual. In fact, the public health approach is somewhat paradoxical [51]; it is aimed at the majority who are at moderate individual risk rather than the individuals at highest risk because “a large number of persons with moderately increased risk levels contribute more cases than a small number with extreme risk levels.” This dilutes the effect of the message to each individual, making it seem impersonal, less relevant “for me,” and therefore often ignored. A more individualized approach also is required because, as the studies presented in this chapter demonstrate, genetic variation influences response to diet, and while a general MD may be healthy for the population, a personalized MD will be more healthy for the individual. The MD is not expected to be the panacea for all of the growing lifestyle-related health problems facing the Western world; indeed, there are some who argue that rather than focusing on a single regional diet it may be more appropriate to promote healthy nutritional aspects of local diets for different populations [52]. How much of the benefits are related to the Mediterranean lifestyle itself? [53]. Should the Scandinavian population follow the Mediterranean or the Nordic diet? [54]. These are good questions. Nutrigenetics and nutrigenomics are unlikely to be able to determine which individual will respond best to which diet; nutrition and genetics are too complex to expect, or promise, such simplistic answers. The benefits of nutritional genetics will most likely be found in the fine tuning of generally healthy diets and certainly in improving the reliability and consistency of all nutritional research. The concept of personalized nutrition is not new, but the application of nutrigenetics now—and eventually nutrigenomics—is making it a growing reality. There is still a healthy debate about how much can be achieved with nutrigenetics [55], but there is good evidence that it can be beneficial, and there is little doubt that this knowledge may provide the basis for successful public health as well individual approaches for disease prevention [6]. SUMMARY POINTS l l l l l l l Nutrigenetics studies common genetic variants (usually SNPs) that affect the activity of proteins involved in the assimilation, transport, and metabolism of dietary nutrients. Nutrigenomics studies the direct or indirect effects nutrients can have on gene expression. Gene–diet interactions have a strong influence on long-term health, especially in the development of the common complex diseases of aging (e.g., CVD, T2DM). In Western society these common diseases are becoming more prevalent at an earlier age, affecting personal, social, and economic well-being. The MD has been shown to be effective at reducing risk factors associated with certain genetic variations interacting with a nonoptimal nutrition. The MD itself is also not “one size fits all,” and there is evidence that it can be optimized for the individual by making specific modifications according to genotype. Components of the MD (especially MUFAs and PUFAs from olive oil, fish, and nuts) have been demonstrated to have a positive effect on the expression of genes involved in inflammation and oxidative stress. REFERENCES [1] Schwarzfuchs D, Golan R, Shai I. Four-year follow-up after two-year dietary interventions. N Engl J Med 2012;367:1373–4. [2] Estruch R, Ros E, Salas-Salvado J, Covas MI, Corella D, Aros F, et al. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med 2013;368:1279–90. 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Chapter 11 The Mediterranean Diet Quality Index (KIDMED) and Nutrition Knowledge Semra Akar Sahingoz, PhD University of Gazi, Ankara, Turkey. INTRODUCTION Practices to protect one’s health should begin during youth and continue throughout adulthood. Health protection measurements can be categorized into three stages: primary, secondary, and tertiary. Primary protection involves measures taken before developing a disease; secondary protection involves preemptive screening for an early diagnosis of disease; the treatment/control of a disease after it emerges to prevent impairment and secondary health issues falls under the tertiary protection category. Primary protections are the measures taken before the disease emerges and thus are the most effective of the protection methods. The following are widely accepted primary protection measures: developing healthy behavior, preemptive vaccination, preventing accidents, and providing social assistance. Healthy behavior involves having a sufficient and balanced nutrition plan, being a nonsmoker, and remaining physically active across all age groups. A sufficient, healthy, and balanced nutrition plan is a basic requirement for sustaining one’s health and preventing disease. Physical activity is also an important factor because it has a role in preventing accidents, disabilities, and conditions such as cardiovascular diseases, hypertension, osteoporosis, and diabetes. Individuals should be encouraged to engage in physical activity from childhood to adulthood. For an average individual without any disabilities, becoming educated regarding healthy habits and nutrition, as well as engaging in 30 min of medium-level physical activity a day, is incredibly important [1]. The studies in the relevant literature also show that the level of the effectiveness of nutrition education is highest among early age groups. Societal nutritional habits do not change radically because they are rooted within the culture and customs of a society. These habits form over long periods of time according to a group’s social processes. Therefore, traditions, societal norms, religious beliefs, and local habits all have a part in shaping a child’s nutritional education. By this reasoning, malnutrition is actually a result of societal and social factors. Correcting such mistakes in preexisting nutritional styles cannot, therefore, be a short-term goal; it requires time and consistent effort. Different geographical forms and climate conditions are the basis for different nutritional habits, resulting in various nutritional models in each culture. However, in time these nutritional models may result in the emergence of numerous health problems. For instance, pellagra disease almost became an epidemic in societies consuming excessive amounts of corn, which in turn helped the niacin vitamin be discovered. Currently, the nutritional style derived from fast food is related to diabetes, cardiovascular diseases, and obesity. The Mediterranean Diet (MD) is a nutritional style with a focus on healthy nutrition and the prevention of cardiac diseases. It is based around the eating habits of countries along the Mediterranean coastline and is the result of centuries of culinary tradition. These countries include Morocco and Tunisia in northern Africa and Greece, Portugal, Spain, southern France, and some parts of Italy in Europe. With the addition of some parts of the Balkans, Turkey, and Middle Eastern countries such as Lebanon and Syria, these regions have all helped develop the original form of the MedDiet, which stems from the rural traditions and agricultural products within these regions. Many nutrition experts and their respective studies determined that this diet is one of the healthiest in terms of increasing average life span and general health. Studies showed that the MD is effective in the prevention and treatment of many diseases [2]. Adherence to the MD has been associated with lower rates of cardiovascular disease [3], cancer [4], diabetes mellitus [5], hypertension [6,7], inflammation, depression [8], and all-cause mortality [9–13]. The MD has been shown to have protective effects against cognitive functional decline and dementia in older individuals, resulting from a combination The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 115 116 SECTION 1 The Mediterranean Diet: Concepts and General Aspects of several potentially protective foods and nutrients such as fish, monounsaturated fatty acids, vitamin B12 and folate, antioxidants (vitamin E, carotenoids, flavonoids), and moderate amounts of alcohol [14–19]. A recent analysis of the Invecchiare in Chianti (In Chianti) Study indicated a protective effect of the MD against decreasing mobility over time [20]. In their study Milaneschi et al. [20] stated that the relationship between the MD pattern, physical functioning, and age-related disability in non-Mediterranean countries has yet to be examined. MD was previously shown to be associated with longevity [11,12,19] in cognitive function and the prevention of Alzheimer’s disease, loss of physical function, and other chronic diseases [3,6,14,16,20,21]. A study conducted among 2339 physically active, nonsmoking individuals with good health who consume little alcohol in 11 European countries over 10 years applied a MD consisting of fruit, vegetables, olive oil, and fish. The results of the study determined a 60% decrease in diseases such as cancer and cardiovascular diseases, which are common causes of death among the elderly community [12]. As the only herbal oil obtained fresh through pressing fruits without refinement, olive oil is differentiate from other herbal oils: the important natural compounds of olive oil are not artificially removed. It is nearly the only oil that can be consumed “roughly,” that is, without refinement [22]. An individual’s lifestyle is an important factor in determining their quality of life in old age. Every individual should be conscious of their nutritional habits and should be taught how to control and maintain their diet. THE IMPORTANCE OF NUTRITIONAL EDUCATION AND THE MD Societal nutritional problems result from food production and distribution technologies, environmental health conditions, insufficient purchasing power, negative cultural factors, and excessive food consumption. All these problems are based on the failure to develop accurate nutrition awareness, which stems from insufficient nutritional education. Nutritional education is the most important factor in removing and minimizing the factors causing health problems. The first humans ate animals they hunted and foraged for seasonal and regional fruits. Social changes such as urbanization caused drastic changes in nutritional behaviors. Together with sociologic and technologic developments, rapid developments in the food industry have altered the nutrition styles of individuals, causing them to become distant from natural food materials and to increase their fast-food consumption [23,24]. The literature includes studies examining knowledge regarding the topics of nutrition and health, as well as the nutritional states of individuals of different age, sex, and ethnic groups from different professions throughout countries around the world [25,26]. One common thread of these studies is a relationship between levels of nutritional knowledge and the state of the individual’s food consumption. In addition, all of them suggest nutritional education is necessary for individuals. In their study of high school students, Akhtar Khan et al. [27] determined that 62% of students had insufficient knowledge about maintaining their health and consumed vegetable and fruits at insufficient rates while consuming fat and energy at high rates. Health and disease prevention is strongly related to food selection. Sahingoz and Sanlıer [28] found that adolescents had low knowledge scores regarding fruit and vegetable consumption. In addition, they found relationships between nutritional knowledge levels and the food consumption habits of children [28], which are similar to the results of a study by Gibson et al. [29]. A study of physicians in England revealed that most rated their nutrition knowledge as “poor” or “very poor” [30]. Nutritional knowledge can be seen as a key prevention strategy for overweight and obesity [31]. Trichesa and Giugliani [31] speculated that there are two distinctive patterns in the relation between nutritional knowledge and obesity: First, nutritional knowledge could prevent obesity, in which case the greater the nutritional knowledge, the lower the probability of being obese. Second, nutritional knowledge could also be reactive to obesity, in which case obesity may lead to greater nutritional knowledge. Kruger et al. [32] believes that nutritional knowledge is an important factor in promoting healthier eating habits and, consequently, maintaining an appropriate body weight, thus preventing obesity. People who are aware of the connection between poor nutrition and certain health conditions are more likely to follow a balanced diet and avoid excessive weight gain. Overall health and disease prevention has been significantly linked to food selection. Diets high in fiber may help to moderate caloric intake and benefit blood lipid profiles, thus decreasing the prevalence potential obesity-related diseases [33]. Adolescent and School Health Centers for Disease Control and Prevention explained potential health risks of obesity in childhood in the future. (a) Obese youth are more likely to have risk factors for cardiac disease, such as high cholesterol or high blood pressure. In a population-based sample of 5- to 17-year-olds, 70% of obese youth had at least one risk factor for cardiovascular disease. (b) Obese adolescents are more likely to have prediabetes, a condition in which blood glucose levels indicate a high risk for development of diabetes. (c) Children and adolescents who are obese are at greater risk for bone and joint problems, sleep apnea, and social and psychological problems such as stigmatization and poor self-esteem. Long term health effects are also reported. (a) Children and adolescents who are obese as adults are therefore more at risk for adult health problems such as heart disease, type 2 diabetes, stroke, several types of cancer, and osteoarthritis. For example, children who become obese as early as age 2 were more likely to be obese as adults. (b) Overweight and obesity are associated The Mediterranean Diet Quality Index (KIDMED) and Nutrition Knowledge Chapter 11 117 TABLE 1 KIDMED Test to Assess Mediterranean Diet Quality [27] Practice Score Eats one serving of fruit or drinks fruit juice every day +1 Eats a second serving of fruit every day +1 Eats fresh vegetables (salads) or cooked vegetables once a day +1 Eats fresh vegetables or cooked vegetables more than once a day +1 Eats fish regularly (at least 2 or 3 times/week) +1 Visits a fast-food establishment at least once a week 1 Likes vegetables +1 Eats pasta or rice almost daily (5 days/week) +1 Eats cereals or derivatives (bread, etc.) for breakfast +1 Eats dried fruit regularly (at least 2 or 3 times/week) +1 Uses olive oil at home +1 Does not eat breakfast 1 Eats a dairy product (milk, yogurt, etc.) for breakfast +1 Eat mass-produced cakes for breakfast 1 Eat two portions of yogurt and/or 40 g of cheese every day +1 Eat sweets and confectionery several times a day 1 with increased risk for many types of cancer, including cancer of the breast, colon, endometrium, esophagus, kidney, pancreas, gall bladder, thyroid, ovary, cervix, and prostate, as well as multiple myeloma and Hodgkin’s lymphoma [34]. To determine whether individuals’ daily diets are consistent with Mediterranean dietary patterns, the Mediterranean Diet Quality Index (KIDMED) was developed. KIDMED detects whether individuals’ daily diets are practically consistent with Mediterranean dietary patterns. The development of the KIDMED index is based on principles of Mediterranean dietary patterns while also acknowledging and drawing attention to principles that undermine it. The index ranges from 0 to 12, and is based on a 16-question test that could be self-administered or conducted via an interview with, for example, a pediatrician or dietitian. Questions denoting a negative connotation with respect to the MedDiet were assigned a value of 1, and those with a positive aspect were scored +1 [24] (Table 1). The following results were obtained through the accumulation of these points. 8 points indicates “optimal” diet quality 4–7 points indicates “average” diet quality (improvement needed) 3 points indicates “very low” (minimal) diet quality According to these results, people’s daily diets can easily be evaluated. The MD is advocated because of its health-promoting and disease-preventing characteristics; yet, ironically, during recent decades the MD has been gradually abandoned by Mediterranean populations, particularly younger generations [6,9–12]. In Spain, where the MD is widely adopted, it has recently been observed that adolescents have begun to abandon the MD. To address this, the development of educational programs aimed at parents and teachers is recommended; this in turn will allow the MD to spread among children and adolescents [35]. A relationship between the implementation of the MD among adolescents and their nutritional knowledge levels was found in Turkey, which has coasts along the Mediterranean Sea. Individuals with higher scores of nutritional knowledge were found to have parallel MD scores [29] (Figure 1). This situation does not differ by sex. Another study reported that there was no significant difference between KIDMED scores of children according to gender (P > 0.05) (Table 2) [36]. When nutritional knowledge and MedDiet scores of people older than age 35 were compared, it was found that those having a high nutritional knowledge score had higher MD scores as well (Figure 2) [37]. Montero [38] investigated the quality of sight-disabled children’s diets and found that only 11.9% had good-quality food consumption. 118 90 SECTION 1 The Mediterranean Diet: Concepts and General Aspects 82.15 FIGURE 1 KIDMED scores and nutritional knowledge scores of adolescent according to gender. Average scores of Mediterranean Diet Quality (KIDMED) and nutrition knowledge (NK) among boys and girls. Data is from Sahingoz and Sanlier [28]. 82.37 80 70 60 50 KIDMED score 40 NK score 30 20 10 5.72 5.57 0 Boys Girls TABLE 2 Diet Quality of Children by Sex (%) KIDMED Index Categories Boys Girls Poor-quality diet (score 3) 48.4 51.6 Average diet (score of 4-7) 49.3 50.7 Good-quality diet (score 8) 51.7 48.3 P ¼ 0.59. This material is adapted from Farajian et al. [36]. 40 36.9 35.3 32.9 33.8 35 30.9 FIGURE 2 Mediterranean Diet Quality scores by level of nutrition knowledge (NK) as a percentage of the total score. Data is from Bonaccio et al. [37]. 30.2 30 25 Low 0–4 20 High >4 15 10 5 0 Low NK Med NK High NK In another study, anthropometric measurements of children and adolescents were compared according to very low diet, need for dietary improvement, and an optimal KIDMED index; the study found that those with a low KIDMED index had a body mass index of 22.3 4.7 kg/m2; the body mass index of those with a moderate KIDMED index was 21.5 4.3 kg/m2, and for those with a good KIDMED index the body mass index was 19.1 3.47 kg/m2. The difference between those with good, moderate, and low scores were statistically significant (P < 0.05) (Figure 3) [39]. Lazarou et al. [40] reported that obese children had lower MD quality scores than other children, and the difference was found to be statistically significant (Table 3). The results of a comprehensive study conducted in European countries demonstrated that children living in Sweden had the highest MD quality score, and those living in Cyprus had the lowest score [41] (Figure 4). Belen and Perez [42] The Mediterranean Diet Quality Index (KIDMED) and Nutrition Knowledge Chapter 11 119 FIGURE 3 The lowest and the highest KIDMED scores according to body mass index (BMI). Data has been adapted from Kabaran and Gezer [39]. TABLE 3 Diet Quality of Children by Weight (%) KIDMED Index Categories Normal Weight (n ¼ 638) Overweight and Obese (n ¼ 188) Poor-quality diet (score 3) 40.7 37.8 Average diet (score of 4-7) 52.4 60.3 7.0 1.9 Good-quality diet (score 8) P < 0.05. This material is adapted from Lazarou et al. [40]. 60 50 40 30 Mediterranean score >3 20 10 0 n de we S (n y l Ita 9) ) ) 00 44 7 =1 (n = y an G m er 1 n= ( in a Sp (n 4 =1 y H un r ga 0) ) ) 86 60 97 22 (n = m u gi B el ) 1 n= ( ia Es n to ) 44 82 24 (n 6 =1 s 37 (n 6 =1 ru p Cy FIGURE 4 Mediterranean Diet Quality scores (percentages) among various countries. Data has been adapted from Tognon et al. [41]. investigated KIDMED indices of Spanish and migrant youth living in Spain according to their nationality. They showed that Asian young people were far from consuming an optimal diet, and northwest African young people had high KIDMED index values. However, differences between KIDMED indices were not statistically significant (P > 0.05), and it was emphasized that the nutrition of young people should be improved (Table 4). It also was reported that KIDMED indices of children living in rural and urban areas were not different, and optimal KIDMED indices were low in both populations [43]. In a similar study, KIDMED indices of children living in urban and semi-urban places were investigated. Scores of children living in semi-urban areas were higher, and the difference was statistically significant (P < 0.03) [36] (Table 5). In their study of 1140 children in Cyprus, Lazarou et al. [44] discovered that 37% of participants had a very low MD score. The study stressed the necessity of educating children about the MedDiet to improve the quality of their diets. 120 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 4 KIDMED Indices Among Migrant Youth in Spain Very Low-Quality Diet (%) (Score 3) Needs to Improve (%) (Score 4–7) Optimal Diet (%) (Score 8) Spain 6.6 55.7 37.7 Eastern Europe 0 66.7 33.3 Northwest Africa 0 57.1 42.9 Asia 16.7 83.3 0 Latin America 13.7 58.7 27.5 Data are adapted from the EAA Summer School eBook [42]. TABLE 5 KIDMED Scores of Children According to Place of Living KIDMED Score t P Urban 3.56 2.25 2.96 0.003* Semi-urban 3.75 2.28 This material is adapted from Farajian et al. [36]. Diseases such as cancer, cardiovascular diseases, obesity, and hypertension are strongly related to healthy eating patterns, behaviors, and dietary practices [45,46]. Studies indicated that individuals who have basic nutrition knowledge and a positive attitude apply these principles when selecting foods. Therefore, improving nutrition knowledge, attitude, and dietary practices through nutritional education may help to prevent or mitigate the aforementioned diseases [47–49]. Nutritional education is a lifelong education. An accurate nutritional education should start from the early ages of life and continue into adulthood until it becomes integrated into one’s natural behavior and lifestyle. Its implementation throughout life is important despite changing conditions. In these contemporary times, the spread of processed foods and the surge of fast-food consumption increases the importance of nutritional education. Knowing the requirements of one’s body and choosing the most appropriate foods to suit its needs allows one to be more effective with their energy intake. By preparing foods in accordance with healthy cooking principles, one can achieve the least nutritional loss possible, and having the appropriate knowledge of how to store and maintain these foods enables one to meet their body’s needs at that highest level using limited sources [50]. The MD is the culmination of centuries of tradition and acts as a perfect contribution to one’s health. Not only is it nourishing, it constitutes an important part of the world’s cultural heritage. Countries anywhere outside of the Mediterranean region can adopt the MD. This model can be suggested for countries aiming to develop their own healthier diet alternatives; from Americans to northern and eastern Europeans, all can benefit. Its standards of food consumption will help individuals to sustain a quality life by preventing obesity and other diseases. For any society aiming to develop a new nutritional style, the MD is an ideal choice not only for its nourishment but also for the delicious trademark taste of the Mediterranean. Parents and children alike should be taught about the importance of the consumption of olive oil, fruits, vegetables, and legumes. In addition, parents should increase their awareness about the importance of balanced nutrition. Using the MD to develop healthy eating habits in children could be the primary protective measure for minimizing the health problems they encounter as they grow older. The MD is also important for increasing one’s quality of life. Physical activity, which is a foundation of the MD pyramid, should be taught and spread via youth education programs. Individuals should be aware of the fact that physical activity is just as much a part of daily life as food and water. Doing some type of physical fitness as a hobby allows one to be not only physically, but also spiritually, healthy. SUMMARY POINTS l l Nutritional education is an education for life. Learning about the MedDiet through nutritional education may help to prevent or mitigate cancer, cardiovascular diseases, obesity, and hypertension. The Mediterranean Diet Quality Index (KIDMED) and Nutrition Knowledge Chapter 11 l l l 121 Training programs should be organized to increase the awareness of people regarding their dietary preferences. Fruit and vegetable consumption among the young and children should be encouraged using written and visual media. Fruits and vegetables must be processed as little as possible and consumed fresh within their season. Consumption of olive oil and fish should be encouraged. It can be suggested that meat should be consumed once or twice a month at most. REFERENCES € [1] Uçku R, Arslantaş D, Ozbabalık D, Sayıner D, Aslan D, Arslantaş A, et al. Yaşlı ve Hasta Bakım Hizmetleri. T.C. Anadolu Üniversitesi Yayını No: 2491. Açık€ oğretim Fakültesi Yayını No: 1462; 2012. 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Nutrition and food technology. Ankara, Turkiye: Detay Yayıncılık; 2009. Chapter 12 Socioeconomic Factors Affecting Adherence to the Mediterranean Diet in North Africa Karima El Rhazi, MD, PhD1, Vanessa Garcia-Larsen, PhD2 and Chakib Nejjari, MD, PhD1 1 Department of Epidemiology and Public Health, Faculty of Medicine of Fes, University Sidi Mohamed Ben Abdillah, Fes, Morocco. 2 Respiratory Epidemiology, Occupational Medicine, and Public Health, National Heart and Lung Institute, Imperial College London, London, UK. ABBREVIATIONS DES MD NA NCDs SES TEI UNESCO dietary energy supply Mediterranean diet North Africa noncommunicable diseases socioeconomic status total energy intake United Nations Educational, Scientific and Cultural Organization INTRODUCTION The Mediterranean diet (MD) is primarily characterized by an abundance of plant-based foods (fruits, vegetables, cereals, legumes, nuts, and seeds). Other key dietary elements are consumption of olive oil as a main source of fat, a low to moderate intake of dairy products, a moderately high intake of fish, a low to moderate amount of poultry and eggs, a low intake of meat and processed meat products, and a regular but modest consumption of wine, preferably during meals [1]. There are several dietary components of the MD that are recognized as having a beneficial effect on health and on the prevention of chronic diseases. The content of fats is largely comprised of monounsaturated fatty acids, with a small proportion coming from saturated fats. Intake of carbohydrates is mainly in the form of whole grains, legumes, and brown cereals, which are predominant sources of complex carbohydrates, and have a low glycemic index. Fruits and vegetables provide an excellent source of fiber and antioxidant components, such as vitamins and minerals, and a wide range of polyphenols (also found in olive oil) [2]. These antioxidants have been strongly associated with improved health status in several noncommunicable chronic diseases. Adherence to the MD has, therefore, been proposed as a protective factor in the prevention and modulation of chronic diseases, the most documented being cardiovascular (including markers metabolic disfunction), respiratory, and renal diseases [3]. The definition of MD is mostly documented for countries on the north side of the Mediterranean Sea, namely Greece, Spain, France, Italy, Malta, Croatia, Bosnia, Albania, and Cyprus. In spite of their geographic similarity, dietary patterns are heterogeneous between and within these Mediterranean countries [4]. Food habits have been well defined in most of the countries along the Mediterranean coast, and we know that neighboring countries have a more similar food pattern than those on opposite sides of the Mediterranean Sea [5]. WHAT IS THE ASSOCIATION BETWEEN ADHERENCE TO THE MD AND SOCIOECONOMIC STATUS? Assessing the factors that influence choices in dietary intake in a given population is important for supporting the development of public health policies on health and disease. Several studies relate socioeconomic status (SES) to nutritional status, which is usually considered a sensitive indicator of an individual’s quality of life [6,7]. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 123 124 SECTION 1 The Mediterranean Diet: Concepts and General Aspects Several studies have consistently associated dietary patterns in Mediterranean countries with emerging economies with better remunerated jobs [8–12] and with better educational level [13] independent of the method used to define diet. Parental educational level and SES have a marked effect on children’s and adolescents’ lifestyles and dietary habits [14]. Maternal educational level has been associated with diet quality [15] and with adherence to the Mediterranean dietary pattern [16]. Conversely, low maternal SES and educational level have been related to high consumption of sweets, high-fat bakery products, and sugary and salty snacks [17,18]. Food cost is another factor that may influence people’s dietary choices: following a Mediterranean dietary pattern is usually more expensive than following a Western one [19]. In some developed countries [20–22], fruit and vegetable intake was positively associated with a higher SES. Younger and more sedentary women, as well as those with a poorer educational level and background SES, tend to have a worse diet quality [23]. Conversely, others studies in less developed countries have reported that a higher SES was linked to a higher consumption of non-MD products (such as dairy, meat, and meat products), fish, and beverages and a lower consumption of precooked products [24,25]. Cultural habits are also a determinant of diet quality. Food cultures can vary to a great extent according to geographical area and populations. These differences include food availability, food choice, as well as culinary preparations. The United Nations Educational, Scientific and Cultural Organization (UNESCO) recently recognized the MD as an Intangible Cultural Heritage of Humanity; this together with the scientific evidence, reinforces the concept of the MD as a cultural and health model [2]. Indeed, scientific evidence emphasized that SES influences quality of diet but in different ways depending on the level of income of the country we all as local aspects that determine differences within the country. This is because of the current rapid nutritional transition that is occurring faster in today’s developing countries than was earlier observed in many industrialized countries. Moreover, the number of people who are overweight and obese is rapidly increasing in developing countries [26]. The situation in high-income countries, where the prevalence of obesity and related diseases is disproportionately high among groups with lower SES, is being repeated in middle-income countries. This variation in the impact of SES on the MD drew our attention to the question of how the interaction between dietary recommendations and sociodemographic characteristics observed in middle-income countries might also be influenced by socioeconomic variables in north Africa (NA). To date, this has been seldom studied. OBJECTIVE In this review, we describe the MD and the impact of SES on food habits in NA countries using available national dietary data, and we discuss the differences and similarities of the MD in NA with that in north Mediterranean countries. We mean by NA countries, the countries which are located at the south Mediterranean side. DEMOGRAPHIC AND SOCIOECONOMIC PROFILE OF THE SOUTHERN MEDITERRANEAN REGION According to the United Nations classification of geographical regions, NA comprises seven countries, namely Algeria, Egypt, Libya, Morocco, Sudan (and eventually South Sudan), Tunisia, and western Sahara. Although Egypt is a transcontinental country (sometimes referred to as Middle Eastern), the bigger part of the land stretches along the Nile River in NA, whereas the Sinai Peninsula is in Asia. In this review we refer to the NA countries next to the border of the Mediterranean Sea: Algeria, Morocco, Tunisia, Libya, and Egypt. According to the gross domestic product and other developmental indices [27,28], these are considered middle-income countries (Table 1). They also have in common a current demographic, epidemiologic, and nutritional transition, with increasing prevalence of obesity and other noncommunicable diseases (NCDs) (Table 2) [28–31]. The life expectancy at birth in NA has improved considerably in the past three decades, increasing between 10 and 20 years, depending on the country. For example, the current life expectancy in Egypt is 68.3 years and in Libya and Tunisia is 73 years. Urbanization levels vary across countries in NA. The percentage of people living in urban areas also has dramatically increased in the past 30 years. While in Libya 80% of the population lives in urban areas, in Egypt the urban population is 48%, and in Morocco the urban population is 58%. It is expected that the urbanization rate of the southern-rim countries will increase by nearly 13%, from 61.9% in 2000 to 74.4% in 2025. These indices have been accompanied by economic progress, which has been demonstrated by an increase in the global quality of life of the population, revenue, acquisition of material items, consumption of selected products, and energy [27]. Mediterranean Diet in North Africa Chapter 12 125 TABLE 1 Per Capita Gross Domestic Product (GDP), Purchasing Power Parity (PPP), and Socioeconomic Status (SES) in 2007 in the Middle East and North Africa Actual Per Capita GPD 2007 ($US) Gross National Income PPP 2007 ($US) SES Algeria 4011 7670 Upper-middle income Egypt 1630 5110 Lower-middle income Libya 11,639 16,130 Lower-middle income Morocco 2373 3960 Lower-middle income Tunisia 3483 7130 Lower-middle income Data are from the World Bank and Ref. [28]. TABLE 2 The Burden of Noncommunicable Diseases (%) over the Past Two Decades in North Africa Overweight Obesity Country 1990-2003 2010 1990-2003 2010 Algeria - 42.8 16.4 17.5 Egypt 47.5 69.8 14.5 34.6 Libya - 65.4 Morocco 28.8 48.5 16.0 17.3 Tunisia 52.5 55.9 24.3 23.8 30.8 Data taken from Ref. [28]. PATTERNS OF FOOD CONSUMPTION IN NA COUNTRIES The dietary habits and cuisine of NA has been greatly influenced by its history over the past 10 centuries. The Phoenicians are regarded as having brought sausages, whereas semolina (a key foundation wheat) was introduced by the Carthaginians. The Berbers (a traditional ethnic indigenous group spread across NA) adapted semolina into couscous, which is an almost ubiquitous food in the NA diet today. Olives and olive oils were introduced before the arrival of the Romans. The Arabs introduced a wide range of spices such as saffron, nutmeg, cinnamon, ginger, and cloves. The Ottoman Turks brought sweet pastries and other bakery products. Vegetables such as tomatoes, courgettes, and potatoes were introduced from Central and South America. This rich combination of cultural influences has shaped the MD in NA and partly explains why several of its components differ from the known European models of the MD. In addition to the historical evolution of the introduction of foods in NA, some ethnic groups, for example, the Barbers and the Copts, have helped to preserve local food traditions, but the effect of the Western diet has now reached most of NA. This effect has been gradually observed in the past 50 years; foods such as pizza, hamburgers, and carbonated drinks have been incorporated into the usual diet of children and adolescents in most NA countries. This shift in food choice has been accompanied by a decrease in the intake of whole-grain foods and an increase in the intake of red meat and its derivatives. Similarly, total energy intake (TEI) has increased some 1000 kcal in the past 5 decades [32]. Table 3 summarizes the traditional foods commonly consumed in NA. The combination of the traditions in the food habits and culinary preparations and the current trends in fast food intake make adherence to the MD in NA difficult to achieve. The MD pattern in NA also differs slightly from the most traditional concepts of the MD used in Europe (i.e., high intake of vegetables, oils, and fish and lower intake of foods with a high glycemic index and high in saturated fats). The main components of the south Mediterranean countries, particularly the Maghreb countries, are cereals, legumes, fruits, and vegetables. However, meats and meals prepared with the traditional butter, as well as pastries and sweets, are also staple foods. The main staple foods in the NA diet include cereals (grains), legumes, vegetables, fruits, and meats. Table 4 illustrates some of the typical dishes and meals prepared in these countries and their key ingredients. Plant-based foods are widely 126 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 3 Traditional Foods Commonly Consumed in North African Countries Food Group Foods Cereals Mhamsa soup Rice (whole grain, white, vermicelli) Kshari, Msemen Bram rice Wheat (semolina, couscous) Breads Khabz (Algerian flat bread) Zwane bread Semolina bread Whole wheat bread Assida/smida Thcicha/belboula Herbel, karantita (farinata, flat bread made from chickpea flour) EishMasri (glutinous pita bread) (Egypt) AishMerahrah (Egypt) Oils and fats Argan oil Corn oil Smen Olive oil Sesame seed oil Traditional fruits and nuts Fig, raisin, date, almond, groundnuts (peanuts), cashew nut, hazelnut, pistachio, chestnut, arcon (fruit oak) (usually considered to be spices for the type of flavor they add to dishes) Fruits Sweet and sour varieties of the same fruit, for example, sweet or sour pomegranate, sweet or sour apple Figs, dates, oranges, olives, apricots Dairy products Raib Lben (alone and combined with couscous) Camel milk Akkawi cheese Kessiah Ghreyba (butter biscuits in Libya) Cakes and sweets Keabkgzal Briouates Cakes in semolina (bessboussa, maqroute) Selou Zmita Chbakia Mkherqa, Sfenj Legumes Poichiches (whole and ground) Karann Bissara Fulmedames Lablabi Shahanful Vegetables Corchorus Bettrave Red courgette Maquoda Aromatic plants Mushrooms (white/black) Brussel sprouts Broccoli Cabbage Continued Mediterranean Diet in North Africa Chapter 12 127 TABLE 3 Traditional Foods Commonly Consumed in North African Countries—cont’d Food Group Foods Stuffed vegetables Onion Garlic (reduced intake in NA countries) Mulukhiyah (green leaves) Torly Potatoes Pastries Brik (stuffed), Algeria Briouat (stuffed-sweet), Morocco Fig roll (filled sweet pastry), Egypt Qatayef Soft/ hot drinks/ infusions Oualmasse (orange blossom water and beet juice) “Cooked coffee” (pan or zizwa) Herbal infusions (aelabdy, salmiya, cheeba) Meats Sheep, goat, camel, smoked meat (mhhalaa), chicken liver, beef liver, Usban (traditional Libyan sausage) Fish Fesikh (Egypt), sardines Eggs Home sourced (farmers) or industrial Other traditional foods Brik, sorghum Sugar/sweeteners White (ground), leaves, palm sugar, honey, Rub (Libya, syrup made from dates) TABLE 4 Traditional Meals, Sauces, and Beverages Commonly Consumed in North Africa Food Group Type of Meal and Its Main Components Semolina pasta and breads Couscous with meat and vegetables or raisins Couscous seffa with sugar and cinnamon Couscous with lben Mesfouf (couscous with butter) (Tunisia) Chakhchoura (Algeria); the dish consists of tearing small pieces of rougag (thin round flatbread) and mixing them with marqa, a stew Pastries Brik (stuffed) (Algeria) Briouat (stuffed-sweet) (Morocco) Fig roll (filled sweet pastry) (Egypt) Qatayef Soft/hot drinks Oualmasse (orange blossom water and beet juice) Pan or zizwa (“cooked coffee”) Herbal infusions (aelabdy, salmiya, cheeba) Meats Kocha (bread stuffed with meat) Kabab; meat that is cooked over or next to flames; large or small cuts of meat, or even ground meat; it may be served on plates, in sandwiches, or in bowls; sually made from lamb but other meats also used Chawarma; meat (cow, sheep, chicken, or goat) is grilled and then saved and served on a plate with side salads or in a wrap with tabbouleh, fattoush, taboon bread, tomato, and cucumber Smoked meat (mhhalaa) Continued 128 SECTION 1 The Mediterranean Diet: Concepts and General Aspects TABLE 4 Traditional Meals, Sauces, and Beverages Commonly Consumed in North Africa—cont’d Food Group Type of Meal and Its Main Components Hawawshi (Egypt); meat minced and spiced with onions and pepper, parsley, and sometimes hot peppers and chilies, placed between two circular layers of dough, then baked in the oven Chakhchoukha (Algeria); lamb stew with tomato and flat bread Cholent; a slow-cooked stew of meat, potatoes, beans, and barley Méchoui; whole sheep or a lamb spit-roasted on a barbecue Merguez; A very spicy, red sausage of mutton or beef Mrousia (Morocco); sweet and salty tajine with honey, cinnamon, and almonds Pastilla (Algeria and Morocco); traditional Berber Moroccan dish. Meat pastry made with squab (young domestic pigeon) Shishtaouk marinated and grilled, skewered cubes of chicken (Usba, Tunisia); a traditional kind of Tunisian sausage stuffed with a mixture of rice, herbs, lamb, chopped liver, and heart Tagines Tagine; one of the most typical Berber dishes, the Moroccan tagine is a rich stew of meat, chicken, or fish and most often includes vegetables or fruit. Vegetables can also be cooked alone. This preparation takes its name from the clay pot in which it is cooked. It is worth noting that the Tunisian “tagine” is a baked omelet/quiche-like dish. Eggs Shakshouka (Maghreb); eggs poached in a sauce of tomatoes, chili peppers, onions, often spiced with cumin Appetizers Bichak (Morocco); a stuffed and baked appetizer. It can be stuffed with pumpkin and jam for a sweet taste, or meat and cheese for a savory addition to a main meal Sauces/herb mixtures/pastes Chermoula; marinade of oil, lemon juice, pickled lemons, herbs, garlic, cumin, and salt most often used to flavor seafood Duqqa (Egypt); a mix of herbs and spices Tabil (Tunisia); Tunisian spice mixture consisting of ground coriander seed, caraway seed, garlic powder, and chili powder. The term can also refer to coriander by itself Tahini ; paste made from ground sesame seeds Toum (Egypt); garlic sauce with herbs, olive oil, lemon Tabbouleh; a sauce made with burgul, parsley, mint, spring onions, lemon juice Ras al-hanut (“the best of the shop”); considered the most famous mix of herbs in North Africa. It is a combination of at least 12 spices including pepper, meleguetta pepper, lavander, thyme, rosemary, cumin, ginger, nutmeg, mace, cardamon, fenugreek, and cinnamon, among others Baba Ghannoug; condiment made with eggplants, chickpeas, lemon juice, salt, pepper, parsley, cumin, and oil Shahanful; slow-cooked fava beans in water and crushed into a paste, which is then served alongside a diverse variety of foods Fragrant spices Cardamon, cinnamon, cloves, fenugreek, galangal, mastic, nard, nutmeg, rose petals, saffron, coriander Sour spices/chilies Vinegar from grapes, herbal vinegar (homemade), ground sumac, lemon juice, dried red chili, caraway (meridian fennel), harissa (chili and Serrano peppers) Soups Harira (Maghreb) consumed. Wheat is a staple food, eaten mainly in the form of bread (and its varieties) as well as its derivative semolina and couscous. In addition, a wide variety of seasonal vegetables and fruit are included in daily menus in NA. Citrus fruits, dates, and legumes also are commonly consumed, the most common being chickpeas, lentils, dried beans, and fava beans. Olive oil is produced locally and is the main sources of fat for either cooking or dressing. Dairy and meat are important components of the diet in NA, although these are not usually considered to be part of what is normally included in the European style of the MD. The main dairy products consumed are obtained from cows, goats, sheep, or camels and are a favorite choice during Ramadan. Fermented and acidified milk (lben), as well as curd, are occasionally consumed. In spite of the extensive coastline of the Maghreb countries, fish and seafood products are seldom consumed, probably with the exception of sardines. This is largely because of the high cost, which influences the irregular Mediterranean Diet in North Africa Chapter 12 129 supply of fish to the inland areas of the countries, which are the most populated. Meals often are prepared with variations of vegetable fats and butter, making them have a high caloric density. Intake of traditional foods coexists with a growing trend in the consumption of Westernized products—such as white bread, dairy products (major increases in milk and yogurt consumption, less so for cheese), sugars (major increase in sweet beverages), and added fats (mostly from butter)—and sedentary lifestyles among populations, communities, and people in the same household. The simply prepared foods of the traditional diet gave way to elaborate dishes requiring complex, lengthy preparation and generous amounts of clarified butter, with less attention given to the consumption of fresh vegetables. Deep-frying; the use of rich, composite sauces; and the growing popularity of rich sweetmeats and desserts containing nuts and soaked in heavy syrup considerably increased the overall intake of sugars and saturated fats. In many instances white bread replaced the traditional high extraction local bread. This type of bread is also a white bread which is made in the traditional way: we grind and sift the wheat to completely remove the crust. External influences leading to changes in food habits are more evident in larger urban communities and coastal towns. These influences affect socioeconomic groups in different ways: In high-income groups, some lifestyle-related risk factors have increased as a result of economic prosperity and development. At the same time, in low- and middle-income groups many unhealthy food consumption patterns emanate from poverty and undernutrition, especially during childhood. Thus, malnutrition problems caused by deficiency (avitaminosis, mineral deficiency, energy-protein deficiency), together with overweight and obesity, exist in the same populations and in the same individuals [33]. These countries must face the negative effects of the nutritional transition, with a rapid increase in overweight adults and children and in the prevalence of comorbidities [30,31,33–35], and at the same time keep up the fight against hunger, undernourishment, and infectious disease, coined “the double burden of disease” [33]. For some authors, the obesity epidemic observed in recent decades is due to a sociocultural and economic phenomenon [36,37]. Contrary to the health aspirations of developed countries, the prevalence of obesity in NA countries is still seen as a positive feature, which is probably reflected in the higher rates of overweight and obesity observed among the more privileged and most educated sectors of the population. The positive view of female obesity is particularly strong in the Sahara and Morocco. Even in urban areas, women tend to show more appreciation for a heavier corpulence rather than slimness. Several reasons converge to explain this trend. Some traditional communities see thinness in females as a sign of economic distress and low income. There is also the assumption that slimmer women might experience psychological problems derived from social expectations to fit certain models of misunderstood beauty. From a public health point of view, women in NA often are dedicated to looking after the family, cooking, and staying at home, leading to low levels of physical activity that might explain the increasing prevalence of overweight in NA [38]. SOCIOECONOMIC DETERMINANTS OF ADHERENCE TO THE MD IN NA Determinants of MD adherence in NA countries may be different from those observed on the north side of the Mediterranean Sea and even between these NA countries. This difference is related to the process of nutritional transition that is occurring more quickly and depends on the socioeconomic characteristics of population groups, which include, among others, accessibility to certain foods, differences in habits and lifestyles related to energy expenditure, and higher prevalence of overweight and obesity among more privileged groups [39]. Factors such as cultural differences, personal taste and traditions, education, geographic location, access to technology, and health and health attitudes influence food preferences and food availability. Notwithstanding, income and the economic environment assume a crucial role in the context of dietary pattern changes [40]. We summarize below the evidence of SES in NA with reference to dietary habits. The evidence is largely derived from observational studies and reports [41–45]. Studies about the MD are rare or nonexistent for some countries such as Lybia and are not primarily aimed at comparing dietary habits among countries. From a methodological point of view, and taking into account the cross-sectional nature of the evidence, these observations are coherent with the literature about countries on the north side Mediterranean Sea and reflect the chronological process of the food transition observed south of Mediterranean Sea. In Morocco, adherence to an MD is often poor, independent of age and education, especially in rural areas and among people living alone and those living in luxurious housing [44]. For people living in more rural areas, which are usually farther away from food markets, the low availability of a variety of foods and the many years of drought could explain their departure from traditional food habits. The exception to this are the traditional houses located in old medinas, for example, where people still keep their traditional lifestyle with easy availability and affordability of plant-based foods. Marital status seems to be an important determinant of adherence to traditional dietary habits. Married individuals have high adherence to traditional diets, which is observed often among single, widowed, and divorced people. In Moroccan 130 SECTION 1 The Mediterranean Diet: Concepts and General Aspects culture, although there is a huge cultural and historical reduction in the proportion of stay-at-home mothers and an increase in women working outside the home, the notion of a nuclear family and its related social obligations are very important. This has a strong influence on the regular preparation of traditional meals prepared and eaten with the family group. The dietary habits of younger people are more influenced by parental education. Adolescents whose parents have a low educational level have shorter height/age and have lower body mass index/age than those whose fathers have a high educational level. The energy provided by lipids is higher in adolescents whose fathers have a high educational background. The quality of fats consumed is better among boys whose fathers have low education [39]. In Tunisia [42], dietary habits seem to be more modernized in urban areas, in more affluent households, or in homes where the mother has a higher educational level. This modernized diet is more common among males, in Tunis and the center-east versus center-west of the country, particularly among adolescents whose mothers work outside the home. On the other hand, a more traditional diet is more associated with older subjects, to the middle eastern versus the middle western regions, and to the middle versus low economic level. Adolescents with a mother working outside the home with a higher education level followed this dietary pattern less often, whereas those attending school (vs. not) were more adherent to it. From a descriptive point of view, there is a gradient of nutritional transition correlated to a gradient of socioeconomic development within the country. For now, however, this main traditional-to-modern gradient is a characteristic dimension underlying the variability of food-based dietary intake among Tunisian youth. In Egypt, the higher social classes were much more readily influenced by the food habits of the foreign ruling elite. For people living in poverty or those with limited incomes, changes were related more to the need to cope with increasing food costs and to making nutritionally correct choices. By necessity, they depended on cheaper traditional foods and the subsidized local bread. For this group, the continued consumption of fresh dark green leafy and other vegetables was a nutritionally valuable traditional habit. A series of changes in the food habits and lifestyles of the rural populations followed the acceleration of rural development programs (and modernization), which gradually ended the traditional isolation of the fellaheen. Rural populations became exposed to a host of unfamiliar external influences and lifestyles with little prior preparation or guidance on how to deal with new situations and new food products [37]. In Algeria [41], during the 1990s, reduced purchasing power inherent in the economic crisis resulted in changes in household behavior in terms of food consumption. According to a survey of a sample of 2000 households conducted in 1998, 56% of households have, since 1993, restricted or even abandoned the use of some food products such as meat, fruit, and oil. From 1988 to 1998 commodity prices in all sectors have increased fivefold, whereas wages have not experienced this rate increase. Thus, during the period 1993–1996, prices increased annually an average of 25%, whereas wages in the public sector registered an average annual increase of 19%. Since 1998, the inflation rate has fallen (5% in 1998 to <1% in 2000). The share of spending on food decreased from 53% of household expenditure in 1998 to 45% in 2000–2001, according to data from the Survey of Household Spending. A nutritional policy aiming to provide for the basic food needs of people all over the country by lowering food prices through subsidizing major food groups that supply energy, such as cereals, vegetable oils, and sugar, has been established in Lybia [45]. Because cereals supply 45% of dietary energy, strategies for cereal fortification with iron should be considered. OTHERS RELATED FACTORS TO CHANGES IN DIETARY HABITS Urbanization affects food consumption patterns. Countries with higher gross national products and more urbanized populations consume larger amounts of energy from fats, sweeteners, and protein [46,47], and a larger amount of fruit and red meat, probably because of increased availability and affordability in urban areas [48]. This diet change can increase body weight, which is an independent risk factor for the development of many NCDs. Mass migration from rural to urban and peri-urban areas probably accounts for the high prevalence of NCDs among low-income groups living in urban and peri-urban areas. Mediterranean culture is centered on a strong patriarchal family. This has lessened in recent years, but family ties are still strong. Customs and family traditions greatly influence nutrition. CONCLUSION This review provides evidence regarding dietary habits and their socioeconomic determinants in emerging NA countries. Adherence to the MD is associated not only with SES but also with cultural attitudes and food availability in the Mediterranean area, which is quite heterogeneous. Between-country differences are present; these probably are linked to differences in natural and economic resources, as well as inherited cultural and religious traditions. Toward the end of the last century it became evident that the healthy diet of NA countries was progressively losing some of its essential elements and rapidly disintegrating. Mediterranean Diet in North Africa Chapter 12 131 SUMMARY POINTS l l l l l l l l l l The MD in the countries of NA comprises a different variety of foods than those normally reported in Europe. Traditions and cultural habits strongly coexist with more modern habits of feeding. Social structure, particularly what refers to the perceived role of women in the family and social expectations, are an important determinant of the way people eat in NA. Although the traditional diets in NA have many beneficial components related to an MD profile, economic progress is influencing the introduction of a more “modernized” or Western diet. NA countries show a rural-urban gradient in the access to plant-based foods, which is partly influenced by the remoteness of some areas and the dramatic geographical access. In spite of their proximity to the sea, consumption of fish is rare in NA countries. This is heavily determined by the cost of seafood in these countries, which increases if it is transported to the inner valleys where most people live. NA countries with more advanced economic development, such as Tunisia, also show a stronger influence of Westernized and processed foods in their diets. As observed in European countries with an MD, the prevalence of overweight and obesity are increasing in most NA countries. Although some of the etiological factors for this are common (higher TEI and sendentarism), NA countries have the added factors of cultural habits (daily family dinners) and perception of social status, which are strong determinants that are difficult to change. The strongest influence of Westernized dietary habits is observed in young adults and adolescents in NA. Older people tend to maintain a more traditional dietary pattern. The higher the educational level of adolescents and their mothers, the more prevalent processed food and Westernized dietary habits become. ACKNOWLEDGMENTS The authors are indebted to James Potts for his careful reading and editing of this chapter. El Rhazi is supported by the University of Fes. REFERENCES [1] Willett WC, Sacks F, Trichopoulou A, Drescher G, Ferro-Luzzi A, Helsing E, et al. Mediterranean diet pyramid: a cultural model for healthy eating. Am J Clin Nutr 1995;61:1402S–6S. [2] Serra-Majem L, Bach-Faig A, Raidó-Quintana B. 