European Review for Medical and Pharmacological Sciences 2010; 14: 521-526 Relation of phase angle tertiles with blood adipocytokines levels, insulin resistance and cardiovascular risk factors in obese women patients D.A. DE LUIS, R. ALLER*, E. ROMERO, A. DUEÑAS, J.L. PEREZ CASTRILLON Institute of Endocrinology and Nutrition, Medicine School and Unit of Investigation, Hospital Rio Hortega; Hospital Clinico*, University of Valladolid, Valladolid (Spain), RETICEF RD 056/0013 Abstract. – Background: Few studies have evaluated the relation between phase angle (PA) and metabolic syndrome. As long as we know, there are not studies of association between phase angle and adipocytokines. The aim of our study was to evaluate the association of adipocytokines levels and classical cardiovascular risk factors with tertiles of phase angle in obese women. Material and Methods: A cross-sectional study was designed to establish whether phase angle from 228 adult female patients with obesity are related with adipocitokynes and cardiovascular risk factors. These patients were studied in a Nutrition Clinic Unit after signed informed consent. All patients with a 2 weeks weight-stabilization period before recruitment were enrolled. Weight, blood pressure, basal glucose, C-reactive protein (CRP), insulin, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides blood and adypocitokines (leptin, adiponectin, resistin Interleukin-6 and TNF-alpha) levels were measured. The phase angle α was determined by bioimpedance with the equation [PAº=(Xc/R)x(180º/π)]. Results: Two hundred and twenty-eight females gave informed consent and were enrolled in the study. The mean age was 38.2±14.7 years and the mean BMI 35.27±6.5. Patients were divided by tertiles of phase angle. Fat mass was higher in first tertile than third tertile (43.6±12.6 vs 40.9±15 kg: p<0.05). HOMA (2.4±1.6 vs 1.46±1.6: p<0.05), insulin (14.4±8.5 vs 11.3±9.4 mUI/L: p<0.05) and glucose (102.1±20 vs 90±19.5 mg/dl: p<0.05) levels were higher in first tertile than second and third tertiles. Leptin (167.3±98 vs 104.5±80 ng/ml: p<0.05) and IL-6 (3.84±5.7 vs 1.8±2.9 pg/ml: p<0.05) levels were higher in first phase angle tertile than third tertile phase angle. Conclusion: Obese women with a low PA tertile have high fat mass with a secondary high level of glucose, HOMA, IL-6 and leptin. Perhaps, a low tertile of phase angle could be a new subrogate cardiovascular risk factor to categorize the obese patients. Key Words: Adipocytokines, Cardiovascular risk factors, Obesity, Insulin resistance, Phase angle α. Introduction Whole-body impedance represented by Z vector is a combination of resistance (R) and reactance (Xc). Impedance is a measurable property of electric ionic conduction of soft tissue of electric ionic conduction of soft tissue; fat and bone are poor conductors1. In bioelectrical impedance analysis (BIA), the arc tangent of Xc/R is called the phase angle2. Phase angle alpha can be easily obtained from BIA, this angle is one of best single predictor of survival in HIV infected patients3. Interestingly, the phase angle of the impedance vector was lower because of a decreased Xc component patients with hemodynamic instability 4, in hemodialysis patients with poorer prognosis5 and in critically ill patients6. Obesity and insulin resistance are associated with cardiovascular risk factors, including altered levels of inflammatory markers and adipocytokines7. The incidence of obesity and associated comorbidities is dramatically increasing worldwide. Obesity is characterized by a low grade systemic inflammation. Epidemiologic evidence of this rising tide of obesity and associated pathologies has led, in the last years, to a dramatic increase of researches on the role of adipose tissue as an active participant in controlling the body’s physiologic and pathologic processes8. Adipocytokines are proteins produced mainly by adipocytes9. These molecules have been shown to be involved in the pathogenesis of the meta- Corresponding Author: Daniel A. de Luis, MD; e-mail: [email protected] 521 D.A. de Luis, R. Aller, E. Romero, A. Dueñas, J.L. Perez Castrillon bolic syndrome and cardiovascular disease. Few studies have evaluated the relation between phase angle and metabolic syndrome. Mara et al. 10 showed a significant predictor of phase angle in the metabolic rate in severely obese patients. Guida et al. 11 have demonstrated that obese women with