Protein Quantity and Quality in Infant Formula: Closer to the Reference

Transcription

1 infant Formula: Closer to the Reference, edited by Niels C. R Ra'ihii and Firminci F' Ruhallelli. Nestle Nutrition Workshop Series. Pediatric Program. Vol. 47 Supplement. Nestec Ltd.. Vevey/Lippincott Williams & Wilkins. Philadelphia Protein Quantity and Quality in Infant Formula: Closer to the Reference Niels C. R. Raiha, *A. Fazzolari Nesci, *C. Cajozzo, *G. Puccio, tlolanda Minoli, tguido E. Moro, t A. Monestier, ^Elisabeth Haschke-Becher, Anne-Lise Carrie, and ^Ferdinand Haschke Department of Pediatrics, University of Lund, Malmii, Sweden; *Clinica Ostetrica Ginecologia B Dell'universita Di Palermo, Italy; jdepartment of Perinatal Pathology, Macedonio Melloni Maternity Hospital, Milan, Italy; ^Department of Clinical Chemistry, University of Lausanne, Lausanne, Switzerland; Nestec Ltd., Vevey, Switzerland; and f Nutrition Division, Nestle South Africa, Randburg, South Africa In 1915 Gerstenberger and Ruh (1) from Western Reserve University in Cleveland, Ohio, USA, developed the first commercially manufactured infant formula called "Synthetic Milk Adapted" or SMA (1). The formula was based on skimmed milk, and in the original test formula, the protein content was close to that of human milk, or only 9 g/1. When the formula was commercially produced, the protein content was 12.5 g/1, and in 1945, it was increased to the 15 g/1 or 2.2 g/100 kcal, which is the usual value in infant formulas on the market today. The increase in the protein content was in response to the growing recognition that the protein in processed cow's milk was not so nutritionally adequate as that of fresh human milk. In 1961, a major breakthrough occurred in the development of infant formulas. The electrodialysis process of milk proteins offered a practical solution to the demineralization of whey protein. With the addition of equal parts of protein from skimmed cow's milk and demineralized whey from cow's milk, it was possible to manufacture an infant formula with a protein composition more like that of human milk. Terms such as "casein predominant," "whey predominant," "humanized," or "adapted" came into use. Casein-predominant formulas are made from nonfat dry milk and have a protein composition similar to that of bovine milk, or 82% casein and 18% whey proteins. Whey predominant is the term used to describe infant formulas that have >50% of the protein as bovine whey proteins. Human and bovine milk proteins differ in their composition, and the addition of bovine whey proteins to cow's milk in formula does not make the amino acid composition equal to that found in human milk. Thus although the terms casein predominant or whey predominant correctly describe the protein composition of formula, the term "humanized" can be misleading. Ill

2 112 PROTEIN QUANTITY AND QUALITY IN FORMULA TABLE 1. Revised estimates of protein intake in exclusively breast-fed infants Age (mo) Milk consumption (g/day) Adapted from Dewey etal. (5). Adjusted protein intake (g/kg/day) The protein composition of human milk is unique, and to date it has not been possible to duplicate it in infant formulas, either in quantity or in quality. However, when the composition and quality of human milk proteins and the requirement for protein of the infant at different ages became more precisely known, it was possible to develop infant formulas with a modified cow's milk protein composition more closely resembling human milk proteins. PROTEIN REQUIREMENT OF HEALTHY TERM INFANTS It has been assumed on teleologic grounds that the milk of a given species is best adapted to the nutritional requirements of its young. Thus it is generally accepted that the normal human infant fed by a healthy mother is the nutritional norm for infant feeding. Several studies have shown that the growth pattern of breast-fed infants is quite different from that of formula-fed infants, particularly for weight. However, protein intake is not likely to be a limiting factor for growth in exclusively breast-fed infants for the first 4 to 6 months (2,3), and the increased weight gain in formula-fed infants may be explained by excessive energy and protein intakes. Human milk proteins change dramatically in both quantity and quality during lactation, and in mature milk, the nutritionally available protein may be as low as 8 to 9 g/1 or 1.3 g/100 kcal (4). Protein requirement during the first months of life is usually estimated by using the normal breast-fed infant as a model (5). As seen in Table 1, the protein intake decreases from ~2 g/kg/day during the first month to ~1 g/kg/day during the sixth month (Table 1). COMPARISON OF PROTEI IN INFANT FORMULA AND HUMAN MILK The total nutritionally available protein content in mature human milk and conventional infant starting formulas differs considerably. Human milk has from 8 to 9 g/1 (1.3 g/100 kcal), whereas formulas usually have 15 g/1 or 2.2 g/100 kcal. The differences in protein quality between mature human milk and either caseinor whey-predominant infant formulas can best be illustrated by comparing the

