Protein Requirements in Infancy

Transcription

1 Infant Formula: Closer to the Reference, edited by Niels C. R. Raiha and Firmino F. Rubaltelli. Nestle Nutrition Workshop Series, Pediatric Program. Vol. 47 Supplement. Nestec Ltd.. Vevey/Lippincott Williams & Wilkins, Philadelphia Protein Requirements in Infancy Ekhard E. Ziegler Fomon Infant Nutrition Unit, Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA REQUIREMENTS AND RECOMMENDED INTAKES Adults need nutrients to replace inevitable losses. Infants also need nutrients to replace inevitable losses, but in addition, infants need nutrients for growth. In the case of protein, the needs of the infant for growth are large relative to total needs. Fomon (1) estimated that during the first month of life, as much as 52% of the total protein requirement is needed for growth, and Dewey et al. (2) provided an even higher estimate (64%). As the rate of growth slows with increasing age, the amount of protein needed for growth also decreases, and with it the proportion of protein requirement accounted for by growth. By age 9-12 months, the proportion reaches 18% of total protein requirement. The rapid change of requirements with increasing age explains why defining protein requirements of infants has been such a difficult task (3). The requirement for a nutrient is defined as the minimal amount that satisfies the needs of an individual. Because individuals differ in their requirements, groups of individuals have a range of requirements. This is particularly true for infants, who show considerable interindividual and gender-related differences in rate of growth. It is, however, seldom possible to determine the needs of individual subjects and to establish the mean requirement for groups of individuals. However, the mean requirement is seldom of interest. It defines a level of intake that meets the needs of only half of all individuals. What is of interest is an intake that meets the needs of all or almost all individuals of a given class. Such an intake is strictly not a requirement but rather a "recommended dietary intake" or a "safe level of intake." In common parlance, however, such an intake is usually referred to as a "requirement." The fact that individuals differ with regard to requirements has important implications when experimental approaches are used for the establishment of group "requirements." As intake is reduced from an adequate level to a slightly inadequate level, individuals with the highest requirement will be affected before individuals with lower requirements. Conversely, if intake is increased from an inadequate level toward an adequate level, individuals with the lowest requirements will have their needs satisfied before individuals at the opposite end of the spectrum. The greater the proportion of affected individuals, the more likely it is that a group response is detectable. 97

2 98 PROTEIN REQUIREMENTS IN INFANCY Referencing requirements for specific nutrients to requirements for energy is useful because interindividual differences in nutrient requirements are usually paralleled by differences in energy requirements. This is particularly true for infants, who are very adept at regulating food intake to satisfy energy needs. Variation in growth rate affects energy needs almost as much as protein needs. Referencing protein requirements to energy requirements (protein/energy ratio) is therefore the preferred way of expressing protein requirements of infants. THEORETICAL APPROACHES Among the approaches available for determining protein requirements, the two "theoretical" approaches must be distinguished from the experimental approaches. The factorial approach obtains estimates of protein needs for maintenance and growth. These are combined to yield estimates of total protein requirement. In the other theoretical approach, the average protein intake of the breast-fed infant is estimated, and the assumption made that the protein needs of the breast-fed infant are met at all times. The great advantage of these approaches is that they provide age-specific estimates of requirements and thus make transparent how dramatically requirements of the infant change with age. Conversely, the theoretical approaches yield average requirements only, and it is difficult if not impossible to deduce recommended intakes that is, intakes that meet the needs of most individuals. Estimates of Requirements by the Factorial Method In this approach, separate estimates are obtained for maintenance needs that is, needs determined by inevitable losses and needs for formation of new tissues (growth). The estimates obtained with this approach depend somewhat on the assumptions selected. The factorial estimates presented by Fomon (1) and Dewey etal. (2) differ slightly because of differences in assumptions. Table 1 illustrates the Age interval (mo) TABLE 1. Protein requirements estimated by the factorial approach Growth a Fomon (1) (g/kg/d) Maintenance Total Growth Dewey etal. (2) (g/kg/d) Maintenance 01 Total a Increments from Fomon et al. (3) and assuming 90% efficiency. " Inevitable losses. c Increments for male breast-fed infants from WHO Working Group (4) and assuming 70% efficiency (4). "Assuming maintenance needs to be 90 mg/kg/d.

