Enteral nutrient supply for preterm infants. A comment of the ESPGHAN
Committee on Nutrition.
ESPGHAN Committee on Nutrition and invited expert guests: 1C. Agostoni; 2Buonocore G, 3Carnielli VP
4
5
M. De Curtis, 5Darmaun D, 6T. Decsi; 7M. Domellöf, 8N.D. Embleton, 9C. Fusch, 10Genzel-Boroviczeny
O, 11O. Goulet;12Kalhan S.C. 13S. Kolacek; 14* #B. Koletzko, 15A. Lapillonne, 16W. Mihatsch, 17L. Moreno;
18
Neu J, 19Poindexter B, 20J. Puntis, 21Putet G , 22*J.Rigo, 23Riskin A, 24Salle B, 25Sauer P, 26R. Shamir;
27§
H. Szajewska; 28Thureen P, 29D. Turck, 30*J.B. van Goudoever, 31Ziegler E.
*Project steering committee, # Committee on Nutrition Chair, §Committee on Nutrition Secretary
10
1
Department of Paediatrics, San Paolo Hospital, University of Milan, Italy; 2Pediactrics, Obstetrics and
Reproductive Medicine, University of Siena, Italy;
3
Division of Neonatology, Salesi Hospital,
Polytechnical University of Marche, Ancona, Italy; 4University of Rome, Italy; 5Centre Hospitalier,
Universitaire de Nantes, France; 6Department of Paediatrics, University of Pecs, Hungary; 7Department of
paediatrics Umeå University, Sweden; 8Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom;
15
9
Ernst-Moritz-Arndt-University, Greifswald, Germany; 10Neonatologie Klinikum der Universität München,
Germany;
11
Hôpital Necker Enfants-Malades, University of Paris Descartes, Paris, France;
12
Cleveland
Clinic Lerner College of Medicine, Case Western Reserve University, USA; 13Children’s Hospital, Zagreb
Medical University, Croatia;
20
14
Dr von Hauner Children’s Hospital, University of Munich, Germany;
15
Hôpital Saint-Vincent de Paul, Paris, France;
16
17
Escuela Universitaria de Ciencias de la Salud, Zaragoza, Spain;
Deaconry Hospital, Schwaebisch Hall, Germany;
18
Department of paediatrics, University
of Florida, Gainesville, USA; 19Section of neonatal, Indiana University, School of Medicine, Indianapolis,
USA; 20Leeds General Infirmary, Leeds, UK;
de la Croix Rousse, Lyon, France;
Center, Haifa, Israel;
25
25
24
22
21
Service de Néonatologie Réanimation néonatale, Hôspital
CHR Citadelle, University of Liege, Belgium;
23
Bnai Zion Medical
Service de Medicine de la Reproduction, Hôspital Edouard Herriot, Lyon, France;
Department of paediatrics, University Medical Centre Groningen, The Netherlands;
Children's Medical Center, Tel-Aviv University, Tel Aviv, Israel;
Poland;
28
27
30
Schneider
The Medical University of Warsaw,
University of CO Health Sciences Center, Denver, USA;
Hospital/University of Lille, France;
26
Jeanne de Flandre Children’s
29
Erasmus MC - Sophia Children's Hospital, Rotterdam, the
Netherlands; 31Fomon Infant Nutrition Unit, Children’s Hospital, University of Iow, USA
30
1
Acknowledgements: A scientific workshop held to discuss the draft recommendations with invited expert
guests was financially supported by unrestricted educational grants donated by Danone Baby Nutrition
(then Nutricia Baby Foods), Mead Johnson Nutritionals, and Nestlé Nutrition to and administered by the
Charitable Child Health Foundation, Munich, Germany (www.kindergesundheit.de). All meetings and the
35
writings of the manuscripts were performed without any participation of representatives or employees of
commercial enterprises, and subjects and contents of the guideline were in no way influenced by the
supporting companies.
Correspondence:
40
Prof. Dr. J.B. van Goudoever, MD PhD
Division of Neonatology, Department of Peadiatrics, Sophia Children’s Hospital – Erasmus Medical
Center, Rotterdam, The Netherlands, Tel: +31-10-7036077, Fax:+31-10-7036811
2
45
Abstract ............................................................................................................................................ 4
Introduction ...................................................................................................................................... 4
Fluid ................................................................................................................................................. 5
Energy .............................................................................................................................................. 6
50
Protein ............................................................................................................................................. 8
Lipids .............................................................................................................................................. 9
Carbohydrates< .............................................................................................................................. 12
Minerals ......................................................................................................................................... 14
Trace elements ............................................................................................................................... 20
55
Vitamins ......................................................................................................................................... 24
Pre- and Probiotics ......................................................................................................................... 32
Nucleotides .................................................................................................................................... 34
Choline ........................................................................................................................................... 35
References ...................................................................................................................................... 37
60
Tables ............................................................................................................................................. 50
3
Abstract (235 words)
The number of surviving children born prematurely have increased substantially over the last two decades.
65
The major goal of enteral nutrient supply to these infants is to achieve growth similar to foetal growth
coupled with satisfactory functional development. The accumulation of knowledge since the previous
guideline on nutrition of preterm infants from the Committee on Nutrition of the European Society of
Paediatric Gastroenterology and Nutrition in 1987, has made a new guideline necessary. Thus, an ad hoc
Expert Panel was convened by the Committee on Nutrition of the European Society of Paediatric
70
Gastroenterology, Hepatology and Nutrition in 2007 to make appropriate recommendations. The present
guideline is consistent with, but not identical to, recent guidelines from the Life Sciences Research Office
of the American Society for Nutritional Sciences published in 2002 and recommendations from the
handbook "Nutrition of the preterm infant. Scientific basis and practical application", edited by Tsang et al,
2nd ed. published in 2005. The preferred food for premature infants is fortified human milk from the infant's
75
own mother, or alternatively formula designed for premature infants. This guideline aims provides
proposed advisable ranges for nutrient intakes for stable growing preterm infants up to a weight of
approximately 1800 gram, since most data are available for these infants. These recommendations are
based on a considered review of available scientific reports on the subject, and on expert consensus where
the available scientific data is considered inadequate.
80
Key words: Child Development, Embryonic and Fetal Development, *Premature infant feeding,
*Nutritional Requirements
4
85
Introduction
In 1987 the European Society of Paediatric Gastroenterology and Nutrition published recommendations on
nutrition and feeding of preterm infants [1] to provide guidance on feeding of preterm infants. Even though
extensive reviews on the topic have recently been published [2, 3], the ESPGHAN Committee on Nutrition
considered it necessary to review the recommendations on nutrient needs of preterm infants.
90
An expert group reviewed the existing evidence and prepared draft manuscripts on advisable intakes of
macro- and micro-nutrients for preterm infants. These proposals were reviewed and discussed in detail at a
scientific workshop organised by the charitable Child Health Foundation (www.kindergesundheit.de) in
March 2007. This meeting was attended by observing experts in infant formula design and manufacturing
(listed in footnote 1) who were asked to provide advice on the feasibility of producing food products based
95
on the recommendations made.
The aim of this report is to provide guidance on quantity and quality of nutrients needed for preterm
infants, so as to achieve growth similar to foetal growth coupled with satisfactory functional development.
The recommendations relate to ranges of enteral intakes for stable growing preterm infants up to a weight
of approximately 1800 gram, since most data are available for these infants. No specific recommendations
100
are provided for infants with a weight below 1000 gram as data is lacking for this group for most nutrients,
except for protein needs. The needs of infants with specific diseases (e.g. bronchopulmonary dysplasia,
congenital heart disease or short bowel syndrome) and those receiving parenteral nutrition have been
reviewed recently [4] and are not specifically addressed in this report.
The Committee advocates the use of human milk for preterm infants as standard practice, provided it is
105
fortified with added nutrients where necessary to meet requirements. Parents and health care providers
should be aware that human milk composition may vary over the duration of lactation, within the day and
even during one expression. Also the treatment following expression, e.g. storage or pasteurisation, may
influence composition. Alternatively to human milk, preterm formula may be used. This comment focuses
on providing guidance on appropriate nutrient intakes with fortified human milk or formula.
110
Footnote 1: Observers from the dietetic industry at the scientific workshop held to discuss the draft
recommendations with invited expert guests (in alphabetical order): H. Böckler, G. Boehm, C. Garcia
F. Haschke, J. Wallingford
5
115
Recent extensive reports on this topic [2, 3] as well as recommendations on nutrient supply for term infants
[5] have been taken into account in preparing this report. A Medline search was performed for publications
on preterm nutrition. For several nutrients, however, there is insufficient evidence on which to base
definitions of lower and upper intake levels. When no sufficient data were available, intakes provided with
human milk feeding and currently available human milk fortifiers and with preterm infant formulae were
120
considered.
Ranges of advisable nutrient intakes are expressed both per kg bodyweight per day and per 100 kcal (table
1). Calculation of the latter values was based on the minimum energy intake of 110 kcal/kg/d that we chose
to recommend. Thereby, the ranges of nutrient intakes per 100 kcal will ensure the infant receives the
minimum or maximum of each specific nutrient at an intake of 110 kcal/kg/d. One should be aware that at
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higher energy intakes, the individual nutrient should not exceed an acceptable maximum level of intake.
While the recommended ranges of nutrient intakes are considered reasonable, a high degree of uncertainty
remains and hence the provision of nutrient intakes outside of the specified ranges is not discouraged if
justified by good reasons. Nevertheless, it must be noted that using levels found in available commercial
products without apparent problems as the basis for providing guidelines is less than satisfactory, since
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subtle adverse affects may not be detected without conducting adequate randomised controlled trials. Such
trials can also be aimed at obtaining data on suitability and safety of intakes that are outside the specified
ranges.
6
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Fluid
Ad libitum feeding studies as in healthy breastfed infants have not been performed in very low birth weight
infants in view of their lack of autonomy with respect to fluid intake. Randomized controlled trials on
enteral fluid intake of preterm infants are also lacking as are studies comparing different fluid volumes (e.g.
140 vs. 200 ml/kg/d) providing identical nutrient intakes. Measured values of fluid intake established by
140
dietary protocols or stable isotopes are available for 6-week-old healthy ex preterm neonates (140 – 180
ml/kg/d) [6], term infants (160 ml/kg/d) [7-9] and non-ad libitum fed preterm infants (160 +/- 30 ml/kg/d)
[10, 11].
Extrapolation from measured enteral intake at term to preterm needs in addition to a factorial approach
should account for higher preterm growth rates with storage of intra- and extra cellular water and
145
electrolytes, and for higher losses via more immature skin and reduced renal concentration capacity. Thus,
the preterm infant’s enteral fluid needs are about 15 – 25 % higher than that of the term infant. [6-11]
From data of combined parenteral/enteral regimens, and assuming full enteral absorption, it follows a) that
fluid volumes between 96 to 200 ml/kg/d are tolerated and that these values may serve as lower and upper
limits [12]; b) that postnatal intakes at the lower range is likely to minimise risk of long-term morbidity
150
such as BPD and PDA. It is important to note that fluid volumes needed for enteral nutrition are influenced
by osmolarity, renal solute load and are not synonymous with actual water needs.
On the basis of these data sources, recommendations made in this comment and based upon final
osmolarity and previous recommendations [3, 4] we regard 135 ml/kg/d as the minimum fluid volume
and 200 ml/kg/d as a reasonable upper limit. For routine feeding, rates of 150 - 180 ml/kg/d nutrient
155
intakes when standard formula or breast milk is used are likely to achieve meeting nutrient requirements.