3 PROTEIN QUANTITY AND QUALITY IN FORMULA 113 essential amino acid patterns of these milks. Table 2 illustrates the amino acid content in mature human milk and whey-predominant and casein-predominant conventional formulas with a label claim of 15 g/1 of protein (6). The low cystine content of casein results in a very low methionine-to-cystine ratio in human milk (1:2) as compared with bovine milk (2:1). A methionine-to-cystine ratio of <1 is seen only in vegetable proteins and in human whey proteins. In casein- and whey-predominant formulas, the ratio is modified from the unprocessed bovine milk but is still > 1, being higher in the casein-predominant formula. Another striking difference is the content of the phenolic amino acids phenylalanine and tyrosine, which are much lower in whey proteins, and thus are lower in human milk than in casein-predominant formulas. Threonine is present in high concentration in whey proteins. However, if the nutritionally partially nonavailable whey proteins in human milk, such as secretory immunoglobulin A (IgA) and lactoferrin (3-10%), are subtracted from the "true" protein content of human milk, then the whey/casein ratio of the nutritionally available proteins will change, and the amino acid profile will be different, so that wheypredominant bovine formulas will have a much higher threonine content than does human milk (7). Low plasma total tryptophan concentrations have been reported in infants fed casein-predominant formulas when compared with breast-fed infants (8,9), because of the low content of this essential amino acid in bovine casein (9). The nonprotein nitrogen (NPN) fraction of milk contains a number of free amino acids including glutamic acid, glycine, alanine, valine, leucine, aspartic acid, serine, threonine, proline, and taurine. Glutamic acid is the predominant amino acid, and taurine, which is almost absent from the milk of the dairy cow, is the second most TABLE 2. Protein amino acid content (p.mol/dl) in human milk and conventional formulas Amino acid Protein (g/dl) Glutamate Proline Leucine Aspartate Lysine Valine Serine Alanine Isoleucine Threonine Glycine Tyrosine Phenylalanine Arginine Cystine Histidine Methionine Human milk 0.9 1, Adapted from Jarvenpaa et al. (6). a Whey/casein ratio. 1.5 g Protein formula (60/40) a 1.5 2,196 1,234 1,334 1, g Protein formula (18/82) a 1.5 2,277 1,425 1,