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INTRODUCTION Olive tree cultivation and olive oil production have accompanied humankind since time immemorial. In classical antiquity in the Mediterranean basin, no other plant was so important, useful, and revered as the olive tree; its olives, its wood, and the olive oil were indispensable goods for the wealth and well-being of the various peoples. The Holy Land and the island of Crete, in which were found the first presses, are considered to be the country of origin of olives. In ancient times, olive oil was marketed as edible oil, especially as a balsamic oil often enriched with essences. Olive oil was used for daily care at home, in the thermal baths, or in the gyms. Throughout the various civilizations, the olive tree and olive oil have occupied pivotal positions in the agricultural economy of Mediterranean countries and in their commerce with neighboring populations. OLIVE TREE The olive tree (Olea europea L.) belongs to the botanical family of Oleaceae. It is an evergreen plant that presents different varieties, called “cultivars,” according to the geographic areas in which it grows. The first traces of olive tree cultivation have been individualized in Palestine, and they go back to 3500 BC. The characteristic area of growth is that of the Mediterranean basin included between the 35 and the 45 parallel of north latitude; nowadays, however, olive groves can also be found in regions far from there, such as in the United States, or some countries in the southern hemisphere such as Argentina, New Zealand, and Australia [1]. The olive fruit is a drupe, but it differs from other drupes because it contains much lower sugar content and a higher concentration of oil. Drupe size and shape depend on the cultivar and growing conditions, with a weight at physiological maturity ranging from 1.5 to 4.5 g [2]. The olive tree’s fruit is composed of three parts (Figure 1): an external part called the exocarp or skin; a middle part, the monocarp or pulp, which is more or less colored and represents 70-80% of the weight of the whole fruit to physiological maturity, from which we obtain about 70% of oil; and an internal part called the endocarp, or stone, which represents 15–25% of the fruit and contains the seed (2.5–4%) and produces the remaining 30% of oil. In general, the olive presents the following composition: 50% water, 18–25% oil or greasy material, 20% carbohydrates, 6% cellulose, 1.5% proteins, and 1.5% ash (Figure 2). The olive ripening process proceeds through the following phases: around the months of May and June occurs the period of “mignolatura,” the formation of small, white-green flowers called “mignole” (olive blossoms); at the beginning of summer, is the “allegagione,” when the first green fruits develop; and at about the end of September is the “invaiatura,” when the classical drupes change to black-plum colored, typical of the ripe fruit. The trees bear on a 2-year cycle. A plentiful olive crop one year is followed by a dearth the next. New techniques and equipment for removing the oil from the fruit have been developed, but the object is the same as it was in the days of the Roman Empire: to exert sufficient pressure to obtain the oil without at the same time extracting impurities and deleterious substances. Olive oil in general is ideal for cooking, especially for searing and frying foods, because it can be heated to a higher temperature than almost all other cooking oils and fats without burning and without undergoing harmful chemical modifications. FROM OLIVE TO OLIVE OIL Olive oil is a vegetable oil that can be obtained directly from olive fruit using only mechanical extraction and that can be consumed without further treatments. The steps of the olive oil production process include collecting, washing, and crushing olives, malaxation of olive paste, centrifugation, storage, and filtration (Figure 3) [3,4]. Most attributes of olive oil quality are determined by the chemical composition and biochemical status of the olive fruit. To produce high-quality The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 135 136 SECTION 2 Components of the Mediterranean Diet Endosperm Mesocarp Episperm Endocarp Embryo Seed Pericarp Exocarp FIGURE 1 Structure of olive fruit. Esocarp or skin Drupe Physical composition Mesocarp or pulp (about 70% of the oil) Endocarp or stone (about 30% of the oil) Water 50% Drupe Chemical composition Oil 18-25% Other 30% FIGURE 2 Physical and chemical composition of olive oil drupe. FIGURE 3 Olive oil extraction. Olive harvesting Washing Crushing Extraction with traditional stone mill Extraction with continous hammer mill Malaxation Centrifugation Change of the “fiscoli” Mechanical pressure Marc Marc Oil + water Oil + water Centrifugation Centrifugation Oil Oil Water of vegetation Olive Oil Chapter 13 137 oil, the olives must be harvested without breaking the fruit skins, and fruit should be processed within 12–24 h of harvest. Olives are washed only if they have been harvested from the soil or have spray residues on the skin. The extra moisture can reduce extraction efficiency because water–oil emulsions form. Oils produced from washed olives are usually less desirable due to a reduction in bitterness and pungency and a less fruity flavor. Crushing olives is a physical process used to break the fruits’ tissues and release the oil contained in the vegetable cell vacuoles. The olive paste is currently prepared in industrial oil mills with either a traditional discontinuous stone mill or a continuous hammer crusher. The latter is mainly used in the olive oil industry, where oil is usually extracted by centrifugation [5]. Stone mills, the oldest method for crushing olives, consist of a stone base and upright millstones enclosed in a metal basin, often with scrapers and paddles to spread the fruit under the stones and to circulate and expel the paste. The slow movement of the stone crushers does not heat the paste and results in less emulsification, so the oil is easier to extract. The disadvantages of this method are the bulky machinery and its slowness, its high cost, and its inability to be continuously operated. Most stone mills have been replaced during the past 20 years because of their inefficiency. Hammer mills generally consist of a metal body that rotates at a high speed, hurling the olives against a metal screen. The major advantage of hammer mills is their speed and continuous operation, which translate into high output, compact size, and low cost. The rapid crushing of the fruit, however, creates more emulsification of the oil and water within the paste and higher temperatures. Oil produced from a hammer mill generally has a stronger flavor because the pulp is broken up more. The size of the mesh screen in a hammer mill is normally adjusted as the season progresses and the fruit becomes riper and softer [3]. Malaxation of the olive paste is carried out with a stainless steel device made of a semicylindrical vat with a horizontal shaft, rotating arms, and blades of different shapes and sizes. This vat is equipped with a heating jacket, circulating hot water to warm the olive paste [6]. The efficiency of malaxation depends on the rheological characteristics of the olive paste and the technological parameters of the operation, such as temperature and time. Malaxation prepares the paste for separation of the oil. It is done to reverse the emulsification that occurs during the crushing process and is particularly important if the paste was produced in a hammer mill. The mixing process optimizes the amount of oil extracted through the formation of larger oil droplets and a reduction of the oil–water emulsion. Optimally, the malaxator is designed to ensure thorough mixing, leaving no portion unmixed. The paste is slowly stirred for 30–60 min. The temperature of the paste during malaxation is very important. It should be warm, 80–86 F (26.6–30 C), which is still cool to the touch, to improve the viscosity of the oil and improve extractability. Temperatures more than 86 F (30 C) can cause problems such as loss of fruit flavors, increased bitterness, and increased astringency. The newest trend in the management of olive oil paste is to exclude oxygen, which can be done by either flooding the surface of the mixing tanks with nitrogen or vacuum exclusion of oxygen in special malaxation tanks. Limiting oxygen exposure is believed to reduce enzyme activity that can break down polyphenols, which are major flavor compounds of olive oil [7,8]. The next step is extracting the oil from the solids and fruit-water. The oil can be extracted by pressing, centrifugal decanters, selective filtration, or through combinations of the different methods. Pressing is one of the oldest methods of oil extraction. This method involves applying pressure to stacked filter mats, each of which is covered with about 0.5 inch (1.27 cm) of paste, that alternate with metal disks. A central hollow spike allows the expressed oil and water (olive juice) to exit in both directions. This process requires more labor than other extraction methods, the cycle is not continuous, and the filter mats can easily become contaminated, introducing fermentation and oxidation defects into the oil. Consequently, the use of traditional presses is becoming obsolete. Centrifugation is usually applied for a primary separation of the olive oil fraction from the solid vegetable material and vegetation water. This step may be carried out using the combination of two different systems: horizontal centrifugation (three- and two-phase decanters) and vertical centrifugation. Horizontal centrifugation using a three-phase decanter requires the addition of warm water to dilute the olive paste to facilitate the separation described above, whereas the two-phase decanter consists of “no-water” centrifugation plants for separating the oily phase from malaxed pastes without requiring the addition of warm water. It should be considered that the two-phase decanter requires a minimal moisture value in the olive paste (50%) to facilitate the separation process. When this value is not reached, a small amount of water is loaded into the decanter [9]. A vertical centrifugation system is used to separate the oily must obtained from horizontal centrifugation. In the Mediterranean area, olive oil is generally produced from September to February and stored in the mill until filtration and commercialization. As expected, after storage for 9 months the peroxide values increase and the total phenol content and oxidative stability of olive oil decrease [10]. OLIVE OIL PRODUCTION AND CONSUMPTION Olive oil comprises 4% of total vegetable oil production; its production worldwide is around 2,000,000 tons/year, and the Mediterranean countries contribute more than 95% of the world production. Today, olive plantations worldwide cover 138 SECTION 2 Components of the Mediterranean Diet Other Portugal Marocco Syria Turkey Tunisia Greece Italy 4% 1,6% FIGURE 4 Worldwide production of olive oil. Libya, Egypt, Israel, Jordan, Lebanon, Yugoslavia, Croatia, France, USA, Mexico, Argentina, Chile, Brazil, Peru, South Africa, China, New Zealand, Australia 2,5 % 3,7 % 4% 7,2 % 17 % 24 % 36 % Spain % of olive oil production about 9.4 million hectares, producing 1.5 million tons of table olives and 16 million tons of olives that are processed into 2.56 million tons of olive oil. Spain has about one-quarter of the world’s acreage, with 2.42 million hectares of olive trees under cultivation and 36% of oil production ( 800,000 tons/year), most of which comes from the region of Andalucı́a, which ranks Spain as the top producer. Italy is ranked second, with 1.43 million hectares and 24% of the world’s oil production ( 520,000 tons/year). Major Italian producers come from the regions of Tuscany, Umbria, Liguria, and Apulia. Greece is third, with 17% ( 400,000 tons/year) of the world’s oil production, and is fourth in world acreage, with 2.55 million acres of olive trees (1.03 million hectares). Together the big three produce 77% of the world’s olive oil (Figure 4) [1]. Olive oil consumption is linked to its production; Greece consumes the largest per capita amount of olive oil—over 26 L per person per year—Spain and Italy around 14 L, Tunisia, Portugal, Syria, Jordan, and Lebanon, around 8 L. Because of the increasing popularity of the Mediterranean diet, however, olive oil production and consumption is now expanding to nontraditional producing countries, such as the United States, Canada, Australia, South America, and Japan. High production costs, especially for harvest, have accelerated the redesign of olive orchards during the past 30 years [1]. The old, traditional olive production system in dry-farmed areas around the Mediterranean range in tree spacing from 25 to 60 feet (7.6–18.3 m) apart, giving 12–70 trees/acre (30–173 trees/ha). Their yields were somewhere between 0.5 and 2 tons/acre (1.1–4.5 tons/ha), with a long delay (15–40 years) before full production and severe alternate bearing. The trees are almost always harvested by hand or by beating the fruit off the trees with long poles onto nets. The modern high-density production system started around the early 1980s to reach full production faster in irrigated orchards, with tree densities of about 100–340 trees/acre (250–840 trees/ha). The overall benefits have been significant increases in yields per acre, usually double to triple what had been achieved previously. The orchards also come into full production sooner (>7–10 years), suffer less alternate bearing problems, and are more efficient to harvest. Superhigh-density production systems uses specific varieties of trees planted at spacings of 3–5 feet (0.9–1.5 m) within the row and 12–13 feet (3.7–3.9 m) between rows, for 670–1210 trees/acre (1655–2990 trees/ha). Here the varieties are very precocious, tend to produce a good crop every year, start bearing at an early age, and have excellent oil quality characteristics. However, the long-term production of the superhigh-density orchard system is not known, and this production systems required requires fairly flat ground and requires the ability to control tree vigor through pruning, fertility, management, and controlled deficit irrigation, with important capital investment. OLIVE OIL VARIETY AND COMPOSITION Composition of olive oil is related to its physical, chemical, and organoleptic characteristics [11]. The chemical composition of olive oil consists of major and minor components. The major components constitute the saponificable fraction, Olive Oil Chapter 13 139 TABLE 1 Fatty Acids Composition of Olive Oil Fatty Acids Common Name Numerical Symbol % Saturated Myristic acid 14:0 0–0.05 Palmitic acid 16:0 7.5–20 Margaric acid 17:0 0–0.3 Stearic acid 18:0 0.5–5 Arachidic acid 20:0 0–0.6 Behenic acid 22:0 0–0.2 Lignoceric acid 24:0 0–0.2 Total saturated 8–26.8 Monounsaturated Palmitoleic acid 16:1n7 0.3–3.5 Heptadecenoic acid 17:1 0–0.3 Oleic acid 18:1n9 55–83 Eicosenoic acid 20:1n9 0–0.4 Total monounsaturated Polyunsaturated Total polyunsaturated 55.3–87.2 o-6 Linoleic acid 18:2n6 3.5–21 o-3 a-Linolenic acid 18:3n3 0–0.9 3.5–21.9 which comprises 98–99% of the total weight of the oil and is formed mainly by triacylglycerides. Oleic acid (18:1n9) is the main component (55–83%) of the saponificable fraction; other fatty acids are palmitic, stearic, linoleic, and a-linolenic acids (Table 1). The fatty acid composition of olive oil, which is high in monounsaturated fatty acids, confers oxidative resistance to olive oil. Minor components, which amount to about 2% of the total oil weight, include more than 230 chemical compounds, for example, aliphatic and triterpenic alcohols, sterols, hydrocarbons, volatile compounds, and antioxidants [8,12]. The main antioxidants of olive oil are carotenes and phenolic compounds including lipophilic and hydrophilic phenols. While the lipophilic phenols, including tocopherols, can be found in other vegetable oils, some hydrophilic phenols of olive oil are not generally present in other oils and fats [13–15]. Minor components, mainly polyphenols, have been recently related to the healthy properties of olive oil and are associated with the oxidative stability and flavor characteristic of virgin olive oil. The content of the minor components of olive oil varies depending on the cultivar, climate, ripeness of the olives at harvesting, the processing system used, and storage conditions [3,16]. Consequently, rigorous controls of all olive oil processes are recommended to produce olive oil with a high phenolic content. During ripening, several metabolic processes take place in olives, followed by variations in the phenolic composition caused by the different biosyntheses and biotransformation pathways of phenolic compounds. The main phenolic compounds and their derivatives, including hydroxytyrosol, ligstroside aglycon, oleuropein aglycon, acetoxy-pinoresinol, and elenolic acid, increased in extra-virgin olive oil at the early stages of olive harvest, followed by a reduction of their concentrations at more advanced stages of maturity. Therefore, fruit harvested early produces olive oil with high polyphenol content and high oxidative stability. Moreover, after crushing the olives, several enzymes that can be activated are involved in the generation and transformation of phenolic compounds through the action of endogenous b-glucosidases followed by other chemical reactions. Malaxation of paste at a temperature lower than 30 C and time shorter than 60 min, centrifugation of paste using a two-phase decanter followed by vertical centrifugation with a minimum amount of water added, storage of extra-virgin olive oil for a short time and at a low temperature, and filtration using inert gases contributed to creating olive oil rich in phenolic compounds and to preserving its positive sensory attributes. Quality-related parameters of olive oil include acidity, peroxide index, and K232 and K270 parameters, together with total phenolic and fatty acid profiles. Table 2 shows characteristics of types of olive oil, including acidity, peroxide index, and K232 and K270 parameters. The level of acidity of olive oil is a measure of the free fatty acids, and it is conventionally expressed as a percentage of oleic acid. Acidity is 140 SECTION 2 Components of the Mediterranean Diet TABLE 2 Types of Olive Oil Type Free Acidity (% Oleic Acid) Peroxides Index (mEq O2/kg) K232 K270 DK Extra-virgin olive oil 0.8 20 2.50 0.22 0.01 Virgin olive oil 2.0 20 2.60 0.25 0.01 Ordinary virgin olive oil 3.3 20 N/A 0.30 0.01 Lampante virgin olive oil >3.3 No limit N/A N/A N/A Refined olive oil <0.3 5 N/A 1.10 0.16 Olive oil <1.0 15 N/A 0.90 0.15 Refined olive pomace oil <0.3 5 N/A 2.00 0.15 Olive pomace oil <1.0 15 N/A 1.70 0.20 influenced by olive ripeness, integrity, and harvest systems. Increased acidity changes the oil quality and decreases its commercial value. The peroxides index is an important indicator of good conservation of olive oil. Indeed, unsaturated fatty acids react with oxygen to form peroxides, releasing volatile compounds that give the characteristic rancid off-flavors. So, oil is good if the number of peroxides is low. Spectrofotometric indices of olive oil, K232 and K270, determined according to The Commission of the European Communities (1991, 2011), allow an understanding of whether oil is altered. Sensory or organoleptic analysis of olive oil has a significant importance in judging the quality of the final product. In fact, an unacceptable oil from an organoleptic point of view can be downgraded. Sensory or organoleptic analysis are carried out by specialized organizations. The committees are made up of a panel leader and a minimum of 8 and a maximum of 12 tasters. The set of different sensory perceptions allows these members to formulate a final judgement, which takes into account of the overall harmony of sensations. At the end of the assessments, each taster completes the data sheet and assesses the presence and the intensity of the good qualities (fruity, bitter, spicy) and the defects (rancid, mold, heating, winey, metal, etc.). Fruity is an important sensory characteristic that is derived from healthy, fresh, green or ripe fruit. The olive oil composition profile allows it to be distinguished from other plant oils [17]; however, a wide diversity can be found in composition among different varieties of olive oils [18–20]. Also, environmental and seasonal effects, as well as the olive oil processing methods, have been reported to affect olive oil composition [21], Different processing methods produce extra virgin, virgin, and ordinary olive oil. Virgin and extra-virgin olive oil are produced by direct first-pressing or centrifugation of the olives. Four criteria should be satisfied for an olive oil to be certified as extra-virgin olive oil: it must contain no more than 0.8% oleic acidity; it must be produced by mechanical extraction methods without chemicals and hot water; it must come from first cold-pressing; and it must have a perfect taste (Table 2). Virgin olive oils have a maximum free acidity of 2%, whereas olive oils with an acidity greater than 3.3% are submitted to a refining process in which some components, mainly phenolic compounds and, to a lesser degree, squalene, are lost [22]. By mixing virgin and refined olive oil, an ordinary olive oil is produced and marketed. After virgin olive oil production, the rest of the olive drupe and seed is processed and submitted to a refining process, resulting in pomace olive oil, to which a certain quantity of virgin olive oil is added before marketing. Olive trees present different varieties, called “cultivars,” according to the geographic areas in which they grow. The most common cultivars of olive tree are the Spanish Arbequina, which produces a very aromatic oil with a very light pungency and bitterness; the Italian Tuscan Frantoio, with a strong pungency and fruity flavor; the Apulian Coratina, which produces stable oils very high in polyphenols; as well as the Greek Koroneiki [16,23–26]. CONCLUSIONS The consumption of olive oil is associated with a low incidence of cardiovascular diseases, neurological disorders, and breast cancer. Several minor components have recently been related to the healthy properties, mainly polyphenols, of olive oil. These compounds also are associated with the oxidative stability and flavor characteristics of virgin olive oil. However, the phenolic composition of olive oil is influenced by complex multivariate interactions of genotype and agronomical, environmental, and technological factors. The qualitative and quantitative phenolic composition of olive oil is widely affected by many variables related to production processes, from the ripening stage of olive fruits to storage conditions. Olive Oil Chapter 13 141 In the past 20 years, the olive oil production has spread to nontraditionally producing countries, leading to an increased olive and olive oil trade supported by high- and superhigh-density production systems. However, this is not always accompanied by the production and marketing of high-quality olive oil, and many efforts have to be made to obtain olive oil with preserved content of minor components that are mainly responsible for the healthy properties of olive oil. SUMMARY POINTS l l l l l Minor components contribute to olive oil quality. Among vegetable oils, olive oil has unique nutritional and sensory characteristics. The chemical composition of olive oil consists of triacylglycerols and antioxidant compounds. Extra-virgin and virgin olive oil are oils obtained from the fruit of the olive tree solely by mechanical or other physical means. The chemical composition of olive oil is influenced by genotype and agronomical, environmental, and technological factors. REFERENCES [1] Vossen P. Olive oil: history, production, and characteristics of the world’s classic oils. Hortscience 2007;42(5):1093–100. [2] Nergiz C, Engez Y. Compositional variation of alive fruit during ripening. Food Chem 2000;69(1):55–9. [3] Di Giovacchino L, Sestili S, Di Vincenzo D. Influence of olive processing on virgin olive oil quality. Eur J Lipid Sci Technol 2002;104 (9–10):587–601. [4] Di Giovacchino L, Mucciarella MR, Costantini N, Ferrante ML, Surricchio G, Sestili S. Virgin olive oil storage and stability, In: Proceedings of the fourth international symposium on olive growing; 2002. p. 567–9, Vols 1 and 2, No. 586. [5] Servili M, Piacquadio P, De Stefano G, Taticchi A, Sciancalepore V. 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Chapter 14 Moderate Red Wine Consumption in Cardiovascular Disease: Ethanol Versus Polyphenols Marcello Iriti, PhD1 and Elena M. Varoni, DD, PhD1,2,3 1 Milan State University, Milan, Italy. 2 McGill University, Montreal, QC, Canada. 3 University of Eastern Piedmont ‘A. Avogadro’, Novara, Italy. INTRODUCTION Although a unique Mediterranean diet does not exist, some common traits in the dietary patterns of Mediterranean populations can be identified: high consumption of fruit, both fresh and dried (e.g., figs, raisins); raw and cooked seasonal vegetables; whole-cereal products; fish and seafood; dairy products, including goat and ewe milk in the fermented forms of cheese and yogurt; nuts; lean meat such as rabbit and chicken; aromatic herbs and spices; virgin olive oil as the main dressing; and moderate alcohol intake, particularly red wine (Tables 1 and 2). In the southern Mediterranean region, however, because of cultural and religious reasons, wine is replaced with black tea as a main source of polyphenols. The particular composition and variety of traditional Mediterranean diets have been proposed to explain the low prevalence of cardiovascular diseases and the improved life expectancy in some Mediterranean countries [1]. Even if wine is an alcoholic beverage, in the past two decades a plethora of beneficial biological and pharmacological activities have been ascribed to some grape and wine metabolites. In particular, polyphenols, a large group of phenylalanine derivatives, have been extensively investigated. These phytochemicals—divided into flavonoids (including anthocyanins), stilbenes (e.g., resveratrol), and proanthocyanidins (or condensed tannins) (Figures 1 and 2)—possess different molecular and biochemical targets in both healthy and pathological cells [2,3]. Furthermore, the diversity of grape and wine chemistry has been recently improved by the discovery of new bioactive molecules in these products, for example, melatonin and phytosterols (Figures 2 and 3). Melatonin is an indolamine traditionally considered a vertebrate neurohormone and is significantly present in grapevine products [4] (see Chapter 19) (Figures 2 and 3). In addition, three main phytosterols, that is, b-sitosterol, stigmasterol, and campesterol, which are effective hypocholesterolizing agents, have been detected in grapes and wine [5] (Figures 2 and 3). Therefore, it can be hypothesized that these and possibly other phytochemicals may maximize the healthy properties of polyphenols through additive and/or synergistic effects. Although polyphenols are considered the archetype of health benefits attributed to moderate red wine consumption, as demonstrated in a large number of in vitro and in vivo experimental models [2,3], evidence from humans is still inconclusive, possibly because of the presence of ethanol in wine, which can counteract or nullify to some extent the protective effects of polyphenols in conditions of heavy or binge (episodic heavy) wine intake [6]. In this chapter we briefly review the health-promoting effects of a moderate red wine intake in humans, focusing on the association with cardiovascular protection (Figure 4). INTERVENTIONAL STUDIES Interesting studies of humans have recently been conducted to elucidate the relation between several kind of alcoholic drinks (wine, spirits, beer) and cardiovascular risk. The different effects of red wine and gin consumption on inflammatory biomarkers of atherosclerosis were investigated in a randomized, crossover, single-blind clinical trial. For 28 days 40 healthy men (mean age, 37.6 years) consumed 30 g ethanol/day as either red wine (two 160-mL glasses) or gin (100 mL), a beverage with a very low polyphenol content. Blood sample analyses indicated that both wine and gin drinks had anti-inflammatory effects by reducing plasma fibrinogen (by 9% and 5%, respectively) and cytokine interleukin-1a The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 143 144 SECTION 2 Components of the Mediterranean Diet TABLE 1 Distribution of Recorded Adult Per Capita Consumption of Alcoholic Beverages (%) by World Health Organization Regions and Worldwide in 2005 WHO Region Spirits (%) Beer (%) Wine (%) Other (%) Africa 12.0 34.1 5.6 48.2 Americas 32.9 54.7 12.0 0.6 Eastern Mediterranean 25.2 37.8 5.7 31.3 European 34.6 37.1 26.4 2.5 Southeast Asia 71.0 25.5 2.5 1.0 Western Pacific 54.0 35.5 3.6 6.9 Worldwide 45.7 36.3 8.6 10.5 From the “Global Status Report on Alcohol and Death,” Geneva: WHO; 2011. TABLE 2 Recorded Adult (15 Years Old) Alcohol Consumption by Type of Alcoholic Beverages (% of Pure Alcohol) in Selected European Countries in 2005 Country Beer (%) Wine (%) Other (%) 5 22 73 0 France 20 17 62 1 Greece 26 24 49 1 Spain 13 45 36 6 Portugal 10 31 55 4 Turkey 35 60 5 0 Egypt 33 56 11 0 Morocco 13 50 37 0 5 63 32 0 Germany 20 53 27 0 Switzerland 18 31 50 1 United Kingdom 21 43 30 6 Norway 20 47 31 2 Sweden 17 39 44 0 Russian Federation 63 33 1 3 Ukraine 61 32 7 <1 Italy Tunisia Spirits (%) From the “Global Status Report on Alcohol and Death,” Geneva: World Health Organization; 2011. levels (by 21% and 23%, respectively). However, wine had two additional effects: (1) a 21% decrease in C-reactive protein (CRP); and (2) a 27% reduction of monocyte and endothelial adhesion molecules, that is, lymphocyte function-associated antigen 1 and Mac-1, as well as very late activation antigen 4 (32%), monocyte chemoattractant protein (46%) in monocytes, soluble vascular cell adhesion molecule-1 (17%), and intercellular adhesion molecule-1 (9%) [7]. Similarly, 4-week supplementation of red wine decreased plasma fibrinogen concentrations in 15 healthy male volunteers, who were instructed to drink 250 mL red wine/day (12% alcohol, 1.2 g/L total polyphenols, 0.64 g/L total anthocyanins, and 1.0 g/L Moderate Red Wine Consumption in Cardiovascular Disease Chapter 14 OMe OH OH OH OH O+ HO OH OMe HO O HO OH OH OH OH Malvidin 145 trans-Resveratrol O Quercetin OH HO O OH OH OH OH OH OH HO HO O O OH OH (+)-Catechin OH OH OH OH HO n O OH OH OH O Proanthocyanidins FIGURE 1 Typical bioactive polyphenols present in red wine include anthocyanidins (malvidin), stilbenes (resveratrol), flavonols (quercetin), flavan-3ols [(+)-catechin], proanthocyanidins or condensed tannins, which are oligo and polymeric derivatives of flavan-3-ol units. FIGURE 2 Major polyphenols (anthocyanins, resveratrol, and proanthocyanidins), phenolic acids (hydroxycinnamates), and new phytochemicals (phytosterols) are present in grape berries and seeds. total proanthocyanidins). However, the authors also found an increase of mean platelet volume as well as serum concentrations of inflammatory and endothelial cell activation markers, such as intercellular adhesion molecule-1, E-selectin, and interleukin-6 [8]. On the other hand, 150 mL red wine/day (15 g alcohol/day) for 3 weeks failed to reduce serum CRP concentrations and only slightly decreased fibrinogen concentrations in 87 Norwegian healthy volunteers 35–70 years old (mean age, 50.0 years) [9]. In another randomized, cross-over clinical trial, 20 g ethanol/day (1 glass [100 mL] of red or white wine at lunch and at dinner) were administered to 35 healthy women aged 20–50 years (mean age, 38.0 years) for 4 weeks. Both wines reduced 146 SECTION 2 Components of the Mediterranean Diet FIGURE 3 Recently discovered bioactive phytochemicals in wine are indoleamines (melatonin) and the phytosterols (b-sitosterol, stigmasterol, and campesterol). H N O O N H HO b-Sitosterol Melatonin HO HO Stigmasterol Campesterol FIGURE 4 Cardioprotective effects attributed to proper red wine consumption are caused by both alcoholic and polyphenolic components. Bioactive phytochemicals may also counteract the detrimental effects of ethanol. LDL, low-density lipoprotein; HDL, high-density lipoprotein; Apo A-1, apolipoprotein A-1. serum biomarkers of inflammation and endothelial activation in subjects, even if anti-inflammatory effects associated with moderate red wine consumption were higher than those produced by white wine, probably because of a different polyphenol content [10]. The moderate intake of red wine or an alcoholic beverage with a very low polyphenol content was investigated in terms of influence on serum antioxidant vitamins, antioxidant status, lipid profile, and oxidability of low-density lipoprotein (LDL) particles [11]. For 28 days 40 healthy men aged 30–50 years (mean age, 38.0 years) received 30 g ethanol/day as red wine (two 160-mL glasses at dinner) or gin (100 mL at dinner). In particular, compared to gin, wine intake reduced plasma superoxide dismutase activity, malondialdehyde, and oxidized LDL levels. Moderate Red Wine Consumption in Cardiovascular Disease Chapter 14 147 Using a similar study design (a randomized, cross-over clinical trial), Chiva-Blanch et al. [12] also demonstrated that red wine exerted greater protective effects on glucose metabolism and lipid profile than other alcoholic beverages. Sixtyseven men aged 55–75 years were instructed to consume gin (100 mL/day, containing 30 g ethanol); red wine (272 mL/day, containing 30 g ethanol and 798 mg total polyphenols); or de-alcoholized red wine (272 mL/day, containing 1.14 g ethanol and 733 mg total polyphenols) for 4 weeks. Participants were at high risk of cardiovascular disease, reporting a family history of premature cardiovascular disease and/or the presence of diabetes, hypertension, dyslipidemia, and overweight/ obesity. Plasma insulin and insulin resistance decreased after interventions with both wines; high-density lipoprotein (HDL) cholesterol, apolipoprotein (Apo) A-1, and ApoA-2 increased after red wine and gin intake. On the other hand, only red wine reduced lipoprotein plasma concentrations [12]. In a study using a similar design, the same authors showed that both ethanol and nonalcoholic compounds of red wine may regulate soluble inflammatory mediators in patients at high cardiovascular risk, whereas only phenolic compounds may modulate leukocyte adhesion molecules [13]. De-alcoholized red wine also decreased systolic and diastolic blood pressure in patients at high cardiovascular risk, and these variations correlated with increased concentrations of plasma nitric oxide, a potent vasodilator [14]. The relationship between body mass index (BMI) and plasma triglycerides was investigated in 42 Brazilian individuals (64% men; mean age, 46.0 years; mean BMI, 25.13 kg/m2) consuming 250 mL/day of red wine at meals for 2 weeks. In general, red wine increased plasma concentrations of triglycerides and the ratio of triglycerides to HDL-cholesterol. When subjects were divided into three categories according their BMI, the authors reported that individuals with higher BMI, although not obese, might be at higher risk for elevation of plasma triglycerides and the triglycerides-to-HDLcholesterol ratio after a short period of red wine consumption [15]. Possible prebiotic effects associated with red wine intake were recently suggested. Ten healthy male volunteers received red wine (272 mL/day), de-alcoholized red wine (272 mL/day), or gin (100 mL/day) for 20 days. Total fecal DNA analysis using real-time quantitative polymerase chain reaction revealed that, compared with baseline, daily consumption of red wine polyphenols significantly increased the number of Enterococcus, Prevotella, Bacteroides, Bifidobacterium, Bacteroides uniformis, Eggerthella lenta, and Blautia coccoides-Eubacterium rectale groups. In parallel, systolic and diastolic blood pressure and triglyceride, total cholesterol, HDL-cholesterol, and CRP concentrations decreased significantly. In addition to promoting the growth of probiotic bacteria, red wine polyphenols also inhibited pathogenic bacteria from the human microbiota, such as Clostridium spp. [16]. As previously mentioned, the health benefits of moderate wine consumption have been attributed, at least in part, to grape polyphenols. Protection against oxidative damage of a Mediterranean diet compared with a Western (or US) diet, with or without the concomitant intake of red wine, was assessed in 42 young adults (20-27 years old). Volunteers were randomly assigned either to the Mediterranean diet group or the Western diet group for 3 months, and they received 240 mL red wine/day only during the second month. The Mediterranean diet increased plasma vitamin C, b-carotene, and total antioxidant activity, whereas the Western diet increased vitamin E content. Wine supplementation, analyzed combining both diet groups, increased plasma vitamin C, b-carotene, uric acid, total antioxidant activity, plasma and urinary total polyphenols, and red blood cell glutathione and decreased plasma vitamin E and glutathione. The Western diet group also showed higher concentrations of 8-hydroxy-20 -deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, in DNA from peripheral blood leukocytes and plasma nitro-tyrosine, a marker of oxidative protein damage, when compared with the Mediterranean diet group. Wine intake significantly reduced 8-OHdG and plasma nitro-tyrosine in both diets, particularly in the Western diet group. The authors concluded that moderate red wine consumption counteracted the oxidative damage caused by the Western diet [17]. Furthermore, even if on few participants, it was shown that 300 mL/day of alcohol-free red wine for 1 week increased the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione reductase) in eight volunteers aged 25–40 years (mean age, 28.0 years) following a low phenolic diet [18]. However, despite a general consensus attributing antioxidant benefits in healthy volunteers to sustained wine consumption, in a review article, Covas and colleagues [19] concluded that there is no evidence at present that supports wine consumption as being associated with antioxidant benefits other than counteracting a possible pro-oxidative effect of the alcohol. On the contrary, data on the antioxidant protective effect of red wine in oxidative stress situations are promising. These conclusions are consistent with our recent results showing that drinking red wine was not associated with a reduced salivary antiradical capacity, which conversely was greatly improved by the intake of a capsule of red wine extract [20]. Finally, perspectives on moderate red wine intake in secondary prevention of cardiovascular diseases were evaluated. Thirty-nine patients after myocardial infarction (32 men and 7 women; mean age, 65.0 years) were divided into two groups: red wine drinkers, who were administered 250 mL red wine/day (3.81 g/L total polyphenols) at main meals for 2 weeks, and water drinkers. During this period, all subjects received a Western prudent diet inspired by the Mediterranean diet principles. Moderate red wine intake reduced total cholesterol and LDL-cholesterol and improved erythrocyte membrane fluidity [21]. 148 SECTION 2 Components of the Mediterranean Diet OBSERVATIONAL STUDIES The beneficial properties of red wine have been known for more than three decades. In 1979, St. Leger and colleagues [22] first described a significant inverse relationship between wine consumption and risk of death from coronary heart disease. More recent evidence of a health-promoting effect of red wine on cardiovascular health emerged from many epidemiological studies. In a prospective study, Djoussé and collaborators [23] investigated the association between alcohol consumption and risk and mortality for cardiovascular diseases in 26,399 “non-Mediterranean” participants from the Women’s Health Study, with a mean follow-up of 12.2 years. Compared with abstainers, alcohol intake of 5–15 g/day was associated with 26%, 35%, and 51% lower risk of cardiovascular disease, total mortality, and cardiovascular disease-related mortality, respectively. In moderate drinkers, 86% of reduced cardiovascular disease risk was explained by effects on lipids, glucose metabolism, inflammatory/hemostatic factors, and blood pressure. Furthermore, no differential association for beverage type (i.e., beer, wine, or spirits) and studied outcomes was observed by the authors [23]. Similarly, in a cohort (N ¼ 31,367) of US adult men from the prospective Aerobic Center Longitudinal Study, fewer than six drinks/ week reduced the risk cardiovascular disease mortality by about 29%. When alcohol consumption was stratified by type (wine, beer, and liquor), no difference was observed [24]. The relationship between alcohol intake and cardiovascular risk factors was investigated in a large French population, the urban Paris-Ile-De-France Cohort, composed of 149,773 subjects (97,406 men and 52,367 women with a mean age of 47.6 15 and 47.0 12 years, respectively). The subjects were divided into four groups according to alcohol consumption: never, low (<10 g/day), moderate (10-30 g/day), and high (>30 g/day). With the exception of the subgroup of young subjects (<30 years old), alcoholic beverages mainly consisted of wine. In addition, wine consumption increased with age, whereas that of beer and appetizers decreased. Moderate alcohol drinkers showed more favorable clinical and biological profiles associated with lower cardiovascular risk, including low BMI, waist circumference, heart rate, blood pressure, fasting triglycerides, fasting glucose, and plasma LDL-cholesterol and higher levels of plasma HDL-cholesterol [25]. The Italian Longitudinal Study on Aging is a cross-sectional, multicenter study of 1896 men aged 65–84 years, mainly those who consume moderate intake of wine (98%) as a lifelong habit. Participants were divided into six alcohol consumption groups: lifelong abstainers and those who drink 12, 13–24, 25–47, 48–96, and >96 g/day. Among drinkers, mean daily intake of alcohol from wine, beer, and spirits were 26.7, 0.6, and 3.8 g/day, respectively. Long-term moderate drinkers showed lower levels of systemic inflammatory markers, better hematological parameters, and safer metabolic and glycemic profiles. In particular, alcohol consumption in older age was associated with lower values of fibrinogen and insulin resistance and higher levels of HDL-cholesterol and ApoA-1, even if it was also associated with higher LDLcholesterol and systolic blood pressure [26]. The relative importance of the main components of the Mediterranean diet in producing the inverse association between increased adherence to this dietary style and all-cause mortality was investigated in the Greek segment of the European Prospective Investigation into Cancer and nutrition (EPIC). The population of this prospective cohort study consisted of 23,349 men and women without a previous diagnosis of cancer, coronary heart disease, or diabetes. After a followup of 8.5 years, the individual components of the Mediterranean diet that contributed to this association were moderate ethanol consumption (23.5%; >10 and <50 g/day for men, >5 and <25 g/day for women, mostly in the form of wine at main meals); low intake of meat and meat products (16.6%); high consumption of vegetables (16.2%), fruit and nuts (11.2%), and legumes (9.7%); and high monounsaturated-to-saturated lipid ratio (10.6%) [27]. More specifically, the association between alcohol consumption and coronary heart disease was investigated in the Spanish EPIC cohort. Participants included 15,630 men and 25,808 women, and the median follow-up period was 10 years. Subjects who reported an alcohol intake of >5 g/day at either 20, 30, 40, or 50 years of age, but not during the 12 months before recruitment, were categorized as former drinkers. Never-drinkers were defined as subjects who did not report any consumption >5 g/day at any age and 0 g/day in the 12 months before recruitment. Alcohol intake then was classified in six categories for men (former drinkers, never-drinkers, low intake of 0–5 g/day, moderate intake of 5–30 g/day, high intake of 30–90 g/day, and very high intake of >90 g/day) and five categories for women (former drinkers, never-drinkers, low intake of 0–5 g/day, moderate intake of 5–30 g/day, and high intake of 30–90 g/day). Among men aged 29–69 years, moderate, high, and very high ethanol consumption was associated with reduced cardiovascular risk and a more than 30% lower incidence of coronary heart disease. This association was only statistically significant in men, probably because of the low number of coronary heart disease cases in women (n ¼ 128). The type of alcohol consumed (wine, beer, fortified wines) did not affect hazard ratios [28]. The Turkish Adult Risk Factor study prospectively assessed the long-term effects of alcohol consumption on different outcomes in 3443 men and women (mean age, 47.6 years) included at baseline and followed-up for a mean of 7.4 years. End points included overall mortality, coronary heart disease, diabetes, and metabolic syndrome. Daily alcohol intake Moderate Red Wine Consumption in Cardiovascular Disease Chapter 14 149 was categorized as follows: light drinking, <1 drink (1 unit of alcohol, i.e., 30 mL spirits or 300 mL beer or 120 mL wine); moderate drinking, 1–3 units of alcohol; and heavy drinking, >3 units. Heavy alcohol consumption increased the risk for diabetes, coronary heart disease, and, in men, all-cause mortality and metabolic syndrome, but there was no strong statistical significance. Conversely, moderate intake of alcohol was not associated with any adverse outcome, but it was associated with a borderline decrease in cardiovascular risk and overall mortality and, in women, with a slightly significantly lower risk for metabolic syndrome. Interestingly, 62% of ethanol consumed in Turkey is beer, more than 30% as spirits, and only 5% as wine—a very different drinking pattern compared with populations of southern Europe, where wine predominates [29]. Association of alcohol consumption and cardiovascular disease outcomes has been investigated in systematic reviews and meta-analyses. Among moderate alcohol drinkers compared with nondrinkers, data from 84 eligible studies reported a relative risk of 0.75, 0.71, and 0.75 for cardiovascular disease mortality, incident coronary heart disease, and coronary heart disease mortality, respectively [30]. In another meta-analysis, a previously reported J-shaped dose-response relationship between wine and alcohol consumption and the risk of cardiovascular events and all-cause mortality was confirmed [30]. In a J-shaped curve, regular and moderate wine drinking at the nadir of the J, is beneficial, whereas abstinence and heavy intake, on the short and long arms, respectively, are both detrimental, although to a different extent [31]. A meta-analysis of 34 prospective cohort studies, with more than 1 million subjects and almost 100,000 deaths by any cause, showed a J-shaped association between self-reported alcohol consumption and mortality [32]. Another study reported that the maximum intake of wine at which protection was still apparent decreased from 72 to 66 or 41 g/day when either combined fatal and nonfatal vascular events or cardiovascular mortality or total mortality were considered as end points, respectively. The minimal doses of wine at which the maximal protection could be obtained were 21, 24, and 10 g/day for the three respective end points [33]. The same authors described a J-shaped dose-response curve in patients with cardiovascular risk; light to moderate alcohol consumption (5-25 g/day) was significantly associated with a lower incidence of cardiovascular and all-cause mortality [34]. Finally, a meta-analysis of interventional studies was also recently carried out. The effects of alcohol consumption on 21 biomarkers associated with risk of coronary heart disease in adults without known cardiovascular disease were investigated. Alcohol significantly increased the levels of HDL-cholesterol, ApoA-1, and adiponectin, but a dose-response relationship was observed only for alcohol and HDL-cholesterol. Alcohol decreased fibrinogen concentrations but did not affect triglyceride levels. Analyses were stratified by type of beverage (wine, beer, and spirits), but results were similar [35]. CONCLUSIONS The studies examined in this chapter suggest that red wine may have an “added value” compared to other alcoholic beverages, particularly spirits. Regarding cardioprotection, experiments using moderate intake of wine, de-alcoholized wine, and spirits show related to health benefits deriving from polyphenols and, possibly, other bioactive phytochemicals that may counteract the detrimental effects of ethanol. On the other hand, because of the positive effects of low amounts of alcohol on HDL-cholesterol and hemostatic factors, ethanol and polyphenols may also exert additive and/or synergistic protective effects on the cardiovascular system [36,37]. Even if results from clinical trials represent the highest level of evidence in medicine, however, studies of humans have some intrinsic and environmental limitations. For interventional studies, wine administration is obviously subject to many ethical limitations in both healthy and diseased subjects. Similarly, observational studies have to be properly interpreted because of many biases and confounding factors so the random effect and cause–effect relationships are not misinterpreted. For instance, Mediterranean populations seem to be more physically active than the north European counterpart, probably because of better climatic conditions predisposing open-air physical activity. Another example is related to wine drinkers, who seem to be more fanatical about health than those who drink beer or spirits. These aspects may “confound” the scenario in which the clinical studies are conducted. To conclude, we will further clarify the issue of “regular, moderate red wine consumption at main meals.” This phrase indicates that both pattern and amount of wine intake are relevant. Episodic heavy (binge) drinking is considered more harmful that consumption of two or fewer drinks per day (but more than zero drinks) for men and one or less than one drink per day (but more than zero drinks) for women [38,39]. Last, red wine polyphenols, independent of ethanol, reduced the pro-oxidant effects of fat-rich meals, reducing the postprandial susceptibility of LDLs to oxidation [40]. SUMMARY POINTS l Red wine is an important component of the Mediterranean diet that may contribute to the healthy properties of this traditional dietary style. 150 l l l l SECTION 2 Components of the Mediterranean Diet Regular low to moderate red wine consumption at main meals may reduce the risk of cardiovascular morbidity and mortality. Two major components of red wine are relevant for human health: ethanol and polyphenols. It seems that, in conditions of proper drinking, polyphenols and alcohol may exert an additive/synergistic activity on cardiovascular systems. Phytochemicals recently discovered in grapes and wine, that is, melatonin and phytosterols, may maximize the health benefits of polyphenols through additive/synergistic interactions. REFERENCES [1] Iriti M, Vitalini S. Health-promoting effects of traditional Mediterranean diet—a review. Polish J Food Nutr Sci 2012;62:71–6. [2] Iriti M, Faoro F. Health-promoting effects of grape bioactive phytochemicals. 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Circulation 2002;105:2836–44. [33] Di Castelnuovo A, Costanzo S, Bagnardi V, Donati MB, Iacoviello L, de Gaetano G. Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies. Arch Intern Med 2006;166:2437. [34] Costanzo S, Di Castelnuovo A, Donati MB, Iacoviello L, de Gaetano G. Alcohol consumption and mortality in patents with cardiovascular disease. J Am Coll Cardiol 2010;55:1339–47. [35] Brien SE, Ronksley PE, Turner BJ, Mukamal KJ, Ghali WA. Effect of alcohol consumption on biological markers associated with risk of coronary heart disease: systematic review and meta-analysis of interventional studies. Br Med J 2011;342:d636. [36] Hansel B, Kontush A, Bruckert E. Is a cardioprotective action of alcohol a myth? Curr Opin Cardiol 2012;27:550–5. [37] Li H, F€ orstermann U. Red wine and cardiovascular health. Circ Res 2012;111:959–61. [38] Stockley CS. Is it merely a myth that alcoholic beverages such as red wine can be cardioprotective? J Sci Food Agric 2012;92:1815–21. [39] Chiva-Blanch G, Arranz S, Lamuela-Raventos RM, Estruch R. Effects of wine alcohol and polyphenols on cardiovascular disease risk factors: evidences from human studies. Alcohol Alcohol 2013; http://dx.doi.org/10.1093/alcalc/agt007. [40] Natella F, Ghiselli A, Guidi A, Ursini F, Scaccini C. Red wine mitigates the postprandial increase of LDL susceptibility to oxidation. Free Radic Biol Med 2001;30:1036–44. Chapter 15 Beer: Beneficial Aspects and Contribution to the Mediterranean Diet Sara Arranz, PhD1,2, Gemma Chiva-Blanch, PhD1,2, Palmira Valderas-Martinez1,2, Rosa Casas1,2 and Ramon Estruch, MD, PhD1,2 1 Department of Internal Medicine, Hospital Clínic, Institut d’Investigacions Biome´diques August Pi i Sinyer (IDIBAPS), University of Barcelona, Barcelona, Spain. 2 CIBER CB06/03 Fisiopatologıá de la Obesidad y la Nutrición, (CIBERobn), Girona, Spain. ABBREVIATIONS BP CHD CVD HDL LDL MD MetS PAD T2DM blood pressure coronary heart disease cardiovascular disease high-density lipoprotein low-density lipoprotein Mediterranean diet metabolic syndrome peripheral arterial disease type 2 diabetes mellitus MEDITERRANEAN DIET Definition of the Mediterranean Diet “The Mediterranean diet constitutes a set of skills, knowledge, practices and traditions ranging from the landscape to the table, including the crops, harvesting, fishing, conservation, processing, preparation and, particularly, consumption of food.” This is the definition of Mediterranean Diet (MD), which was recognized by the UNESCO as an Intangible Cultural Heritage in 2010 [1]. UNESCO does not consider the MD as merely a collection of some selected foods but ascribes a cultural promotion role to the eating habits typical of the Mediterranean basin, which deserve special mention, as plainly stated in the description of the MD: “The Mediterranean diet encompasses more than just food. It promotes social interaction, because communal meals are the cornerstone of social customs and festive events. It has given rise to a considerable body of knowledge, songs, maxims, tales and legends. The system is rooted in respect for the territory and biodiversity, and ensures the conservation and development of traditional activities and crafts linked to fishing and farming in the Mediterranean communities of which Soria in Spain, Koroni in Greece, Cilento in Italy and Chefchaouen in Morocco are examples. Women play a particularly vital role in the transmission of expertise, as well as knowledge of rituals, traditional gestures and celebrations, and the safeguarding of techniques.” The MD is considered a healthy eating pattern typical of Mediterranean countries (Crete, Greece, southern Italy, Spain, Morocco, and all countries along the Mediterranean coast). Although there is no single MD, its main characteristics are (a) relatively high consumption of fats (up to 40% of total energy intake), mainly as olive oil/monounsaturated fatty acids (MUFA; more than 20% of total energy intake) used both for cooking and dressing dishes; (b) high consumption of unrefined cereals/grains, fruits, vegetables, legumes, and nuts; (c) moderate to high consumption of fish; (d) moderate to low consumption of white meat (poultry and rabbit) and dairy products, usually as yogurt or cottage cheese; (e) low consumption of red meat, processed meats, and meat products; and (f) moderate consumption of alcohol, usually in the form of red wine or beer with meals. This dietary pattern and proportions of different foods are shown graphically as a food pyramid, which is periodically updated and includes other aspects of lifestyle such as exercise, sociability, and sharing food around a table with family and friends [2]. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 153 154 SECTION 2 Components of the Mediterranean Diet The MD as a Dietary Pattern in Health Promotion: Clinical Evidence It has been estimated that chronic diseases such as coronary heart disease (CHD), cerebrovascular disease, diabetes mellitus (DM), and lung, colorectal, breast, and stomach cancers are responsible for over 40% of all deaths in developed countries [3]. Numerous epidemiological studies support evidence that all these conditions are largely preventable with hygienicdietary measures [4,5]. For this reason, measures aimed at achieving a healthy diet and lifestyle should always be an essential ingredient of any prevention or treatment. Although this seems obvious, these measures are frequently omitted in clinical practice [6]. Specifically, in the field of primary care, clinical practice usually focuses on the prevention of hard end points using treatments such as lipid-lowering drugs, antihypertensive drugs, and others instead of educating toward modifying lifestyles. A more widespread use of hygienic-dietary measures not only decreases drug spending but also reduces the adverse effects arising from the extensive use of drugs, which would result in a huge benefit for the public health system [7]. The positive results of trials with hard end points (incident cases of cardiovascular disease [CVD], cancer, or DM) as the primary outcome variable and usually funded by industry have undoubtedly contributed to the widespread use of “preventive” drugs. However, there are scarce trials of the same type with results focused on not only intermediate markers but also on these hard end points based on hygienic-dietary measures to promote a healthy diet. Although medicine is currently based on evidence, it is paradoxical that usual clinical practice recommendations for a healthy diet are based on decreasing total fat in the diet [8]. However, epidemiological evidence still remains insufficient to recommend the reduction of all fats, as in the National Cholesterol Education Program of the United States. Moreover, a MD rich in vegetable fats (MUFAs and polyunsaturated fatty acid) may constitute a theoretically excellent healthy diet. Indeed, a diet low in total fat may even be counterproductive. If there is an excessive reduction in fat intake, the main source of energy becomes carbohydrates, and given the abundant availability of refined carbohydrate-rich foods with a high glycemic load, this kind of diet increases the risk of the development of insulin resistance and DM, two very important risk factors in CVD [9,10]. To reduce total fat intake, major reviews of diet and CVD recommend dietary patterns that are high in MUFAs as the main source of lipids [11–14], are rich in whole grains as the main source of carbohydrates, have high intake of fruit and vegetables, and have adequate consumption of omega 3 fatty acids, as in the MD; these dietary patterns provide greater protection against ischemic heart disease and stroke. The best evidence available to examine the association between fat intake and the incidence of CHD is the results of several observational studies. A cohort of 44,000 health professionals followed for 6 years showed that the increased risk of CHD associated with the consumption of saturated fat disappeared after adjusting for fiber intake [12]. In a female cohort of nurses including 80,000 women followed for 14 years, similar results were found, reporting only a small negative, albeit nonsignificant, effect for saturated fat (relative risk 1.17, 95% confidence interval 0.97–1.41) and absolutely no effect for total fat, whereas MUFAs decreased the risk of heart disease [11]. Later, in the same female cohort but with more than 1.5 million person-years of follow-up, neither total nor saturated fat was found to influence cardiovascular risk, and only high consumption of artificially hydrogenated (trans) fats increased the risk of CVD [13]. A meta-analysis of prospective studies published in 2010 [14] confirmed that there is no evidence to argue that saturated fat intake is a cardiovascular risk factor, supporting the ineffectiveness of the paradigm of a low-fat diet recommended by the National Cholesterol Education Program. Greece is also a country with a traditional diet rich in fats, fruits, and vegetables but low in meat, similar to traditional Spanish cuisine. Trichopoulou et al. [5] conducted a prospective population-based investigation involving 22,043 adults in Greece and reported that greater adherence to the traditional MD was associated with a significant reduction in total mortality. In Spain, Martı́nez-González et al. [7] included 342 subjects in a case-control study to quantify the risk reduction of incident myocardial infarction provided by a Mediterranean dietary pattern. The results support the hypothesis that an MD that emphasizes the intake of olive oil, fiber, fruits, vegetables, fish, and alcohol; reduces meat and meat products; and excludes refined cereals can be an effective measure for reducing the risk of myocardial infarction. Subsequently, two observational cohort studies in Spain confirmed the inverse association between adherence to the MD and the incidence of fatal and nonfatal CVD in initially healthy middle-aged adults [15] and significantly reduced CHD risk, supporting its role in the primary prevention of CHD in healthy populations [16]. The next step in evidence-based medicine to assess the protective role of a healthy diet is to conduct randomized intervention studies that provide the highest scientific level of evidence. Accordingly, the Lyon Diet Heart Study [17] significantly contributed to the effectiveness of the Mediterranean dietary pattern. Despite being a highly referenced randomized clinical trial, it has serious methodological limitations [18] that restrict its usefulness as a basis for public health recommendations. These limitations are that (a) it is only applicable to secondary prevention because of the analyses of Beneficial Contribution of Beer to the Mediterranean Diet Chapter 15 155 heart attacks and coronary deaths in patients who had already had a coronary event, (b) the fat source used was linolenic acid present in a margarine not available in any supermarket, (c) the diet of the control group was richer in fat than that of the intervention group, (d) the sample size was small (14 events in one group and 44 in another), (5) the follow-up was short (discontinued at 27 months), and (e) the dietary assessment during follow-up was not complete. The Women’s Health Initiative Dietary Modification Trial [19] included 48,835 postmenopausal women aged 50–79 years who were randomly assigned to an intervention group with a low-fat diet (19,541 [40%]) or a control group (29,294 [60%]) in a free-living setting during a mean follow-up of 8.1 years. The results showed that a dietary intervention reducing total fat intake and increasing the intake of vegetables, fruits, and grains did not significantly reduce the risk of CHD, stroke, or CVD in postmenopausal women and achieved only modest effects on CVD risk factors, suggesting that more focused diet and lifestyle interventions may be needed to reduce CVD risk factors. Although these results were not expected by the authors, they are consistent with previous data obtained from observational studies [11,12]. The study therefore concludes that the key to cardiovascular risk reduction should not focus on a reduction of total fat intake. The current paradigm in nutritional epidemiology is to analyze dietary patterns instead of isolated foods or nutrients because these patterns can capture the cumulative effects of the overall diet. Among dietary patterns, the MD may be the healthiest. A recent meta-analysis of 18 prospective cohort studies showed that adherence to the MD confers consistent protection against CVD, neurodegenerative disorders, cancer, and overall mortality [20]. In randomized clinical trials, Mediterranean-style diets have provided beneficial effects on cardiometabolic markers such as body weight, blood pressure (BP), lipid profile, insulin resistance. and inflammation [21]. The MD fits the concept of a healthful eating pattern with overall nutrient adequacy [22]. Given that the MD is plant-based and rich in antioxidants, there are fair grounds to believe that long-term adherence might protect against different diseases, mainly CVD. However, this effect on large samples of patients at high risk has been scarcely assessed. Sánchez-Taı́nta et al. [23] carried out a cross-sectional assessment of baseline characteristics of participants in a primary prevention study and concluded that following an MD was inversely associated with the clustering of hypertension, diabetes, obesity, and hypercholesterolemia among high-risk patients. Numerous clinical trials have found that an MD pattern reduces arterial pressure [24] and improves the lipidic profile [25,26] and endothelial function [27]. Moreover, transversal studies [28] as well as long interventions or short trials [29] observed that subjects with higher adherence to the traditional Mediterranean dietary pattern have a lower concentration of inflammatory markers associated with arteriosclerosis. All of these beneficial effects on cardiovascular risk markers add intermediate biological plausibility to the results of epidemiological studies. Wine and Beer in the MD The overall effects of the Mediterranean dietary pattern on the cardiovascular system make it difficult to assess the effects of the different components of the MD on different body functions and pathologies. Thus, in epidemiological studies it is not easy to separate the effects of individual foods (wine or beer) from other sources in the diet or especially lifestyles, despite the statistical analysis being adjusted for multiple variables such as age, sex, social class, education, smoking, and physical activity. In this regard, it was noted that regular wine drinkers have a healthier dietary pattern (in Anglo-Saxon countries) than beer or spirit drinkers, which could explain some of the additional effects of wine compared with other alcoholic beverages observed in some studies [30]. Johansen et al. [31] analyzed 3,500,000 purchase receipts collected from two Danish supermarkets in 6 months. Wine buyers preferably bought olives, fruit, vegetables, chicken, olive oil, meat, and cheese and dairy products low in fat compared to beer buyers, who more frequently bought sugar, chips, sausages, pork, butter, margarine, and cola. The results of this study indicate that people who buy (and presumably drink) wine also acquire healthier foods than those who buy beer (Figure 1). These results support other studies from the United States, Denmark, and France that found that the dietary habits of wine drinkers are different from those of beer consumers [32]. Many of the beneficial effects attributed to moderate wine consumption on cardiovascular morbidity and mortality and the incidence of some cancers are actually caused by the healthier lifestyles of its consumers. Because most of these studies have been conducted in Anglo-Saxon countries, it would be interesting to compare them with results obtained from studies in Mediterranean countries. The relation between moderate alcohol consumption and health effects has mostly been studied in Nordic countries [33,34], yet this relation may be different in Spain and other Mediterranean countries. In Spain, the consumption of alcohol takes place principally at mealtime (in contrast with the Nordic pattern). Moreover, most characteristic lifestyle habits of the Spanish population (diet, moderate physical activity, frequent sun exposure, warm temperature, high degree of family-based and low degree of community-based social support), might well lead to the determinants of subjective health being different from those in Nordic countries [35]. 156 SECTION 2 Components of the Mediterranean Diet 0.4 Olives 0.3 Beer and wine 0.2 Oil Veal 0.1 Chips Potatoes Wine Beef Dried fruit Spice Organic Coffee Sauce Cheese Flour Poultry Tea Biscuits 0 Pasta Milk Low fat meat Cereal Fruits or vegetables Bread Sweets Low fat cheese Low fat milk Low fat Cold cuts −0.1 Beer Jam Fish Eggs Tinned goods Rice Pork Sugar Margarine or butter Soft drinks Ketchup or mustard Ready cooked dish Lamb Sausages Cold cuts Non-alcohol −0.2 −0.15 −0.10 −0.05 0 0.05 0.10 FIGURE 1 Food items bought by wine and beer buyers. Food items that are highly correlated and more likely to be bought together are closer to each other [31]. A long-term prospective study carried out by Núñez-Córdoba et al. [36] assessed the association between alcohol consumption (including drinking patterns during the week and type of alcoholic beverage) and the incidence of hypertension in a Mediterranean cohort. The results revealed that the consumption of beer or spirits, but not wine, was associated with an increased risk of hypertension. However, the weekly pattern of alcohol consumption did not have a significant effect on the risk of hypertension. The hazard ratio associated with consuming >0.5 drinks of beer per day was 1.53 (95% confidence interval 1.18–1.99) compared with abstainers. In contrast, there was a nonsignificant inverse association between red wine intake and the risk of hypertension. Another cross-sectional population study of a Mediterranean cohort evaluated the hypothesis that alcohol consumption has a protective effect on the prevalence of the metabolic syndrome (MetS), type 2 DM (T2DM), CHD, stroke, peripheral arterial disease (PAD), and overall CVD. Moreover, these effects might be dose-related and dependent on the type of alcoholic beverage [37]. The results showed that wine consumption was associated with a slightly better effect on the prevalence of total CVD than beer or spirit consumption, and beer consumption was associated with a better effect than spirit consumption. Moderate alcohol consumption is associated with a lower prevalence of the MetS, T2DM, PAD, CHD, and overall CVD but not stroke compared with no alcohol use in the Mediterranean population, and heavy drinking has been associated with an increase in the prevalence of all of these diseases states [37]. Therefore, advice on alcohol consumption should probably mainly be aimed at reducing heavy drinking. In summary, it should be emphasized that the benefits associated with red wine and beer depend on regular and moderate consumption. Although general recommendations are one drink (10 g alcohol) daily for women and two drinks (20 g alcohol) daily for men, individual ideals may vary based on age, sex, genetics, body type, and drug/supplement use. These different recommended daily doses of alcohol between sexes are explained by the fact that women are more sensitive to the effects of alcohol on the body. In addition, any healthy effects of wine and beer are greater in combination with a healthy diet. The health benefits associated with the MD, which combines moderate wine and beer consumption with a diet rich in fruits, vegetables, and whole grains, suggests that polyphenols have synergistic effects with compounds found in other groups of foods. BEER CONSUMPTION AND CONTRIBUTION TO THE HEALTH BENEFITS OF THE MD Bioactive Compounds and Mechanisms of Action Beer is one of the most consumed alcoholic beverages around the world; it is rich in nutrients such as carbohydrates, amino acids, minerals, vitamins, and other compounds such as polyphenols. Hop (Humulus lupulus L.) is one of the raw materials of beer and serves as an important source of phenolic compounds. Polyphenols—mainly phenolic acids, prenylated chalcones, flavonoids, catechins, and proantocianidins—comprise about 14.4% of dried hop cones [38]. Around 30% Beneficial Contribution of Beer to the Mediterranean Diet Chapter 15 157 of polyphenols in beer comes from hops and 70–80% originates from malt [39]. Moreover, hops provide a resin containing monoacyl phloroglucinols, which become bitter acids such as a-acids (humulones) and iso-a-acids during the development process of beer. Table 1 details the polyphenols found in beer [41]. The structural classes of polyphenols in beer include simple phenols, benzoic acid derivatives and cinnamic acid, coumarins, catechins, di- and tri-oligomeric proanthocyanidins, prenylated chalcones, and a- and iso-a-acids derived from hops. Different profiles of in vitro biological activities have been described for these compounds, which, in combination, exert a synergistic effect. However, to extrapolate these results and evaluate the in vivo physiological effects of beer consumption TABLE 1 Phenolic Compounds in Beer Phenolic compounds (mg/100 mL) Alkylmethoxyphenols 4-Vinylguaiacol 0.15 Phenolic compounds (mg/100 mL) Syringic acid 0.02 Gentisic acid 0.03 Cinnamic acids Alkylphenols 3-Methylcatechol 1.00e–04 p-Coumaric acid 0.10 4-Ethylcatechol 6.00e–04 m-Coumaric acid 0.02 4-Vinylphenol 4.53e–03 o-Coumaric acid 0.15 5-Caffeoylquinic acid 0.08 Caffeic acid 0.03 4-Caffeoylquinic acid 0.01 5-Caffeoylquinic acid 0.08 Ferulic acid 0.26 0.02 Hydroxybenzaldehydes Vanillin 0.02 Hydroxybenzoketones 2,3-Dihydroxy-1-guaiacylpropanone 3.40e–03 Hydroxycoumarins Esculin 0.02 Sinapic acid Umbelliferone 1.67e–03 Chalcones 4-Hydroxycoumarin 0.11 Xanthohumol 1.41e–03 Flavanones Hydroxyphenylacetic acids 4-Hydroxyphenylacetic acid 0.03 Isoxanthohumol 0.04 Homovanillic acid 0.05 Naringin 7.50e–04 8-Prenylnaringenin 1.04e–03 Alquilphenols 3-Methylcatechol 1.00e–04 6-Prenylnaringenin 2.59e–03 4-Ethylcatechol 6.00e–04 6-Geranylnaringenin 4.29e–04 4-Vinylphenol 4.53e–03 Hydroxyphenylacetic acids 4-Hydroxyphenylacetic acid 0.03 0.3 Homovanillic acid 0.05 2,6-Dihydroxybenzoic acid 0.09 Tyrosol 0.32 2-Hydroxybenzoic acid 0.20 Flavanols 3-Hydroxybenzoic acid 0.03 (+)-Catechin 0.11 3,5-Dihydroxybenzoic acid 0.03 (−)-Epicatechin 0.06 Syringic acid 0.02 Procyanidin dimer B3 0.16 Protocatechuic acid 0.05 Prodelphinidin trimer GC-GC-C 0.04 Vanillic acid 0.07 Prodelphinidin trimer GC-C-C 1.00e–02 Gallic acid 0.07 Prodelphinidin trimer C-GC-C 0.02 Benzoic acid derivatives Gallic acid 3-O-gallate Continued 158 SECTION 2 Components of the Mediterranean Diet TABLE 1 Phenolic Compounds in Beer—cont’d Phenolic compounds (mg/100 mL) Flavonols Phenolic compounds (mg/100 mL) Flavones Quercetin (3-O-arabinoside) 5.83e–04 Apigenin 4.17e–03 Quercetin 6.67e–03 α-acids (humulones) 0.17 3,7-Dimetilquercetin 2.50e–04 Iso-α-acids (iso-humulones) 0.06–10 Myricetin 6.67e–04 Other polyphenols Quercetin (3-O-rutinoside) 0.09 Catechol 1.10e–03 Pyrogallol 4.70e–03 Isoflavonoids Daidzein 0.005 Genistein 0.01 Biochanin A 0.005 Mean value of bibliographic data from the Phenol-Explorer Database, Version 1.5.7 (INRA in collaboration with the Wishart Research Group) [40]. it is necessary to study their bioavailability in the body. The compounds found in beer have different biological activities demonstrated in vitro as antioxidant, anticarcinogenic, anti-inflammatory, estrogenic, and antiviral activities. However, further studies of humans are needed to determine whether the circulating or tissue metabolites of these compounds derived from moderate consumption of beer have the same bioactivity in vitro. Consumption of Beer in Mediterranean Countries and Overall Morbidity/Mortality According to the World Health Organization (WHO), countries have been classified into four scoring categories (1–4) reflecting the risk of mortality and morbidity associated with different amounts of alcohol intake. For example, category 1, characterized by the drinking pattern with the lowest risk (light to moderate alcohol consumption with meals and without heavy bouts of drinking) is associated with a lower burden of mortality and morbidity. Mediterranean Europe, Australia, and Japan are included in this category. Category 4, however, is the drinking pattern with the highest risk, characterized by the highest level of irregular drinking and associated with a high burden of morbidity and mortality. This drinking pattern was found in Central America (e.g., Guatemala and Nicaragua). The lowest risk categories (1 and 2) were associated with the consumption of fermented beverages such as wine and beer, whereas the highest risk categories (3 and 4) were associated with the consumption of distilled spirits [42]. The type of alcoholic beverages consumed differed between sexes and between people from different countries. The European Prospective Investigation into Cancer and Nutrition (EPIC) study [43] evaluated the patterns of alcohol consumption in 10 European countries, mostly from the Mediterranean area. Sex was a strong determinant of drinking patterns in all 10 countries. Among men, wine was consumed mainly among the Italian and Spanish subjects with some regional differences. In both cases more wine was consumed in the southern than in the northern regions. By contrast, beer was consumed mainly by men in Germany, Holland, and Denmark (almost 400 g/day), whereas the lowest beer consumption was in Italy (about 16 times lower than that of the highest consumers in northern European countries). In general, women drank less than men and consumed different types of beverages. Large differences were found among countries in terms of the quantity of alcohol consumed by women, whereas differences in wine consumption were less marked. The largest quantities of beer were consumed by women in Murcia (Spain), Copenhagen (Denmark), Heidelberg (Germany), and Malm€ o (Sweden). These findings are only in partial agreement with those of a previous study of alcohol consumption in the European Community Study [44], which evaluated the frequency and the context of consumption of beverage types and analyzed whether subpopulations defined by sex, age, and educational level differ in the adoption of beverage types. Mediterranean countries were characterized mainly by wine consumption, whereas people in northern Europe mainly drank beer [45]. In the EPIC study this was only really true for men; in all the areas studied except Murcia (Spain), wine contributed more to the total alcohol intake among women than all other sources combined. Possible reasons for these FIGURE 2 Curves for wine (solid lines) and beer intake (dotted lines) of relative risk (95% CI; dashed line) of fatal and nonfatal vascular events extracted from 13 independent relationships using random models. From Costanzo et al. [40]. Relative risk of fatal and nonfatal vascular events Beneficial Contribution of Beer to the Mediterranean Diet Chapter 15 2 159 Wine consumption Beer consumption 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 10 40 50 20 30 Alcohol consumption (g/day) 60 70 discrepancies may be differences in the sampling or assessment methods reported. In a meta-analysis evaluating whether different alcoholic beverages would protect against CVD, Costanzo et al. [40] reported that wine drinkers, at least in some countries, tend to have a healthier lifestyle profile than beer drinkers. However, the analysis between wine or beer consumption and vascular events in Mediterranean and non-Mediterranean countries found comparable results among them (Figure 2). Athyros et al. [37] investigated the relationship between alcohol consumption and the prevalence of different cardiovascular risk factors in a representative sample of Greek adults (Mediterranean cohort) and concluded that wine consumption was associated with a slightly better effect than beer or spirit consumption on the prevalence of total CVD. Beer consumption also was associated with a better effect than spirit consumption. According to the type of alcoholic beverage, the patterns of drinking, and the social reactions to alcohol, the European community can be divided into three different patterns, as reported by Rehm et al. [46]. l l l The Mediterranean pattern: wine-making countries in the south are traditionally characterized by almost daily drinking of alcohol—most often wine and most often consumed with meals. The central European pattern: beer is the dominant alcoholic beverage, and its consumption is similar to the Mediterranean style. However, there is more consumption outside of meals, and there are more alcohol-related problems. The northern European pattern: the drinks of choice are vodka and spirits, whose production only began after the invention of the distillation process; hence it has a substantially shorter tradition than wine drinking in the Mediterranean region. The pattern of drinking in these countries is characterized by nondaily drinking, with irregular episodes of heavy and very heavy drinking. Figure 3 shows the per capita alcohol consumption in liters of pure alcohol recorded for all EU adults (15 years old) since 2000. While the consumption of total alcohol for the European Union as a whole has been stable, different trends can be observed for different regions. Southern European countries have decreased their alcohol consumption since 1999. In a traditional wine country such as Spain, for example, beer has replaced wine as the beverage of choice. There also have been some decreasing trends in central-west and western Europe, except for the British Isles. However, the Nordic countries and the central-east and eastern European countries have increased their beer consumption [44]. Beer in the PREDIMED Clinical Trial Although there is a substantial body of evidence linking the MD to the reduction and prevention of cardiovascular risk, small clinical trials have uncovered plausible biologic mechanisms to explain the salutary effects of this food pattern [24,28]. The largest multicenter randomized clinical trial in Spain, the PREDIMED study, was designed to evaluate the effects of a traditional MD on the primary prevention of CVD [47]. A total of 7447 volunteers were included (age range, 160 SECTION 2 Components of the Mediterranean Diet FIGURE 3 Per capita alcohol consumption in liters of pure alcohol recorded for all EU adults (15 years old) since 2000. Modified from Rehm and Shield [44]. 12 Liters of pure alcohol 10 Beer 8 Wine 6 Spirits Other 4 Total 2 0 1999 2001 2003 2005 Years 2007 2009 55–80 years). Patients had no CVD at enrollment and had either T2DM or at least three of the following major risk factors: smoking, hypertension, elevated low-density lipoprotein (LDL) cholesterol levels, low high-density lipoprotein (HDL) cholesterol levels, overweight or obesity, or a family history of premature CHD. Participants were randomly assigned in a 1:1:1 ratio to one of three dietary intervention groups: a Mediterranean diet supplemented with extra virgin olive oil, a Mediterranean diet supplemented with nuts, or a control diet. The high number of participants included in the PREDIMED study, with complete nutritional study and vascular risk factors, provided the opportunity to study the relationship between moderate beer consumption, dietary habits, and CVD in a Spanish population at high cardiovascular risk. The most recent results from this trial concluded that an energy-unrestricted MD supplemented with either extra virgin olive oil or nuts resulted in an absolute risk reduction of approximately three major cardiovascular events per 1000 person-years, which is relative risk reduction of 30% [47]. A preliminary substudy of 1249 participants from the PREDIMED cohort also assessed the effects of beer consumption on the main cardiovascular risk factors, evaluating dietary habits and physical activity levels. Results showed that moderate beer consumers follow a healthier eating pattern and have greater adherence to the traditional MD than nonbeer drinkers (Table 2). In fact, the beer drinkers in the PREDIMED tended to consume this drink accompanied even by meals. Thus, moderate beer drinkers consumed more vegetables, legumes, fish, cereals, and olive oil and fewer dairy products than nondrinkers. However, the study also showed a greater consumption of meat and meat products among beer drinkers than nondrinkers, although the overall dietary pattern of beer drinkers was closer to that of the traditional MD [17], being completely different from the pattern observed in the Anglo world where beer drinkers typically consume fewer fruits and vegetables and more ready-made meals, sugar, chips, sausages, pork, butter, margarine, and soft drinks [34]. Moreover, beer drinkers also referred moderate or higher consumption of protein and carbohydrates (including fiber) than nondrinkers, while total fat intake was similar in both groups. Finally, beer drinkers also consumed significantly more folic acid, vitamins B1, B6, B12, E, and D, iron, and calcium than nondrinkers. Therefore, the first conclusion of this substudy was that the eating pattern of the beer drinkers included in the PREDIMED cohort is much healthier than that of nondrinkers and totally different from that of beer drinkers in Anglo countries. Moreover, in this substudy beer drinkers had a lower incidence of T2DM and hypertension than nondrinkers. Although there was no difference in the incidence of dyslipidemia in the two groups, it was found that moderate beer drinkers received less statin therapy than nondrinkers. These data are in accordance with the results of numerous epidemiological studies that have shown that regular and moderate alcohol drinkers have a lower incidence of diabetes and hypertension and have increased HDL and decreased LDL-cholesterol [27,37]. Another important aspect to analyze is the usual relationship between beer consumption and body weight. Traditionally, it has been noted that excessive alcohol consumption is associated with an increase in body weight in the early stages, but in later stages many alcoholics show clinical and biological signs of malnutrition [48]. Moderate beer drinkers in the PREDIMED study had a significantly lower body mass index than nondrinkers, with no differences in waist circumference as a measure of visceral obesity. Regarding BP and lipid profile, moderate beer drinkers showed a lower BP and higher HDL-cholesterol levels than nondrinkers, but in this case the differences were not statistically significant. However, blood glucose concentrations Beneficial Contribution of Beer to the Mediterranean Diet Chapter 15 161 TABLE 2 Baseline Characteristics of 1249 Volunteers Included in the PREDIMED Substudy Beer Consumption Characteristics 0 mL (n ¼ 827) 22 mL (n ¼ 161) 203 mL (n ¼ 261) Mean age SD (years) 68.7 5.7 66.8 5.7 65.8 6.1a Male sex 273 (33) 81 (50)a 188 (72)a Family history 149 (18) 27 (17) 47 (18) a Smoking 83 (10) 26 (16) 68 (26)a Diabetes mellitus 463 (56) 71 (44)a 126 (48) Hypertension 687 (83) 127 (79) 200 (77)a Dyslipidemia 546 (66) 106 (66) 173 (66) Obesity/overweight 728 (88) 148 (91) 231 (88) Data are n (%) unless otherwise indicated. SD, standard deviation. a P < 0.05, compared with nondrinkers. and glycated hemoglobin were significantly lower when comparing the moderate beer drinkers with the nondrinkers. Finally, moderate beer drinkers also had lower plasma concentrations of homocysteine compared with nondrinkers, confirming the results of other studies [49,50] in which lower homocysteine concentrations also were found in beer drinkers. This effect has been attributed to the high content of folic acid and vitamin B in beer. In summary, contrary to the results obtained in studies conducted in the Anglo world, those of the PREDIMED substudy showed that moderate beer drinkers have healthier lifestyle habits and an eating pattern closer to the traditional MD than those of nondrinkers. In addition, moderate beer drinkers have fewer cardiovascular risk factors than nondrinkers, a fact that supports the protective role of moderate beer consumption against the onset and development of atherosclerosis. CONCLUSIONS Clinical and epidemiological studies indicate that wine may protect against CVD, atherosclerosis, hypertension, certain types of cancer, T2DM, neurological disorders, and the metabolic syndrome. However, sufficient evidence also supports a significant inverse association between regular and moderate beer consumption and cardiovascular risk. There is evidence that polyphenols provide an abundance of health benefits. In beer, xanthohumol and its metabolites, isoxanthohumol and phytoestrogen 8-prenylnaringenin, also provide healthy properties such as antioxidant, anticarcinogenic, anti-inflammatory, estrogenic, and antiviral activity. It must be emphasized that the benefits associated with beer depend on regular and moderate consumption. Although general recommendations are one drink (220 mL beer or 10 g alcohol) daily for women and two drinks (440 mL beer or 20 g alcohol) daily for men, individual ideals may vary based on age, sex, genetics, body type, and drug/supplement use. The health benefits associated with the MD, which combines moderate wine and beer consumption with a diet rich in fruits, vegetables, and whole grains, suggest that polyphenols have synergistic effects with compounds found in other groups of foods. SUMMARY POINTS l l l l l Clinical and epidemiological studies indicate that wine may protect against CVD, atherosclerosis, hypertension, certain types of cancer, type DM, neurological disorders, and the metabolic syndrome. Evidence also supports a significant inverse association between regular and moderate beer consumption and cardiovascular risk. There is evidence that polyphenols provide an abundance of health benefits. In beer, xanthohumol and its metabolites, isoxanthohumol and phytoestrogen 8-prenylnaringenin, provide healthy antioxidant, anticarcinogenic, anti-inflammatory, estrogenic, and antiviral properties. The benefits associated with beer depend on regular and moderate consumption. 162 l l SECTION 2 Components of the Mediterranean Diet General recommendations are one drink (220 mL beer or 10 g alcohol) daily for women and two drinks (440 mL beer or 20 g alcohol) daily for men; however, individual ideals may vary based on age, sex, genetics, body type, and drug/supplement use. The health benefits associated with the MD, which combines moderate wine and beer consumption with a diet rich in fruits, vegetables, and whole grains, suggest that polyphenols have synergistic effects with compounds found in other groups of foods. ACKNOWLEDGMENTS The authors are grateful for the support granted by the Spanish Ministry of Health (RETIC G03/140 and RD06/0045); the Spanish Ministry of Science and Innovation (AGL2006-14228-C03-01/02-ALI, AGL2007-66638-C02-02/ALI, AGL200913906-C02-02, AGL2010-22319-C03-02); the FIS PI07/0473; Centro Nacional de Investigaciones Cardiovasculares (CNIC06); Fundación Mapfre and CIBEROBN, an initiative of the Instituto de Salud Carlos III, Spain. S. Arranz is grateful to the Sara Borrell postdoctoral program (CD10/00151), supported by the Instituto de Salud Carlos III, Spain. G. Chiva-Blanch is grateful for the Manuel de Oya fellowship from the Spanish Information Center of Beer and Health, and P. Valderas is grateful to the APIF fellowship program from the University of Barcelona. REFERENCES [1] http://www.unesco.org/culture/ich/en/RL/00394. [2] http://dietamediterranea.com. [3] Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. 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Eur Neuropsychopharmacol 2013;23(2):89–97. 164 SECTION 2 Components of the Mediterranean Diet [47] Estruch R, Ros E, Salas-Salvadó J, Covas MI, Corella D, Arós F, et al. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med 2013;368(14):1279–90. [48] Estruch R, Nicolás JM, Villegas E, Junqué A, Urbano-Márquez A. Relationship between ethanol-related diseases and nutritional status in chronically alcoholic men. Alcohol Alcohol 1993;28:543–50. [49] Ubbink JB, Fehiliy AM, Pickering J, Elwood PC, Vermaak WJH. Homocysteine and ischemic heart disease in the Caerphilly cohort. Atherosclerosis 1998;140:349–56. [50] Mayer Jr O, Simon J, Rosolova H. A population study of the influence of beer consumption on folate and homocysteine concentrations. Eur J Clin Nutr 2001;55:605–9. Chapter 16 The Contribution of Fish to the Mediterranean Diet Ermelinda Prato and Francesca Biandolino CNR—Institute of Coastal Marine Environment (IAMC), Taranto, Italy. ABBREVIATIONS ARA DHA EPA MeHg PUFAs Arachidonic acid (20:4 o6) Docosahexaenoic acid (22:6n-3) Eicosapentaenoic acid (20:5n-3) Methylmercury Polyunsaturated fatty acids INTRODUCTION Nutrition is a key element in maintaining overall physical well-being and its importance is nothing new. In 400 BC Hippocrates said, “Let food be your medicine and medicine be your food.” Another famous phrase is by the German philosopher Ludwig Feuerbach in the nineteenth century: “We are what we eat”; both quotes point out that to be fit and healthy one needs to eat good food. Nutritional regulation occurs both by macro- and micronutrients, leading to changes in cell growth, differentiation, or metabolism. Carbohydrates, fats, and proteins are the macronutrients essential for health maintenance, growth, reproduction, immunity, and healing. Any nutrient deficiencies or excess can be dangerous to metabolism, which is why a healthy and balanced diet is fundamental in giving the body all it needs to living at its best. Never before has the focus on the health benefits of food or food components been so strong. It is known that the Mediterranean diet is a healthy eating pattern that provides foods that ensure the proper functioning of the body. The Mediterranean dietary pattern has gained enormous popularity lately, mainly because numerous epidemiological studies have shown that the people living along the Mediterranean Sea in southern Europe have a longer life expectancy and a lower risk of suffering from certain chronic diseases. Marine foods, especially fish, are an important part of the Mediterranean diet, which recommends consuming fish (on a weekly basis) more often than meat (on a monthly basis). BIOCHEMICAL COMPOSITION OF FISH The role of fish in the Mediterranean diet shows marked variations across continents, regions, and countries, reflecting different eating habits and traditions, the availability of fish and other foods, prices, socioeconomic levels, and seasons. Consumption is usually higher in coastal areas. Fish are characterized by low fat content, in particular cholesterol, and are one of the most significant sources of high-value proteins. Moreover, they contain a wide range of essential micronutrients, including various vitamins (D, A, and B) and minerals (e.g., calcium, iodine, zinc, iron, and selenium), which makes them a good choice for a healthy and balanced diet. However, health benefits from fish consumption have been primarily related to polyunsaturated fatty acids (PUFAs), especially the omega 3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that occur in large amounts in most fish [1–3]. The structures of these fatty acids (FAs) are illustrated in Figure 1. Humans (like most animals) have a very limited capacity to synthesize these FAs from the precursor a-linolenic acid, which is essential; therefore dietary intake of these FAs is an important aspect of human nutrition [4,5]. For these reasons the MD does not limit total fat consumption, but rather recommends making wise choices about the types of fat consumed, discouraging intake of The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 165 166 w SECTION 2 Components of the Mediterranean Diet 17 3 14 11 8 5 a C OH 9 6 FIGURE 1 Chemical structure of eicosapentaenoic acid and docosahexaenoic acid. O Eicosapentaenoic acid (EPA) 20:5 O w 16 19 3 6 13 10 7 4 a 9 C OH Docosahexaenoic acid (DAH) 22:6 H3C COOH H3C COOH Linoleic acid (18:2w6) FIGURE 2 Structures of linoleic and a-linolenic acids. a-Linolenic acid (18:3w3) saturated fats and hydrogenated oils (trans fats), both of which contribute to heart disease. The simplest members of the omega 6 and omega 3 fatty acid families are linoleic (18:2) and a-linolenic (18:3) acids, respectively (Figure 2). However, it should be noted that the net content of omegas 3 and 6 is low because of the low lipid content in fish species, and only regular consumption of fish can help to increase the amount of omega 3 in the diet. Figure 3 gives an overview of the metabolic steps (desaturation plus elongation reactions) by which linoleic acid is metabolized to arachidonic acid (ARA) and by which a-linolenic acid is metabolically converted to long-chain products, including EPA and DHA, the physiologically essential omega 3 fatty acids. THE IMPORTANCE OF OMEGA 3 FATTY ACIDS FOR HUMAN HEALTH In recent years the medical and public health communities have shown increased interest in the advantageous FA composition of fish. Indeed, it has been the topic of numerous studies, which reported that long-term and regular fish consumption (omega 3 PUFA intake) reduces the risk of contracting many diseases (such as cancer, lung diseases, Alzheimer’s disease). In particular, epidemiological and experimental studies supported the beneficial activity of these omega 3 PUFAs in the prevention of cardiovascular diseases [6]. Indeed, pioneering studies demonstrated that the low incidence of heart disease, diminished platelet aggregation, and prolonged clotting times in Greenland Eskimos and Japanese were correlated with the high intake of fish and fish oils [7]. EPA is an important essential omega 3 FA in the human diet because it is the precursor to the 3-series eicosanoids, such as prostaglandins, thromboxanes, and leukotrienes—hormone-like chemicals with short-lived but powerful effects [8]. Eicosanoids play significant roles in immune function, inflammation, thrombosis, proliferation, reproduction, gastroprotection, and hemostasis, in addition to other functions. DHA and EPA were reported to be interchangeable by retrogradation [9]. In the prevention of cardiovascular disease it is important to consider the ratio of PUFA to saturated FAs. The UK Department of Health recommends a minimum value of this coefficient of 0.45 [10]. Populations that consume 0.5–0.7 g DHA/day have a lower incidence of heart disease. The general recommendation for daily intakes of DHA/ EPA is 0.5 g/day for infants and 1 g/day for adults and patients with heart disease [11]. Also, omega 3 PUFAs play a vital role in decreasing the incidence of diabetes, and they seem to alleviate symptoms of rheumatoid arthritis [12], inflammatory bowel diseases (ulcerative colitis and Crohn’s disease), systemic lupus erythematosus, septicemia, and septic shock [13]. They exert a fundamental action in photoreception (vision); in the development and function of the nervous system (brain); in infants, during pregnancy, and during the first years after birth; and in decreasing the concentration of low-density lipoprotein cholesterol in plasma [6]. Figure 4 summarizes the health benefits of long-chain omega 3 FAs (EPA and DHA). Fish oils also contain PUFAs from the omega 6 family, which have an antagonistic effect on omega 3 acids. However, all fish species had low levels of ARA, which may be advantageous to cardiovascular health because of the antagonistic effects on the health benefits of omega 3 FAs [3]. This is why the ratio of omega 6 to omega 3 acids is so important and is a Fish in the Mediterranean Diet Chapter 16 167 FIGURE 3 Synthesis of omega 3 and omega 6 fatty acids. good index for comparing the relative nutritional value of fish oils. A dietary intake of fish with a high ratio of omega 3 to omega 6 would therefore be beneficial. The UK Department of Health recommends a maximum omega 6-to-omega 3 ratio of 4.0 [10]. Values higher than the maximum are harmful to health and may promote cardiovascular diseases. Because this ratio in food is generally higher, however, fish consumption is excellent at lowering it. Considering all these facts, increasing the consumption of fish and its products, which are rich in omega 3 PUFAs and poor in omega 6 PUFAs, is important for human health. 168 SECTION 2 Components of the Mediterranean Diet Maternal health CVD and hypertension Inflammation Kidney health Development EPA Arthritis Depression DHA Brain health Immunity Fertility Aging Cancer Obesity Insulin resistance FIGURE 4 Summary of health benefits of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). CVD, cardiovascular disease. FATTY ACIDS OF COMMERCIALLY IMPORTANT FISH SPECIES Fish can be grouped into four categories according to their fat content: lean (<2%), low fat (2–4%), medium fat (4–8%), and high fat (>8%) [14]. Fish species, namely hake, mackerel, common sole, anchovy, sea bass, sardine, gilthead sea bream, and other species, are a main food component in the diet of the Mediterranean population. The fillet is the most important edible part of fish and it is strongly affected by lipid content from both a nutritional point of view and an organoleptic perspective (flavor, texture, etc.). Fish with high fat content traditionally has been considered to be nutritionally important species because they have a relatively high content of omega 3 FAs. However, it has been demonstrated that there is an inverse relationship between amount of omega 3 FAs and the total fat content [15]. This implies that it is important from the nutritional point of view to pay attention to species with high proportions of omega 3 FAs (e.g., eel, Anguilla anguilla) instead of focusing only on the fat content (Table 1). In addition to the size, age, and reproductive status of fish, diet, location, and season are theP major factors affecting fatty acid composition [14,37]. Table 1 shows the known composition of total lipids, the sum of PUFA ( PUFA), DHA, EPA, DHA-toP P EPA ratio, the sum of omega 3 ( omega 3), the sum of omega 6 ( omega 6) FAs, and omega 3-to-omega 6 ratio of some commercial fish. There is a noticeably large variation in the FA compositions of different fish of the same species, although all species contain higher proportions of omega 3 than omega 6 PUFAs, except for sea bass from Turkey (eastern Mediterranean) [8] and eel from Spain (western Mediterranean) [23]. With regard to the omega 3-to-omega 6 ratio, it is noteworthy that it is variable; indeed, it ranges from 0.73 in sea bass [8] to 39 in Salema porgy [23]. A very favorable omega 3-to-omega 6 ratio also has been found in bogue, mackerel salmon, anchovy, sardine, pickerel, hake, swordfish, and sole (Table 1). Numerous studies have shown that the different species of economically important fish have different FA composition, so it is advisable that consumers become aware of the species with the best nutritional quality for use in the diet. FAT COMPOSITION OF FARMED FISH Another important aspect to consider is farmed fish. Indeed, because fish stocks are limited, consumers farmed fish are now being proposed as an alternative. Fish farming, or aquaculture, is a fast-growing sector and is expected to fill the gap in Fish in the Mediterranean Diet Chapter 16 TABLE 1 Total Lipid, Commercial Fish Species Gilthead sea bream (Sparus aurata) Salema (Sarpa salpa) P PUFA, DHA, EPA, DHA/EPA Ratios, v3, v6 Fatty Acids, v3/v6 Ratios of Some Important Total Lipid % SPUFA DHA EPA DHA/EPA Sv3 Sv6 v3/v6 Ratio References - 38 17.6 12.7 1.4 28.7 9.3 3.1 [16] 7.4 16.5 3.3 5 0.7 12.1 4.4 2.8 [17] 0.1 38.6 9.2 7.6 1.2 19.4 19.3 1 [18] 12.4 34.5 17.4 6.8 2.6 26.2 8.1 3.2 [19] - 26.2 6.8 2.9 2.3 13.6 12.6 1.1 [8] - 19.6 3.1 5.3 0.6 10.3 4.3 2.4 [20] 0.86–1.20 29–51 15.5–35 5–6.3 2.7–7 21–41 2.8–9.9 2.6–10.9 [5] - 35.2 8 4.7 1.7 0:00 16.1 1.2 [21] - 23.1 9.5 0.3 34.1 15.9 7.2 2.2 [22] 0.62 44 33.3 8.6 3.9 42.9 1.1 39.0 [23] 0.4–3.6 14–35 0.8–6.0 2.0–4.1 0.4–1.4 9.5–20.4 6–14.5 1.6–1.4 [24] 1.7 32.1 17.4 7.2 2.4 26.8 4.8 5.6 [3] Two-banded seabream (Diplodus vulgaris) - 52.1 20.3 18.7 1.1 42.4 9.6 4.4 [4] 1.0 36.1 21.9 5.3 4.1 27.6 7.9 3.5 [25] 3.6 34.0 17.2 7.4 2.3 27 6.8 3.9 [3] Seabass (Dicentrarchus labrax) 2.2 31.5 12.1 7.1 1.7 23.7 7.8 3 [17] 3.0 39.3 14.7 7.0 2.1 24.2 14.4 1.7 [19] Bogue (Boops boops) Sand smelt (Atherina boyeri) Mackarel (Scomber scombrus) Golden gray mullet (Liza aurata) 169 - 20.9 2.1 3.9 0.5 8.8 12.1 0.7 [8] - 44.9 24.3 7.8 3.1 34.4 10.5 3.3 [26] 2.3 32.4 13.8 6.9 2 23.1 9.3 2.5 [3] 1 39.1 16.6 12.2 1.4 29.9 9.2 3.3 [27] 3.6 27.5 18.7 5.1 3.7 26 1.2 21.0 [28] - 36.4 20.7 4.5 4.6 30.6 5.8 5.3 [8] 2.5–1.0 31.6–47.2 14.7–31.2 6.7–6.4 2.2–4.9 25.5–41.1 6.09–6.1 4.2–6.7 [29] 5.6 29.9 15.9 6.4 2.5 24.0 5.1 4.7 [3] - 49.9 24.8 12.8 1.9 39.9 9.4 4.2 [30] 6.2 26.6 8.5 6.1 1.4 18.2 6.0 3.0 [31] 1.8 36.6 12.1 9.9 1.2 26.0 10.5 2.5 [32] - 40.3 16.7 5.1 3.3 25.2 6.3 4.0 [20] 2.9 34.7 15.7 8.6 1.8 26.1 6.9 3.7 [3] 7.4 33 15.9 8.7 1.8 28.2 3.6 7.8 [23] - 39.4 25.3 5.9 4.3 33.3 5.7 5.8 [30] - 33.6 12.7 10.2 1.2 28.9 4.7 5.6 [4] 1.2 48.2 35.2 4.7 7.4 43.6 4.1 10.6 [19] 0.95 53.4 36.4 7.8 4.7 46.7 6.1 7.6 [1] 2.3 21.7 3.3 10.0 0.3 13.8 7.8 1.8 [25] - 22.0 3.5 2.2 1.6 14.5 7.4 1.9 [33] 4.9 14.2 3.9 7.5 0.5 11.7 2.5 4.7 [34] - 28.1 4.4 7.3 0.6 17.0 5.0 3.4 [20] 5.96 28.9 10.8 6.1 1.7 21.5 6.9 3.1 [3] Continued 170 SECTION 2 Components of the Mediterranean Diet P TABLE 1 Total Lipid, PUFA, DHA, EPA, DHA/EPA Ratios, v3, v6 Fatty Acids, v3/v6 Ratios of Some Important Commercial Fish—cont’d Total Lipid % SPUFA DHA EPA DHA/EPA Sv3 Sv6 v3/v6 Ratio References Salmo (Salmo salar) 13.1 49.6 26.6 3.8 7.0 43.7 5.9 7.4 [2] Wild 7 27.3 13.1 6.6 1.98 25.0 2.3 11.0 [35] Species Farmed 7.4 41.0 15.2 7.9 1.9 31.1 9.8 3.6 [35] - 35.8 20.7 6.0 3.4 29.7 6.0 4.9 [4] Wild 6.3 26.2 12.5 6.5 1.92 24.4 1.8 13.5 [36] Farmed 12.3 30.4 8.4 5.5 1.53 21.2 9.2 2.3 [36] Anchovy (Engraulis encrasicolus) - 35.4 16.2 9.3 1.7 29.1 5.5 5.3 [30] - 34.3 21.1 7.7 2.7 32.0 2.3 13.7 [37] - 45.4 24.6 10.4 2.4 39.7 5.6 7.0 [8] 0.97 56.0 41.4 7.5 5.5 51.2 4.4 11.6 [1] 35.1 15.8 10.4 1.5 30.3 4.8 6.9 [38] 33.0 14.7 8.6 1.7 27.3 4.4 6.2 [30] 30.4 11.3 6.5 1.7 20.9 7.6 2.7 [39] 38.1 20.8 10.7 1.9 35.3 2.7 13.0 [37] Sardine (Sardina pilchardus) - - Picarel (Spicara smaris) Red mullet (Mullus barbatus) - 46.0 26.0 10.5 2.5 41.3 5.7 7.2 [8] 4.15 43.5 25.8 9.7 2.7 39.1 3.9 10.0 [1] - 34.0 18.4 9.3 2.0 31.6 2.1 14.8 [37] 2.8 32.1 14.7 7.2 2.0 25.2 5.7 4.4 [3] 5.3 32.4 13.7 10.0 1.37 27.1 5.3 5.1 [23] 3.7–5.7 17.3–20.1 4.4–10.9 4.6–7.9 0.5–2.4 12.9–16.5 1.2–1.3 9.0–14.0 [40] 8.1 27.7 12.0 7.2 1.7 20.87 6.4 3.3 [3] Black goby (Gobius niger) 1.9 34.2 12.5 6.1 2.0 23.4 9.5 2.5 [3] Grass goby (Zoosterisessor ophiocephalus) 2.1 31.8 12.3 5.0 2.40 22.9 7.7 3.0 [3] Gray wrasse (Symphodus cinereus) 4.1 28.7 9.8 6.8 1.4 21.1 7.3 2.9 [3] Sole (Solea solea) 0.4 32.1 18.5 3.7 5.0 22.4 9.7 2.3 [41] 0.7 33.6 18.7 7.7 2.4 31.1 1.9 16.4 [28] Hake (Merluccius merluccius) Eel (Anguilla anguilla) Swordfish (Xiphias gladius) 0.9 43.9 16.1 11.3 1.4 38.7 5.2 7.9 [1] 0.7–1.1 34–43 22–30 0.2–6.2 3.6–125 27–34.5 5.1–7 3.9–5.8 [5] 0.9 35.1 22.4 6.0 3.7 31.4 3.7 8.5 [23] 0.3–1.23 25.0–36.3 16.4–22.9 3.9–7.3 3.1–4.2 20.9–30.5 4.1–5.8 5.2–5.4 [42] 0.9 52.3 35.0 8.7 4.0 46.1 5.8 8.0 [1] 8.7 18.9 3.1 2.6 1.2 8.8 10.1 0.9 [23] 27.2 18.5 5.0 2.4 2.0 12.8 5.6 1.3 [43] 19.7 9.3 3.5 2.7 16.3 2.2 7.4 [23] 44.3 28.1 2.9 9.7 36.6 4.8 7.6 [44] PUFA, polyunsaturated fatty acids; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid, ratio of docosahexaenoic to eicosapentaenoic acid; So3, sum of omega 3 fatty acids; So6, sum of omega 6 fatty acids; o3/o6 ratio, ratio of omega 3 to omega 6 fatty acids; ww, wet weight. Fish in the Mediterranean Diet Chapter 16 171 supplies of fish as food for humans as demand continues to increase [45]. Data from the literature suggest that the nutrient content of farmed fish is more uniform than that of wild fish and that fat content of farmed fish exceeds that of wild fish [17]. Farmed fish are fed a controlled diet, usually based on fish oil and fish meal. This diet is not subject to the seasonal variations found in the diets of wild fish, causing more constant lipid levels than those of wild fish. Indeed, the acquisition of food by wild fish is related to food availability, ecosystem productivity and energy expended to catch prey [46]. Farmed fish have the advantage of being reared and harvested under controlled conditions, so hazards associated with fish consumption can more easily be controlled. Therefore, the knowledge of the compositional and nutritive differences between farmed and wild fish is of considerable interest, for the farming industry and consumers. Regarding differences in FAs, in general, most of the data reported by Hossain [47] showed higher content of total PUFAs and omega 3 PUFAs in wild than in farmed fish. However, these results are in contrast to those of other authors [17,18] who found higher proportions of total PUFAs and omega 3 PUFAs in farmed fish than in wild specimens of different fish species. The difference in omega 3 PUFA composition of lipids between farmed and wild fish observed in different studies can be explained by the FA composition of the diet of both wild and farmed fish. Most farmed fish are carnivorous species, such as Dicentrarchus labrax and Sparus aurata, and eat small fish and crustaceans in which the PUFAs are nearly all omega 3 PUFAs and the proportion of omega 6 PUFAs is very low. In contrast, commercial feeds usually use cereal and vegetable oils that contain more of the omega 6 FAs and fewer omega 3 PUFAs. However, commercial feeds that use high proportions of fish meal and fish oil usually contain a higher proportion of omega 3 PUFAs and fewer omega 6 PUFAs. Ackman and Takeuchi [48] reported that the percentage of omega 3 FAs in cultured marine fish is usually lower than in their wild counterparts, presumably because of the lack of lipids originating from phytoplankton and other marine organisms. Moreover, aquaculture provides greater control during the production and processing of the organisms to optimize omega 3 PUFAs content. Consumers take wild fish as they come, but the body composition of farmed fish can be altered. Fish feed can be supplemented with protein and lipids to make the body composition more favorable for human health and nutrition. FISH CONTAMINATION AND HEALTH RISKS TO CONSUMERS In contrast to the potential health benefits of dietary fish intake, the potential risks associated with the excessive consumption of fish should also be taken into account. Several studies have reported that high intake of omega 3 FAs may negatively affect immune response by causing prolonged bleeding and oxidative damage to various tissues [49]. Another important aspect of concern associated with frequent fish consumption is the risk derived from exposure to chemical pollutants contained in fish, such as methylmercury (MeHg), dioxins and polychlorinated biphenyls, brominated flame retardants, chlorinated pesticides, and organotin compounds. Contaminants in fish are derived predominantly from their diet and are conserved as they pass from organism to organism in a food chain or food web, possibly resulting in progressively higher concentrations at high trophic levels; this process is called biomagnification (Figure 5). For most chemical contaminants, the lipids serve as the primary storage compartment. Fat is stored mainly in the liver but also in the muscles and the perivisceral and subcutaneous adipose tissues. In lean fish, for instance, the amount of FAs in the liver can reach as much as 40–70 g/100 g tissue, whereas the muscles contain only little fat (5 g/100 g tissue). On the other hand, fatty fish have large amounts of fat in muscles (more than 10% of the total fat), exceeding the fat in the liver [44]. Consequently, the fish at the top of the food chain and fish with large amounts of fat in muscle are those that potentially accumulate the highest levels of contaminants. Species, season, diet, location, life stage, and age have a major effect on both the nutrient and contaminant levels of fish. These levels vary broadly within and between species of both wild and farmed fish. Consequently, assessing the health risks of these chemicals is currently a very difficulty—if not impossible—task. Studies of pollutant bioaccumulation in fish is widespread, but in recent years the calculation of risk factors for the population have become of great importance because although the contaminants sometimes exceed the legal limits for food set by European regulations, they do not always represent a risk for human health. For that reason, the weekly intake is estimated with the provisional tolerable weekly intake recommended by the European Food Safety Authority and the target hazard quotients provided in the United States Environmental Protection Agency region III risk-based concentration table [50], in order to evaluate possible alerts regarding human health hazards. However, the type of fish, the frequency of consumption, and the meal size are essential issues in balancing the health benefits and risks. Certainly, public health warnings identify tuna, swordfish, and marlin (species at the top of food chain) as foods to be eaten in moderation because of possible pollution with MeHg, but it is unlikely that the risk of exposure to toxins would outweigh the potential benefits from omega 3. The regular fish consumption suggested by Mediterranean Diet 172 SECTION 2 Components of the Mediterranean Diet FIGURE 5 The concentration of contaminants tends to increase in the tissue of organisms at higher levels in the marine food chain (biomagnifications). Pattern should not mean special concern for health risks. On the other hand, replacing fish with meat, cereals, or vegetables will not inevitably lead to decreased dietary exposure to these contaminants. SUMMARY POINTS l l l l l l l The Mediterranean diet recommends consuming fish at least twice a week. Benefits from fish consumption have been primarily related to PUFAs. 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[43] Polak-Juszczak L, Komar-Szymczak K. Fatty acid profiles and fat contents of commercially important fish from Vistula Lagoon. Pol J Food Nutr Sci 2009;59(3):225–9. [44] Ben Smida MA, Marzouk B, El Cafsi M. The composition of fatty acids in the tissues of Tunisian swordfish (Xiphias gladius). Food Chem 2009;115:522–8. [45] FAO. The State of World Fisheries and Aquaculture. Rome, Italy: Fisheries and Aquaculture Department, Food and Agriculture Organization of the United Nations; 2008. [46] Sutton SG, Bult TP, Haedrich RL. Relationships among fat weight, body weight, water weight, and condition factors in wild Atlantic salmon parr. Trans Am Fish Soc 2000;129:527–38. [47] Hossain MA. Fish as source of n-3 polyunsaturated fatty acids (PUFAs), which one is better-farmed or wild? Adv J Food Sci Technol 2011;3(6):455–66. [48] Ackman RG, Takeuchi T. Comparison of fatty acids and lipids of smolting hatchery-fed and wild Atlantic salmon (Salmo salar). Lipids 1986;21:117–20. [49] Kelley DS, Taylor PC, Nelson GJ, Mackey BE. Dietary docosahexaenoic acid and immunocompetence in young healthy men. Lipids 1998;33:559–66. [50] US-EPA. Risk-based concentration table. Philadelphia: United States Environmental Protection Agency; 2000. Chapter 17 Contribution of Nuts to the Mediterranean Diet Emilio Ros, MD, PhD1,2 1 Lipid Clinic, Endocrinology & Nutrition Service, Institut d’Invesigacions Biome`diques, August Pi Sunyer, Hospital Clinic, Barcelona, Spain. 2 CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III (ISCIII), Barcelona, Spain. ABBREVIATIONS ALA CHD MUFA NO PREDIMED PUFA RCT SFA a-linolenic acid coronary heart disease monounsaturated fatty acids nitric oxide PREvención con DIeta MEDiterránea polyunsaturated fatty acids randomized clinical trial saturated fatty acids INTRODUCTION By definition, tree nuts are dry fruits with one seed in which the ovary wall becomes hard at maturity. Common edible tree nuts include almonds, Brazil nuts, cashews, hazelnuts, macadamias, pecans, pine nuts, pistachios, and walnuts, but the consumer definition also includes peanuts, which botanically are legumes but have a nutrient profile similar to tree nuts are thus identified as part of the nuts food group [1]. Chestnuts are tree nuts as well, but they are different from all other common nuts because of being starchier and having a different nutrient profile. For the purpose of this chapter, the term nuts comprises all common tree nuts (with the exception of chestnuts) plus peanuts. Extensive research on nuts and health outcomes has been conducted during the 20 years since the publication of the results from the pioneering Adventist Health Study, relating nut consumption to a lower risk of coronary heart disease (CHD) in 1992 [2]. That soon was followed by a landmark randomized clinical trial (RCT) by Sabaté et al. [3], showing an association of walnut intake with reduction of serum cholesterol levels. Nuts, seeds, and pulses are all nutrient-dense foods and have been a regular constituent of mankind’s diet since preagricultural times [4]. In the past century, however, nut consumption in most industrialized nations followed a downward trend, becoming only a marginal source of energy in the daily diet, except for vegetarians and other health-conscious populations such as Seventh Day Adventists [5]. Nevertheless, there has been a recent upsurge of nut intake in Western countries following both the inclusion of this food group in many guidelines for healthful eating and wide media advertisement of accruing evidence of their beneficial health effects. Thus, leading experts in nutritional epidemiology have proposed nuts as a component of optimal diets for the prevention of CHD [6]. Furthermore, in the summer of 2003, the US Food and Drug Administration issued a health claim for nuts and nut-containing products because of the link of nut consumption with a reduced risk of heart disease [7]. Nuts were included in the American Heart Association report setting goals for health promotion and disease reduction for 2020 [8] and are an integral part of the plant-based dietary patterns recommended by dietary guidelines [9]. The scientific evidence behind the proposal of nuts as heart-healthy foods stem from both epidemiological observations suggesting the frequency of nut intake relates inversely to incident CHD and diabetes and from numerous RCTs showing beneficial effects of nut intake on blood lipids and other intermediate markers of CHD [9,10]. The mechanism for these salutary effects probably lies in the interaction of the many bioactive constituents of nuts, which may all favorably influence human physiology. Thus, nuts contain large amounts of vegetable protein and fat, which is made up mostly of unsaturated fatty acids; The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 175 176 SECTION 2 Components of the Mediterranean Diet they are also dense in dietary fiber; vitamins (e.g., folic acid, niacin, tocopherols, vitamin B6); minerals (e.g., calcium, magnesium, potassium, zinc); and many other bioactive constituents such as phytosterols and phenolic compounds [1]. Contrary to expectations because of the high energy content of nuts, epidemiological studies and clinical trials suggest that their regular consumption is unlikely to promote weight gain [11]. This chapter summarizes current knowledge on the expanding topic of nuts and health and on their contribution to the healthful properties of the Mediterranean diet. NUTS IN THE MEDITERRANEAN DIET The oldest evidence of cultivation of the common tree nuts almonds (Prunus amygdalus), hazelnuts (Corylus avellana), walnuts (Juglans regia), and pistachios (Pistachia vera) are from Asia, mainly the Anatolian peninsula (modern Turkey). From there, cultivars of these trees were introduced in Greece, then in Italy during the Roman Empire, and extended to all of Europe during the Middle Ages [12]. Common pine nuts (Pinus pinea) are not usually collected from plantations but from natural forests, mostly in the Mediterranean area. The most popular edible tree nuts are precisely the Asian/Mediterranean nuts: almonds, walnuts, hazelnuts, and pistachios. In Europe, where comparisons across different countries have been made, nut supply is reported to be highest for Mediterranean countries [13]. In the European Prospective Investigation into Cancer and Nutrition, wherein 24-h food records were collected for nearly 37,000 subjects from 10 countries [14], a clear north-to-south gradient was apparent; Swedish individuals had a mean whole nut and peanut intake of 0.61 g/day and average portion size of 15.1 g/day, whereas Spanish subjects had a mean intake of 4.83 g/day and average portion size 34.7 g/day. Of note, the proportion of subjects reporting nut consumption on the day of the 24-h food recall followed a similar pattern (Sweden 2.5% and Spain 11.9%). Thus, nuts are an important component and part of the definition of the Mediterranean diet, the traditional dietary pattern found in Crete, Greece, Italy, and Spain in the early 1960s. This dietary pattern is characterized by a high intake of cereals, vegetables, fruits, nuts, and olive oil; a moderate intake of fish and alcohol (mostly wine); and a low intake of dairy products, red meat and meat products, and sweets [15]. Nuts are incorporated in the Mediterranean diet in various ways: they are consumed as snacks, desserts, or part of a meal or are eaten whole (fresh or roasted), in spreads (almond paste), as oils, or hidden in commercial products, mixed dishes, sauces, pastries, ice creams, and baked goods. Almond-based desserts, such as nougat and marzipan, are customary in many Mediterranean areas for almost ritual consumption during holiday seasons such as Christmas. In the past decade there has been a slowly increasing pattern of nut consumption in both Mediterranean areas and Western countries, likely reflecting increased awareness of the health-promoting properties of nuts and their lack of fattening power. NUTRIENT CONTENT OF MEDITERRANEAN NUTS As mentioned, nuts are nutrient-dense foods. With the exception of chestnuts, which contain little fat, nuts have a high total fat content, ranging from 46% in pistachios to 68% in pine nuts, and they provide 23 to 27 kJ/g (Table 1). Thus, nuts are one of the natural plant foods richest in fat after vegetable oils. However, the fatty acid composition of nuts is beneficial because the saturated fatty acid content is low (range, 4–6%), and nearly one-half of the total fat content is made up of unsaturated fat, monounsaturated fatty acids (MUFAs) in most nuts, a predominance of polyunsaturated fatty acids (PUFAs) over MUFAs in pine nuts, and mostly PUFAs, both linoleic acid and a-linolenic acid (ALA), the plant omega 3 fatty acid, in walnuts [1]. With regard to walnuts, it must be noted that of all edible plants they are the whole food with the highest content in ALA. As discussed below, the particular lipid profile of nuts in general and walnuts in particular is likely to be an important contributor to the beneficial health effects of frequent nut consumption. Nuts are also rich sources of other bioactive macronutrients that have the potential to beneficially affect metabolic and cardiovascular outcomes. They are an excellent source of protein (25% of energy) and often have a high content of the amino acid L-arginine, which is the substrate for endothelium-derived nitric oxide synthesis, a principal regulator of vascular tone and blood pressure [16]. This explains why nut intake might help improve vascular reactivity, as discussed below in the section “Nut Consumption and Health Outcomes” subheading “Randomized Clinical Trials.” Nuts also are a good source of dietary fiber, which ranges from 4 to 11/100 g nuts (Table 1), and standard servings provides 5–10% of daily fiber requirements [1]. Among the constituents of nuts are significant amounts of essential micronutrients that are associated with an improved health status when consumed at doses beyond those necessary to prevent deficiency states. Mediterranean nuts contain sizeable amounts of the B vitamin folate (Table 2). They are also rich sources of antioxidant vitamins (e.g., tocopherols) and polyphenols, necessary to protect the germ from oxidative stress and preserve the reproductive potential of the seed but also bioavailable after consumption and capable of providing a significant antioxidant load [17]. Almonds in particular are especially Contribution of Nuts to the Mediterranean Diet Chapter 17 177 TABLE 1 Average Nutrient Composition of Mediterranean Nuts (Per 100 g) Nuts Energy (kJ) Protein (g) Fiber (g) Fat (g) SFAs (g) MUFAs (g) PUFAs (g) LA (g) ALA (g) Almonds 2418 21.3 8.8 50.6 3.9 32.2 12.2 12.2 0.00 Hazelnuts 2629 15.0 10.4 60.8 4.5 45.7 7.9 7.8 0.09 Pine nuts 2816 13.7 3.7 68.4 4.9 18.8 34.1 33.2 0.16 Pistachios 2332 20.6 9.0 44.4 5.4 23.3 13.5 13.2 0.25 Walnuts 2738 15.2 6.4 65.2 6.1 8.9 47.2 38.1 9.08 Data are for raw nuts. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; LA, linoleic acid; ALA, a-linolenic acid. Source: US Department of Agriculture Nutrient Data Base at: http://www.nal.usda.gov/fnic/cgi-bin/nut_search.pl [accessed 20.09.12]. TABLE 2 Average Composition of Mediterranean Nuts in Selected Micronutrients, Minerals and Phytochemicals (Per 100 g) Nuts Folate (mg) a-Tocopherol (mg) Potassium (mg) Magnesium (mg) Calcium (mg) Phytosterols (mg) Polyphenols (mg) Almonds 29 25.9 728 275 248 120 287 Hazelnuts 113 15.0 680 163 114 96 687 Pine nuts 34 9.3 597 251 16 141 58 Pistachios 51 2.3 1025 121 107 214 1420 Walnuts 98 0.7 441 158 98 72 1576 Data are for raw nuts. Sources: US Department of Agriculture Nutrient Data Base at: http://ndb.nal.usda.gov/ndb/foods/list [accessed 20.09.12]; National Institute for Health and Welfare, N.U. Fineli. Finnish Food Composition Database at: http://www.fineli.fi [accessed 26.09.12]; and Phenol-Explorer 2.0. Data base on polyphenol content in foods at: http://www.phenol-explorer.eu [accessed 25.09.12]. rich in a-tocopherol, whereas walnuts contain significant amounts of its isomer g-tocopherol, which has been investigated much less than a-tocopherol but is increasingly recognized as a relevant antiatherogenic molecule [18]. Remarkably, most of the antioxidants in all nuts are located in the pellicle or outer soft shell, as shown for almonds [19], and 50% or more of them are lost when nuts are peeled or roasted [17]. These facts, usually overlooked in prior studies of nuts, should be considered when giving advice on nut consumption in healthful diets. Walnuts are an exception because they are almost always consumed as the raw, unpeeled product; moreover, among nuts, walnuts have the highest polyphenol content [20]. Nuts are cholesterol free, but their fatty fraction contains sizeable amounts of chemically related noncholesterol sterols belonging to a heterogeneous group of compounds known as plant sterols or phytosterols [1] (Table 2), which are non-nutritive components of all plants that play an important structural role in plant membranes, where they serve to stabilize phospholipid bilayers, just as cholesterol does in animal cell membranes [21]. Phytosterols interfere with cholesterol absorption and thus help lower blood cholesterol when present in gram doses in the intestinal lumen. The mechanism of action of phytosterols has been linked to their hydrophobicity, which is higher than cholesterol because of a bulkier hydrocarbon molecule and entails a higher affinity for micelles than has cholesterol. Consequently, cholesterol is displaced from micelles and the amount available for absorption is limited [22]. The phytosterol content of nuts very likely contributes to their cholesterol-lowering effect (see “Nut Consumption and Health Outcomes” subheading “Randomized Clinical Trials”). Compared to other commonly consumed foods, nuts have an optimal nutritional density regarding beneficial minerals such as calcium, magnesium, and potassium (Table 2). Like that of most vegetables, the sodium content of nuts is very low. A high intake of calcium, magnesium, and potassium, together with a low sodium intake, is associated with protection against hypertension, insulin resistance, and overall cardiovascular risk [23], as well as bone demineralization. 178 SECTION 2 Components of the Mediterranean Diet ↓ Cholesterol Unsaturated fatty acids Phytosterols Fiber ↓ Oxidation ↓ Inflammation ↑ Glucose control Minerals (K, Mg, Ca) Tocopherols Vegetable protein (L-arginine) Polyphenols ↑ NO ↓ Blood pressure ↑ FMD ↓ CVD and diabetes FIGURE 1 Potential mechanisms of cardiometabolic protection by nut constituents. The consumption of nuts ameliorates cardiovascular health because of their unique composition, including bioactive nutrients and phytochemicals, which synergize to beneficially affect cardiometabolic pathways. The main known nutrients of nuts are represented, together with their principal biological targets (thin arrows). The net effects of nuts on intermediate markers of cardiovascular risk that have been demonstrated in clinical trials are lowered cholesterol, improved glycemic control, decreased blood pressure, improved vasomotion, and antioxidant and anti-inflammatory actions. The overall result is reduced cardiovascular disease and/or type 2 diabetes (thick arrows), as suggested in prospective cohort studies and observed in the PREDIMED trial. See text for details. K, potassium; Mg, magnesium; Ca, calcium; NO, nitric oxide; FMD, flow-mediated vasodilation; CVD, cardiovascular disease. In summary, the macronutrient, micronutrient, and phytochemical components of nuts have all been documented to contribute to a reduced risk of CHD and related metabolic disturbances. As shown in Figure 1, most bioactive nut components synergize to affect multiple metabolic and vascular physiology pathways, leading to protection from cardiovascular disease and type 2 diabetes. For these reasons, whole, raw, unpeeled, and otherwise unprocessed nuts may be considered natural health capsules, where the whole is always better than the parts. NUT CONSUMPTION AND HEALTH OUTCOMES Nut consumption has been and still is the focus of intense epidemiological and clinical research. Prospective studies have examined clinical cardiovascular end points, e.g., CHD, type 2 diabetes, stroke, heart failure, hypertension, and obesity, whereas RCTs have generally explored intermediate biomarkers of cardiovascular disease risk. An exception is the PREvención con DIeta MEDiterránea (PREDIMED) trial of primary cardiovascular prevention, which includes nut supplementation in one study arm and has hard cardiovascular end points as the main study outcome [24]. The scientific evidence acquired from these studies is summarized below. Epidemiological Studies As reviewed by Ros et al. [10], four large prospective studies examining associations of dietary components with health outcomes in the United States reported a beneficial effect of nut consumption on fatal and nonfatal CHD after follow-up of large cohorts of healthy subjects ranging from 6 to 18 years old (Figure 2). A pooled analysis of these studies indicated that subjects in the highest category of nut and/or peanut butter intake had a 37% reduction in multivariable-adjusted risk of fatal CHD [24]. A dose-response relationship between nut consumption and reduced CHD mortality was observed in all studies. The dose–response relationship between nut consumption and CHD risk resulted in an average 8.3% reduction for each weekly serving of nuts [25]. The consistency of findings in all studies points to a causal association between nut consumption and reduced CHD and suggests that nuts are probably one of the most heart-healthy foods in the usual diet. Findings from the Nurses’ Health Study suggested that nut and peanut butter consumption was associated with a reduced incidence of type 2 diabetes in women [26]. A subsequent report from a Chinese cohort of nearly 64,000 women also suggested a protective effect of nuts against diabetes risk [27]. These findings, however, were not confirmed in male participants in the Physicians’ Health Study [28]. Figure 3 illustrates the findings of the main prospective studies relating nut consumption to diabetes risk. Thus, regular consumption of nuts is clearly beneficial for CHD risk, but confirmation of any protective role against diabetes risk must await further studies. Additional reports from the Physicians’ Health Study showed no association between the frequency of nut intake and ischemic stroke [29] or heart failure [30]. While epidemiologic data on the relationship between nut consumption and Contribution of Nuts to the Mediterranean Diet Chapter 17 1.2 Relative risk (adjusted) FIGURE 2 Incidence of fatal coronary heart disease by frequency of nut intake. The graph presents the results of four large prospective studies, all conducted in the United States, of nut consumption and risk of death from coronary heart disease. 179 1.0 Adventists Health Study 1992 0.8 Iowa Women’s Health Study 1993 Nurses’ Health Study 1998 0.6 Physicians’ Health Study 2002 0.4 0.2 0 1-2/mo 3-4/mo 1-4/wk >5/wk Frequency of nut intake 1.2 Relative risk (adjusted) FIGURE 3 Incidence of type 2 diabetes by frequency of nut intake. Results of three large prospective studies of nut consumption and risk of diabetes. The two US studies considered the frequency of consumption of all nuts, including peanuts, whereas the Chinese study considered exclusively quintiles of peanut consumption. 1.0 Nurses’ Health Study 2002 Shanghai Women’s Health Study 2008 Physicians’ Health Study 2009 0.8 0.6 0.4 0.2 <1/mo 0.1 g/d <1/wk 0.4 g/d 1-4/wk 0.7 g/d 5-6/wk 1.4 g/d >7/wk Tree nuts and peanuts 3.1 g/d Peanuts Frequency of intake incident hypertension is limited and inconclusive, at least three studies have reported an association between increasing nut consumption and reduced circulating levels of inflammatory biomarkers, as reviewed by Ros et al. [10]. Randomized Clinical Trials The epidemiologic evidence reporting benefits of nut consumption on CHD risk was the impetus for RCTs designed to investigate effects on cardiovascular risk factors and begin to understand the underlying mechanisms for cardioprotection in observational studies. Most RCTs using nuts have been short term and compared diets supplemented with nuts with control diets for outcomes on blood lipid changes in healthy subjects or patients with moderate hypercholesterolemia. Few studies have investigated glycemic control in addition to the lipid profile using nuts in patients with diabetes. Some studies have focused on the relevant question of whether unrestricted nut intake has an effect on body weight. Other RCTs have examined the effects of nut diets on intermediate risk markers such as insulin sensitivity, blood pressure, endothelial function, and inflammatory status. Of the Mediterranean nuts, almonds and walnuts have been the most studied, followed by pistachios and hazelnuts, whereas there are no studies of pine nuts. The long-term PREDIMED trial targeted both the effects of nut consumption on intermediate cardiometabolic markers and clinical outcomes, such as the metabolic syndrome, diabetes, and cardiovascular events, among others. Given the relevance of this study for the issue of nuts and health outcomes, its results are dealt with in a separate section, “Health Effects of Mediterranean Nuts in the PREDIMED Trial.” 180 SECTION 2 Components of the Mediterranean Diet The effects of nut consumption on blood lipids and lipoproteins have been investigated in many RCTs. A recent pooled analysis of 25 RCTs conducted in seven countries and examining nut-enriched diets versus control diets for outcomes on blood lipids found that nuts had a cholesterol-lowering effect that was related to both dose and baseline cholesterol level, similar by sex and across all age groups, and independent of the type of nut examined [31]. Specifically, consumption of 67 g (2.4 oz) of nuts daily was associated with mean reductions of 10.9 mg/dL (5.1%) in total cholesterol, 10.2 mg/dL (7.4%) in low-density lipoprotein (LDL) cholesterol, and 0.22 (8.3%) in LDL-to-high-density lipoprotein (HDL) ratios (P < 0.001 for all). Nuts had no significant effect on HDL-cholesterol or triglycerides, except in participants with serum triglycerides >150 mg/dL, in whom a significant 10.2-mg/dL reduction was observed. A separate meta-analysis of 13 RCTs examined the effect of walnut-enriched diets on blood lipid levels [32]. Compared with control diets, diets containing walnuts in amounts varying from 30 to 108 g/day (10–24% of energy) were associated with weighted mean reductions of total cholesterol and LDL-cholesterol of 10.3 and 9.2 mg/dL, respectively (P < 0.001 for both), which concur with the results of the pooled analysis for different types of nuts [31]. Acute feeding studies indicate that nuts reduce postprandial glucose responses when consumed with foods that have a high glycemic index, suggesting that they may be useful in diabetic control [33]. Even though it seems counterintuitive to recommend an energy-dense food such as nuts to people with diabetes, it is important to underline that there is no evidence that their frequent consumption is associated with increased adiposity, as shown in a recent meta-analysis of RCTs using nut diets versus control diets [34]. Indeed, the common perception that fatty foods provide excess energy and thus promote obesity has had a negative effect on the image of nuts. The question of whether increasing the intake of nuts and therefore calories could lead to unwanted weight gain and related health problems, such as diabetes, is a critical one. Mechanistically, the lack of weight gain after consuming nuts is largely attributable to their prominent satiating effect, which results in food compensation accounting for up to 75% of the energy they provide [35]. An additional factor contributing to offset energy acquisition after eating nuts is fat malabsorption, documented as increased fecal fat excretion, which may be due in part to the high fiber content of nuts or to incomplete digestion of their matrices—the fat of nuts is enclosed within cell membranes, which are not readily available to digestive enzymes even after thorough mastication [36]. By virtue of their unique composition, nuts are likely to affect cardiometabolic markers other than blood lipids and glycemic control. Thus, studies have examined the effects of nuts on oxidative stress, inflammation, and vascular reactivity, as reviewed by Ros et al. [10,11]. Limited evidence from small RCTs suggests that nuts, particularly walnuts, have beneficial effects on blood pressure and endothelial function, which is attributable to their richness in L-arginine, PUFAs, and polyphenols [37]. Regarding oxidative stress, it is well known that PUFAs are more susceptible to oxidation than MUFAs [38], but nuts are a rich source of antioxidants. Available evidence suggests that MUFA-rich nuts (almonds, hazelnuts, pistachios) can moderately improve oxidative status, whereas PUFA-rich nuts (walnuts) have a neutral or slightly beneficial effect [39]. In any case, there is no evidence that frequent nut consumption reduces antioxidant defenses. Finally, nut feeding studies have documented reduced circulating concentrations of inflammatory cytokines but no consistent changes in C-reactive protein [11]. In summary, the emerging picture is that frequent nut consumption has beneficial effects on cardiovascular risk factors beyond well-established cholesterol lowering effects without incurring any undue increase in body weight. Health Effects of Mediterranean Nuts in the PREDIMED Trial The PREDIMED study is a large, parallel group, multicenter, controlled, 5-year RCT conducted in Spain. Eligible participants were men 55 to 80 years of age and women 60 to 80 years of age who were at high risk for but had no cardiovascular disease at enrolment. Candidates for the study had to fulfill at least one of two criteria: type 2 diabetes or three or more cardiovascular risk factors, including current smoking, hypertension, hypercholesterolemia, low HDL-cholesterol, body mass index 25 kg/m2, or family history of premature CHD. Participants were randomized to three groups: two Mediterranean diets supplemented with either extra virgin olive oil or mixed nuts or a control diet (advice on a low-fat diet). They received quarterly individual and group educational sessions and, depending on group assignment, free provision of extra virgin olive oil (1 L/week), mixed Mediterranean nuts (30 g/day; 15 g walnuts, 7.5 g almonds, and 7.5 g hazelnuts), or small nonfood gifts. Of note, the diets were ad libitum and increased physical activity was not promoted; thus the full effect of dietary intervention by itself, without decreased energy intake or increased energy expenditure, could be ascertained. The primary end point was the rate of major cardiovascular events (myocardial infarction, stroke, or cardiovascular death). The detailed protocol has been published elsewhere [24]. Since its inception in October 2003, the trial has provided a continuous flow of data on the beneficial health effects of Mediterranean diets enriched with both supplemental foods, culminating with the recent publication of results on the main cardiovascular outcome [40]. The results have generally been similar Contribution of Nuts to the Mediterranean Diet Chapter 17 181 for both the extra virgin olive oil- and nut-supplemented Mediterranean diets in comparison with the control diet. The principal results concerning the Mediterranean diet enriched with nuts are summarized below. Results of a pilot study involving the first 772 participants completing the intervention for 3 months [41] showed that, compared with the control diet, the two enhanced Mediterranean diets resulted in significant reductions of both systolic and diastolic blood pressure and total and LDL-cholesterol; increased HDL-cholesterol; decreased fasting glucose levels in diabetic participants and increased insulin sensitivity in those without diabetes; and reduction in circulating levels of inflammatory cytokines. Furthermore, the nut-enriched diet also reduced fasting triglyceride levels [41]. While most results confirmed prior data, the blood pressure-lowering effect of the nut diet was novel. Another PREDIMED report provides insight into the possible mechanism by showing that both the nut- and olive oil-enriched diets were associated with reduced cholesterol-to-phospholipid ratios of erythrocyte membranes, which would translate into increasing membrane fluidity [42]. Another report from the 3-month pilot study showed that both supplemented Mediterranean diets were associated with reduced oxidized LDL levels [43], which support the beneficial effects of nut diets on oxidative stress. A further substudy analyzed 3-month changes in both circulating inflammatory biomarkers and in the expression of ligands for inflammatory molecules in circulating monocytes after the study diets [44]. The findings indicate reductions in both circulating inflammatory mediators and, importantly, reduced monocyte expression of proinflammatory ligands after both extra virgin olive oil- and nut-enriched Mediterranean diets, thus beginning to unravel the molecular bases for the anti-inflammatory effects of nut consumption. Regarding cardiometabolic outcomes, the PREDIMED trial has provided first-level evidence for the protection afforded by long-term consumption of Mediterranean nuts in the context of a healthful diet against metabolic syndrome [45], type 2 diabetes [46], and cardiovascular diseases [40]. In the first 1224 trial participants completing the intervention for 1 year, the nut-enriched Mediterranean diet was associated with a 14% reduction in the prevalence of metabolic syndrome, which was mainly due to reduced visceral adiposity [45]. Because there were no weight changes, this suggests fat redistribution away from the abdominal compartment. In one of the 11 PREDIMED recruiting centers, oral glucose tolerance tests were performed yearly in nondiabetic participants to accurately detect incident diabetes. Results of this substudy of 418 subjects followed for a mean of 4 years showed that both enhanced Mediterranean diets protected against development of diabetes, and the reduction of incident diabetes in the nuts arm was 52% compared with the control diet [46]. Again, this beneficial effect took place in the absence of weight loss or increased energy expenditure during physical activity. The final results of the PREDIMED trial have recently been published, showing for the first time a reduction of incident cardiovascular diseases after long-term consumption of a diet enriched by nuts [40]. After a median follow-up of 4.8 years in 7447 participants, those assigned the two Mediterranean diets showed a 30% reduction in cardiovascular disease events (myocardial infarction, stroke, or cardiovascular death) compared with the control diet. The nut diet also was associated with a significant 49% reduction in stroke risk. No effects of the intervention diets could be discerned on myocardial infarction alone or total mortality. Of note, the interventions were intended to improve the overall dietary pattern, but the major between-group differences in food intake were for the supplemental items. Thus, nuts were probably responsible for most of the observed benefits in the Mediterranean diet with nuts group. The results of the PREDIMED trial show the full potential of nuts and other healthy foods, such as extra virgin olive oil, to improve cardiovascular health. CONCLUSION Nuts are energy-dense foods rich in bioactive macronutrients, micronutrients, and phytochemicals. The unique composition of nuts is critical to their health effects. Indeed, there is consistent evidence from epidemiologic and clinical studies of the beneficial effects of nut consumption and their constituents on cardiovascular risk, as well as on diabetes and major and emerging cardiovascular risk factors, as summarized in Table 3. The evidence to-date that including nuts in a healthy dietary pattern affords protection against cardiometabolic disorders beyond that attributable to the other components of the diet is convincing. Importantly, these effects take place without undue weight gain, or even with reduced adiposity, and target multiple cardiovascular risk factors and mechanisms, which help explain why nuts so potently reduce cardiovascular risk. The PREDIMED trial has already demonstrated that long-term adherence to a healthful diet enriched with one daily serving of Mediterranean nuts reduces the incidence of type 2 diabetes and cardiovascular disease, particularly stroke. Ongoing research within this landmark trial will eventually prove whether the beneficial properties of nuts extend to the prevention of other prevalent chronic diseases, including cancer, heart failure, and neurodegenerative disorders. 182 SECTION 2 Components of the Mediterranean Diet TABLE 3 Effects of Nut Consumption on Cardiovascular Diseases and Risk Factors: Summary of the Scientific Evidence Disease/Factor Effect Level of Evidence Coronary heart disease Decrease ++ Ischemic stroke No change + Heart failure No change + Hypertension Decrease Diabetes No change/decrease Inflammatory markers Decrease + Body weight No change/decrease ++ Decreasea ++ a ++ Epidemiologic studies Clinical studies Blood lipid profile Total cholesterol LDL-cholesterol HDL-cholesterol Triglycerides Insulin sensitivity Decrease a + Increase Decrease a a + + Increase Decrease a + Oxidation Decrease a + Inflammation Decrease + Vascular reactivity Increase + Body weight No change Blood pressure Visceral adiposity Metabolic syndrome Type 2 diabetes Cardiovascular diseases Stroke ++ Decrease a + Decrease b + Decrease b + Decrease b + Decrease b + , equivocal evidence; +, limited evidence from few studies; ++, evidence from several studies. a Evidence collected from the PREDIMED trial, among others. bEvidence collected only in the PREDIMED trial. SUMMARY POINTS l l l l l Nuts, particularly almonds, hazelnuts, pine nuts, pistachios, and walnuts, are integral components of the Mediterranean diet. All nuts are nutrient-dense foods rich in unsaturated fatty acids, protein, fiber, minerals, potent antioxidants, and other beneficial phytochemicals. Prospective studies have associated exposure to nuts with a reduced incidence of CHD in both sexes and of diabetes in women. In RCTs, nut-enriched diets have consistently reduced total and LDL-cholesterol independent of the background diet. The PREDIMED trial has convincingly shown that long-term adherence to a Mediterranean diet supplemented with nuts results in reduced rates of cardiovascular disease, particularly stroke. Contribution of Nuts to the Mediterranean Diet Chapter 17 l l 183 A beneficial effect of the nut-enriched Mediterranean diet on metabolic syndrome and diabetes also has been reported by the PREDIMED study. Increasing nut intake does not promote obesity largely because of a pronounced satiating effect. ACKNOWLEDGMENTS This work was supported in part by grants from ISCIII, Spanish Ministry of Economy and Competitiveness (FIS Thematic Research Networks C03/01 and G03/140), and the California Walnut Commission, Sacramento, CA. CIBERobn is an initiative of ISCIII, Spain. REFERENCES [1] Sabaté J, Salas-Salvadó J, Ros E. Nuts: nutrition and health outcomes. Br J Nutr 2006;96(Suppl. 2):1–102. [2] Fraser GE, Sabaté J, Beeson WL, Strahan TM. A possible protective effect of nut consumption on risk of coronary heart disease. the Adventist Health Study. Arch Intern Med 1992;152:1416–24. [3] Sabaté J, Fraser GE, Burke K, Knutsen SF, Bennett H, Lindsted KD. Effects of walnuts on serum lipid levels and blood pressure in normal men. N Engl J Med 1993;328:603–7. [4] Eaton SB, Konner M. Paleolithic nutrition. A consideration of its nature and current implications. N Engl J Med 1985;312:283–9. 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Eur J Clin Nutr 2010;64:75–9. 184 [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] SECTION 2 Components of the Mediterranean Diet Djoussé L, Gaziano JM, Kase CS, Kurth T. Nut consumption and risk of stroke in US male physicians. Clin Nutr 2010;29:605–9. Djoussé L, Rudich T, Gaziano JM. Nut consumption and risk of heart failure in the Physicians’ Health Study I. Am J Clin Nutr 2008;88:930–3. Sabaté J, Oda K, Ros E. Nut consumption and blood lipid levels: a pooled analysis of 25 intervention trials. Arch Intern Med 2010;170:821–7. Banel DK, Hu FB. Effects of walnut consumption on blood lipids and other cardiovascular risk factors: a meta-analysis and systematic review. Am J Clin Nutr 2009;90:56–63. Kendall CWC, Josse AR, Esfahani A, Jenkins DJA. Nuts, metabolic syndrome and diabetes. Br J Nutr 2010;104:465–73. Flores-Mateo G, Rojas-Rueda D, Basora J, Ros E, Salas-Salvadó J. Nut intake and adiposity: meta-analysis of clinical trials. Am J Clin Nutr 2013;97:1346–55. Mattes RD, Kris-Etherton PM, Foster GD. Impact of peanuts and tree nuts on body weight and healthy weight loss in adults. J Nutr 2008;138:1741S–5S. Ellis PR, Kendall CW, Ren Y, Parker C, Pacy JF, Waldron KW, et al. Role of cell walls in the bioaccessibility of lipids in almond seeds. Am J Clin Nutr 2004;80:604–13. Casas-Agustench P, López-Uriarte P, Ros E, Bulló M, Salas-Salvadó J. Nuts, hypertension and endothelial function. Nutr Metab Cardiovasc Dis 2011;21(Suppl. 1):S21–33. Reaven PD, Witzum JL. Oxidized low density lipoproteins in atherogenesis: role of dietary modification. Annu Rev Nutr 1996;16:51–71. López-Uriarte P, Bulló M, Casas-Agustench P, Babio N, Salas-Salvadó J. Nuts and oxidation: a systematic review. Nutr Rev 2009;67:497–508. Estruch R, Ros E, Salas-Salvadó J, Covas MI, Corella D, Arós F, et al. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med 2013;368:1279–90. Estruch R, Martı́nez-González MA, Corella D, Salas-Salvadó J, Ruiz-Gutiérrez V, Covas MI, et al. Effects of a Mediterranean-style diet on cardiovascular risk factors. A randomized trial. Ann Intern Med 2006;145:1–11. Barceló F, Perona JS, Prades J, Funari SS, Gomez-Gracia E, Conde M, et al. Mediterranean-style diet effect on the structural properties of the erythrocyte cell membrane of hypertensive patients: the Prevención con Dieta Mediterránea Study. Hypertension 2009;54:1143–50. Fitó M, Guxens M, Corella D, Sáez G, Estruch R, de la Torre R, et al. Effect of a traditional Mediterranean diet on lipoprotein oxidation. A randomized, controlled trial. Arch Intern Med 2007;167:1195–203. Mena MP, Sacanella E, Vázquez-Agell M, Morales M, Fitó M, Viñas C, et al. Inhibition of circulating immune cell activation: a molecular antiinflammatory effect of the Mediterranean diet. Am J Clin Nutr 2009;89:248–56. Salas-Salvadó J, Fernández-Ballart J, Ros E, Martı́nez-González MA, Fitó M, Estruch R, et al. Effect of the Mediterranean diet supplemented with nuts on metabolic syndrome status. One-year results of the PREDIMED randomized trial. Arch Intern Med 2008;168:2449–58. Salas-Salvadó J, Bulló M, Babio N, Martı́nez-González MA, Ibarrola N, Basora J, et al. Reduction in the incidence of type 2 diabetes with the Mediterranean diet: results of the PREDIMED-REUS dietary intervention trial. Diabetes Care 2011;34:14–9. Chapter 18 The Mediterranean Diet and Mineral Composition Marta Mesı́as, PhD, Isabel Seiquer, PhD and M. Pilar Navarro, PhD Consejo Superior de Investigaciones Ciencı´ficas, (CSIC), Spain. ABBREVIATIONS AAS DR FCT FFQ MD MDP RDA WFR atomic absorption spectroscopy dietary recall food composition tables food frequency questionnaire Mediterranean diet Mediterranean dietary patterns Recommended Dietary Allowances. weighed food records INTRODUCTION The traditional Mediterranean diet (MD) is considered to be one of the best dietary models for human health, and its benefits have been demonstrated in numerous studies. Adequate mineral intake is known to be necessary for overall health; in addition, certain healthy dietary patterns have been correlated with the suitable intake of specific minerals [1]. The MD is highly caloric and rich in minerals and vitamins derived from vegetables, fruits, whole-meal cereals, fish, and virgin olive oil; therefore, close adherence to Mediterranean dietary patterns (MDPs) makes the risk of deficient mineral intake quite improbable [2]. Although several authors have studied the adherence of population groups to an MDP [2–4], it is also important to estimate the nutritional value of the diet and its proximity to dietary recommendations. Accordingly, there is a need to evaluate the dietary factors associated with the MD that may affect the absorption and retention of minerals. This chapter focuses on the mineral composition of the MD and examines whether it satisfies both intake and bioavailability requirements of minerals. MINERAL INTAKE IN THE MD Prior research concerning the mineral content of the MD and the intake of minerals according to MD adherence is detailed below and summarized in Tables 1 and 2. Calcium Dairy products are the major source of calcium in conventional diets, although other foods such as cereals, fruit and vegetables, nuts and legumes—which are typical of the MDP—also provide significant quantities of calcium. Moreover, calcium intake may be increased by the consumption of moderate amounts of foods of animal origin, including small fish consumed as a whole [13,14]. Contribution of the main food sources to daily calcium intake among Spanish male adolescents consuming a diet based on MDPs, according to data reported by Seiquer et al. [6], is represented in Figure 1. Because the MD is associated with a low to moderate consumption of dairy products, closer adherence to this diet could imply reduced calcium intake. Corroborating this hypothesis, Feart et al. [4] reported an inverse association between MD adhesion and calcium intake among French women aged 65 years and older, although this association was of borderline significance The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 185 186 SECTION 2 Components of the Mediterranean Diet TABLE 1 Studies Evaluating Mineral Content in Diets and Adherence to the Mediterranean Diet Low Adherence to the MD High Adherence to the MD FFQ + 24-h DR + FCT Low MD score (score 0–3) High MD score (score 6–9) Evaluation of nutrient composition of Cretan MD and current typical diet of Greek adolescents 7-day WFR + AAS Greek adolescents’ diet Traditional Cretan MD [6–10] Evaluation of mineral intake in Spanish adolescents. Comparison between habitual diet and diet based on the MDP 24-h DR + 48-h WFR + FCT (adolescents’ diet) 7-day duplicate menu + AAS (MD) Spanish adolescents’ habitual diet (KIDMED index ¼ 1) Diet based on the MDP (KIDMED index ¼ 7) [11] Evaluation of nutrient intake among Spanish adolescents (first line: 6–14 y; second line: 15–24 y). Comparison between diets according to MD adherence 24-h DR + FCT Poor adherence to the MD (KIDMED index 3) High adherence to the MD (KIDMED index 8) [2] Evaluation of nutrient intake among Spanish population. Comparison between diets according to MD adherence FFQ + FCT Low MD score (quintile 1) High MD score (quintile 5) [3] Evaluation of nutrient intake among the Spanish population. Comparison between diets according to MD adherence FFQ + FCT Low MD score (quartile 1) High MD score (quartile 4) [12] Evaluation of mineral content in diets provided at Spanish University residence Duplicate meals + AAS - Diet close to the MDP(KIDMED index ¼ 9) References Methodology Mineral Content [4] Evaluation of nutrient intake in French older community dwellers. Comparison between diets according to MD adherence [5] This table details information about methodology, mineral content evaluation, and characteristics of the diets according to low and high adherence to the Mediterranean diet from studies evaluating mineral content in diets. AAS, atomic absorption spectroscopy; DR, dietary recall; FCT, food composition table; FFQ, food frequency questionnaire; MD, Mediterranean diet; MDP, Mediterranean dietary pattern; WFR, weighed food records. in men (Table 2). After consuming diets scoring high in the MD category, neither men (862 mg/day) nor women (773 mg/ day) reached the calcium intake recommendations for this age group (1000–1200 mg/day) [15]. Similarly, Kafatos et al. [5] showed that the calcium intake associated with the traditional MD in Crete (826 mg/day) was lower than the 1062 mg calcium/day consumed by Greek adolescents in their typical diet, characterized by the consumption of fast foods, bakery products, and carbonated drinks and by a low intake of legumes, fruit, and vegetables. A positive association between adherence to the MD and calcium intake has, however, been reported by other authors. Seiquer et al. [6] reported a slight increase in calcium intake among a group of Spanish adolescents who consumed a diet based on the MDP (1024 mg/day; KIDMED index ¼ 7) compared with their habitual diets (935 mg/day; KIDMED index ¼ 1). With the MD, the percentage of calcium provided by dairy products decreased but that provided by cereals, legumes, and fruit and vegetables increased. Nevertheless, despite this higher mineral intake, it remained lower than the calcium dietary reference intake for this age group, estimated to be 1300 mg/day [15]. In this respect, Serra-Majem et al. [11] observed a significant positive relationship between calcium intake and adherence to the MD among Spanish adolescents. In their study, calcium intake did not reach recommended levels, but the closer adherence to the MDP decreased the percentage of subjects with a low calcium intake. Studies of the global Spanish population have reported no variation in calcium intake when subjects consumed diets that closely adhere to an MDP compared with diets with a lower score in this respect [2,3]. In both of the latter studies, calcium intake was close to or even above recommended levels. Ultimately, a diet based on an MDP may provide an adequate amount of calcium, but mineral intake should be increased among population groups with higher needs to meet recommended levels and prevent diseases related to calcium deficiency. Mediterranean Diet and Minerals Chapter 18 187 TABLE 2 Daily Mineral Intake According to Adherence to Mediterranean Dietary Patterns Men Women Low Adherence to MD High Adherence to MD Low Adherence to MD High Adherence to MD 947.7 (481.9) 862.1 (415.7) 892.9 (471.9a) 773.5 (381.4) Global Population Low Adherence to MD High Adherence to MD 1062 826 [2] 1102 (350) 1409 (497) [3] 1235 (479.7) 1244 (436.3) 1488 1322 1702 (296) 2220 (460) 295 483 352 (53) 497 (98) 15 20 14.4 (2.2) 20.3 (4.3) 11 9 13 (6) 24 (16) Minerals by Reference Calcium (mg) [4] [5] [6] 935 (42.3) 1024 (23.7) [11] 936.3 (141.6) 1007.6 (181.4) 763.9 (149.7) 863.8 (148.5) 834.6 (224.2) 992.3 (233.1) 735.0 (130.7) 843.6 (153.1) Phosphorus (mg) [4] 1240 (420) 1220 (350) 1020 (390) 1040 (360) [5] [11] 1398.6 (185.8) 1505.1 (230.3) 1226.8 (161.7) 1294.0 (146.0) 1478.3 (210.1) 1625.1 (236) 1183.1 (160.6) 1334.8 (185.5) [2] Magnesium (mg) [4] 276.0 (78.5) 301.2 (80.0) 221.0 (71.5) 252.6 (83.7) [5] [11] 268.6 (42.0) 292.0 (51.2) 240.2 (23.3) 252.5 (29.4) 279.2 (40.7) 312.7 (50.5) 236.5 (32.8) 266.3 (40.0) [2] Iron (mg) [4] 12.53 (5.56) 14.44 (8.14) 8.79 (4.53) 10. 88 (5.62) [5] [7] 15.40 (0.65) 16.49 (0.32) [11] 13.2 (2.5) 14.6 (2.4) 11.8 (1.5) 12.3 (1.5) 15.5 (2.6) 15.8 (2.5) 11.6 (1.7) 12.7 (2.1) [2] [12] 18.50 Zinc (mg) [4] 6.41 (4.98) 7.21 (5.76) [5] [8] [11] 9.75 (0.66) 7.30 (7.51) 8.40 (7.42) 11.36 (0.31) Continued 188 SECTION 2 Components of the Mediterranean Diet TABLE 2 Daily Mineral Intake According to Adherence to Mediterranean Dietary Patterns—cont’d Men Minerals by Reference Women Global Population Low Adherence to MD High Adherence to MD Low Adherence to MD High Adherence to MD 2900 (880) 3130 (830) 2370 (760) 2720 (880) Low Adherence to MD High Adherence to MD 2748 4504 3749 (732) 6132 (1501) 4178 (2023) 3616 (1926) 305 (178) 397 (244) 86 (25) 107 (35) Potassium (mg) [4] [5] [9,10] 2045 (131) 3229 (71) [11] 2568.7 (326.0) 2945.9 (465.3) 2277.7 (355.4) 2534.6 (346.4) 2873.4 (408.1) 3128.4 (395.9) 2221.6 (377.2) 2678.5 (472.5) [2] Sodium (mg) [11] 2507.4 (489.7) 2596.1 (710.3) 2114.7 (537.0) 2129.7 (436.4) 2863.9 (583.8) 2882.4 (512.8) 2057.5 (437.5) 2056.2 (488.4) [2] Iodine (mg) [2] Selenium (mg) [2] Chromium (mg) [12] 110 This table details information about mineral intake reported in several studies according to adherence of diets to Mediterranean dietary patterns. Values are mean (standard deviation). MD, Mediterranean diet. Legumes 3.4 Others 4.6 Meat, fish and eggs 5.0 Cereals 8.9 Fruits and vegetables 9.4 68.7 Dairy products 0 20 40 60 80 100 % contribution FIGURE 1 Contribution (%) of the main food sources to daily calcium intake among Spanish male adolescents aged 11–14 years consuming a diet based on Mediterranean dietary patterns. This figure represents the percentages of contribution of the different sources of food to the daily calcium intake among Spanish male adolescents consuming a diet based on Mediterranean dietary patterns. Data are based on results reported by Seiquer et al. [6]. Mediterranean Diet and Minerals Chapter 18 189 Phosphorus Phosphorus is abundant in all foods, and deficiency situations are not common. In general, phosphorus intake is close to or even exceeds the maximum requirement for this mineral, estimated to be 1250 mg/day for periods when higher levels are needed, such as during adolescence, pregnancy, and lactation [16]. Although in some cases closer adherence to the MD can significantly increase phosphorus intake up to 2220 mg/day, requirements are usually satisfied even when diets are distant from the MD model [2,4,5,11] (Table 2). Nutritional studies of adolescents have shown that phosphorus intake increases when subjects consume a Mediterranean-like diet compared with their habitual diets (1387 and 1227 mg/day, respectively) [17], mainly because of the increased intake of legumes and cereals—foods that are rich in phytates. Cereals and legumes may provide more than 30% of the total daily intake of phosphorus, a contribution similar to that of dairy products [17]. Figure 2 shows the contribution of the main food sources to daily phosphorus intake among Spanish male adolescents consuming a diet based on an MDP [17]. Magnesium Cereals, legumes, vegetables, and nuts are the main sources of magnesium in the diet. It is also present, although in smaller quantities, in meat, dairy products, and fruits. Therefore, the MD is expected to contribute important quantities of magnesium. The distribution of daily intake of magnesium between the different food sources in a typical MD is shown in Figure 3 [17]. Feart et al. [4] reported a positive significant association between MD adherence and magnesium consumption, which was close to the recommendations of 420 and 320 mg/day for men and women older than 65, respectively [16]. In this respect, Kafatos et al. [5] showed that the traditional Cretan MD presents a higher magnesium intake (483 mg/day) than does the typical diet of Greek adolescents (295 mg/day) (Table 2). According to Serra-Majem et al. [2,11], a closer Fish 4.9 Legumes 5.5 Fruits and vegetables 6.7 Others 8.6 Meat 15.0 Cereals 26.1 Dairy products 33.2 0 20 40 60 80 100 % contribution FIGURE 2 Contribution (%) of main food sources to daily phosphorus intake among Spanish male adolescents aged 11–14 years consuming a diet based on Mediterranean dietary patterns. This figure represents the percentages of contribution of the different sources of food to the daily phosphorus intake among Spanish male adolescents consuming a diet based on Mediterranean dietary patterns. Data are based on results reported by Mesıás et al. [17]. Fish 2.7 Meat 6.8 Others 7.4 Legumes 10.7 Dairy products 15.0 Fruits and vegetables 22.0 Cereals 35.4 0 20 40 60 80 100 % contribution FIGURE 3 Contribution (%) of main food sources to daily magnesium intake among Spanish male adolescents aged 11–14 years consuming a diet based on Mediterranean dietary patterns. This figure represents the percentages of contribution of the different sources of food to the daily magnesium intake among Spanish male adolescents consuming a diet based on Mediterranean dietary patterns. Data are based on results reported by Mesıás et al. [17]. 190 SECTION 2 Components of the Mediterranean Diet adherence to the MD both among Spanish adolescents and the whole population increases magnesium intake compared with diets distant from the MD; moreover, it also reduces the percentage of subjects with inadequate magnesium intake, with the exception of girls aged 6–14 years. Magnesium intake associated with close adherence to an MDP exceeds the recommendations for the adult population [2,16]. Iron Iron deficiency is considered the most common single-nutrient deficiency disease in the world, particularly affecting women, children, and adolescents [18]. Iron is mainly obtained from cereals, legumes, nuts, and foods of animal origin. Several studies have determined that diets following an MDP provide adequate amounts of iron, thus helping to prevent iron deficiency. One study showed that elderly French men and women consuming diets that closely adhere to the MD had an iron intake of 14.44 and 10.88 mg/day, respectively [4] (Table 2), exceeding the recommendations for people aged 65 and older (8 mg/day) [19]. Kafatos et al. [5] showed that the traditional Cretan diet provides 20 mg iron/day, thus meeting the iron recommendations for all age groups. Similarly, according to several authors [2,7,11,12], among Spanish adolescents, young women, and the global population, iron recommendations are surpassed when MDPs are followed, with the exception of women aged 15–24 years, whose iron intake (12.7 mg/day) does not reach the 15–18 mg/day recommended for this population group.[19] Furthermore, it has been shown that the contribution of food sources to iron intake varies depending on whether the diet is closer to or more distant from an MDP. The Mediterranean style significantly increases the percentage of iron provided by cereals, legumes, and fruits and vegetables and decreases that contributed by meat [7]. Thus, in Mediterranean-type diets, cereals are the main contributors to daily iron intake, whereas meats are the second contributor, according to data from a study by Mesı́as and coworkers [7] (Figure 4). Therefore, the MD provides an adequate amount of iron, sufficient to meet global population requirements, although special attention should be paid to groups with higher nutritional needs, such as adolescents. Zinc Zinc deficiency is a serious nutritional problem that negatively affects growth and intellectual and sexual development [20]. This deficiency is mainly caused by insufficient mineral intake or by the consumption of diets with low zinc bioavailability. Zinc is mostly found in seafood products, meat, and dairy products, although it also is found in nuts and cereals. Opinions about the suitability of the MD to meet zinc recommendations differ. Mesı́as et al. [8] reported a zinc intake of 11.36 mg/ day among male adolescents consuming diets based on the MD style (Table 2), which satisfies the requirements for this population group (11 mg/day) [19]. Cereals were the major contributors of dietary zinc (39%), followed by meat (21%) and dairy products (20%) (Figure 5). When these subjects followed their usual diets, their zinc intake was 9.75 mg/day, with meat (35%), cereals (28%), and dairy products (21%) being the major contributors to the mineral intake. These findings support the view that the MD provides adequate amounts of zinc, allowing mineral intake recommendations during adolescence to be met. In agreement with this study, Serra-Majem et al. [2] reported a suitable zinc intake in the global Spanish population when the diet consumed is close to the MDP (24 mg/day); a low percentage of participants did not achieve the recommended zinc intake (1%) compared with those consuming diets with a low adherence to the Mediterranean model Fish 2.6 Eggs 2.6 Dairy products 2.9 Others 4.1 Legumes 11.1 Fruits and vegetables 12.9 18.1 Meat 45.7 Cereals 0 20 40 60 80 100 % contribution FIGURE 4 Contribution (%) of main food sources to daily iron intake among Spanish male adolescents aged 11–14 years consuming a diet based on Mediterranean dietary patterns. This figure represents the percentages of contribution of the different sources of food to the daily iron intake among Spanish male adolescents consuming a diet based on Mediterranean dietary patterns. Data are based on results reported by Mesıás et al. [7]. Mediterranean Diet and Minerals Chapter 18 Fish 3.0 Others 4.0 Legumes 4.0 Fruits and vegetables 191 9.0 Dairy products 20.0 Meat 21.0 Cereals 39.0 0 20 40 60 80 100 % contribution FIGURE 5 Contribution (%) of main food sources to daily zinc intake among Spanish male adolescents aged 11–14 years consuming a diet based on Mediterranean dietary patterns. This figure represents the percentages of contribution of the different sources of food to the daily zinc intake among Spanish male adolescents consuming a diet based on Mediterranean dietary patterns. Data are based on results reported by Mesıás et al. [8]. (8%). Zinc intakes below recommended levels have, however, been reported among elderly people whose diet is close to an MDP (7.21 and 8.40 mg/day for men and women, respectively) and in people consuming a traditional MD in Crete (9 mg/ day) [4,5]. In both cases, the recommended levels of mineral intake were achieved by women, but not by men, which suggests that the latter group should increase their consumption of foods rich in zinc to prevent zinc deficiency. Sodium and Potassium It is well known that the population’s dietary habits have changed in recent decades and that large quantities of snacks, bakery products, fast foods, fried, and reheated foods are consumed, especially during adolescence [21]. The increased consumption of processed foods and the decreased intake of natural foods low in sodium and high in potassium, favor sodium intake and decrease that of potassium and, consequently, alter the sodium-to-potassium ratio. The association of dietary sodium intake with the development and prevalence of hypertension in adults is well documented, as is the inverse relationship between dietary potassium and blood pressure. Potassium can counteract the negative effects of sodium in relation to hypertension and cardiovascular disease, which are being observed at increasingly early ages, even among the preschool population [22], probably because of current dietary habits. Moreover, high consumption of sodium, increases urinary calcium excretion, enhancing calcium bone resorption and, consequently, bone loss [23]. This outcome may be counteracted by an adequate potassium intake, which has a buffering effect in the organism [24]. Concern about the high consumption of sodium is widespread, so achieving lower sodium intake among the general population is one of the objectives of the Spanish Society of Community Nutrition [25]. The MD involves the consumption of large amounts of natural foods, which is associated with a greater intake of potassium and a lower intake of sodium. Thus, studies of French men and women consuming diets close to an MDP have reported potassium intakes of 3.13 and 2.72 g/day, respectively, significantly higher than those observed when the subjects consumed diets distant from an MDP (2.90 and 2.37 g/day for men and women, respectively) (Table 2), although these amounts are still insufficient to achieve the recommended potassium intake for this population (4.7 g/day) [4,26]. Potassium intakes lower than recommended levels also have been observed in Spanish adolescents consuming diets based on the MD [9,11]. These findings may be because of adolescents’ tendency to refuse fruit and vegetables. Intervention studies have shown that servings of fruit and vegetables per day are higher in diets based on an MDP compared with the adolescents’ usual diet, although the subjects did not reach the recommended levels for fruit (3 servings/day) or vegetables (2 servings/day) according to the Spanish Society of Community Nutrition [9,10]. On the other hand, some studies reported that adults following the MD meet potassium recommendations [2,5]. Although adherence to the MD may produce a lower sodium intake, Serra-Majem et al. [2,11] reported that diets close to an MDP consumed by the Spanish population were associated with a sodium intake that was higher than the recommendations (1500 mg/day) [26]. Therefore, although the traditional MD includes abundant plant foods that are minimally processed, culinary techniques and a certain evolution in this respect seem to favor sodium intake and restrict that of potassium. Contribution of main food sources to daily potassium and sodium intake among Spanish male adolescents consuming a diet based on MDPs, according to data reported by Seiquer et al. [10] and Mesı́as et al. [17] is represented in Figure 6. 