Some infants may need higher volumes in order to meet requirements of substrates other than fluid.
Energy
Recommendations for energy intake are based on the assumption that growth and nutrient retention similar
160
to intrauterine references are appropriate. Yet we must make allowances for extra uterine environment and
differences in nutrient supply and metabolism (e.g. the foetus receives only a small proportion of energy as
fat). Using intrauterine growth as a standard should involve not only achieving similar weight gain but also
body composition, even though a higher extra uterine fat deposition may be needed to provide thermal and
mechanical protection.
7
165
Studies in the two decades since the last ESPGHAN recommendations[13] have provided data on longer
term outcomes, and there are indications that rapid infant weight gain in term infants may be associated
with adverse outcomes [14], adding to the uncertainty regarding optimal energy provision for preterm
infants.
Energy requirements for otherwise healthy preterm infants will depend on the post conceptional age (higher
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per kg body weight at 24 than at 36 weeks post conceptional age), accumulated nutrient deficits (both preand postnatal growth restriction), alterations in body composition, and differences in resting energy
expenditure (REE). As REE is affected by sleep state and activity levels, environment (thermoregulation),
genetic differences in basal metabolic rate and demands for tissue synthesis, energy needs may vary
considerably amongst populations of healthy preterm infants. Synthesis of new tissue is energy intense and
175
strongly affected by the intake of protein and other nutrients, thus achieving an adequate energy:protein
ratio is as important as providing adequate energy intake.[15]
Energy required for protein deposition approaching intrauterine dimensions is approximately 5.5-7.75
kcal/g protein deposited, and for fat deposition 1.55-1.6 kcal/g fat deposited, excluding the amount of
energy which is stored in the process.[16, 17] REE is approximately 45 kcal/kg/d and does not seem to vary
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much with gestational age, but may be lower in some babies[18]. Estimated average energy requirements
for depositing new tissue (13% protein, 20 to 30% fat) are 3.3-4.7 kcal/g,[16, 19], so achieving an
intrauterine weight gain of 17 g/kg/d[20] will require about 56-80 kcal/kg/day on top of REE (~45
kcal/kg/d). Thus, a total metabolizable energy intake (including protein) of approximately 100-125
kcal/kg/d is needed, corresponding to 110-140 kcal intake for an energy absorption rate of 85% with human
185
milk and 90% with a well-absorbed formula.
Clinical studies suggest that energy intakes ≤100 kcal/kg/d will not meet the needs of some preterm infants
prior to discharge. Where protein:energy ratios are adequate (>3-3.6g/100kcal) in a formula providing well
absorbed nutrients, an energy intake >100 kcal/kg/day is generally appropriate[21] and may result in a fat
mass (FM) percentage closer to both intrauterine references and normal term infants. SGA infants might
190
need a higher energy intake than AGA infants [21], however, a focus on achieving an optimal lean mass
(LM) accretion rather than FM may be more appropriate. Whilst higher intakes (140-150 kcal/kg/d) appear
generally safe in the short term, there is limited evidence of improved linear growth (as a proxy for LM
accretion) but FM deposition appears excessive.[21-24]
Nitrogen retention is affected by source of non-protein energy. Carbohydrate appears more effective at
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sparing protein oxidation and may result in faster linear growth, but studies at energy intakes of 155
kcal/kg/d with high carbohydrate:fat ratios showed substantially greater fat deposition than intrauterine
8
references.[24] A reasonable range of energy intake for healthy growing preterm infants with
adequate protein intake is 110-135 kcal/kg/d. Increasing energy intake may not be appropriate for infants
whose growth appears inadequate (without evidence of fat malabsorption) as it is more likely that other
200
nutrients (e.g. protein) are rate limiting.
When considering other nutrient intakes it is important that recommended minimum intakes meet needs. At
an intake of 110 kcal/kg/day, formula compositions should ensure that basic minimum intakes of other
nutrients are met, and this figure should then be used in determining nutrient ratios of enteral feeds.
Protein
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There is a lack of data on long-term outcome effects of different protein intakes from randomized
controlled trials, but there are some indications that a suboptimal intake of protein, energy and other
nutrients may lead to lower cognitive achievements. [25] Dietary protein requirements can be estimated by
the factorial method or by empirical approach. The factorial method estimates requirements from the sum
of obligatory losses with urine, faeces and skin and the amount deposited in newly formed tissue. However,
210
this method is subject to misinterpretation since losses might be underestimated and the exact composition
of the newly formed tissue is not known. The empirical approach measures biochemical or physiological
responses to graded intakes. Individual amino acid requirements can also be determined by both these
methods, but the use of indicator amino acid methodology may be more accurate.[26]
Compositional analysis of foetal tissues has been a valuable data source for our understanding of the
215
nutrient needs of the foetus, and by analogy, those of the growing preterm infant. Protein accretion has
been estimated at approximately 1.7 g/kg/d for foetuses throughout the second half of gestation but is lower
at the end of gestation.[27] Obligatory protein losses are at least 0.7 g/kg/d but may be higher if nitrogen
losses from skin and breath could be measured. Nevertheless, this value is close to that found necessary to
reach a nitrogen equilibrium.[28] Clinical practice, however, regularly shows deficits in protein supply
220
relative to estimated requirements in the first few weeks of life, particularly in more immature preterm
infants, depending on feeding policy, tolerance and illness. [29]
Faecal nitrogen loss is related to the protein source fed and to feeding effects on endogenous nitrogen
losses. Net absorption equals nitrogen intake minus faecal loss (either secreted or non-absorbed). The
highest nitrogen absorption rate (% of intake) is observed with powder whey predominant formula (90%).
225
Absorption rate with ready-to-use liquid whey predominant premature formulae (86.0 %) is slightly higher
than with human milk supplemented with fortifiers (82.7 %) or hydrolysed preterm formula (84.3%) Table
1.[30] Some hydrolysed proteins in preterm formulae tend to shorten gastrointestinal transit time, which
might accelerate feeding advancement [31, 32] but could also reduce utilisation.
9
Compared to current preterm formulae, human milk contains a higher proportion of non-protein nitrogen
230
(20-25% of total nitrogen). The degree of utilisation of non-protein nitrogen in preterms is not entirely
known and may vary considerably, contributing to the lower nitrogen absorption rate of human milk.
Furthermore, the quality of the provided protein might interfere with the recommended intake, since the
infant does not require proteins but specific amino acids. Little is known about optimal intakes of specific
amino acids. A different composition of the proteins administered might change the quantity of proteins
235
required.
However, based on the above figures for protein needs and nitrogen utilisation, the protein intake should at
be at least 3.0 g/kg/d. Empirical data show that weight gain approximating to that in utero can be achieved
at approximately 3 g/kg/d protein intake [3, 28, 33, 34], and that weight gain rates are linearly related to
protein intakes up to 4.5 g/kg/d. Intrauterine weight gain can be matched at protein intakes less than 3 - 3.5
240
g/kg/d accompanied by a very high energy intake, but body fat percentage will then be much higher than
observed in the foetus.
Protein supply needs to compensate for the accumulated protein deficit observed in almost all small
preterm infants, and can be increased to a maximum of 4.5 g/kg/d, depending on the magnitude of the
accumulated protein deficit. Intakes in the range of 3-4.5 g/kg/d will achieve acceptable plasma albumin
245
and transthyretin concentrations [35]. Some excess of protein intake over requirements was not shown to
cause detrimental effects in preterms, but, on the other hand, a small deficit will impair growth. We
therefore recommend to aim at 4.0 - 4.5 g/kg/d protein intake for infants up to 1000 g, and 3.5 - 4.0 g
for infants from 1000-1800 g which will meet the needs of most very preterm infants. Protein intake
can be reduced towards discharge if the infant’s growth pattern allows for this. The recommended
250
range of protein intake is therefore 3.6-4.1 g/100 kcal for infants weighing less than 1000 g and 3.2-3.6
g/100 kcal for infants from 1000-1800 g.
Lipids
Dietary lipids provide the preterm infant with much of its energy needs, essential polyunsaturated fatty
255
acids and lipid soluble vitamins [3]. Amount and composition of dietary lipids affect both growth pattern
and body composition. The availability and metabolism of long-chain polyunsaturated fatty acids (LCPUFA) have direct implications for cell membrane functions and the formation of bioactive eicosanoids.
Brain grey matter and the retina are particularly rich in LC-PUFA, and complex neural functions are related
to energy supply and the composition of dietary fatty acids.
10
260
Limits for dietary fat intake are determined by the minimal requirements and maximal tolerance for protein
and carbohydrate intake, and possibly by practical limits with respect to achieving a desirable energy
density at acceptable osmolarity of the feed. A desirable minimal fat intake is derived from the concept that
the dietary intake of metabolisable fat (with long-chain fatty acids) should at least equal body fat deposition
of the foetus during growth. Assuming a daily intrauterine fat deposition of 3 g/kg [3], 10-40 % loss from
265
fat malabsorption, and 15 % loss from unavoidable oxidation and conversion of absorbed triglyceride to
deposited triglyceride in tissue, the minimum fat intake to meet is estimated at 3.8-4.8 g/kg/day. On this
basis, a minimal dietary fat intake of 4.8 g/kg per day is suggested. At an energy intake of 110 kcal/kg, this
fat intake is met by a total fat content in preterm formula of at least 4.4 g/100 kcal (or 39.5 % of energy;
E%), as previously recommended by the ESPGAN Committee on Nutrition) [36] and the expert committee
270
convened by the US Life Science Research Office [2]. The same two expert committees recommended fat
intake upper limits of 6.0 g/100 kcal (54 % of energy; E%) [36] and of 5.7 g/100 kcal (51 E%) [2], which
are similar to the upper end of the range usually observed in human milk samples. The LSRO report
discussed the possibility that very high fat contents of preterm infant formulae could be excessive given the
somewhat limited fat digestion and absorption of preterm infants, whereas formulae fat contents of 5.1-5.4
275
g/100 kcal seem to be tolerated reasonably well [2]. Definitive scientific data on this issue are not yet
available. While some infants with restricted fluid and feed intakes may need high fat intakes to meet
energy needs, for most preterm infants a reasonable range of fat intake is 4.8 – 6.6 g/kg per day or
4.4 – 6.0 g/100 kcal (40-55 E%).
Medium chain triglycerides (MCT)
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MCT containing primarily octanoic and decanoic acids are produced by fractionation of coconut oil and are
used in preterm formulae to facilitate the absorption of fat and calcium [3]. A 40 % contribution of MCT to
fat intake was found to enhance fat absorption by about 10 % relative to formulae based on long-chain
triglycerides [37]. Still, the degree of enhancement of fat absorption is determined by the lipid composition
of the formulae used. The above effect of MCT does not necessarily lead, however, to increased supply of
285
metabolisable energy, due to the shorter chain length of the fatty acids in MCT and hence about 15 % lower
energy content [3]. Clinical studies so far failed to find any favourable effect of replacing part of the fat in
low birth weight infant formulae with MCT on energy or nitrogen balance, or weight gain.[3]
Replacing the long chain saturated fatty acids 16:0 and 18:0 in preterm infant formulae by MCT oils can
increase calcium and magnesium absorption [3, 38], but other formula modifications have achieved similar
290
benefits on mineral bioavailability. Formulae for stable growing preterm infants need not necessarily
include MCT oils if they contain other well absorbed fats; there is indeed insufficient evidence for benefit
11
of including more than 40% of total fat as MCT in preterm formulae. We therefore support that the
MCT content in preterm formulae, if added, should be in the range of up to 40% of the total fat
content.
