The daily requirements for infant nutrition have been studied and standardized on the basis of their content in human milk (1-4). Commercial infant formulas are generally designed to contain similar concentrations of the major and minor nutrients. The major ingredients are listed on the label and vary little between brands. However, other important nutrients for infant growth, such as free amino acids (FAAs) and free fatty acids, are not listed individually.
Understanding of the amino acid (AA) requirements of the infant from birth to weaning is incomplete. During this developmental period milk generally provides the sole food source. Protein deposition must be achieved with high nutritional efficiency for maturation of individual tissues. AA equilibrium is tightly regulated by transfer of nitrogen from AA taken in excess to those that are deficient (5,6). The daily rate of protein deposition from birth to age 2 has an obvious biexponential fall in various tissues that reflects a progressively weaker anabolic response to nutrient intake, including the capacity for protein synthesis. As this process occurs, AA requirements in those tissues decreases (5-10). Although AA are released from milk proteins, they are also supplied by FAAs in the milk. The milk of each mammalian species has a distinctive FAA, which may reflect the relative importance of FAAs during early postnatal development. When an inadequate mixture of AAs is fed, the rate of protein synthesis and ultimately growth will be adversely affected (11,12).
There have been very few studies on the FAA content of human milk or infant formulas. Clarifying the FAA content of human milk may promote the manufacture infant formulas with a composition closer to that of human milk. We designed this study to compare the milks of mothers of pre-term and full-term infants with two brands of infant formulas in regular and pre-term formulations. We also investigated the changes in FAA composition of human milk through the transition from colostrum to transitional to mature milk.
MATERIALS AND METHODS
A total of 67 human milk samples were collected from 44 healthy mothers of term infants and 23 mothers or pre-term infants with gestations ranging from 29 to 36 weeks (mean 33 weeks). Milk was collected in sterile polypropylene tubes (5 to 10 mL) at the end of a feeding. The intervals between collections was adjusted in order to obtain samples of colostral milk (0 to 7 days), transitional milk (8-21 days) and mature milk (>21 days). We obtained colostral milk from 16 term mothers (FTHM), 9 pre-term mothers (PTHM). We obtained transitional milk samples from 15 term mothers and 6 pre-term mothers. We obtained mature milk from 13 term mothers and 8 pre-term mothers. Human milk samples were frozen immediately after collection. Two brands of infant formula (IF) were selected, each of which had a formulation for term infants (IF-A, Similac, Abbott Laboratories, Granada, Spain; IF-B, Enfalac, Mead Johnson Nutritionals, Nijmegen, The Netherlands; 10 samples of each) and a formulation for preterm infants (PTIF-A: Similac SC and PTIF-B: Enfalac PM, 5 samples of each). Formulas were prepared according to the manufacturer instructions.
All milk samples were thawed at room temperature before analysis. They were analyzed by a method used for analysis of AA in plasma (13,14). Total protein in each sample was measured by the Lowry method at a wavelength of 500 nm (15). The AA analysis was performed using an ion exchange chromatography amino acid analyzer (System 6300, Beckman Instruments, Fullerton, CA) with a method we described previously (14). A completely mixed milk sample of 200 μL was placed in a 0.6-mL centrifuge tube, and 20 μL of aqueous sulfosalicylic acid solution (35%) was added to eliminate residual protein. The supernatant was filtered using a microspin filter reservoir (0.2 μm). Finally, the filtrate was diluted and injected into a Beckman AA analyzer for quantitation of individual AAs. Statistical comparison of individual AA concentrations in the various milks was performed using one-way analysis of variance and post hoc tests (16).
The total protein and FAA in human milks and infant formulas are listed in >Table 1. The average total protein in the two PTIFs was 3.82 ± 0.32 g/dL, higher than that of IF-A (2.50 ± 0.35 g/dL), IF-B (2.84 ± 0.14 g/dL), FTHM (1.19 ± 0.27 g/dL) and PTHM (1.11 ± 0.12 g/dL). The highest concentration of FAAs was in colostral PTHM (16,722 μmol/L). Other values were as follows: colostral FTHM (3,831 μmol/L), transitional FTHM (3,065 μmol/L), mature FTHM (3,492 μmol/L), transitional PTHM (4,196 μmol/L), mature PTHM (3,500 μmol/L), PTIF-A (820 μmol/L), PTIF-B (788 μmol/L), IF-A (720 μmol/L), and IF-B (696 μmol/L). The mean FAA concentration in all human milks was significantly higher than that in any of the formula samples (one-way analysis of variance, P < 0.01). There were significant differences in the mean concentration of total FAA in colostral versus transitional milk and colostral versus mature milk (Figure 1, >Table 2). There was no significant mean difference between IF-A and IF-B, between PTIF-A and PTIF-B or between IF and PTIF.
