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2. Protein requirements of infants

2.1. Using the breastfed as a model (0-6 months)

2.1.1. Rationale for this approach. In the 1985 report, protein requirements during the first 6 months were based on estimated intakes of breastfed infants. The explicit assumption of this approach was that 'the protein needs of an infant will be met if its energy needs are met and the food providing the energy contains protein in quantity and quality equivalent to that of breast milk' (p. 98). This approach is often justified using the evolutionary argument that human milk has been adapted to be ideally suited to the nutritional needs of the human infant; therefore its protein content by definition should meet or exceed the required amounts.

The above argument should be carefully examined because it is the underpinning of most calculations of nutrient requirements (not just for protein) during early infancy. In criticising this argument, Fomon (1993) has suggested that the composition of human milk represents an evolutionary compromise between the needs of the infant and the needs of the mother. Theoretically, if the 'true' needs of the infant required a milk protein concentration that would result in excessive depletion of the mother's reserves, it is argued that natural selection might result in milk protein levels somewhat less than optimal from the infant's point of view. The observation that, after the first 2-3 months, breastfed infants gain weight less rapidly than do formula-fed infants leads some to question whether breastfed infants are really meeting their protein requirements.

This 'evolutionary compromise' theory deserves further scrutiny. In the case of protein, it is useful to calculate how much of a 'saving' the mother might achieve if her milk were somewhat lower in protein than the amount truly required by the infant. If one assumes an average production of about 800 ml/d, and a milk protein concentration of 9 g/1, the average woman secretes approximately 7 g/d of protein (Butte et al, 1984; Heinig et al, 1993). If the infant's true requirement were closer to the typical intakes of formula-fed infants (approximately 11 g/d at 3 months (Heinig et al, 1993)), the 'saving' to the mother of producing a lower-protein milk would be about 4 g/d. This represents about 8% of the current RDA (in the US) of 50 g/d protein for an adult woman during the period of exclusive breast-feeding. After other foods are introduced to the infant and breast milk production decreases, this 'saving' would be even less. During most of our evolutionary past, it is believed that the typical human diet was based heavily on wild game and fish and was thus very high in protein (Eaton and Konner, 1985). Therefore, it is unlikely that there was much evolutionary pressure to limit protein secretion in human milk. After the agricultural revolution of about 12 000 years ago, a relatively recent event in evolutionary terms, humans began to rely more extensively on plant foods as the dietary staples. It is possible that a decrease in availability of animal protein preceded (perhaps even prompted) the agricultural revolution. Fomon (1993) argues that if selection pressure is strong enough, 12-20000 years is sufficient time for the spread of a favorable gene. He provides as an example the maintenance of lactase activity among adults of dairying cultures. However, it is debatable whether a reduction in protein secretion by adult women in agrarian societies that represents < 8% of total daily needs would confer enough of a selective advantage to result in a decrease in milk protein concentrations over such a time frame. In modern-day agrarian societies with heavy reliance on plant foods there is little evidence that adult protein intakes are marginal (although some would argue that intakes of certain amino acids may be low). Current thinking is that it is generally the poor availability of micro nutrients (such as iron and zinc) from predominantly plant-based diets that is the most serious nutritional problem in such societies, rather than protein deficiency per se (Beaton et al, 1992; Allen, 1993, 1994).

In terms of outcomes such as immune function and behavioral development, breastfed infants apparently do better than formula-fed infants despite their lower protein intakes (Institute of Medicine, 1991; Rogan and Gladen, 1993; Lucas et al, 1992). Of course, it could be claimed that this is an unfair comparison, given that there are many other differences between human milk and infant formula (and potentially, in parental behaviors associated with breast- vs bottle-feeding). However, even when functional measures such as morbidity and activity level are compared within a breastfed cohort, there is no evidence that lower protein intakes (expressed either as g/d or as percent of energy) are associated with adverse outcomes (Heinig et al, 1993). In fact, in the study by Heinig et al, a higher protein intake at 6-9 months was significantly associated with greater morbidity. With respect to growth, the differences that exist between breastfed and formula-fed infants are greater for weight than for length (Dewey et al, 1992; WHO, 1994), indicating that the level of fatness differs more than doe, the rate of linear growth (Dewey et al, 1993). It does not appear that protein intake is responsible for these differences: when controlling for energy intake, protein intake (i.e. protein density of the diet) was not associated with weight or length gain during any quarter of the first year of life within a breastfed cohort (Heinig et al, 1993). If protein concentration of human milk were marginal, one would have expected a positive association between protein density and growth. Thus, with the evidence in hand there is little reason to suspect that the amount of protein consumed by breastfed infants in the first 4-6 months is inadequate.