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Essential fatty acids
There are no data yet to reliably establish minimal or optimal intakes of the essential fatty acid linoleic
acid, in preterm infants. Previous expert reviews have recommended ranges of 500-1200 mg/100 kcal (4.510.8 % E%) [36] and of 352-1425 mg/100 kcal (3.2-12.8 E%) [2], equivalent to about 8-25 % of the fatty
acids in a preterm formula. There is no evidence of linoleic acid deficiency or of adverse effects from high
300
intakes in infants fed current preterm formulae. Linoleic acid intakes of 385-1540 mg/kg per day or 3501400 mg/100 kcal (3.2-12.6 E%) are considered acceptable.
Current understanding suggests that the essential fatty acid alpha-linolenic acid plays an essential role as a
precursor for synthesis of eicosapentaenoic acid and docosahexaenoic acid. Definitive quantitative
requirements have not yet been established and may depend on docosahexaenoic acid content in the milk or
305
formula diet. In the absence of more information, a reasonable minimum intake of alpha-linolenic
acid for preterm infants of 55 mg/kg per day, or 50 mg/100 kcal (0.45 E%) has been suggested, which
is equivalent to about 0.9 % of total fatty acids [3]. Lower ranges of intakes might also be adequate for
formulae that provide preformed docosahexaenoic acid, but there is no evidence to support this
recommendation.
310
The ratio between linoleic and alpha-linolenic acids appears important because these two fatty acids
compete for the same desaturase enzymes. It may be more important for formulae that provide no or very
little long chain polyunsaturated fatty acids (LC-PUFA) than for formulae supplemented with arachidonic
acid and docosahexaenoic acid. Little clinical data are available for preterm infants fed different ratios of
linoleic and alpha-linolenic acid, yet one study found lower growth among term and preterm infants fed
315
formula with a ratio of 4.8:1 when compared to a ratio of 16:1 [39]. It seems reasonable that the linoleic
acid to alpha-linolenic acid ratio is in the range of 5-15:1 (wt/wt) [3].
Clinical trials in preterm infants fed formulae containing both arachidonic acid (AA) and docosahexaenoic
acid (DHA) have shown beneficial effects on the developing visual system and measures of cognitive
development during the first year of life, as well as on immune phenotypes [3, 40-42]. There was no
320
evidence of adverse effects including growth among infants fed formulae containing up to 0.5 % DHA and
up to 0.7 % AA of total formula fatty acids. Although the long-term effects on visual and neural
development are not fully known, it would seem prudent to provide LBW infants with a dietary source of
these fatty acids. Lower growth has been observed in LBW infants fed formulae supplemented with DHA
12
and EPA and not with AA. Nevertheless, subsequent clinical trials in LBW or term infants fed formulae
325
with both DHA and AA did not show lower growth rates. Eicosapentaenoic acid competes with AA, and
eicosapentaenoic acid levels are very low in human milk. These considerations lead to the conclusion that
both AA and DHA should be included in preterm formulae, and that oils containing significant amounts of
eicosapentaenoic acid should be avoided. Preterm formulae providing DHA intakes of at least 0.2 % of
formula fatty acids (equal to about 12 mg/kg per day at an energy intake of 120 kcal/kg and a formula fat
330
content of 5 g/100kcal), along with a provision of AA, were found to enhance visual function and other
neurodevelopmental and immune outcomes in preterm infants [3, 40-42]. Some clinical evaluations are
available for formulae providing DHA intakes of up to about 0.5 % of fatty acids or 30 mg/kg per day, and
AA intakes of about 0.3-0.7 % of fatty acids or about 18-42 mg/kg per day. Higher amounts of AA and
DHA could possibly be more advantageous to growth and development of VLBW infants, an assumption to
335
be addressed in clinical trials A reasonable range for the balance between AA and DHA appears to be 1.02.0 : 1, i.e. the range of most preterm formulae clinically tested, and similar to that found in many studies
on fatty acid composition of human milk [2]. Adverse effects of preterm formulae providing high amounts
of eicosapentaenoic acid have been reported; therefore eicosapentaenoic acid contents should not exceed
30% of the amount of DHA [2].
340
Recommended intakes are for DHA (22:6n-3) 12-30 mg/kg/day or 11-27 mg/100 kcal and for AA
(20:4n-6) 18-42 mg/kg/day or 16-39 mg/100 kcal. The ratio of AA to DHA should be in the range of
1.0-2.0 to 1 (wt/wt), and eicosapentaenoic acid (20:5n-3) supply should not exceed 30 % of DHA
supply.
345
Carbohydrates
Carbohydrates are a major source of energy. Glucose is the principal circulating carbohydrate and the
primary source of energy for the brain. It is an important carbon source for de novo synthesis of fatty acids
and several non-essential amino acids.
Preterm infants have a higher rate of basal glucose production than full-term infants (11.5 – 12.9 g/kg/d
350
compared with 7.2 g/kg/d) [43, 44] They have the capacity to oxidize large amounts of glucose to meet
energy demands, so that at high rates of glucose infusion nearly all energy expenditure comes from
oxidation of glucose or other carbohydrates [45].
The upper limit of carbohydrate intake has been calculated as the glucose equivalent of the total energy
expenditure minus the calories from the minimum requirements for protein and fat. The minimum protein
13
355
concentration in formula recommended in this report is 3.2 - 3.6 g/100 kcal, or approximately 12% of
calories. The minimum lipid concentration in formula recommended in this report is 4.4 g/100 kcal, or
approximately 40% of calories. The maximum caloric intake from carbohydrate is therefore 100% minus
12% (for protein) minus 40% (for fat), or 48% of total caloric intake. Therefore a maximum
carbohydrate content of preterm infant formula (glucose or nutritionally equivalent di-, oligo-and
360
polysaccharides) of 12.0 g/100 kcal is recommended.
The lower limit for carbohydrate intake has been defined based on energy requirements of the brain and
other glucose dependent organs, minimizing the irreversible loss of protein and nitrogen by limiting
gluconeogenesis, and preventing ketosis. For preterm infants, such estimates have been derived from the
minimum endogenous rate of glucose production (11.5 – 12.9 g/kg/d). These estimates do not separate the
365
very preterm infants from the less preterm infants. Preterm formulae containing 10.5 g carbohydrate/100
kcal provide 11.5 g/kg/d at an energy intake of 110 kcal/kg/d. A minimum content of 10.5 g
carbohydrate/100 kcal (glucose or nutritionally equivalent di-, oligo-or polysaccharides) in preterm
infant formulae is recommended.
Lactose
370
Incomplete digestion of lactose in the small intestine of the preterm infant limits the availability of energy
from carbohydrate. Lactase activity gradually increases with advancing gestation until about 36 weeks GA,
when it reaches the level of term newborns [46]. Lactose digestion of 79 +/- 26% (mean +/- SD) and a
percentage of lactose fermentation of 35 +/- 27% have been observed in preterm infants [47]. Early feeding
and early achievement of full feeds increased the intestinal lactase activity [48].
375
Undigested lactose (and possibly other dietary carbohydrates) is fermented in the colon, where much of the
constituent energy can be absorbed as short-chain fatty acids, lactate and other organic acids [47]. This
process of colonic salvage or retrieval of carbohydrate energy compensates, at least in part, for the
inefficiency of dietary energy utilization. Some of this source of energy is lost as heat during fermentation.
However, there is controversy concerning whether the production of short chain fatty acids, such as butyric
380
acid, is advantageous to the preterm infant or is toxic to the colon or the small intestine if excessive
fermentation occurs at these sites [47, 49]. The fermentation process does not salvage all the energy from
lactose: a double blind, randomized controlled trial in 130 infants at 26-34 weeks of gestation showed that
adding lactase to the diet increased weight gain [50]
Although lactose is the only source of dietary galactose and the major carbohydrate in human milk, the
385
available evidence does not justify the recommendation that preterm infant formula must contain lactose.
The widely held belief that lactose increases calcium absorption has been questioned.[51] However, there is
14
long standing experience of successful enteral feeding of preterm infants using human milk or cows’ milk
formulae containing lactose. Although it has been suggested that reduction of lactose intake might improve
feeding tolerance, current evidence is insufficient to recommend a maximum level of tolerance. Present
390
formulae contain usually 4-7 g lactose/100kcal and human milk contains approximately 10 g lactose/100
kcal so that higher amounts of lactose should be studied prior to routine use.
Glucose
Glucose absorption is well developed in enterally fed preterm infants. The absorption rate appears to
change with type of feeding, administration of glucocorticoids, and duration of enteral feeding [52-54].
395
Glucose has not been used as the only carbohydrate in preterm infant formula because this would markedly
increase the osmolarity of the formula.
Galactose
Human milk contains much galactose, but the requirements and benefits for the infant remain unknown
[55]. Hydrolysis of lactose at the intestinal brush border results in the release of glucose and galactose, both
400
of which are readily absorbed. Most of the enterally absorbed galactose is taken up by the liver during the
first pass [56, 57], so plasma galactose concentration shows little change after a feed. Galactose in the liver
is either converted to glucose or deposited as glycogen [58]. The rate of endogenous synthesis of galactose
is substantial, but there does not appear to be an obligatory requirement for galactose. There is no evidence
to justify a firm recommendation for galactose in preterm infant formula.
405
Glucose polymers and maltose
Glucose polymers consist of polymers of glucose of various chain lengths, but predominantly of medium
length (6–10 glucose units). The proportion of free glucose is usually less than 2%. The glucose polymers
are mainly linear, with the glucose residues attached to each other by α-1,4-glucosidic bonds. They have
been used as nutritional supplements for infants because they have lower osmolarity per kilocalorie than
410
glucose. Glucose polymers are hydrolyzed by salivary, pancreatic, and intestinal amylases and maltases to
free glucose. In the newborn, glucose polymers are primarily digested via intestinal glucoamylase and
isomaltase [59, 60]. Glucose polymers appear to be rapidly hydrolyzed and absorbed by the newborn, and
the carbohydrate energy not absorbed in the small intestine is rapidly salvaged by colonic bacteria [59-61].
Reduction or elimination of lactose and replacement with more readily digestible carbohydrate such as
415
maltose or glucose polymers has been reported to improve feeding tolerance, weight gain and calcium
absorption [62-66].
15
Current commercial preterm infant formulae regularly contain glucose polymers as a replacement for
lactose. Glucose polymers, maltose, or other potentially more readily digestible carbohydrates as a partial
alternative to lactose may have beneficial effects. Nevertheless, glucose polymers have also been used as an
420
energy source in human milk fortifiers. They were partly hydrolysed, thereby increasing osmolarity, due to
the amylase content in human milk, which was resistant to pasteurisation [67].
Minerals
Sodium
425
Sodium is a cation that has several roles. Sodium is the major regulator of extracellular fluid
volume and important for blood pressure regulation. Furthermore, it is importance for bone and
nervous tissue development. No new data have been accumulated during the last few years that
would question previous recommendations [2, 3]. A minimum sodium intake of 63 mg/100 kcal
(2.7 mmol/100 kcal) and a maximum sodium intake of 85 mg/100 kcal (4.6 mmol/100 kcal)
430
is recommended.
