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Free Amino Acid Content in Standard Infant Formulas: Comparison with Human Milk

Department of Pediatrics, San Paolo Hospital, Milan (C.A., E.R.)
Istituto Superiore di Sanità, Rome (B.C., C.B., E.S.), ITALY

Address reprint requests to: Dr. Carlo Agostoni, Department of Pediatrics, San Paolo Hospital, 8 Via A. di Rudini, 20142 Milano, ITALY. E-mail: agostoc{at}tin.it.

	ABSTRACT

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Objective: To compare the concentration of non-protein nitrogen (NPN) and free amino acids (FAA) in powdered and liquid commercial formulas with that in human milk.

Methods: The non-protein nitrogen and FAAs in pooled breast milk was compared with that in 11 protein-modified starting infant formulas (seven powdered, four liquid whey-predominant formulas) and one powdered soy-formula. Human milk was collected at the end of each feeding (hindmilk) over 24 hours in a group of 40 healthy lactating women after delivery of full-term infants at age one month.

Results: In human milk glutamic acid plus glutamine and taurine were the prevalent amino acids, accounting for around 50% total FAA. In the analysed formulas the total FAA fraction was 10% or even less than in human milk, mostly represented by taurine, while methionine was high in soy formula. The sum of glutamic acid and glutamine in all the formulas was much lower than in human milk.

Conclusions: Breastfed infants are supplied with FAA, mainly glutamic acid and glutamine, compared to formula-fed counterparts. The different FAA intake might be the origin of some functional differences at the enteral level between breast- and formula-fed infants.

Key words: human milk, infant formulas, free amino acids, glutamic acid, glutamine

Abbreviations: FAA • free amino acid • NPN • non-protein nitrogen • PN • protein nitrogen • AaN • total free amino acid nitrogen • TN • total nitrogen

	INTRODUCTION

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Free amino acids (FAA) fall within the non-protein nitrogen (NPN) fraction of human milk, which also includes peptides, urea, uric acid, ammonia, creatinine, creatine, nucleic acids, carnitine, amino sugars and other compounds [1,2]. Since these moieties have recently been given functional roles, the NPN components of human milk should not be considered equivalent to whole protein nitrogen when estimating the true protein content of human milk. In the 60’s and 70’s several authors studied the pattern of FAA in human milk [3–5]. The comparison of these studies is difficult, since the data were of dissimilar lactational intervals and the number of subjects considered was often limited. On the whole, most FAA seemed to decrease significantly with progressing lactational stage, while the sum of glutamate plus glutamine has been reported to increase with progressing lactation [6]. The biological significance of the FAA quota of human milk is still obscure. However, FAA are known to contribute to the total body pool of utilizable nitrogen and to the initial changes of plasma FAA concentrations following a feed since they may be more easily absorbed than protein-derived amino acids [7, 8].

The total amount of NPN in human milk is significantly higher than in cow’s milk, where it averages about 5% total nitrogen, while in infant formulas it varies widely [9]. The large variation of the NPN content in infant formulas is due to differences in nitrogen composition of the protein sources (skim milk, whey) used for manufacturing cow’s milk formulas. The proportion of NPN, as well as its composition, may also change considerably depending on the treatment of the raw materials and the concentrations used [10, 11]. On the other hand, NPN composition and FAA content of infant formulas have been poorly investigated so far, with these investigations often including a restricted number of reference human milk samples [4].

The aim of the present study was to determine and compare the NPN and FAA content of powdered and liquid commercial formulas with that of human milk from a representative number of Italian mothers one month after delivery.

	MATERIAL AND METHODS

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Milk Samples
Human milk from all the feedings over 24 hours was obtained from 40 healthy well-nourished mothers of term infants at the end of the first month of lactation. Mothers were instructed to express breast milk at the end of each feed (hindmilk) into separate sterile polypropylene vials. Samples were frozen immediately after collection and delivered to us for analysis the following day, when the participants brought their infants in for clinical examination. Aliquots from all the milk samples collected by each mother on the same day were pooled for analysis.

Seven powdered and four liquid whey-predominant starting infant formulas in line with the European indications [12] and one soy-formula were used for comparison. Duplicates of the same formula, from different commercially available lot numbers, were selected for analysis. Formulas analyzed were produced by Nutricia N.V. (Zoetermer, Netherlands), Milupa S.A. (Colmar, France), Societé des Produits Nestlè S.A. (Vevey, Switzerland), Guigoz (Milan, Italy), Dieterba (Latina, Italy), Plasmon Dietetici Alimentari S.p.A. (Latina, Italy), STAR S.p.A. (Steenwoorde Usine G6, France) and Abbott Laboratories BU, Ross Products (Zwolle, Netherlands).

Biochemical Determinations
Total nitrogen (TN) was subdivided into protein nitrogen (PN), NPN and total free amino acid nitrogen (AaN). TN was determined according to Kjeldahl’s method [13] applied to the whole sample. PN was determined according to Kjeldahl after precipitation and separation of the NPN fraction by filtration [14]. NPN was calculated as the difference between TN and PN. AaN was derived by calculating the nitrogen content of each amino acid on the basis of its nitrogen percentage.

Three hundred µL of milk (powdered formulas were prepared according to manufacturer’s specifications) was slowly added to 2 mL of methanol. The solution was mixed on a vortex type mixer and centrifuged at 2500g for five minutes [15]. The supernatant phase was removed and analyzed for amino acids after filtration with a 0.45 µm filter.