2.1.2. Assumptions of the 1985 recommendations for 0-6 months. The 1985 report estimated the protein intakes of breastfed infants from data available in the literature at that time. The average protein content of human milk was assumed to be 1.15 g/100 ml after the first month post partum, calculated from total nitrogen content × 6.25. It was recognized that a substantial proportion of human milk nitrogen is contributed by non-protein nitrogen, but it was assumed that the non-protein fraction is fully utilized. It was also assumed that the protein concentration of human milk remains stable after the first month, which is not the case. Section 2.1.3 discusses these issues in light of more recent data.

To calculate total protein intake, the 1985 report utilized data on average breast milk intake from two studies (Wallgren, 1944/45; Whitehead and Paul, 1981). However, in those studies the systematic bias (3-6%) caused by insensible water loss from the infant while test-weighing was not taken into account, resulting in an underestimate of the amount of milk actually consumed. Section 2.1.4 presents data on breast milk intake with an adjustment for insensible water loss;

To estimate protein intake per kg body weight, the 1985 report used average weights of infants in the NCHS reference (which are based on the Fels Longitudinal Study). However, the majority of infants in the Fels study were bottle-fed. Given the above-mentioned differences in growth between breastfed and bottle-fed infants, it would be preferable to estimate protein intake per kg on the basis of actual weights of breastfed infants whose intake has been measured. Section 2.1.4 utilizes this approach in revising the estimates in Table 29 of the 1985 report.

Finally, the estimates presented in the 1985 report extended only to 4 months of age because there was insufficient information on the intakes of exclusively breastfed infants beyond that age. Section 2.1.4 includes data up to 6 months of age for exclusively breastfed infants.

In the 1985 report, the average intake of breastfed infants was assumed to approximate the mean requirement. However, it has since been pointed out (Beaton and Chery, 1988; Waterlow, 1990, 1992a) that this approach was incorrect, as it will automatically define half of breastfed infants as having 'deficient' intakes. If it is assumed that nearly all breastfed infants are meeting their protein requirements, then their average intake should be above the safe level for protein intake, i.e. > 2 s.d. higher than the mean requirement. This issue is discussed in more detail in section 2.2.8.

2.1.3. Protein content of human milk and utilization of non-protein nitrogen. The protein content of human milk decreases during the course of lactation (Lonnerdal et al, 1976a). This decrease is most pronounced during the very early part of lactation; values for colostrum (approx. 1-5 days of lactation) can be as high as 20-30 g/1, but by day 10 they are around 13- 15g/l and at one month about 1012g/l. Subsequently, there is a slow decline, with values in mid-lactation being around 8-9 g/1. It should be emphasized that these are concentrations of 'true protein'; i.e. (total nitrogen- non-protein nitrogen) × 6.25, which is equivalent to protein analyzed by amino acid analysis (Lonnerdal et al, 1976b). Various protein assays have also been used and although they can give reasonable estimates when care is taken to use appropriate standards, they all have a tendency to over-estimate the true protein content (Lonnerdal et al, 1987).

Human milk proteins are usually divided into two major classes, casein and whey proteins. Overall, caseins are easily digested and utilized, while the digestibility of some whey proteins is more limited (Lindberg et al, 1982) and intact proteins have been found in the stool of breast-fed infants (Davidson and Lonnerdal, 1987). It should be noted that the changes in protein content of human milk described above are net changes; the patterns for these two classes are quite different (Kunz and Lonnerdal, 1992). The casein content of colostrum is initially very low (or casein is absent) and it then rapidly increases to reach a peak at about 8-10 days of lactation; thereafter, concentrations decline. In contrast, whey proteins are very high in concentration in early colostrum and they then immediately decrease in concentration. Thus, the commonly used whey: casein ratio is not constant for human milk, but varies from about 80: 20 in early milk, to 60: 40 in mid-lactation and about 50: 50 in late lactation (Kunz and Lonnerdal, 1992). These ratios are important when considering digestibility of human milk proteins, and, as a consequence, amino acid utilization and requirements.