3 PROTEIN REQUIREMENTS IN INFANCY 99 differences in assumptions. Inevitable losses were assumed by Fomon to be 1.17 g nitrogen/kg body protein/day, whereas Dewey et al. assumed losses to be 90 mg N/kg body weight/day. Protein accretion was calculated by Fomon on the basis of the growth of formula-fed infants, whereas Dewey et al. based it on the somewhat slow growth of breast-fed infants. Fomon assumed 90% efficiency of conversion of dietary protein to body protein, whereas Dewey et al. assumed the efficiency to be 70%. Not too surprising, the estimates of total protein requirements show some differences (Table 1). Aside from the differences, both estimates show a rapid decline in protein requirements during the early months of life. Protein Intake of the Breast-fed Infant As with the factorial method, certain assumptions have to be made with this approach, and differences in assumptions explain differences in estimates. Ample information is available regarding the volume of intake of breast milk and breastmilk nitrogen concentration. Choosing the most representative from the available data presents some difficulty. A larger problem is presented by the nonprotein nitrogen fraction of breast milk, which accounts for 24% of total nitrogen in mature milk (5). The difficulty arises from uncertainty surrounding the "bioavailability" of the various components of the nonprotein nitrogen fraction. In other words, there is disagreement over what proportion of nonprotein nitrogen can be considered to be equivalent to protein. Fomon (1) made the assumption that 27% of nonprotein nitrogen is available, whereas Dewey et al. (2) assumed that somewhere between 46% and 61% is available. As the comparison in Table 2 shows, the estimates of protein intakes differ remarkably little, given the differences in assumptions as well as the data on milk intake and composition. What is very evident is the marked decrease in protein intake with advancing age of the infant. This decrease largely reflects the decrease in protein concentration of breast milk and, to a lesser degree, the decrease in volume of milk intake per unit of body weight. TABLE 2. Estimates of protein intake of breast-fed infants Age interval Fomon (1) Dewey et al. (2) (mo) (g/kg/d) a (g/kg/d)" ^t NPN, nonprotein nitrogen. a "Protein" intake includes 27% of NPN. " "Adjusted" protein intake includes 41-61% of NPN.

4 700 PROTEIN REQUIREMENTS IN INFANCY EXPERIMENTAL APPROACHES In experiments designed to define protein requirements of groups of individuals, dietary protein intake is manipulated, and a relevant biologic response is measured. Among the many available responses, nitrogen balance has been used extensively to determine protein needs in the short term. In the case of the infant, nitrogen balance cannot be used to establish protein requirement, although nitrogen balance is very useful for comparative purposes. In the infant, the establishment of requirements hinges on the demonstration of normal growth. Growth is arguably the most important test of long-term adequacy. For the establishment of requirements, it is necessary that one of the experimental groups receives protein at a level below the presumed requirement. Only then is it possible for another group or groups to show a response when receiving higher levels of intake. Because this approach necessitates the provision of subrequirement levels of protein, it must be used with the utmost caution. Often the objective is not to establish a requirement but to demonstrate that an experimental protein level yields a response indistinguishable from the response obtained with a level known to be above requirement. In other words, the objective is to demonstrate equivalency. In this case there is no necessity to include a subrequirement level. A comparison is made between two levels of protein intake (control and experimental), or two types of protein, which are fed at similar levels; if similar responses are obtained, equivalency is presumed to have been demonstrated. The equivalency approach is suitable for assessing not only different levels of protein but also different types of protein that is, the assessment of protein quality. It is important to keep in mind the limitations of experimental approaches. One set of limitations resides in the chosen end point and the accuracy with which it can be measured. For example, if infant growth is used as the outcome, the quality of the anthropometric measurements, together with the degree of control over experimental variables such as age intervals, determines the detection limit of the test. Experimental approaches also have theoretical limitations, given the fact that groups of individuals by definition have a range of requirements. Most experiments that are designed to explore the "requirement" for a given nutrient use a design whereby the experimental treatment provides an intake at or close to the presumed requirement, while the control group receives an intake that is known to be adequate. Inasmuch as all individuals in the experimental group receive the same intake, whether it be per unit body weight or per unit of dietary energy, as the intake is reduced, those individuals with the highest requirement will be the first to show evidence of inadequate intake. The more individuals fail to have their needs met, the more the response of the entire experimental group will be affected. Thus when the response of the experimental group is detectably different from that of the control group, a proportion of its individuals will have an inadequate intake, whereas others will not. In other words, when there is a detectable difference, it can be said with certainty that not all individuals of the experimental group had their requirements met.

