Human Milk Fortifiers for Preterm Infants: Do We Offer the Best Amino Acid Mix?

Abstract

For preterm and small-for-gestational age infants on enteral nutrition, the best solution is to add human milk fortifier (HMF) to human milk (HM) which is provided by the mother or a milk bank. HMF provides a means to add additional protein, energy, and micronutrients, while maintaining HM as the main source of nutrition. Because of their rapid increase of lean body mass, preterm infants have much higher protein requirements than term infants. 
Recommendations on protein requirements of preterm infants are available, but protein quality - i.e. the amino acid (AA) profile in HMFs has not been systematically assessed. Present guidelines for enteral nutrition recommend protein intakes around 4 g/kg body weight (BW) for preterm infants <1,500 g, an intake that is not achievable with unfortified HM intakes <200 mL/kg BW/day. It is generally assumed that the AA profile of HM is the best reference for the AA profile of HMF. We calculated advisable intakes of AAs for preterm infants between 400-2,500 g which are based on AA increments of the fetus. Corrections for absorption, inevitable losses, oxidation, and variation of AAs in HM were introduced. Our calculations indicate that extremely low birth weight (ELBW <1,000 g) and very low birth weight (VLBW <1,500 g) infants have substantially higher AA requirements than low birth weight (LBW) infants growing from 1,900 to 2,400 g. In ELBW infants, daily intakes of the different indis-pensable AAs (IAA) with 4 g of (term) HM protein/kg BW range between 59 and 125% of the respective advisable intakes. Intakes of 7 lAAs and 3 conditionally indispensable AAs (CIAA) are below advisable intakes. On the other hand, with 4 g HM protein per kg BW/day, the IAAs isoleucine and leucine and some dispensable AAs are already supplied in abundance. In VLBW infants, daily intakes of the IAA methionine and 3 CIAAs are still below the advisable intakes. In LBW infants (<2,000 g) receiving 3.5 g HM protein per kg BW daily intakes of 1 IAA and 3 CIAAs would be too low. Preterm infants should receive HMFs which provide adequate amounts of AAs which are needed for their rapid growth and development while avoiding excessive intakes. In particular, very high AA requirements of ELBW infants are a challenge. AA composition of present HMFs for preterm infants should be reconsidered: spiking HMF protein with the AAs which are presently undersupplied or providing targeted AA-based HMF are options to further improve the AA profile in fortifiers.

Protein and Amino Acids in Nutrition of Preterm Infants: What We Know and Need to Know

Mother's own milk is the first choice when enteral nutrition of extremely low birth weight (ELBW), very low birth weight (VLBW), and low birth weight (LBW) infants starts. Alternatively, banked human milk (HM) can be offered whenever mothers own milk is not available. HM has a strong trophic effect on the immature gut. This maturational effect not only enables earlier establishment of full feedings, it also provides protection against necrotizing enterocolitis (NEC) and late-onset sepsis [1] and has beneficial effects on brain development [2]. Proteins are an important constituent of HM, but also peptides and free amino acids (AAs) (5-10% of the total amount), molecules that also carry nitrogen atoms, are found in HM. All of these molecules can be used for tissue growth once absorbed. Besides their function as precursor for protein synthesis, HM contains many functional and immune-protective proteins and peptides [3-5]. Free AA are absorbed easily and may also serve a non-nutritive role as signaling molecules [6, 7]. Besides free AAs, peptides, and proteins, HM also contains non-protein-bound nitrogen such as urea molecules that may serve microbes in the intestines. In total, non-protein-bound nitrogen accounts for 20-25% of the total amount of nitrogen in HM.