an altered body composition can be identified and monitoring using bioimpedance. As long as we know, there are not studies of association between phase angle and adipocytokines. The aim of our study was to evaluate the association of adipocytokines levels and clasical cardiovascular risk factors with tertiles of phase angle in obese women. Materials and Methods Study Design A cross-sectional study was designed to establish whether phase angle from 228 adult female patients with obesity are associated with adipocytokines and cardiovascular risk factors. These patients were studied in a Nutrition Clinic Unit after signed informed consent. All patients with a 2 weeks weight-stabilization period before recruitment were enrolled. Weight, blood pressure, basal glucose, lipoprotein (a), C-reactive protein (CRP), insulin, total cholesterol, LDLcholesterol, HDL-cholesterol, triglycerides blood and adypocitokines (leptin, adiponectin, resistin and TNF-alpha) levels were measured. Exclusion criteria included; severy impaired hepatic function (total bilirubin concentration >3.5 mg/dl) and renal function (serum creatinine concentration >2.5 mg/dl), ongoing infections, major gastrointestinal disease, autoimmune disorders, steroids treatment, and medication could modulate body composition or weight. Biochemical Parameters Serum total cholesterol and triglyceride concentrations were determined by enzymatic colorimetric assay (Technicon Instruments, Ltd., New York, NY, USA), while HDL cholesterol was determined enzymatically in the supernatant after precipitation of other lipoproteins with dextran sulfate-magnesium. LDL cholesterol was calculated using Friedewald formula. Plasma glucose levels were determined by using an automated glucose oxidase method (Glucose analyser 2, Beckman Instruments, Fullerton, CA, USA). 522 Insulin was measured by enzymatic colorimetry (Insulin, WaKo Pure-Chemical Industries, Osaka, Japan) and the homeostasis model assessment (HOMA) for insulin sensitivity was calculated using these values12. Adipocytokines Resistin was measured by ELISA (Biovendor Laboratory, Inc., Brno, Czech Republic) with a sensitivity of 0.2 ng/ml with a normal range of 412 ng/ml. Leptin was measured by ELISA (Diagnostic Systems Laboratories, Inc., Texas, USA) with a sensitivity of 0.05 ng/ml and a normal range of 10-100 ng/ml. Adiponectin was measured by ELISA (R&D systems, Inc., Minneapolis, MN, USA) with a sensitivity of 0.246 ng/ml and a normal range of 865-21424 ng/ml. Interleukin 6 and TNF-alpha were measured by ELISA (R&D systems, Inc., Minneapolis, MN, USA) with a sensitivity of 0.7 pg/ml and 0.5 pg/ml, respectively. Normal values of IL-6 was (1.12-12.5 pg/ml) and TNF-alpha (0.5-15.6 pg/ml). Anthropometric Measurements Body weight was measured to an accuracy of 0.5 kg and body mass index (BMI) computed as body weight/(height2). Waist (narrowest diameter between xiphoid process and iliac crest) and hip (widest diameter over greater trochanters) circumferences to derive waist-to hip ratio (WHR) were measured, too. Tetrapolar body electrical bioimpedance was used to determine body composition13. An electric current of 0.8 mA and 50 kHz was produced by a calibrated signal generator (Biodynamics Model 310e, Seattle, WA, USA) and applied to the skin using adhesive electrodes placed on right-side limbs. After introduction of an 800 mA (50 kHz) excitation current, BIA measures the geometrical components of electrical impedance Zc, i.e., resitance R (the sum of in phase vectors) and the capacitive component, reactance X (the sum of out-phase vectors) derived from Z2=R2+Xc2. The phase angle α is determined by the equation [PAº=(X c/R) × (180º/π)]. Precautions taken to insure valid BIA measurements were: no alcohol within 24 hours of taking the test, no exercise or food for four hours before taking the test. Dietary Intake Patients received prospective serial assessment of weight, nutritional intake with 3 days written food records. All enrolled subjects received in- Phase angle and obesity struction to record their daily dietary intake for three days including a week end day. Handling of the dietary data was by means of a personal computer equipped with personal software, incorporating use of food scales and models to enhance portion size accuracy. Records were reviewed by a registered dietitian and analyzed with a computer-based data evaluation system. National composition food tables