The proportion of non-protein nitrogen (NPN) in human milk, about 20-27% of total nitrogen, is large as compared to most other species (Hambraeus et al, 1978). When determining the true protein content of human milk, this NPN fraction is subtracted from the total nitrogen to obtain protein nitrogen. Although this is formally correct, this fraction must be considered when assessing the amino acid requirements of breastfed infants. Amino acids in free form or bound in smaller peptides are likely to be utilized and together comprise about 30% of the NPN (Donovan and Lonnerdal, 1989a).

The major component of the NPN fraction in human milk is urea; as much as 50% of the NPN belongs to this fraction (Donovan and Lonnerdal, 1989a). The level of urea in human milk can be affected by maternal protein intake (Forsum and Lonnerdal, 1980). Studies utilizing stable isotopes have attempted to assess the utilization of urea nitrogen for amino acid synthesis in breast-fed infants (Donovan et al, 1990; Heine et al, 1986; Fomon et al, 1988). These have shown that about 10-20% of this nitrogen is utilizable. However, results vary among studies. For example, Heine et al (1986) found higher utilization rates of urea nitrogen (3943%), but their studies were performed in children recuperating from infection, in whom there may have been rapid regeneration of the intestinal mucosa. It is not clear whether these results could be applied to healthy children.

Other components of the NPN fraction include choline, carnitine, creatine, creatinine, nucleotides and nitrogen-containing carbohydrates (such as sialic acid) and oligosaccharides (Atkinson et al, 1989). Several of these compounds are involved in lipid and nucleic acid metabolism and are likely to be used for amino acid synthesis; some are present in low concentrations and the amounts of nitrogen contributed are very small as compared to the requirements of infants.

2.1.4. Revised estimates of protein intake of breastfed infants. Table 1 provides estimates of the protein intake of exclusively breastfed infants from 1 to 6 months, in a similar format to that of Table 29 in the 1985 report. Data were taken from two studies in the USA in which breast milk intake was carefully measured by test weighing with electronic balances for periods of 24 (Butte et al, 1984) or 96 hours (Heinig et al, 1993). In both studies, milk samples were collected over a 24-h period at each age using the alternate breast expression method, and pooled samples were analyzed for protein concentration. In the Butte et al study, the actual breakdown of protein nitrogen and non-protein nitrogen (NPN) was provided; the latter averaged 26.6% of total nitrogen. In the study by Heinig et al, it was estimated that 25.5% of the total nitrogen was NPN. Although several other studies have presented data on breast milk intake (with values similar to those observed in these two studies), they were not included here either because milk protein concentration was not measured, the samples were not representative of a 24-h period, or the weights of the infants were not stated.

In Table 1, the data from the above two studies were used to calculate total nitrogen intake, 'crude' protein intake (total N × 6.25) end 'adjusted' protein intake. To estimate 'adjusted' protein intake it was assumed that, of the NPN fraction, all of the µ-amino nitrogen and glucosamines (together representing about 35% of NPN; Fomon, 1993) and 17-40% of the remaining NPN can be utilized. Thus, the values are given for a range representing 46-61% utilization of the NPN in human milk.

Table 1 shows that adjusted protein intake (g/d) declines sharply between 1 and 2 months as the protein concentration of human milk decreases, and then levels off at 7.5-8.4 g/d. When expressed per kg body weight, the adjusted protein intakes range from 1.95-2.04g/kg/d at 1 month to 1.05-1.16g/kg/d at 4-6 months. These values are about 0.20-0.46 g/kg/d less than the values listed in Table 29 of the 1985 report, a difference of 1026%, depending on age (this comparison takes into account the fact that Table 29 in the 1985 report used age ranges, rather than specific ages; the latter was chosen for Table 1 because the two studies cited generally measured intake within a few days of the age specified). Most of this difference is due to the change in assumption regarding the proportion of the NPN fraction utilized, but part of the difference is due to higher means for infant weight in Table 1 than in the previous Table 29, and another part is related to the decrease in milk protein concentration over time (which was not accounted for in the 1985 report).