5 PROTEIN REQUIREMENTS IN INFANCY 101 Nitrogen Balance Studies Nitrogen balance studies are an indispensable tool for the establishment of protein adequacy. However, balance studies permit assessment of only short-term adequacy. Therefore, just as balance studies are unsuitable for the establishment of long-term adequacy, they are unsurpassed for comparative evaluation of different protein sources or different levels of dietary protein. Nitrogen balance studies are especially useful when comparison is made with a protein source of established adequacy, preferably by using a balanced crossover design. Metabolic Balance Studies with Reformulated NAN Recently the commercially available formula NAN was compared with a similar formula that, however, contained a somewhat different protein at a lower concentration than regular NAN. The objective of the study was to demonstrate equivalency. It used a balanced crossover design in which each infant was studied once while being fed the control formula and once while being fed the experimental formula. The order in which the formulas were studied was random and predetermined, and the investigators were blinded in that the formulas were identified only by code, which was revealed after the conclusion of the study. Eight normal infants (two girls, six boys) served as the subjects. They were between 39 and 139 days old at the start of the first balance study. Methods The balance studies were of 72 hours' duration. Feces and urine were collected between carmine markers using methods described by Fomon (6). The formulas were fed for > 11 days before the start of a balance study. Formula intake was determined by carefully weighing feeding bottles before and after each feeding. Formulas, feces, and urine were analyzed by methods described previously (7,8). Nutrient intakes were calculated from formula intakes and measured nutrient concentrations. Statistical treatment included standard procedures for a 2 X 2 crossover design. Formula Composition Formula (NAN) had a protein concentration of 2.24 g/100 kcal. Protein was provided by demineralized whey and skim milk. Formula (reformulated NAN) had a protein concentration of 1.83 g/100 kcal. Its protein was provided by modified whey proteins, unmodified whey proteins, and skim milk. Formula also contained added L-arginine and L-histidine. The carbohydrate of both formulas was lactose and a small amount of maltodextrin. The fat of both formulas was provided by palm olein, low-erucic-acid rapeseed, coconut, and sunflower oils. Formula contained in addition small amounts of fish oil and fermentation oil as sources of long-chain polyunsaturated fatty acids. Both formulas contained added taurine and added nucleotides.