192 SECTION 2 Components of the Mediterranean Diet Fish Potassium 4.0 Legumes 7.7 Others 9.4 Meat and meat products Sodium 10.3 Fruits 14.4 Cereals 14.8 Dairy products 15.4 Vegetables 20 1.1 Fruits and vegetables 1.6 Others 1.8 Meat and meat products 13.3 Dairy products 40 29.3 Cereals 24.0 0 Fish 60 80 100 52.9 0 20 40 60 80 100 % contribution % contribution FIGURE 6 Contribution (%) of main food sources to daily potassium and sodium intake among Spanish male adolescents aged 11–14 years consuming a diet based on Mediterranean dietary patterns. This figure represents the percentages of contribution of the different sources of food to the daily potassium and sodium intake among Spanish male adolescents consuming a diet based on Mediterranean dietary patterns. Data are based on results reported by Seiquer et al. [10] and Mesıás et al. [17]. Other Micronutrients Few studies have evaluated the intake of microelements as a result of adherence or otherwise to Mediterranean dietary habits. A nutritional intervention assay performed among Spanish adolescents consuming an MD measured an average copper intake of 1230 mg/day, which is higher than the recommended intake of 700–890 mg/day for this age group [17]. Serra-Majem et al. [2] reported an iodine intake of 397 mg/day in the Spanish population as a whole when the diet consumed was close to an MDP (Table 2), and this intake exceeded the recommendations for this micronutrient for adults (150 mg/day), even in special situations such as pregnancy (220 mg/day) and lactation (290 mg/day) [19]. In this population, diets with a profile close to the MD provide a selenium intake of 107 mg/day, which, like iodine, exceeded recommendations for this micronutrient for the adult population (55 mg/day), including during pregnancy (60 mg/day) and lactation (70 mg/day) [27]. Cabrera-Vique and Mesı́as [12] evaluated chromium content in the meals provided at a Spanish university residence, which closely adhered to the MD according to the KIDMED index (score ¼ 9). This diet was found to contribute 110 mg chromium/day, which greatly exceeded the recommended intake for young people (24–25 mg/day) [19]. It must be highlighted that, although the MDP seems to increase the intake of trace elements to levels above recommendations, tolerable upper intake levels (according to reports of dietary reference intakes [19,27]) are never reached when a diet highly adhering to the MD is consumed. MINERAL CONTENT IN TYPICAL MEDITERRANEAN MENUS Most currently available data concerning mineral evaluation in the MD correspond to epidemiological studies performed using consumer surveys and transformed into mineral content using food composition tables (FCT). There are few data on the analysis of minerals in Mediterranean meals performed using laboratory methods, which are much more accurate than FCT. Our research group analyzed the mineral content of various meals typically consumed in the MD, such as Spanish omelettes and paella. Eight different menus containing starters, a main dish, and a dessert, equivalent to a lunch menu, were analyzed (Table 3). Taking into account that these menus are just one part of the daily food intake, it may be concluded that typical Mediterranean meals are adequate to satisfy mineral requirements, even in special situations of increased needs (Figure 7). The mineral contribution of MDP lunch menus to the daily intake may be classified as low (calcium), moderate (potassium and zinc), and high (phosphorus, magnesium, iron, sodium, and copper); some of these minerals reach over 100% of the daily requirements for both adults and adolescents, even during pregnancy and lactation. It should be taken into account that calcium is mostly provided by dairy products that are usually consumed at breakfast and with the afternoon meal in the MD. Similarly, cereals may provide an important amount of zinc in the diet; fruits and vegetables, as mentioned earlier, are rich sources of potassium, and all three—cereals, fruits, and vegetables—may also be consumed during meals other than lunch. The Mediterranean menus analyzed do not seem to provide enough iron to meet the requirements of pregnant women, although iron supplementation is generally recommended during pregnancy [28]. Regarding sodium, the menus meet or greatly exceed recommended intakes. In a previous assay we evaluated the content of sodium in menus using FCT, and the results were compared with those obtained from TABLE 3 Mineral Content in Typical Mediterranean Lunch Menus and Daily Recommended Intakes Menua Ca (mg) P (mg) Mg (mg) Fe (mg) Zn (mg) K (mg) Na (mg) Cu (mg) 1 418 63 758 129 223 1 10.1 1.6 6.6 1.3 1842 208 3029 671 963 8 2 284 3 592 17 204 3 9.8 0.2 5.2 0.1 2201 292 2795 45 1063 22 3 146 5 487 6 186 9 3.6 0.2 2.8 0.1 1204 35 1351 43 471 6 4 300 3 603 26 210 3 9.9 4.0 6.5 0.2 1824 22 2400 45 675 7 5 380 8 85 7 286 3 9.0 0.3 5.3 0.2 2079 42 3670 254 1035 12 6 267 37 997 24 157 3 4.5 0.8 2.9 0.5 1224 29 2643 218 538 6 7 290 2 690 14 208 4 11.7 1.3 7.1 0.3 1466 19 1788 107 687 10 8 173 11 582 17 162 4 15.2 0.4 3.8 0.4 1270 10 1056 159 503 9 b Daily recommended intakes Adolescents 1300 1250 240–410 8–15 8–11 4500–4700 1500 700–890 Adults 1000–1200 700 310–420 8–15 8–11 4700 1500 900 Pregnant women 1000–1300 700–1250 350–400 27 11–12 4700 1500 1000 Lactating women 1000–1300 700–1250 310–360 9–10 12–13 5100 1500 1300 This table details mineral content (mean standard deviation) in typical Mediterranean menus and daily recommended intakes for the different collectives. Menu composition: (1) chickpeas with chicken and vegetables, lettuce salad, and banana; (2) lentils with potatoes, Spanish omelette, rice with milk and cinnamon; (3) tropical salad, breaded fish with rice, orange; (4) sautéd vegetables, griddled loin of pork with fried potatoes, torrija (fried bread with milk, sugar, and cinnamon); (5) empanadillas (small tuna-filled breaded pasties), beans with rice, yogurt; (6) consommé with noodles, breaded fish with fried potatoes, pear; (7) omelette with ham and potatoes, meatballs with vegetables, custard; (8) lettuce salad, paella, apple. Olive oil was the only added fat source. bDaily recommended intakes are based on the dietary reference intakes [15,16,19,26]. a SECTION 2 Components of the Mediterranean Diet 250 250 200 200 %RDA adults %RDA adolescents 194 150 100 50 0 Ca P Mg Fe Zn K Na 100 50 0 Cu Ca P Mg Fe Zn K Na Cu Ca P Mg Fe Zn K Na Cu 250 %RDA lactating 250 %RDA pregnants 150 200 150 100 50 0 200 150 100 50 0 Ca P Mg Fe Zn K Na Cu FIGURE 7 Ranges of contribution of some typical Mediterranean lunch menus to daily recommended intakes of minerals. Figure represents ranges of contribution of some typical Mediterranean lunch menus to daily recommended intakes of minerals for different collectives. RDA: Recommended Dietary Allowance, according to the dietary reference intakes [15,16,19,26]. laboratory analysis of the menus. It was observed that sodium content by analytical evaluation was >50% higher than when composition tables were used. Because composition tables do not usually include the salt added to foods during culinary processes, we concluded that the sodium values obtained are always lower than the real ones [29]. Therefore, differences between the sodium intake from food sources and that provided by added salt should be considered to estimate the contribution of meals to the total intake of this mineral. MINERAL BIOAVAILABILITY To meet nutrient requirements, diets should not only contribute a sufficient quantity of nutrients but also enable adequate digestive and metabolic utilization and, consequently, adequate bioavailability. In this sense, the bioavailability of dietary iron seems to be an important determinant of iron status [30], and zinc utilization is greatly influenced by both zinc concentration and the presence of enhancers and inhibitors of zinc absorption in the diet [31]. Phytates, oxalates, and tannins are known to form insoluble complexes with minerals that are not absorbed by the gastrointestinal tract, and thus the presence of these compounds in the diet may reduce the bioavailability of minerals [32]. Cereals, legumes, and vegetables are foods containing large amounts of these compounds; therefore, a high intake of these sources of food, together with a moderate consumption of dairy products, as is usual in the MD, could compromise the bioavailability of calcium, iron, zinc, and magnesium in the MD. However, the negative effect of phytate on dietary calcium seems to occur only if the diet is unbalanced [33], which is not the case with the MD. Thus, digestibility of calcium and magnesium has been reported to increase or even double with the consumption of diets based on the MDP in comparison with those distant from these dietary patterns [6,17]. According to Brown et al. [31], the zinc absorbed from the diet depends on the phytate-to-zinc molar ratio. It is accepted that diets with a phytate-to-zinc ratio <15 have a medium level of zinc bioavailability and a high level when the ratio is <5. In the MD this ratio is often between 10 and 2 [8], thus corresponding to moderate or even high zinc bioavailability. It is quite possible that the special characteristics of the MD counteract the negative effect of phytates on iron availability. The MD provides higher proportions of nonheme iron in comparison with conventional diets [7], and its absorption may vary up to 10-fold depending on enhancer or inhibitor factors. Ascorbic acid, which is significantly high in the MD, is one of the key enhancers of nonheme iron absorption. Its effect is known to be more marked in the presence of inhibitors such as phytate or iron-binding polyphenols, probably because of its ability to reduce and to bind iron, preventing the formation of less soluble iron compounds [30]. In meals containing medium levels of inhibitors, a molar ratio of 2:1 (ascorbic Mediterranean Diet and Minerals Chapter 18 195 acid:iron), very similar to that found in Mediterranean-like diets [7], promotes iron absorption and overcomes the inhibitory effect of phytic acid [34]. In a similar way, citric acid, both alone and especially when bound to phytase, significantly increases the solubility of calcium, magnesium, zinc, and manganese [35]. This probably occurs in the MD because of the high consumption of citrus fruits, which are very common in Mediterranean regions. In 1991, Torre et al. [36], reported that the consumption of a varied diet with appropriate protein and mineral intake protects against the detrimental effects of phytate on mineral utilization and that this could be the case with the MD. Dietary fiber, an essential component of plant foods, may bind metal cations, and earlier studies have suggested that diets rich in fiber might unfavorably affect mineral absorption [37]. However, the role of fiber on mineral availability has not been fully clarified, as its effect depends on the type of fiber and varies according to the presence of other compounds that can compete in binding minerals. Moreover, the conjoint presence of fiber and phytates in the diet makes it difficult to separate the effects of the two compounds in the utilization of minerals [36]. This finding is argued by L€onnerdal [32], for whom the negative effect of fiber on zinc absorption is probably caused by the phytate content. It has even been proposed more recently that fiber could be beneficial to the absorption of some minerals because of the positive effect of fermentable carbohydrates acting through various mechanisms [38]. Finally, the culinary treatment of plant foods before their consumption may affect mineral utilization; for example, soaking legumes in a basic solution of water with bicarbonate, which is a frequent practice in Spanish kitchens, improves the digestibility of calcium, phosphorus, and magnesium [39]. Animal tissues such as red meat, poultry, and fish are considered to be enhancers of iron absorption [30]. Moreover, Gibson et al. [40] reported that the decreased consumption of red meat may compromise the intake and bioavailability of zinc. Low levels of consumption of red meat certainly could be a negative factor in the MD for both iron and zinc bioavailability. In the case of iron, it would represent a reduction in heme iron, which is highly available, and a decrease in the capacity of the meat to enhance nonheme iron absorption [30]. Nevertheless, the MD is associated with moderate to high consumption of fish, and fish is a relatively good source of available iron [41] and zinc. Moreover, fish enhances iron absorption from plant foods in a similar way to meat [30]. Feeding rats diets containing fish as the protein source has been shown to significantly increase dietary iron utilization compared with control diets [42]. Another enhancer factor of mineral bioavailability is dietary fat. The use of olive oil as a culinary fat may be associated with higher iron availability, and it has been suggested that oleic acid promotes iron absorption [43]. Moreover, the interaction between protein and fat digestion products may be involved in enhancing intestinal iron absorption [44], and it has been reported that sardine protein plus olive oil (fried sardines, common in the MD) has beneficial effects on dietary iron bioavailability [42]. Most authors agree that a high ratio of unsaturated to saturated fatty acids, as is found in the MD, enhances calcium absorption [45]. Martinez-Ramirez et al. [46] suggested that monounsaturated fatty acids have beneficial effects on bone health. Therefore, it could be hypothesized that the special protein-fat combination of the MD contributes to increasing mineral availability. In this sense, adolescents consuming diets based on an MDP have been shown to present adequate zinc bioavailability [8] and significantly increased iron digestibility and bioavailability compared with their usual diets [7]. Regarding dietary calcium bioavailability, other enhancer factors can be found in the MD. For instance, certain nondigestible oligosaccharides improve calcium absorption in diets supplemented with inulin and oligofructose [47]. The intake of potassium and magnesium, which is relatively high in the MD, decreases urinary calcium excretion, probably as a consequence of reduced endogenous acid production [24]; on the other hand, an excessive intake of sodium increases urinary calcium excretion because almost all ingested sodium is excreted in the urine, whereas the renal clearance of calcium is linked to that of sodium [33]. Thus, some authors consider that the special characteristics of the MD may optimize calcium retention and bone accretion [48]. Our research group observed an important increase in calcium retention in male adolescents when a diet close to the MDP is consumed compared with their usual diet, without significant differences in calcium intake [6]. This outcome may enhance the peak bone mass achieved during adolescence, thus preventing diseases such as osteoporosis. Rivas et al. [49] indicated that traditional Mediterranean food patterns are associated with a higher body mass density in pre- and postmenopausal women. These findings are in agreement with the lower rates of osteoporosis in Mediterranean countries reported by Puel et al. [50]. Seiquer et al. [6] and Mesı́as et al. [7,8,17] extensively studied the effects of the MD on mineral bioavailability among adolescents compared with their usual diets. The findings of this group reflect important improvements in the absorption and bioavailability of minerals associated with MD intake (the effects are summarized in Table 4). It should be taken into account that mineral availability can also be influenced by the Mediterranean lifestyle, which is associated with outdoor activities and greater exposure to sunshine and, therefore, higher levels of vitamin D synthesis. Both factors are critical to bone health and, consequently, should be preserved. 196 SECTION 2 Components of the Mediterranean Diet TABLE 4 Effects of a Varied Diet Based on Mediterranean Patterns on Mineral Bioavailability in Adolescents Versus Their Habitual Diet Digestibilitya Bioavailabilityb References Calcium " "" [6] Phosphorus " "" [17] Magnesium "" "" [17] Iron "" "" [7] Zinc " " [8] Sodium ¼ " [17] Potassium "" "" [17] This table summarizes the effects of a varied diet based on Mediterranean patterns on mineral availability compared to adolescents’ habitual diet, according to the results reported by Seiquer et al. [6] and Mesı́as et al. [7,8,17]. ¼, no effect; ", effect is statistically nonsignificant; "", effect is statistically significant. a Calculated as a percentage of absorbed mineral from total intake. bCalculated as a percentage of retained mineral from total intake. CONCLUSIONS The special food composition of the traditional MD, rich in vegetables, fruits, whole-meal cereals, and nuts, provides significant amounts of minerals; therefore, close adherence to an MDP makes mineral deficiencies quite improbable. This statement is supported by the epidemiological and nutritional intervention studies reviewed in this chapter: consumption of the MD is associated with improved intake of nearly all nutritional minerals, particularly phosphorus, iron, zinc, and potassium. Lower intake of calcium is observed, consistent with the low content of dairy products in the MD; thus, there is a need to promote calcium consumption in Mediterranean countries, especially in situations of increased need. This chapter includes important results concerning mineral bioavailability: consumption of the MD clearly improves the absorption and bioavailability of most minerals compared with non-MDs. Thus, the positive effect on mineral bioavailability would be another benefit obtained from the MD, supporting the view that this diet is one of the healthiest in the world. SUMMARY POINTS l l l l l The MD has a high mineral content, derived from vegetables, fruits, whole-meal cereals, and fish. As well as supplying sufficient minerals to meet nutritional recommendations, the MD has special dietary factors that affect mineral absorption and bioavailability. Menus typically consumed in the MD adequately satisfy daily mineral requirements, even in special situations of increased need. The special composition of the MD is clearly beneficial to mineral bioavailability. The positive effect on mineral bioavailability seems to be another benefit obtained from the MD. REFERENCES [1] Román-Viñas B, Ribas-Barba L, Ngo J, Martinez-Gonzalez MA, Wijnhoven TMA, Serra-Majem L. Validity and reproducibility of dietary patterns to assess nutrient intake adequacy. Br J Nutr 2009;101:S21–8. 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Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model. J Nutr 1998;128:1555–61. 198 SECTION 2 Components of the Mediterranean Diet [42] Seiquer I, Aspe T, Pérez-Granados AM, Navarro MP. Consumption of raw and fried sardine (Clupea pilchardus) as protein source of diets: effects on iron metabolism in rats. J Sci Food Agric 2002;82:1497–503. [43] Kapsokefalou M, Miller DD. Lean beef and beef fat interact to enhance nonhaem iron absorption in rats. J Nutr 1993;123:1429–34. [44] Kapsokefalou M, Miller DD. Iron speciation in intestinal contents of rats fed meals composed of meat and nonmeat sources of protein and fat. Food Chem 1995;52:47–56. [45] Haag M, Magada ON, Claassen N, Boéhmer LH, Kruger MC. Omega-3 fatty acids modulate ATPases involved in duodenal Ca absorption. Prostaglandins Leukot Essent Fatty Acids 2003;68:423–9. 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Chapter 19 Melatonin: A New Perspective on the Mediterranean Diet Marcello Iriti, PhD and Elena M. Varoni, DD, PhD Milan State University, Milan, Italy. INTRODUCTION Healthy properties of the Mediterranean diet have been attributed to the phytochemical diversity of this traditional dietary style. In the past two decades, a plethora of in vitro and in vivo studies have emphasized the many biological activities of non-nutrient components of plant foods, with polyphenols representing the archetype of bioactive phytochemicals. Because of many confounding factors that make it difficult to differentiate between causal or casual relationships, however, epidemiological and human studies failed to unquestionably associate adequate plant food consumption with a reduced risk of chronic-degenerative diseases. A new element has recently contributed to improving the phytochemical diversity of typical Mediterranean foods and, possibly, their healthy potential. This new factor is melatonin (Figure 1); among the Mediterranean typical food plants, it was first discovered in grapes and then in grape products [1,2]. In animals, particularly in vertebrates and mammals, melatonin is a hormonal, endogenous molecule that modulates circadian and circannual physiological functions, such as the sleep/wake cycle, reproductive function, bone metabolism, and turnover, via cell receptor-mediated mechanisms. Receptor-independent processes also have been reported, mainly because of melatonin’s powerful antioxidant activity. Melatonin can directly scavenge free radical species (both reactive oxygen and nitrogen species) and stimulate the activity of antioxidant enzymes; thus it is involved in immune response and the pathogenesis of chronic-degenerative disorders. Melatonin biosynthesis was first described in the pineal gland (epiphysis), but it also occurs in other tissues outside of the central nervous system, such as the gastrointestinal tract, bone marrow, and lymphocytes. The aromatic amino acid tryptophan is the precursor of melatonin and other indoleamines, including serotonin (Figure 2). Animal organisms are not able to produce this amino acid and therefore they depend on exogenous plant tryptophan for the synthesis of endogenous melatonin [3]. Outside the animal kingdom, melatonin was first discovered in the photosynthesizing unicellular alga Lingulodinium polyedrum (Stein) J. D. Dodge sin. Gonyaulax polyedra Stein, belonging to the phylum Dinoflagellata [4]. Since then, the presence of melatonin in food plants and medicinal herbs has been extensively reported. The first complete publications reporting melatonin in tracheophytes (vascular or higher plants) were independently provided by two research groups, which found this indoleamine in a number of edible plants [5,6]. So far, melatonin has been detected and quantified in roots, shoots, leaves, flowers, fruits, and seeds of a considerable variety of spermatophyte species, and its presence in plants has been unequivocally confirmed [7]. In flowering plants (angiosperms), the occurrence of melatonin in a number of families belonging to both the mono- and dicotyledons, which are relevant as food and medicinal plants, has been described [8] (Figure 3). MELATONIN IN THE MEDITERRANEAN DIET The traditional Mediterranean diet originated in areas where olives (Olea europaea L.) and grapevines (Vitis vinifera L.) were cultivated and where olive oil and wine are produced and regularly consumed. In addition to these foodstuffs, other main components of the Mediterranean diet include whole grains, fruits, vegetables, legumes, and nuts; yogurt and ricotta cheese as dairy products; and fish and white meat as sources of protein. Among typical Mediterranean products, melatonin was first reported in grapes and then in olive oil. In the berry exocarp (skin) of different Italian and French wine grape cultivars grown in northwestern Italy, the highest melatonin concentrations were detected in Nebbiolo and Croatina varieties (0.9 and 0.8 ng g 1, respectively), whereas the lowest concentrations occurred in the Cabernet Franc cultivar (0.005 ng g 1) [9]. Differences were also found when refined olive oils and extra virgin olive oils designation of origin The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 199 200 SECTION 2 Components of the Mediterranean Diet H N O O N H FIGURE 1 Structure of melatonin. NH2 N H COO– Tryptamine HO NH2 NH3+ N H Tryptophan COO– HO N H Serotonin NH3+ N H 5-Hydroxytryptophan SNAT HO N H N H CH3O N H COCH3 N-Acetylserotonin COCH3 N H Melatonin FIGURE 2 Biosynthetic pathway of melatonin in mammals. Enzymes involved include tryptophan decarboxylase (TrpDC); tryptophan 5-hydroxylase (Trp5H); 5-hydroxytryptophan decarboxylase (5HTDC); tryptamine 5-hydroxylase (T5H); serotonin N-acetyltransferase (SNAT); and hidroxyindole-Omethyltranferase (HIOMT). MELATONIN (endogenous) Animals (from invertebrates to vertebrates) Plants (from unicellular algae to higher plants) Dietary or exogenous melatonin Medicinal and food plants FIGURE 3 Melatonin in food and medicinal plants may represent an endogenous or dietary source of bioactive melatonin in humans. Melatonin Chapter 19 201 (DO) were compared. Among DO oils 71 pg mL 1 and 119 pg mL 1 were measured in DO Bajo Aragón and DO Baena, respectively. Diversities in the heat treatment or chemical processing of the products may explain the diverse concentrations measured [10]. MELATONIN IN GRAPE PRODUCTS A huge disproportion between studies of the melatonin content in grapevine products and in other Mediterranean plant foodstuffs exists (Figure 4). As previously mentioned, an intraspecific variation of the melatonin content in berry skin tissues of different cultivars (Nebbiolo, Croatina, Barbera, Cabernet Sauvignon, Sangiovese, Merlot, Marzemino, and Cabernet Franc) was found, with levels ranging from 0.005 to 0.96 ng g 1 [9]. Similar results (from 0.6 to 1.2 ng g 1) were reported by Stege and colleagues [11] for the same tissue of Malbec, Cabernet Sauvignon, and Chardonnay varieties cultivated in Argentina. In Merlot berry skin, we found 17.5 and 9.3 ng g 1 of melatonin at prevéraison and véraison, respectively, the two main phenological stages of grapes [12]. Conversely, the transition from prevéraison to véraison increased the melatonin content both in Merlot berry seeds (from 3.5 to 10 ng g 1) and flesh (from 0.2 to 3.9 ng g 1) [12]. In berry skin of the Malbec variety cultivated in Argentina, melatonin concentration showed similar values during the night (around 10 ng g 1), reaching a strong peak in the morning (159 ng g 1) [13]. However, much higher melatonin concentrations— between 100 and 150 mg g 1, depending on the phenological stage—were measured in the whole berry (i.e., skin, flesh, and seeds analyzed together) of a Merlot cultivar grown in Canada [14]. Melatonin was recently found at 1.5 and 1.2 ng g 1 in the whole berry of Sangiovese red and Albana white grapes, respectively, from central Italy. Further studies ascertained the occurrence of melatonin in wine (Table 1). Mercolini et al. [15,16] detected this indoleamine at 0.6, 0.5, and 0.4 ng mL 1 in Albana white, Sangiovese red, and Trebbiano white wines, respectively. Stege and colleagues [11] reported melatonin at 0.16, 0.24, and 0.32 ng mL 1 in Chardonnay, Malbec, and Cabernet Sauvignon wines, respectively. Our results showed that the levels of melatonin in Groppello and Merlot red wines varied between 5.2 and 8.1 ng mL 1, depending on agrochemical treatments [17], whereas Rodriguez-Naranjo and colleagues [18,19] measured higher melatonin concentrations (up to 150 and 400 ng mL 1) in racked wines. In a recent screening of Italian mono- and polyvarietal red, white, and dessert wines from different geographical areas, we measured melatonin at concentrations around or less than 0.5 ng mL 1 [20]. Intriguingly, in this study the linear correlation coefficient between the melatonin content in wines and their antiradical capacity was higher compared with the weak correlation found between trans-resveratrol-3-O-glucoside (trans-piceid) and the same activity, even though the levels of stilbenes (around or <1.0 mg mL 1) were of three orders of magnitude higher then the levels of indoleamine [20]. In these terms, it can be hypothesized that melatonin possesses a higher antioxidant activity than resveratrol. In other grapevine products, the presence of melatonin was reported in traditional balsamic vinegars of Modena DO (around 0.1 ng mL 1), in grape juice (0.5 ng mL 1), and in Albana grappa (0.3 ng mL 1) [14,20] (Table 1). FIGURE 4 Presence of melatonin in grape berry and seed tissues. 202 SECTION 2 Components of the Mediterranean Diet TABLE 1 Melatonin Content in Some Typical Mediterranean Plant Foods Mediterranean Product Melatonin References Grape products Red wines < 0.5 ng mL 1 Mercolini et al. [16], Stege et al. [11], Vitalini et al. [20] White wines < 0.5 ng mL 1 Mercolini et al. [16], Stege et al. [11], Vitalini et al. [20] Dessert wines < 0.5 ng mL 1 Vitalini et al. [20] Grape juice (undisclosed cultivar) < 0.5 ng mL 1 Mercolini et al. [14] 1 Albana grappa 0.3 ng mL Modena balsamic vinegar (DO) 0.1 ng mL Mercolini et al. [14] 1 Vitalini et al. [20] Olive oil Extra virgin (DO) Refined 1 71–119 pg mL 53–75 pg mL 1 de la Puerta et al. [10] de la Puerta et al. [10] DO, designation of origin. Three melatonin isomers were recently detected in different grape products: Italian mono- and polyvarietal red, white, and dessert wines from different geographical areas; Modena balsamic vinegars; and grape juices. In particular, isomer 1 and isomer 2 were the most recurring and abundant in all wines and vinegars assayed, respectively, with levels much higher than those of melatonin and maximum concentrations of 68.4 and 40.3 ng mL 1 in red wine and vinegar, respectively. The emergence of naturally occurring melatonin isomers has opened a new, exciting, and fascinating topic in the field of melatonin research, as recently emphasized in the excellent review article by Tan et al. [21] Certainly, it would be of great interest to ascertain in the near future the nutritional significance of these isomers, comparing their biological activity with that of melatonin, particularly in terms of interaction with melatonin receptors. Many endogenous and external factors may noticeably influence the melatonin levels in grapes and their products; these factors include the genetic traits of the cultivars and their geographic origin, the berry tissues/plant organs analyzed, the difference between thin- and thick-skinned grapes, the phenological stages, day/night fluctuations, pathogen (mainly fungal) infections and agrochemical treatments, agro-meteorological conditions and environmental stresses, altitude, ultraviolet radiation and high light irradiance, the vintage, and winemaking procedures [13,15,17,18]. BIOLOGICAL SIGNIFICANCE OF MELATONIN IN PLANT FOODS The possibility of modulating the circulating levels of melatonin in mammals, which are basically very low (200 pg mL 1 at the maximum nighttime peak and lower than 10 pg mL 1 during the day) [22] compared to melatonin in grape products (near 1 ng g 1 and 0.5 ng mL 1 in berry skin and wine, respectively), through the intake of plant foods represents and exciting and relevant topic. Efficient uptake and bioavailability of dietary melatonin have been demonstrated in both animals and humans. In a pioneering study, feeding chicks with plant foods rich in melatonin increased their serum melatonin levels, and melatonin extracted from plants inhibited the binding of labeled melatonin to cell membrane receptors in rabbit brain [6]. In rats fed 3 g of walnuts (Juglans regia L.), containing 10.5 ng of melatonin, the serum concentration of indoleamine were augmented from 11.5 to 38.0 pg mL 1, as was the serum antioxidant capacity [23]. The effects of melatonin-rich plant foods also were studied in humans. Both melatonin levels and total antioxidant status of serum samples from healthy volunteers were increased 45 min after drinking beer [24]. Likewise, consumption of tropical fruits (including oranges) increased the serum melatonin concentration and antioxidant capacity in healthy subjects [25]. In premenopausal women, urinary excretion of 6-sulfatoxymelatonin (a major metabolite of melatonin) (Figure 5) was considered a biomarker of vegetable intake and was inversely correlated to breast cancer risk [26,27]. The intake of 200 mL of grape juice from the Tempranillo cultivar twice a day for 5 days significantly increased urinary 6-sulfatoxymelatonin and total antioxidant capacity in young, middle-aged, and elderly healthy individuals [28]. Similar results were obtained after ingesting Jerte Valley sweet cherry products, Japanese plums, and tropical fruits (including oranges) [29–32] Jerte Valley cherry products also improved mood and sleep parameters in healthy participants [33]. Finally, we recently reported that administration of red wine (125 mL) did not decrease the salivary antiradical capacity in humans, thus suggesting that the pro-oxidant effects of ethanol may be counteracted by melatonin present at 0.23 ng mL 1 and polyphenols [34]. Melatonin Chapter 19 203 H N O O N H O O S O OH FIGURE 5 Structure of 6-sulfatoxymelatonin, the main urinary metabolite of melatonin in mammals. CONCLUSIONS With regard to grape products, the levels of melatonin can be defined near 1 ng g 1 and 0.5 ng mL 1 in grape berry skin and wine, respectively (Table 1), in agreement with Rayne [35], although data from studies of berry tissues are still fragmentary and somewhat contrasting. In any case, neither polyphenols nor melatonin possess thaumaturgic properties, but their synergy with other bioactive phytochemicals (e.g., carotenoids, glucosinolates) contributes to maximizing the benefits of healthy dietary styles. Similarly, Mediterranean foods are not a panacea. The Mediterranean diet is a philosophy of life that also includes nondietary lifestyle factors, such as a moderate physical activity (walking every day) and resting in the middle of the day after an enjoyable family meal (in Spanish, a siesta). SUMMARY POINTS l l l l l l Health-promoting properties of the Mediterranean diet have been, at least in part, attributed to the chemical diversity of plant foods. Melatonin and possibly other indoleamines recently discovered in some relevant Mediterranean foods may represent new factors contributing to the elucidation of the protective effects of diets rich in plant products. In synergy with polyphenols and other bioactive phytochemicals (e.g., carotenoids, glucosinolates), melatonin may contribute to maximizing the benefits of healthy dietary habits. Melatonin content in grape products has been extensively investigated compared with other plant foods. Melatonin in plant foods is absorbed in the gastrointestinal tract and transported in the bloodstream. Dietary melatonin represents an emerging and exciting topic in the field of nutritional sciences. REFERENCES [1] Iriti M, Varoni EM, Vitalini S. Melatonin in Mediterranean diets. J Pineal Res 2010;49:101–5. [2] Iriti M, Vitalini S. Health-promoting effects of traditional Mediterranean diet—a review. Pol J Food Nutr Sci 2012;62:71–6. [3] Reiter RJ, Tan DX, Rosales-Corral S, Manchester LC. The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives. Mini Rev Med Chem 2013;13:373–84. [4] Balzer I, Hardeland R. Photoperiodism and effects of indoleamines in a unicellular alga, Gonyaulax polyedra. Science 1991;253:795–7. [5] Dubbels R, Reiter RJ, Klenke E, Goebel A, Schnakenberg E, Ehlers C, et al. Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry. J Pineal Res 1995;18:28–31. [6] Hattori A, Migitaka H, Iigo M, Itoh M, Yamamoto K, Ohtani-Kaneko R, et al. Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem Mol Biol Int 1995;35:627–34. [7] Paredes SD, Korkmaz A, Manchester LC, Tan D-X, Reiter RJ. Phytomelatonin: a review. J Exp Bot 2009;60:57–69. [8] Tan DX, Hardeland R, Manchester LC, Korkmaz A, Ma S, Rosales-Corral S, et al. Functional roles of melatonin in plants, and perspectives in nutritional and agricultural sciences. J Exp Bot 2012;63:577–97. [9] Iriti M, Rossoni M, Faoro F. Melatonin content in grape: myth or panacea? J Sci Food Agric 2006;86:1432–8. [10] de la Puerta C, Carrascosa-Salmoral MP, Garcı́a-Luna PP, Lardone PJ, Herrera JL, Fernández-Montesinos R, et al. Melatonin is a phytochemical in olive oil. Food Chem 2007;104:609–12. [11] Stege PW, Sombra LL, Messina G, Martinez LD, Silva MD. Determination of melatonin in wine and plant extracts by capillary electrochromatography with immobilized carboxylic multi-walled carbon nanotubes as stationary phase. Electrophoresis 2010;31:2242–8. [12] Vitalini S, Gardana C, Zanzotto A, Simonetti P, Faoro F, Fico G, et al. The presence of melatonin in grapevine (Vitis vinifera L.) berry tissues. J Pineal Res 2011;51:331–7. 204 SECTION 2 Components of the Mediterranean Diet [13] Boccalandro HE, Gonzáles CV, Wunderlin DA, Silva MF. Melatonin levels, determined by LC-ESI-MS/MS, deeply fluctuate during the day in Vitis vinifera cv Malbec. Evidences for its antioxidant role in fruits. J Pineal Res 2011;51:226–32. [14] Murch SJ, Hall BA, Le CH, Saxena PK. Changes in the levels of indoleamine phytochemicals during véraison and ripening of wine grapes. J Pineal Res 2010;49:95–100. [15] Mercolini L, Addolorata Saracino M, Bugamelli F, Ferranti A, Malaguti M, Hrelia S, et al. HPLC-F analysis of melatonin and resveratrol isomers in wine using an SPE procedure. J Sep Sci 2008;31:1007–114. [16] Mercolini L, Mandrioli R, Raggi MA. Content of melatonin and other antioxidants in grape related foodstuffs: measurement using a MEPS-HPLC-F method. J Pineal Res 2012;55:21–8. [17] Vitalini S, Gardana C, Zanzotto A, Fico G, Faoro F, Simonetti P, et al. From vineyard to glass: agrochemicals enhance the melatonin and total polyphenol contents and antiradical activity of red wines. J Pineal Res 2011;51:278–85. [18] Rodriguez-Naranjo MI, Gil-Izquierdo A, Troncoso AM, Cantos E, Garcia-Parrilla MC. Melatonin: a new bioactive compound in wine. J Food Compos Anal 2011;24:603–8. [19] Rodriguez-Naranjo MI, Gil-Izquierdo A, Troncoso AM, Cantos-Villar E, Garcia-Parrilla MC. Melatonin is synthesized by yeast during alcoholic fermentation in wines. Food Chem 2011;126:1608–13. [20] Vitalini S, Gardana C, Simonetti P, Fico G, Iriti M. Melatonin, melatonin isomers and stilbenes in Italian traditional grape products and their antiradical capacity. J Pineal Res 2013;54:322–33. [21] Tan DX, Hardeland R, Manchester LC, Rosales-Corral S, Coto-Montes A, Boga JA, et al. Emergence of naturally occurring melatonin isomers and their proposed nomenclature. J Pineal Res 2012;53:113–21. [22] Bonnefont-Rousselot D, Collin F. Melatonin: action as antioxidant and potential applications in human disease and aging. Toxicology 2010;278:55–67. [23] Reiter RJ, Manchester LC, Tan DX. Melatonin in walnuts: influence on levels of melatonin and total antioxidant capacity of blood. Nutrition 2005;21:920–4. [24] Maldonado MD, Moreno H, Calvo JR. Melatonin present in beer contributes to increase the levels of melatonin and antioxidant capacity of the human serum. Clin Nutr 2009;28:188–91. [25] Sae-Teaw M, Johns J, Johns NP, Subongkot S. Serum melatonin levels and antioxidant capacities after consumption of pineapple, orange, or banana by healthy male volunteers. J Pineal Res 2013;55(1):58–64. http://dx.doi.org/10.1111/jpi.12025. [26] Nagata C, Nagao Y, Shibuya C, Kashiki Y, Shimizu H. Association of vegetable intake with urinary 6-sulfatoxymelatonin level. Cancer Epidemiol Biomarkers Prev 2005;14:1333–5. [27] Oba S, Nakamura K, Sahashi Y, Hattori A, Nagata C. Consumption of vegetables alters morning urinary 6-sulfatoxymelatonin concentration. J Pineal Res 2008;45:17–23. [28] González-Flores D, Gamero E, Garrido M, Ramı́rez R, Moreno D, Delgado J, et al. Urinary 6-sulfatoxymelatonin and total antioxidant capacity increase after the intake of a grape juice cv Tempranillo stabilized with HHP. Food Funct 2012;3:34–9. [29] Garrido M, Espino J, González-Gómez D, Lozano M, Cubero J, Toribio-Delgado AF, et al. A nutraceutical product based on Jerte Valley cherries improves sleep and augments the antioxidant status in humans. e-SPEN 2009;4:e321–3. [30] Garrido M, Paredes SD, Cubero J, Lozano M, Toribio-Delgado AF, Munoz JL, et al. Jerte Valley cherry-enriched diets improve nocturnal rest and increase 6-sulfatoxymelatonin and total antioxidant capacity in the urine of middle-aged and elderly humans. J Gerontol A Biol Sci Med Sci 2010;65:909–14. [31] Gónzalez-Flores D, Velardo B, Garrido M, González-Gómez D, Lozano M, Ayuso MC, et al. Ingestion of Japanese plums (Prunus salicina Lindl. cv. Crimson Globe) increases the urinary 6-sulfatoxymelatonin and total antioxidant capacity levels in young, middle-aged and elderly humans: nutritional and functional characterization of their content. J Food Nutr Res 2011;50:229–36. [32] Johns NP, Johns J, Porasuphattana S, Plaimee P, Sae-Teaw M. Dietary intake of melatonin from tropical fruit altered urinary excretion of 6-sulfatoxymelatonin in healthy volunteers. J Agric Food Chem 2013;61:913–9. [33] Garrido M, Espino J, González-Gómez D, Lozano M, Barriga C, Paredes SD, et al. The consumption of a Jerte Valley cherry product in humans enhances mood, and increases 5-hydroxyindoleacetic acid but reduces cortisol levels in urine. Exp Gerontol 2012;47:573–80. [34] Varoni EM, Vitalini S, Contino D, Lodi G, Simonetti P, Sardella A, et al. Effects of red wine intake on human salivary antiradical capacity and polyphenol content. Food Chem Toxicol 2013;58:289–94. [35] Rayne S. Concentrations and profiles of melatonin and serotonin in fruits and vegetables during ripening: a mini-review. Nat Proceed 2010; http://dx. doi.org/10.1038/npre.2010.4722.1. Chapter 20 Hydroxytyrosol as a Component of the Mediterranean Diet and Its Role in Disease Prevention Mª del Carmen Ramı́rez-Tortose, PharmD, PhD1, Mario Pulido-Moran, MSc1, Sergio Granados, PhD2, José J. Gaforio, MD, PhD3 and José L. Quiles, PhD1 1 Granada University, Granada, Spain. 2 The Methodist Hospital Research Institute, Houston, TX, USA. 3 University of Jaeń, Jaeń, Spain. ABBREVIATIONS ADME COX HT LOX OA OO Ty VOO absorption, distribution, metabolism, and elimination cyclooxygenase hydroxytyrosol lipooxygenase oleic acid olive oil tyrosol virgin olive oil INTRODUCTION It is well known that Mediterranean countries have lower rates of incidence of and mortality from different diseases such as cardiovascular diseases and cancer. Knowledge about these facts link them to the so-called Mediterranean diet, which is a concept emphasizing not only healthy nutrition but also lifestyle, together with regular physical activity. This typical diet has its origin mainly in three countries: Spain, Italy, and Greece. Many people have contributed to establishing the current Mediterranean diet, namely Iberia, Celts, Greeks, Romans, and Arabs. With this evolution through time, the Mediterranean triad concept emerged; it is mainly based on products derived from wheat, olives, and grapes, for example, bread, olive oil (OO), and wine. Therefore, the Mediterranean diet supplies a high level of antioxidants, especially those derived from the high consumption of fruit and vegetables. The most appreciated element and characteristic of the Mediterranean diet is undoubtedly OO. This oil originates from olive tree fruit (Olea europea L.). Using pressure, different types of OO with different tastes, acidity, and levels of compounds can be obtained. Using chemical methods, refined OO is obtained. All types of OO are interesting because of their aroma and palatability but only those obtained by pressure are interesting because of its their high hydroxytyrosol (HT) content. HT is a phenylethanoid, a type of phenolic compound characterized by a phenethyl alcohol structure (Figure 1), that forms part of the minor compound fraction of extra virgin olive oil and is considered a powerful antioxidant. Because of these characteristics, HT has a lot of interesting activities related to the prevention, treatment, and improvement of diverse pathologies. Abundant information about the benefits of OO is available in the scientific literature. Classically, many of these studies strictly focused on the benefit of oleic acid (OA) without taking into account the minor compound fraction of OO [1]. Other studies have reported that OA was also in other foods such as pork (porcine meat derivates); nevertheless, OA lacks the beneficial properties that OO has. A lot of information about HT is currently available and its antioxidant, anti-inflammatory, antiatherogenic, or antiplatelet properties are well documented, but the functions that initially were assigned to OA and OO are actually targeted to HT. This information is explained in this chapter. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 205 206 SECTION 2 Components of the Mediterranean Diet FIGURE 1 The chemical structure of hydroxytyrosol. This is a type of phenolic compound characterized by a phenethyl alcohol structure. WHAT IS THE ORIGIN OF HT? As mentioned above, HT is a component of the minor compound fraction of OO. OO is roughly divided into two main fractions: the saponificable fraction, mainly comprised of triacylglycerides, and the minor compound fraction, which is constituted by two subfractions—an unsaponificable fraction (extracted with solvents) and a soluble fraction, which represents 2% of the total oil weight of polyphenols; more than 230 different chemical compounds are included in the last fraction. It has to be taken into account that the phenolic content in OO can be influenced by multiple factors, including the olive variety, environmental conditions, and the method of production. The amount of polyphenols in OO can increase to more than 450 mg/kg, depending on the characteristics previously defined [2–4]. In virgin olive oil (VOO), the average amount of HT is within a wide range of 9–21 mg/kg [2], obviously differing depending on the conditions mentioned before. HT comes from the hydrolysis of oleuropein, and this chemical process takes place during the maturation of olives, oil storage, and the processing of table olives (Figure 2). During oil processing, different compounds such as olive mill wastewater, rinse water, and olive pomace are obtained. All of these fractions are rich in phenolic components, including HT. HT is part of the OO soluble fraction. This element has an amphipathic character, and therefore it can be identified in the previously mentioned liquids. Consequently, these fractions contain a large amount of different polyphenols and other phenolic compounds. Olive tree leaves have classically been used to treat different pathologies such as fevers or parasitic diseases because of its antioxidant, hypoglycemic, antihypertensive, antimicrobial, antiatherosclerotic, and antiviral effects. Nowadays, antitumor activity has been reported. The average concentration of HT in these samples is about 10-17 mg/g [5]. Since 1998, HT has been studied in other products of the Mediterranean diet, such as wine. Many studies have shown firmly that HT is contained in both red and white wine, although in a higher concentration in the red wine [6,7]. HT was found for the first time while studying the composition of some Italian wines. Later, HT was highlighted in Greek wines; HT was found in two types of wines in different concentrations [6]. The average concentration in red wines ranged from 1.8 to 3.1 mg/L [7]; the concentration in white wines was approximately half that of the red [6]. The amount of HT present in aged red wines markedly increases to 25 mg/L [6]. All the facts mentioned above give an idea about the importance of HT in the Mediterranean diet, and it has been identified in at least two of the three components of the Mediterranean triad (Table 1). O HO O O COOCH3 b-Glucosidase OH O HO COOCH3 + Glucose OH O O Oleuropein aglycone O–Glc OH Oleuropein Hydrolysis HO O COOCH3 + O OH HO OH Elenolic acid FIGURE 2 Biosynthesis of hydroxytyrosol. OH Hydroxytyrosol Hydroxytyrosol and the Mediterranean Diet Chapter 20 207 TABLE 1 Range of Concentrations of Hydroxytyrosol in Different Components Content in HT White wine 1,50–2.70 mg/l Red wine 2–3.9 mg/l Aged red wine 25 mg/l Virgin olive oil 9–21 mg/Kg Olive leaves 10–17 mg/g HT THROUGH THE BODY: WHAT ARE ITS ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION PROCESSES? The absorption, distribution, metabolism, and elimination processes (ADME) of HT are currently being studied. The majority of these studies have been performed using animals models, although human research has been increasing in the past two decades, and these trials are commonly designed to elucidate the effects of HT in OO—they are not focused on HT in wine. Chemical Characteristics Taking into account the International Union of Pure and Applied Chemistry formulation, HT is also known as 4(2-hydroxyethyl)-1,2-benzenediol or ((3,4,dihydroxyphenyl)ethanol) (Figure 1). According to this structure, the high antioxidant activity of HT is related to O–H bond dissociation enthalpy (o-dihydroxyl) and has a stronger correlation with oxidative stability than other derivates [8]. All phenolic conjugates are very quickly converted into HT and another compound called tyrosol (Ty), albeit with lower efficiency. This compound and HT have some similar antioxidant, antiplatelet, or as anti-inflammatory functions in the body, but the activity of Ty is lower compared with HT. HT is included in phenolic alcohols from OO. The ratio between the HT and the Ty in OO is around 0.5:1, and this ratio depends on the origin of the OO, growth conditions, geographic area, or the method of production [3,9]. The only difference between Ty and HT lies in the hydroxyl group featured in HT but not in Ty. According to this, the activities of HT are mainly due to the ortho-hydroxyl group in HT. With its catechol structure, HT has a much higher antioxidant potential than Ty [8]. From Food to Blood: The Absorption Process It is well known that hydrophilic phenolic compounds of OO are absorbed in a dose-dependent form in both animals and humans [3,10a,b] and all of them (including HT) have a strong metabolism phase I (where it is hydrolized) and phase II (where it is conjugated into glucuronidated, methylated, and sulfated derivatives) to facilitate its absorption [11,12]. The absorption of HT and that of the rest of the phenolic compounds in OO takes place in the small intestine and colon, with an average uptake of 66% with respect to their total amounts [13], and it has been suggested that transport through the intestinal epithelium can occur by passive bidirectional diffusion [14]. The absorption of HT is rapid, and a maximum plasma concentration is reached 5–10 min after intake, followed by rapid decline. The absorption of HT varies according to the vehicle in which it is carried, ranging between 75% and 99%, depending on the nature of the vehicle (aqueous or oily); obviously, an aqueous vehicle leads to lower absorption than an oily one. Finally, after postprandial absorption, HT binds to circulating lipoproteins (as do other phenols from OO). Transport in the Body: The Distribution Process Distribution studies of the intravenous administration of radioactive HT in rats demonstrated that its half-life in blood is around 1–2 min and that, 5 min after injection, most of the marked HT is found in the kidney, where 10-fold more radioactive HT accumulates than in other organs, such as the skeletal muscle, liver, lungs, or heart. Similar levels of HT are found in all of them [15]. HT can cross the blood–brain barrier and easily reach the brain. 208 SECTION 2 Components of the Mediterranean Diet It has been proposed that our understanding of the function of HT is hampered by the fact that HT is a metabolite of dopamine and its concentration in body fluids is a combination of exogenous and endogenous sources [16]. This is the main reason why it is not possible to wholly evaluate its bioavailability in the organism. Because of the first metabolic step that HT undergoes in the gut and liver, its bioavailability is poor because of the formation of sulfate and glucuronide conjugates [11,12], to the extent that concentrations of its free form in body fluids are almost undetectable. The most important form that can be found in the body is the conjugated form with sulfate, which could be used as a good biomarker [10b,12,16]. In fact, HT is metabolized rapidly, and plasma concentrations return to baseline values in 6 h at most. The first metabolic step takes place in the enterocytes and subsequently in the liver; metabolites are found in blood 5 min after intravenous injection [15]. In plasma, HT alone, orthomethyl derivatives (homovanillic alcohol), glucuronide derivatives, and their glutathionyl conjugates can also be found, indicating that perhaps the derivatives of glucuronic acid and glutathionyl conjugates are synthesized in the enterocytes and liver, whereas homovanillic alcohol appears after HT metabolism in the liver. Transformations: What are the Metabolic Processes? HT has two different types of metabolism: the first-pass metabolism and a transformation metabolism in the liver. Both of those are equally important because HT undergoes different modifications along the body, supposedly endowing it with its properties. The first modification is performed in the enterocytes, and it is necessary to uptake HT through gastrointestinal lumen, as summarized above. All the processes are described in Figure 3 [12]. According to different studies [15], the process can be schematized in three abbreviated forms: 1. Oxidation through the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, giving rise to dihydroxyphenylacetic acid 2. Methylation by the enzyme catechol-o-methyltransferase, rendering homovanillic alcohol 3. Methylation-oxidation, a reaction that occurs to form homovanillic acid FIGURE 3 Metabolism of hydroxytyrosol and different derived components. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; COMT, catechol-O-methyl transferase; SULT, sulfotransferase. Hydroxytyrosol and the Mediterranean Diet Chapter 20 209 In addition, a high number of conjugates from HT and its metabolites with glucuronide and sulfate are generated, which are detectable in both plasma and urine [10b,17]. Taking into account the nature of HT and the metabolic pathways taking place, we can predict the form of elimination. Elimination As previously described, HT is a compound that forms part of the soluble fraction of OO (it is an amphipathic component), and its natural method of elimination from the body is by urine. It should to taken into account that HT has been studied as a pharmacological or nutraceutical component and can be administrated as an isolate compound or as part of OO or wine; because of this, excretion could differ about the amount, amount of metabolites, or even the method of excretion, depending on the administration in humans or animals, or even according to the vehicle [18], the elimination can be different. The time required for HT and its metabolites to be totally eliminated through the urine in humans is roughly 6 h [10b,12]. The endogenous production of HT, related to dopaminergic metabolism, could be the reason because it tends to accumulate in urine for a long time, independent of the dosage administered, as occurs with Ty [3]. Many studies of animals and humans have been performed, with differences in the results obtained. We have to be cautious when trying to extrapolate the results of absorption/excretion from experiments in rats. Obviously, all this affects the dosage of HT used for nutraceutical of pharmacological purposes. DOES HT HAVE SIDE EFFECTS? There are many reasons to study HT metabolism, essentially because of the wide range of properties associated with this component and its possible benefits in human health. The presence of HT in OO could lead one to think that it has no capacity to damage the body, but this has to be ratified by different studies of toxicity because HT is still a drug that can be used in different diseases and treatments. It is well known that there are many active ingredients that are used in medicine in their natural form (within a plant extract, for example) but that could be lethal or highly toxic without control. For these reasons, it is extremely important to perform additional toxicity studies. Acute/Subchronic Toxicity In terms of toxicity and safety profile, HT has only been investigated as part of OO or olive mill wastewater. To study acute toxicity, some researchers administered a very large single dose (2 g/kg) in rats and did not find any toxic effects or macroscopic alterations in organs. They recorded piloerection only 2 h after administration (maybe caused by the injection itself), and this effect disappeared in <48 h. Furthermore, other toxicity studies have been performed: aqueous olive-pulp extracts in which the HT content ranged from 50% to 70% of the total quantity of phenols [19]. Other studies used oral administration in rats, giving a single dose of solid olive-pulp extract until HT levels reached 2000 mg/kg/day (the equivalent dose of 1000–1400 mg of pure HT), without any adverse effect except soft or liquid feces [20]. At the maximum dose, no signs of acute toxicity (either mutagenic and teratogenic effects) were recorded. This fact suggests that the mean lethal dose of the extract is >2000 mg/kg. As part of a micronucleic assay, Sprague-Dawley rats (five of each sex) were administered a single gavage dose of 5000 mg olive-pulp extract/kg and observed for 6 days, after which a 5000 mg/kg dose was given for 29 consecutive days. No mortality or clinical signs of toxicity were noted. This study demonstrated that the mean lethal dose of the solid olive-pulp extract was >5 g/kg (2.5–3.5 g HT/kg), suggesting that the extract is practically nontoxic [20]. Another recent study in which the potential toxicity of HT was evaluated using a pure extract of this component in rats has been published. The toxicity of pure HT has never been properly investigated [21]. Different doses of HT were administered using oral administration (by gavage) for 13 weeks at levels of 5, 50, and 500 mg/kg/day. According to other previous studies, this dose would be innocuous for rats, but the simple use of pure HT changes the characteristics of these studies, which were able to report much different results than those expected. No mortality or micro- or macroscopic alterations were found. No toxicological effects were observed in all examinations. According to the authors, based on the results, HT does not induce effects that can be considered of toxicological relevance. The 500-mg/kg/day dose is proposed as the no observed adverse effect level [21]. 210 SECTION 2 Components of the Mediterranean Diet Establishing a Dose Taking into account all the concepts described above, it is easy to understand why establishing doses in humans is a really controversial issue. As previously mentioned, most studies were undertaken using animals because of the ease of modifying the dose or the length or method of administration. It also must be remembered that the ADME processes are different in animals and humans, which is why the dose has to be carefully selected. There are some available studies of humans in which HT is administrated within both common OO and enriched OO [22,23]. In 2011 the European Food Safety Authority pointed out the possible use of HT in different formulations. They recommend that 5 mg of HT and its derivates in OO should be consumed daily to get the beneficial properties of low-density lipoprotein (LDL) oxidation. This fact is essential so that researchers will start or continue their different studies related to HT, promoting its possible use as a pure component in humans. Actually, it is well known that many assays are being carried out in several innovation and development centers and hospitals in which HT is used as a supplement in the diets of patients with different pathologies. Because of the early approval of HT by the European Food Safety Authority, these studies will probably soon see the light of day. ROLE OF HT IN DIFFERENT DISEASES: HT AGAINST THEM AND AS PROTECTOR OF LIFE Nowadays it is known that several compounds different from OA could be related to the healthy properties of OO. In the past two decades, many studies with the sole purpose of determining which components in OO are responsible for its healthy properties have been proposed. One study [24] described the relative importance of the saponificable and unsaponificable fractions of VOO for lipid peroxidation in mitochondria of rabbit hearts. Other research found that OA was not the only compound responsible for the antioxidant effect of OO in relation to the formation of atheroma plaques [25]. Other studies demonstrated the influence of the minor components of VOO and noted that the phenolic fraction of OO considerably improves the lipid peroxidation of LDL, which is related to an increase in antioxidant capacity [26,27]. HT is currently the gold standard in the studies related to OO. It is considered one of the most important components of OO and is responsible for a large number of its healthful properties, thanks to its antioxidant characteristics. All properties and studies proposed in the following sections have the great aim of highlighting the importance of certain components of the Mediterranean diet to understand that they are really responsible for the healthy benefits of OO. Everything is proposed with the only objective of understanding why the Mediterranean diet contributes to a lower incidence of and mortality from different diseases, trying to express which are the molecular pathways involved in all activities associated with the Mediterranean diet, specifically those related to HT. Antimicrobial Effect of HT In this decade, some studies have demonstrated that extracts from olive leaves have antimicrobial activity against some microbes such as Escherichia coli, Candida albicans, Kluyveromyces marxianus, Clostridium perfringens, Streptococcus mutans, Shigella sonnei, and Salmonella enterica [28]. This fact has been known for centuries. In Egyptian and Roman civilizations, extracts from olive leaves were used in solutions, soaps, and ointments. Later knowledge about the main components of olives and olive leaves responsible for their antimicrobial effects was gained, showing that these are the dialdehyde and decarboxymethyl forms of elenolic acid, together with HT [29]. In vitro, HT also possesses antimicrobial properties against infectious agents of the respiratory and gastrointestinal tracts, such as Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhi, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, at low inhibitory concentrations, even lower than dose used for certain antibiotics such as ampicillin [30]. The Mediterranean basin is an endemic area for leishmaniasis. This pathology is caused by a protozoon called Leishmania spp. Many species generate different varieties of the disease. Pure extracts of HT were tested, showing antiparasitic activity against L. infantum and L. donovani, which are responsible for visceral leishmaniasis, as well as against L. major, which causes the cutaneous form of the disease [31]. How Can HT Protect the Skin? It is well known that human skin has very efficient antioxidant systems, such as superoxide dismutase, catalase, and glutathione peroxidase. The skin is inevitably exposed to environmental factors such as ultraviolet radiation (UVR), which Hydroxytyrosol and the Mediterranean Diet Chapter 20 211 increases intracellular free radicals. This intracellular overexpression modifies the redox status in the cell and renders unusable the antioxidant cellular systems. Despite polyphenolic HT being touted as an effective antioxidant, biocatalytically produced derivatives may prove to be even more efficacious in their endogenous antioxidant ability to protect against long-wavelength UVR [32,33] and 8-hydroxydeoxyguanosine level [33]. The results of a study by Zwane et al. [32] demonstrate that both HT and its metabolites have a potent radical scavenging activity in skin cells. The results induce the idea that HT and its metabolites could decrease the intracellular accumulation of reactive oxygen species (ROS) through a free radical scavenging pathway, thus decreasing the prejudicial effects produced by UVR, among others. HT can also significantly reduce the DNA strand breaks caused by ultraviolet B. Furthermore, HT attenuated the expression of p53 and nuclear factor-kB in a concentration-dependent manner [33]. Antiatherogenic and Cardioprotective Factor Several studies have demonstrated that one of the most important factors linked to atherogenic processes is an imbalance in the redox status in cells. An excess of free radicals inside the cell can originate different molecules, such as LDL, that are easily oxidized. In particular, this oxidized LDL is largely responsible for this one process. In multiple studies HT featured a high capacity of scavenging free radicals from the inside of the cell as superoxide anions, hydrogen peroxide, and hypochlorous acid. HT also has a potent metal-chelating activity, as occurs with ferrous cations. Because of this redox potential, HT is able to decrease ROS, both directly and because of its chelating action—it can prevent the appearance of ROS produced in the reactions derived from the metal. Nowadays it is known that macrophages are responsible for LDL oxidation, and for this reason, HT avoids this oxidation in these cells and prevents the reservoir of these lipoproteins inside the arterial intima, thus impeding the formation of atherosclerotic plaque [4,34–36b]. Some extracts enriched in HT also have shown a capacity to reduce blood total cholesterol [36,37] and lipid concentrations [36,38] as well as blood pressure (in both hypertensive and normotensive animals) [39]. All these actions are intimately linked with cardiovascular health, so it is easy to understand that HT has an important role as cardioprotector—above all understanding that postprandial lipemia is a risk factor for atherosclerosis. The cardioprotective effect of HT lies, in part, in the capacity to reduce the expression of proteins related to aging in cardiac cells (sirtuins, Forkhead box subfamily O proteins, and pre-B cell colony-enhancing factor), although to a lesser extent than other molecules, such as resveratrol or Ty [40]. Does HT Have Anti-Inflammatory and Antiplatelet Actions? Platelets play a key role in hemostasis and wound healing, contributing to the formation of vascular plugs. They also are involved in formation of atherosclerotic plaque through platelet aggregation. HT can be considered antithrombotic because it significantly reduces platelet aggregation [41] and is able to inhibit platelet function and eicosanoid formation. HT was shown to be able to decrease serum thromboxane B2 (TxB2) production after blood clotting in an in vivo model [42a,b]. In in vitro studies, platelet-rich plasma was incubated in the presence of HT, obtaining maximal inhibition in aggregation induced by adenosine diphosphate or collagen and TxB2 production by collagen or thrombin stimulation. A reduction in the synthesis of thromboxane A2 (TxA2), measured by the reduction of its metabolite TxB2, also was found. This decline was mainly attributable to the inhibition of activity of the enzyme cyclooxygenase (COX). This finding suggests that these antithrombotic effects would be helped by the decline in the production of vascular prostacyclin, effects similar to those featured by acetyl salicylic acid [43], which is able to inhibit the synthesis of prostaglandin E2 by indirectly blocking the enzymes inducible nitric oxide synthase, COX-2, and 5-lipoxygenase. Some studies have featured the anti-inflammatory properties of HT by suppressing the expression of COX-2 and inducible nitric oxide synthase in human monocytic cells [44]. Moreover, HT also encouraged the action of cells of the immune system, both increasing its antioxidant capacity (protecting neutrophils against oxidation mediated by hydrogen peroxide) and decreasing the DNA damage in monocytes, as mentioned above. In addition, HT augments the cytosolic levels of Ca2+, activating T and B lymphocytes [45]. 