4 /14 PROTEIN QUANTITY AND QUALITY IN FORMULA abundant amino acid in the NPN fraction of human milk. The technique used to demineralize bovine milk whey protein will affect the NPN fraction in a whey-predominant formula. Ultrafiltration will remove most of the NPN fraction, and there also are differences in the whey preparations produced by electrodialysis and ion exchange. Thus the taurine concentration in formulas can vary depending on the processing technique used to make whey proteins, but it is never > 10-15% of that of human milk. Most recent formulas on the market are therefore supplemented with taurine. RECOMMENDATIO FOR PROTEIN CONTENT IN INFANT FORMULAS The European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) (10) proposed a protein content in standard infant formula between 1.8 and 2.8 g/100 kcal, which would correspond to between 12 and 19 g/1 in a 67-kcal/dl formula. The Committee on Nutrition of the American Academy of Pediatrics (11) gave a wider range, from 1.8 to 4.5 g/100 kcal, and the United States Food and Drug Administration (FDA) (12) gave the same limits as the American Academy. The upper limit of 4.5 g/100 kcal has been criticized as being much too high (13). Fomon (14) recommended a minimal level of protein in infant formulas of 2.2 g/100 kcal (15 g/1) for infants younger than 3 months and 1.6 g/!00 kcal (11 g/1) for infants older than 3 months. This recommendation is similar to the recommendation of Beaton and Chery (15) of 1.7 g/100 kcal (11.4 g/1) for infants aged 3 to 4 months. INFANT FORMULA WITH REDUCED PROTEIN CONTENT The international recommendation for the minimal protein content of an infant starter formula is thus 1.8 g/100 kcal, and no precise recommendation is given for the whey/casein ratio. To test whether a whey modified infant formula with a protein-energy ratio of 1.8 g/100 kcal is adequate and safe from birth, we recently performed a prospective, randomized multicenter clinical study (16). infants were compared with infants receiving either a standard infant formula with 2.2 g protein/100 kcal, whey/casein ratio, 60:40, or a formula with 1.8 g protein/100 kcal, whey/casein ratio, 70:30. The protein concentration was calculated from the analyzed nitrogen concentration of the formulas multiplied by the factor The "true" protein was estimated by assuming that 40% of the NPN (10% in both formulas) was composed of a-amino nitrogen and was nutritionally available. The amino acid profiles of the two formulas are presented in Table 3. Modifications of the proteins in the 1.8-g/100 kcal formula resulted in a decrease in the threonine concentration and an increase in the concentrations of cystine, arginine, histidine, and tryptophan when compared with the standard formula (Table 3). The study was conducted from birth to 4 months in a controlled and blind design (except for the breast-fed group). Formula-fed infants were randomly assigned to one of the formula groups, and all study feedings were initiated in the maternity hospital

5 PROTEIN QUANTITY AND QUALITY IN FORMULA 115 Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan TABLE 3. Amino acid composition of diets Breast milk Formula Values expressed as grams amino acyl/16 g N. Values for breast milk are from reference 20. Formula before the infants were discharged. To assure that infants would complete the study in each feeding group, a total of 34 infants was recruited into each group. All infants recruited into the study fulfilled the following criteria: they were healthy term infants; gestation was &37 weeks and <42 weeks; and birth weight was >2,500 g and <4,200 g, female and male infants combined. The volume of daily formula intakes was similar in both formula groups at 30, 60, 90, and 120 days. The protein and energy intakes are presented in Table 4. No differences were found in energy intakes between formula groups, whereas protein intakes were less in infants fed. All intakes were within the safe level of protein intake, as presented by Dewey et al. (5). Table 4. Protein and energy intakes Age (d) Feeding group n Values expressed as mean ± standard deviation. Protein intake (g/kg/d) 2.71 ± ± ± ± ± ± ± ± 0.44 Energy intake (kcal/kg/d) ± ± ± ± ± ± ± ± 23.1