Potassium
Potassium, the major intracellular cation, maintains the transmembrane electrical potential and intestinal
ionic strength and therefore is of pivotal importance in contractility, blood pressure regulation and nerve
435
function. No new data have been accumulated the last few years to challenge previous recommendations [2,
3]. A minimum potassium intake of 60 mg/100 kcal and a maximum potassium intake of 120 mg/100
kcal (1.5-3.1mmol/100 kcal) is recommended.
Chloride
440
Inadequate chloride intake will result in failure to thrive and decreased growth of head circumference, body
length and delayed mental development [68] Hypochloridaemia results in hypercalceamia,
hyperphosphataemia and metabolic acidosis. [69] . No new data have appeared in the last few years to
challenge previous recommendations [2, 3]. A chloride intake of 95 – 161 mg/100 kcal (2.7-4.6
mmol/100 kcal) is recommended.
445
16
Calcium
Current mineral recommendations are based on healthy premature infants and aim at providing a
postnatal mineral accretion, during the ‘stable-growing’ period, equivalent to that of a normal
foetus’ [1, 70]. The Life Sciences Research Office (LSRO) committee suggested premature milk
450
formulae should contain 123–185 mg/100 kcal of calcium and 80–110 mg/100 kcal of
phosphate[2]. Atkinson and Tsang in 2005 proposed similar figures [3]. Since knowledge of
perinatal bone physiology has recently improved, we now have reason to question these
recommendations. The preterm infant’s bone is exposed to different conditions before and after
birth. First and foremost, there is a rapid change in hormonal environment: estrogen environment is
455
high during foetal life and decreases after birth [71]; calciotropic hormones [PTH and
1,25(OH)2D)] levels are low during the last trimester and are followed, after birth, by a surge in
plasma concentrations of 1,25(OH)2 D, remaining elevated until the second month of life [72].
Although intrauterine gravity is low, mechanical stimulation in utero is important, for example, the
regular kicks against the wall of the uterus representing a form of ‘training’ not available in the
460
post-natal period [73, 74]. Newborn premature infants suffer from diminished accessibility to
minerals required for proper bone accretion, partly due to limited availability and partly to poor
gastrointestinal absorption. The latter clearly has implications for feed composition[75].
The various possible influences on absorption include vitamin D status, solubility and
bioavailability of calcium salts, quality and quantity of fat intake, as well as various production
465
processes such as heat treatment of liquid formulae promoting Maillard reaction products that alter
calcium absorption [76, 77]. Therefore, increasing the mineral supply does not necessarily increase
mineral accretion. It should be noted that higher mineral supply has been associated with high
faecal calcium, impaired fat absorption, increased stool hardness and prolonged gastrointestinal
transit time, which are all potential risk factors for gastro-intestinal disorders and potentially for
470
necrotizing enterocolitis [75, 78-80].
A number of mineral balance studies have been performed in premature infants fed human or
formula milk (reviewed in [81]). Collectively, these studies showed that calcium absorption
depends on calcium and vitamin D intakes, and that calcium retention is additionally related to
absorbed phosphorus. They suggests that calcium retention ranging between 60 to 90 mg/kg/d
475
decreases the risk of fractures, diminish the clinical symptoms of osteopenia and assure appropriate
mineralization in VLBW infants Thus, a calcium absorption rate of 50 to 65 % will lead to a
calcium retention of 60 to 90 mg/ kg/d at an intake of 120 to 140 mg/kg/d.
17
Osteopenia or rickets of prematurity seems to be a self resolving disease quite similar to that
observed during adolescence after the initial growth spurt. Bone mineral content improves
480
spontaneously in most infants and catch-up mineralization is soon observed in VLBW infants after
discharge. At 6 months, age-corrected, spine and total bone mineral density adjusted for
anthropometric variables are in the range of those for normal term newborn infants [82, 83].
Phosphorus and calcium to phosphorus ratio
The calcium to phosphorus ratio may be an important determinant of calcium absorption and
485
retention [75]. In human milk, the Ca to P ratio is approximately 2 in terms of mass and 1.5 as
molar ratio. In preterm infants, supplementation of human milk with phosphorus alone is
associated with a marked decrease in urinary excretion of calcium and improvement of calcium
retention. Indeed, phosphate is not only an important skeletal constituent with a calcium to
phosphorus mass ratio constant from foetal to adult life (2.2:1), but it is also an important
490
intracellular anion with a nitrogen to phosphorus mass ratio of 15:1. During foetal life, 75% of
phosphorus (55 mg•kg−1 d−1) is retained in bone and 25% (20 mg•kg−1 d−1) is retained in
tissues. In premature infants, phosphorus accumulation is related to calcium and nitrogen retention
but with a lower proportion for bone compared to that in the foetus.
Phosphorus absorption is very efficient and exceeds 90% in infants fed human milk. In formula-
495
fed infants, phosphorus absorption may also be close to 90%. Nevertheless, the use of poorly
absorbable calcium salt, such as calcium triphosphate, is associated with significant reduction in
phosphorus absorption [75].
Owing to the relative poor availability of calcium, preterm formula frequently provides much more
phosphorus than needed, and phosphaturia of over 20 mg/kg/d has been observed. By contrast,
500
when calcium absorption is satisfactory, urinary phosphorus excretion is reduced. The present
recommendation for preterm formula is a calcium to phosphorus ratio close to 2:1, but ideally this
should be adapted taking into account nitrogen retention as well as bioavailability of the calcium
salt. Considering a nitrogen retention ranging from 350 to 450 mg/kg/d and a calcium retention
from 60 to 90 mg/kg/day, the adequate phosphorus intake represents 65 to 90 mg/kg/d of a highly
505
absorbable phosphate source (90%) with a Ca to P ratio between 1.5 and 2.0.
In conclusion, newly acquired understanding of bone physiology [82] makes it advisable to review
the current recommendations of mineral content for preterm formula, thereby promoting the use of
calcium sources with better fractional absorption rate as well as mechanical stimulation of the
skeleton during the neonatal period. Considering that a calcium retention level ranging from 60
18
510
to 90 mg/kg/day assures appropriate mineralization and decreases the risk of fracture, an
intake from 120 to 140 mg/kg/day (110-130 mg/100 kcal) of highly bioavailable calcium salts
and 60 to 90 mg/kg/day (55-80 mg/100 kcal) of phosphate is recommended.
Magnesium
515
The skeleton represents the largest magnesium (Mg) store (60%) divided in two compartments:
firmly bound to apatite and non mobilisable, and absorbed to the surfaces of the mineral crystals
and contributing to Mg homeostasis [84]. The remaining Mg is distributed throughout skeletal
muscle, the nervous system and other organs with high metabolic rates. As the second most
abundant intracellular cation, Mg is crucial to many physiological functions. Mg crosses the
520
placental barrier freely and accumulates in the foetus during the whole gestation at a daily rate of
3 to 5 mg [85, 86].
About 40% of ingested Mg is absorbed, mainly in proximal parts of the small intestine. Newborn
infants, and especially preterm infants, have a high capacity for Mg intestinal absorption [84, 86].
The factors regulating Mg intestinal absorption are largely unknown. Concentration in the
525
digestive tract is the major determinant of the amount of Mg absorbed. Substances increasing Mg
solubility favour its absorption, whereas substances that form insoluble complexes tend to decrease
it. Recent data show that there is no competition between magnesium and calcium for absorption,
as calcium supplementation does not decrease Mg absorption. By contrast, phosphates can inhibit
Mg absorption through the formation of insoluble Mg complexes in the intestine. While higher
530
values have been reported in preterm infants fed fortified human milk, Mg absorption appears to be
similar in human milk and formula fed infants. Preterm infants fed human milk providing a low
Mg intake 5.5 to 7.5 mg/kg/d, showed limited retention, i.e. lower than intrauterine accretion, with
reduced urinary excretion, suggesting a relative intake deficit. By contrast, preterm infants fed
formula providing a mean of 8 to 12 mg/kg/d showed Mg retention in the range of intrauterine
535
accretion, with slightly higher urinary excretion. [87].
Considering foetal accretion,, a magnesium intake of 8 to 15 mg/kg/d (7.5-13.6 mg/100 kcal)
appears appropriate and is in line with previously suggested requirements [2, 3].
Iron
19
540
Iron is essential to brain development, and prevention of iron deficiency is important. Many observational
studies have shown an association between iron deficiency anaemia and poor neurodevelopment in infants
[88]. In contrast to most other nutrients, however, there is no mechanism for regulated iron excretion from
the human body. Excessive iron supplementation of infants may lead to increased risk of infection, poor
growth and disturbed absorption or metabolism of other minerals [89]. Furthermore, iron is a potent pro-
545
oxidant and non protein bound iron has been suggested to cause formation of free oxygen radicals and to
increase the risk of retinopathy of prematurity, especially when given in high doses as a component of
blood transfusions or as an adjunct to erythropoietin therapy [90-93]. Thus, one must not only prevent iron
deficiency but also iron overload.
Total body iron in foetuses and newborns is approximately 75 mg/kg [94]. At a growth rate of 15-20
550
g/kg/d, this translates to an iron accretion rate of 1-1.5 mg/kg/d. This does not apply to neonates born at
term since the normal decline in haemoglobin concentration after birth significantly increases iron stores in
other tissues.. A healthy, term infant is initially independent of external iron supply and can double its birth
weight before iron stores are depleted. To some extent this is true for preterm infants as well. However, the
relative size of iron stores at birth in preterm infants is not known. Furthermore, while a term infant will
555
double its birth weight in about 5 months, a preterm infant will do so in 1-2 months. In VLBW infants, iron
losses due to phlebotomy can amount to 6 mg/kg per week [95]. On the other hand, each red blood cell
transfusion typically adds about 8 mg/kg of iron. Variation in practices regarding blood sampling, blood
transfusions and erythropoietin treatment will therefore greatly influence iron requirements of preterm
infants.
560
Iron absorption from iron supplements given between feedings is about 25-40% in preterm infants, which is
higher than in term infants [96-100]. Iron absorption from preterm formula was reported to be 11% [101].
Studies are lacking on iron absorption from multinutrient fortified human milk or from iron supplements
given with human milk. Yet, absorption from these sources might be higher than that from preterm
formula, considering reports of higher iron absorption from human milk than from formula in term infants
565
[102], but human milk has a very low iron content (about 0.3 mg/L) [103].
A meta-analysis of studies published prior to 1992 showed that prophylactic iron supplementation given to
preterm infants significantly reduced incidences of anaemia at 6 months [104]. Enteral iron dosages of
about 2 mg/kg/d had been used in these studies [105, 106]. Two more recent randomized studies have
compared different doses of iron supplementation in preterm infants [107, 108]. Hall et al [107]
570
administered 0.3 or 1.3 mg/kg/d to infants with an average birth weight of 1.4 kg. Even at higher intakes,
one third of the infants showed insufficient serum ferritin levels. The most comprehensive trial of iron
20
supplementation in preterm infants was published by Friel et al [108]. Infants with an average birth weight
of 1.46 kg received an iron intake of 5.9 vs. 3.0 mg/kg/d at discharge and about 3 vs. 2 mg/kg/d at 3-9
months. There was no difference between the two groups in anaemia prevalence or neurodevelopment at 12
575
months, but the high-iron group had higher glutathione peroxidase concentrations (a marker of oxidative
stress), lower plasma zinc and copper levels, and more respiratory tract infections, suggesting possible
adverse effects from the higher intake.