were used as reference14. Drinking and smoking habit were recorded as dicotomic variables. Statistical Analysis The results were expressed as means ± standard deviation. The distribution of variables was analyzed with Kolmogorov-Smirnov test. Quantitative variables with normal distribution were analyzed with a two-tailed, paired Student’s-t test and ANOVA test. Non-parametric variables were analyzed with the Friedman and Wilcoxon tests. Qualitative variables were analyzed with the chisquare test, with Yates correction as necessary, and Fisher’s test. Pearson test and Spearman’S test were used to assess correlation analysis. A pvalue under 0.05 was considered statistically significant. Results Two hundred and twenty-eight females gave informed consent and were enrolled in the study. The mean age was 38.2±14.7 years and the mean BMI 35.27±6.5. Baseline characteristics of patients were presented in Table I. All subjects were weight stable during the 2 weeks period preceding the study (body weight change, 0.3±0.09 kg). Anthropometric measure- Table I. Clinical and epidemiological characteristics of study population. Characteristics Age (years) BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Glucose (mg/dl) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) CRP (mg/dl) Insulin (UI/L) HOMA Values 38.2 ±1 4.7 35.3 ± 6.5 126. 3 ± 17 81.9 ± 12.8 98.1 ± 18 207.5 ± 43.1 123.9 ± 41 57 ± 1 6.6 ± 9.9 15.2 ± 8.6 2.4 ± 1.6 BP: blood pressure. CRP: C-reactive protein. HOMA: homeostatic model assessment [glucose (mmol/L) x insulin (µU/ml)/22.5]. ments showed an average waist circunference (109.2±14 cm), waist-to hip ratio (0.89±0.9), and average weight (89.5±17.8 kg). Tetrapolar body electrical bioimpedance showed the next data: fat free mass (FFM) (45.8±14.4 kg) and fat mass (43.1±13.5 kg). Serial assessment of nutritional intake with 3 days written food records showed a caloric intake of 1672±488 kcal/day, a carbohydrate intake of 169.4±64 g/day, a fat intake of 73.5±13 g/day and a protein intake of 83.9±22.9 g/day. Anthropometric variables by phase angle tertile groups (tertile 1: phase angle below 6.17º), (tertile 2: phase angle between 6.18º and 7.07º) and (tertile 3: phase angle above 7.08º) are shown in Table II. Average of age was similar in all tertiles. Fat mass was higher in first tertile than third tertile and FFM was lower in the first tertile than third tertile. Table II. Anthropometric characteristics by tertiles of phase angleº. Phase angle Characteristics Weight (kg) BMI (kg/m2) Fat free mass (kg) Fat mass (kg) Waist circumference Waist to hip ratio (1) (n = 75) (2) (n = 76) (3) (n = 77) 89.2 ± 18 35.1 ± 6.4 44.8 ± 6.5 43.6 ± 12.6 106.6 ± 15 0.89 ± 0.08 88.8 ± 15.2 35 ± 5.7 45.7 ± 4.6 42.2 ± 12.5 105.6 ± 13.5 0.89 ± 0.07 88.9 ± 18 35.5 ± 7.1 47.4 ± 9.9* 40.9 ± 15* 105.7 ± 15 0.89 ± 0.08 BMI: body mass index. *(p<0.05), significant difference between first and third ALT tertiles. 523 D.A. de Luis, R. Aller, E. Romero, A. Dueñas, J.L. Perez Castrillon Table III. Cardiovascular risk factors by tertile of phase angleº. Phase angle Characteristics Systolic BP (mmHg) Diastolic BP (mmHg) Glucose (mg/dl) Total Ch. (mg/dl) LDL Ch. (mg/dl) HDL Ch. (mg/dl) Triglycerides (mg/dl) Insulin (mUI/L) CRP (mg/dl) HOMA-IR (1) (n = 75) (2) (n = 76) (3) (n = 77) 130.1 ± 16 81.4 ± 1 102.1 ± 20 206.1 ± 50 28.6 ± 45 58.1 ± 13 117.9 ± 46 14.4 ± 8.5 6.3 ± 6.4 2.4 ± 1.6 124.5 ± 18 79.7 ± 11 93.8 ± 14 202.4 ± 44 118.6 ± 44 56.8 ± 12 113 ± 58 13.1 ± 7.4 7.1 ± 4.6 3 2.37 ± 11.3 126.9 ± 19.2 81 ± 11 90 ± 19.5* 213.5 ± 35.9 128.3 ± 34 54.7 ± 13 124.8 ± 60 11.3 ± 9.4* 5.6 ± 6.1 1.46 ± 1.6* *Statistical differences with first tertile (p<0.05) BP: blood pressure; Ch.: cholesterol; HOMA-IR: homeostatic model assessment [glucose (mmol/L) x (nsulin (µU/ml)/22.5]. Cardiovascular risk factors variables by phase angle tertiles are shown in Table III. HOMA, insulin and glucose levels were higher in first tertile than second and third tertiles. Caloric intake, smoking habit and physical activity were similar in the three phase angle tertiles (Table IV). Table V shows the blood adipocytokines levels. Leptin and IL-6 levels were higher in first phase angle tertile than third tertile phase angle. Correlation analysis shows the next associations with phase angle, with FFM (r=0.3; p<0.05), fat mass (r=–0.13; p<0.05), leptin (r=–0.2; p<0.05), IL-6 (r=–0.13; p<0.05). Other parameters that did not reach