Table 1 Revised estimates for Table 29 in the 1985 FAO/WHO/UNU report on Energy and Protein Requirements

^a Exclusively breastfed infants; data for milk intake from Butte et al and Heinig et al were corrected for insensible water loss ( + 5.7%, Heinig et al 1993).
^b From Butte et al (1984).
^c From Heinig et al (1993).
^d Including non-protein nitrogen (NPN).
^e Based on milk protein concentration plus 46-61% of the NPN (protein = 6.25 nitrogen).

If one is interested in estimating the true amount of protein that is utilizable by the breastfed infant, the adjusted protein values in Table 1 are most appropriate. However, when comparing intakes of breastfed infants to factorial models of requirements, it is generally more appropriate to use total nitrogen intake. This is because the factorial models usually include assumptions regarding the efficiency of utilization of dietary nitrogen. If this has already been adjusted for when calculating intake, there is a risk of adjusting twice for the same phenomenon (incomplete utilization of dietary nitrogen).

2.2. Using the factorial approach

2.2.1. Basis of the 1985 recommendations. A modified factorial method was used in the 1985 report to estimate protein requirements of infants and children after the age of 6 months. This approach requires the estimation of both maintenance nitrogen needs and the amount of nitrogen required for growth. The factorial method necessitates several assumptions regarding (a) the adequacy of nitrogen balance data for estimating maintenance requirements, (b) the rate of growth and the composition of tissue gain during growth, (c) the degree of daily (intra-individual) variation in growth rate, (d) the efficiency of conversion of dietary protein to body protein and (e) the amount of inter-individual variability in nitrogen needed for maintenance and growth. The following sections examine each of these assumptions.

2.2.2. Estimated maintenance nitrogen requirement. Although there are many criticisms of using nitrogen balance studies to estimate protein requirements (FAO/ WHO/UNU, 1985), at present there are no satisfactory alternative data to estimate maintenance nitrogen needs of infants (see section 2.5 for a discussion of amino acid requirements). In the 1985 report, the values for maintenance nitrogen needs of infants and children were based primarily on short-term balance studies, generally performed in children who were healthy but had recently recovered from malnutrition and were still short in stature though normal in weight for height. Energy intake was maintained at a level assumed to be adequate, and protein was fed at various levels. The maintenance requirement (nitrogen intake needed to achieve balance, assuming no growth) was calculated from the regression of nitrogen balance on nitrogen intake, allowing 10 mg N/kg/d for sweat and miscellaneous losses.

The only data cited in the 1985 report for maintenance protein needs of infants were from a study of children 917 months of age (Huang et al, 1980) (see Table 2). Data from other studies were shown, but none of the children were younger than 12 months. In the Huang et al study, the infants' energy intake was only 77 kcal/kg/d. far below both the recommended level (> 100 kcal/kg/d) for this age range and the average intake of breastfed infants at 12 months (9O kcal/kg/d; Heinig et al, 1993). The authors explained that the children's usual intake was 100 kcal/kg/d, but when they were confined on the metabolic beds their appetite decreased and their intake during the study was much lower than normal. Maintenance nitrogen needs were calculated to be 113 mg N/kg/d on the milk diet (including the extra 10 mg N/kg/d for unmeasured losses). The 1985 report chose 120 mg N/kg/d as the average maintenance requirement.

Given that this key value included only one study of infants, and that study involved energy intakes below usual requirements, it is worthwhile to reconsider maintenance nitrogen needs of infants. In a free-living population consuming higher energy intakes, one might expect that maintenance nitrogen needs would be considerably lower than 120 mg N/kg/d. Balance data for infants 4-6 months of age reported by Fomon (1986: p. 64; Fomon et al, 1965), but not cited in the 1985 report, appear to support this conclusion. In two studies of infants fed very low protein diets for 6 days, total nitrogen excretion was 68 mg/kg/d when fed 0.16 g/kg/d protein and 93 mg/kg/d when fed 0.39 g/kg/d protein. When higher levels of protein were given (1.26 g/kg/d), total nitrogen losses were considerably higher (141 ma/ kg/d). However, if one defines inevitable nitrogen losses in the same fashion as used in balance studies with adults, one would conclude that they should be less than the losses observed when the lowest protein diet was given to the infants (i.e. < 68 mg/kg/d).