6 102 PROTEIN REQUIREMENTS IN INFANCY Results All eight infants completed the study as planned. The results are summarized in Table 3. Data for intake, absorption, urinary excretion, and retention of nitrogen are presented for individual infants in Figs. 1 and 2. Nitrogen intake was lower with Formula (mean intake, 284 mg/kg/day) than with Formula (mean, 349 mg/kg/day), a reflection of the difference in protein content between the formulas. Because percentage of nitrogen absorption was similar (, 89.3%, vs., 89.5%), nitrogen absorption (mg/kg/day) was lower with Formula than with Formula (p = 0.002; Fig. 1). However, urinary excretion also was significantly (p = 0.006) lower with Formula than with Formula (Fig. 2). Consequently, net retention of nitrogen (milligrams per kilogram per day) was similar with the two formulas (p = 0.73; Fig. 2). As Table 3 shows, absorption and retention of calcium, magnesium, and phosphorus were similar with the two formulas. Absorption of fat averaged 92.2% with Formula and 92.1% with Formula. Conclusions Despite the lower protein content of reformulated NAN (Formula ), infants achieved similar nitrogen retention as when they were fed commercial NAN (Formula ). The two formulas were equivalent with regard to meeting the protein needs to the study infants. It was evident that equal retention was achieved through lower urinary nitrogen excretion with Formula. The lower urinary nitrogen excretion with Formula was adequately explained by the lower protein intake provided by that formula. Therefore inferences regarding the biologic value of the protein of Formula could not be drawn from the data. Nitrogen Calcium Magnesium Phosphorus Fat TABLE 3. Results of metabolic balance studies with formulas blue and yellow* Intake (mg/kg/d) 349 ± ± ± ± ± ± ±8 33 ± 6 (g/kg/d) 5.40 ± ± 1.04 Absorption (mg/kg/d) 312 ± 70 s 253 ± 46 a 33 ± ± ± ± ± 7 30 ± 6 (g/kg/d) 4.96 ± ± 0.88 Urinary excretion (mg/kg/d) 194 ± 21 a 136 ± 25 a 5±3 4 ± ± ± ± 5 13 ± 4 (mg/kg/d) 117 ± ± ± ± ± ± ±6 17 ± 7 Retention * Study used eight normal infants in crossover design. a Values with same superscript within columns are statistically different at p < (% no. of intake) 32.2 ± ± ± ± ± ± ± ± 12.3

7 PROTEIN REQUIREMENTS IN INFANCY 103 a I > c 0) O) o o intake Absorption FIG. 1. Intake (mg/kg/d) and absorption (mg/kg/d) of nitrogen by infants fed Formula (open circles) or Formula (solid circles). Each symbol indicates the result of a metabolic balance study. Horizontal lines indicate group means. 300 'kg/d ogen o o o Urinary Excretion Retention FIG. 2. Urinary excretion (mg/kg/d) and retention (mg/kg/d) of nitrogen by infants fed Formula (open circles) or Formula (solid circles). Each symbol indicates the result of a metabolic balance study. Horizontal lines indicate group means.

8 104 PROTEIN REQUIREMENTS IN INFANCY Growth Studies When growth of infants is used as the biologic response, the implicit assumption is made that all needs have been met when the needs for growth have been met, as evidenced by the infant showing normal growth. Although theoretically there might be needs that are not necessarily met when growth needs have been met for example, for optimal immune status or neurocognitive or behavioral development evidence for this possibility is lacking. Hence normality of growth is widely accepted as a proxy for global adequacy of protein intake (4). Protein Quality The report by Fomon et al. (9) provides an example of the use of growth studies to answer questions of protein quality. Formulas with protein derived from isolated soy protein (ISP) are customarily fortified with methionine. The question Fomon et al. tried to answer was whether ISP alone (without methionine fortification) was able to meet the infant's needs for sulfur-containing amino acids, and what level (protein/energy ratio) of ISP would be required for that to be the case. In a series of studies, the growth performance of normal infants, together with serum concentrations of albumin and urea, was used to define the lowest level at which ISP alone would be satisfactory. The series did not attempt to define the lowest level at which ISP with methionine was satisfactory. The standard against which performance of infants fed the various experimental formulas was judged was the growth of infants fed formulas with cow's milk protein at concentrations of 2.1 to 2.7 g/100 kcal. On the basis of growth performance and serum chemical responses, it was concluded that formulas with ISP concentrations of 2.8 and 3.0 g/100 kcal supported normal growth, even in the absence of methionine supplementation. At ISP concentrations of 2.6 and 2.2 g/100 kcal, there was clear evidence from weight gain and/or serum chemical values that methionine supplementation was beneficial. As mentioned earlier, at the level of 2.6 and 2.2 g/100 kcal, nitrogen balance studies failed to show an effect of methionine supplementation on nitrogen retention. It was therefore concluded that growth is more sensitive as an indicator of protein and amino acid adequacy in infants than is nitrogen balance. Protein Requirement Two growth studies were specifically designed for establishing protein requirements of normal infants. The somewhat different objectives are reflected in the different designs the studies used. The first of these studies (10) had the objective of assessing the validity of the estimates derived by the factorial method (Table 1). It was designed so that protein intakes decreased with age and approximately matched the estimated requirements. To achieve the desired protein intakes, two formulas with protein/energy ratios of 1.70 and 1.24 g/100 kcal, respectively, were fed in changing proportions to the experimental group (n = 15 normal male infants). A control group of male infants (n = 13) was fed throughout a formula with protein/energy ratio of