Amino acids were determined by an automatic precolumn derivatization with fluorenylmethyl-chloroformate and reversed-phase high-performance liquid chromatography with UV and fluorescence detection [16]. Cystine-cysteine cannot be detected with this method.

The percentage of recovery has been calculated as 96% by the addition of fixed amounts (comparable to human milk content) of standard taurine to human milk. The repeatability has been carried out by analyzing ten methanol solutions obtained by extraction from the same samples of human milk (coefficient of variation = 5.7%). The limit of detection was 3.3 µm/L of milk.

Human milk values are reported as means and standard deviations (SD). Analyses on each formula were carried out in duplicate (mean values are reported).

	RESULTS

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Nitrogen distribution is shown in Table 1 (powdered infant formulas) and in Table 2 (liquid infant formulas). In infant formulas the quota of AaN, as well as its percentage when compared to NPN, are lower than in human milk. NPN values are similar to those of human milk. Soy milk shows a higher content of NPN and AaN because of the enrichment with methionine (lacking in soy protein) and taurine.

	DISCUSSION

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It has recently been shown that the whey sources of formulas may contain different levels of NPN depending on the method of preparation, e.g. ultrafiltration, electrodialysis, ion-exchange chromatography. These methods separate the differ-ent compounds with varying degrees of efficiency, depending on their physical chemical characteristics [10, 11]. The utilization of ultrafiltered whey proteins is declared only on the label of sample No. 3. Ultrafiltration removes most of the NPN fraction, thus influencing the NPN and FAA content of the product. In fact, samples No. 3, 4 and 12 (from the same manufacturer) show low contents of NPN and total FAA. The similarity of NPN amounts in most formulas and human milk can be explained by the nitrogen enrichment of infant formulas with components such as carnitine and choline and/or by the different technological treatment of the protein matrix; whereas the varying FAA content of the examined infant formulas is due to differences in nitrogen composition of the raw protein sources. Human milk contains a pool of FAA twofold to fivefold higher than that in formulas. This component may significantly contribute to the initial changes in the plasma FAA concentrations occurring as the infant begins to nurse and to the total body pool of metabolizable nitrogen [8].

We have found that taurine, glutamic acid and glutamine are the most abundant FAA in human milk in a consistent number of lactating women at a stage in which mature milk composition is well established. We may thus confirm the previous findings from heterogenous studies on the FAA quota of human milk, extending the observation to the FAA content of standard infant formulas. Since there is agreement that each component of breast milk should a priori be beneficial to the infant, the nutritional relevance of the FAA quota should not be underestimated.

Taurine has been more extensively studied so far, but its role in the diet of the preterm and term infants has not been clearly understood. This amino acid enters the bile acid conjugation pathways in the infant gut, and it may play a relevant role in the structure and function of retinal photoreceptors [17, 18]. Taurine appears to have a neuroprotective role in infants and children deriving most of their calories from total parenteral nutrition [19] and should, therefore, be considered among the "conditionally essential" amino acids in infant nutrition. Its addition to a high-protein formula for healthy growing term infants reduced blood urea levels and the plasma and urine amino acids to concentrations similar to those found in breast-fed infants through some as yet unknown mechanisms [20].

Glutamic acid and glutamine account for most FAA in human milk and their sum represents the 50% of total FAA. The total quantity of free glutamic acid and glutamine in cow milk is about 300 µm/L (personal unpublished data). This amount is lower than that found in human milk, but higher than in examined formulas, in agreement with the literature [21]. This is probably due to the technological treatment cow’s milk is subject to. Accordingly, a healthy, breastfed one-month-old, 4-kg infant supplied with 600 mL of human milk per day would ingest around 120 mg of free glutamine plus glutamic acid (i.e. more than 30 mg per kg of body weight). The meaning of the higher amounts of free glutamic acid in human milk (around fourfold the amounts of glutamine) is open to speculation. Glutamic acid provides a source of ketoglutaric acid for the citric acid cycle [22]. Moreover, it may act as a neurotransmitter in the brain [23]. Mucosal intestinal cells preferentially transaminate glutamic acid to yield alanine entering the gluconeogenic pathway, and both glutamic acid and glutamine from the lumen constitute major energy substrates for the intestinal cells [24, 25]. New functions for glutamine in the metabolism of enterocytes and immune cells have recently been shown in humans [26, 27]. In rat studies the addition of NPN mixture to either human milk or infant formula did not improve the net protein utilization [28], but in very low-birth-weight infants the plasma amino acid response to glutamine supplementations (from 80 to 300 mg/kg/day) has been interpreted as evidence of decreased tissue catabolism and enhanced gluconeogenesis [29]. We speculate that the major FAA of human milk, as well as other characteristic compounds such as long-chain polyunsaturated fatty acids and oligosaccharides [30], might have a double role of protecting the intestinal growth while supplying functional substrates to the nervous tissues.

Therefore, the enrichment of infant formulas with nitrogen components (particularly glutamic acid and glutamine) could be helpful not just for protein anabolism, but also to promote the adaptation and growth of highly-specialized tissues. While these effects have been addressed by some studies in low-birth-weight infants and high-risk patients, no data are available on healthy term infants at present.

	ACKNOWLEDGMENTS

 
We thank Anna Maria Lammardo, PhD, for the contribution in collecting and preparing the human milk samples.

Received June 28, 1999. Accepted June 8, 2000.

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