In a more recent open trial, Franz et al randomized 204 infants with an average birth weight of 0.87 kg into
an early iron group receiving 2-4 mg/kg/d of iron supplements from about 2 weeks and a late iron group
580
that did not receive iron supplements until 2 months of age [109].. There were no differences in serum
ferritin and hematocrit at 2 months of age but infants in the late iron group had received more blood
transfusions.
Iron intakes of <2 mg/kg/d are likely to result in iron deficiency in preterm infants, at least in those with
birth weights less than 1800 g. Since high enteral iron intakes have been associated with possible
585
adverse effects, an intake of 2-3 mg/kg/d, corresponding to 1.8-2.7 mg/100 kcal is recommended.
Prophylactic enteral iron supplementation (given as a separate iron supplement, in preterm formula or in
fortified human milk) should be started at 2-6 weeks of age (2-4 weeks in ELBW infants). Infants who
receive erythropoietin treatment and infants who have had significant, uncompensated blood losses may
initially need a higher dose, requiring a separate iron supplement in addition to preterm formula or fortified
590
human milk. Enteral iron doses >5 mg/kg/d should be avoided in preterm infants because of the possible
risk of retinopathy of prematurity. Iron supplementation should be delayed in infants who have received
multiple blood transfusions and have high serum ferritin concentrations [110]. Iron supplementation should
be continued after discharge, at least until 6-12 months of age depending on diet.
595
Trace elements
Zinc
Zinc is essential for a multitude of enzymes and plays an important role in cellular growth and
differentiation. Zinc deficiency leads to stunted growth, increased risk of infection, skin rash and possibly
poor neurodevelopment [111]. Marginal zinc deficiency is difficult to diagnose due to the lack of a reliable
600
biomarker [112]. Zinc homeostasis is maintained by regulation of absorption and endogenous excretion to
the gastrointestinal tract; this regulation seems to function to some extent also in moderately preterm
21
infants [113]. The fractional absorption in preterm infants is about 30-40 % and is higher from breast milk
than from formula [2].
The zinc content in colostrum is high (5.4 mg/L), decreasing to 1.1 mg/L in mature milk at 3 months [114].
605
The high concentration of zinc in early breast milk in combination with the release of stored zinc from
hepatic metallothionein helps protect the infant from zinc deficiency during early infancy. These
mechanisms are insufficient, however, in infants with birth weights less than 1500-2000 g. The foetal
accretion rate of zinc has been estimated at about 0.85 mg/d during the last 3 months of gestation [115],
corresponding to 35 µg/g weight gain. Using the factorial method, dietary zinc requirements for preterm
610
infants have been calculated as 1-2 mg/kg/d [2].
Three randomized studies have examined effects of different zinc intakes in preterm infants with average
birth weights of 1.1-1.7 kg [116, 117] [118]. Two of the studies compared post discharge formulae with
different zinc concentrations: 0.7-1.0 mg/100 kcal in the low zinc formulae and 1.5-1.6 mg/100 kcal in the
high zinc formulae. In the high zinc groups, plasma zinc concentrations were higher at 3 months, and linear
615
growth was higher at 6 months. One of these studies found higher motor development scores in the high
zinc group [117]. The third study [118] was a comparison between two human milk fortifiers. In this study,
zinc intakes were lower (about 0.3-0.6 mg/100 kcal) and there was no difference in average serum zinc
between the groups. The growth results from this study are difficult to interpret since the two fortifiers also
differed in the contents of other nutrients.
620
These clinical trials suggest that an intake of > 1 mg/100 kcal may be needed in order to achieve optimal
growth in preterm infants.
Zinc is a relatively non-toxic nutrient which, in contrast to iron and copper, does not have a pro-oxidant
effect. There is one described case in which several months of oral zinc supplementation at a dose of 3.6
mg/kg/d led to symptomatic copper deficiency in a young child [119]. The assumed mechanism was
625
inhibition of copper absorption by zinc supplementation. In clinical studies, zinc doses up to 1.8 mg/kg/d
have been used in preterm infant formula without adverse effects on the metabolism of copper or other
minerals [120]. The maximum recommended zinc concentration in term formula is 1.5 mg/100 kcal [5,
121]. For preterm infants, a zinc intake of 1.1-2.0 mg/kg/d or 1.0-1.8 mg/100 kcal is recommended.
630
Copper
Copper is an essential nutrient that functions as a cofactor of different enzymes, e.g. in the electron
transport chain, in collagen formation, and in neuropeptide synthesis [122]. It is also a component of
22
antioxidant enzymes, e.g. copper/zinc superoxide dismutase (CuZn-SOD) [123]. Low birth weight is a risk
factor for copper deficiency [124]. Severe copper deficiency is a rare condition associated with anaemia,
635
neutropenia, and osteoporosis [124]. Little is known about the prevalence and possible health impact of
marginal copper deficiency [125]. However, a recent paper describes an association of copper status with
head circumference at birth [126].Copper status can be assessed from the plasma concentration of copper or
of ceruloplasmin, the main copper binding protein in plasma. Both concentrations are decreased in severe
copper deficiency but are insensitive biomarkers for marginal copper deficiency [127]. The activity of
640
CuZn-SOD in erythrocytes is considered to be the most sensitive marker of Cu deficiency [128].
The intrauterine accretion rate of copper is approximately 50 µg/kg/d [114, 129]. Similar to iron and zinc,
there is a hepatic store of copper at birth, bound to metallothionein, which is utilized during early infancy.
Fractional copper absorption is about 60% from breast milk but as low as 16% from bovine milk. Copper
homeostasis is maintained by regulation of both intestinal absorption as well as biliary excretion. Using a
645
factorial method, the minimum enteral copper requirements for preterm infants have been estimated to be
100 µg/kg/d [2].
The copper content of human milk declines from 600 µg/L during the first week of lactation (800 µg/L in
preterm milk) to 220 µg/L by 5 months [114, 130].
Iron and especially zinc may, in sufficient doses, impair copper absorption. It has therefore been suggested
650
that the zinc to copper molar ratio in infant formulae should not exceed 20 [2].
High doses of copper can damage the liver, kidneys and the central nervous system [131]. Infant rhesus
monkeys fed infant formula with a high copper concentration (6.6 mg/L, corresponding to about 1000
µg/100 kcal) from birth to 5 months did not show any clinical evidence of copper toxicity and there was no
histological damage to the liver [132]. On the other hand, a number of human cases have been reported
655
where infants and young children developed cirrhosis due to high chronic copper exposure from drinking
water from copper pipes or copper utensils for preparation of foods [133]. The risk of copper induced liver
damage appears to be particularly high in young infants in the first 3 months of life, and also in children
with some degree of cholestasis. Preterm infants might thus be particularly susceptible to toxic effects of
high copper supply. There are very few clinical trials of different copper intakes in preterm infants. Enteral
660
feeding of 41-89 µg/kg/d of copper in preterm infants has been associated with copper deficiency [134].
Tyrala showed no benefit of a copper intake of 260 µg/100 kcal compared with 120 µg/100 kcal in preterm
infants as assessed by copper balance, serum copper and ceruloplasmin [135]. For that matter, no adverse
effects were observed in the high copper group. A daily copper intake of 100-132 µg/kg or 90-120
23
µg/100 kcal is recommended; the local copper content of the drinking water should be taken into
665
account when powdered formula is used.
Selenium
Selenium is an important antioxidant of which a deficiency is associated with increased morbidity in
preterm infants [136, 137]. A review of published postnatal concentrations of selenium shows that
670
selenium supplementation of at least 5 µg/kg/d is necessary to maintain blood selenium concentration close
to the range of that of umbilical concentrations [138]. Since very high intakes may cause untoward effects,
the maximum concentration of selenium in preterm infant formula should not exceed that of term infants
[5]. The recommended selenium intake for preterm infants is 5-10 µg/kg/day or 4.5 to 9 µg /100 kcal.
675
Manganese
There is no evidence that currently used manganese supply to preterm infants is inadequate .
Postconceptional and postnatal age, iron status, and calcium or iron intake significantly alter manganese
status. Using a ratio of 50 mg calcium to 1 µg manganese [2], a minimum manganese intake of 2.5 µg/kg/d
has been recommended. Foetal manganese accretion, however, is estimated at 7 µg/d [139], which suggests
680
that manganese intake should be higher. However, very high manganese intakes should be avoided, since
they may cause accumulation in tissues, including the brain, and induce neurodevelopmental abnormalities
[140]. As for copper, manganese is secreted in the bile, and, although not demonstrated in clinical trials,
high intake of manganese may enhance accumulation in the presence of cholestasis. A maximum
manganese intake of 27.5 µg/kg/d is recommended, which is the highest manganese intake shown to be
685
safe [141] and associated with positive manganese balance. Whey protein, calcium salts and ferrous
sulphate may be contaminated with manganese [142], which has to be taken into account before adding
manganese to formula. The recommended intake of manganese should not exceed 27.5 µg/kg/d.
Preterm infant formula should contain 6.3 to 25 µg /100 kcal.
690
Fluoride
There is insufficient firm evidence to recommend a minimum requirement of fluoride for preterm infant
feeds, considering infants may be exposed to an additional fluoride intake, e.g. from fluoride containing
water [5]. Fluoride content of human milk is highly variable and ranges from 2-19 µg/L [2]. Fluoride
24
content of human milk is highly variable and ranges from 2-19 µg/L [2]. A very high intake during early
695
infancy may carry the risk of dental fluorosis. VLBW children seem to be at a higher risk of dental enamel
defects [143]. In older children, the ‘no observed adverse effect’ level was set at 60 µg/kg/d (US
Environmental Protection Agency, 1989). A reasonable minimal intake of fluoride is set at 1.5 µg/kg/d
(10 µg/L) and a reasonable maximum intake at 60 µg/kg/d or 1.4 to 55 µg/100 kcal.
700
Iodine
Iodine deficiency in preterm infants may exacerbate transient hypothyroxinaemia and may be associated
with adverse respiratory or neurological outcomes, even though the available data are insufficient to draw
firm conclusions [144]. Considering the effects on thyroid function of various iodine intakes in preterm
infants born in countries with presumably normal or endemic low iodine intakes and available balance
705
studies [145, 146], the recommended iodine intake is 11 - 55 µg/kg/d or 10 - 50 µg /100 kcal.
Chromium
Chromium concentration in human milk ranges from 200-8200 ng/L. In general, infant formulae contain
higher amounts [147]. Chromium potentiates the action of insulin, but plasma chromium concentrations
710
were not found to be lower in hypoglycaemic infants [148]. A firm basis is lacking for recommending
intakes other than that found in human milk. Thus the chromium intake should be in the range of 30 1230 ng/kg/d or 27 - 1120 ng/100 kcal.
Molybdenum
715
Human milk supplies 0.3 µg/kg/d of molybdenum, and it has been shown to be absorbed at a high rate
[149]. Retention in preterm infants increased as much as 36-fold upon an increased intake from average
0.024 to 0.284 µmol/kg/d, but the median intake of 2.3 µg/kg/d did not ensure positive molybdenum
balance in two groups of preterm infants studied [150]. These results suggest that a minimum provision of
molybdenum intake close to this level is needed, but that there is no need to provide more than 5 µg/kg/d
720
[151].
Based on the concentration in human milk and the available data in preterm infants, the expert panel
recommends that molybdenum intake should be in the range of 0.3 - 5 µg/kg/d or 0.27 to 4.5
µg/100kcal.