Once full enteral nutrition is established or even earlier [1, 2], HM needs to be supplemented with HM fortifier (HMF) to meet requirements for growth. Because of their exceedingly high rate of growth, preterm infants, and specifically ELBW and VLBW infants have very high needs for all nutrients, among which protein is the most important one [2, 8]. After having adapted to extra- uterine life and being in stable condition, these infants can grow as fast as the87fetus [9]. Daily protein gain/kg BW (kg body weight) of the fetus between 23-26 and 26-29 gestational weeks is about 2.7 and 2.0 times higher, respectively (Table 1, bottom line) than the protein gain of term infants during the first month of life [10-12]. As a result, a continuing challenge in the neonatal intensive care units is the need to nourish these infants to achieve healthy growth, often as much as 2-3 kg of body mass over a 12-16-week period. Rapid growth of lean body mass - for example muscle, bone, brain, and organ mass - cannot happen without the necessary high intakes of protein of high quality, since these organs are built on a protein matrix. Most importantly, brain growth and later life cognitive function are directly related to protein intake during the neonatal period in preterm infants. Although other factors such as illness may play a role in the causation of growth failure, inadequate intake of protein of the best quality can result in growth failure of the extent and with the consequences observed: growth failure is strongly associated with poor neurodevelopment and cognitive outcome, which has been well documented [13-15].

The recommendations on the amounts of protein needed by ELBW and VLBW infants [1, 2, 8, 16] are based on the reference fetus [11] and clinical trials which have recently been reviewed and form the basis of evidence-based guide-lines [17]. The presently available powdered and liquid HMFs [18] deliver of 1.4-1.8 g/100 mL of milk and protein intakes are, therefore, nowadays close to the recommended protein target. The protein sources utilized for HM fortification are intact or hydrolyzed cow's milk whey fractions and casein. All cow's milk-based fortifiers on the market try to copy the AA profile of HM by spiking the fortifiers with AA. In addition, fortifiers which are based on HM protein came to the market during the last decade. However, the optimal protein quality in HMFs is still uncertain because it has not been systematically investigated [17]. The concept that the AA profile of HM is optimal for the rapidly growing premature infant was first challenged in a symposium on preterm nutrition almost half a century ago [19], on the grounds that protein requirements should not be separated from AA requirements. At that time, nothing was known about AA requirements of the preterm infant. Two recent studies which measured the protein concentration in HM provided to the VLBW and LBW infants indicated that delivering 1.4-1.8 g of fortifier protein with 100 mL of milk might still not be sufficient. [20, 21]. The authors of those studies speculated that the AA profile of the HMF which is close to HM is not perfect.

When aiming to meet all indispensable AA (IAA), conditionally indispensable AA (CIAA) [22], and dispensable AA (DAA) needs of preterm infants, in particular of ELBW and VLBW infants, the quality of the protein which is provided by HMF is important. It is assumed that the AA pattern in HMFs should be close to the pattern of HM protein. However, Fenton et al. [17] recently published evidence-based nutrition practice guidelines for preterm infants and concluded that no recommendations can be made on the quality of protein in HMF, because there are no studies in the literature. The quality of the protein may modify the recommended intake because the infant does not require protein but requires specific AAs. Individual AAs do not only serve as building blocks for protein synthesis and net protein balance [23], they also provide the basis for growth of all cells, tissues, and structural links between cells (e.g., dendrites among neurons), function as signaling molecules and neurotransmitters, and stimulate the secretion of growth-promoting hormones (i.e., insulin, insulinlike growth factor 1, IGF-1). If one or more of the IAAs or CIAAs become limiting for lean body mass gain and metabolism, specific body proteins may be degraded to free the limiting AAs. Consequently, the remaining AAs from the degraded protein become available as well and contribute to the excess of these AAs. They will be oxidized [24, 25] and thus serve as energy source (1 g = 4 kcal). AAs that are oxidized will yield ammonia, the source of urea, as well as CO2. Especially during fetal life, AAs function as fuel source besides functioning as building blocks for protein. Following term birth, lipids become a more important source of energy. The increasing urinary urea nitrogen excretion in preterm infants during increasing protein supply may be due to their immaturity but may also indicate an increasing AA oxidation rate [26], suggesting a disbalance in the ratio of the supplied AAs [27].

Here, we reviewed the methods which were employed to measure protein and AA requirements of ELBW and VLBW infants. Moreover, we analyzed the available data on AA gains (increments) [10, 28] of the fetus between 23 and 40 weeks and present a mathematical model to calculate advisable AA intakes - in particular for ELBW and VLBW infants.