212 SECTION 2 Components of the Mediterranean Diet 45 % Ki67 Cellular proliferation 40 35 30 25 * * BCHTY BCADR FIGURE 4 Cell proliferation in rats with breast cancer treated with hydroxytyrosol or adriamycin: breast cancer control group (BCC) (n ¼ 10 rats); breast cancer adriamycin group (BCADR) (n ¼ 10 rats), and breast cancer hydroxytyrosol group (BCHTY) (n ¼ 10 rats). Values are expressed as percentage of Ki67 as mean SEM. *Significant differences between the control group and treated groups (P < 0.01). 20 15 10 5 0 BCC Cancer Studies There are several studies that have been mainly performed on cell cultures and animal models because of the complexity of handling research with humans and this pathology. HT has been studied because of its properties as an antioxidant, among others, and it is for these properties that most studies are directed at demonstrating the chemopreventive activities of HT. It is well known that HT can promote apoptosis and inhibit the proliferation and growth of different types of tumor cells such as HL60 (promyelocytic leukemia) and HT29 (colon adenocarcinoma). There is controversy about the effect of HT in nontumor cells such as health lymphocytes. A possible pathway by which HT triggers apoptosis has been established. This is involved in the activation of c-jun by c-jun NH2-terminal kinase, which under certain circumstances causes cell death and inactivates the antiapoptotic protein B-cell lymphoma protein 2 [3,46]. Other studies have demonstrated that HT is able to inhibit cellular proliferation, blocking the G1 phase of the cell cycle and increasing the proportion of cells in the G0/G1 phase, with a corresponding decrease in the S and G2/M phases. Some proposed mechanisms are as follows: (1) Direct blockage of cyclin-dependent kinases. (2) Induction of cyclin-dependent kinase inhibitors. (3) Blockage of messengers involved in cell proliferation, such as ROS. Furthermore, HT induces apoptosis-dependent stress in the endoplasmic reticulum. This leads to activation of the proapoptotic pathway endoplasmic reticulum to nucleus signaling 1 (Ire1)/c-jun NH2-terminal kinase/c-jun/activator protein1/NADPH oxidase 4 (Nox4) [3]. HT can inhibit human epidermal growth factor receptor 2 (HER2) expression in breast cancer cells resistant to trastuzumab, which overexpress this oncogene (SKBR3); in addition, it can cause cytotoxic activity on these SKBR3, MCF7 cells (breast cancer cells with natural HER2 expression) and MCF7/HER2 (HER2-induced breast cancer cells) [47]. Our studies have shown that HT exerts antitumor properties in Sprague-Dawley rats with experimental mammary tumors, inhibiting cell growth and proliferation (associated with a less nuclear Ki-67 immunostaining) in these tumors (Figure 4). Moreover, HT alters several genes associated with cell proliferation, apoptosis, and the Wnt signaling pathway, promoting a high expression of Sfrp4 [48]. HT is a powerful inhibitor of the fatty acid enzyme synthase, a key enzyme involved in carcinogenesis in SKBR3 and MCF7 cells, so that it is presented as a strong chemopreventive. Another mechanism described recently involves catechol quinones from HT metabolism and from reaction with hydrogen peroxide. These are reactive arylating electrophilic species that produce Michael adducts with cellular thiol nucleophiles in glutathione and proteins, particularly on cysteinyl proteins. In this sense, they exert their cytotoxic, anti-inflammatory, and anticarcinogenic properties, very probably via the inhibition of nuclear factor-kb by these quinones [3,8]. These compounds bind to cysteine residues, forming arylating/alquilant adducts and hampers the bonding of this transcription factor with DNA; this impedes the start of transcription of COX-2 and 5-lipoxygenase, reducing the synthesis of prostaglandin E2 and therefore chronic inflammation. In addition, antitumor activity is expressed [3,8]. Table 2 summarizes the most important mechanisms of HT described for cancer and microbial and cardiovascular diseases [3]. Hydroxytyrosol and the Mediterranean Diet Chapter 20 213 TABLE 2 The Biochemical and Cell-Signaling Effects of Hydroxytyrosol Capacity of Hydroxytyrosol Biochemical Effects Cell-Signaling Effects Reduction of LDL oxidation (high antioxidant capacity and lipid peroxidation decrease) Adhesion molecules (VCAM-1 and ICAM-1) reduced expression Hypocholesterolemic effect NF-kb, AP-1, GATA, and NAD(P)H oxidase inactivation In cardiovascular diseases Antiatherogenic effect Antithrombotic and anti-inflammatory Vascular endothelium protection Cardioprotective effect Increase in HDL action Diminishes the synthesis of thromboxane A2 and B2, leukotriene B4, and vascular prostacyclin VCAM-1 and ICAM-1 reduced expression Reduces cAMP and cGMP platelet phosphodiesterase NF-kb, IRF-1, and STAT-1a transcriptional activation prevention Inhibits 5- and 12-LOX Inflammatory cytokines (IL-1b and TNF-a) reduced expression Lowers synthesis of PGE2 Blocks COX-2 and iNOS Nitric oxide increase Lowers size of infracted region and apoptosis of cardiomyocytes Reduced expression of ageing-related proteins (Sirts, FoxO, and PBEF) Not found Cell cycle arrest (G0/G1, S, G2/M) In cancer Antiproliferative Increased expression of p21WAF/Cip1 and p27kip1 CDK6 inhibition HER2 oncogene inhibition Fatty acid synthase inhibition Proapoptotic Not found Cytochrome c release Caspase 3 and c-jun activation Bcl-2 activation Ire 1/JNK/c-jun/AP-1/Nox-4 pathway activation P13K/Akt/NF-kb pathway inhibition NF-kb inhibition Akt, v-akt murine thymoma viral oncogene; AP-1, activator protein-1; Bcl-2, B-cell lymphoma protein 2; CDK, cyclin-dependent kinase; COX-2, cyclooxygenase-2; GATA, a transcription factor that regulates T lymphocyte differentiation and maturation; HDL, high-density lipoproteins; ICAM-1, intercellular adhesion molecule-1; IL-1b, interleukin-1b; iNOS, inducible nitric oxide synthase; IRF-1, interferon regulatory factor-1; JNK, c-jun NH2-terminal kinase; LDL, low-density lipoprotein; LOX, lipooxygenase; NAD(P)H, nicotinamide adenine dinucleotide phosphate; NF-kb, nuclear factor-kb; PBEF, pre-B cell colony-enhancing factor; PGE2, prostaglandin E2; PI3K, phosphoinositol 3 kinase; Sirts, sirtuins; STAT-1a, transducer and activator of transcription1alpha; TNF-a, tumor necrosis factor-a; VCAM-1, vascular cell adhesion molecule-1. SUMMARY POINTS l The Mediterranean diet is responsible for the lower rates of incidence of and mortality from different pathologies, such as cardiovascular diseases or cancer, because of the high level of antioxidants, especially those derived from the high consumption of fruit and vegetables, in this diet. 214 l l l l l SECTION 2 Components of the Mediterranean Diet Currently, much information about HT (a minor component in OO) is available, and its properties as an antioxidant, anti-inflammatory, antiatherogenic, or antiplatelet are well documented, being the target of different studies that report that HT performs the activities described above. HT is located in OO, olive leaves, and in red and white wines in varying concentrations. HT is an amphipathic element that needs to be metabolized in the small intestine and colon (first metabolic step) to be absorbed, with an average uptake of 66%. HT is quickly metabolized and excreted from the body. HT presents no adverse effects. 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Elevated circulating LDL phenol levels in men who consumed virgin rather than refined olive oil are associated with less oxidation of plasma LDL. J Nutr 2010;140(3):501–8. Hydroxytyrosol and the Mediterranean Diet Chapter 20 215 [24] Ochoa JJ, Huertas JR, Quiles JL, Olvera AB, Mataix J. Relative importance of the saponified and unsaponified fractions of dietary olive oil on mitochondrial lipid peroxidation in rabbit heart. Nutr Metab Cardiovasc Dis 1990;9:284–8. [25] Ramirez-Tortosa MC, Urbano G, López-Jurado M, Nestares T, Gomez MC, Mir A, et al. Extravirgin olive oil increases the resistance of LDL to oxidation more than refined olive oil in free-living men with peripheral vascular disease. J Nutr 1999;129:2177–83. [26] Ochoa-Herrera JJ, Huertas JR, Quiles JL, Mataix J. Dietary oils high in oleic acid, but with different non-glyceride contents, have different effects on lipid profiles and peroxidation in rabbit hepatic mitochondria. J Nutr Biochem 2001;12:357–64. [27] Ochoa JJ, Quiles JL, Ramı́rez-Tortosa MC, Mataix J, Huertas JR. Dietary oils high in oleic acid but with different unsaponifiable fraction contents have different effects in fatty acid composition and peroxidation in rabbit LDL. Nutrition 2002;18:60–5. [28] Medina E, de Castro A, Romero C, Brenes M. Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity. J Agric Food Chem 2006;54:4954–61. [29] Medina E, Brenes M, Romero C, Garcı́a A, de Castro A. Main antimicrobial compounds in table olives. J Agric Food Chem 2007;55:9817–23. [30] Bisignano G, Tomaino A, Lo Cascio R, Crisafi G, Uccella N, Saija A. On the in vitro antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacol 1999;51:971–4. [31] Kyriazisa JD, Aligiannisb N, Polychronopoulosb P, Skaltsounisb AL, Dotsikaa E. Leishmanicidal activity assessment of olive tree extracts. Phytomedicine 2013;20(3–4):275–81. [32] Zwane RE, Parker A, Kudanga T, Davids LM, Burton SG. Novel, biocatalytically produced hydroxytyrosol dimer protects against ultravioletinduced cell death in human immortalized keratinocytes. J Agric Food Chem 2012;60(46):11509–17. [33] Guo W, An Y, Jiang L, Geng C, Zhong L. The protective effects of hydroxytyrosol against UVB-induced DNA damage in HaCaT cells. Phytother Res 2010;24(3):352–9. [34] González-Santiago M, Martı́n-Bautista E, Carrero JJ, Fonollá J, Baró L, Bartolomé MV, et al. One-month administration of hydroxytyrosol, a phenolic antioxidant present in olive oil, to hyperlipemic rabbits improves blood lipid profile, antioxidant status and reduces atherosclerosis development. Atherosclerosis 2006;188(1):35–42. [35] Di Benedetto R, Varı̀ R, Scazzocchio B, Filesi C, Santangelo C, Giovannini C, et al. Tyrosol, the major extra virgin olive oil compound, restored intracellular antioxidant defences in spite of its weak antioxidative effectiveness. Nutr Metab Cardiovasc Dis 2007;17(7):535–45. [36] (a) Poudyal H, Campbell F, Brown L. Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats. J Nutr Dis 2010;140(5):946–53. (b) Granados-Principal S, Quiles JL, Ramirez-Tortosa CL, Ochoa-Herrera J, Perez-Lopez P, Pulido-Moran M, et al. Squalene ameliorates atherosclerotic lesions through the reduction of CD36 scavenger receptor expression in macrophages. Mol Nutr Food Res 2012;56(5):733–40. [37] Fki I, Bouaziz M, Sahnoun Z, Sayadi S. Hypocholesterolemic effects of phenolic-rich extracts of Chemlali olive cultivar in rats fed a cholesterol rich diet. Bioorg Med Chem Lett 2005;13:5362–70. [38] Jemai H, Bouaziz M, Fki I, El Feki A, Sayadi S. Hypolipidimic and antioxidant activities of oleuropein and its hydrolysis derivative-rich extracts from Chemlali olive leaves. Chem Biol Interact 2008;176:88–98. [39] Khayyal MT, el-Ghazaly MA, Abdallah DM, Nassar NN, Okpanyi SN, Kreuter MH. Blood pressure lowering effect of an olive leaf extract (Olea europea) in L-NAME induced hypertension in rats. Arzneimittelforschung 2002;52:797–802. [40] Mukherjee S, Lekli I, Gurusamy N, Bertelli AA, Das DK. Expression of the longevity proteins by both red and white wines and their cardioprotective components, resveratrol, tyrosol, and hydroxytyrosol. Free Rad Biol Med 2009;46:573–8. [41] Cicerale S, Conlan XA, Sinclair AJ, Keast RS. Chemistry and health of olive oil phenolics. Crit Rev Food Sci Nutr 2009;49:218–36. [42] (a) Leger CL, Carbonneau MA, Michel F, Mas E, Monnier L, Cristol JP, et al. A thromboxane effect of a hydroxytyrosol-rich olive oil wastewater extract in patients with uncomplicated type I diabetes. Eur J Clin Nutr 2005;59:727–30. (b) de Roos B, Zhang X, Rodriguez GG, Wood S, Rucklidge GJ, Reid MD, et al. Anti-platelet effects of olive oil extract: in vitro functional and proteomic studies. Eur J Nutr 2011;50:553–62. [43] González-Correa JA, Navas MD, Muñoz-Marı́n J, Trujillo M, Fernández-Bolaños J, de la Cruz JP. Effects of hydroxytyrosol and hydroxytyrosol acetate administration to rats on platelet function compared to acetylsalicylic acid. J Agric Food Chem 2008;56:7872–6. [44] Zhang X, Cao J, Zhong L. Hydroxytyrosol inhibits proinflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells. Naunyn Schmiedebergs Arch Pharmacol 2009;379:581–6. [45] Zbidi H, Salido S, Altarejos J, Perez-Bonilla M, Bartegi A, Rosado JA, et al. Olive tree wood phenolic compounds with human platelet antiaggregant properties. Blood Cells Mol Dis 2009;42:279–85. [46] Della RF, Cucciolla V, Criniti V, Indaco S, Borriello A, Zappia V. Antioxidants induce different phenotypes by a distinct modulation of signal transduction. FEBS Lett 2002;532:289–94. [47] Menendez JA, Vazquez-Martin A, Colomer R, Brunet J, Carrasco-Pancorbo A, Garcia-Villalba R, et al. Olive oil’s bitter principle reverses acquired autoresistance to trastuzumab (Herceptin™) in HER2-overexpressing breast cancer cells. BMC Cancer 2009;7:80–99. [48] Granados-Principal S, Quiles JL, Ramirez-Tortosa C, Camacho-Corencia P, Sanchez-Rovira P, Vera-Ramirez L, et al. Hydroxytyrosol inhibits growth and cell proliferation and promotes high expression of sfrp4 in rat mammary tumours. Mol Nutr Food Res 2010;54:1–10. Chapter 21 Frying: A Cultural Way of Cooking in the Mediterranean Diet Sara Bastida, PhD and Francisco J. Sánchez-Muniz, PhD Universidad Complutense de Madrid, Madrid, Spain. ABBREVIATIONS CVD CYP4A1 GPx MUFA PUFA SFA sICAM-1 SOD VOO cardiovascular disease cytochrome P450 4A1 glutathione peroxidase monounsaturated fatty acids polyunsaturated fatty acids saturated fatty acids soluble intercellular adhesion molecule-1 superoxide dismutase virgin olive oil INTRODUCTION: CULTURAL AND GASTRONOMIC IMPORTANCE OF FRYING IN THE MEDITERRANEAN DIET Evidence from archeological remains shows that humans took advantage of primitive wild vegetables such as the Aligustre (Ligustrum vulgare), considered the probable father of the olive tree [1]. More than 6000 years ago, together with the civilization flowering in the Mediterranean basin around the Mare Nostrum, the cultivation of olive trees (Olea europaea var. sativa, L.) started in Asia Minor, and from Syria it went to Greece. Since the third millennium BC, in diverse Mediterranean areas such as Palestine, Egypt, and Crete, olive oil has been implemented for quite different uses: religious, esthetic, alimentary, and home lighting. According to oral tradition, the technique of frying was discovered more than 6000 years ago and extensively used by the ancient Egyptians as early as 1600 BC. References to frying date from the earliest period in which the use of pots and pans is recorded [1–4]. The use of oil for cooking created a new and fast culinary practice that produced tasteful, safe, stable, easy-to-prepare food and made it possible to store foods in that warm/hot environment [1]. This is why frying is a common culinary practice in all Mediterranean countries. Although in Greco-Roman antiquity olive oil was used to fry foods, frying was much less common than today because the procedures to produce olive oil were rudimentary and its quality was worse [1]. After the fall of the Roman Empire, Mediterranean people gradually accepted the culture of northern inhabitants, decreasing the consumption of olive oil and therefore frying with olive oil [1]. However, in Moorish Spain, olive oil was the omnipresent fat. Fried fish was very popular; breaded foods such as greens, vegetables, eggs, cheeses, and pastries (doughnuts, pastry rings, almond pastries, etc.) also were fried. Using the characteristic Andalusı´preparation of food, frying gained notable nutritional and cultural importance [1]. Different circumstances changed the acceptability of oil in Mediterranean and non-Mediterranean countries in the following centuries. At the end of the sixteenth century, priests from the Jesuit order exported the Mediterranean tradition of frying to Japan and other eastern countries that integrated frying in their gastronomies (e.g., tempuras). It was one of the longest travels for Mediterranean frying. In the nineteenth and twentieth centuries, olive tree cultivation underwent a general expansion because of the strong demand for olive oil in industrialized countries. The explosion of leisure culture in the twentieth and twenty-first centuries pushed people from different parts of the world to visit the Mare Nostrum— attracted by its weather, culture, and gastronomy. Figures 1–3 show examples of fried foods, some of them corresponding to typical foods produced by non-Mediterranean cultures that have integrated frying in their gastronomies. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 217 218 SECTION 2 Components of the Mediterranean Diet 1 2 3 5 4 6 7 9 8 FIGURE 1 Pictures of several fried foods. Some correspond to non-Mediterranean typical foods produced by other cultures that have adapted and integrated frying into their gastronomies (e.g., tempuras). 1: fine tortes of Asiatic noodles; 2: vegetable fries; 3: seafood (fried crab); 4: fried pickles; 5: fried wonton; 6: Uruguayan empanada (pastries); 7: shrimp fried in butter; 8: spring rolls; 9: fried banana. Source of pictures 1–4; 7, and 8: http://www.bing.com/ images/search?q¼Picture+of+frying+foods; source of picture 5: http://diaryfoods.blogspot.com.es/2010/09/fried-wonton-recipes.html; source of picture 6: http://www.oocities.org/heartland/7187/empanada-recipe-uruguay.htm; source of picture 9: http://www.preventionrd.com/2009/11/plantains-treesand-pancakes. CULINARY OILS: FRYING PROCEDURE Oil Types, Selection, and Uses Oil selection in the kitchen is a complex issue influenced by factors such as price, taste, health, and efficiency [4,5]. Oil selection is a key part of the cuisine, and several sociocultural and historic factors have been integrated into our gastronomic culture. However, the factors’ hierarchy is quite different at the domestic or the restaurant/hotel level (Figure 4). In such selection, fatty acids and minor compound profiles are of main importance because they condition an adequate balance between nutrition and stability. Thus, oils that are very stable at high temperatures could be inadequate from a nutritional point of view because they would enrich food with saturated fatty acids (SFAs). On the other hand, oils rich in essential fatty acids can be adequate when used raw but become very unstable at high temperatures [3,4,6–9]. Frying and the Mediterranean Diet Chapter 21 1 3 219 2 4 5 FIGURE 2 Pictures of several fried foods. 1: Filled eggplants, onions, aromatic herbs, tomato, and minced Spanish ham are included (source: http://www. plazavea.com.pe/mundococina/recetas-de-cocina/platos-de-fondo-1/berenjenas-rellenas-de-jamon-y-queso.html). 2: Fish fritters, normally containing minced cod with herbs. 3: Little pastries that frequently contain tuna brined in oil or meat accompanied by tomato sauce. 4: Croquettes (source: www.pequerecetas.com). 5: Squid rings (source: http://www.espanita-frankfurt.com). FIGURE 3 Pictures of several fried foods. Sweets doughnuts (rosquillas; left) and fried flowers (typical desserts for special days; right). Humans and animals search for food and eat to make up the loss of energy and nutrients. However, eating is something besides consuming food; sociocultural aspects enrich the alimentary behavior contributing to the finding of pleasure and well-being [10]. The authonomic nervous system (sympathicus and parasympathicus) answers mediated by the hypothalamus in response to a plethora of signals from internal and external body receptors (corporal senses, central organs, or the brain), are controlled by signals coming from the limbic system (archaic part of the nervous system related to behavior). The limbic system continuously learns from our actions, and it is highly influenced by rewards and punishments 220 SECTION 2 Components of the Mediterranean Diet Taste Health Taste Price Price Efficiency and Shelf-life Tradition Natural Efficiency and Shelf-life Health Homes Restaurants/catering FIGURE 4 Factors influencing the oil choice at home and in restaurants or caterings. SIGNALS Hunger Satiety SOCIOCULTURAL INFLUENCES Rewards Punishments Food symbol HYPOTHALAMUS LIMBIC SYSTEM Alimentary behavior (basic) Modified/ mediated alimentary behavior (enriched) FIGURE 5 Summary of factors influencing alimentary behavior. Notice that innate behavior (basic behavior) of food searching responds mostly to information from external and internal body receptors (volume, pH, osmolality, concentration of nutrients, etc.). Information about the presence or absence of available substrates arrives at the hypothalamus, sending hunger or satiety signals. This basic response can be modified (enriched) by information coming from other parts of the brain such as the limbic system, an archaic part of the brain that controls behavior in response to learned information, most of which is based on rewards and punishments, that has being received throughout life in the form of sociocultural (news, family and group norms, etc.) signals. by social, religious, and cultural factors that make the consumption of food enjoyable or disgusting (Figure 5). According to Cruz [11], man searches for and eats those foods that form part of his cultural heritage. Thus, in colloquial terms, “we eat what we are,” an aphorism that is quite different from the common one: “we are what we eat.” The cuisine of a society is a language in its structure, which is unconsciously transmitted from generation to generation, especially in the form of typical foods and dishes produced by a family or local group; it can be paella, gazpacho, or fritos. Oil as a Mediator During Frying According to Lévi-Strauss [12], in all civilizations foods are distributed in similar categories, mostly following schemes of simple structures. Raw food is transformed into cooked food by means of a mediator (air, fire, water) and a recipient (pot, pan, grill). In the words of Cruz [11], Lévi-Strauss [12] has designed a new relationship between food and cuisine called the “culinary tetrahedron” (Figure 6): oil is the fourth mediator and “fried” is the new cooking category. When considering oil as a cooking mediator, the most appropriate oil for each type of food must be selected. Food preferences are based on a cultural tradition, making such a choice difficult. Thus, Mediterranean people are accustomed to the bitterness and pungency of olive oils, whereas non-Mediterranean people may dislike those attributes. According to gastronomic information, fruity oils with some bitterness strengthens the taste of fried food, mainly in the case of frying potatoes. They improve almost all sofritos and the base of stews. The quality of the fat eaten seems to be highly affected by the oil available at home. After a careful review of recipes in several Spanish cuisine books, we found a large proportion (about 30%) of recipes in which frying (in the total preparation or in one or some steps) was used. Although there is limited information about the exact amount of oil used for frying and that of fried foods consumed, a rough calculation suggests that 50% of the fat consumed by Mediterranean people belongs to oil used for cooking and dressing, and 25–60% of this amount is used for frying [13]. Accepting that 10–20% of fat used for frying in Spain is not consumed—instead being discarded after frying—final calculations show that between 25 and 60 g oil/day are “absorbed” by the fried food consumed in Spain. Frying and the Mediterranean Diet Chapter 21 221 Raw Boiled Fried Smoked/roasted FIGURE 6 Oil as a mediator in the transformation of raw food into fried food. Notice differences from other cooking procedures by the mediation of air and water. Performed from comments and suggestions made by Cruz [11]. Frying Technique Frying can be defined as the process used to succeed in making a raw food edible by keeping it in boiling oil or fat for a sufficient period of time. Although frying seems to be a rather simple process—dehydration accompanied by the entry of hot oil into the food and superficial browning—it is rather complex from a scientific point of view. Two principal frying techniques exist: shallow frying and deep-fat frying. Shallow frying is performed in relatively flat pots or pans containing little oil, in which the product is not completely immersed. The food in direct contact with the hot oil is fried. It is a common system in Mediterranean cuisine, preferentially used to prepare first dishes into which tomato, green pepper, garlic, onion, and cucumbers, among other vegetables, are placed. Thus, performing this type of frying permits the consumer access to several “healthy” foods that mixed together make sofritos such as pisto and Tumbe´, and that raw or boiled would be less pleasant and thus consumed less. Moreover, the oil is just used once, decreasing the negative effect that repeated frying could have on the oil and on the food. In deep-fat frying all the food is immersed in oil and the cooking process takes place throughout the entire product simultaneously. This kind of frying is performed in domestic and industrial fryers or in pans containing large amounts of oil. According to Bognár [14], uptake of oil in meat and fish fried using the deep-fat frying method is lower than that which occurs in the shallow frying. Frying can be done in a continuous or discontinuous manner, depending on whether the oil is left to cool between fryings or sets of fryings. It has been reported that continuous frying of potatoes displayed a lower degree of oil alteration than discontinuous frying [3,7]. Frying can also be classified as being characterized by frequent, slow, or null replenishment of fresh oil. As discussed in this chapter, frequent addition of fresh oil minimizes alteration of the oil (see reviews by Sánchez-Muniz et al. [3,7]). Factors influencing the quality and alteration of frying oil are summarized in Figure 7. Breadings and coatings help to perform adequate frying: on one hand these reduce the entrance of excess oil into the food; on the other hand they decrease dehydration of and lixiviation in the foods. CHANGES THAT OCCUR DURING FRYING As the fat or oil is heated, the quality decreases, as evidenced by a decrease in heat capacity and surface and interfacial tension and an increase in specific gravity, viscosity, acid values, anisidine values, and polymer content. Surface tension and interfacial tension are reduced by oxidative polymers with low-polarity and high-polarity, causing the uptake of excessive amounts of oil by the food [15]. Unsaturated fatty acids are prone to autoxidation, a free radical chain process consisting mainly of three steps: initiation, propagation, and termination (Figure 8). These phases are described in detail in different reviews [3,6–9]. Heating oil (in the absence of food) at 100-200 C speeds up all initiation and propagation reactions, and the amount of altered compounds formed, mostly secondary oxidation products, depends on the heating time. At temperatures get close to 200 C, hydroperoxide decomposition more than hydroperoxide formation occurs. In this case, the major compounds generated are dimers and polymers of triglycerides. Thermal alteration also is responsible for the formation of cyclic 222 SECTION 2 Components of the Mediterranean Diet Frying method Process Continuous Discontinuous Pan Fryer Domestic Industrial Temperature Time Vessel material Composition Fatty acids/minor compounds Additives (antioxidant, antifoam) Oil/fat type Smoke point Organoleptic properties Stability Food size and shape Breading, battering Preparation facilities Type of food Direct effect On thermolabile nutrients Interaction between nutrients and non-nutrients Nutrient interchange between food and frying oil FIGURE 7 Factors influencing the alteration of frying fats. Modified from Sánchez-Muniz FJ. Oils and fats: changes due to culinary and industrial processes. Int J Vitam Nutr Res 2006;76:230–7. With permission, Verlag Hans Huber; and from Sánchez-Muniz FJ, Bastida S, Márquez-Ruiz G, Dobarganes G. Effects of heating and frying on olive oil and food fatty acids. In: Chow CK, editor. Fatty acids in foods and their health implications. 3rd ed. Boca Raton, FL: CRC Press; 2008. p. 511–43. With the publisher’s permission. O2 Scavenger RH Initiation FIGURE 8 Major steps in oxidation. RH, unsaturated fatty acid. R, alkyl radical; OH Hydroxyl radical; RO, alkoxyl radical; ROO, alkylperoxyl radical; scavenger, radical scavenger. Scavenger • R • Propagation ROO• ROOH RH ROOH OH• Termination Monomeric compounds (epoxy-, hydroxy-, ketoderivatives) RO• Polymeric compounds (RR, ROOR, ROR) Volatile compounds (aldehydes, hydrocarbons, alcohols, ketones) compounds, such as triglyceride monomers, showing intermolecular or intramolecular cyclization. Because the propagation reaction also ends when peroxyl radicals combine with a radical scavenger such as vitamin E, the presence of antioxidants in the oil can help to stop or significantly decrease oil degradation (Figure 8). Frying clearly differs from heating because the presence of food in the fryer oil makes the situation rather more complex (Figure 9). During frying the fat is exposed to the action of moisture from the foodstuff, oxygen from the atmosphere, and the temperature at which the operation takes place. Because of the relatively high temperature of the frying process, autoxidation is accelerated, producing modified triglycerides in which at least one of the three fatty acyl chains is altered. Moisture from foods may induce hydrolytic alteration, thus yielding diglycerides and free fatty acids, and very reduced amounts of monoglycerides and glycerol that all together are commonly classified as hydrolytic products. All these Frying and the Mediterranean Diet Chapter 21 FIGURE 9 Major changes during frying. (1) Interior of food. Cooking is performed at 100 C due to vapor production. (2) Crust. The fat content increases and the water content decreases. A well-defined crust prevents dehydration and excessive penetration of grease in food. (3) Surface. Total dehydration occurs. Food becomes crispy and with typical odors. Browning and caramelizing take place. Capital letters indicate that mayor pathways are occurring. Letter size suggests the amount of originated products. Modified from Sánchez-Muniz FJ. Oils and fats: changes due to culinary and industrial processes. Int J Vitam Nutr Res 2006;76:230–7. With permission, Verlag Hans Huber. 223 Antioxidants Smoke Flavors Oxygen Light Volatiles Vapor HYDROLYSIS Metals 2 Oil Diglycerides Free fatty acids Monoglycerides Glycerol 1 3 AUTOXIDATION Heat Peroxides Alcohols Aldehydes Ketones Acids etc. Free radicals Residuals CYCLIZATION POLYMERIZATION Lipid solubilization Cyclic monomers Dimers Polymers Flavor Fat Color thermally oxidized and hydrolytic compounds are present in different quantities, depending on a considerably high number of frying variables (Figure 7). Finally, in addition to the migration of lipids from the food into the frying oil, the presence of some compounds from the foods can substantially affect the thermal oxidation reactions [7,16] (Figure 9). Thus, (1) amphiphilic compounds such as phospholipids and emulsifiers can contribute to early foaming; (2) lipid-soluble vitamins and trace metals leaching into the frying oil may inhibit or accelerate oil oxidation, depending on their antioxidant or pro-oxidant effects; (3) cholesterol and its oxidation-derived compounds from fatty animal foods can be transferred to vegetable frying oils and then incorporated into nonfatty foods during subsequent frying operations; (4) pigments and Maillard browning products can modify the susceptibility of frying oils to oxidation and contribute to darkening; (5) phenolic compounds present in the foods or in added spices can increase the stability of the frying oil; and (6) volatile compounds coming from strongly flavored foods such as fish or onions can contribute to specific off-flavors. During frying, crust formation occurs, and the substrate surface is almost totally dehydrated (Figure 9). This crust acts as a barrier, avoiding excessive fat uptake and food dehydration. Furthermore, monounsaturated or unaltered oils produce a thinner and more defined crust than polyunsaturated and thermal oxidized oils. Therefore, the crust contributes to producing leaner, safer, and healthier foods. ADVANTAGES AND DISADVANTAGES OF FRYING VERSUS OTHER COOKING PROCEDURES Some major advantages and disadvantages of frying are summarized in Table 1. In addition to being one of the most performed cooking techniques, frying has cultural and economic importance and transforms perishable food into more stable ones. Frying is a process of mass and heat interchange. The heat, as in other culinary techniques, reduces alimentary risk related to microbiological pathogens [3,17,18]. Entrance of oil into the foodstuff can increase some organoleptic properties of the food (texture, flavor, aroma, taste, crispiness) and improve or impair it; the fatty acid profile gives rise to a most balanced SFA-to-monounsaturated fatty acids (MUFAs)-to-polyunsaturated fatty acids (PUFAs) ratio. When the frying oil is rich in fat-soluble vitamins and/or minor compounds, it can improve these substances in foods [3,5]. For example, frying sardines or salmon with different oils and fats induces deep fatty acid changes and thus changes in the omega 6-to-omega 3 ratio; the fried fish becomes enriched in the major fatty acid of the oil used for frying [3,5,19]. After six 224 SECTION 2 Components of the Mediterranean Diet TABLE 1 Advantages and Disadvantages of Frying Advantages The most common culinary process in Mediterranean countries; helps maintain adherence to Mediterranean foods and diet Cultural importance; food as a symbol; emotional choice of traditional foods (“we eat what we are”) Very little cooking time required No greater damage to food quality than that produced by other culinary techniques Home frying could decrease the consumption of industrial snacks and fritters Improves food palatability (texture, taste, flavor); this is very important during childhood and adolescence for increasing consumption of vegetable (e.g., tempura) and fish and meeting nutritional health guidelines and recommendations Improves food composition; it can produce more balanced and cardio-healthy foods with regard to their fatty acid composition Food can be enriched in fat-soluble vitamins and bioactive compounds from oils (e.g., carotenoids, tocopherols, polyphenols) Disadvantages Often related to poor frying performance (e.g., addition of no or little fresh oil during a large number of frying operations); however, a proper frying technique does not give rise to a concentration of altered compounds above the level established by the present legislation (>25% polar material) Decreases palatability related to poor-quality fats and inadequate frying conditions Dehydrates and enriches fat in foods; thus food becomes highly enriched in energy, which is not recommended in overweight and obese individuals Changes in food composition: liberation of important fatty acids, such as eicosapentaenoic and docosahexaenoic acids, from foodstuffs into the fryer media; possible losses of nutritional value (essential fatty acids, antioxidants, thermal-sensitive vitamins); losses by lixiviation of hydrosoluble compounds Food adsorption of alteration compounds from frying fats (oxidized and polymerized triglyceridesa) could induce toxicity Contributes to the formation of acrylamide in food rich in carbohydrates and asparagine For more details on advantages and disadvantages see the text. a Triglycerides also are called triacylglycerides or triacylglycerols. discontinuous fish fryings, the presence of altered fatty acids was higher in lard than in sunflower oil and higher in sunflower oil than in olive oil [3,7]. The process requires high temperatures, implying thermal aggression with its disadvantages and limitations (Table 1). However, it has to be pointed out that frying is normally performed at 170–180 C and for a very short time (<10 min); thus it can be expected that frying does not produce higher alterations than, for example, grilling and baking, during which the cooking time is longer and the temperature higher [14,16] (Figure 10). Most of the inconveniences of frying are related to bad practices such as reuse of the oil a large number of times, null or low oil turnover, and overheating (Table 1). There is also concern about the consumption of fried foods because of their potential high fat content. Results found by Varela and Ruiz Roso [20] support the idea that frying does not increase the fat contents more than other cooking methods (Figure 11). However, quality of the frying oil and the breading or cutting Vitamin C Loss (%) Vitamin B6 Loss (%) 80 60 60 Loss (%) Loss (%) 40 40 20 20 0 0 Potatoes Red meat Fish Boiled Deep frying Peppers Stewed Chips/Potatoes Shallow frying Deep frying FIGURE 10 Percentages of losses of vitamin B6 (left) and vitamin C (right) in different foods after being cooked using different procedures. Notice the tendency of frying to induce minor vitamin losses than other cooking methods. Modified from [14]. With permission from Grasas and Aceites. Frying and the Mediterranean Diet Chapter 21 60 Fat (g/100 g dry matter) FIGURE 11 Fat content of some selected foods prepared according different cooking systems. Modified from [20]. With permission, Grasas and Aceites. 225 50 40 30 20 10 0 Chicken Hake Fried Minced meat Stewed influence oil uptake [3,18,21]. During repeated frying in the same oil, the polarity of the oil increases. The increase of such polar substances decreases the interfacial tension, facilitating oil uptake [21]. Thus the average amount of fat in some fried foods is quite ample (e.g., 10–27% in beef, pork, and meatballs; 15–36% in potatoes and potato products; 60–80% in breaded mushrooms; and 20–42% in breaded fish and shrimp) [18]. In pan-frying, with no large amounts of oil, oil smoking must be avoided, mostly during preheating. Pan-frying permits a reduced number of fryings—normally fewer than three—because the oil volume is not large and the oil can easily be overheated. However, studies by our group suggest that the oil “disappears” before the alteration becomes relevant [4]. In Mediterranean homes, where pan-frying is a normal culinary practice, oil resting in the pan is gathered with utensils and reused once or twice more [4]. Frying in a fryer or in deep pans permits the reuse of oil more often because the fryer contains a high volume of oil (e.g., domestic fryers can contain 1–3 L of oil), permitting the dilution of alteration products. It is difficult to set criteria for the adequate maximum number of fryings because many factors are at work: type of food (fatty, lean, medium-fat); type of oil (saturated, monounsaturated, polyunsaturated); type and characteristics of the fryer or the deep pan (aluminum, steel, coated or not, heater resistance location, with or without the bottom water chamber); frying temperature (low [<170 C], medium [170–190 C]; high [>200 C]); frying time; continuous or discontinuous frying; among others (Figure 7). Based on previous studies by our group, the use of oil for 10 to 20 fryings at 180 C when cooking with relatively low turnover (adding oil to replenish the decreased oil volume every ten fryings) is recommended when frying different foods in the same oil. The number of uses will be much higher when fresh oil is added after each frying [3,4,7]. ADVANTAGES OF FRYING WITH OLIVE OILS Frying with olive oil presents important advantages with regard to other oils. This is discussed in detail in this section and is summarized in Table 2. Oil Composition The fatty acid (a high proportion of MUFAs and a moderate percentage of SFAs and PUFAs); minor compounds (vitamins, phytosterols, squalene, phenolic compounds composition); and a well-balanced PUFA-to-tocopherol ratio make virgin olive oil (VOO) unique. Although the fatty acid profile largely contributes to the prevention of certain degenerative diseases [22–25], well-designed studies suggest that diets differing in the type of olive oil (rich, medium, and poor polyphenol contents) induce differential effects on cardiovascular disease (CVD) markers that are clearly dependent on the amount of minor compounds [26]. The whole oil composition is influenced by hydrolytic and oxidative reactions and by cultivar and pedoclimatic environment, irrigation, fly attacks, ripening, milling, malaxation, filtration, and storage. Information on the health effects of minor compounds of VOO is available in other chapters in this book, some of which suggest that the biological activities of these compounds extend beyond their antioxidant capacity; for example, squalene has been found to exert in vitro antitumoral effects, b-sitosterols act as hypocholesterolemics, hydroxytyrosol inhibits platelet aggregation, accumulation of thromboxane in human serum, and the production of proinflammatory leukotrienes [27]. However, it is interesting that, in the frame of a Mediterranean diet, VOO contributes a large percentage of tocopherols, a medium percentage of carotenes, but only a low percentage of the total polyphenol content (<1.5%), all from the secoiridoid family, 226 SECTION 2 Components of the Mediterranean Diet TABLE 2 Advantages of Frying Food with Olive Oil (Virgin and Extra Virgin Olive Oils, in Particular) Compared with Other Edible Oils a. Oil composition High percentage of monounsaturated fatty acids (oleic acid) with well-established health properties Modest percentage of n-6 polyunsaturated fatty acids (linoleic acid), whose percentage in the diet should not be too high High amount of phenolic compounds and phytosterols that display antioxidant attributes and other properties beneficial to health Acceptable levels of tocopherols, which are antioxidants b. Low formation of polar material and thermal oxidized compounds during frying due to the fatty acid profile and minor compound content of olive oil Low hydroperoxide production The hydroperoxides formed break down at frying temperatures, originating volatiles and contributing to improved food palatability Low production of secondary alteration compounds (e.g., polymers, dimers, and cyclic monomers) c. Olive oil’s high stability implies a long frying life, and the oil can be used during a large number of occasions for frying d. Formation of a well-defined crust, with good texture and palatability, that prevents excess penetration of fat e. Obtaining of more cardio-healthy foods Foods fried in olive oils have a more balanced fatty acid composition (more appropriate ratio of saturated, monounsaturated, n-6 polyunsaturated, and n-3 polyunsaturated fatty acids) Food is enriched with antioxidants and bioactive compounds from the frying oil Food has a small amount of alteration compound, and therefore a low potential toxicity These advantages should be considered only when fryings are performed in unused virgin or extra virgin olive oils or after a smaller number of successive fryings with frequent oil turnover. For more details see the text. which is absent in other Mediterranean foods, suggesting that secoiridoids are very active independent of other dietary compounds present in the diet. Frying Temperature Frying is the process of cooking food by immersing it in hot oil. Unfortunately, when pan-frying the oil temperature is not controlled and, most of the time, the cook considers the temperature to be adequate when oil is smoking. Friedman [28] reported that “Frying at lower temperatures shows that foods will be as crisp and crusty, or even better, if they are fried at only 325 and 335 F.” Our group has shown that frying at 160 C is the correct temperature because the oil undergoes less alteration than at 180 C and food presents adequate organoleptic properties [29]. Taking those circumstances into account, it has to be pointed out that VOO has a lower smoking point (170 C) than some seed oils (e.g., sunflower oil [210 C] and soybean oil [232 C]) (Table 3). Nonetheless, the oil variety, the oil refining process, and the presence of different stabilizing compounds do not permit us to clearly ascertain the smoking point. To accelerate the frying process, and to get more crunchy and browned foods, some bars and restaurants set the oil temperature at 200 C or higher, and although the seed oil still does not smoke, that temperature is high enough to produce unwanted thermal oxidations. Thus, polymerizations and cyclizations are much more abundant in overheated oils than at lower temperatures. Domestic and semi-industrial fryers equipped with a thermostat permit the unhealthy overheating of oil to be avoided. Crust Formation Crust formation is a property of the food that permits the concentration of oil and products on a fine surface [3,17,18]. Monounsaturated oils in fried foods form a more defined crust than polyunsaturated oils [3,7,17,20]. Such a crust prevents food from absorbing fat and dehydration and improves its palatability. Sánchez-Muniz et al. [3,7] found less fat absorption in sardines fried with olive oil than those fried with sunflower oil or lard. The lower degree of alteration in olive oil during frying contributes to the formation of a more defined crust and permits frying at relatively low temperatures, decreasing oil penetration. Resistance to Thermal Oxidation of Olive Oil During Frying or at Frying Temperatures VOO displays great stability when used for frying and other culinary purposes [3,6,7]. Dobarganes and Márquez-Ruiz [6] reported that frying olive oil displays fewer alterations than frying seed oils. Unpublished data from our group suggest that this Rancimat induction period was high but clearly distinct in three Picual olive oils differing in their polyphenol and tocopherol concentrations. Our group found that heating olive oil at 180 C, despite whether in presence of extracts that Frying and the Mediterranean Diet Chapter 21 227 TABLE 3 Smoke Points of Different Oils Used for Frying IMIDRA (1) Cocina y Arte (2) USDA (3) Oil Type Refined Nonrefined Linseed - 107 C - - Hemp - 165 C - - Canola 204 C 107 C - 435 F Olive 210 C - 160 C 410 F Virgin olive - 160 C - - Pork lard - 182 C - Palm olein - - 180 C - Grape seed 204 C - - 445 F Sesame seed 210 C 176 C - - - - - Cotton seed Mani a 215 C 320 F - - a Corn 232 C 160 C - 410 F Sunflower 232 C 107 Ca - 410 F Soybean 232 C 160 Ca - a 450 F Peanut 232 C 160 C 160 C Coconut - 232 C - - - - Pomace - 238 C Notice that the consulted sources do not inform of added products (antifoam, antioxidants, etc.). Also, conditions for refining or final oil characteristics are not indicated. a In some circumstances the oil is mixed with oils with a high coking point to permit cooking at low temperatures. Modified from (1) Instituto Madrileño de Investigación y desarrollo Rural, Agrario y Alimentario (IMIDRA) (Ed). TDC Olive Project. In: Enciclopedia del Olivo. Grasas comestibles en los hábitos de comida europeos, vol. 7. Madrid: IMIDRA; Consejerı́a de Economı́a e Innovación Tecnológica; 2006. p. 27–8. (2) Cocina y Aceite. http://apuntesdecocina.com/2009/08/08/aceites-y-puntos-de-humo/. (3) US Department of Agriculture. Food Safety and Inspection Service (USDA). Food Safety Information. Deep Fat Frying and Food Safety. http://www.fsis.usda.gov. were very rich in nonextractable tannins, produced lower thermal oxidation after 48 h than sunflower oil or a homogeneous mix of olive oil plus sunflower oil [30]. Compared with other vegetable oils such as sunflower, cotton, corn, and soybean oils, olive oil presents a lower degree of alteration, as demonstrated by measuring viscosity, polar material, and tocopherol losses (for a review see SánchezMuniz et al. [3,7]). A comparative study of the frying behavior of extra VOO, sunflower oil containing a large amount of oleic acid, and “conventional” sunflower oil used repeatedly to fry potatoes is presented in Figure 12. A plausible explanation for the resistance to rapid deterioration of extra VOO at high temperatures may be its fatty acid composition. The production of hydroperoxides and the generation of potentially toxic thermal oxidation compounds in the frying oil is lower with oleic acid than with linoleic acid. That, in turn, implies less uptake of such compounds by the food during frying and thus lower toxicity and healthier foods. As already mentioned, VOO contains a large amount of minor compounds (a-tocopherol, squalene, and phenolic compounds such as diphenols, phenolic acid, and hydroxytyrosol) with powerful antioxidant capacity. VOO is rich in phytosterols, such as D5-avenasterol, a potent antipolymerizing agent [7,27]. Tocopherols undergo gradual degradation during frying or storage. Therefore, supplementation with phenolic compounds has been recommended to preserve the original level of tocopherols in the oil [3,7,25,27,30]. Our group has demonstrated that frequent additions of fresh oil or antioxidants lengthen the frying life of oil, helping to maintain the original quality of the oil because it dilutes the altered compounds and adds minor compounds with antioxidant and antipolymerizing properties. Nonetheless, current legislation in Spain and other countries permits only the addition of antioxidants (heading with VOO) to refined olive oil, not to VOO or extra-VOO. Olive oil and VOO are more stable than other oils, implying that they can be used more often for frying than other extensively used oils before reaching the 22–25% polar material or the 10–12% polymers cutoff points [31] 228 SECTION 2 Components of the Mediterranean Diet (Figure 13). Figure 12 shows less polymer formation in olive oil than in other oils during frying and therefore suggests the potentially lower toxicity of products fried in olive oil. Data in Figure 12 and Table 4 suggest that the shelf-life of frying oil depends on how frequently the oil is replenished. The alteration of polymer and cyclic monomer contents of different oils used to fry fresh potatoes and frozen prefried foods is summarized in Table 5. Frying fresh potatoes produces less alteration of the oil. Moreover, oils replenished frequently displayed less alteration than oils to which fresh oil was not added. In conclusion, the use of extra VOO for frying and the practice of frequent oil replenishment reduce oil and food alteration and therefore produce quality foods. Obtaining more Cardio-Healthy Food Because of the entrance of oil into food during frying, the higher the thermal oxidation within the oil, the more thermal oxidized compounds are present in the fried food. Thus, frying with abused oils (>25% polar material; >12% polymers) or Oil discarding limit Polar material (g/100 g oil) 25 20 FIGURE 12 Change in the amount of polar material (g/100 g oil) in extra virgin olive oil, highly oleic acid sunflower oil, and sunflower oil used in 75 fryings of fresh potatoes performed with the frequent addition of fresh oil (FR) and in 75 fryings of fresh potatoes in sunflower oil performed with no oil turnover (NR). Source: Modified from [7]. With the publisher’s permission. 15 10 5 0 0 10 20 30 40 50 Fryings (n) -·- FR Sunflower oil 60 70 80 ··· FR High oleic sunflower oil – – FR Extra virgin olive oil NR Sunflower oil 30 Discarding limit Oligomers (mg/100 mg oil) Polar material (mg/100 mg oil) 35 25 20 15 10 5 0 (a) 0 5 10 20 Fryings (n) 30 20 15 Discarding limit 10 5 0 40 (b) 0 5 10 20 30 40 Fryings (n) Sunflower oil Olive oil + sunflower oil Olive oil FIGURE 13 Changes in polar material (mg/100 mg oil) (a) and oligomers (b) of olive oil, sunflower oil, and a blend of both oils during the frying of different fresh and frozen prefried foods performed with slow oil turnover. The line at 25 mg polar material/100 mg oil or at 12 mg oligomers/100 mg oil reflects the cutoff point selected for discarding oil in many countries (German Society for Fat Research) [31]. Modified from Bastida S, Sánchez-Muniz FJ. Polar content vs. TAG oligomer content in the frying-life assessment of monounsaturated and polyunsaturated oils used in deep frying. J Am Oil Chem Soc 2002;79:447–51. With the publisher’s permission. Frying and the Mediterranean Diet Chapter 21 229 TABLE 4 Cyclic Monomer and Oligomer Contents of Oils Before and After Frying in Different Conditions Various Types of Foods Oil Type Food Type Number of Fryings Extra virgin olive oil Frozen prefried 20 Sunflower oil Highly oleic sunflower oil Oil Added? No a Cyclic Monomers (mg/kg Oil) Oligomers (g/100 g Oil) Final Initial 0 684 0.08 7.59 Initial Final Frozen prefried 20 Yes 0 574 0.08 5.41 Potatoes 75 No 0 195 0.03 2.55 Frozen prefried 20 No 71 855 1.40 11.40 a Frozen prefried 20 Yes 71 697 1.40 8.58 Frozen prefried 20 No Frozen prefried Potatoes 20 64 706 0.27 7.15 a 64 608 0.27 5.91 a 64 334 0.21 3.36 Yes 75 Yes a Oil was added before each frying to keep constant the food-to-oil ratio. Modified from Sánchez-Muniz FJ. Oils and fats: changes due to culinary and industrial processes. Int J Vitam Nutr Res 2006;76:230–37. With permission, Verlag Hans Huber. TABLE 5 Oil Addition and Alterations in the Frying Oil and in the Fat Extracted from Fried Foods Polar Material (g/100 g Oil) Trans-Fatty Acids (g/100 g Oil or Fat) 8th Frying 20th Frying 8th Frying 20th Frying 8.08 17.3 0.22 0.42 Yes 7.13 13.5 0 0.24 No 9.23 19.3 0.22 0.49 8.14 14.1 0.19. 