9 PROTEIN REQUIREMENTS IN INFANCY g/100 kcal. All formulas provided whey-predominant bovine milk proteins and were supplied in ready-to-feed form. Data also were compared with data from a reference group of 261 male infants fed other milk-based formulas with protein/energy ratios between 1.98 and 2.67 g/100 kcal. As Table 4 shows, the protein/energy ratio of the experimental group was 1.56 g/100 kcal between ages 8 and 28 days and decreased gradually to 1.25 g/100 kcal by days. Corresponding protein intakes were 1.85 g/kg/d between ages 8 and 28 days, decreasing to 1.18 g/kg/day by age days. It is evident that these protein intakes were slightly less than the estimated requirements. The rate of weight gain was slightly less for the experimental group than for the control group or the reference group, but the differences were not statistically significant. The same was true for rates of length gain (data not shown), although for the entire interval of days, length gain in the experimental group was less (p = 0.007) than that in the reference group. Serum albumin concentrations tended to be slightly but not significantly lower in the experimental group. Urea nitrogen concentrations of the experimental group, conversely, were very low at each age and were significantly different (p < 0.001) from both the control and the reference group. Based on the slightly slower rates of growth and the markedly lower serum urea nitrogen concentrations, TABLE 4. Energy and protein intakes, growth, and serum chemical values Energy intake (kcal/kg/d) Experimental Control Reference Protein intake (g/kg/d) Experimental Control Reference Protein-energy ratio (g/100 kcal) Experimental Control Reference Gain in weight (g/d) Experimental Control Reference Serum urea nitrogen (mmol/l) Experimental Control Reference ± 14 a 116 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.35 Age interval (d) ±8 102 ± ± ± ± ± ± ± ± ± ± ± ± ± ±8 95 ± 6 94 ± ± ± ± ± ± ± ± ± ± ± ± 0.47 Male infants (experimental, n = 15; control; n = 13; reference, n = 261. From Fomon et al. (10). a mean ± SD.

10 106 PROTEIN REQUIREMENTS IN INFANCY it was concluded that protein intakes achieved by the experimental group were slightly below requirement. This finding provided support for the validity of the estimates based on the factorial method. The second growth study had the objective of testing the adequacy of a formula with a protein/energy ratio of 1.7 g/100 kcal (11). The formula provided casein-predominant cow's milk protein and was fed during the entire study period from age 8 to 112 days. The protein/energy ratio was set slightly above the ratio of 1.53 g/100 kcal that was provided in the previous study between ages 8 and 28 days and found to be inadequate. Sixteen male infants were fed the experimental formula, and data were compared with those from a reference group of 344 male infants fed other milkbased formulas. Results of this study are summarized in Table 5. Protein intake averaged 2.14 g/kg/d between ages 8 and 56 days, and energy intake averaged 128 (SD, 12) kcal/kg/d, significantly higher than that in the reference group (p < 0.001), where it was 116 (SD, 13) kcal/kg/day. At an energy intake of 116 kcal/kg/day, protein intake of the experimental group would have been 1.94 g/kg/day. As Table 5 shows, weight gain but not length gain was significantly greater in the experimental group than in the reference group. Plasma concentrations of albumin and urea nitrogen were less (p < 0.001) in the experimental group than in the reference group. It was concluded that infants in the experimental group received adequate intakes of protein, but only because energy intakes were unusually high. The study raised the possibility that a diet with a marginally low protein/energy ratio stimulates increased energy intake. As pointed out, with a normal energy intake, the protein intake would have been slightly Energy intake (kcal/kg/d) Mean SD Protein intake (g/kg/d) Mean SD Weight gain (g/d) Mean SD Length gain (mm/d) Mean SD TABLE 5. Energy and protein intakes, gain in weight, and gain in length a a Experimental c a a a Reference C " a Male infants (experimental n = 16, reference n = 344). From Fomon etal. (11). a Values with the same superscript within a row and corresponding age column are statistically different p < " Values with the same superscript within a row and corresponding age column are statistically different at p < c Values with the same superscript within a row and corresponding age column are statistically different at p < 0.05.