Amino Acids - Functions and Requirements

The specific roles, requirements, and metabolism of individual AAs and risks of deficiency have been described in detail [29]. As far as growth is concerned, all IAAs are fundamental for production of protein and key regulatory products. Especially leucine stimulates protein synthesis but, when provided in excess may become an important oxidative substrate [30, 31]. The branched-chain lAAs leucine, isoleucine, and valine, and the CIAA arginine stimulate insulin secretion, which in turn augments protein synthesis and protein accretion [32], but a recent Cochrane review [33] indicated that no clinical studies so far have been performed on the effect of branched-chained AA supplementation on growth of preterm infants. Lysine is primarily used for protein synthesis and a deficiency
in lysine intake, like for all essential AAs, limits protein synthesis and causes weight loss in young infants [34]. CIAAs and other DAAs are built from precursors (e.g., cysteine from methionine) but at present it is not clear under which conditions the formation of some CIAAs is limited in the preterm infant [29, 35]. Arginine, glycine, and proline require attention. Arginine is essential for ammonia detoxification through the urea cycle and is a precursor of nitric oxide, which is important for endothelial cell vasodilation and therefore for blood flow to growing organs. Preterm infants receiving low arginine intakes demonstrate elevated plasma ammonia levels and impaired nitric oxide synthesis during hy- poargininemia [36]. Low arginine supply has been found to be associated with increased incidence of necrotizing enterocolitis [37]. A Cochrane review did indeed suggest that arginine supplementation may decrease the risk of necrotizing enterocolitis and the risk of mortality due to this disease [38]. Glycine, besides being a precursor of the major intracellular antioxidant glutathione, serves as an inhibitory neurotransmitter too. Enterally fed preterm infants, in particular SGA infants may have increased glycine requirements in case of oxidative injury such as during critical illness [22]. Proline is among the most abundant AAs in (connective) tissue protein (Table 1). Since preterm infants are unable to syn-thesize proline from glutamate and because of their high protein turnover and tissue accretion, they may have a particularly high proline requirement [22]. Glutamate, serine, and other DAAs promote fetal metabolism [22] and might be important for growth as well. Taurine is the most abundant free AA in HM but is not among the protein-bound AAs [6, 7]. It might contribute to development of brain function (vision, hearing), intestinal fat absorption, bile acid secretion. It is endogenously synthesized from cysteine, but at low rates in preterm infants. Despite the lack of evidence of a benefit from randomized clinical trials, taurine is considered to be a CIAA in preterm infants and is added to formulas for preterm infants and HMF.

AA requirements and metabolism of preterm and term infants can be studied by metabolic balance studies [38] and stable isotope techniques [23]. Balance studies are valuable and relatively easy to perform in larger groups of infants but are prone to errors because of their long duration when intake should be measured exactly, or additional errors as urine is sometimes difficult to collect and nitrogen may be lost through feces and skin. Furthermore, they do not specify intermediate metabolism or synthesis rates of certain AAs [22, 39]. The latter require stable isotope studies which can be performed safely in preterm infants [23]. The stable isotope technology has been employed to estimate protein turnover and requirements in rapidly growing premature infants who were appropriate or small for gestational age. Estimates of protein requirements were made based on measured turnover rates of the stable isotope-labelled glycine [23]. Studies of AA requirements of LBW infants with those technologies are available in the case of phenylalanine, lysine, and cysteine [34, 35, 40, 41]. Phenylalanine requirements of LBW infants (>1.5 kg) were estimated to be 80 mg/kg/day (95% CI 40-119 mg/kg/day; [40]. Corresponding minimum requirements of term infants were 58 mg/kg/day (95% CI 38-78 mg/kg/day). Balance studies in VLBW infants [39] indicated a mean lysine retention of 291 mg/kg/ day (weight 1.3, SD 0.2 kg), lysine requirements of term infants were 130 mg/kg/ day [34, 41]. Thus, requirements in VLBW infants were approximately 2.2 times higher than those in term infants. If generous amounts of methionine are provided to LBW infants, <18 mg/kg/day of cysteine is required (n = 25; weight 1.78 kg, SD 0.32 [34]). It must be mentioned that estimates of phenylalanine, lysine, and cysteine requirements were established in LBW infants who were fed elemental formulas or preterm formulas under standardized conditions. Elemental formulas are known to yield higher AA oxidation rates than intact protein formulas. The weight of the LBW infants in those studies was between 1.3 and 1.8 kg. Therefore, information on AA requirements of ELBW/VLBW infants employing the stable isotope method and balance technology is scarce. Estimates on tryptophan requirements are available from 30 term neonates (gestational age 39 ± 1 weeks) who were fed an elemental formula at 9±4 days. Mean requirement was determined to be 15 mg/ kg/day (upper confidence interval 31 mg/kg/ day [42]). In term infants during the first month of life, methionine (38 mg/kg/ day; 95% CI 27-48 [43]), threonine (68 mg/kg/day; 95% CI 32-104; [44]), isoleucine (105 mg/kg/day), leucine (140 mg/kg/day), and valine requirements (110 mg/kg/day) have been published [45].