Table 2 Various estimates of basal and/or maintenance nitrogen needs in infants and children

^a Values include measured fecal losses. They do not include integumental (unmeasured) N losses, generally taken at 10 mg N/kg d.
^b Milk based formula.
^c Soybean isolate based formula.
^d Calculated using × = N intake and y = retention.
^e Calculated using × = [N intake - fecal N] and y = [retention - fecal N], adding back 'basal' N losses of 20 mg/kg/d (the mean value for losses on the lowest protein intake).

Regression equations based on the data from Fomon et al (1965) were calculated to estimate both basal losses (the y intercept when intake = 0) and maintenance needs (the × intercept when retention = 0), as shown in Table 2. Depending on the method of calculation, basal needs were 50-58 mg N/kg/d, not including integumental losses. This estimate is very close to the estimates of 50-55 from Torun et al (1981a) and Torun and Viteri (1981a) for children 17-31 months of age, which again suggests that the estimate from Huang et al was too high. Assuming integumental losses of about 10 mg N/kg/d, the total basal need would be 60-68 mg N/kg/d. 'Maintenance' needs calculated from the regressions for the infants in the Fomon et al study range from 64 to 102 mg N/kg/d, depending on the method of calculation. However, neither of the slopes obtained (0.49 and 0.91) is consistent with the expected 'efficiency' of utilization of dietary nitrogen, which is generally 73-80% in both animal and human studies, even in rapidly growing individuals. The low slope obtained using the conventional balance calculation (retention against absolute intake) occurs because there is a relatively strong relationship between nitrogen intake and fecal nitrogen excretion in the Fomon et al data, whereas most other studies generally show a weak relationship between these two variables. The high slope obtained using the alternative calculation (using apparent nitrogen absorption for the × axis, as shown in footnote e, Table 2) probably occurs because that method assumes that all fecal nitrogen is unabsorbed (which is not the case - endogenous fecal nitrogen losses can be substantial), and thus may underestimate nitrogen absorbed from the diet. Because of this discrepancy, when estimating maintenance needs it is probably more prudent to apply an assumed 'slope' of 0.73 to the range of basal needs calculated from the actual data (6068 mg N/kg/d). This results in an estimate of 82-93 mg N/kg/d. A value of 90 mg N/kg/d was chosen for the factorial model presented in section 2.2.7.

2.2.3. Estimated needs for growth. During infancy, the amount of protein required for growth is an important component of total protein needs. As shown in Table 3, this proportion declines from about 64% in the first month to 24% in the second six months of life.

To estimate body protein gain during growth, the 1985 report used data from Fomon et al (1982). Those data were based on formula-fed infants during the first 112 days of life, and on NCHS reference data from 3 to 10 y of age. Between 112 days and 3 y the values were interpolated, 'with the requirement that they parallel as closely as possible the shape of the NCHS curves' (p. 1170). During infancy, the NCHS curves are also based primarily on formula-fed infants.

Given that the growth patterns of breastfed and formula-fed infants differ considerably, it is worthwhile to examine how the estimates in Table 32 of the 1985 report might differ if they were based on growth of breastfed infants. Table 4 shows the estimated daily increment in body protein of male breastfed infants, based on growth data for 109 males who were breastfed throughout the first year of life. These data were derived from a pooled analysis of studies from the US, Canada, Denmark, Sweden, Finland and the UK (WHO Working Group on Infant Growth, 1994). The percentage of weight gain contributed by the fat free body mass (FFBM), and the percentage of FFBM assumed to be protein, are taken from the estimates provided by Fomon et al (1982), although it is recognized that body composition of breastfed and formula-fed infants also may differ (see below). The resulting estimates of body protein gain range from 1.00 g/kg/d in the first month to 0.15 g/kg/d from 9 to 12 months of age. For reference, the values recently published for formula-fed infants by Fomon (1991) (which are almost identical to the values in Table 32 of the 1985 report) are shown in the last column. These are generally similar to the estimated values for breastfed infants in Table 4 from 0 to 6 months, but are 1520% higher after 6 months of age because of the higher growth rate of formula-fed infants from 6 to 12 months.

Continued

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