Enteral nutrient supply for preterm infants



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 4M. 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, 17 L. Moreno; 18Neu 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

1Department of Paediatrics, San Paolo Hospital, University of Milan, Italy; 2Pediactrics, Obstetrics and Reproductive Medicine, University of Siena, Italy; 3Division 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; 9Ernst-Moritz-Arndt-University, Greifswald, Germany; 10Neonatologie Klinikum der Universität München, Germany; 11Hôpital Necker Enfants-Malades, University of Paris Descartes, Paris, France; 12Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, USA; 13Children’s Hospital, Zagreb Medical University, Croatia; 14Dr von Hauner Children’s Hospital, University of Munich, Germany; 15Hôpital Saint-Vincent de Paul, Paris, France; 16Deaconry Hospital, Schwaebisch Hall, Germany; 17Escuela Universitaria de Ciencias de la Salud, Zaragoza, Spain; 18Department of paediatrics, University of Florida, Gainesville, USA; 19Section of neonatal, Indiana University, School of Medicine, Indianapolis, USA; 20Leeds General Infirmary, Leeds, UK; 21Service de Néonatologie Réanimation néonatale, Hôspital de la Croix Rousse, Lyon, France; 22CHR Citadelle, University of Liege, Belgium; 23Bnai Zion Medical Center, Haifa, Israel; 24Service de Medicine de la Reproduction, Hôspital Edouard Herriot, Lyon, France; 25Department of paediatrics, University Medical Centre Groningen, The Netherlands; 26 Schneider Children's Medical Center, Tel-Aviv University, Tel Aviv, Israel; 27The Medical University of Warsaw, Poland; 28University of CO Health Sciences Center, Denver, USA; 29Jeanne de Flandre Children’s Hospital/University of Lille, France; 30Erasmus MC - Sophia Children's Hospital, Rotterdam, the Netherlands; 31Fomon Infant Nutrition Unit, Children’s Hospital, University of Iow, USA

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 (kindergesundheit.de). All meetings and the 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:

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

Abstract 4

Introduction 4

Fluid 5

Energy 6

Protein 8

Lipids 9

Carbohydrates< 12

Minerals 14

Trace elements 20

Vitamins 24

Pre- and Probiotics 32

Nucleotides 34

Choline 35

References 37

Tables 50

Abstract (235 words)

The number of surviving children born prematurely have increased substantially over the last two decades. 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 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 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.

Key words: Child Development, Embryonic and Fetal Development, *Premature infant feeding, *Nutritional Requirements

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.

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 (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 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 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 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.

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

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 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 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 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.

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 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 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 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 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 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.

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 per kg body weight at 24 than at 36 weeks post conceptional age), accumulated nutrient deficits (both pre- and 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 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 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 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 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 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 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 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

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, 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 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 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%). 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.

Compared to current preterm formulae, human milk contains a higher proportion of non-protein nitrogen (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 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 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 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 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 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 (LC-PUFA) 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.

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 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 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 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)

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 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 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 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.

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 (4.5-10.8 % 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 intakes in infants fed current preterm formulae. Linoleic acid intakes of 385-1540 mg/kg per day or 350-1400 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 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.

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 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 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 and EPA and not with AA. Nevertheless, subsequent clinical trials in LBW or term infants fed formulae 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 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 be addressed in clinical trials A reasonable range for the balance between AA and DHA appears to be 1.0-2.0 : 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].

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.

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 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 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 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 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

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].

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 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 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 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 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]. 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 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.

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 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 maltose or glucose polymers has been reported to improve feeding tolerance, weight gain and calcium absorption [62-66].

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 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

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) 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 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

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.

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 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 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 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 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 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 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.

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 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 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 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-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, 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 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 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

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 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 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 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 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

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-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 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 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.

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 [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] 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 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 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 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 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.

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 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 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]. 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 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 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.

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 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.

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 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, 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 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 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 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 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 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 µg/100 kcal is recommended; the local copper content of the drinking water should be taken into 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 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.

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 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 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.

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 content of human milk is highly variable and ranges from 2-19 µg/L [2]. A very high intake during early 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.

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 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 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

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 [pic]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 [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.

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 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 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 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.

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 preterm infants is considered to be 140-300 µg/kg/day or 125-275 µg/100 kcal.

Riboflavin (vitamin B2)

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 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 adequate riboflavin intake recommended for preterm infants is 200-400 µg/kg/day or 180-365 µg/100 kcal.

Niacin (vitamin B3)

There is insufficient evidence for defining quantitative niacin requirements of preterm infants. 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 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].

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.

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 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 [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.

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 0.1-0.77 µg/kg/day or 0.08-0.7 µg/100 kcal is recommended. However, the upper end of the 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].

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 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 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 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.

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 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 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.

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 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.

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 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 equivalent (µg RE; 1 µg RE = 3.33 IU vitamin A). The efficacy of retinol synthesis from ß-carotene 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 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].

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.

Vitamin D

Vitamin D is important for supporting a vast number of physiological processes such as 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.

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 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 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 [176-178]. 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 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].

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 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 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 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 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 D-regulated mechanisms are expressed during early infancy.

Bronner’s retrospective study was not initially designed to evaluate the influence of vitamin D on 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 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 trans-cellular 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 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 [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 (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 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 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 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 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 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.

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 used to adapt the minimal vitamin E content to the formula fatty acid composition: 0.5 mg (alpha-TE/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).

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 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 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 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 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 oligosaccharides may have anti-infection roles in the intestinal, respiratory, and urinary tracts. One of the 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 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 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 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.

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 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 Bifidobacterium bifidus reduced the combined incidence of NEC including stage one disease [226]. 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 [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 (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 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 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 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 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 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 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 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 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 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 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].

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 small proportion of dietary choline intake is required for the synthesis of acetylcholine, but 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 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 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 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 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 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 exceed that found in mature human milk (48 mg/100 kcal) are recommended.

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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 50 |

| DHA (mg) |12 – 30 |11 – 27 |

| AA (mg)** |18 – 42 |16 – 39 |

|Carbohydrate (g) |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 |

|Fluoride (µg) |1.5 – 60 |1.4 – 55 |

|Iodine (µg) |11 – 55 |10 - 50 |

|Chromium (ng) |30 – 1230 |27 – 1120 |

|Molybdenum (µg) |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 |

|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 |

|Nucleotides (mg) | |( 5 |

|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 to copper molar ratio in infant formulae should not exceed 20.

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