Calculating Advisable Amino Acid Intakes

We calculated advisable AA intakes (requirements) of ELBW and VLBW infants based on gains of the fetus. Such an approach has been established (factorial ap-proach) for advisable protein- [11, 16] but not for AA intakes. Data on gain of fetal IAAs, CIAAs, and DAAs based on chemical analyses of 38 fetuses with ges-tational age between 11 and 40 weeks and birth weights 0.11-3.440 kg are in the literature [10, 28]. The data are good proxies to estimate how much of each IAA is required for gain of lean body mass and metabolism of the LBW infant. Data on tryptophan- and cysteine gains were not reported. We found that fetal protein content [10, 28] and therefore also protein gain between 28-40 weeks are very close to the reference fetus [11]. Employing curve fitting programs, we calculated best estimates for gains of IAAs (Fig. 1) and CIAAs/DAAs (Fig. 2) per kg BW. All AA gains declined in an exponential way with increasing BW. The contribution of each AA to the increments in total AAs in the body did not change appreciably between 0.5 and 3.4 kg, which corresponds to the period of gestational age between 160 (approximately 23 weeks) and 280 days (term-equivalent age). This makes calculation of the advisable intakes of AAs based on gains much easier than they would otherwise be. Calculated daily gains of AAs/kg BW between 0.5-0.9 and 0.9-1.4 kg were approximately 87 and 37% higher, respectively, than gains between 2.4-2.9 kg. They were also 77 and 30% higher than of LBW infants growing from 1,900-2,400 g. Higher daily increments of AAs/kg BW of ELBW and VLBW infants are caused by higher weight gain/kg BW (Table 1, bottom line) but also by the fact that growth consists mainly of gain of fat- free mass [11]. After a BW of 1.5 kg is reached, weight gain rate becomes slower as the percentage of fat starts to increase in the body of the fetus. This is reflected in disproportionately lower increments of protein and AAs.

For calculation of advisable daily enteral intakes for each IAA/kg BW, we used the daily fetal gain as the basis requirement, added 30% to compensate for low absorption rates [39, 41, 46] and 20% for oxidation [46]. AAs which are delivered to the fetus are bypassing the intestine that is known to use AAs for (glycol)pro- tein synthesis and as fuel. Consequently, fetal needs are lower than those of their corresponding enterally fed prematurely born counterparts [46]. For this reason, the advisable AA intakes were corrected for absorption and oxidation when calculated from AA gains in the fetus. 40% was added to compensate for inevitable losses [16, 18, 26, 39, 40]. Finally, we added 20% to compensate for assumed variation [7] of the AA concentrations in term HM. The calculated advisable intake of each AA corresponded to 210% of fetal gain. We assumed daily tryptophan requirements of ELBW infants to correspond to 2.5 times the upper confidence interval of term infants, which was established by the stable isotope method [42] and also corresponded to the intake with HM. For calculation of advisable daily enteral intakes for each CIAA and DAA (per kg BW), we first made an estimate how much could be synthesized in the body of preterm infants. The ratios of IAAs:DAAs (mg:mg) in the body between 0.5 and 3.4 kg BW are almost constant, around 2:1 (Table 1 [10, 28]). Corresponding ratios in milk of mothers who delivered preterm, as well as in transitory and mature HM of mothers who delivered at term are between 1.38-1.33 to 1.0 [6, 7]. Based on those calculations, we assumed that the preterm infants can synthesize at least 30% of DAA in the body and that 70% of AA gain is needed with enteral nutrition. The requirement for each DAA was therefore calculated as fetal gain (mg per kg BW/day) X 0.7. Corrections for absorption, inevitable losses, oxidation, and assumed variation of AA concentration in milk were made as described for lAAs. Cysteine requirement of ELBW was assumed to be 3 times the minimum requirement of LBW infants during the first month [35] and 60% higher than in term HM.