0.37 Oil Added? Oil/Fat Extra virgin olive oil No a Fat from potatoes fried in extra virgin olive oil a Yes a Oil was added before each frying to keep constant the food-to-oil ratio. Modified from Sánchez-Muniz FJ. Oils and fats: changes due to culinary and industrial processes. Int J Vitam Nutr Res 2006;76:230–37. With permission, Verlag Hans Huber. overheated oil give rise to unhealthy and potentially toxic foods [3,6,7,32,33]. The alteration of food (measured by the amounts of polar material and trans-fatty acids) is higher than that of the oil, although the thermal oxidation compound profiles are similar (Table 3). The consumption of food fried in VOO supports some bioactive compounds and fatty acids of VOO in the diet that would help to correct certain imbalances between ratios of SFAs, MUFAs, omega 6 PUFAs, and omega 3 PUFAs in today’s diets [3,4,17]. FRIED FOOD CONSUMPTION AND HEALTH There is general concern, mostly in non-Mediterranean countries, about the convenience of consuming fried foods. Most of it comes from the results and conclusions of studies performed decades ago (for a review see Refs. [3,6,7,33,34]). Overheating but not true frying was performed, producing a very large amount of potentially toxic compounds (e.g., cyclic monomers) in the oil. In addition, large amounts of isolated altered compounds or whole altered oils were obtained and tested in animals, sometimes at a level equivalent to 1.4 kg in a man weighing 70 kg [32]. Our group found no significant changes in weight gain, conceptus weight, number of fetuses, or maternal lipoprotein levels in pregnant rats that received as the only fat source in their diets olive oil used in potato frying; the alteration of the frying used oil was 9%, as polar content, while that of the fresh oil was 3% [35]. At present, several aspects of the digestion, absorption, metabolism, and elimination of thermal oxidized products remain unknown. Our group and others found results suggesting that thermal oxidation compounds are absorbed, 230 SECTION 2 Components of the Mediterranean Diet Dietary altered lipid Tg-polymers Ox-Tg-monomers Secondary products Lipase Phospholipase Cholesterol oxidase Intestine Chylomicrons Ox-Tg-monomers Tg-polymers? Secondary products Ox-Tg-monomers Tg-polymers? Oxysterols Lyso-phospholipids SOD GPx GR Cit P450 Others Ox-phospholipids Lysophospholipids Oxysterols DAG MAG Free fatty acids Bile Intestinal bacteria Feces Ox-Tg-monomers Tg-dimers? Oxysterols Lyso-phos-pholipids Cells/tissues Arylesterase Polymers? Ox-Tg-monomers Secondary products Enzymes Secondary products Bile acids Esteroids Others LDL/HDL FIGURE 14 Possible metabolic fate of dietary oxidized and polymerized fats/oils. The scheme illustrates dietary compounds and possible body pathways for eliminating the altered products observed. Secondary products include short- or medium-chain alkyl groups, carbonyl, hydroxyl, aldehyde, ester, epoxy, carboxyl, etc. Adducts include hydroxynonenal (HNE) adducts and HNE conjugates. ADH, aldehyde dehydrogenase; EH, epoxy hydrase; GPx, glutathione peroxidase; GR, glutathione reductase; Ox, oxidized; Tg, triacylglycerols. Source: Modified from [34]. With the publisher’s permission. SOD GPx GR Cit P450 isoenz Others (ADH, EH, etc.) Urine HNE Adducts Others H2O Lungs CO2 H2 O contributing to a decrease in antioxidant defense [33–36]. Figure 14 illustrates dietary compounds and possible body pathways for eliminating altered compounds, clearly suggesting that the “frying picture” is rather complex. Oxidized LDL (oxLDL) and its component hydroxy fatty acids have been shown to activate peroxisome proliferatoractivating receptor alpha (PPARa) and gamma (PPARg). Chao et al. [36] tested the hypothesis that lipid oxidation products in oxidized frying oil obtained by frying wheat dough sheets in soybean oil at 2055 C for 24 h can activate PPARa and upregulate its target genes on Sprague–Dawley male weanling rats. Oxidized frying oil dose dependently and significantly increased mRNA of acyl-CoA oxidase and cytochrome P450 4A1 (CYP4A1) in liver of rats. Dietary oxidized frying oil also dose dependently increased liver microsomal CYP4A protein. The activity of hepatic acyl-CoA oxidase of the group consuming high percentage of oxidized oil was sixfold that of the group consuming high percentage of fresh oil. The results of Chao et al. [36] support the hypothesis that dietary oxidized frying oil, by activating PPARa, up-regulates the expression of PPARa downstream genes. On the other hand, although research on the effect of fatty acids in cell signaling and gene regulation is very active, few studies have investigated the effect of oxidized fat ingestion and gene regulation of enzymes related to the antioxidant defense. As an example, Chao et al. [36] reported that oxidized frying oils dose dependently increased in liver the microsomal cytochrome P450 4A1 (CYP4A1) protein and the messenger RNA of acyl-CoA oxidase, and CYP4A1 of Sprague– Dawley rats, supporting the hypothesis that oxidation products from frying oils can up-regulate genes activated by the peroxisome proliferator-activated receptor alpha. The effect of ingesting highly thermally oxidized sunflower oil on antioxidant levels and enzyme activity and expression in the small intestine of fed and fasted rats has been studied by our group [34]. Ingestion of altered oil under fasting conditions increases the amount of intestinal thiobarbituric acid reactive substance; decreases the activity of superoxide dismutase (SOD), selenium-dependent glutathione peroxidase (GPx), and non-selenium-dependent GPx; and increases the activity of catalase. Expression of SOD, GPx, and tumor necrosis factor-a increased significantly in fasting test rats. Most of these effects were partially diminished in fed rats, suggesting that both long fasting and consumption of food containing oxidized fat should be avoided to prevent intestinal oxidative stress. The information about the effects of fried products on different aspects of health in humans is rather low. Table 6 summarizes different studies on this topic. More than one decade ago, we studied the dietary characteristics and CVD and hematological markers in a group of nuns who followed for years a semi-vegetarian-Mediterranean diet in which meat and meat products were absent. Frying was used in about 20% of their recipes. This sedentary group showed good profile markers, with high levels of antioxidants and high-density lipoprotein (HDL) cholesterol and low blood pressure [46]. Pérez Herrera et al. [47] investigated the effect of consuming frying oil to find an oil model for deep-frying that prevents oxidative stress. Following a crossover design, 20 obese received four breakfasts containing different oils submitted to 20 fryings: VOO, sunflower oil, sunflower oil plus canola oil supplemented with dimethylpolysiloxane, or sunflower oil supplemented with natural antioxidants from olive oil. The authors concluded that oils with naturally present or added phenolic compounds reduced postprandial oxidative stress compared with sunflower oil. Frying and the Mediterranean Diet Chapter 21 231 TABLE 6 Summary of Some Trials Assessing the Effect of Fried Food Consumption Population, Patients; Main Outcome Number of Cohort (Age), Detail Duration Results and Conclusions; Comments References EPIC cohort; CHD events, death from all causes 40,151 (29–69 years), free CVD 11 years’ follow-up Fried food consumption associated with general and central obesity GuallarCastillon et al. [37] SUN study; BMI, weight gain 9850 (38.1 11.4 years) 6.1-year follow-up The association between fried food consumption and higher weight gain was not significant (differences of small magnitude) Sayon-Orea et al. [38] 6621 with BMI <25 kg/m2 Incidence of overweight/ obesity OR for developing overweight/obesity, 1.00 (fried foods <2 times/week); 1.37 for fried foods >4 times/week (P for trend ¼ 0.02) Total fried food consumed at home and away home These ORs were nonsignificant when separating for frying in olive oil (OR, 1.12) or sunflower oil (OR, 1.26) EPIC cohort; CHD events and death from all causes 40,757 (29–69 years), free CVD 1992–1996, follow-up till 2004 (11 years) 606 CHD events, 1135 deaths from all causes INTERHEART; risk of myocardial infarction 52 countries Fried food consumption was associated with a higher risk of acute myocardial infarction 5761 cases and 10,646 control subjects. Nonquantitative assessment of fried food consumption; only nine fried foods were studied and no type of oil was indicated No differences between oils; no association was observed between fried food consumption and all-cause mortality GuallarCastillon et al. [39] Iqbal et al. [40] Costa Rica; myocardial infarction 482 case and 482 controls 4 years’ follow-up Palm and hydrogenate oil; no association between consumption of fried food and risk of nonfatal acute myocardial infarction Kabagambe et al. [41] Pizarra study 1226 adults 6 years’ follow-up Fried food in reused oils were associated with higher prevalence of arterial hypertension Soriguer et al. [42] Cardiovascular Health study; allcause mortality 3910 (older people, >65 years old) No association between fried fish or fish sandwich consumption and mortality from CHD or cardiac arrhythmia or with incidence of nonfatal acute myocardial infarction Mozaffarian et al. [43] Multi Ethnic Study of Atherosclerosis (MESA); carotid and lipid markers 5677 (45–84 years), African Americans, Caucasians, Chinese, Hispanics 2 years No association between fried fish consumption and subclinical atherosclerosis (carotid, intima media thickness); no association of fried fish consumption and change of cholesterol, HDLc, LDL-c, and triglycerides He et al. [44] 2 years Consumption of fried fish was associated with the levels of sVCAM-1; not observed in no-fried fish consumption He et al. [45] MESA study; subclinical markers of atherosclerosis BMI, body mass index; CHD, coronary heart disease; CVD, cardiovascular disease; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; OR, odds ratio; sVCAM-1, soluble vascular cell adhesion molecule-1. Effects on Body Weight Some time ago it was demonstrated that olive oil did not induce negative effects on body weight in the frame of Mediterranean diets; however, with respect to fried food consumption, controversial results are available. Some studies suggest an association between fried food consumption and body weight gain [37], whereas others do not. In the Sun study [38] (Table 6), the consumption of fried food was associated with weight gain after a 6-year follow-up, although differences were small and nonsignificant. People in the highest consumption group also ate more total energy, fast foods, snacks 232 SECTION 2 Components of the Mediterranean Diet between meals, and sugared drinks. However, the incidence of new overweight/obese cases during the follow-up displays differences between both groups of individuals. Those negative effects disappear when the individuals were separately tested for food fried in olive oil or in sunflower oil. Gesteiro et al. [48] observed that frying was used in 30% of the recipes included in the diets of mothers in the Mérida Study. These authors, in agreement with others, found a positive association between adequate neonatal birthweight and malondialdehyde. Effects on Mortality and Degenerative Diseases Data from some studies of effects of frying on mortality and degenerative diseases are summarized in Table 6. Consumption of fried food is associated with a higher risk of acute myocardial infarction in the INTERHEART study [40]; however, in the EPIC study, definite coronary disease and all-cause mortality were not significantly different between individuals in the higher quartile for fried food consumption and those in the lowest quartile [39]. Moreover, in a cross-sectional analysis of the Multi Ethnic Study of Atherosclerosis (MESA), no association was found between the consumption of fried fish and subclinical atherosclerosis. Nor was fried fish consumption associated with significant changes in total cholesterol, HDL cholesterol, or triglycerides [44]. The Cardiovascular Health Study, including a cohort of about 4000 people aged >65 years, did not find any association between consuming fried fish or fish sandwiches and both mortality from cardiac arrhythmia or CHD and acute myocardial infarction [41]. No association between the consumption of fried food and risk of nonfatal acute myocardial infarction was found in a case-control study performed in Costa Rica [43]. The MESA study found that fried fish consumption was negatively associated with the level of soluble intercellular adhesion molecule-1, a marker of endothelial damage [43]. These results were not expected because it was not evidenced for the consumption of nonfried fish. In another study, food rich in oxidized oils previously used for frying did not affect endothelial function but increased postprandial triglyceridemia [45]. As we have stated, the consumption of frying oils is different than that of fried foods because foods are not only fat; in addition, fast foods are not typical components of Mediterranean diets, and they are usually fried in seed oils that become more easily and rapidly altered than olive oil or VOO. In the Pizarra study a higher prevalence of hypertension among people with higher consumption of foods fried in reused oils was recorded [42]. Donfransesco et al. [49] found that the consumption of oxidized food was associated with lower HDL cholesterol levels and larger waist circumference. CONCLUSIONS Several aspects of frying will be studied in future works. (1) Pan-frying has been studied much less than deep-frying. (2) Imprecise information about the type of frying performed (with or without turnover, continuous or discontinuous) is normally given. (3) No information on the consumption of altered fats is available and only numbers of fried food rations are normally taken into consideration. (4) The degree of alteration of fried food consumed is normally missed; thus a general conclusion is misleading because oils perform differently at the same temperature, depending on the oil used and frying conditions. (5) The application of objective techniques quantifying total alteration in the fat of fried foods (e.g., polar artefacts, polymers) is demanded to clearly compare results from different studies. (6) New studies comparing different oils at the same experimental conditions—and the fried foods obtained from those oils—will be performed. (7) Studies of frying must be centered on typical Mediterranean foods and not on other nontypical Mediterranean foods (e.g., fried red meat). (8) The convenience of consuming fried food in the frame of individualized diets of candidates carrying different gene polymorphisms for degenerative diseases should be tested. SUMMARY POINTS l l l l Fried foods, mainly pan-fried, are common foods in the Mediterranean diet. Deleting the consumption of fried foods would cause Mediterranean people to perform wrong dietary practices, decreasing the consumption of fried fish, sofritos, and traditional fritos and increasing that of other foods richer in SFAs. The use of VOO or extra VOO for frying, together with the practice of frequently replenishing oil, reduce oil and food alteration and, therefore, produce quality foods. Recent results suggest that no deleterious effects were observed when the number of fried food rations consumed is not large. Frying and the Mediterranean Diet Chapter 21 l l l 233 Some interesting effects were observed after consuming fried fish in relation to some CVD markers. 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[48] Gesteiro E, Rodrı́guez-Bernal B, Bastida S, Sánchez-Muniz FJ. Maternal diets with low healthy eating index or Mediterranean diet adherence scores are associated with high cord-blood insulin levels and insulin resistance markers at birth. Eur J Clin Nutr 2012;66:1008–15. [49] Donfrancesco C, Lo Noce C, Brignoli O, Riccardi G, Ciccarelli P, Dima F, et al. Italian network for obesity and cardiovascular disease surveillance: a pilot project. BMC Fam Pract 2008;9:53. Chapter 22 The Mediterranean Diet and Obesity from a Nutrigenetic and Epigenetics Perspective Marta Garaulet, PhD University of Murcia, Murcia, Spain. ABBREVIATIONS MUFA SFA PUFA PPARg FTO APOA2 CLOCK NUGENOB SNP DNA RNA ADRB3 GWAS BMI SREBP HNF4 LPL MetS GOLDEN SIRT1 HDL PER CpG00 PTMs HAT BMAL1 or ARNTL monounsaturated fatty acid saturated fatty acid polyunsaturated fatty acid peroxisome proliferator-activated receptor gamma fat mass and obesity associated apolipoprotein A2 circadian locomotor output cycles kaput nutrient-gene interactions in human obesity single-nucleotide polymorphisms deoxyribonucleic acid ribonucleic acid adrenoceptor beta 3 genome-wide association studies body mass index sterol regulatory element binding protein hepatocyte nuclear factor 4 lipoprotein lipase metabolic syndrome genetics of lipids lowering drugs and diet network sirtuin 1 high-density lipoprotein period citosine–phosphate–guanine post translational modifications histone acetile transferase aryl hydrocarbon receptor nuclear translocator-like INTRODUCTION The Mediterranean Diet in the Treatment of Obesity Evidence points toward a role of the Mediterranean diet in preventing obesity. It also seems to be a safe strategy for treating metabolic syndrome and reducing associated cardiovascular risk. Moreover, results suggest that promoting eating habits consistent with Mediterranean dietary patterns may be a useful part of efforts to combat obesity. Indeed, the Mediterranean diet has been proposed to induce weight loss. It covers most nutritional recommendations indicated for achieving adequate weight loss, such as small amounts of refined carbohydrates, high fiber content, moderate fat content (mostly unsaturated), and moderate to high content of vegetable proteins. The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 237 238 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet Several types of Mediterranean diets have been demonstrated to be effective in the treatment of obesity [1,2]. Flynn and Colquhoun [3] suggested that the Mediterranean diet is as effective as any other diet in patients seeking to follow a weight-loss program. Furthermore, Fernandez de la Puebla and coworkers [4] found that isocaloric substitution of a diet rich in saturated fatty acids with one with the properties of the Mediterranean diet reduced body fat proportion. In this regard, our group performed a study of 1500 patients and found an average weight loss of 10% of initial weight and a significant improvement in blood-related parameters, such as glucose, cholesterol, and uric acid [2]. More important, dropping out was reduced in obesity treatments based on the Mediterranean diet compared to other hypocaloric diets. Results of a weight loss program based in the Mediterranean Diet principles administered by our group showed attrition rates of 6–9%; these numbers were much lower than those found in other clinical studies of different diets, which ranged from 36% to 60% of attrition [2] (Figure 1). The low rates of attrition associated with a Mediterranean-style dietary approach could be due in part to the fact that subjects find this diet tastier than low-fat regimens tried before [5]. However, several considerations should be taken into account when recommending the Mediterranean diet for losing weight. First, we must remember that the application of the Mediterranean diet for weight loss purposes must be accompanied by reduced energy intake (reductions of 500–1000 kcal/day); fat must not exceed 30% energy; and oleic acid must constitute at least 55% total fat at the expense of saturated fats—this can be attained by using olive oil as the culinary fat. On the other hand, it is important to bear in mind that the ingestion of fat has been shown to be a main factor in obesity. In this regard, nutritional recommendations from Greece based on the Mediterranean pyramid were criticized because fat intake has been increasing in this country since the 1950s because of the ingestion of olive oil, leading to a substantial overall weight gain [6]. Therefore we must be cautious with recommendations about the intake of olive oil in the context of a weight-loss treatment. Although it should be recommended that olive oil should be the only fat added during cooking, in salads fat intake should represent no more than the 30% or 35% of the total energy intake; in a hypocaloric diet (1200–1800 kcal), this implies that to lose weight in a Mediterranean diet context the patient should not use more than three or four tablespoons of olive oil per day. Another important consideration is that we must be careful because the low intake of meats that characterize the Mediterranean diet could be accompanied by an insufficient amount of heme iron in a hypocaloric diet. In this sense, hypocaloric diets in which vegetal products (lentils, chickpeas, beans) make a considerable contribution to total protein must be varied, and care must be taken to provide sufficient consumption of heme iron. In doing so, iron-related hematological parameters will be maintained within normal values. Apart from these considerations, the Mediterranean diet presents many advantages in the treatment of obesity. It is highly satiating because of high fiber intake. It is composed of high-volume foods with low caloric density. Given its high carbohydrate content, it does not trigger specific hunger and therefore binge eating. For the same reason, it is not ketogenic; even though it can be hypocaloric, it maintains adequate nutrient proportions. More important, our research group, among others, have demonstrated that the Mediterranean diet as a whole as well as some of its components independently are able to interact with different obesity-related genes, protecting us from the establishment of obesity or helping to achieve better success in weight-loss programs. The explanation of this gene-diet interaction is the main objective of this chapter. Attrition (%) FIGURE 1 Percentage of attrition in different weight-loss treatments. Mediterranean Diet Nutrigenetics and Obesity Chapter 22 239 DEALING WITH OBESITY: A COMPLEX DISEASE In 2009 Shadan [7] defined obesity as a complex disorder based on the contributions of environmental as well as many genetic factors. Corella and Portolés [8] hypothesized that many causes interact at the same time, contributing to the development of obesity, for example, genetic inheritance, diet, meal times, social stress, lifestyle habits, and physical activity. In the words of Jose Marı́a Ordovás [9]: “We could say that our genome and our environment are having a constant dialogue.” Among the many gene-environment interactions, diet plays a prominent role. Gene-nutrient interactions are attracting the attention of numerous studies because of their complexity and potential therapeutic value. However, interindividual variability complicates the treatment of obesity. The relationship of “nature versus nurture” has been widely reported among twin studies regarding obesity and its traits. Genetic and environmental influences on the final phenotype of an individual are markedly related to lifestyle habits (diet, physical activity, social support, education, etc.). Furthermore, recent studies [10] indicate that the chronobiology of the individual seems to contribute significantly to the metabolic alterations related to obesity. In these studies, lifestyle habits such as shift work, sleep deprivation, and exposure to bright light at night have been associated with increased adiposity. NUTRIGENETICS: THE KNOWLEDGE TRANSITION The success of the Human Genome Project in 2003 and the development of molecular biology techniques have opened new doors for the understanding of medicine and nutrition. Nowadays, however, a huge amount of scientific literature seems to clarify, even for specialists, the number of new terms involved in the so-called Omics. Nutrigenetics studies the effect of genetic variation on the interaction between diet and disease; the study of this effect on dietary response is the main objective of nutrigenetics. Nowadays, the Nutrient–Gene Interactions in Human Obesity: Implications for Dietary Guidelines (NUGENOB) project represent the most complete range of studies regarding gene-nutrient interactions. Critical mutations in the genome, such as single nucleotide polymorphisms (SNPs), the most common cause of genetic diversity between individuals (i.e., affecting response to a drug or diet), provide useful information about nutrient metabolism and its pathways. On the other hand, in 2009 Corella and Ordovás [11] described nutrigenomics as the science that focuses its attention on the role of nutrients and bioactive food compounds in gene expression. Metabolic changes (metabolomics) and protein expression (proteomics) could also provide interesting information about future therapies. However, mutations are not always required to modify gene expression. Depending on environmental conditions, epigenetic studies show how and when certain genes can be turned on or off. In this sense, a new science has emerged: epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. Hence the name: epi- (the Greek epί, meaning “over, above, outer”) -genetics. It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such changes are DNA methylation and histone modification, both of which serve to regulate gene expression without altering the underlying DNA sequence. In 2011 it was demonstrated that the methylation of messenger RNA has a critical role in human energy homeostasis. This opened the related field of RNA epigenetics. Genetics of Obesity and Weight Loss Obesity is described in general as a disease with a complex multigenic base, implicating various numbers of genes as well as interactions between them and the environment (e.g., diet) [8]. Variations in candidate genes implicated in different physiological pathways related ti obesity traits first were studied in experimental animal models. These studies reported that the suppression of some candidate genes is able to confer resistance to obesity relative to a control group of mice (“wild type,” or nongenetically modified, mice) with the same energy intake. Data regarding weight loss are contradictory. The results of the NUGENOB Consortium in 2006 reported that some studies found no associations between weight loss and candidate genes [12]. Others studies showed that changes in fat mass were predicted by polymorphisms at several obesity-related candidate genes. Furthermore, different genes such as leptin, G protein, ADRB3, PPARs, and perilipin have been proposed as genes related to changes in fat mass. Our research proposes a polymorphism in CLOCK, the 3111T/C, as a genetic variant implicated in weight loss in the context of a Mediterranean diet [13]. Nowadays, the list of genes implicated in obesity has dramatically increased, and genome-wide association studies (GWASs) have been demonstrated that multiple gene variants are related to obesity. However, there is still and important 240 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet work to do to demonstrate the physiological mechanism that underlies these associations. Moreover, replications are difficult because of the complexity of the interactions, but they are particularly necessary to establish the bases of this relative new science of nutrigenetics and its relevance in obesity. Hainer et al. [14] proposed in 2008 that the potential obesity candidate genes could cause either a predisposition to obesity (the so-called obesogenic genes) or to leanness (leptogenic genes). Authors suggested that these genes participate in several kinds of interactions: (1) genes interact with each other (gene–gene interactions); (2) genes interact with various environmental factors (gene–environment interactions); and (3) interactions between biological factors (genes, hormones, and neurotransmitters, etc.), psychobehavioral factors, and environmental factors. These interactions influence body fat accumulation and fat distribution as well as obesity-related health risks. Among the gene–environment interactions, diet has been the most studied. In this regard, different works demonstrate that, apart from the classical advantages of the Mediterranean diet, in those subjects who follow Mediterranean dietary recommendations there are relevant gene–diet interactions that may participate in the protection that the Mediterranean diet exerts against obesity. GENE–DIET INTERACTIONS FOR OBESITY IN THE CONTEXT OF A MEDITERRANEAN DIET Peroxisome Proliferator-Activated Receptor Genes and the Mediterranean Diet Regarding nutrient–gene interactions, a wide range of experimental models have shown that polyunsaturated fatty acids (PUFAs), as well as monounsaturated fatty acids (MUFAs)—but in a minor proportion—are able to regulate gene expression. Genes codifying the enzymes responsible for lipogenesis and fatty acid oxidation are the most widely studied. In an elegant review, Sánchez-Muñiz and Nus [15] affirmed that PUFAs could act directly or through their metabolites on a family of transcription factors called peroxisome proliferator-activated receptors (PPARs). PUFAs are able to act on other transcription factors as well (sterol regulatory element binding protein, hepatocyte nuclear factor 4, thyroid hormone receptor, and the estrogenic receptors between others, modulating their activity and levels. The PPARs are transcription factors involved in the regulation of glucidic and lipid metabolism, as well as in adipocyte differentiation and inflammatory response. They belong to the superfamily of hormone nuclear receptors, and their natural ligands are fatty acids. A point mutation found on the B exon of the NH2-terminal of PPARl2, substituting alanine for proline at position 12 (PPARl Pro12Ala SNP) (rs1801282), has been shown to decrease receptor activity. This mutation has been associated with higher insulin sensitivity and a more favorable lipid profile. This observation is consistent with previous work showing that heterozygous-deficient mice have increased insulin sensitivity [16]. However, in spite of this evidence, multiple studies have shown no association with or no increase in the risk for type 2 diabetes mellitus or metabolic syndrome in people with the Ala12 variant. PPARl2 is also related to adipogenesis [17]. Based on this evidence, its association with obesity parameters and, more precisely, its contribution to weight loss has been a research focus during recent years. Again, however, conflicting results have been reported. For example, in some studies Ala carriers seem to be more responsive to lifestyle interventions that promote beneficial health effects, whereas others have reported that the PPARl rs1801282 Pro12Ala SNP is associated with resistance to weight loss or with no effect on response to a weight-loss intervention. These discrepancies may be related to differences in intervention protocols, age of the participants, sample sizes, gene–gene interactions, or gene–diet interactions. In this regard, our group decided to look at potential interactions between fat intake and the PPARl polymorphism in a Spanish overweight/obese population enrolled in a behavioral treatment program for obesity based on a Mediterranean Diet. Consistent with numerous prior reports, we did not observe direct associations between the PPARl2 Pro12Ala genotype and body mass index (BMI). However, we did observe an obesity-related gene–diet interaction between the PPARl2 genotype and MUFA intake. In this context obesity was attenuated in carriers of the reduced-activity Ala12 allele, but this association was significant only in subjects with high MUFA intake (56% of total fat intake) (Figure 2). Three other groups have examined the interaction between PPARl2 Pro12Ala and fatty acid intake for obesity-related traits in humans. Memisoglu et al. [18] were the first to find an interaction identical to ours in which the PPARl2 Pro12Ala polymorphism interacted with MUFA intake for the outcome of BMI. Furthermore, an earlier study [19] detected an interaction between the Pro12Ala genotype and a dietary PUFAs-to-saturated fatty acids (SFA) ratio (PUFA:SFA), such that carriers of the Ala12 allele were more obese than Pro12 homozygotes when the dietary PUFA:SFA was low, whereas the opposite was true (i.e., Ala12 allele carriers were less obese than Pro12 homozygotes) when the PUFA:SFA was high. Mediterranean Diet Nutrigenetics and Obesity Chapter 22 241 MUFA% of fat <56% 33,5 ≥56% 33 BMI (kg/m) 32,5 P = 0.031 P = 0.351 32 31,5 31 30,5 30 29,5 29 28,5 CC CG + GG <11.8% 104 Waist circumfernce (cm) CC CG + GG PPARl2 Pro12 Ala (rs1801282) (a) SFA% of energy >11.8% 102 100 (P = 0.84) 98 (P = 0.005) 96 94 92 AA AG + GG (P = 0.017) FIGURE 2 Examples of gene–nutrient interactions for obesity. (a) Monounsaturated fatty acids as a percentage of fat. (b) Saturated fatty acids as a percentage of energy. BMI, body mass index. (b) AG + GG AA Clock 3111T/C These results were similar to ours, although in our study of obesity the Pro12Ala genotype interacted not with PUFA: SFA but with the percentage of MUFAs. Notably, studies were performed in populations that habitually consumed vegetable oils that were rich in PUFAs but not in oleic acid or MUFAs. However, results from our group of Mediterranean individuals who habitually consume large amounts of olive oil are consistent with those from a previous report performed in another southern Spanish population [20]. In both our study and the previous cross-sectional Spanish study, MUFAs consumed in the context of a Mediterranean diet were beneficial for PPARl2 Ala12 allele carriers, although in the earlier study the outcome was insulin resistance rather than obesity. It has been suggested that direct PPAR activation by oleic acid may promote oxidation or sequestration of palmitate, thereby preventing deleterious effects related to SFAs, such as insulin resistance or cardiovascular risk [21]. Moreover, PPARl2 activation mediates the expression of several target genes implicated in adipose tissue accumulation, such as lipoprotein lipase, and also plays a role in adipose triglyceride lipase saturation [17]. Our detection of a gene–diet interaction may partly explain the discrepancies among different studies and populations because diets vary widely in their fatty acid composition across different countries and even within the same country. FTO and the Mediterranean Diet A large number of genes have been implicated in obesity-related phenotypes, but among the most consistently reported obesity candidates is the fat mass and obesity-associated gene (FTO) located on chromosome 16q12.2 of the human genome. The relationship between FTO and obesity-related traits was originally detected through a GWAS and has been subsequently replicated in other GWASs. The mostly widely studied of the FTO variants is a SNP located in the first intron 242 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet of this gene (rs9939609 T > A). To date, the association between the rs9939609 T > A SNP and BMI has been extensively, but not uniformly, supported in both adults and children. For this reason, our group aimed to investigate associations and interactions with physical activity and dietary patterns that underlie FTO associations with obesity in a Mediterranean population of 1465 subjects. Our preliminary data indicate that the FTO rs9939609 T/A SNP is associated with physical activity and BMI. Although FTO rs9939609 was not associated with total energy intake, a significant gene–nutrient interaction between the FTO SNP and amount of dietary fat was detected. Specifically, increased adiposity was associated with the A allele only in individuals with high intakes of PUFAs and SFAs. Of note, we did not detect an interaction between the FTO genotype and MUFA intake for obesity-related traits. These results could be related to the dietary characteristics of the population, which traditionally have included high intake of MUFAs, particularly from olive oil, as a primary fat source. Garaulet et al. [22] described MUFAs as a type of fatty acid that protects against body fat accumulation, with an inverse correlation between MUFAs and the number of fat cells. Moreover, in a 3-year intervention using a Mediterranean diet supplemented with virgin olive oil, subjects had less body weight gain than did control subjects without this supplementation [23]. Therefore, our preliminary studies suggest that a high proportion of MUFAs, with olive oil as the main fat in the diet, in Mediterranean countries could contribute to protection against obesity traits, even among carriers of the FTO risk allele. CLOCK Gene, Obesity, and the Mediterranean Diet Daily timekeeping in many organisms depends on internal circadian clocks that temporally organize biological functions relative to each other as well as to the environment. These clocks generate physiological and behavioral rhythms using circuits of gene expression that are organized in negative feedback loops. The great interindividual differences in chronotype, responses to sleep curtailment, and association with obesity point to an underlying genetic component, and some limited data suggest that genetic variation at loci involved in circadian regulation may underlie these large phenotypic differences. In this regard, genetic variation at the circadian locomotor output cycles kaput gene(CLOCK), a key driver of the circadian rhythm, has been related to psychological alterations and eveningness. Since 2008, following the study of an Argentinean population, Sookoian et al. [24] and those of a European population carried out by Scott et al. [25], it has been known that different CLOCK variants are associated with obesity—in particular abdominal obesity—and metabolic syndrome. Subsequently, our group replicated these data in a North American sample of 540 men and 560 women who participated in the Genetics of Lipids Lowering Drugs and Diet Network (GOLDN) [26]. In this American population, four of five CLOCK SNPs (Figure 3) were associated with BMI, energy intake, and different variables related to obesity [27]. In fact, our results showed that individuals carrying the gene variants or polymorphisms ate more, slept less, ate more fat, and were more obese. Some of these associations may be functionally explained, such as the CLOCK polymorphism rs3749474, potentially leading to a change in messenger RNA structure that makes CLOCK less viable, so that its expression loses efficacy. CLOCK Interacts with Dietary Fat Quality Perhaps one of the most interesting results obtained was that these associations between gene polymorphism and abdominal obesity or impaired glucose metabolism were shown only in individuals with an unbalanced diet that included a high proportion of saturated fat; factory-made pastries, sausages, and other processed foods; and a low proportion of MUFAs FIGURE 3 Different CLOCK polymorphisms related to obesity. % Name Localization Minor allele frequency Major allele Minor allele rs3749474 3¢-UTR 40 C T rs1801260 3¢-UTR 37 G A rs4580704 Intron 9 33 G C rs4864548 Promoter 41 G A Mediterranean Diet Nutrigenetics and Obesity Chapter 22 243 (olive oil). In other words, results indicated that CLOCK polymorphisms interacted with fatty acids to modulate metabolic syndrome traits. We identified significant gene–diet interactions associated with metabolic syndrome at the CLOCK locus. By dichotomizing MUFA intake, we found different effects on glucose and insulin resistance across rs4580704 genotypes. The protective effect of the minor allele on insulin sensitivity was present only when MUFA intake was high. Different effects were found across CLOCK 3111TNC genotypes for SFA intake. The deleterious effect of gene variants on waist circumference was found only with high SFA intake [26] (Figure 2). These results suggest that dietary source and membrane content of MUFAs are implicated in the relationship between alterations in the circadian system and metabolic syndrome and, as a consequence, we can say that the Mediterranean diet is able to interact with genes, protecting subjects from obesity. Other CLOCK Genes We found similar gene–diet interactions related to MUFAs and obesity with other CLOCK genes such as sirtuins (SIRT1). Considering the tight connections between SIRT1 and CLOCK, and the role of SIRT1 in transducing signals originated by metabolic components of the circadian clock, we developed a SIRT1 and CLOCK combined genotype to assess its associations with the chronotype of subjects (morning or evening types) and their potential resistance to weight loss in a behavioral treatment for obesity based on a Mediterranean diet [28]. Results indicated that the SIRT1 and CLOCK combined genotype has an additive effect on resistance to weight loss that could be related to less adherence to Mediterranean dietary patterns. Indeed, dietary habits indicated that carriers of the resistance genotype had a lower intake of total carbohydrates and MUFAs and a higher intake of SFAs than those carrying the intermediate and the protective genotype [28]. We are currently working on another CLOCK gene called REV-ERB that functions as an integral driver of the circadian clock. Despite the solid implication of REV-ERB in obesity, the information about whether genetic variations at this locus may be associated with those traits is scarce. Our preliminary results show novel associations between the REV-ERBALPHA1 rs2314339 genotype and obesity in two independent populations. Of note, a significant interaction between the REV-ERB-ALPHA1 variant and MUFA intake for obesity was detected in a Mediterranean population. Other Components in Addition to MUFAs Although most of the gene–diet interactions implicated in obesity are related to MUFA intake, other components of the Mediterranean diet influence the connection between the circadian genes and obesity. A recent study of experimental animals published by our group [29] demonstrated that resveratrol, a component of red wine, reverses the change in the expression of REV-ERBa in adipose tissue induced by high-fat feeding. This change seems to be related to a reduced lipogenesis, which might be involved in the body fat-lowering effect of this molecule. SNPs and Obesogenic Behaviors in the Context of a Mediterranean Diet Other polymorphisms have been related to the dietary behaviors more than to specific components of the diet, such as MUFAs or resveratrol. This is the case of the interaction between APOA2 and PERIOD2 SNPs with obesogenic behaviors in the context of a Mediterranean diet. APOA2 and Skipping Meals One example of genetic variations that have recently produced interesting results regarding the genetic predisposition to obesity via altered energy intake is the apolipoprotein A-2 gene (APOA2). Apolipoproteins combine with lipids to form several classes of lipoprotein particles with different densities, ranging from chylomicrons and very-low-density lipoproteins to very-high-density lipoproteins. More concretely, APOA2 is considered the major component of high-density lipoprotein particles and a regulator of triglyceride and postpandrial metabolism. In addition, nutrigenetic interactions have focused the interest of numerous studies: the interaction between the APOA2 m265 genotype and SFAs for obesity traits has been more extensively demonstrated than any other locus. In 2011 Corella et al. [30] replicated these results in two different populations (Mediterranean and Asian). The results showed that SFA intake modulates body weight-related traits under the influence of the m265T/C polymorphism. In a recessive model, a Mediterranean population that was homozygous for the minor allele (CC) was classified into two groups depending on their SFA intake (low and high, with 22 g/d as cutoff point). The CC carriers consuming high rates of SFAs reported 6.8% higher BMI than the CC carriers with low SFA intake [30]. The trend was maintained in the Chinese and the Asian Indian populations. Furthermore, we recently replicated these results in a Mediterranean population from southeast 244 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet ApoA2 Odds ratios 3 2 Major (TT) Minor (TC+CC) 1 P<0.05 Do you skip Do you plan meals? meals? FIGURE 4 Associations between APOA2 and obesogenic behaviors in a Mediterranean diet context. Data shown are positive responses to each question. 0 Spain [31]. In spite of all this knowledge, this particular polymorphism is not widely studied in GWASs because it is not included in habitual microchips. Moreover, the mechanisms implicated in its association with obesity are still unknown. When we studied more deeply which behaviors could connect this SNP with obesity or weight loss, we discovered that homozygous minor (CC) subjects were more likely to exhibit behaviors that impede weight loss (response of “yes” to the question “Do you skip meals?”) and less likely to exhibit protective behaviors (response of “yes” to the question, “Do you plan meals in advance?”) (Figure 4). Therefore we concluded that the APOA2 m265 genotype may be associated with eating behaviors that are far different than the Mediterranean diet characteristics. PERIOD2 (PER2) and Attrition Another SNP related to obesogenic behaviors is the circadian clock-related gene PERIOD2 (PER2), which was associated to attrition in patients prone to withdrawal from a behavioral weight-reduction program based on the Mediterranean diet [32]. A total of 454 overweight/obese participants, aged 20 to 65 years, who attended outpatient clinics specializing in obesity were studied. Results indicate that the PER2 polymorphisms rs2304672C > G and rs4663302C > T were associated with abdominal obesity. Moreover, the frequency of the rs4663307 minor allele was significantly greater in those who withdrew from the study than in those who successfully completed treatment. Logistic regression analysis showed that carriers of the rs2304672 C > G minor allele had a greater probability of dropping out, displaying extreme snacking, experiencing stress with dieting, eating when bored, and skipping breakfast than noncarriers (Figure 5). We concluded that PER2 was implicated in attrition in weight-loss treatment and may modulate eating behavior-related phenotypes. EPIGENETICS IN THE INTERNAL CLOCK AND THE MEDITERRANEAN DIET As we introduced at the beginning of this chapter, one important revolution in genetics science is epigenetics. We now know that we do not have a predetermined a genome. What we eat, how much we sleep, whether we exercise, and even how we use our mind may change our genome. In other words, epigenetics does not change DNA but decides how much or whether some genes are expressed in different cells in our bodies. Per2 (rs2304672) 3 FIGURE 5 Associations between PER2 and obesogenic behaviors in a Mediterranean diet context. Odds ratios 2.5 2 Major (CC) 1.5 Menor (GC+GG) 1 0.5 0 on iti A ttr ng ki c na g S n he l hi s re St sw Ea ti ng d re in et i ed w bo st fa k ea o N br Mediterranean Diet Nutrigenetics and Obesity Chapter 22 245 The molecular basis of epigenetics is complex. It involves modifications of the activation of certain genes but not the basic structure of DNA. One way that gene expression is regulated is by the remodeling of chromatin (the complex of DNA and histones). Chromatin proteins associated with DNA may be activated or silenced. Histones can change how tightly or loosely the DNA wraps around them by modifying their amino acids. If the amino acids that are in the chain are changed, the shape of the histone sphere might be modified. A second way chromatin is remodeled is the addition of methyl groups to the DNA, mostly at CpG sites, which are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide. CpG is shorthand for –C–phosphate– G–, that is, cytosine and guanine separated by only one phosphate. Methylation converts cytosine to 5-methylcytosine. Some areas of the genome are methylated more heavily than others, and highly methylated areas tend to be less transcriptionally active, although the mechanism not fully understood. Epigenetic control can be exerted through a variety of mechanisms, including not only DNA methylation but also microRNA-mediated metabolic pathways, histone variants, and posttranslational modifications of histone. Different studies point out the association between epigenetic changes and several illnesses, for example, cancer, in which methylation of CpG sites within the promoters of genes can lead to the silencing of tumor suppressor genes. In contrast, the hypomethylation of CpG sites has been associated with the overexpression of oncogenes within cancer cells. Epigenetics and Circadian Rhythms The connection between epigenetics and the clock machinery was made in a study by Crosio et al. [33], who demonstrated that chromatin remodeling was involved in circadian gene expression. It has been hypothesized that because a large number of transcripts oscillate in a circadian manner, there must be a widespread program of dynamic changes in chromatin remodeling that accompany circadian gene expression. This has been described as the “circadian epigenome” and probably includes cycles of chromatin transitions that allow a highly dynamic chromatin structure to be temporally permissive to transcription. Histones can be modified at more than 30 sites within the N-terminal tails. There are several modifications in the histones, such as acetylation and phosphorylation, among others. The finding that CLOCK has an intrinsic histone acetile transferase activity reveals one link between epigenetic control and the circadian clock. CLOCK may acetylate histones, particularly the lysine residues in the histones 3 and 4. Interestingly, CLOCK can also acetylate nonhistone substrates. This is the case with BMAL1, which is acetylated by CLOCK in a lysine residue, an event that is crucial for the circadian transcriptional program. Another substrate susceptible to acetylation by CLOCK is the glucocorticoid receptor, whose function is regulated by this process. This acetylation activity by CLOCK has been demonstrated to be essential for circadian expression. Another important actor in the circadian epigenome is sirtuin, particularly SIRT1 and SIRT6. Sirtuins possess deacetylase activity and are implicated in the induction of gene silencing. In particular, SIRT1 physically interacts with CLOCK, and it has been defined as a circadian enzymatic rheostat because it controls via different mechanisms the balance of acetylation and chromatin remodeling by the circadian clock. Although all this knowledge is useful, there are no studies about the connection between this circadian epigenome and obesity. Our group identified novel associations between methylation patterns at three circadian clock genes and metabolic syndrome factors as well as changes in body weight resulting from dietary intervention [34]. In addition, our data support a modulation of these associations by dietary intake of MUFAs and PUFAs. In fact, in the study performed by our group, the close direct associations found between methylation of several CpGs of the CLOCK gene and waist circumference, serum glucose and triglyceride concentrations, blood pressure, homeostasis model assessment index, and the metabolic syndrome score, as well as the inverse association with circulating adiponectin—together with the similar results obtained with BMAL1 and PER2 genes—strongly suggest that some of these factors could affect the methylation pattern of CLOCK genes or even that this methylation could be a causative factor of obesity and metabolic syndrome. This report was the first demonstrating that epigenetic mechanisms related to cytosine methylation in CLOCK gene promoters might be involved in the origin of obesity and metabolic syndrome. Finally, an outstanding result of our study was the association between the type of fatty acids (particularly MUFAs and PUFAs) in the diet and the methylation patterns in adults. Indeed, high intake of MUFAs was related to a decreased degree of methylation. SUMMARY AND CONCLUSIONS The Mediterranean diet is protective against obesity, and following a hypocaloric diet based on Mediterranean dietary principles may be a good therapy for losing weight. Furthermore, new nutrigenetics studies are demonstrating that Mediterranean diet components or behaviors may interact with different genes and exert a protective role against obesity or 246 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet PUFA and SFA MUFA Mediterranean diet MUFA SFA Obesogenic behaviors FTO PPARs FIGURE 6 Interactions between different genes and Mediterranean diet characteristics for obesity traits. MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid. CLOCK APOA2 Per2 metabolic syndrome traits. This is the case of different SNPs related to obesity; PPARl, FTO, APOA2, and CLOCK variants lose their obesogenic action when the subject highly adheres to Mediterranean diet patterns (Figure 6). In most studies, in those subjects who have a MUFA intake higher than the median of the population, the genetic variant is not associated to obesity traits. Other components, such as resveratrol from red wine, also seem to be protective against the obesogenic effect of REV-ERBa. However, it is important to elucidate whether this gene-diet interaction are a consequence of the MUFA intake itself or whether MUFAs act as a marker of Mediterranean diet adherence. In any case, the Mediterranean diet protects form the deleterious effects of several genetic variants in obesity. Moreover, our results of CLOCK gene variants suggest that the Mediterranean diet is a protective factor in the important connections existing between the circadian clock and obesity. This new knowledge should be considered in the treatment of obesity. SUMMARY POINTS l l l l l l Results suggest that promoting eating habits consistent with Mediterranean dietary patterns maybe a useful part of efforts to combat obesity. Indeed, the Mediterranean diet has been proposed to induce weight loss. New nutrigenetics studies demonstrate that Mediterranean diet components or behaviors may interact with different genes and exert a protective effect against obesity or metabolic syndrome traits. We observed a gene-diet interaction between the PPARl2 genotype and MUFA intake for obesity. In this context obesity was attenuated in carriers of the reduced-activity Ala12 allele, but this association was significant only in subjects with high MUFA intake (56% of total fat intake). CLOCK polymorphisms interacted with fatty acids to modulate metabolic syndrome traits, and we found similar gene– diet interactions for obesity between MUFAs and other CLOCK genes such as sirtuins (SIRT1). Other polymorphisms have been related to dietary behaviors more than to specific components of the diet, such as MUFAs or resveratrol; this is the case of the interaction between APOA2 and PERIOD2 SNPs and obesogenic behaviors in the context of a Mediterranean diet. Our results from CLOCK variants suggest that the Mediterranean diet is a protective factor in the important connections existing between the circadian clock and obesity. REFERENCES [1] Vranešić Bender DZ. Nutritional and behavioral modification therapies of obesity: facts and fiction. Krznaric Dig Dis 2012;30(2):163–7. [2] Corbalán MD, Morales EM, Canteras M, Espallardo A, Hernández T, Garaulet M. Effectiveness of cognitive-behavioral therapy based on the Mediterranean diet for the treatment of obesity. Nutrition 2009;25(7–8):861–9. [3] Flynn G, Colquhoun D. Successful long-term weight loss with a Mediterranean style diet in a primary care medical centre. Asia Pac J Clin Nutr 2004;13:S139. [4] Fernández de la Puebla RA, Fuentes F, Pérez-Martı́nez P, Sánchez E, Paniagua JA, Lopez-Miranda J, et al. A reduction in dietary saturated fat decreases body fat content in overweight, hypercholesterolemic males. Nutr Metab Cardiovasc Dis 2003;13:273–7. [5] McManus K, Antinoro L, Sacks F. A randomized controlled trial of a moderate-fat, low-energy diet compared with a low fat, low-energy diet for weight loss in overweight adults. Int J Obes Relat Metab Disord 2001;25(10):1503–11. [6] Ferro-Luzzi A, James WPT, Kafatos A. The high-fat Greek diet: a recipe for all? Eur J Clin Nutr 2003;57:S2–7. Mediterranean Diet Nutrigenetics and Obesity Chapter 22 247 [7] Shadan S. Obesity: fat chance. Nature 2009;457(7233):1095. [8] Corella D, Portolés O. Avances en el conocimiento de las bases genéticas de la obesidad. In: Edimsa SA, editor. Genética, nutrición y enfermedad, vol. II. Madrid: Instituto Tomás Pascual Sanz y Consejo Superior de Investigaciones Cientı́ficas; 2008. [9] Ordovás JM. XL Semanal 1257; 2011. [10] Garaulet M, Madrid JA. Chronobiology, genetics and metabolic syndrome. Curr Opin Lipidol 2009;20:127–34. [11] Corella D, Ordovás JM. Nutrigenomics in cardiovascular medicine. Circ Cardiovasc Genet 2009;2:637–51. [12] Sørensen TI, Boutin P, Taylor MA, Larsen LH, Verdich C, Petersen L, et al. Genetic polymorphisms and weight loss in obesity: a randomised trial of hypo-energetic high- versus low-fat diets. PLoS Clin Trials 2006;1:e12. [13] Garaulet M, Corbalán MD, Madrid JA, Morales E, Baraza JC, Lee YC, et al. CLOCK gene is implicated in weight reduction in obese patients participating in a dietary programme based on the Mediterranean diet. Int J Obes (Lond) 2010;34(3):516–23. [14] Hainer V, Zamrazilová H, Spálová J, Hainerová I, Kunesová M, Aldhoon B, et al. Role of hereditary factors in weight loss and its maintenance. Physiol Res 2008;57:S1–S15. [15] Sánchez-Muniz F, Nus M. Importancia de la interaccion dieta-genetica en la prevencion cardiovascular. In: EDIMSA SA, editor. Genética, nutrición y enfermedad, vol. VII. Madrid: Instituto Tomás Pascual Sanz y Consejo Superior de Investigaciones Cientı́ficas; 2008. [16] Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, Gonzalez FJ, et al. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev 2002;16(1):22–6. [17] Tontonoz P, Hu E, Spiegelman BM. Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma. Curr Opin Genet Dev 1995;5:571–6. [18] Memisoglu A, Hu FB, Hankinson SE, Manson JE, De Vivo I, Willett WC, et al. Interaction between a peroxisome proliferator-activated receptor gamma gene polymorphism and dietary fat intake in relation to body mass. Hum Mol Genet 2003;12(22):2923–9. [19] Luan J, Browne PO, Harding AH, Halsall DJ, O’Rahilly S, Chatterjee VK, et al. Evidence for gene–nutrient interaction at the PPARgamma locus. Diabetes 2001;50(3):686–9. [20] Soriguer F, Morcillo S, Cardona F, Rojo-Martı́nez G, de la Cruz Almaráz M, Ruiz de Adana MdeL, et al. Pro12Ala polymorphism of the PPARG2 gene is associated with type 2 diabetes mellitus and peripheral insulin sensitivity in a population with a high intake of oleic acid. J Nutr 2006;136(9):2325–30. [21] Nolan CJ, Larter CZ. Lipotoxicity: why do saturated fatty acids cause and monounsaturates protect against it? J Gastroenterol Hepatol 2009;24(5):703–6. [22] Garaulet M, Hernandez-Morante JJ, Lujan J, Tebar FJ, Zamora S. Relationship between fat cell size and number and fatty acid composition in adipose tissue from different fat depots in overweight/obese humans. Int J Obes (Lond) 2006;30:899–905. [23] Razquin C, Martinez JA, Martinez-Gonzalez MA, Bes-Rastrollo M, Fernandez-Crehuet J, Marti A. A 3-year intervention with a Mediterranean diet modified the association between the rs9939609 gene variant in FTO and body weight changes. Int J Obes (Lond) 2010;34:266–72. [24] Sookoian S, Gemma C, Gianotti TF, Burgueño A, Castaño G, Pirola CJ. Genetic variants of Clock transcription factor are associated with individual susceptibility to obesity. Am J Clin Nutr 2008;87(6):1606–15. [25] Scott EM, Carter AM, Grant PJ. Association between polymorphisms in the Clock gene, obesity and the metabolic syndrome in man. Int J Obes (Lond) 2008;32(4):658–62. [26] Garaulet M, Lee YC, Shen J, Parnell LD, Arnett DK, Tsai MY, et al. CLOCK genetic variation and metabolic syndrome risk: modulation by monounsaturated fatty acids. Am J Clin Nutr 2009;90(6):1466–75. [27] Garaulet M, Lee YC, Shen J, Parnell LD, Arnett DK, Tsai MY, et al. Genetic variants in human CLOCK associate with total energy intake and cytokine sleep factors in overweight subjects (GOLDN population). Eur J Hum Genet 2010;18(3):364–9. [28] Garaulet M, Esteban Tardido A, Lee YC, Smith CE, Parnell LD, Ordovás JM. SIRT1 and CLOCK 3111 T > C combined genotype is associated with evening preference and weight loss resistance in a behavioral therapy treatment for obesity. Int J Obes (Lond) 2012;36(11):1436–41. [29] Miranda J, Portillo MP, Madrid JA, Arias N, Macarulla MT, Garaulet M. Effects of resveratrol on changes induced by high-fat feeding on clock genes in rats. Br J Nutr 2013;28:1–8. [30] Corella D, Tai ES, Sorlı́ JV, Chew SK, Coltell O, Sotos-Prieto M, et al. Association between the APOA2 promoter polymorphism and body-weight in Mediterranean and Asian populations. Replication of a gene-saturated fat interaction. Int J Obes (Lond) 2011;35:666–75. [31] Smith CE, Ordovás JM, Sánchez-Moreno C, Lee YC, Garaulet M. Apolipoprotein A-II polymorphism: relationships to behavioural and hormonal mediators of obesity. Int J Obes (Lond) 2012;36(1):130–6. [32] Garaulet M, Corbalán-Tutau MD, Madrid JA, Baraza JC, Parnell LD, Lee YC, et al. PERIOD2 variants are associated with abdominal obesity, psycho-behavioral factors, and attrition in the dietary treatment of obesity. J Am Diet Assoc 2011;111(4):626. [33] Crosio C, Cermakian N, Allis CD, Sassone-Corsi P. Light induces chromatin modification in cells of the mammalian circadian clock. Nat Neurosci 2000;3(12):1241–7. [34] Milagro FI, Gómez-Abellán P, Campión J, Martı́nez JA, Ordovás JM, Garaulet M. CLOCK, PER2 and BMAL1 DNA methylation: association with obesity and metabolic syndrome characteristics and monounsaturated fat intake. Chronobiol Int 2012;29(9):1180–94. Chapter 23 Mediterranean Diet: Antioxidant Nutritional Status Elena Azzini and Giuseppe Maiani National Institute for Food and Nutrition Research, Rome, Italy. ABBREVIATIONS AC CAT FRAP GPx IL-10 LDL MD MDA MDS SOD TAC TRAP antioxidant capacity catalase ferric reducing ability of plasma glutathione peroxidase interleukin-10 low density lipoprotein Mediterranean diet malondialdehyde Mediterranean diet score superoxide dismutase total antioxidant capacity total TNF-a: tumor necrosis factor alpha INTRODUCTION Scientists have focused their attention on the traditional Mediterranean diet (MD), designated an Intangible Cultural Heritage by UNESCO, for its proven health benefits. The overall objective of this chapter is to reexamine the role of the MD in human nutrition, particularly its effect on human antioxidant nutritional status. We emphasize the attention on plant-origin foods rich in vitamins, minerals, dietary fiber, and phytonutrients that contribute to general well-being, satiety, and the maintenance of a balanced diet. A solid scientific background seems to support the MD as an optimal dietary pattern for healthy eating and the prevention of a wide spectrum of disease including cardiovascular disease [1,2]. neurodegenerative [3–5] and inflammatory disease [6], metabolic disorders [7,8], and cancer [9]. ANTIOXIDANT DEFENSES AND OXIDATIVE STRESS Several international health authorities [10] have indicated that some foods and food components are powerfully beneficial for human health because of their role in slowing the progression of the aging process and preventing several pathological conditions related to oxidative stress (inflammation, metabolic disorders, reperfusion damage, atherosclerosis, and carcinogenesis). Dietary habits can play a key role in regulating the redox status of human plasma, improving the defense against oxidative damage. It is well known that oxidative stress represents an imbalance between humans’ protective molecules, the antioxidants, and molecules that are able to damage all sorts of cellular components, the free radicals (Figure 1). Endogenous production of free radicals occurs during normal physiological processes; moreover, environmental factors including diet, air pollution, ultraviolet radiation, and cigarette smoking can also contribute to the accumulation of free radicals in the body. The role of oxidative stress is clearly different depending on the pathological conditions involved, but it can be attributed to a series of changes in several biological molecules including polyunsaturated fatty acids, nucleic acids, proteins, and carbohydrates, resulting in altered metabolic and functional processes (Figure 2). The human body responds to oxidative injury through an interacting network of antioxidant enzymes including superoxide dismutases, The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 249 250 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet Aging; toxin; stress; environmental insults Normal; young Antioxidants Reactive oxygen species Minimal oxidative damage Reactive oxygen species Antioxidants Increased oxidative damageaging, age-related diseases FIGURE 2 Relationship between ROS and diseases. Dominguez LJ, Barbagallo M. G Gerontol 2007;55:231–238. FREE RADICALS LIPIDS Peroxidation FIGURE 1 Diagrammatic representation of the oxidative stress by healthy status and age-related diseases develop. From Reiter RJ. Prog Neurobiol 1998;56:3. PROTEIN ENZYMES Degeneration Deactivation From DNA Damage of cell membranes and other cellular components (mitochondria, lipoproteins) Cancer Heart attack, stroke, atherosclerosis Aging catalase, and various peroxidases (glutathione, glutathione reductase, glutathione peroxidases, and glutathione S-transferases) and nonenzymatic low-molecular-weight antioxidants, including glutathione, ubiquinol, and uric acid, that are produced during normal metabolism in the body [11] (Figure 3). Other antioxidants that are able to counteract the cell damage and homeostatic disruption caused by free radicals are dietary antioxidants and combinations of protective substances in plants called phytochemicals, such as vitamin C, vitamin E, carotenoids, and polyphenols, including phenolic acids and flavonoids (Figure 4). Dietary antioxidants also interact with the endogenous antioxidant network and may have synergistic effects on total antioxidant activity [12,13]. Moreover, a homeostatic mechanism in which endogenous antioxidants compensate for the influx of dietary antioxidants has been indicated [14]. MEDITERRANEAN DIET A diet should contribute adequate amounts of nutrients to meet the metabolic requirements of an individual and to give the consumer safety, quality, and variety. As has been widely documented, fruit and vegetable consumption has been closely associated with a lower risk of degenerative diseases [15]. The MD seems to provide a balanced diet that is suitable for all ages and seems to be a valid model of sustainability from a health point of view, both supporting food quality and promoting sustainable resource management through environmentally sound farming systems linked to territorial characterization and to local cultural heritage [16]. Locally grown, wild, and seasonal edible plants contribute a considerable portion of the daily Mediterranean Diet: Antioxidant Nutritional Status Chapter 23 251 Signaling messenger Gene expression Prooxidant? Antioxidants NOX, LOX, COX MPO, CYP, cyt c 1st line 2nd line Metal chelating GPx, Trx, Prx CAT, SOD 3rd line Repair de novo excision Radical scavenging Oxidative stress Free radicals ROS/RNS/ROCI Disturbance of redox balance Oxidative damage 4th line Adaptation Diseases aging Oxidation of biological molecules Induction of defense enzymes FIGURE 3 In vivo defense. From Niki [11]. FIGURE 4 Main dietary phytochemicals. Phytochemicals Catechins Isoflavones Flavonoids Flavones Polyphenols Phenolic acids Flavonols Stilbenes Lignans Flavanols Flavanones Glucosinolates Carotenoids Anthocyanins Phytoestrogens diet in certain Mediterranean areas, and it has been suggested that the beneficial effects of the MD on human health originate in part from antioxidant-rich food plants [17]. A traditional Mediterranean dietary pattern rich in fruits, vegetables, whole-grain cereals, and virgin olive oil seem to support the important role of foods in improving physical and mental well-being, maintaining a state of health, and reducing the risk of illness [18]. Dried fruits and berries have an important role in the MD, too. Following a MD style [19,20], the higher consumption of raw foods, production of fewer cooking-related oxidants, and a consequent decreased waste of nutritional and endogenous antioxidants associated with the high intake of phytochemicals and dietary fiber help to scavenge even small amounts of oxidants or oxidized compounds. Thus the effect of peroxides and pro-oxidants present in processed food on antioxidant status should be limited even if cooking and food processing can enhance the bioavailability of antioxidants, such as some carotenoids in vegetables [21]. The cumulative activities of phytochemicals 252 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet present in food and their synergistic action, known as dietary total antioxidant capacity (TAC), defines a food’s nutritional characteristics and healthy properties. Furthermore, as shown by several studies [22,23], vitamins can improve DNA repair enzymes, such as enzymes involved in DNA methylation or base excision repair, contributing indirectly to a decrease in oxidative damage. Alleva et al. [24] demonstrated that foods rich in ascorbic acid provide dual protection against oxidative stress, enhancing plasma antioxidant concentrations and stimulating genes involved in cell detoxification. The effect of a Mediterranean dietary pattern on plasma TAC has been evaluated in several studies as the measure of antioxidant capacity (AC), reflecting the synergistic action of all the antioxidants present in plasma and body fluids, thus providing an integrated parameter rather than the simple sum of measurable antioxidants [25], even if the assay does not account for in vivo antioxidant enzyme activities [26,27]. The effect that diet exerts on total plasma AC has been the source of much debate over the past decade. There currently is growing interest in studying overall dietary patterns because food items and nutrients could have synergistic or antagonistic effects when they are consumed in combination. Overall patterns represent the current practices found in the assessed population and therefore provide useful epidemiological information [28]. HUMAN ANTIOXIDANT STATUS Human antioxidant status acts as a system and can be evaluated by direct measurement of several endogenous or dietary components in tissues or body fluids, such as glutathione, superoxide dismutase, sulfur-containing amino acids, ascorbate, tocopherol, carotenoids, and others. The TAC of body fluids, especially plasma, can be used as an index of redox status of the human organism in both healthy individuals and those suffering from different diseases [29], representing a parameter summarizing the overall activity of all types of antioxidants in living systems. The TAC of biological samples can also be evaluated in clinical studies that measure the end products of the free radical damage of endogenous compounds such as lipids or DNA. Changes from the baseline levels of these products could then be ascribed to changes in the AC of the diet. The AC of biological samples can be monitored by a variety of simple, nonspecific, high-throughput screenings assays, which do not necessarily reflect the human physiological mechanisms in vivo [30]. As suggested by Niki [31], the term antioxidant capacity means different things to different people and on different occasions, and it may mean the capacity of scavenging free radicals, the capacity of inhibition of oxidation, or the capacity to prevent a disease. Consequently, it is perhaps best to define the overall plasma AC as a “concept” rather than a simple analytical determination because it results from the simultaneous presence of many poorly defined pro-oxidant and antioxidant compounds (Figure 5). Although much of the research to-date focuses on the potential benefit of single antioxidant nutrients, as reported by Jacob [32], the best protection against oxidative stress comes from a wide assortment of interrelated antioxidants and antioxidant cofactors [33]. The antioxidant value of a particular food component, by simple measure of ability or how well it can quench free radicals in a chemically defined reaction system, is an important factor; nevertheless, we also need to consider the coexistence of other compounds in the food matrix that may take part in inhibitory, additive, or synergistic interactions in vitro. In humans the bioavailability of the compound; its metabolism and distribution through the body; the effectiveness of the compound within the cell, after a single dose, and when administered on a regular basis; and the effect of the whole diet on properly chosen biomarkers could provide an overview of antioxidant effectiveness in reducing oxidative stress [34]. FIGURE 5 The multivariables feature of total antioxidant capacity: “the TAC concept.” From Serafini and Del Rio [14]. Mediterranean Diet: Antioxidant Nutritional Status Chapter 23 253 HUMAN STUDIES Increased oxidative stress at the cellular level could be caused by many factors, including exposure to alcohol, drugs, trauma, cold, infections, poor diet, toxins, radiation, or vigorous physical activity. Protection against all of these processes depends on the adequacy of various antioxidant substances that are derived either directly or indirectly from the diet. Therefore, inadequate intake of antioxidant nutrients may compromise antioxidant potential, thus compounding overall oxidative stress. The best way to ensure the adequate intake of phytonutrients is to eat a diet rich in a wide variety of fresh fruits and vegetables; adherence to the MD could represent a good choice to help in the persistence of health-protective attributes. The development of a tool, and its variants, to allow the semiquantitative assessment of adherence to the traditional MD underlies the shift from ecological to the more reliable analytical epidemiological studies and the exponential increase of publications in the international scientific literature of studies evaluating the relation of adherence to the MD with health and disease. In an observational study conducted in a healthy Italian population, the effects of adherence to an MD model, mainly on antioxidant nutritional status, the cellular immune response, as well as oxidative stress, were evaluated [16]. The results show that subjects with high adherence to a Mediterranean dietary pattern had greater plasma AC than those with low adherence (Table 1), indicating that the quality of the diet could have a different effect using a single food matrix. Even if pigments and other phytochemicals contribute to total antioxidant activity, results have shown that, after consuming a high-quality diet, subjects exhibited an increase in TAC and a decrease in malondialdehyde, as well as a decrease in tumor necrosis factor-a and an increase in interleukin-10. In addition, TAC values correlate with its major small-molecule contributors (urate, ascorbate, and a-tocopherol). In this study, the increased circulating levels of endogenous and exogenous antioxidants and the synergistic effects of bioactive food constituents, improving the immune system, seem to explain the overall quality of the MD, as well as the increased consumption of fruits and vegetables as a focal point TABLE 1 Antioxidant Status, Immune Status and Lipid Peroxidation Indicator (Mean SEM) by MedDietScore (MDS) Diet Quality Low (MDS 3) Medium (MDS 4–5) High (MDS 6) FRAP (mM) 976 42 883 22 953 28 TRAP (mM) 842 34 855 22 886 26 Uric acid (mmol/L) 0.34 0.02 0.35 0.01 0.38 0.02 SH (mM/L) 546 31 524 29 520 32 GPx (UI/g Hb) 41.2 2.5 48.8 2.1 49.2 2.7 SOD (U/L) 37.0 3.7 41.3 3.6 40.0 5.1 Vitamin A (mmol/L) 1.87 0.08 1.89 0.06 2.05 0.08 Vitamin E (mmol/L) 23.22 0.93 23.44 0.46 24.15 0.70 Vitamin C (mmol/L) 49.97 2.27 52.24 1.70 48.27 2.27 Lutein + Zeaxanthin (mmol/L) 0.57 0.05 0.63 0.04 0.64 0.05 Cryptoxanthin (mmol/L) 0.16 0.02 0.15 0.02 0.20 0.02 Lycopen (mmol/L) 0.79 0.02 0.83 0.02 0.83 0.02 a-carotene (mmol/L) 0.07 0.02 0.07 0.01 0.08 0.01 b-carotene (mmol/L) 0.55 0.07 0.57 0.06 0.58 0.07 TNF-a (pg/ml) 42.4 9.0 a 35.7 7.0 14.8 2.9b IL10 (pg/ml) 8.3 3.3a 9.98 1.3a 19.5 5.2b MDA (mM/L) 118 7 104 7 110 7 Statistic: Anova P < 0.05 a versus b. From Azzini et al. [16]. a 254 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet of the MD. Plant-origin foods provide key nutrients, dietary fiber, and protective substances that contribute to general wellbeing, satiety, and the maintenance of a balanced diet; thus they should be consumed frequently in high proportions. This MD core—a basis of plant-origin foods—is responsible for the prevention of many chronic diseases and for weight control [20]. In a case-control study [35] in Cyprus, an island where the diet closely resembles the MD, there was no association with breast cancer risk for either score of MD adherence; however, higher consumptions of vegetables, fish, and olive oil were independently associated with a decreased risk of breast cancer. The association between total dietary AC and plasma antioxidant status in healthy young adults was reported by Wang et al. [36]: dietary TAC was positively correlated with plasma TAC. In a cross-sectional study, Hermsdorff et al. [37] showed that dietary TAC values are inversely associated with glucose and lipid biomarkers as well as with central adiposity in healthy young adults, indicating dietary AC as a useful epidemiological tool to assess the health benefits of the cumulative AC from food intake. In addition, the independent and inverse relationships of oxidized low-density lipoprotein concentrations with dietary and plasma TAC suggest a putative role for an antioxidant-rich diet in the link between redox state and early stage atherogenesis. Results from the ATTICA epidemiological study [38] demonstrated that, in an apparently healthy population, plasma TAC was positively associated with the consumption of fruit and vegetables. Greater adherence to the MD is associated with elevated TAC levels and low oxidized low-density lipoprotein cholesterol concentrations (Table 2), and the traditional MD enhances antioxidant defenses, whereas a diet high in saturated fats induces oxidative stress. Moreover, Figure 6 shows that increased physical activity and greater adherence to the MD were associated with increased TAC [39]. On the other hand, in a crossover TABLE 2 Biochemical Characteristics of the Participants, According to Mediterranean Diet Score Tertile of Diet Score Men (n 1514) Women (n 1528) 1st (0–20) 2nd (21–35) 3rd (36–55) 1st (0–20) 2nd (21–35) 3rd (36–55) Pa Hypercholesterolemia (%) 45 39 36 51 47 25 0.08 Oxidized LDL cholesterol (U/L) 62 21 56 18* 51 17* 63 22 52 25* 51 23* 0.03 TAC (mmol/L) 225 33 242 31* 251 32* 231 26 239 29** 255 44* 0.002 Hypertension (%) 51 27* 20* 50 36* 10* 0.001 TAC, total antioxidant capacity; No significant interactions were observed between tertile of diet score and sex. Significantly different from 1st tertile (Bonferroni correction for multiple comparisons): *P < 0.01, **P < 0.01. a Derived from ANOVA. Reflect the association between tertiles of diet score and the investigated variables, after adjustment for sex. From Pitsavos et al. [38]. FIGURE 6 TAC levels and physical activity status (inactive, minactive, and HEPA active) by MedDietScore that assessed adherence to the Mediterranean diet (away, close, and very close). From Kavouras et al. [39]. Total antioxidant capacity (mmol/L) 288 300 280 260 240 220 200 180 160 140 120 100 257 253 245 245 228 232 240 230 Very c lose Close Away tive Inac Min e iv act A HEP e activ Mediterranean Diet: Antioxidant Nutritional Status Chapter 23 255 intervention study Valtueña et al. [40] investigated the effects of a diet naturally rich in antioxidants compared with a diet low in antioxidants (both containing the same amount of fruit, vegetables, alcoholic beverages, and fiber) on markers of antioxidant status, systemic inflammation, and liver dysfunction. Selecting foods according to their TAC markedly affects antioxidant intake and modulates hepatic contribution to systemic inflammation without affecting traditional markers of antioxidant status. Thus positive results in human metabolic function without improvement of antioxidant status were reported. The foregoing considerations lead us to note that levels of dietary antioxidants in human tissues are strictly related to the amount and food composition of the diet and to their bioavailability, highlighting the importance of interindividual genetic variation. These genetic variants may also interact with chemical form, chirality, absorption, bioavailability, and biochemical interactions between dietary antioxidants and other factors to determine an individual’s overall antioxidant status. Therefore, a healthy food and/or diet should ensure the right amount of nutrients in a bioavailable form and in relation to various intrinsic factors. Indeed, novel results have suggested gene–diet interactions and high adherence to the Mediterranean dietary pattern seem to counteract a genetic predisposition to type 2 diabetes [41]. Furthermore, in a well-controlled study of twins, Dai et al. [42] showed a robust association between adherence to the MD and lower oxidative stress by plasma concentrations of reduced and oxidized glutathione, supporting the hypothesis that the MD exerts cardioprotective effects through lowering oxidative stress. Diverse traditional methods of food preparation within and between countries and the cutoffs used to score food items, based on median dietary values from a particular population, could yield the conflicting findings of MD score application and the erroneous data interpretation. In summary, the beneficial effects from diets rich in fruits and vegetables could be due to the presence of a more complex set of micronutrients, phytochemicals, and fiber (these substances have great bioavailability) and by eating fruits and vegetables to replace less healthful foods in the diet [43]. CONCLUSION A range of factors contribute to achieving a state of wellness, including nutritional factors, a positive lifestyle, physical fitness, environmental factors (e.g., pollution and contaminants), human biology (inherited conditions), and inadequacies in the health care system. The increase in average life expectancy, living in an urban environment, and the westernization of lifestyle are the most important risk factors for the global increases in morbidity and mortality. Diet represents one of the main determinants of health, giving everyone the best chance of being able to fight off the onset of specific pathologies. Numerous epidemiological studies have shown that greater adherence to the MD affects longevity, improves the state of health in general, and significantly reduces the incidence and mortality of chronic degenerative diseases and those associated with aging. In this framework, prevention represents the main goal of public health strategies to counteract the etiology of various diseases, so a well-balanced overall dietary pattern and a healthy lifestyle contribute to a higher quality of life. Although new epidemiological and intervention clinical trials could improve our knowledge of the role of antioxidant status in health and disease, the MD should be recommended as a daily diet for a healthy lifestyle. SUMMARY POINTS l l l l l l Oxidative stress is one of the most significant risk factors for degenerative disease. Dietary habits can play a key role in regulating the redox status of human plasma, improving the defense against oxidative damage. Oxidative stress represents an imbalance between protective molecules, the antioxidants, and molecules that are able to damage all sorts of cellular components, the free radicals. Oxidative stress can cause a series of changes in several biological molecules, including polyunsaturated fatty acids, nucleic acids, proteins, and carbohydrates, resulting in altered metabolic and functional processes. The human body responds to oxidative injury through an interacting network of antioxidant enzymes including superoxide dismutases, catalase, and various peroxidases and nonenzymatic low-molecular-weight antioxidants including thiols, ubiquinol, and uric acid. Other antioxidants that are able to counteract the cell damage and homeostatic disruption caused by free radicals are dietary antioxidants and combinations of protective substances in plants, called phytochemicals, such as vitamin C, vitamin E, carotenoids, and polyphenols, including phenolic acids and flavonoids. The cumulative phytochemical activities present in food and their synergistic actions are known as dietary TAC. Plasma TAC reflects the synergistic action of all the antioxidants present in plasma and body fluids, thus providing an integrated parameter rather than the simple sum of measurable antioxidants, even if the assay does not account for in vivo antioxidant enzyme activities. 256 l l l l SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet In humans the bioavailability of an antioxidant; its metabolism and distribution through the body; the effectiveness of the compound within the cell, after a single dose, or when administered on a regular basis; and the effect of the whole diet on properly chosen biomarkers could provide an overview of antioxidant effectiveness to reduce oxidative stress. An inadequate intake of antioxidant nutrients may compromise antioxidant potential, thus compounding overall oxidative stress. The best way to ensure an adequate intake of phytonutrients is to eat a diet rich in a wide variety of fresh fruits and vegetables. The levels of dietary antioxidants in human tissues are strictly related to the amount and food composition of the diet and to their bioavailability, highlighting the importance of interindividual genetic variation. These genetic variants may also interact with chemical form, chirality, absorption, bioavailability, and biochemical interactions between dietary antioxidants, as well as other factors, to determine an individual’s overall antioxidant status. A well-balanced overall dietary pattern and a healthy lifestyle contribute to a higher quality of life, and although new epidemiological and intervention clinical trials could improve our knowledge of the role of antioxidant status in health and disease, the MD should be recommended as a diet for a healthy life. REFERENCES [1] Buckland G, González CA, Agudo A, Vilardell M, Berenguer A, Amiano P, et al. Adherence to the Mediterranean diet and risk of coronary heart disease in the Spanish EPIC cohort study. 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[7] Martı́nez-González MA, de la Fuente-Arrillaga C, Nunez-Cordoba JM, Basterra-Gortari FJ, Beunza JJ, Vazquez Z, et al. Adherence to Mediterranean diet and risk of developing diabetes: prospective cohort study. BJM 2008;336(7657):1348–51. [8] Esposito K, Maiorino MI, Ciotola M, Di Palo C, Scognamiglio P, Gicchino M, et al. Effects of a Mediterranean-style diet on the need for antihyperglycemic drug therapy in patients with newly diagnosed type 2 diabetes: a randomized trial. Ann Intern Med 2009;151(5):306–14. [9] Buckland G, Agudo A, Luján L, Jakszyn P, Bueno-de-Mesquita HB, Palli D, et al. Adherence to a Mediterranean diet and risk of gastric adenocarcinoma within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study. Am J Clin Nutr 2010;91(2):381–90. [10] WHO/FAO. Diet, nutrition and the prevention of chronic diseases. Tech. Rep. no. 916, Geneva, Switzerland; 2003. [11] NiKi E. Assessment of antioxidant capacity in vitro and in vivo. Free Radic Biol Med 2010;49:503–15. [12] Carlsen H, Myhrstad MCW, Thoresen M, Moskaug JÃ, Blomhoff R. Berry intake increases the activity of the y-glutamylcysteine synthetase promoter in transgenic reporter mice. J Nutr 2003;133:2137–40. [13] Breinholt V, Lauridsen ST, Daneshvar B, Jakobsen J. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett 2000;154:201–10. [14] Serafini M, Del Rio D. Understanding the association between dietary antioxidants, redox status and disease: is the Total Antioxidant Capacity the right tool? Redox Rep 2004;9:145–52. [15] Visioli F. Antioxidant in the Mediterranean diets. World Rev Nutr Diet 2000;87:43–55. [16] Azzini E, Polito A, Fumagalli A, Intorre F, Venneria E, Durazzo A, et al. Mediterranean diet effect: an Italian picture. Nutr J 2011;10:125. [17] Schaffer S, Schmitt-Schillig S, Müller WE, Eckert GP. Antioxidant properties of Mediterranean food plant extracts: geographical differences. J Physiol Pharmacol 2005;56(1):115–24. [18] Sofi F, Cesari F, Abbate R, Gensini GF, Casini A. Adherence to Mediterranean diet and health status: meta-analysis. BMJ 2008;337:1344. [19] Ghiselli A, D’Amicis A, Giacosa A. The antioxidant potential of the Mediterranean diet. Eur J Cancer Prev 1997;6(1):S15–9. [20] Bach-Faig A, Berry EM, Lairon D, Reguant J, Trichopoulou A, Dernini S, et al. Mediterranean diet pyramid today. Science and cultural updates. Public Health Nutr 2011;14(12A):2274–84. [21] Maiani G, Periago Castón MJ, Catasta G, Toti E, Cambrod Goni, Bysted A, et al. Carotenoids: actual knowledge on food sources, intakes, stability and bioavailability and their protective role in humans. Mol Nutr Food Res 2009;53:S194–218. [22] Go VL, Butrum RR, Wong DA. Diet, nutrition, and cancerprevention: the postgenomic era. J Nutr 2003;133:3830S–6S. [23] Tudek B, Swoboda M, Kowalczyk P, Olin’ ski R. Modulation of oxidative DNA damage repair by the diet, inflammation and neoplastic transformation. J Physiol Pharmacol 2006;57(S7):33–49. [24] Alleva R, Di Donato F, Strafella E, Staffolani S, Nocchi L, Borghi B, et al. Effect of ascorbic acid-rich diet on in vivo-induced oxidative stress. Br J Nutr 2012;107(11):1645–54. Mediterranean Diet: Antioxidant Nutritional Status Chapter 23 257 [25] Ghiselli A, Serafini F, Natella Scaccini C. Total antioxidant capacity as a tool to assess redox status: critical view and experimental data. Free Rad Biol Med 2000;29:1106–14. [26] Sies H. Total antioxidant capacity: appraisal of a concept. J Nutr 2007;137:1493–5. [27] Bartosz G. Non-enzymatic antioxidant capacity assays: limitations of use in biomedicine. Free Radic Res 2010;44(7):711–20. [28] Jacques PF, Tucker KL. Are dietary patterns useful for understanding the role of diet in chronic disease? Am J Clin Nutr 2001;73(1):1–2. [29] Bartosz G. Total antioxidant capacity. Adv Clin Chem 2003;37:219–92. [30] Ndhlala AR, Moyo M, Van Staden J. Natural antioxidants: fascinating or mythical biomolecules? Molecules 2010;15:6905–30. [31] Niki E. Antioxidant capacity: which capacity and how to assess it? Journal of Berry Research 2011;1:169–76. [32] Jacob RA. The integrated antioxidant system. Nutr Res 1995;15(5):755–66. [33] Lichtenstein AH, Russell RM. Essential nutrients: food or supplements? Where should the emphasis be? JAMA 2005;294:351–8. [34] Collins AR. Assays for oxidative stress and antioxidant status: applications to research into the biological effectiveness of polyphenols. Am J Clin Nutr 2005;81(1):261S–7S. [35] Demetriou CA, Raaschou-Nielsen O, Loft S, Møller P, Vermeulen R, Palli D, et al. Biomarkers of ambient air pollution and lung cancer: a systematic review. Occup Environ Med 2012;69(9):619–27. [36] Wang Y, Yang M, Lee SG, Davis CG, Koo SI, Chun OK. Dietary total antioxidant capacity is associated with diet and plasma antioxidant status in healthy young adults. J Acad Nutr Diet 2012;112:1626–35. [37] Hermsdorff HH, Puchau B, Volp AC, Barbosa KB, Bressan J, Zulet MÁ, et al. Dietary total antioxidant capacity is inversely related to central adiposity as well as to metabolic and oxidative stress markers in healthy young adults. Nutr Metab (Lond) 2011;8:59. [38] Pitsavos C, Panagiotakos DB, Tzima N, Chrysohoou C, Economou M, Zampelas A, et al. Adherence to the Mediterranean diet is associated with total antioxidant capacity in healthy adults: the ATTICA study. Am J Clin Nutr 2005;82:694–9. [39] Kavouras SA, Panagiotakos DB, Pitsavos C, Chrysohoou C, Arnaoutis G, Skoumas Y, et al. Physical activity and adherence to Mediterranean diet increase total antioxidant capacity: the ATTICA Study. Cardiol Res Pract 2011;248626. [40] Valtueña S, Pellegrini N, Franzini L, Bianchi MA, Ardigò D, Del Rio D, et al. Food selection based on total antioxidant capacity can modify antioxidant intake, systemic inflammation, and liver function without altering markers of oxidative stress. Am J Clin Nutr 2008;87(5):1290–7. [41] Ortega-Azorı́n C, Sorlı́ JV, Asensio EM, Coltell O, Martı́nez-González MÁ, Salas-Salvadó J, et al. Associations of the FTO rs9939609 and the MC4R rs17782313 polymorphisms with type 2 diabetes are modulated by diet, being higher when adherence to the Mediterranean diet pattern is low. Cardiovasc Diabetol 2012;11:137. [42] Dai J, Jones DP, Goldberg J, Ziegler TR, Bostick RM, Wilson PW, et al. Association between adherence to the Mediterranean diet andoxidative stress. Am J Clin Nutr 2008;88(5):1364–70. [43] Mozaffarian DL, Appel J, Van Horn L. Recent advances in preventive cardiology and lifestyle medicine. Circulation 2011;123:2870–91. Chapter 24 The Mediterranean Diet and Body Iron Stores Luca Mascitelli, MD1, Mark R. Goldstein, MD, FACP2 and Leo R. Zacharski, MD3,4 1 Comando Brigata alpina “Julia”, Multinational Land Force, Udine, Italy. 2 NCH Physician Group, Naples, FL, USA. 3 Veterans Affairs Hospital, White River Junction, VT, USA. 4 Geisel School of Medicine at Dartmouth College, Hanover, NH, USA. Let food be thy medicine and medicine be thy food. (Hippocrates, 460-377 BC) ABBREVIATIONS CI CR-LIPE FeAST HR confidence interval carbohydrate-restricted, low-iron available, polyphenol-enriched Iron (Fe) and Atherosclerosis Study hazard ratio INTRODUCTION The concept of the Mediterranean diet originated from the Seven Countries Study initiated in the 1950s and proposed by Keys et al. [1] in the mid-1980s. The study showed that the higher life expectancy observed in Mediterranean countries compared with northern Europe or the United States was in great part attributed to the Mediterranean diet. The term Mediterranean diet reflects the dietary pattern prevalent in the olive-growing areas of the Mediterranean region. It is characterized by high intake of plant foods; fresh and varied fruits as the main and usual dessert; moderately high intake of fish; moderate wine consumption with meals; a small amount of red meat; low to moderate consumption of dairy products; and, most important, olive oil as the main source of fat (commonly used for salads and cooking). Adherence to a traditional Mediterranean diet has been shown to be associated with significantly lower total mortality, mortality from coronary heart disease, and mortality from cancer [2]. To measure adherence to this diet, a score that incorporated the single components of the Mediterranean dietary pattern was constructed. Intriguingly, despite a robust inverse association between the overall Mediterranean diet score and mortality, no appreciable associations for most of the individual dietary components used to construct the score were seen, and it has been suggested that the effects of single nutrients or food may be too small to detect, whereas the cumulative action of multiple dietary components may be substantial [3]. Therefore, methodological approaches to studying dietary patterns in relation to risk of major degenerative diseases should consider overall food patterns instead of evaluating isolated nutrients. In this setting, it has been proposed that lower body iron stores, induced by dietary components of the Mediterranean diet, may be involved in its beneficial effects [4]. OVERVIEW OF IRON METABOLISM Iron is an essential nutrient in humans, and states of both iron deficiency and iron excess result in deviation from optimal health [5]. Iron is a fundamental cofactor for several enzymes involved in oxidation-reduction reactions because of its ability to exist in two ionic forms: ferrous (Fe2+) and ferric (Fe3+) iron. The ability of iron to be converted between these oxidation states through the acceptance or donation of an electron is a key factor in allowing it to perform a range of biological functions. Most of the iron in the body is contained within two proteins: hemoglobin, the oxygen transport protein of red blood cells, and myoglobin, the oxygen storage protein present in muscle cells. Iron metabolism differs from the metabolism of The Mediterranean Diet © 2015 Elsevier Inc. All rights reserved. 259 260 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet other metals in that there is no physiologic mechanism for iron excretion, and close to 90% of daily needs are obtained from an endogenous source, especially recycling it from the breakdown of circulating erythrocytes. There are iron losses, however, which include obligatory losses in all population groups (from skin, intestine, urinary, and airways tracts) and menstrual blood losses in fertile women. To maintain iron balance, the sum of the losses plus the iron required for the body’s needs must be provided by the diet. DIETARY SOURCES OF IRON There are two types of dietary iron: nonheme iron, which is present in both plant foods and animal tissues, and heme iron, which comes from hemoglobin and myoglobin in animal foods. There are three sources of iron in the diet [6]: native food iron (either heme or nonheme iron); fortification iron (nonheme iron intentionally added to food during its manufacture); and contaminant iron (nonheme iron incorporated into foods as a result of either mechanical or chemical reactions occurring during food production or preparation). All fortification iron that is soluble in the gut enters a common nonheme iron pool and is absorbed into the intestinal cells, similar to native food nonheme iron. It has been estimated that up to 25% of the total iron intake in Sweden and the United States comes from fortification iron [7]. However, iron fortification of food in Sweden, considered the highest in the world, was withdrawn at the beginning of 1995 because the effect on target groups was considered to be uncertain. Nonheme iron may also be inadvertently added to food: a substantial increase in the iron content of foods cooked in cast iron or steel cookware has been reported, particularly when acidic foods are cooked for extended periods of time [8]. However, modern high-quality stainless steel tends not to dissolve into iron that can be taken up in the body. Heme iron is estimated to contribute 10–15% of total iron intake among meat-eating populations; however, because of its higher and more uniform absorption (estimated at 15–35%), it could contribute 40% of total absorbed iron [6]. The relative heme iron content in commonly ingested meats is shown in Table 1 [9]. Nonheme iron is usually much less well absorbed than heme iron (estimated at 5–15%). All food iron that enters the common iron pool in the digestive tract is absorbed to the same extent, which depends on the balance between the absorption inhibitors and enhancers and the iron status of the individual [10]. These mechanisms may be involved in determining and maintaining lower body iron stores in people consuming a Mediterranean diet [4]. Inhibitors of Iron Absorption Phytates In plant-based diets, phytate is the main inhibitor of iron absorption. The inhibitory action of phytates on iron absorption has been found to be dose dependent and starts at concentration as low as 2–10 mg/meal [11]. Phytates are found in grains, seeds, nuts, vegetables, and roots. Chemically, phytates are inositol hexaphosphate salts and are a storage form of phosphates and minerals. Food processing and preparation methods, which include milling, heat treatment, soaking, fermentation, and germination, may remove or degrade phytates to a varying extent [12]. Polyphenols Polyphenols occur in various amounts in plant food and beverages, such as vegetables, fruit, olive oil, some cereals and legumes, tea, coffee, and wine. Polyphenolic compounds are released from food or beverages during digestion and can combine with iron in the intestinal lumen, making it unavailable for absorption. Polyphenols are such powerful inhibitors of nonheme iron that substantial changes in the amount of iron absorbed are more likely to occur if the timing of consumption rather than the quantity consumed is altered [6,13]. There is limited information on the effect of bioactive polyphenols on heme iron absorption. However, it has been reported that 46 mg/L of both ()-epigallocatechin-3-gallate and grape seed extract almost completely blocked heme iron absorption by decreasing the basolateral iron export in human intestinal Caco-2 cells [14]. Because many dietary factors modulate iron absorption in a dose-dependent manner, whether these selected bioactive polyphenolic compounds retained the ability to reduce heme iron absorption when added at lower concentrations was investigated. Indeed, it was recently found that both ()-epigallocatechin-3-gallate and grape seed extract inhibit heme iron absorption in a dose-dependent manner in the same human intestinal cells [15]. Therefore, all major types of food polyphenols can strongly inhibit dietary iron absorption, and a dose-dependent inhibitory effect of polyphenol compounds on iron absorption has been demonstrated. In particular, it has been reported that any beverage providing 20–50 mg total polyphenols reduces iron absorption from a bread meal by 50–70%, whereas beverages Mediterranean Diet and Body Iron Stores Chapter 24 261 TABLE 1 Relative Heme Iron (in Milligrams of Iron) in Commonly Ingested Meats (Per 75-g Serving) Food Iron (mg) Liver Pork 13.4 Chicken 9.2 Beef 4.8 Shellfish Oysters 6.42 Mussels 5.0 Clams 2.0 Shrimp 2.2 Beef 2.4 Lamb 1.7 Turkey 1.2 Chicken 0.9 Pork 0.8 Fish Sardines 2.0 Tuna 1.2 Salmon 0.5 Flatfish 0.3 Adapted from http://www.healthlinkbc.ca/healthfiles/hfile68d.stm. containing 100–400 mg total polyphenols reduce iron absorption by 60–90% [16]. Of note, in cereals and legumes, polyphenols add to the inhibitory effect of phytates on nonheme iron absorption. Calcium Calcium, consumed as a salt or in dairy products, interferes significantly with the absorption of both heme and nonheme iron [17], which makes it different from other inhibitors that affect mostly nonheme iron absorption. The mechanism of action for calcium’s inhibition of iron absorption is unknown, but mounting evidence suggests that the inhibition is located within the mucosal cell itself at the common final transfer step for heme and nonheme iron. Dose-dependent inhibitory effects were found at doses of 75–300 mg when calcium was added to bread rolls and at doses of 165 mg calcium from milk products [17]. Proteins Whereas animal tissues enhance iron absorption, animal proteins such as milk proteins, egg proteins, and albumin inhibit iron absorption [18]. The two major bovine milk protein fractions, casein and whey, have been clearly found to inhibit iron absorption in humans [19]. Enhancers of Iron Absorption Ascorbic Acid Ascorbic acid is the most potent enhancer of nonheme iron absorption [20]. Synthetic vitamin C increases the absorption of iron to the same extent as the native ascorbic acid in fruits, vegetables, and juices. The enhancing effect occurs largely because of its ability to reduce ferric to ferrous iron [21]. 262 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet In fruits and vegetables, the enhancing effect of ascorbic acid is often canceled out by the inhibitory action of polyphenols [22]. Furthermore, cooking, industrial processing, and storage may degrade ascorbic acid and remove its enhancing effect on iron absorption [23]. However, several derivates of ascorbic acid are less sensitive to heat and oxygen. Erythorbic acid, an ascorbic acid derivate, is widely used as an antioxidant in processed foods in industrialized countries. Although it has little vitamin C activity, its enhancing action on iron absorption seems to be almost double that of ascorbic acid [24]. Muscle Tissue The ingestion of fish, poultry, and especially red meat has been found to enhance iron absorption from vegetarian meals [25]: 30 g muscle tissue is considered equivalent to 25 mg ascorbic acid. The nature of the “meat factor” that enhances iron absorption is unknown. Most evidence indicates that it may be within the protein fraction of muscle tissue; however, it also is possible that other muscle tissue components are involved, such as the cysteine-containing peptides, which are rich in digests of myofibrillar proteins [26]. Iron Status Beyond genetic disorders, there is an inverse correlation between iron status and iron absorption: that is, more iron is absorbed in an iron-deficient state and less iron is absorbed in an iron-replete state. However, in a study of iron-replete women and women with iron-deficiency anemia, although the overall difference in iron absorption continued to be dictated by iron status, the strong inhibitory effects of polyphenols and the enhancing action of ascorbic acid persisted in both groups of women [27]. On the other hand, it has been reported that about 13% of elderly American individuals from the Framingham Heart Study had high iron stores, defined as serum ferritin concentration >300 mg/L in men and >200 mg/L in women [28]. This prevalence of high iron stores was in marked contrast to the prevalence of iron deficiency (2.7%) and iron-deficiency anemia (1.2%) and was still evident after the exclusion of subjects with possible pathologically increased serum ferritin concentrations, such as those found in anemia of chronic disease due to inflammation, infection, and liver disease [28]. Notably, among the elderly Framingham Heart Study cohort, individual dietary patterns, as determined by food-frequency questionnaires, were significantly associated with the risk of large body iron stores [29]. MEDITERRANEAN DIETARY PATTERN AND REDUCED BODY IRON STORES Body iron stores, which are relatively low during childhood and the premenopausal years, increase with aging because dietary intake exceeds loss [30]. The typical pattern of change in ferritin concentrations with aging for men and women is shown in Figure 1 [30]. Reduction of these iron stores can be induced by the use of a low-iron-available diet, that is, a diet containing a low amount of iron and in which iron absorption inhibitors (polyphenols, phytates, and dairy products) prevail over enhancers (ascorbic acid and red meat). FIGURE 1 Patterns of change in serum ferritin concentrations (micrograms per liter) and percentage transferrin saturation according to age and sex. From Zacharski et al. [30]. 200 Women Men 100 50 50 0 0 –8 + 90 80 9 9 –7 9 9 –6 70 60 –5 9 9 –3 –4 50 40 30 9 9 –2 –1 20 17 Age (years) Mean transterrin saturation(%) Mean ferritin (µg/L) 150 Mediterranean Diet and Body Iron Stores Chapter 24 263 The promise and challenge of investigating these interactions have been well illustrated in a clinical trial evaluating whether a carbohydrate-restricted, low-iron available, polyphenol-enriched (CR-LIPE) diet might improve outcome in diabetic nephropathy to a greater extent than standard protein restriction [31]. Patients with type 2 diabetes (n ¼ 191) were randomized to either CR-LIPE or standard protein restriction. Over a mean follow-up of 3.9 1.8 years, serum ferritin concentration in patients following a CR-LIPE diet decreased from 301 162 to 36 31 mg/L (P < 0.001), whereas it was unchanged in control subjects. As shown in Figure 2, the serum ferritin in individuals following the CR-LIPE diet reached its nadir by 2 years and remained essentially unchanged at the lower level for the balance of the observation period [31] (Figure 2c). The substantial reduction in iron storage level was achieved without chelation or phlebotomy and was associated with a significant protection of renal function and a significant survival benefit in subjects randomized to the CR-LIPE diet [31] (Figure 3). The Iron (Fe) and Atherosclerosis Study (FeAST) [32] gave a perspective on the potential survival benefit of iron reduction therapy. FeAST was a randomized controlled trial of mild iron reduction therapy through controlled phlebotomies in patients with peripheral vascular disease. In the initial analysis from this trial, full iron depletion was not achieved, and no overall mortality benefit was found. However, in the youngest age quartile randomized to iron reduction (age 43–61 years) there was a 54% reduction in total mortality (P ¼ 0.019), the primary end point, and a 57% reduction in death plus nonfatal myocardial infarction and stroke (P < 0.001), the secondary end point, compared with control patients. The age-intervention interaction was statistically significant for both the primary (P ¼ 0.042) and secondary (P < 0.001) end points. Further analysis of the age effect on outcomes demonstrated an interaction between age and both entry and mean follow-up ferritin concentrations that seemed to mask benefits [33]. The substantial mortality benefit in the youngest quartile was seen in association with a phlebotomy-induced reduction in serum ferritin from a mean entry value of 122.5 to 79.7 mg/L after 3.5 years of observation—a modest 35% decrease. FIGURE 2 Effect of a low-iron diet over follow-up (horizontal axis) showing no change in mean arterial pressure (a) or glycosylated hemoglobin (b) but marked changes in serum ferritin concentration (c; P < 0.001) in subjects randomized to a low-iron diet (diamonds) versus control subjects (squares). From Facchini and Saylor [31]. 264 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet FIGURE 3 The graphs show the cumulative percentage of individuals in whom serum creatinine concentrations doubled compared to baseline (a; P < 0.01) and of individuals who either died or reached end-stage renal disease (ESRD) (b; P < 0.01) among those randomized to a low-iron diet (diamonds) versus control (squares). From Facchini and Saylor [31]. FIGURE 4 The association between the log-relative hazard for all-cause mortality in the entire FeAST trial cohort (vertical axis) versus the mean followup ferritin concentration (horizontal axis). P for slope ¼ 0.037. From Zacharski et al. [33]. The significant improvement in all-cause mortality observed in FeAST for the total cohort with lower follow-up ferritin concentrations is shown by linear regression analysis in Figure 4 [33]. The roughly fivefold reduction in serum ferritin in the CRLIPE trial [31] greatly exceeded the iron reduction achieved in the FeAST trial [32] and could have been responsible in itself for a major proportion of the highly significant survival benefit observed among the diabetic subjects in the CR-LIPE trial. Indeed, the balance between the average bioavailability of dietary iron and the overall effects of inhibitors and enhancers of iron absorption may also lead to lower iron stores in people consuming a Mediterranean diet. In fact, it has been reported that elderly men from Crete, in the Mediterranean south of Europe, had consistently lower levels of indicators of oxidative stress and iron status than elderly men from Zutphen, The Netherlands, in the north of Europe [34]. In particular, serum ferritin was twofold lower in men from Crete than in men from Zutphen (69.8 and 134.2 mg/L, respectively). Mediterranean Diet and Body Iron Stores Chapter 24 265 Mediterranean Diet and Iron Homeostasis in the Metabolic Syndrome and Diabetes Recent evidence has shown that the Mediterranean diet may be associated with lower prevalence and progression of the metabolic syndrome [35]. Greater adherence to the Mediterranean diet was associated with favorable effects on the components of the metabolic syndrome. In particular, the Mediterranean diet had a beneficial effect on abdominal obesity, lipid levels, glucose metabolism, and blood pressure levels—all of which are components of the metabolic syndrome—and risk factors for the development of cardiovascular disease, insulin resistance, and diabetes. A meta-analysis of different dietary approaches to the management of type 2 diabetes confirmed the beneficial metabolic effect of a Mediterranean dietary pattern [36]. Examining the merits of low-carbohydrate, low-glycemic index, high-fiber, high-protein, Mediterranean, vegetarian, and vegan diets by the inclusion of randomized controlled trials lasting at least 6 months, it was found that the Mediterranean diet was associated with the greatest improvement in glycemic control [36]. In particular, comparing a Mediterranean diet with a conventional diet (i.e., no change to participants’ current diets) and an American Diabetes Association diet, better glycemic control, greater weight loss, and a more favorable lipid profile were reported in the Mediterranean diet arm. Lower iron stores induced by a Mediterranean dietary pattern could offer a possible explanation for the aforementioned findings. Excess body iron has frequently been found in patients with metabolic syndrome [37,38], with serum ferritin showing a linear increase with the increasing number of metabolic syndrome features [37]. The elevated risk for metabolic abnormalities could not be explained merely by excess adipose tissue or an underlying inflammatory status [38]. Epidemiological evidence has confirmed a positive association between large body iron stores and the risk of type 2 diabetes [39], independent of established diabetes risk factors and a range of diabetes biomarkers. Iron overload may have a crucial role in metabolic diseases, even in countries with a relatively high prevalence of iron deficiency [38]. On the contrary, removing excess iron may have a beneficial effect on the extent and course of conditions associated with insulin resistance [40]. An intervention study of patients with type 2 diabetes with elevated ferritin levels provided evidence that bloodletting, which resulted in a 50% reduction of serum ferritin concentrations, improved glycemia and insulin sensitivity [41]. Iron depletion via phlebotomy in nonalcoholic fatty liver disease, the hepatic manifestation of the metabolic syndrome, has been found to reduce hepatic insulin resistance and to improve pancreatic insulin sensitivity, even in addition to successful lifestyle modifications [42]. Mediterranean Diet, Lower Iron Stores, and Cardiovascular Prevention Prospective primary and secondary prevention studies have clearly shown that adhering to a Mediterranean diet protects against coronary heart disease mortality [2,43]. Furthermore, a Mediterranean diet, as followed by a Mediterranean population, has been found to reduce the incidence of coronary heart disease in the healthy population [44].The effect of the Mediterranean diet on iron homeostasis may partially explain the beneficial action of this dietary pattern on cardiovascular prevention. In 1981, it was proposed that a state of sustained iron depletion or mild iron deficiency exerts a primary protective action against ischemic heart disease [45]. As previously mentioned, during late adolescence men begin a steady accumulation of stored iron with age, but women fail to acquire significant iron stores because of their continual losses of iron in menstrual blood, pregnancies, and deliveries (Figure 1). An escalation of risk follows the initial acquisition of significant stored iron after cessation of menses due to natural menopause or to surgical removal of the uterus and/or the ovaries. A protective effect of iron depletion that may have multiple beneficial consequences is decreased availability of redox-active iron in vivo. The amount of free iron available at sites of oxidative or inflammatory injury seems to be a function of the stored iron level. Removal of stored iron from the body by phlebotomy, systemic iron chelation treatment, or dietary iron restriction has been shown to decrease the amount of iron deposition within atherosclerotic lesions in animal studies [46]. In addition, epidemiological observations suggest a role for iron depletion in cardiovascular protection: (1) lower stored iron level mediated by cyanosis-induced hypoxia may explain why cyanotic patients with congenital heart disease might be protected from atherosclerosis [47]; (2) the protection against ischemic cardiovascular disease in individuals with impaired hemostasis might be related to the decrease of stored tissue iron caused by recurrent bleeding [48].Recent evidence confirms the possible effect of iron metabolism on plaque destabilization [49]. Mediterranean Diet, Iron, and Cancer It is well known that cancer is, in part, a preventable and largely environmentally determined disease, with diet as an important variable. Several aspects of the Mediterranean diet have been related to a reduced risk of cancer mortality, and it has been suggested that up to 25% of colorectal cancers, 15% of breast cancers, and 10% of prostate, pancreatic, 266 SECTION 3 Health and Nutritional Aspects of the Mediterranean Diet and endometrial cancers could be prevented by shifting to a Mediterranean diet [50]. Reduced body iron stores in people consuming a Mediterranean diet may be involved in this protective action. Several pathways for iron-induced carcinogenesis have been proposed [51]. Iron-induced oxidative stress may cause lipid peroxidation and direct damage to DNA and proteins. Excess iron may also block some of the protective mechanisms that cells are believed to use to limit the damaging effects of oxidative stress. Along these pathways, iron has predominantly been considered an early stage carcinogen. However, it has also been suggested that iron may influence carcinogenesis at later stages, perhaps as a nutrient and a facilitator of tumor growth. Numerous studies across a variety of populations have found a positive correlation between body iron stores and risk of the development of a range of cancers including colorectal, liver, kidney, lung, and stomach cancers [52]. Mounting evidence suggests that reducing body iron stores may lead to cancer protection. Analyzing cancer incidence and mortality of the FeAST trial has confirmed an effect of iron loss in reducing cancer risk. [53]. FeAST [32] represents the first, and so far the only, randomized trial of the effects of reduction of stored iron by controlled phlebotomies on cancer mortality. Favorable effects of iron reduction in the FeAST trial are illustrated in Figure 5. As mentioned earlier, the decrease of mean serum ferritin in the iron reduction group (from 122.5 to 79.7 mg/L—a 35% decrease) is within normal reference ranges for serum ferritin and, although it does not equate to iron depletion, it is noteworthy that it is of the same order of magnitude as the decrease of storage iron reported with the regular consumption of a polyphenol-rich tea with meals [54] and lower than the difference found when comparing ferritin levels between men from Zutphen, in the north of Europe, and men from Crete (134.2 vs. 69.8 mg/L—a 48% difference) [34]. During the 4.5 years of follow-up in the FeAST trial, the risk of new malignancy was significantly lower in the iron reduction group than in controls. Among patients with new cancers, those with iron reduction had highly significant lower cancer-specific and all-cause mortality. In particular, the 636 patients who were randomly assigned to iron reduction, followed for a range of 2.5–6 years, were found to have a lower rate of visceral cancer occurrence (hazard ratio [HR], 0.65; 95% confidence interval [CI], 0.43–0.97; P ¼ 0.036) than the 641 patients in the control arm. Patients who developed cancer in the iron reduction group had higher mean ferritin concentrations across all follow-up visits than those in the same intervention arm who did not develop cancer, suggesting that the treatment advantage could have been even larger if compliance had been better. Furthermore, the 38 patients in the iron reduction group who developed cancer during follow-up had lower risks of death from cancer (HR, 0.39; 95% CI, 0.21–0.72; P ¼ 0.003) and all-cause mortality (HR, 0.49; 95% CI, 0.29–0.83; P ¼ 0.009) in the fairly short period that passed between cancer diagnosis and end of follow-up than the 60 control patients who also were diagnosed with cancer. The FeAST trial results clearly suggest that reduction of stored iron may have a broad antitumor effect. FIGURE 5 The graph shows the cumulative incidence of cancer as the primary cause of death for the entire FeAST trial cohort according to randomization to control or iron reduction groups (P ¼ 0.002). The numbers below the horizontal axis represent the numbers of patients observed at each follow-up time point. From Zacharski et al. [53]. 0.10 0.09 Cumulative Incidence Estimates 0.08 Control 0.07 0.06 0.05 0.04 Iron Reduction 0.03 0.02 0.01 0.00 Years 1 Control (641) 626 Iron reduction (636) 607 2 562 547 3 482 460 4 320 331 5 191 178 6 59 52 Time to Event in Years and Number of Patients at Risk for each Group 7 Mediterranean Diet and Body Iron Stores Chapter 24 267 CONCLUSIONS Iron is an essential nutrient in humans. It has a key role in oxygen transport and in enzymes involved in mitochondrial respiration, DNA biosynthesis, and the citric acid cycle via its capacity to change the redox state. However, this characteristic also renders excess iron detrimental, mostly via the formation of reactive oxygen species, which may lead to the development of degenerative diseases. Body iron stores, which are relatively low during childhood and the premenopausal years, increase with aging as dietary intake exceeds loss [30]. Humans have no physiologic mechanism for sensing and excreting excess iron. Elevated iron stores can be prevented by dietary means or corrected by removing blood or iron chelation. Because iron homeostasis is regulated primarily at the level of dietary intake [29], benefits of reduced iron burden could be achievable by diet, as occurs in individuals living in Mediterranean countries [34], who exhibit low concentrations of ferritin and few markers of lipid oxidation, as well as reduced mortality. Efforts to implement a Mediterranean-style diet in the United States and in northern Europe may be less than fully successful because of population-wide exposure to iron overdosing from the consumption of processed foods and iron-containing vitamins and minerals [7]. Evidence from iron balance studies suggests the existence of a natural brake on iron absorption at ferritin concentrations of 60–80 mg/L, above which absorption slows [55]. However, most adults in Western countries seem to have artificially exaggerated iron intake capable of overcoming this brake, resulting in increasing ferritin concentrations [7] that presumably overwhelm endogenous antioxidant mechanisms, leading to increased disease risk. An optimal balance between the average bioavailability of dietary iron and the overall effects of inhibitors and enhancers of iron absorption, as observed in people following a Mediterranean dietary pattern, may lead to lower iron stores. Preserving optimal iron nutrition may be a safe and cost-effective strategy for managing and preventing diseases of aging. SUMMARY POINTS l l l l l l l Adherence to the Mediterranean diet is associated with longevity, decreased cardiovascular mortality, and decreased cancer mortality. Iron excess, as determined by serum ferritin concentrations, is very common and has been associated with cardiovascular disease, cancer, diabetes, and the metabolic syndrome. Iron is a necessary nutrient but in excess is pro-oxidant and leads to degenerative diseases. Humans have no physiologic mechanism of eliminating excess iron. Iron excess can be prevented by diet and treated by therapeutic phlebotomy or chelation. Elimination of excess iron stores has been associated with atherosclerotic plaque stabilization, cancer reduction, and improved insulin sensitivity. The Mediterranean diet, compared to the traditional Western diet, is lower in iron and replete in iron absorption inhibitors such as phytates, polyphenols, and dairy products. These prevail over iron absorption enhancers such as ascorbic acid and red meat. Over time, this dietary pattern leads to physiologically reduced iron stores and, consequently, a reduction in degenerative diseases. REFERENCES [1] Keys A, Menotti A, Karvonen MJ, Aravanis C, Blackburn H, Buzina R, et al. The diet and 15-year death rate in the seven countries study. Am J Epidemiol 1986;124:903–15. 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