6 116 PROTEIN QUANTITY AND QUALITY IN FORMULA TABLE 5. Gain in weight Age interval From birth to 60 d From birth to 90 d From birth to 120 d From 30 d to 120 d Feeding n Values expressed as g/day, mean ± standard deviation. ANOVA, analysis of variance;, not significant. Gain in weight 30.1 ± ± ± ± ± ± ± ± ± ± ± ± 6.5 P (ANOVA) Gains in weight and length were normal, and there were no statistically significant differences between the feeding groups (Tables 5 and 6). There also were no deviations in the weight gain in the study groups when compared with the Euro-Growth standards (Table 7). The data on body mass index are presented in Table 8. At birth, and at ages 30,60, and 120 days, there were no differences between the feeding groups. As could be anticipated, the serum urea concentrations were higher in the infants receiving the standard infant formula than in those receiving breast milk or the reduced-protein test formula (1.8 g/100 kcal) at 30 days and at 60 days. At 120 days, there were no differences between the feeding groups in the serum urea concentrations (Table 9). The serum albumin concentrations were normal, and no differences could be found between the study groups (Table 9). Age interval From birth to 60 d From birth to 90 d From birth to 120 d From 30 d to 120 d TABLE 6. Feeding Gain in length n Values expressed as mm/d (mean ± standard deviation). ANOVA, analysis of variance;, not significant. Gain in length 1.35 ± ± ± ± ± ± ± ± ± ± ± ± 0.2 P (ANOVA)

7 PROTEIN QUANTITY AND QUALITY IN FORMULA 117 Age interval 30-60d 30-90d d, not significant TABLE 7. Gain in weight versus Eurogrowth values (Z-scores) Feeding Z-score p value TABLE 8. Change in body mass index Age interval At birth At30d At60d At90d At 120 d Feeding n Values expressed in kg/m 2 (mean ± standard deviation). ANOVA, analysis of variance;, not significant. Body mass index ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.39 P (ANOVA) Urea (mmol/l) Albumin (g/l) Urea (mmol/l) Albumin (g/l) Urea (mmol/l) Albumin (g/l) TABLE 9. Serum urea and albumin 30 d 2.97 ± 0.7 a 45.1 ± d 3.59 ± 0.5 b 42.4 ± d 2.71 ± 0.9 a 42.9 ± 1.8 (n = 22) 60 d 2.16 ± 0.4 a 47.0 ± 3.1 (n = 19) 60 d 3.45 ± 0.7 b 46.9 ± 3.1 (n = 18) 60 d 2.83 ± 0.7 b 47.0 ± d 2.16 ± ± d 2.88 ± ± d 2.88 ± ± 1.9 Values for the same age with different superscript letters differ significantly at p <

8 /18 PROTEIN QUANTITY AND QUALITY IN FORMULA It can be concluded that whey-modified, whey-predominant formula with a protein-energy ratio of 1.8 g/100 kcal meets the needs of normal term infants during the first month of life and also thereafter to age 4 months without causing a compensation in increased volume and energy intakes, which has been shown to cause an increased body mass index (17). The decreased protein intake compared with that of a standard infant formula is reflected in lower serum urea concentrations, which were similar to those found in breast-fed infants during the first months of life. CONCLUSIO Mature human milk has a nutritionally available "true" protein content of only 8-9 g/1 and an energy content of between 650 and 700 kcal/1. The protein-energy ratio is thus 1.3 g/100 kcal. Standard infant formulas on the market have a protein content of 15 g/1 or a protein-energy ratio of 2.2 g/100 kcal and are either casein or whey predominant. Thus they have a protein content that is almost twice that of human milk. International recommendations are that the minimal protein content of an infant formula should be 1.8 g/100 kcal. However, previous clinical studies on the adequacy and safety of such formulas from birth are conflicting and not convincing (18). The results of the clinical study presented here show that a whey-modified, whey-predominant formula with a protein-energy ratio of 1.8 g/100 kcal and an amino acid profile close to that of human milk provides a mean protein intake of 2.26 g/kg/d during the first month of life. This is somewhat greater than that provided by breast milk during the same period. Such a formula will produce normal growth rates, which are not different from those in the recently presented Euro-Growth study (19). The formula will not cause increased volume or energy intakes or increase in body mass index. Serum urea levels are closer to those found in breast-fed infants than to those in infants receiving conventional infant formula. It can be concluded that a whey-modified, whey-predominant formula with a protein-energy ratio of 1.8 g/100 kcal is adequate and safe for term infants from birth. Such a formula will decrease the metabolic stress on the developing organs. REFERENCES 1. Gestenberger HJ, Ruh HO. Studies in the adaptation of an artificial food to human milk, II: a report of three years clinical experience with the feeding of S.M.A. AmJDis Child 1919; 171: Salmenpera L, Perheentupa J, Siimes M. Exclusively breast-fed healthy infants grow slower than reference infants. Pediatr Res 1985; 19: Dewey KG, Cohen RJ, Rivera LL, Canahuati J, Brown KH. Do exclusively breast-fed infants require extra protein? Pediatr Res 1996; 39: Raiha NCR. Protein content of human milk from colostrum to mature milk. In: Raiha' NCR, ed. Protein metabolism during infancy. Nestle' Nutrition Workshop Series, Vol 33. New York: Raven Press, 1994: Dewey KG, Beaton GH, Fjeld B, Lonnerdal B, Reeds P. Protein requirement of infants and children. EurJClin Nutr 1996; 50(suppl 1): Jarvenpaa A-L, Rassin DK, Raiha NCR, Gaull GE. Milk protein quality and quantity in the term infant, II: effects on acidic and neutral amino acids. Pediatrics 1982; 70: Davidson LA, Lonnerdal B. Persistence of human milk proteins in the breast-fed infant. Ada Paediatr Scand 1987; 76:

9 PROTEIN QUANTITY AND QUALITY IN FORMULA Fazzolari-Nesci A, Domianello D, Sotera V, Raiha NCR. Tryptophan fortification of adapted formula increases plasma tryptophan concentrations to levels not different from those found in breast-fed infants. J Pediatr Gastroenterol Nutr 1992; 14: Lonnerdal B, Chen C-L. Effect of formula protein level and ratio on infant growth, plasma amino acids and serum trace elements. Ada Paediatr Scand 1990; 79: ESPGAN Committee on Nutrition. Guidelines on infant nutrition, I: Recommendations for the composition of an adapted formula. Ada Paediatr Scand Suppl 1977; 3: American Academy of Pediatrics Committee on Nutrition. Commentary on breast feeding and infant formulas, including proposed standards for formulas. Pediatrics 1997; 57: Food and Drug Administration, Rules and Regulations. Nutrient requirements for infant formulas (21 CFR, Part 107). Fed Reg 1985; 50: Young VR, Pelletier VA. Adaptation to high protein intakes, with particular reference to formula feeding of healthy term infants. J Nutr 1989; 119: Fomon SJ. Requirements and recommended dietary intakes of protein during infancy. Pediatr Res 1991; 30: Beaton GH, Chery A. Protein requirements of infants: a reexamination of concepts and approaches. Am J Clin Nutr 1988; 48: Raiha NCR, Fazzolari A, Cajozzo C, et al. Whey modified infant formula with protein-energy ratio of 1.8 g/100 kcal is adequate and safe from birth to 4 months. J Pediatr Gastroenteral Nutr 2000; 31 (suppl 2): S Fomon SJ, Ziegler EE, Nelson SE, Rogers RR, Franz JA. Formula with protein-energy ratio of 1.7 g/100 kcal is adequate but may not be safe. J Pediatr Gastroenterol Nutr 1999; : Jonas LM, Picciano MF, Hatch TF. Indices of protein metabolism in term infants fed either human milk or formulas with reduced protein concentration and various whey/casein ratios. J Pediatr 1987; 110: Haschke F, van't Hof MA. Euro-Growth. J Pediatr Gastroenterol Nutr 2000; 31 (suppl 1): S1-S Nayman R, Thomson ME, Scriver CR, Clow CL. Observations on the composition of milk-substitute products for treatment of inborn errors of amino acid metabolism: comparison with human milk: a proposal to rationalize nutrient content to treatment products. Am J Clin Nutr 1979; 32: DISCUSSION Dr. Lead: My question is about glutamine. Although glutamine is not an essential amino acid, in some catabolic situations, it can become one, and particularly for the gut, which uses glutamine as a major substrate. How do these formulas compare with human milk with respect to glutamine concentrations? Dr. Raiha: Maybe Dr. Boza can answer that. Dr. Boza: We have studied glutamine concentrations in milk proteins. Casein contains ~9 g glutamine per 100 g of protein, whereas whey protein contains 6.4 g glutamine per 100 g. From the known whey/casein ratio in human milk, you can easily calculate the actual content of glutamine, which turns out to be somewhat higher in human milk than in a whey-predominant formula. Dr. Rubaltelli: When we see an infant who is not gaining weight adequately, we often measure the serum urea concentration to determine whether the protein intake is adequate. Is this a valid test, or is there a better way? Dr. Raiha: I think the serum urea level and even urinary urea levels are very good indicators of whether you should increase or decrease the intake of protein, especially in preterm infants. We are using these indicators in Malmo to assess the correct protein intake for preterm infants. Dr. Vanderweid: Before accepting your data as a basis for making recommendations, I would like to know more about the statistical power of your study. I wonder whether with cases, you have the statistical power to detect differences on the order of 10-20%. For instance, you showed us that weight gain was faster with traditional NAN, but the difference was not significant. If you had 100 cases, there would surely be a statistically significant difference.