As the next step, we compared advisable intakes with those with HM without and with fortification. Table 1 shows published data on the amounts of AAs in 150 mL of “mature” HM, which corresponds to the amounts which an enterally fed premature infant usually receives/kg BW. We used the calculated data on AAs in “mature breast milk” of Zhang et al. [6] which are based on a systematic review of 26 articles providing 79 mean values from 3,774 subjects for total AAs in HM from a wide geographical distribution. Similar data on AAs in HM from several cohorts of Chinese cities were recently published [7]. Influences of gestational age and lactation stage were also reported in both studies. Calculated means for each AA in HM at different stages of lactation are available [6]. We selected the time interval 21-58 days after delivery (“mature HM”), because most donor milk provided is from that period of lactation or even later. Big differences between advisable intakes/kg BW and intakes with 150 mL of HM are evident both in the case of the lAAs and DAAs (Table 1). It can be seen that with HM, ELBW and VLBW infants do not receive the amount of each AA which would be needed for growth based on fetal gains. The data presented in Table 1 also indicate that the AA profile in HM does not match the profile of AA gains.

Reconsideration of Amino Acid Requirements during Fortification

To get an estimate of the daily IAA, CIAA, and DAA intakes of ELBW, VLBW, and LBW infants (per kg BW/day), we added those AAs (Table 1) which are provided by 2.1 g HM protein from HMF to the amounts in 150 mL term HM (21-58 days after delivery [6]) - i.e. total AA intakes are approximately 4 g/kg/ day. Cow's milk-based HMF try to copy the AA profile of HM; therefore, AA intakes would be similar. In ELBW infants, daily IAA and total AA intakes with fortified HM are 175 and 279 mg/kg BW/day lower than the respective advisable intakes (Table 1). Corresponding cumulative intakes between a gestational age of 23 and 26 weeks (period of 21 days) are 3.7 and 5.9 g/kg BW lower. Intakes of 7 IAAs and 3 CIAAs are below the advisable intakes. IAA intakes range between 59 and 125% of the respective advisable intakes. As already mentioned, big gaps are caused by the high requirements of ELBW infants but also by differences between IAA profiles in HM and requirement profiles for fetal increments (Table 1). The biggest IAA and CIAA gaps between actual and advisable intakes are shown in Figure 3. In a preterm infant growing from 400-1,400 g the cumulative actual intake of lysine, which is important for protein synthesis [29, 34, 41] and the first limiting IAA in mammals, would be 1.6 g/kg BW lower than the advisable intake. HM protein provides only 16% of IAAs as lysine, whereas the contribution of lysine to the AA gains of the fetus is 19%. In VLBW infants, intakes of 1 IAA and 3 CIAAs are still below the advisable intakes, but leucine and isoleucine are already oversupplied. This indicates that the amounts and the profile of AAs in HMF for ELBW and VLBW infants need to be reconsidered. For the most vulnerable groups adequate intake of all AAs, in particular those which are indispensable, during the first period of life is key for normal growth and development as well as for long-term health. In LBW infants infants >1,500 g), there are no more deficits in actual AA intakes if 4 g HM protein are provided (Table 1). However, if the recommended HM protein intake of 3.5 g for those infants [2, 8, 16] is provided, intakes of 1 IAA and 3 CIAAs are still too low. HMF which are based on cow's milk protein or HM protein should be spiked with those AAs where the intake might be too low. As an alternative, AA-based HMF could be developed which can provide tailor-made AA supply. We hope that quantity and profiles of AAs in HMF will be clinically studied in the near future. A tailor-made HMF should be offered in different concentrations to ELBW, VLBW, and LBW infants. However, development of HMF is costly because clinical trials are needed. Those trials are necessary to prove that the advisable intake based on AA gain of the fetus is safe and results in better growth and development than with the present HMF. On the other hand, business options are limited, because most neonatology departments are asking companies to receive HMF based on cow's milk protein fractions for free or at reduced costs.