11 PROTEIN REQUIREMENTS IN INFANCY 107 below the estimated requirement. Findings in two recent studies of postdischarge premature infants (12,13) are consistent with the notion that a marginally low protein/energy ratio is associated with increased energy intake. In each of the studies, feeding a formula with lower (that is, marginally inadequate) protein/energy ratio was associated with substantially higher energy intakes than was feeding a formula with higher protein /energy ratio. REFERENCES 1. Fomon SJ. Requirements and recommended dietary intakes of protein during infancy. Pediatr Res 1991; 30: Dewey KG, Beaton G, Fjeld C, et al. Protein requirements of infants and children. Eur J Clin Nutr 1996; 50(suppl 1): SI Fomon SJ, Haschke F, Ziegler EE, Nelson SE. Body composition of reference children from birth to age 10 years. Am J Clin Nutr 1982; 35: World Health Organization Working Group on Infant Growth. An evaluation of infant growth: a summary of analyses performed in preparation for the WHO Expert Committee on Physical Status: the use and interpretation of anthropometry. Doc WHO/NUT Geneva: WHO, Lonnerdal B, Forsum E, Hambraeus L. A longitudinal study of the protein, nitrogen, and lactose contents of human milk from Swedish well-nourished mothers. Am J Clin Nutr 1976; 29: Fomon SJ. Nutrition of normal infants. St. Louis: Mosby-Year Book, 1993: Haschke F, Ziegler EE, Edwards BB, Fomon SJ. Effect of iron fortification of infant formula on trace mineral absorption. J Pediatr Gastroenterol Nutr 1986; 5: Nelson SE, Rogers RR, Frantz JA, Ziegler EE. Palm olein in infant formula: absorption of fat and minerals by normal infants. Am J Clin Nutr 1996; 64: Fomon SJ, Ziegler EE, Nelson SE, Edwards BE. Requirement for sulfur-containing amino acids in infancy. J Nutr 1986; 116: Fomon SJ, Ziegler EE, Nelson SE, Frantz JA. What is the safe protein-energy ratio for infant formulas? Am J Clin Nutr 1995; 62: Fomon SJ, Ziegler EE, Nelson SE, Rogers RR, Frantz J A. Infant formula with protein-energy ratio of 1.7 g/100 kcal is adequate but may not be safe. J Pediatr Gastroenterol Nutr 1999; 28: Brunton JA, Saigal S, Atkinson SA. Growth and body composition in infants with bronchopulmonary dysplasia up to 3 months corrected age: a randomized trial of a high-energy nutrient-enriched formula fed after hospital discharge. J Pediatr 1998; 133: Carver JD, Wu PY, Hall RT, Ziegler EE, Sosa R, Jacobs J, et al. Growth of preterm infants fed nutrient enriched or term formula after hospital discharge Pediatrics 2001; 107: DISCUSSION Dr. Rdihd: In the studies with Fomon that you published last year on the 1.7 g/100 kcal casein-predominant formula, do you think that one explanation for the increased volume intake (and thus the increased energy intake, increased growth, and increased body mass index) could be to compensate for inadequate protein quality? We know there are problems with limiting amino acids in casein-predominant formulas, especially at that low level of protein intake. Dr. Ziegler: Certainly that is a possibility. I have deliberately not touched on protein quality because it complicates an already complicated matter, but it is true that casein-predominant formulas may be of lower protein quality than whey-predominant formulas when it comes to meeting the requirements of infants. Dr. Zoppi: I agree completely with Dr. Ziegler about the differentiation between requirement and recommended intake. This is an important difference. There is currently a group of scientists and nutritionists who are constantly reducing the protein requirement, but I believe that is wrong we are in danger of going below the minimal protein intake that is necessary