25
725
Vitamins
Water-soluble vitamins are cofactors for enzyme reactions in intermediary metabolism, and as
such, required amounts are related to energy and protein supply as well as the infant’s growth and
energy utilization. The preterm infant is at risk of deficiency because the mechanism of
assimilating and conserving vitamins is still immature, coupled with low tissue stores, rapid
730
growth and high metabolic turnover. With the exception of vitamin B12, water-soluble vitamins
are not stored in the body to any large extent and are rapidly depleted if intake is marginal.
Minimum supplies of each vitamin should ensure the preterm infant’s normal growth and
development and take away the risk of developing metabolic depletion. As human milk may not
provide adequate amounts of many water-soluble vitamins, the requirements for the enterally fed
735
preterm infants cannot be solely determined based on the concentration ranges of vitamins in
human milk. Nevertheless, in the absence of valid data on the minimal requirement of a specific
vitamin, minimum supplies are based on the upper ranges of supplies with human milk. Maximum
levels should avoid the risk of excess, however, very high intakes of water-soluble vitamins in
preterm infants have generally not been systematically evaluated for their biological effects and
740
potential interaction with other formula components, and neither have safety aspects been well
documented. Since the content of several vitamins in formulations may decrease during production
and storage, often overages are added. Nevertheless, it is recommended not to add to preterm
infant formulae excessive amounts of any nutrient that does not serve any nutritional purpose or
provides any other benefit.
745
Thiamin (vitamin B1)
Thiamin (vitamin B1) is a coenzyme in reactions related to energy metabolism and is therefore
related to energy intake. Much higher intake than that provided by human milk is needed to
maintain adequate thiamine status in premature infants [152-154]. An adequate intake for
750
preterm infants is considered to be 140-300 µg/kg/day or 125-275 µg/100 kcal.
Riboflavin (vitamin B2)
26
Based on biochemical indices, a minimal intake of 200 µg/kg/day is needed to ensure normal
riboflavin status in preterm infants. Adequate riboflavin status of preterm infants is supported by
755
an average intake of 300 µg/kg/d [152-155]. Intakes higher (i.e., 670 µg/100 kcal; [152]) than the
current recommendation of 620 µg/100 kcal[2] result in low erythrocyte glutathione reductase
activity, which may be detrimental [156]. Because flavins are thought to contribute to oxidative
stress through their ability to produce superoxide [156] the expert panel recommends that
riboflavin content in preterm infant formulae should not exceed two times the minimum level. The
760
adequate riboflavin intake recommended for preterm infants is 200-400 µg/kg/day or 180365 µg/100 kcal.
Niacin (vitamin B3)
There is insufficient evidence for defining quantitative niacin requirements of preterm infants.
765
Because the protein intake recommended by the expert panel is higher than the previous
ESPGHAN recommendations, niacin synthesis from tryptophan may be higher as well. Given
there are no data suggesting such an effect, a minimum intake corresponding to the upper limit of
the human milk concentration is recommended. The maximum limit can not be set with accuracy.
The maximum limit set previously by the LSRO panel [2], which corresponds to the upper limit
770
found in available preterm formulae, is most probably safe. There are no known adverse effects
caused by ingestion of such amount of niacin in preterm infants, and the upper limit of safety of 7
mg/d is also not expected to result in any adverse effects [157]. A niacin supply of 380-5500
µg/kg/day or 345-5000 µg/100 kcal is recommended [2].
775
Pantothenic acid (vitamin B5).
There is not enough published evidence to determine quantitative requirements for preterm infants.
No deficiencies for pantothenic acid have been reported for preterm infants fed human milk. In
line with previous guidance [2], 0.33-2.1 mg/kg/day or 0.3-1.9 mg/100 kcal is recommended.
780
Pyridoxine (vitamin B6).
The vitamin B6 content of term breast milk varies markedly, from 70 to 310 µg/L (10.4 to 46.3
µg/100 kcal) [158]. Factors that affect the concentration in mature milk include nutritional status of
27
the mother, stage of lactation, length of gestation, and use of drugs [159]. Providing preterm
infants with 300 µg/kg/d vitamin B6 for a period of one month did not show any adverse effects
785
[160]. In the past, vitamin B6 requirement was found to be related to protein intake (14.7µg/g
protein, [161]) and a lower intake (4.3 µg/g protein) resulted in deficiency symptoms. Assuming a
protein content of 2.7 - 4.1 µg/100 kcal, the vitamin B6 level should be 41 - 61 µg/l00 kcal.
A vitamin B6 intake of 45 - 300 µg/kg/d or 41 - 330 µg/100 kcal is recommended.
790
Cobalamin (vitamin B12)
Even though hepatic storage of vitamin B12 in preterm infants is less than that of term infants
[162], no deficiency has been reported in preterm infants fed human milk. Considering average
human milk contents [163] and previous recommendations [2], a vitamin B12 intake of 0.10.77 µg/kg/day or 0.08-0.7 µg/100 kcal is recommended. However, the upper end of the
795
recommended range should not be considered an absolute maximum intake since under some
circumstances a much higher vitamin B12 intake may be needed. For example, combined
treatment with erythropoietin, intravenous iron, folate, and vitamin B12 (3 µg/kg/day given
subcutaneously or intravenously) during the first weeks seems effective in stimulating
erythropoiesis and in reducing the need for transfusion in VLBW infants [164, 165].
800
Folic acid
Adequate folate status of preterm infants, based on biochemical indices including plasma and RBC
folate concentrations, is usually supported by a minimum intake of 30-40 µg/kg/d [152, 154].
Associated with an erythropoietin, iron and vitamin B12 therapy, 100 µg/kg/d of folate has been
shown to improve red blood cell counts, haemoglobin and hematocrit levels and to reduce needs
805
for blood transfusion [164, 165]. A folic acid intake of 35-100 µg/kg/day or 32-90 µg folic
acid/100 kcal is recommended.
L-ascorbic acid (vitamin C)
A minimum level of 10 mg/100 kcal in infant formula is recommended for term infants [5], and
810
there are no data suggesting that this would not be appropriate for preterm infants. An average
intake of 28 mg/kg/d was associated with adequate plasma vitamin C concentrations [153]. A
vitamin C intake of 40-46 mg/kg/d vs. 20-26 mg/kg/d tended to reduce the rate of
28
bronchopulmonary dysplasia at 36 weeks postmenstrual age [166]. The recommended vitamin C
supply is 11-46 mg/kg/day or 10-42 mg/100 kcal.
815
Biotin
The expert panel found no evidence to define biotin requirements for preterm infants. No
deficiencies have been reported for enterally fed preterm infants [2]. Recent data have shown that
biotin not only acts as a carboxylase cofactor, but also affects several systemic functions, such as
820
development, immunity and metabolism, via transcriptional and post-transcriptional effects or
function related to its attachment to histones [167, 168]. There is no evidence of any toxicity in
preterm infants fed currently available preterm formulae containing large amounts of biotin.
Taking into account reported human milk contents in the range of about 0.75-1.3 µg/100 kcal
[163], a minimal level similar to that for term infants [5] is recommended for preterm infant
825
formulae. Also, considering the rapid growth of preterm infants, and the absence of data
supporting otherwise, the expert panel has set the maximum level as twice that recommended for
term infants. Recommended biotin intakes for preterm infants are 1.7-16.5 µg/kg/d or 1.5-15
µg/100 kcal.
830
Lipid-soluble vitamins (A, E, D, K)
The lipid-soluble vitamins A, E, D and K are absorbed with dietary fat and are stored primarily in
liver and adipose tissue. Preterm infants may show evidence of fat-soluble vitamin deficiency
owing to several factors, including limited tissue storage at birth, intestinal malassimilation, and
rapid growth rates that increase requirements. On the other hand, all lipid-soluble vitamins with the
835
exception of vitamin K are generally excreted more slowly than water-soluble vitamins, and
vitamins A and D can accumulate and cause toxic effects. High intakes over prolonged periods of
time may thus induce untoward effects. Therefore, both too low and too high intakes must be
avoided.
840
Vitamin A
In the VLBW preterm newborn, low vitamin A reserves associated with low retinol binding
protein concentrations cause lower plasma concentrations than in term newborns. In preterm
29
infants (especially ELBW), intraluminal bile acid concentrations are often low, which may affect
not only total lipid but also retinol absorption. The commonly used unit for vitamin A is µg retinol
845
equivalent (µg RE; 1 µg RE = 3.33 IU vitamin A). The efficacy of retinol synthesis from ßcarotene in preterm babies is unsure, therefore, the vitamin A requirement should be met by
providing preformed vitamin A. There is not enough vitamin A in human milk (~120-180 µg
RE/L) to support the needs of the preterm infant. Vitamin A deficiency is considered by many
authors to be a contributing factor to the development of chronic lung disease, primarily in
850
extremely preterm infants [169]. A minimum vitamin A intake can be estimated at 404-530 µg
RE/kg/d (1335-1750 IU/kg/d) [170, 171]. A mean dose of 1000µg RE/kg/d 3000 IU/kg/d) given
enterally did not appear to be toxic[170]. On the other hand, 1200 µg RE/kg/d administered to
infants resulted in some instances in very high retinol binding protein levels, associated with toxic
vitamin A levels [172].
855
A vitamin A intake of 400 -1000 µg RE/kg/d (1330 -3330 IU vit A/kg/d) or 360 - 740 µg/100
kcal (1210 -2466 IU vit A/100 kcal), provided as preformed vitamin A, is recommended.
30
Vitamin D
Vitamin D is important for supporting a vast number of physiological processes such as
860
neuromuscular function and bone mineralization. The intestinal receptor-dependent actions of
calcitriol [1,25(OH)2D] are critical for optimal calcium absorption [173] and the pathways of
vitamin D absorption and metabolism are fully operative in babies less than 28 weeks of gestation
[174, 175]. Nevertheless, the requirements for optimal growth in VLBW and ELBW infants are
still matters of discussion.
865
In 1987 the ESPGAN Expert Panel on Nutrition of the Preterm Infant [1] recommended that
preterm infants formulae do not contain more than 3µg or 120 IU/dL of vitamin D, and that an
additional 800 to 1600 IU be provided to all human milk- and formula-fed preterm infants. By
contrast, in 2002, the Life Sciences Research Office (LSRO) panel of the American Society for
Nutritional Sciences [2] recommended that all vitamin D supply be included in pre-term formulae
870
and human milk fortifiers, with a minimum and a maximum vitamin D content ranging from 75 to
270 IU/100 kcal. More recently, Atkinson et al. [3] suggested a total minimal and maximal daily
intake vitamin D of 5 μg (200 IU) and 25 μg (1000 IU) that could be translated into 150-400
IU/kg/day or 115 to 364 IU/100 kcal.
Maternal nutrition is unequivocally recognized as a determinant factor for the health of the
875
neonate. Studies have consistently demonstrated a direct relationship between maternal and cord
blood 25-OHD concentrations although being 20 to 30% lower in the foetal compartment [176178]. The effect of maternal vitamin D insufficiency may be deleterious for the newborn infant,
especially the premature, and was found to be associated with increased incidence of rickets and
long term reduction in bone mineral content [179, 180]. Compromised vitamin D bioavailability
880
may also contribute to vitamin D deficiency in preterm infants. Decreased cutaneous vitamin D
synthesis due to low UVB light irradiation in the course of prolonged hospitalization, relative
insufficiency in bile salt production that could impair the enterohepatic cycle and fat absorption,
and the use of corticosteroid, caffeine or barbiturates treatments may all theoretically impair
vitamin D metabolism [180, 181].
885
Although the measurement of serum 25-OHD concentration is generally accepted as being the
appropriate means of assessing nutritional status, there is much debate as to the minimum
circulating level required to meet the needs of the growing child. In adults, recent data using
various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and
skeletal density measurements, suggest this minimum level to be 80 nmol/L (32 ng/ml). In
31
890
addition, the relationship between circulating vitamin D3 and 25(OH) D might be more indicative
of healthy optimal level. This relationship has recently been evaluated in significant sun exposed
and in vitamin D supplemented adults. The relationship was prove to be non-linear, but saturable
and controlled, suggesting that 25(OH) D is substrate dependent and that 25 hydroxylase operates
below its Vmax because of chronic substrate deficiency. Such studies suggest a need to increase
895
the vitamin D dietary allowance to 1000-2000 IU/d and the target value of circulating 25 (OH) D
to at least 75 nmol (30 ng/ml) [182-184].
Vitamin D, when given to preterm infants as early as the first week after birth, has consistently
been shown to raise the circulating levels of 25-OHD, independently of the colour of the skin or
mode of nutrition (enteral or parenteral). Several studies [170, 185-191] evaluated the relationship
900
between vitamin D3 intake and the mean circulating concentration of 25-OHD. The findings led to
the consensus that, in pre-term infants of vitamin D-deficient mothers, a vitamin D intake of 800 to
1500 IU/day is necessary to reach a circulating 25-OHD concentration above 75 nmol/L.
The LSRO panel recommendations are mainly based on standard balance studies from Bronner et
al. [192]. These showed that calcium absorption in LBW infants was directly proportional to the
905
daily calcium intake in the range from 40 to 142 mg/kg, and was independent of vitamin D
supplementation of up to 2000 IU daily. These studies led to the concept that most of the calcium
absorption in pre-term infants is probably due to a passive diffusion and that the vitamin Dregulated mechanisms are expressed during early infancy.
Bronner’s retrospective study was not initially designed to evaluate the influence of vitamin D on
910
Ca absorption rate. Results were collated from a large number of different metabolic balance
studies performed over time in preterm infants with the same methodology and in the same
laboratory. All 103 infants were fed human milk or pre-term formula and, received between 1000
to 2000 IU/d of vitamin D, except 11 who were fed human milk with no vitamin D
supplementation. Analysis with vitamin D supply as a co-variable was not performed but the effect
915
of vitamin D was derived mathematically from the relationship between Ca absorption and Ca
intake. In addition, the statement on the independence of the Ca absorption from vitamin D was
also derived from studies in rats in which maturation of the saturable calcium-specific transcellular transport occurs only after birth [193]. Whether these studies can be directly translated to
the human remains to be shown. On the contrary, we now have evidence that, as early as in the
920
20th week of gestation, the human foetal intestine possesses functional calcitriol receptors that
regulate the expression of CaBP and calcidiol-24-hydroxylase [194, 195]. In contrast to that study
32
[192] , Senterre et al. [196] have shown, by performing careful 3-day metabolic balances, that
calcium net absorption increased from 50 to 71% by feeding AGA pre-term infants weighing less
than 1500 grams, banked human milk alone, or supplemented with 30 µg of cholecalciferol/day
925
(1200 IU) without Ca fortification. One year earlier, Senterre and Salle [197] had reported similar
results in a study involving 28 pre-term babies fed banked human milk. These 2 studies
demonstrated that vitamin D indeed affected the Ca absorption rates.
Vitamin D metabolism and intestinal maturation are appropriate in preterm infant, even in the most
premature infants. Nevertheless, vitamin D and Ca bioavailability could be impaired requiring
930
higher supply during the first weeks of life. There is a general consensus to increase the reference
values and the threshold level of circulating vitamin D in infants as in adult with target value for
25-OHD > 80 nmol/L [184]. Considering the prevalence of vitamin D deficiency in pregnant
mothers, higher vitamin D supply in pre-term infants could be necessary to rapidly correct the
foetal low plasma level. A vitamin D intake of 800 to 1000 IU/d (and not per kg) during the
935
first months of life is recommended. This implies that a formula should provide for basic needs
to which a supplement has to be given e.g. in the order of 100-350 IU/100 kcal, avoiding toxic
intakes at high levels of formula consumption. An intake of 800 to 1000 IU/d would improve
serum 25-OHD concentration and the plasma levels of 1,25 OH2 D and subsequently the Ca
absorption rate, allowing to reduce the high calcium content of some formula. This statement holds
940
for both premature infants fed mother’s milk and those fed formula milk.
Vitamin E
Vitamin E is found in all tissues, where it serves as an antioxidant and free radical scavenger. It
blocks natural peroxidation of polyunsaturated fatty acids found in lipid layers of cellular
945
membranes. Preterm infants have less vitamin E reserves since 90% of the tocopherol pool is
located in adipose tissue; early provision of vitamin E is necessary to correct the depleted state and
prevent adverse consequences attributable to insufficient antioxidants. This is usually done
parenterally during the first days/weeks of life. Haemolytic anaemia associated with vitamin E
deficiency is aggravated by iron supply [198]. However, large doses of vitamin E (50 IU/d = 34
950
mg alpha-TE; alpha-TE = alpha-tocopherol equivalent; 1 mg alpha-TE = 1,49 IU)) did not increase
the response to erythropoietin and iron [199]. Preterm infants should be supplied with 2.2 to 11
mg alpha-TE/kg/d [2, 170], or 2 to 10 mg alpha-TE/100 kcal.
33
Since vitamin E requirements have been reported to increase with the number of double bonds
contained in the dietary fatty acid supply [200], the following factors of equivalence should be
955
used to adapt the minimal vitamin E content to the formula fatty acid composition: 0.5 mg (alphaTE/g linoleic acid (18:2n-6), 0.75 mg alpha-TE/ alpha-linolenic acid (18:3n-3), 1.0 mg alpha-TE/g
arachidonic acid (20:4n-6), 1.25 mg alpha- 470 TE/g eicosapentaenoic acid (20:5n-3), and 1.5 mg
alpha-TE/g docosahexaenoic acid (22:6n-3).
960
Vitamin K
Vitamin K is the only vitamin routinely administered in large quantities at birth and no toxicity has
been reported in the premature infant despite the wide range of intakes up to 25 μg/100 kcal [201].
Vitamin K intakes above ~4 µg/100 kcal provide an effective protection against vitamin K
deficiency [202]. Assuming prophylactic vitamin K1 supply given within the first hours after
965
birth, the recommended supply with formulae for preterm infants [2, 202] is 4.4-28
µg/kg/day or 4 - 25 µg/100 kcal.
Pre- and Probiotics
The immune system of preterm infants is still immature. These premature infants are prone to delayed
970
gastrointestinal colonization, reduced microbial diversity in the bowel, acquisition of antibiotic-resistant
strains, loss of strains associated with antibiotic treatment, and increased intestinal bacterial translocation
[203-206]. These effects make the preterm infant more susceptible to antibiotic-resistant infections,
systemic inflammatory response syndrome, and NEC [206-208]. A recent systematic review showed a
significant decrease in NEC after the introduction of different strains and dosages of probiotics. [209]. The
975
authors, however, recommended more studies with regard to the type of strain, dosage and duration.
Prebiotics
Human milk contains more then 130 different oligosaccharides, which are fermented in part in the infant’s
colon. The concentration changes with the duration of lactation, being highest in the colostrum at 20–23
g/L, about 20 g/L on day 4 of lactation, and 9 g/L on day 120 of lactation [210]. Preterm infants show some
980
absorption of intact human milk oligosaccharides; but most resist digestion in the small intestine and
undergo fermentation in the colon [211]. Oligosaccharides are implicated in maintaining normal gut flora
and inhibiting growth of pathogenic bacteria. Their fermentation products, short-chain fatty acids, provide
nutrition and energy for the colonocytes and the body as a whole. Other evidence suggests that human milk
34
oligosaccharides may have anti-infection roles in the intestinal, respiratory, and urinary tracts. One of the
985
monomers of oligosaccharides, sialic acid, is a structural and functional component of brain gangliosides
and could play a role in neurotransmission and memory. The composition of oligosaccharides in human
milk is genetically determined and thus large variability in oligosaccharide composition exist in the
population. Therefore, it is difficult to define the exact oligosaccharide composition of human milk. In
infant formula primarily one type of oligosaccharide mixture (GosFos) has been systematically studied in
990
term and preterm infants [212-217]. GosFos are not oligosaccharides present in human milk but they
represent short and long chain moieties of oligosaccharides: GosFos is a mixture of 90 % short-chain
galactooligosaccharides and 10 % long-chain fructooligosaccharides. Only two randomized trials have been
conducted in preterm infants in which standard preterm formula was supplemented by GosFos, at
concentrations of 8 g/l and 9 g/l respectively. GosFos has been shown to increase faecal bifidobacteria
995
counts, to reduce stool pH, to reduce stool viscosity, and to accelerate gastrointestinal transport [216, 218].
It has been hypothesized that GosFos may accelerate feeding advancement, reduce the incidence of
gastrointestinal complications such as NEC, improve immunological functions, reduce the incidence of
hospital acquired infections, and improve long term outcome, but there are no data available from preterm
studies to support these assumptions. In adults prebiotics have been shown to increase stool volume and
1000
stool water and might possibly have a similar effect in the preterm infant. In randomized trials in infants, no
relevant adverse effects were detected, and weight gain (a secondary outcome measure) was not negatively
affected. Further trials relating to the safety of GosFos should address nutrient bioavailability, intestinal gas
production, intestinal water loss, intestinal flora, and possible interaction with other fermentable
substances.
1005
Probiotics
Several probiotics studied in randomized trials over the past two decades were shown to transiently modify
faecal flora in preterm infants [219-222]. As some clinical outcome studies in preterm infants are available,
we will not review here randomised trials that addressed surrogate outcome parameters, such as intestinal
IgA production, faecal calprotectin, cytokine production, or reduced gut permeability. A large and
1010
frequently cited cohort trial in newborns using historic controls suggested that probiotics might prevent
NEC (stage >2) [223]. Four well designed randomized controlled trials in VLBW infants have been
published so far, with mixed results. Lactobacillus rhamnosus GG failed to protect from NEC (Bell stage >
2) [224]. Bifidobacterium lactis failed to reduce the incidence of nosocomial infections within the first 6
weeks of life [225]. A mixture of Bifidobacterium infantis, Streptococcus thermophilus, and
1015
Bifidobacterium bifidus reduced the combined incidence of NEC including stage one disease [226].