The five most abundant AAs in the samples are shown in >Table 3. Colostral PTHM contained more than four times the amount of FAA as the other HMs. The five most abundant AAs in colostral PTHM were proline (19.3%), alanine (7.8%), valine (7.4%), glutamic acid (7.3%) and taurine (5.1%), whereas the five most abundant AAs in FTHM and PTHM (excluding colostral PTHM) were glutamic acid (32.2%, 45.6%), taurine (17.4%, 16.3%), alanine (8.6%, 6.7%), aspartic acid (4.5%, 3.5%) and serine (3.7%, 3.1%). The results for IF-A and IF-B were similar as follows: taurine (52.3%, 53.6%), phosphoserine (11.7%, 10.2%), glutamic acid (7.5%, 6.6%), glycine (4.8%, 4.7%) and phosphoethanolamine (4.3%, 4.5%). In PTIF-A and PTIF-B, the most abundant AAs were taurine (49.4%, 50.9%), phosphoserine (12.0%, 12.5%), glutamic acid (11.5%, 10.3%), phosphoethanolamine (4.4%, 3.8%) and α-amino-n-butyric acid (4.2%, 3.9%).
Comparing individual AAs among these groups using the post hoc test, most concentrations differed significantly between HM and IF, excepting hydroxyproline, asparagine and homocystine (>Table 1). Most FAAs differed significantly between FTHM and PTHM, with the exception of taurine, phosphoethanolamine, hydroxyproline, asparagine, glutamic acid, citrulline, α-amino-n-butyric acid, cystine, homocystine and tryptophan. There were no differences between IF-A and IF-B or between PTIF-A and PTIF-B. The concentrations of only two FAAs (α-amino-n-butyric acid and tryptophan) differed significantly between IF and PTIF. FTHM contained considerably more essential and non-essential AAs than did PTHM, IF and PTIF. The ratio of total essential AA to total non-essential AA was 1:4.5 for FTHM, which differed significantly from that of PTHM (1:1.6), IF (IF-A, 1:2.0; IF-B, 1:1.8) and PTIF (PTIF-A, 1:3.7; PTIF-B, 1:3.7).
Given the current brief duration of breastfeeding (17,18), selection of a formula containing the same nutritional ingredients as HM is particularly important. In this investigation we observed significant differences between the amounts of FAA in HM and IF.
Several points need to be considered for the accurate interpretation of the variance of FAA between HM and IF. First, the concentrations of many FAAs in HM, such as hydroxyproline, glutamine, proline, cystathionine and tryptophan, may vary considerably from one sample to another. One reason may be variations in the nutritional status of mothers during pregnancy and after delivery (19-23). According to recommendations in the United States in 1990 (24), the suggested weight gain during pregnancy for a woman with body mass index of 19.8 to 26.0 is 1.6 kg in the first trimester and 0.4 kg per week in the second and third trimesters. In our study, the weight gain during pregnancy averaged 2.36 kg (2.36 ± 1.04 kg; range, 0.0 to 3.5 kg) in the first trimester, 0.3 kg per week (3.60 ± 2.39 kg; range, 0.0. to 8.5 kg) in the second trimester and 0.37 kg per week (5.85 ± 1.97 kg; range, 2.8 to 10.0 kg) per week in the third trimester. The variation in weight gain during pregnancy may result in marked differences in concentrations of certain FAAs in the HM samples. In addition, race and dietary habits may minimally affect the total FAA in HM. The total FAA in HM from Taiwanese mothers (3,627 μmol/L in FTHM and 3,848 μmol/L in transitional/mature PTHM) is similar to that from Italian (3,019.7 ± 810.1 μmol/L) and Canadian (3,069 μmol/L in FTHM and 3,397 μmol/L in PTHM) mothers (11,12).
HM contains several times the amount of total FAA as IF, including essential and non-essential AA. The ratios of essential to non-essential AA also differ between HM and IF. Although glutamic acid and taurine are the two most abundant FAAs in HM, accounting for approximately 50% of total FAA, free taurine alone accounts for half the FAA in IF and glutamic acid is third on the list. Similar results have been found by others (4,25). It has been suggested that these differences in glutamic acid, glutamine and taurine may contribute to differences in enteral mucosa protection, neurotransmitters and nitrogen supply in IF-fed infants (25-27).
There were significant differences in FAA between FTHM and PTHM but not between IF and PTIF. Because of their low birth weight and incomplete organ development, sufficient nutrition, whether from HM or IF, is particularly important for preterm infants. PTHM contains the most abundant FAA, with particularly high quantities of glutamic acid and glutamine. These AAs accelerate growth and physiological maturity in low birthweight infants (10,28).
In conclusion, understanding the differences in FAA concentrations between IF and HM reinforces the contention that HM is the best nutritional source for infant growth and development. HM, particularly colostral milk, is rich in FAA. In FTHM, the relative proportions of the five most abundant AA remained constant throughout the evolution from colostrums through transitional to mature milk. HM contains plentiful AA for infants, and IF should be formulated to better match the FAA content of HM. If infant formula is to match the nutritional quality of HM, milk manufacturers should increase the amount of FAA and adjust the relative proportions to resemble that of HM.
The authors would like to express sincere thanks to Dr. Mary Jeanne Buttrey for her revision of this article.
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