12 108 PROTEIN REQUIREMENTS IN INFANCY not only for growth and nitrogen balance but also for other functions such as antibody formation and so on. Dr. Ziegler: I agree with you. We used objective indices that can be measured, such as growth and nitrogen balance. It is certainly possible that requirements could be defined by other end points such as behavior or the immune response. Unfortunately the tools are not available to enable us to use those variables in a quantitative way. I wish we could measure behavior or other measures of well-being of the infant, but we cannot. Dr. Rubaltelli: I wonder if you could comment on the actual advantages of the reduced protein intake with the new lower-protein NAN formula? Dr. Ziegler: The Scandinavians have long thought that formula-fed babies receive an excess of protein, and that may well be true. With this formula, we really do not have an excess of protein. We did not measure urea nitrogen, but my guess is that if you compare this formula with breast feeding, the urea nitrogen would be similar. So if excess protein is indeed harmful, or potentially harmful, then we avoid that problem with this lower protein content. Dr. Rigo: Could you speculate on the risks for the infant of increasing the protein intake during the first year? Dr. Ziegler: I do not think the risk is very great. I think Axelsson from Raiha's group has suggested that there may be increased insulin secretion with excessive protein intake (1). From the point of view of the renal solute load, the additional protein has a trivial effect and is not worthy of concern. However, philosophically I take the view that you should provide only a small excess above requirement, and so I think the move toward lower-protein formulas is a good thing. Dr. Haschke: My question is related to older infants. The data obtained by using the factorial approach clearly show that protein requirement in relation to energy consumption diminishes during later infancy. In the United States, you do not have follow-up formulas, and American children are given infant formulas with a protein concentration of ~ 2 g/100 kcal during the first year of life. However, in Europe, the situation is different because of the regulations imposed by the European Community. We have a two-step system: we have start-up formulas, like the new NAN, and then we have follow-up formulas, in which the companies are forced to put much more protein, which is complete nonsense. The only justification for a high-protein intake would be in developing countries where these formulas may sometimes be the sole source of protein. I think it is high time we be allowed to reduce the protein content substantially in follow-up formulas. Would you agree about that? Dr. Ziegler: I agree completely with you. The only rational change in formula composition would be to go from a higher protein concentration in a start-up formula to a lower protein concentration in a follow-up formula. Traditionally the reason for higher protein concentrations in the follow-up formulas has been that weaning foods are low in protein, and formulas need to supplement that low-protein content. However, in the United States at least, this is not true. The composite protein concentration in weaning foods cereals, fruits, and so on is just about the same as that of a formula, so there is no need for protein supplementation. Dr. Moro: I absolutely agree with Dr. Haschke. I have been saying for a long time that follow-up formulas have a too high protein content. It is high time to produce follow-up formulas with a lower protein content. Dr. Endres: Until now, all attempts at European level to decrease the lower limit of protein in follow-up formulas from 2.25 g to 1.8 g/100 kcal have failed. I suppose this reflects a lack of discussion in the Codex Alimentarius at international level. The currently required protein content of these formulas results in a very high protein intake around 5 g/kg/day, which is about 3 times the value that infants or young children need at that age.