35
Lactobacillus acidophilus together with Bifidobacterium infantis reduced the combined incidence of NEC
or death [227]. These studies have been criticized for various reasons, i.e. unclear power and sample size
calculation [224], no peer reviewed paper published [225], NEC of all stages as primary outcome rather
than stage >2 [226], unusually high local death incidence and use of death and NEC as combined outcome
1020
[227]. In a recent systematic review seven of 12 randomised controlled trials retrieved (n=1393) were
eligible for inclusion. Meta-analysis using a fixed effects model estimated a lower risk of necrotising
enterocolitis (relative risk 0.36, 95% CI 0.20-0.65) in the probiotic group than in controls. Risk of sepsis
did not differ significantly between groups, while risk of death was reduced in the probiotic group (RR
0.47, CI 0.30-0.73). In addition, the time to full feeds was significantly shorter in the probiotic group
1025
(weighted mean difference -2.74 days, 95% CI -4.98 to -0.51). The authors conclude that probiotics might
reduce the risk of necrotising enterocolitis in preterm neonates with less than 33 weeks' gestation[209]. A
study designed to prove NEC (Bell stage >2) incidence reduction from 5% down to 2.5% would require a
sample size of 792 subjects per group (one-sided Mann Whitney U test, a=0.05, ß=0.80). ). Currently the
most effective probiotic or combination of probiotics, dosage and timing are unknown. In addition, the
1030
effect might depend on type of feeding. Although the available studies have not reported any adverse
effects, we counsel caution in the introduction of any potentially infectious agent for immunologically
immature VLBW infants. While potential benefits must be weighed against potential harms, safety cannot
be defined as an absolute risk-free condition [228]. Future randomized probiotic trials should also address
the risk of transformation of probiotics in vivo, infections by probiotics, transposition of antibiotic
1035
resistance, and lasting effects on gut microbiota. Each probiotic strain and potential combinations need to
be characterized separately.
In conclusion, there is not enough available evidence suggesting that the use of probiotics or prebiotics in
preterm infants is safe. Efficacy and safety should be established for each product. We conclude that the
presently available data do not permit recommending the routine use of prebiotics or probiotics as
1040
food supplements in preterm infants.
Nucleotides
Nucleotides represent up to 20% of the non protein nitrogen in human milk; they contain a purine
base (adenine and guanine) or a pyrimidine base (cytosine, thymine and uracil) attached with a
1045
phosphorylated pentose sugar. Nucleosides are nucleotide precursors that lack phosphorylation.
Because nucleotides are structural units of nucleic acids – RNA and DNA – and are essential
compounds of the energy transfer systems (i.e. in adenosine and guanosine triphosphate), they are
36
assumed to play an important role in carbohydrate, lipid, protein and nucleic acid metabolism, and
to act as modulators of many neonatal physiological functions [229]. Dietary nucleotides have
1050
been reported to enhance small intestinal development [230], and to modify intestinal microflora
[231], blood lipids [232] and immune responses.
Nucleotides can be synthesized de novo, and the contribution of dietary nucleotides to the overall
pool of nucleotides is as yet uncertain. A wide range of nucleotide contents in human milk has
been reported which could be due to biological variation and to methodological differences in
1055
collection and measurement. The total amount of potentially available nucleotides in human milk
providing 670 kcal/L is estimated at 9.1 and 7.6 mg/100 kcal in 1 month post partum and in 3
months post partum milk respectively [233], corresponding to a total amount of potentially
available nucleotide content of 61 mg/L in early human milk and 51 mg/L in 3 months milk. It is
not clear, however, what proportions of these potentially available nucleotides can truly be utilized
1060
by the infant in vivo. In bovine milk, non protein nitrogen accounts for only 2-5% of the total
nitrogen, resulting in a nucleotide content in infant formula that is significantly lower than that of
human milk. Due to degradation during heat treatment, concentrations of nucleotides in many
infant formulae are lower than those in cow milk. In addition, cow milk shows a different
nucleotide profile, since cytidine and adenosine derivatives are present in relatively lower
1065
proportion.
Although thirteen nucleotides have been described in human milk, only five have been added to
infant formulae: cytidine 5’-monophosphate, uridine 5’–monophosphate, adenosine 5’ –
monophosphate, guanosine 5’–monophosphate and inosine 5’ –monophosphate. The available
preclinical and clinical studies do not provide convincing justification for the addition of
1070
nucleotides to term or preterm infant formulae.
It is concluded that there is not sufficient evidence to generally recommend addition of
nucleotides to preterm infant formulae. If nucleotides are added, the total amount should not
exceed 5 mg/100 kcal as recommended for term formulae [5].
1075
Choline
Choline is required to synthesize phospholipids (phosphatidylcholine, sphingomyelin) that are
essential components of biological membranes. Choline also is the precursor of the
neurotransmitter acetylcholine and is an important source of transferable methyl groups. Only a
37
small proportion of dietary choline intake is required for the synthesis of acetylcholine, but
1080
alterations in dietary choline will influence brain levels of this neurotransmitter [234]. Under
normal conditions, the exogenous supply of choline is sufficient to maintain acetylcholine
synthesis and release.
Human cells grown in culture have an absolute requirement for choline. Humans fed intravenously
with solutions containing little choline develop liver dysfunction similar to that seen in other
1085
choline deficient mammals[235]. Choline and choline ester concentrations are highest in colostrum
and transitional milk (150 µmol/dl) and lower in mature milk (75-110 µmol/dl). Cow’s milk-based
infant formulae contain 40-150 µmol/dl, depending on the protein source, and soy formulae
contain about 120 µmol/dl. Even though choline is a conditionally essential nutrient for humans of
all ages, there are no data to recommend an optimal intake of choline in premature infants. It is
1090
recommended that preterm infants should receive a choline supply of 8-55 mg/kg/d or 7 – 50
mg/100kcal in line with previous recommendations [2].
Inositol
Inositol is a six-carbon sugar alcohol present in biological systems primarily as myo-inositol. Inositol and
1095
phosphoinositides mediate signalling, activate cell surface enzymes and receptors , serve as growth factors
for human cell lines, are lipotrophic factors that promote lipid synthesis, and serve as a source of
arachidonic acid for the synthesis of eicosanoids [236]. Even though inositol is not listed as an essential
nutrient for humans, and no human deficiency syndrome has been described for inositol, studies indicate
that provision of supplemental inositol may be beneficial for infants born prematurely. Serum inositol
1100
levels are high during neonatal life and decline to adult levels by approximately 8 weeks of age [237].
Levels in foetal and preterm infant blood are significantly higher than in term infant blood. [237, 238].
Preterm infant cord blood levels are more than twice those of term infants and three times higher than
maternal levels [237-239]. These relatively high levels suggest that inositol plays an important role in
prenatal and neonatal development. A potential benefit of inositol supplementation has been reported for
1105
surfactant production and lung development as well as the protection against the development of
retinopathy of prematurity [240, 241]. Human milk inositol level is similar for mothers of term and preterm
infants up to four months of lactation [238] and decreases with time during the first six weeks post partum.
The concentration of myo-inositol in human milk ranges from about 13 to 48 mg/100 kcal.
A minimum content of 4 mg/100 kcal, as previously advised[2], and an upper limit that should not
1110
exceed that found in mature human milk (48 mg/100 kcal) are recommended.
38
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Klein, C.J., Nutrient requirements for preterm infant formulas. J Nutr, 2002. 132(6 Suppl 1): p.
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Tsang R, et al., Nutrition of the preterm infant. Scientific basis and practical application. 2nd ed.
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Koletzko, B., et al., 1. Guidelines on Paediatric Parenteral Nutrition of the European Society of
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Peppard, R.J., et al., Measurement of nutrient intake by deuterium dilution in premature infants. J
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Coulthard, M.G. and E.N. Hey, Effect of varying water intake on renal function in healthy preterm
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1600
1605
1610
1615
1620
1625
1630
51
1635
Table 1: Nitrogen balances according to feeding regimen in premature infants [81].
mg/kg/d
Fortified
Human milka
Liquid
formulaec
Protein hydrolysed
formulaed
Fed powder
formulab
n=88
n=58
n=31
n=49
Intake
517±86d
506±58
553±56 ab
522±70d
Faecal excretion
90±28 bc
71±28 abd
87±26 bc
49±19acd
Absorbed
428±76 bd
434±52 bd
466±51 ac
474±75ac
Urinary excretion
121±45 c
98±21 ad
122±39 c
106±36
Retained
307±56 bcd
337±46 ab
343±42 ab
368±57acd
Absorption %
82.7±4.8 bc
86.0±5.0ab
84.3±4.0 b
90.7±3.3acd
Net Protein utilization*%
59.7±7.7bc
66.6±5.8abd
62.4±6.5bc
71.5±6.5acd
Protein efficiency** %
72.1±7.6bc
77.5±4.4ad
74.0±6.9bc
77.7±6.4ad
*nitrogen retention/nitrogen intake; **nitrogen retention/nitrogen absorption; a,b,c,d p<0.05
52
1640
Table 2: Recommended intakes for macro- and micronutrients expressed per mg/kg/d and per 100 kcal
unless otherwise denoted. Calculation of the range of nutrients expressed per 100 kcal is based on a
minimum energy intake of 110 kcal/kg.
Min-Max
Kg –1 d –1
/100 kcal
Fluid (ml)
135 - 200
Energy (kcal)
110 - 135
Protein (g) < 1 kg body weight
4.0 – 4.5
3.6 – 4.1
Protein (g) 1 – 1.8 kg body weight
3.5 – 4.0
3.2 - 3.6
Lipids (g) (of which MCT  40%)
4.8 – 6.6
4.4. – 6.0
385 – 1540
350 – 1400
 55 (0,9% of fatty acids)
> 50
DHA (mg)
12 – 30
11 – 27
AA (mg)**
18 – 42
16 – 39
11.6 – 13.2
10.5 – 12
Sodium (mg)
69 - 115
63 – 105
Potassium (mg)
66 - 132
60 – 120
Chloride (mg)
105 – 177
95 – 161
Calcium Salt (mg)
120 – 140
110 – 130
Phosphate (mg)
60 – 90
55 – 80
Magnesium (mg)
8 – 15
7.5 – 13.6
Iron (mg)
2–3
1.8 – 2.7
Zinc (mg) ***
1.1 – 2.0
1.0 – 1.8
Copper (µg)
100 – 132
90 – 120
Selenium (µg)
5 – 10
4.5 – 9
Manganese (µg)
 27.5
6.3 – 25
Linolenic acid (mg)*
Alpha linolenic acid (mg)
Carbohydrate (g)
53
Fluoride (µg)
1.5 – 60
1.4 – 55
Iodine (µg)
11 – 55
10 - 50
30 – 1230
27 – 1120
0.3 – 5
0.27 – 4.5
Thiamin (µg)
140 – 300
125 – 275
Riboflavin (µg)
200 – 400
180 – 365
Niacin (µg)
380 – 5500
345 – 5000
Pantothenic acid (mg)
0.33 – 2.1
0.3 – 1.9
Pyridoxine (µg)
45 – 300
41 – 273
Cobalamin (µg)
0.1 – 0.77
0.08 – 0.7
Folic acid (µg)
35 – 100
32 – 90
L-ascorbic acid (mg)
11 – 46
10 – 42
Biotin (µg)
1.7 – 16.5
1.5 – 15
Vitamin A (µg RE) (1 µg ~ 3.33 IU)
400 – 1000
360 – 740
Chromium (ng)
Molybdenum (µg)
Vitamin D (IU/d)
800 – 1000 IU/d
Vitamin E (mg alpha-TE)
2.2 – 11
2 – 10
Vitamin K1 (µg)
4.4 – 28
4 – 25
5
Nucleotides (mg)
Choline (mg)
8 – 55
7 – 50
Inositol (mg)
4.4 - 53
4 – 48
* The linoleic acid to alpha-linolenic acid ratio is in the range of 5-15:1 (wt/wt). ** The ratio of AA to DHA should be in the
range of 1.0-2.0 to 1 (wt/wt), and eicosapentaenoic acid (20:5n-3) supply should not exceed 30 % of DHA supply. *** The zinc
1645
to copper molar ratio in infant formulae should not exceed 20.
54