statistical differences in tertile analysis show significative correlations with phase angle, for example; adiponectin (r=–0.15; p<0.05), resistin (r=0.1; p<0.05) and TNF alpha (r=0.1; p<0.05). Discussion In our study, we have documented a different fat mass, glucose, HOMA, leptin and IL-6 levels in different phase angle tertiles. Phase angle α (PA), which reflects changes in electrical conductivity of the body (indicating either alterations in body composition per se or – more likely – alterations of membrane cell integrity, function, or specific composition, including changes in the intercellular space). Phase angle α can be easily obtained from tetrapolar BIA, which is a noninvasive and safe technique for measuring human electrical tissue conductivity15,16. The overall opposition that a body presents to an alternative electric current has two components. The first is the capacitive reactance (X), resistive effect produced by the tissue interfaces and cell membranes. The second is the re- Table IV. Dietary intake and habits by tertile of phase angleº. Phase angle Characteristics Energy (kcal/d) Carbohydrate (g/d) Fat (g/d) Cholesterol (mg/d) Protein (g/d) % smoking habit Hours. aerobic exercise per week No statistical differences. 524 (1) (n = 75) (2) (n = 76) (3) (n = 77) 1670 ± 440 172.7 ± 60 65.2 ± 23.8 355 ± 144 83.8 ± 26 7.7 0.8 ± 2.1 1762 ± 500 70.1 ± 62 76.1± 31 383 ± 119 88.6 ± 24.7 7.5 0.7 ± 1.6 1645 ± 568 165.9 ± 69 73.4 ± 33.1 343 ± 110 88.4 ± 21 7.6 0.8 ± 1.8 Phase angle and obesity Table V. Circulating adipocytokines by phase angle tertileº. Phase angle Characteristics IL-6 (pg/ml) TNF-alpha (pg/ml) Adiponectin (ng/ml) Resistin (ng/ml) Leptin (ng/ml) (1) (n = 75) (2) (n = 76) (3) (n = 77) 3.84 ± 5.7 4.3 ± 3.1 32.2 ± 36 3.3 ± 1.5 167.3 ± 98 3.34 ± 1.8 5.3 ± 3.7 25.7 ± 18 3.4 ± 1.1 157.6 ± 82 1.8 ± 2.9* 5.4 ± 2.7 26.7 ± 21.6 3.5 ± 1.7 104.5 ± 80* IL-6: interleukin 6. *Statistical differences with first tertile (p<0.05). sistance (R), the restriction to the flow of an electrical current through the body, primarily related to the amount of water present in the tissues. Part of the electric current is stored by the cell membranes, which acts as capacitors, creating a phase shift, quantified geometrically as PA. Although the biological meaning of PA is not completely understood17,18, it reflects not only body cell mass (BCM), but is also one of the best indicators of cell membrane function, related to the ratio between extracellular water and intracellular water. Once PA is representative of BCM and the function of cell membrane, some changes are expected to occur in its values as a result of sex. PA is smaller in women than men19. All our patients are women; effects of sex could no explain our data. Our results showed a relation among PA and glucose, insulin resistance, leptin and IL-6 levels in tertile analysis. This relationship include more parameters (adiponectin, resistin, TNF-alpha). Perhaps, a low tertile of phase angle could be a new subrogate cardiovascular risk factor. However, correlation analysis shows a linear relationship with other cytokines. Adipocytokines are proteins produced mainly by adipocytes, related with fat mass. These molecules have been shown to be involved in the pathogenesis of the metabolic syndrome and cardiovascular disease. Adiponectin is an adipocytederived collagen like protein indentified through an extensive search of adipose tissue. Hypoadiponectinemia increased risk of coronary artery disease togheter with the presence of multiple risk factors, indicating that adiponectin may be a key factor of the metabolic syndrome20. Leptin is a 16 KD protein secreted primarily from adipocytes. Leptin suppresses food intake and increase energy expenditure by enhancing thermogenesis and metabolic rate. Recent reports suggest that leptin contributes to atherosclerosis and cardiovascular disease in obese patients21. Resistin is a 12.5 KD, cysteine-rich protein indentified by screening for the genes that are induced during the differentiation of the adipocytes. However, the role of resistin in linking human obesity with type 2 diabetes mellitus is thus questionable22. Interleukin 6 is increased in most animal and humans models with obesity and insulin resistance23-24. In conclusion, obese women with a low PA tertile have high fat mass with high levels of glucose, HOMA, IL-6 and leptin. 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