Are Advisable Amino Acid Intakes for Preterm Infants Based on a Factorial Approach a Good Reference?
How solid is the estimate of advisable AA intakes based on AA gain in the fetus? (a) Fetal AA gains were published in the 70s of the last century. Chemical analyses of fetuses and stillborn infants would now only be allowed by ethical committees if the research is dedicated to disease prevention and treatment (e.g., cancer, HIV). Therefore, it is extremely unlikely that more data will be created and published. Widdowson's group accumulated a lot of experience in the field of chemical analyses of body components. Procedures and methods which were employed during tissue preparation and AA analysis [10, 28] correspond to today's practices. In addition, data presented to describe gains in fat and lean body mass and its components (e.g., protein, minerals) are close to the reference fetus [11] and therefore solid estimates. Her publication “The Chemical Composition of the Body” [47] is still a standard reference on AA in the human body. (b) It is likely that the AA pattern needed for growth and metabolism of ELBW/VLBW but also of LBW infants differs from that of infants born at term, because postnatal gains in weight and lean body mass/ kg BW are much faster. For those reasons, the needs of those protein fractions which are important for growth - mainly whey and casein - are higher, whereas it is not clear if the needs of non-nutritive proteins are higher as well [3-5]. Under such circumstances, the AA pattern in HM would not be the gold standard for preterm infants. (c) Strong support of the calculation of advisable intakes is provided in the case of lysine and phenylalanine by stable isotope research and metabolic balance studies [34, 40, 41]. The almost identical results that were derived from both approaches for those two IAAs, underline the strength of the reasoning. There is no reason to believe that the requirements should be far off for the remaining lAAs using our estimations. To be specific: our calculated advisable intake of lysine/kg BW is 2.1 times higher than the intake of term infants. Balance and stable-isotope studies indicated that lysine requirements/kg BW of VLBW infants are about 2.2 times higher than of term infants. Our calculation of advisable phenylalanine intake for LBW infants between 29-32 gestational weeks is 130 mg/kg/day. Stable isotope studies indicated that the upper confidence interval for minimal obligatory requirements of phenylalanine requirements for LBW (1.75, SD 0.17 kg; GA 32.5 weeks) was 119 mg/kg/day. Both lysine and phenylalanine are among the undersupplied AA by present HMF. Therefore, similar requirements found by 2 different methods support the request to increase concentrations of those lAAs in HMF, and (d) Banked HM is the backbone of enteral nutrition of preterm infants [48]. The AA profile of mature HM protein which was used in our calculations as an example for AA in HMF is realistic. Such HMF are on the market, and cow's milk-based HMF [49] try to have AA profiles which are close to HM protein.

Conclusions

Infants should receive HMFs which provide all amounts of AAs which are needed for their rapid growth and development. Our calculations which are based on AA gains of the fetus can serve as reference for advisable intakes of preterm infants. Very high AA requirements of ELBW infants are a challenge for enteral nutrition. AA profiles of present HMFs for preterm infants which are close to HM should be reconsidered: spiking HMF protein with the AAs which are presently undersupplied or providing targeted AA based HMF are options to further improve the AA profile in fortifiers.

Acknowledgements

The authors would like to thank Prof. E.E. Ziegler, University of Iowa, for reviewing the manuscript.

Conflict of Interest Statement

F.H. receives honoraria for lectures from the Nestlé Nutrition Institute and different nutrition companies. J.B.v.G. is founder and director of the Dutch National Human Milk bank and member of the National Health Council. Fees for consultancies and lectures are transferred to the Foundation that supports the Emma Children’s Hospital. N.H. receives honoraria for lectures from Nestlé, Baxter, Danone, Novolac, and MUM. D.G. is an employee of Nestec Ltd, Switzerland.

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