10 120 PROTEIN QUANTITY AND QUALITY IN FORMULA Similarly with body mass index, there was no apparent difference, but maybe with more cases, you could have shown differences. Can you tell us about the statistical power of the study? Dr. Raiha: It is very difficult to do studies like this on hundreds of infants, but I think Dr. Haschke would have more to say about the statistics. Dr. Haschke: With subjects, we calculated having a statistical power of 0.8 to detect a difference in weight gain of 3.5 g/day, that is 1 SD. Your difference was ~3 g between the two formula-fed groups, during the first month. A short comment on weight gain. Not all the infants received the formula from the first day of life. As in all studies now, breast feeding was encouraged, and it was only if this was unsuccessful that the infants were enrolled in the study. Thus the day when the infants were enrolled and started on the new formula was not day zero that is almost impossible now with any formula study. Because of this, the data during the first month were not easy to compare between the groups, as the infants started on the test formulas at different ages. However, if you look at the weight-gain and length-gain data between 30 and 120 days, there is definitely no difference between the two formulas (2.2 g and 1.8 g), and one can say that 1.8-g formula achieves growth results that are closer to those of the breast-fed infants. Dr. Rigo: You found quite a high concentration of urea in the nonprotein fraction of human milk, 7% of the nitrogen in human milk. Urea is well absorbed but not very well used by infants, so it contributes to the blood urea nitrogen and to urea excretion in the urine. Therefore when you compare blood urea and urea excretion between the low-protein-content formula and human milk, some of the difference may be due to the urea in human milk. Dr. Raiha: The question of course is how much of the urea in human milk is used. I think this probably depends on the bacterial flora, although there are different views on this. Nevertheless, the protein intake in the reduced-protein formula was definitely higher than that in the human milk-fed infants. Dr. Vigi: I believe that this formula could pose some problems for preterm infants on discharge from hospital, when they are still taking a high-energy, high-protein formula like most of them are if they are not breast-fed. Would it not be advisable to have a transitional period on a conventional formula or a formula with a higher protein content? Dr. Raiha: The feeding of preterm infants after discharge is a very important question. Many preterm infants go home from intensive care units weighing <2 kg, and they are often breast-fed by the time of discharge. It is difficult to say that maternal breast milk is inadequate for such babies, but I think they probably need more nutrients than can be provided by their mothers' milk. More work must be done on this subject, which is discussed by Dr. De Curtis elsewhere in this volume. My own view is that such infants need nutrient supplements. Dr. Fazzolari: I believe it should be stressed that with any new formula produced now, we should be considering not only the overall protein content but also the protein quality.