13 PROTEIN REQUIREMENTS IN INFANCY 109 Dr. Ziegler: If it gives you any comfort, you are not the only ones who have to deal with bureaucratic sclerosis; we have it in the United States, too. United States law recognizes only one infant formula. So in the United States, you cannot have a start-up formula and a followon formula; every formula has to meet the needs of even the youngest infants. Dr. Bachmann: I have a question concerning the solute load and urea. Urea concentrations in the plasma depend on the volume and flow of urine, which is related to the solute load, but also, and principally, on the arginine concentration in the plasma. If you have an increase in plasma arginine because of catabolism, blood urea will increase; thus if you do not supply enough protein and there is a catabolic state, there will be an increase in urea excretion. So urea may not be a reliable indicator of solute load. Dr. Ziegler: Yes, but it is an indicator of protein intake. By the way, healthy babies are seldom catabolic; they are always strongly anabolic. Empirically the relation between blood urea nitrogen and protein intake is a straight line. In breast-fed babies, it is ~6 mg/dl; with formulas, it goes up to 9; with cow's milk, it is 20; so empirically there is a very tight linear relation between protein intake and blood urea concentration because the babies are healthy and have normal renal function. Dr. Rdihd: We did a study some years ago on preterm infants, looking at different protein intakes, and sometimes we noted a sudden increase in urea with no change in the protein intake. We found that these infants developed signs of infection a few days after the urea measurement. I agree the healthy baby is never catabolic, but if the urea starts to go up suddenly, this may be an early sign of infection, and it means that they have become catabolic. Dr. Bohles: Looking at the recommendations for increased protein intake in very premature infants, the classic studies on net acid balance come to mind. Have you considered the influence of protein intake on acid-base metabolism in very premature infants? Do you think those studies on net acid balance done 30 years ago in infants weighing ~ 1,500 g should be repeated in infants weighing < 1,000 g? Dr. Ziegler: We and others have never seen an increase in acidosis with increasing protein intake. Late metabolic acidosis does occur, but nobody makes a big issue of it because most babies get a little citrate, which takes care of it. In the studies in which protein intake has been increased, we have never seen a significant change in base excess or ph in premature babies. Dr. Moro: Nitrogen absorption on Formula was lower than that with Formula. How do you explain this difference? Dr. Ziegler: The nitrogen absorption was lower on Formula because the intake was lower. Dr. Moro: So it was related only to the intake? Dr. Ziegler: It is strictly a function of intake; that is why I showed this straight-line relation. Dr. Bertrand: When you decreased the protein intake of your formula, what did you do about the zinc intake? Was it constant, or did you decrease it? Dr. Ziegler: No, everything was kept as nearly constant as possible calcium, magnesium, zinc, iron, and so on, everything was the same. Dr. Bertrand: And was your zinc balance positive? Dr. Ziegler: Yes, it was positive. Zinc balance is tricky because, as you know, a normal healthy infant occasionally has a negative zinc balance. We do not know why, but presumably it is because there is a temporary increase in endogenous excretion. Average zinc balance was positive with both formulas. Dr. De Curtis: Have you found any difference in protein requirements during the first month of life between male and female infants? Dr. Ziegler: Yes, we have. In the two growth studies that I showed you, and also in the reference data, we used only male infants just because of that difference. Male infants have

14 110 PROTEIN REQUIREMENTS IN INFANCY slightly higher protein requirements than female infants; we have known this for a number of years. We did a series of studies on soy protein-based formulas a while ago, and we realized early on that male infants needed additional methionine at a level of soy protein intake that was adequate for the female infants. Since that time, it has been established that male infants grow faster, so it makes sense that they have a higher protein requirement. Dr. Vigi: Some years ago, we did a study on preterm infants in which we used different concentrations of protein in experimental formulas. One of these formulas was a simple unmodified whey-predominant formula with a protein content of 1.2 g/dl, much like the one you presented. We observed very poor growth during the first month in babies receiving this formula. We were concerned enough to stop the experiment, as we thought we were giving these babies less protein than they needed, and they could not compensate by consuming more of the formula. I thought this was a warning to us that our approach may not have been entirely safe. Dr. Ziegler: Yes, I agree with you that 1.2 g protein/dl is definitely too low for premature infants. REFERENCES 1. Axelsson IE, Ivarrson SA, Raiha NC. Protein intake in early infancy: effects on plasma amino acid concentrations, insulin metabolism and growth. Pediatr Res 1989; 26: