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close this bookEnergy and Protein requirements, Proceedings of an IDECG workshop, November 1994, London, UK, Supplement of the European Journal of Clinical Nutrition (International Dietary Energy Consultative Group - IDECG, 1994, 198 pages)
close this folderProtein requirements of infants and children
View the document(introductory text...)
View the document1. Introduction
View the document2. Protein requirements of infants
View the document3. Protein requirements of children and adolescents
View the document4. Protein needs during catch-up growth
View the document5. protein needs associated with infection
View the document6. Assessment of protein quality of weaning diets
View the document7. Future research needs
View the documentReferences
View the documentDiscussion
View the documentReferences

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





Total nitrogen intaked

Crude 'protein' intake
(N × 6.25/1000)

Adjusted protein intakee

Age (months)

N

Breast milk intake (g/d)a

Weight (kg)

(mg/d)

(mg/kg/d)

(g/d)

(g/kg/d)

(g/d)

(g/kg/d)

1b

37

794

4.76

1723

362

10.8

2.26

9.3 - 9.7

1.95 - 2.04

2b

40

766

5.62

1486

264

9.3

1.65

7.9 - 8.3

1.41 - 1.48

3b

37

764

6.30

1406

233

8.8

1.46

7.5 - 7.9

1.19 - 1.25

3c

61

812

6.24

1472

236

9.2

1.48

7.9 - 8.3

1.27 - 1.33

4b

41

782

6.78

1408

208

8.8

1.30

7.5 - 7.8

1.11 - 1.16

6c

12

881

7.54

1486

197

9.3

1.23

8.0 - 8.4

1.05 - 1.11

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

Authors
(Method)

Age
(months)

Body weight of subjects (kg)

Basala
(y intercept mg nitrogen/kg/d)

Maintenance (mg nitrogen/kg/d)

Slope (B/M)

Fomon et al (1965) (Intercept/slope)

4-6

5-8

50d

102d

0.49d




58e

64e

0.91e

Huang et al (1981) (Low protein)

9-17

10

76

104

0.73

Torun et al (1981a)b (Intercept/slope)

17-31

10.2

50

68

0.74

Torun et al (1981a)c (Intercept/slope)

17-31

10.2

50

82

0.61

Torun and Viteri (1981a) ('Zero' protein)

17-31

10.6

55

-

-

Intengan et al (1981) (Intercept/slope)

18-26

11

61

113

0.54

Egana et al (1984)c (Intercept/slope)

35-62

15

56

102

0.55

Egana et al (1984) (Milk/rice)b (Intercept/slope)

35-62

16

40

78

0.51

Fomon et al 1965) (Intercept/slope)

39-94

12-20

66

-

-

Mean ± s.d.


56 ± 11

93 ± 17

0.60 ± 0.10


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.

Table 3 Protein needs for growth and maintenance at different ages of the human infant

Age

Protein gaina (g/kg/day)

Maintenanceb (g/kg/day)

Maintenance/Total (%)

Growth/Total (%)

0-1 month

1.00

0.56

36

64

1-3 months

0.57

0.56

50

50

3-6 months

0.30

0.56

65

35

6-12 months

0.18

0.56

76

24

1-2 years

0.11

0.56

84

16

2-5 years

0.07

0.56

89

11

a From Table 4, up to 12 months; from Fomon et al (1982) from 1 to 5 years.
b Maintenance assumed to be 90 mg N/kg/d.

Table 4 Daily increment in body protein of male breast-fed infants (pooled dataset, WHO, 1994)







Body protein gain


Age (months)

Wta (kg)

Wt gain (g/d)

% of gain as FFBMb (%)

FFBM gain (g/d)

% of FFBM gain as proteinb (%)

(g/d)

(g/kg/d)

Fomon's estimates

0-1

4.04

31.9

79.5

25.4

15.9

4.0

1.00

0.93

1-2

5.04

35.2

59.9

21.1

16.6

3.5

0.69

0.70

2-3

5.89

25.7

56.9

14.6

17.6

2.6

0.44

0.50

3-4

6.61

20.7

60.6

12.5

18.3

2.3

0.35

0.34

4 5

7.21

17.4

66.9

11.6

18.0

2.1

0.29

0.27

5-6

7.71

14.8

72.4

10.7

18.2

1.9

0.25

0.26

6 9

8.41

10.9

85.7

9.3

18.5

1.7

0.20

0.23

9-12

9.26

8.2

90.7

7.4

18.6

1.4

0.15

0.18

a At midpoint of interval.
b Fomon et al (1982).
c Fomon (1991) for male infants fed milk-based formulas.

Body composition changes rapidly during infancy. The body composition data presented by Fomon et al (1982) were based on measurements at three ages: birth, 6 months and 9-10 y of age, with interpolation used to obtain values for other ages. As they point out, the estimates were considered preliminary 'because of uncertainties about the data and because of the large number of assumptions that have been required' (p. 1172). Thus, there is a need to reevaluate reference values for body composition, utilizing any new data that may be available for the first year of life. For the purposes of this analysis, the major question is whether the estimates of the percentage of weight gain contributed by FFBM are accurate. There is evidence that percentage body fat is lower among breastfed than formula-fed infants during the second six months of life (Dewey et al, 1993); unpublished data (Dewey et: al) indicate that the estimated percentage of weight gain as FFBM is higher from 3 to 12 months in breastfed than in formula-fed infants (the difference is statistically significant at 6-9 months: 88 ± 21% vs 77 ± 9%, respectively, P = 0.001). If the values of percent gain as FFBM for breastfed infants are used in Table 4 from 6 to 12 months, the calculated body protein gain is slightly higher (an increase of 0.01 g/kg/d), but still not as high as the estimates seven by Fomon (1991).

Therefore, if the factorial model were based on growth data. for breastfed infants, the resulting estimates of protein required for growth during the second six months of life would be somewhat less than the values used in the 1985 report.

It should be noted that protein needs for growth during early infancy will depend on birth weight, as infants with lower birth weights generally will gain weight more rapidly and those with higher birth weights will gain weight less rapidly than average. Because of this, protein requirements based on reference growth rates from affluent populations (in which mean birth weight is relatively high) may be an underestimate during early infancy for populations with lower mean birth weights. For example, mean weight gain from birth to 10 weeks of age of 141 exclusively breastfed infants in Honduras, whose mean birth weight was 2.9 kg, was 36.7 g/d (Cohen et al, 1995), compared to 32.0 g/d during the same interval for the pooled dataset shown in Table 4. The difference of 4.7 g/d would represent an additional deposition of about 0.1 g/kg/d body protein, which would require an additional 0.14 g/kg/d protein intake for infants with lower birth weights. This would increase protein needs during early infancy by about 8.5%.

2.2.4. Intra-individual variation in growth. To account for day-to-day variation in the rate of growth, and thus in the need for protein to support that growth, the 1985 report added 50% to the theoretical daily body nitrogen increment. l he reasoning behind this was that there is a very limited storage capacity for amino acids, and thus it is necessary to allow for extra demand on days when growth is particularly rapid. Recent findings by Lampl et al (1992) lend support to the idea that linear growth occurs in spurts, although this hypothesis has been challenged. However, even if linear growth is indeed saltatory, it is by no means clear which nutrients are likely to be most limiting for the deposition of tissue during these brief spurts, and therefore the quantitative impact on daily protein needs has not been estimated. Weight gain is probably also saltatory, but the proportion of lean to fat tissue in each 'spurt', and thus the need for extra protein, has not been estimated. The 50% augmentation used in the 1985 report was arbitrary, and was justified on the basis that the resulting factorial estimate of average requirement was very similar to the estimated intake from breast milk at 4 months. However, as pointed out in section 2.1.2, the assumption that average requirement should be similar to average protein intake of breastfed infants is incorrect. Therefore, the adjustment for day-to-day variability needs to be reconsidered. In Fomon's (1991) estimates, no adjustment for day-to-day variability in growth was utilized.

2.2.5. Efficiency of conversion from dietary protein to body protein. In the 1985 report, it was assumed that the efficiency of conversion from dietary protein to body protein during growth is 70%, the same efficiency assumed for maintenance needs. However, as Waterlow points out (1990), because the reasons for inefficiency are not fully understood, it is difficult to defend this assumption. Considering the potential for salvage of urea nitrogen (Jackson, 1992), it is likely that the efficiency of conversion could be quite a bit higher than 70%. In Fomon's (1991) estimates, he assumed an efficiency of 90%, based on the presumption that efficiency of conversion should be higher from a milk-based diet than from a mixed diet. It could be that the efficiency of utilization of human milk is yet higher than that of cow's milk-based formulas. Serum urea nitrogen and urinary nitrogen levels tend to be lower in breastfed than formula-fed infants (see section 2.3), even though urea content is relatively high in human milk. However, much of this difference may be attributable simply to the lower total protein intake of breastfed infants. Although breastfed infants gain more weight and lean body mass per gram of protein consumed than do formula-fed infants, the slope of the relationship between protein intake and growth does not differ between feeding groups (Heinig et al, 1993); i.e. infants with a lower protein intake will use more of the protein for growth regardless of feeding mode. Taken together, these findings may mean that protein intakes of formula-fed infants are excessive, rather than that breastfed infants are metabolically more 'efficient' at utilizing protein.

More information on the efficiency of conversion of dietary protein to body protein is needed, particularly under conditions of rapid growth. Efficiency of protein utilization (for all needs, not just for growth) can be estimated from balance studies by calculating the slope of the line (by dividing basal needs (the y intercept) by maintenance needs (the x intercept)), as shown in Table 2. In the studies of children fed a predominantly milk based diet, this value was 73-74% (except for the study of very young infants by Fomon et al (1965), as mentioned in section 2.2.2). The appropriate value for infants younger than 6 months of age requires further investigation. Because growth represents a much larger percentage of total protein needs in the early months (see Table 3), and the efficiency of protein utilization for growth may differ from that for maintenance, it is possible that the slope of the line would be considerably different in younger infants. Although differences in diet composition among studies must be taken into account, the data shown in Table 2 are consistent with the hypothesis that efficiency decreases with age. In animal studies, younger animals are apparently more efficient than older animals at utilizing protein, based on the observation that the latter require higher protein intakes to achieve the same rate of growth (Reeds, personal communication).

Waterlow (1992b) recently summarized data on the efficiency of protein deposition from a variety of studies. Efficiency was calculated as N retained/(N intake obligatory losses). Using this definition, values of 80% or less were observed in situations of rapid growth. However, this definition of 'efficiency' yields values that are strongly influenced by the level of protein provided: if this is in excess relative to the anabolic drive (or to other nutrients required for growth), the amount of nitrogen retained as a proportion of intake will be low. When protein is provided closer to the 'requirement' level (i.e. just meeting the anabolic drive), which is likely to be the case for breastfed infants, the calculated 'efficiency' would probably be considerably higher. This does not necessarily imply, however, that the biochemical efficiency of amino acid utilization for growth would differ according to the level of protein intake.

To adjust for efficiency of protein utilization, the factorial model in the 1985 report used a simple additive model (i.e. adding maintenance needs, with an assumed efficiency of 70 %, to needs for growth, also with an assumed efficiency of 70%). However, it is not clear whether an additive approach is appropriate in situations of rapid growth such as during infancy. In such situations, overall efficiency may be greater because the amino acids not needed for maintenance can be readily used for growth. In support of this idea, Chan and Waterlow (1966) found that efficiency of utilization of milk protein was almost 100% in young children recovering from malnutrition.

2.2.6. Inter-individual variability in nitrogen needed for maintenance and growth. To derive safe levels of protein intake for virtually all healthy infants, the average requirement estimates need to be adjusted based on the magnitude of inter-individual variability in the needs for both maintenance and growth. In the 1985 report, the coefficient of variation (CV) in the maintenance requirement was assumed to be 12.5%, the same as the CV found for adults in numerous short-term nitrogen balance studies. As justification for this, it was stated that the range of inter-individual variation in the nitrogen balance studies of children was similar to that of adults, although no actual CV values were presented.

Much more problematic than the maintenance CV is the inter-individual variability in growth. In the 1985 report, data on variability in weight gain over one month intervals from 3.5 to 6.5 months (based on unpublished data of Fomon) were used to estimate a CV of approximately 35% (shown in the footnote to Table 33 of the 1985 report). It was recognized, however, that the CV for growth depends on the interval of measurement: it will be lower over longer periods and higher over shorter periods. The one-month interval CV was 'accepted as a reasonable compromise'. Not mentioned in the report is the fact that the CV for growth will also increase with age during the first year, because the mean growth rate declines disproportionately more than does the standard deviation. This is illustrated in Table 5, which shows CVs for one month and three-month weight increments of breastfed infants from the pooled dataset used for Table 4. With one-month increments, the mean CV increases from 26% at one months to an astonishing 93% at 11-12 months. By contrast, the 3-month CVs range from 24% at 1-4 months to 46% at 9-12 months. Additional analyses with this dataset (data not shown) have indicated that, using the one-month increments and accounting for the effect of age on growth rate, about 70-80% of total variability can be attributed to intra-subject variability (e.g. month to month) and only about 20-30% to inter-subject variability. In other words, there is relatively little 'tracking' in growth velocity by individual infants (in fact, the opposite probably occurs: an individual who grows slowly one month is likely to grow rapidly the next). For this reason, it makes more sense to err on the low side when estimating inter individual variability in growth (i.e. use longer intervals, such as three months). Although the choice of 3-month CVs is admittedly arbitrary, these values were used in calculating the revised estimates of safe levels of protein intake presented in the next section.

Table 5a CVs for one-month weight incrementsa of breast-fed infants (pooled data set, WHO, 1994)

Age (months)

Male

Female

Mean CV (%)

1-2

25

27

26

2-3

31

29

30

3-4

33

30

32

4-5

36

33

35

5-6

38

38

38

6-7

48

44

46

7-8

47

52

50

8-9

55

64

60

9-10

65

65

65

10-11

82

86

84

11-12

80

105

93

a Calculated from absolute weight gain, not gain per kg body weight.

Table 5b CVs for 3-month weight incrementsa of breast-fed infants (pooled data set, WHO, 1994)

Age (months)

Male

Female

Mean CV (%)

1-4

23

24

24

2-5

25

25

25

3-6

26

26

26

4-7

30

28

29

5-8

32

31

32

6-9

33

35

34

7-10

37

37

37

8-11

46

40

43

9-12

48

43

46

a Calculated from absolute weight gain, not gain per kg body weight.

2.2.7. Revised estimates of protein requirements and safe levels of intake. Although there are many unanswered questions regarding the assumptions required to estimate protein requirements, the information presented in the sections above was used to generate a revised factorial model for infants. Table 6 presents data analogous to those shown in Table 32 of the 1985 report, but using the following assumptions: (a) body protein gain was estimated as shown in Table 4; (b) there was no additional augmentation for day-to-day (intra-individual) variability in growth (instead, this was considered to be covered by the CV for growth when calculating safe levels - see Table 7); (c) the efficiency of conversion of dietary protein to body protein was estimated to be 70% (based on the slopes from the balance studies in Table 2) and (d) the maintenance requirement was estimated to be 90 mg N/kg/d. The estimates in Table 6 are 27-35% lower than the 1985 values.

It could be debated whether data for breastfed infants should be chosen for the growth increment calculation. However, because the differences in protein gain per kg between breastfed and formula-fed infants are evident primarily in the second 6 months of life (see Table 4), when protein needs for growth are a relatively small proportion of total protein needs, the impact of this choice is relatively minor, If estimates of protein gain based on formula-fed infants (Fomon et al, 1982) had been used for Table 6, the values at 6-12 months would only be about 4% higher than those shown.

Table 7 shows the revised estimates for the safe level of protein intake, analogous to Table 33 in the 1985 report. In this table, the CVtot is calculated in the same way as in the 1985 report (see footnote to Table 7), but using the 3-month CVs for growth from Table 5 instead of a constant 35%.

Table 6 Revised estimates of average requirements for Table 32 in the 1985 FAO/WHO/UNU report on Energy and Protein Requirements

Age (months)

N incrementa (mg N/kg/d)

N increment corrected
for 70% efficiency
(mg N/kg/day)

Maintenance (mg N/kg/day)

Total as nitrogen (mg N/kg/d)

Total as protein (g protein/kg/d)

1985 Estimate
(g protein/kg/d)

0-1

160

229

90

319

1.99

-

1-2

110

157

90

247

1.54

2.25

2-3

70

100

90

190

1.19

1.82

3-4

56

80

90

170

1.06

1.47

4-5

46

66

90

156

0.98

1.34

5-6

40

57

90

147

0.92

1.30

6-9

32

46

90

136

0.85

1.25

9-12

24

34

90

124

0.78

1.15

a Based on growth data in Table 4.

It is possible to compare mean intakes of breastfed infants with the safe levels in Table 7 (although theoretically, group mean intake should be somewhat greater than the 'safe level' of intake; see section 1). However, such a comparison should be made only on the basis of total nitrogen intake (not estimated protein intake), as the factorial model used for Tables 6 and 7 already takes into account an efficiency of utilization of about 70%, both for the growth component (an assumption of the model) and for maintenance (inherent in the estimate of 90 mg N/kg/d, because the assumed slope was 0.73). In fact, the utilization of nitrogen from human milk is likely to be higher than 70% (see section 2.2.8). Nonetheless, if the safe levels for nitrogen intake during the first six months of life are compared with the average nitrogen intakes shown in Table 1, it is clear that the values are quite similar. This comparison is best made using a probability approach, as illustrated in the following section.

2.2.8. Validation of the revised factorial model using an epidemiological approach. In 1988, Beaton and Chery published a paper challenging the protein requirement estimates of the 1985 report. In that paper, they gathered together fragmentary information relevant to the modelling of distributions of intakes and requirements and asked the questions 'Could requirements be as high as had been estimated?' and if not 'How high could they be before there was major inconsistency between the predicted prevalence of inadequate intakes and the starting assumption that breast fed infants at 3-4 months of age were adequately nourished?' They concluded that the 1985 report had seriously overestimated protein requirements of young infants. The principles of the approach that Beaton and Chery applied have been described in greater detail in a National Research Council report (Subcommittee on Criteria for Dietary Evaluation, 1986) and embody what has become known as the 'probability approach' to assessment of observed intakes - an approach that recognizes that two distributions exist - a distribution of intakes and a distribution of requirements.

Table 7 Revised estimates for safe level of protein intake for infants (Table 33 in the 1985 FAO/WHO/UNU report on Energy and protein Requirements)




Safe level

Age (months)

CVG (%)

CVtota

Nitrogen (mg/kg/d)

Protein
(g/kg/d)

0-1

24

17.6

431

2.69

1-2

24

15.9

326

2.04

2-3

25

14.4

245

1.53

3-4

26

13.9

219

1.37

4-5

29

14.2

200

1.25

5-6

32

14.6

190

1.19

6 9

34

14.2

175

1.09

9-12

46

15.6

163

1.02

a.


where CVM = CV for maintenance, taken as 12.5%, CVG = CV for growth, taken from Table 5 based on 3-month increments.

For the analyses published in 1988, Beaton and Chery could not identify data bases that included in one source all of the variables they needed. They finally approached the problem by assembling information about the likely distribution of protein: energy intake ratios among breastfed infants and then modelling requirements in the same form. This was not their preferred approach but it was workable with the data available at that time. Beaton and Chery suggested that 1.1 g/kg/d was a much more reasonable estimate of the average protein requirement at 34 months than was the 1985 estimate of 1.47 g/kg/d.

In retrospect there were many flaws in the assumptions and estimates that Beaton and Chery were compelled to use. For example, they used the then-existing estimates of energy needs (103 kcal/kg), now known to be considerably higher than the actual intakes of breastfed infants at 3 months. Given that there are now databases which include all of the required information, it was considered worthwhile to reattempt the type of analyses done by Beaton and Chery For this report, data from the Davis Area Research on Lactation, Infant Nutrition and Growth (DARLING) study (Dewey et al, 1991; Heinig et al, 1993) and a subsequent study in the same location (Dewey et al, 1994) were utilized. The dataset included 104 exclusively breastfed infants at 3 months of age. Table 8 shows descriptive data for this cohort. While this cohort is not necessarily representative of all breastfed infants, the dataset allows for a more direct approach to the questions raised in the 1988 paper. Beaton agreed to work with the first author to again ask the question 'How high could infant protein requirements be, given the premise that at three months of age, the breast fed infant is adequately nourished with regard to protein?' In the end, this analysis became an epidemiologic testing of the factorial model presented in Tables 6 and 7.

Table 8 Summary statistics for cohort of 104 exclusively breastfed infants used to test factorial models of protein requirementsa (Mean ± s.d.)

Variable


Weight at 3 months (kg)

6.29 ± 0.75

Rate of weight gain


(g/d)

22.3 ± 6.26

(g/kg/d)

3.54 ± 0.88

Energy intake


(kcal/d)

527 ± 83

(kcal/kg/d)

84.1 ± 11.0

Total nitrogen intake


(mg/d)

1444 ± 241

(mg/kg/d)

230.9 ± 37.8

Sex distribution


(% males)

50.2

a Heinig et al (1993); Dewey et al (1994).

Several components of the factorial model were varied to test the impact of changes in the individual components on the predicted prevalence of inadequate intakes among the 104 infants. These trials made use of the individual infants' observed energy and nitrogen intakes, body weights at 3 months and actual growth rates estimated by linear regression using weights at 2, 3 and 4 months. Each factorial model developed was applied to the known body size of each infant to compute a 'requirement' for that infant. This information was used to ask 'for what proportion of the infants was the observed intake below the estimated requirement?'- a measure of the expected prevalence of inadequacy. Given the starting premise, the expected answer would be 0 - all of the infants should have had intakes above the modelled requirements if the components of the model were correct. It became clear from these modelling exercises that anything affecting estimated mean requirement (e.g. assumed efficiency of utilization) had distinct effects on the estimated prevalence of inadequacy. Changes in the estimates of variability had much less impact.

Table 9 Sample of the testing of factorial models of nitrogen requirements at 3 months of age. Tested with data from 104 exclusively breast fed Californiaa infants

Model





Factorial component

A

B

C

D

Body weight (kg) - actual weights used in fitting models: group mean shown


6.29


Average rate of weight gain (g/kg/d): group mean shown


3.54


Protein concentration (net) associated with weight increase


10.6%


Need for growth (mg N kg/d)b


60


CV of growth need (observed variability of growth)


24.7%


Basal need (ma N kg d)

60

66

CV of basal need

12.5%

12.5%

Total N requirement as utilized intake (mg N/kg d)c

120

120

126

126

CV of requirement

13.8%

13.8%

13.5%

13.5%

Efficiency of utilization of dietary nitrogen

73%

80%

73%

80%

Total requirement as milk nitrogen (mg N/kg/d)d

164.4

150.0

172.6

157.5

Observed breast milk nitrogen intake (ma N kg/d), mean ± s.d.


230.9 ± 37.8


Predicted prevalence of inadequate intakes

5.1%

1.6%

8.1%

3.0%

a Heinig et al (1993); Dewey et al (1994).
b Need for growth (mg N kg/d) = weight gain (g/kg/d) × 0.106/6.25 × 1000.
c Total requirement as utilized N = average basal need + average growth need. Standard deviation estimated as the square root of the weighted
sum of variances and CV calculated as s.d./total requirement (see footnote to Table 7). These estimates were identical for all infants in each model.
d Milk N requirement = Utilized N requirement/Efficiency of utilization.

The factorial components used in the final models, and the associated predicted prevalences of inadequacy, are shown in Table 9. In these models, a basal requirement of 60 or 66 mg N/kg/d was chosen. The lower value (60) corresponds to the lower end of the range calculated from the data of Fomon et al (1965), as explained in section 2.2.2. The higher value (66) is the estimate that would be consistent with the maintenance estimate used in Tables 6 and 7 (90 mg N/kg/d), assuming a slope of 0.73. Two estimates for the efficiency of utilization of dietary nitrogen (for both maintenance and growth) were used: 73% or 80%. The CV for the maintenance requirement was assumed to be 12.5% (as assumed in the 1985 report). The proportion of weight gain attributable to protein accretion was estimated to be 10.6%, based on the data in Table 4 (the average proportion for 2-3 and 3-4 months). The CV for rate of weight gain (24.7%) was based on the actual data (over a 2-month interval).

These estimates of requirements were compared with observed intakes of total nitrogen. Although human milk contains about 25% non-protein nitrogen, not all of which is utilizable (see section 2.1.3), the evidence to date indicates that the percentage utilization of total nitrogen from cow's milk-based formulas and human milk is similar (75-79% in preterm infants fed human milk or formula: Stack et al, 1989; 72-74% in term infants fed human milk: Fomon and May, 1958). The resulting estimated prevalences of inadequate intakes were 1.6-5.1 % when basal requirement was set at 60 mg N/kg/d (Options A and B) and 3.0-8.1% when basal requirement was set at 66 mg N/kg/d (Options C and D).

An important feature to note from these analyses is that the factorial model closest to that used in Tables 6 and 7 (Option C) leads to a predicted prevalence of 'inadequacy' (8.1%) that is higher than expected. Unless one rejects the starting assumption that virtually all breastfed infants meet their protein requirements, this would suggest that one or more of the components in the factorial model is still being overestimated. It is not likely to be the estimates of variability, as they have minimal impact on the prevalence estimate. The most likely components are the estimation of the basal requirement and the efficiency of utilization of dietary nitrogen. Option D illustrates that when the latter is assumed to be 80%, the prevalence of 'inadequacy' is 3.0%, and Option B illustrates that when both of these components are modified, the prevalence of 'inadequacy' is only 1.6%.

Two other potential explanations for the non-zero prevalence of 'inadequacy' were considered but set aside as improbable. The first related to an explicit assumption of the probability approach - the assumption that protein intakes and protein requirements are independent of one another except insofar as both relate to body size. Protein intake is related to energy intake, and it is reasonable to assume that this relates to body size. However, because protein requirements per unit body size were being modelled, this association would not be relevant. Beaton and Chery (1988) considered the possible effect of a correlation mediated through energy requirement (and hence protein intake) for the growth component of protein requirement. This was again tested in the present models and found to have negligible impact.

The second potential explanation relates to within subject (day-to-day) variation in protein intake. The individual intake data used in these analyses, although based on 3-4 days of measurement (average = 3.7 days), would still have a residual 'random' error in relation to 'true' usual intakes. This would act to inflate the distribution of intakes, thereby increasing the estimated prevalence of both low and high intakes. To examine this effect, data for the 71 infants in the DARLING sample were further analyzed. The results indicated that the within-subject component of variance had a CV of about 11.1% and the between-subject variation in 'true intake' had a CV of about 15.1% (actual variance ratio 0.54); this is a substantially lower day-to-day variation than is customarily seen in intakes of older children and adults. With three day means, the remaining random error component would be about 6% while the between subject variation would continue at about 15%, yielding a variance ratio of about 0.18. These estimates were considered in theoretical distribution models, but the impact was too small to explain the non-zero prevalences of 'inadequacy'.

In summary, the inferences drawn from the above analyses seem robust and provide strong support for a recommendation that the estimates of protein requirements for young infants be substantially lower than those published in the 1985 report. The requirement estimate in Table 6 for infants 3-4 months of age is 170 mg N/kg/d, which is similar to Option C in Table 9 but somewhat higher than Options A, B and D, which yielded more reasonable prevalences of 'inadequacy'. This discrepancy arises because Table 6 was designed to present estimates that would be appropriate for a range of ages and feeding modes, and therefore was based on relatively conservative estimates for maintenance requirement and for efficiency of utilization of dietary nitrogen.

2.3. Using a direct experimental approach

A direct experimental approach to estimating protein needs during infancy has been used in several studies of formula-fed infants. Many of these studies have utilized plasma amino acid profiles to help understand protein metabolism and requirements. Postprandial concentrations of amino acids in plasma are reflective of protein intake and synthesis as well as turnover, amino acid metabolism (catabolism, synthesis and excretion) and tissue utilization. Therefore, amino acid concentrations can be viewed as a rough indicator of the balance between intake and utilization, and have been used for assessing deficient or excessive intakes (Heird, 1989). An underlying assumption is that the protein and amino acid intake and the plasma aminogram of exclusively breast-fed infants is optimal. Serum urea nitrogen can be used as an additional parameter in that excessive protein intake will result in elevated serum urea nitrogen levels; however, it should be considered that human milk as well as some infant formulas are high in urea (Donovan and Lönnerdal, 1989b) and that absorbed urea may contribute to the serum urea nitrogen level.

The case for using plasma amino acid and serum urea nitrogen levels to assess protein status is based in part on the potential risks associated with profiles that differ from those of the breastfed infant. For example, it has been cautioned that the high metabolic activity of the liver and the kidney necessary to catabolize and excrete high levels of plasma amino acids and urea nitrogen could cause undue stress to immature organs and potentially lead to long-term consequences (Herin and Zetterström, 1987; Axelsson et al, 1988). Evidence for such consequences later in life is lacking for infants fed high protein formulas, although some animal studies of high protein diets have resulted in tissue damage. However, most of the animal studies used protein levels considerably higher (relative to requirements) than those given to formula-fed infants. It must be emphasized that studies looking at long-term consequences, such as kidney function in adulthood as related to protein intake during early life, are usually not available, making claims of the safety of high protein formulas relatively weak. In terms of specific amino acids, high concentrations of branched-chain amino acids have raised some concern in that the higher than normal transport of these across the blood-brain barrier may interfere with the transport of other essential amino acids (Ginsburg et al, 1985; Scott et al, 1985). The high levels of threonine observed in infants fed high protein whey-predominant formula has also evoked some concern (Rigo and Senterre, 1980; Grugan et al, 1988), although it has been argued that no negative consequences have been associated with hyperthreoninemia. Amino acids that tend to be lower in plasma of formula-fed than breastfed infants include tryptophan and phenylalanine (Janas et al, 1985, 1987; Lonnerdal and Chen, 1990; Hanning et al, 1992). In general, low tryptophan levels are observed in infants fed low-protein whey-predominant (60: 40) formula, while lower phenylalanine levels are observed in infants fed casein-predominant formula. In both cases, hypothetical scenarios can be made for impaired neurotransmitter synthesis, although evidence is lacking. Differences in sleep patterns have been reported between breast-fed and formula-fed infants (Butte et al, 1992); plasma tryptophan levels have been implicated as one possible explanation for this difference (Fernstrom and Wurtman, 1971). However, difficulties arise when judging what constitutes a 'normal' sleeping pattern.

Most experimental studies on the protein requirement of infants have been on formula-fed infants during the first three months of life. Several of these studies have shown that infants fed formula with a true protein level of 15 g/1 have significantly higher plasma levels of several amino acids and urea nitrogen than those found in breast-fed infants (Järvenpää et al, 1982a,b; Janas et al, 1985, 1987; Räihä et al, 1986a,b; Hanning et al, 1992). A true protein level of 13 g/1 has been shown to result in a plasma amino acid profile similar to that of breast-fed infants (Lönnerdal and Chen, 1990), although a few minor differences were observed. One study by Räihä et al (1986a,b) indicated that feeding a formula protein level of 13 g/1 resulted in lower serum urea nitrogen and plasma concentrations of some essential amino acids than found in breast-fed infants. However, the formula used had an unusually high level of urea and the true protein level was actually 11 g/1 (Lonnerdal and Chen, 1990). The low levels of some amino acids were only observed during the first few weeks of life and it is difficult to evaluate if they were truly inadequate even if they were significantly lower than in breast-fed infants. It should be noted that as bovine casein has a different amino acid composition compared to that of human casein, and bovine whey is quite different from human whey (Picone et al, 1989), it is virtually impossible to create an amino acid profile identical to that found in human milk. Even if the amino acid composition of the formula could be made very similar to that of human milk, digestibility and absorption of amino acids and peptides from such a formula would be quite different from that of breast milk, thus resulting in different plasma amino acid profiles.

In a series of studies. Räihä Axelsson and co-workers have evaluated intake and growth during the period from 4 to 6 months, when the study infants were gradually increasing their intake of solid foods (Axelsson et al, 1988; Räihä and Axelsson, 1991). Breastfed infants were compared with those fed experimental formulas varying in protein content. Weight and length gain were significantly higher in infants fed high-protein formulas (ranging from 18 to 27 g/1) than in those who were breastfed or fed a low-protein formula (13 g/1). Nitrogen excretion was much higher in the infants fed the high protein formulas, which the authors interpreted as indicating excessive nitrogen intake. Their conclusion was that a lower-protein formula results in anthropometric and biochemical indices more similar to those of breastfed infants.

A recently completed study by Fomon et al (1995) provides additional information that is useful for understanding protein requirements. By design, one group of male infants (Experimental Group) was provided graded intakes of protein that were intended to match, as closely as possible, the protein requirements for normal male infants estimated by Fomon (1991). Another group of male infants (Control Group) was fed a commercially available formula with a protein concentration of 15 g/1. The source of protein in all formulas was whey-predominant bovine milk proteins. Observed protein intakes are indicated in Table 10 for each 28-d period of the study. Protein intake was calculated assuming that 14% of total nitrogen in the formulas was non-protein nitrogen (Donovan and Lonnerdal, 1989b) and that all of the non-urea portion and 17% of the urea portion (assumed to be 5.6% of total nitrogen) were fully utilizable; this calculation is equivalent to 95.4% of total nitrogen × 6.25. Gains in weight and length by the Experimental Group were slightly less than those by the Control Group, but the differences were not statistically significant. However, in comparison to a larger reference group (n = 380) of formula-fed male infants (Nelson et al, 1989), gain in weight of the Experimental Group was significantly lower for the age intervals 56-112 days, and 8112 days, and gain in length was significantly lower for the intervals 5-56 and 56-112 days. Growth of the Experimental Group was similar to that of male breastfed infants (Nelson et al, 1989). Concentrations of serum albumin of the Experimental Group were normal, but concentrations of serum urea were extremely low. In each age interval, serum urea concentrations were significantly lower than in the Control Group and in either the formula-fed or the breastfed reference group. It was concluded that the lower protein intake had a mild but detectable effect on growth of infants in the Experimental Group. Protein intakes by infants in the Control Group were clearly not limiting for growth.

Table 10 Protein and energy intakes of infants in the experimental (n = 15) and control groups (n = 13); values are means (s.d.) (Fomon et al, 1995)


Protein intakea (g/kg/d)

Energy intake (kcal/kg/d)

Age interval (months)

Exptl

Control

Exptl

Control

0-1

1.81 (0.22)

2.43 (0.33)

118 (14)

116 (16)

1-2

1.69 (0.15)

2.36 (0.29)

116 (11)

113 (14)

2-3

1.40 (0.11)

2.14 (0.19)

100 (8)

102 (9)

3-4

1.16 (0.09)

2.00 (0.13)

95 (8)

95 (6)

a Adapted from Fomon et al (1995), using 6.25 to convert from nitrogen to protein, and adjusting for NPN by multiplying by 0.954 (see text).

The results of Fomon et al suggest that the amount of protein consumed by the Experimental Group was somewhat less than the group mean intake needed to ensure adequacy among essentially all formula-fed infants (theoretically, the latter is somewhat greater than the 'safe' level of intake defined as mean requirement + 2 s.d. of the requirement; see section 1). In comparison with the average adjusted protein intake of breastfed infants (Table 1), the amounts consumed by the Experimental Group were lower from 0 to 1 month, but generally similar thereafter.

Protein requirements of formula-fed infants may be greater than those of breastfed infants if there are differences in the efficiency of utilization of formula vs human milk. Even after accounting for the non-protein nitrogen content of the milk consumed, there may be differences in utilization of the protein and non-protein fractions. As pointed out above, it is virtually impossible to create a formula that matches the digestibility and amino acid composition of human milk. Waterlow et al (1960) compared nitrogen absorption and retention in infants fed either expressed breast milk or a cow's milk formula with a similar concentration of protein (1.1%). Nitrogen absorption and weight gain did not differ significantly between diet periods, but nitrogen retention was 17% higher on human milk than on the formula (P = 0.01). Further studies that include measurements of the composition of weight gain and use infant formulas developed more recently are needed to resolve this issue.

Table 11 Comparison the amino acid composition of whole protein in immature mammals of different species

Amino acid

Human

Cattle

Sheep

Pig

Rat

Lysine

71

69

75

75

77

Phenylalanine

41

39

42

42

43

Methionine

20

18

17

20

20

Histidine

26

27

23

28

30

Valine

47

42

53

52

52

Isoleucine

35

30

33

38

39

Leucine

75

74

79

72

85

Threonine

41

43

47

37

43

Tyrosine

29

27

35

32

34

Glutamate/ine

130

138

137

134

148

Glycine

118

121

100

91

78

Arginine

77

75

71

69

73

Aspartate/ine

90

87

85

117

97

Alanine

73

76

73

72

64

Proline

84

87

84

60

54

Serine

44

47

47

48

50

Values are mg amino acid/g total amino acid excluding cysteine and tryptophan for which few reliable data are available.
Notes:
1. Species are arranged in order of the rate constant of postnatal growth.
2. For source references see Davis et al (1993).
3. Note that as the postnatal growth rate increases (i.e. from the human infant to the rat), the glycine and proline contents decrease, and hence the contribution of collagen to protein mass is higher in the human infant than in the rat pup.

All of the experimental studies described above were conducted with formula-fed infants, because manipulating the protein intake of breastfed infants is of course much more difficult.. However, data are now available from an intervention study in Honduras in which infants were randomly assigned to be exclusively breastfed for the first 6 months (n = 50), or to receive pre-prepared solid foods (with egg yolk as the main source of protein) in addition to breast milk beginning at 4 months (n = 91) (Cohen et al, 1994; Dewey et al, in press). Neither weight gain nor length gain from 4 to 6 months differed between groups despite a 20% higher protein intake in the latter group. The 20 infants with the highest protein intakes in that group were matched to 20 exclusively breastfed infants on the basis of energy intake; protein intake was 33% higher in the solid foods subgroup, but growth rate did not differ between groups. Similarly, the 20 infants with the lowest protein intakes in the exclusively breastfed group were matched (by energy intake) to 20 infants given solid foods; protein intake was very low in the former compared to the latter (0.81 ± 0.13 vs 1.04 ± 0.20 g/kg/d; P < 0.001), yet there was still no difference in growth. Infant morbidity was relatively low and did not influence the results. These analyses indicate that protein intake is not likely to be a limiting factor with regard to growth of breastfed infants from 4 to 6 months of age.

2.4. Using an 'operational' approach

Waterlow (1990) has suggested that the most satisfactory operational approach to assessing protein requirements of infants is by using protein-energy ratios (P: E). Although protein-energy ratios have been used previously (FAO/WHO/UNU, 1985), they required estimating the ratio of the safe level for protein to the mean requirement for energy at each age, with all of the difficulties and assumptions inherent in those calculations (Town et al, 1992). Waterlow proposes instead that one starts with the assumption that breast milk provides sufficient protein for virtually all infants up to about 4 months of age, provided that they consume enough to satisfy their energy needs. The protein-energy ratio of human milk is approximately 8-8.5% at 3-4 months post partum. This estimate is based on (a) a 'protein' concentration of 9.6-100.0 g/l, which includes 46% utilization of the NPN fraction, (b) a gross energy density of 670 kcal/l and (c) a conversion factor of 5.65 kcal per g protein. The question then becomes, what is the appropriate P: E ratio for older infants? To calculate this, one can determine the ratio of requirements at older ages to those of the infant at 3-4 months. For example, the ratio of protein requirements (per kg) at 12 months vs 4 months is approximately 0.7-0.8. Waterlow argues that the 'safe' P: E ratio of the diet of the 12 month infant would thus be (8-8.5) × (0.7-0.8), or about 6%, presuming that the quality of the food is the same as that of breast milk. Adjustments can then be made for the digestibility and protein quality of the diet.

While this approach is attractive in terms of its theoretical simplicity and ease of application, it bypasses the sticky issue of defining minimum protein requirements. This is both a virtue and a limitation. Like the approach based on observed intakes of breastfed infants, it can be used to set 'safe' levels of intake, but the mean requirement level would be at some (unknown) level below the 'safe" level.

2.5. Amino acid requirements

Amino acid needs can be visualized as stemming from two independent pathways of utilization: growth and maintenance. The accretion of some amino acids (glutamine) and their metabolites (e.g. creatine, heme, glutathione and taurine) within the lean body mass should be considered as part of growth, but in the context of amino acid needs, protein deposition is the dominating influence. The basal need for a given amino acid in support of protein deposition is simply the product of the rate of protein deposition and the contribution of the amino acid in question to the proteins being deposited.

Table 11 shows data on the amino acid composition of the body protein of various mammalian 'infants'. With regard to the essential (indispensable) amino acids, the composition of body protein is virtually identical. Thus the relative needs of each essential amino acid (ma amino acid/g total amino acid) for growth will be the same across species. Table 12 compares current definitions (by nitrogen balance) of the amino acid needs for growth of human infants, pigs and rats. To control for differences in protein 'requirements' these are expressed in terms of total essential amino acids (i.e. mg amino acid/mg total essential amino acids). When adjusted on this basis, the patterns are very similar across species. Thus it seems reasonable to conclude that the obligatory or minimum needs for essential amino acids to support growth (protein deposition) are a close function of the growth rate of the individual. It follows that defining amino acid requirements will depend on what is regarded as an appropriate rate of protein deposition (plus the needs for maintenance).

Table 12 The relative amounts of essential amino acids in current estimates of the amino acid requirements for growth


Rata

Pigb

Humanc

Body proteind

Amino acid

(mg amino acid/mg total essential amino acid)e

Lysine

17

15

16

18

Leucine

18

17

15

18

Isoleucine

13

10

10

9

Valine

14

13

12

12

Sulfur

7

8

11

7

Aromatic

14

18

14

17

Tryptophan

3

3

3

3

Threonine

11

10

10

10

Essential/Nonessential

42

44

46

43

a From National Research Council (1978).
b From Fuller et al (1989).
c From Holt and Snyderman (1961).
d From summary of the literature (Davis et al, 1993).
e This method of normalization was used because of the wide differences in the total protein requirement in the three species.

Table 13 Comparison of the composition of total (protein-bound + free) amino acids in different species milks


Human

Great apea

Cattle

Sheep

Pig

Rat

Amino acid

(mg amino acid/g total amino acid)

Lysine

71

70

86

83

79

68

Phenylalanine

37

37

50

48

43

39

Methionine

16

18

26

29

22

25

Histidine

23

23

24

26

24

22

Valine

51

56

52

57

46

44

Isoleucine

53

52

47

49

40

40

Leucine

104

102

99

90

89

92

Threonine

44

41

42

41

37

40

Tyrosine

46

42

47

47

39

36

Cysteine

20

16

9

8

16

26

Glutamate/ine

190

210

208

203

208

221

Glycine

22

21

18

18

32

15

Arginine

36

35

34

34

44

33

Aspartate/ine

86

88

70

75

78

88

Alanine

40

38

32

40

36

59

Proline

95

101

100

102

117

75

Serine

61

44

56

52

51

85

a Mean of values for chimpanzee and gorilla.
Notes:
1. Note the high cysteine/methionine ratio in human and great ape milk.
2. Note the high serine and low proline of rat milk. This reflects the distinct amino acid sequence of rat b-casein
3. Although extensive data are lacking, about 40% of the glutamate seems to be glutamine. Data taken from Davis et al (1994).

It is interesting to compare the relative quantities of total (protein + free) amino acids in milk to the relative quantities of amino acids in body protein. Amino acid composition of milk (ma amino acid/g total amino acid) from various species is shown in Table 13 (taken with permission from Davis et al, 1994), and the ratios of milk amino acids to body amino acids are shown in Table 14. Three main points are of particular interest. First, with the exception of the sulfur amino acids, there is a remarkable commonality among species that differ widely in their postnatal growth rates and functional maturity at birth. Second, three amino acids (leucine, glutamate/glutamine and proline) account for 39.8 ± 1.1% of milk amino acids. Third, as the comparison of milk and body amino acids shows, all milks are markedly 'deficient' in glycine and arginine. Thus, the mixture of amino acids presented to the naturally-fed infant is quite different from its obligatory needs for protein deposition.

The reason for the differences between milk and body amino acid composition is obscure. The additional amino acid needs of the infant for maintenance cannot explain the differences, because maintenance demands relatively large amounts of nonessential amino acids, which are in relatively low concentrations in milk (see below). It could be argued that the unique composition of milk is indicative of a major pathway of amino acid use that has not been identified. What seems more likely is that the amino acid composition reflects a compromise between the evolution of an optimum mixture and the need for specific physical properties such as the maintenance of solubility at < 37°C and in the presence of triacylglycerols.

The central problem in understanding the amino acid needs of human infants arises from their relatively low rate of protein deposition compared with that of other mammals. As a result, amino acid needs for maintenance of existing stores of protein (and amino acid metabolites) are a high proportion of total amino acid needs. As shown in Table 3, it is clear that except for the first month of life, more than half the need is for maintenance.

Table 14 Ratio milk amino acid: body amino acid in various mammalian species

Amino acid

Human

Cattle

Sheep

Pig

Rat

Lysine

1.00

1.24

1.11

1.05

0.88

Phenylalanine

0.90

1.28

1.14

1.02

0.91

Methionine

0.80

1.44

1.70

1.10

1.25

Histidine

0.88

0.88

1.13

0.86

0.73

Valine

1.08

1.23

1.07

0.88

0.85

Isoleucine

1.51

1.56

1.48

1.21

1.13

Leucine

1.51

1.56

1.48

1.21

1.13

Threonine

1.07

0.98

0.89

1.11

0.93

Tyrosine

1.58

1.55

1.34

1.21

1.06

Cysteinea

1.25

0.56

0.50

1.00

1.62

Glutamate/ine

1.46

1.51

1.48

1.55

1.49

Glycine

0.18

0.15

0.18

0.35

0.19

Arginine

0.46

0.45

0.48

0.64

0.45

Aspartate/ine

0.96

0.80

0.88

0.66

0.91

Alanine

0.55

0.42

0.55

0.50

0.92

Proline

1.13

1.14

1.21

1.95

1.38

Serine

1.38

1.19

1.10

1.06

1.70

a Assuming that body protein is on average 16 mg cysteine/g protein.

Formulating this maintenance protein need in terms of amino acids has proved difficult, and is the subject of heated controversy. Animal studies, especially in the pig a species for which very high quality nitrogen balance data are available (Fuller et al, 1989) - suggest that the maintenance amino acid pattern differs markedly from that for growth. According to these data the minimum maintenance needs for leucine, isoleucine, valine, phenylalanine/tyrosine, Lysine and tryptophan are only about 25% of the quantities needed to sustain growth. More than 75% of the total maintenance amino acid need is for non-essential amino acids. There also seems to be a higher relative need for cysteine (maintenance approximately 2.3-fold higher than for growth) whereas the relative threonine need is approximately the same for growth and maintenance. Although these conclusions have been challenged (see below), the same patterns appear in a semi-quantitative way in all available data from animals (including birds) which implies that there is some biological basis for them. Reeds and Hutchens (1994) have proposed that amino acid needs close to nitrogen equilibrium may be dominated by (a) continuing protein loss into the intestine; this will have a specific effect on threonine needs (Fuller et al, 1994), (b) maintenance of some key metabolites of amino acids (creatine and taurine), especially in the skeletal muscle and central nervous systems; this will demand the provision of cysteine and glycine and (c) maintenance of pathways that use amino acids involved in host defenses, e.g. glutathione, nitric oxide and nucleotide synthesis; these will utilize glutamate/ glutamine, cysteine, glycine, arginine, and aspartic acid. It is notable that these latter pathways use non-essential or conditionally essential amino acids. The task at present is to quantify the impact of these pathways (especially glutathione, creatine, nitric oxide and taurine synthesis) on the needs for their respective precursor amino acids. To do this will require novel approaches using stable isotopes.

Until more information is available, the only alternative is to use existing data, based either on nitrogen balance or on amino acid carbon balance. Early nitrogen balance studies by Rose et al (1957) gave results for minimal dietary allowances that proved to be generally similar to data obtained from subsequent nitrogen balance studies in animals. It was these minimum values that were used by FAO/WHO/UNU (1985) to calculate amino acid 'requirements' of adults, and it is these values that have been challenged by the results, and subsequent calculations of Young and co-workers (Young et al, 1989). However, many reviewers of Rose's summary paper have not emphasized that Rose in fact gave two values: a minimum 'requirement' at which some (but by no means all) subjects could maintain nitrogen balance, and a level at which all the subjects maintained nitrogen balance. The latter is generally twice the former. The Young (MIT) patterns were based on experiments in which the oxidation of a single indispensable amino acid was measured as it was progressively removed from the diet. Subsequently these oxidation values were altered to take into account: (a) calculated rates at which they could be mobilized from body protein (based on basal nitrogen loss and the composition of body protein) under conditions where the diet lacked the amino acid and (b) the efficiencies (which contained both a digestibility and utilization efficiency term) with which the diet could supply the need. The various estimates (mg/kg/d) are shown in Table 15. With the exception of Lysine and threonine (which are 50% higher in the MIT pattern) and the sulfur amino acids (which are 50% lower in the MIT pattern), Rose's 'safe' level and the MIT pattern are closer than some recent discussions (Young et al, 1989) have implied.

Tables 16-18 show estimates of the minimum amino acid needs (mg/d in the left hand columns, and mg/kg/d in the far right column) of infants 0-6 months of age. The calculations were made as follows:

1. Median body weights were taken from NCHS reference data. These were averaged over the time intervals chosen i.e. 0-1 months, 1-3 months and 3-6 months.

2. Needs for growth were calculated using data from Table 4 and from Fomon et al (1982) for protein deposition and the amino acid pattern of whole body protein shown in Table 11 column 1. Thus column 1 in Tables 1618 is the product of protein deposition (g/kg/d), body weight and amino acid ,content (ma amino acid/g body protein).

3. Maintenance protein needs were calculated in two ways:

Method 1. Using the value of 120 mg N/kg/d from the 1985 report (this was chosen to be conservative: if a value of 90-100 (see section 2.2.2) were chosen the calculated amino acid needs would obviously be lower).
Method 2. Using 172 mg N/kg0.75/d (i.e. normalized for metabolic body size), calculated from the balance data of the studies shown in Table 2.

4. Amino acid needs for maintenance were calculated in two ways:

Method 1. As a constant quantity (mg/kg/d) using the MIT values (Table 15, column 4).
Method 2. Assuming that at maintenance 30% of total protein needs (Young et al, 1989) are for essential amino acids and that the pattern of amino acids is that defined in Table 15 column 4. This method thus expresses the pattern for each amino acid in terms of percentage of total essential amino acids required:

Lysine

20.9

Aromatic

19.4

Sulfur

8.0

Valine

11.9

Leucine

15.4

Threonine

10.4

Tryptophan

2.9

(note that total essential amino acids are presumed to include histidine and arginine, which would make up the remaining 11%).

5. 'Requirements' for non-essential amino acids were calculated by subtracting the requirements for essential amino acids from the total amino acid requirements for either growth or maintenance.

Table 15 Comparison of the daily needs for maintenance of essential amino acid derived from nitrogen balance by Rose et al (1995) with those derived from carbon balance by Young et al (1989)


Rose

Young


Minimuma
(mg/kg/day)

Safe intakeb
(mg/kg/day)

Minimumc
(mg/kg/day)

Intaked
(mg/kg/day)

Isoleucine

10

20

ND (ND)

22 (1.10)e

Leucine

15

30

22 (1.46)

31 (1.03)

Valine

12

22

11 (0.92)

24 (1.09)

Lysine

11

22

18 (1.64)

42 (1.91)

Sulfur

16

32

11 (0.69)

16 (0.50)

Aromatic

15

30

ND (ND)

39 (1.30)

Threonine

7

14

11 (1.57)

21 (1.50)

Tryptophan

2

4

ND

6 (1.50)

a Level of intake at which some subjects remained in N balance.
b Level at which all subjects were in N balance.
c Calculated carbon catabolic rate after a prolonged period of inadequate amino acid intake.
d Calculated from obligatory nitrogen losses and a presumed efficiency of diet utilization of 70%.
e Values in parentheses are the ratio of the Young (MIT): Rose pattern.

Table 16 Amino acid needs and amino acid supply from breast milk: age 0-1 months; median body weight 3.6 kg



Maintenance

Total



Amino acid (mg/d)

Growth (mg/d)

1 (mg/d)

2 (mg/d)

1 (mg/d)

2 (mg/d)

800 ml milk (mg/d)

Total (mg/kg/d)a

Lysine

258

151

173

409

431

482

116

Aromatic

258

140

162

398

420

564

114

Sulfur

128

58

68

186

195

244

64

Valine

168

86

94

154

261

346

72

Leucine

270

112

130

382

400

707

109

Isoleucine

127

79

92

206

219

360

59

Threonine

146

76

87

222

233

299

63

Tryptophan

57

22

24

79

81

170

22

Glycine

397

ND

ND



150


Arginine

277

ND

ND



244


Alanine

263

ND

ND



238


Total

3600

2700

2809

6300

6409

6800

1765

Essential

1412

724

830

2136

2212

3192

608

Nonessential

2188

1976

1979

4164

4167

3608

1158

a Mean of the two methods of calculation.
Maintenance protein method 1 = 3.6 × 120 × 6.25 = 2700 mg protein; maintenance protein method 2 = (3.6)0.75 × 172 × 6.25 = 2809 mg protein; total maintenance essential amino acids = 2809 × 0.3 = 842 mg.

Table 17 Amino acid needs and amino acid supply from breast milk: age 1-3 months; median body weight 4.7 kg



Maintenance

Total



Amino acid

Growth (mg/d)

1 (mg/d)

2 (mg/d)

1 (mg/d)

2 (mg/d)

800 ml milk (mg/d)

Total (mg/kg/d)a

Lysine

192

197

215

389

497

482

85

Aromatic

222

183

200

405

422

564

88

Sulfur

96

75

82

171

178

244

37

Valine

126

113

113

239

239

346

51

Leucine

200

146

158

346

358

707

75

Isoleucine

94

103

112

197

206

360

43

Threonine

109

99

107

208

216

299

45

Tryptophan

49

28

30

77

79

170

16

Glycine

267

ND

ND




150

Arginine

207

ND

ND




244

Alanine

196

ND

ND




238

Total

2679

3525

3431

6204

6110

6800

1310

Essential

974

994

1064

1861

1981

3196

440

Nonessential

1705

2581

2414

4286

4119

3604


a Mean of the two methods of calculation.
Maintenance protein method 1 = 4.7 × 120 × 6.25 = 3525 mg protein; maintenance protein method 2 =- (4.7)0.75 × 172 × 6.25 = 3431 mg protein; total maintenance essential amino acids = 3431 × 0.3 = 1029 mg.

Table 18 Amino acid needs and amino acid supply from breast milk: age 3-6 months; median body weight 6.3 kg



Maintenance

Total



Amino acid

Growth (mg/d)

1 (mg/d)

2 (mg/d)

1 (mg/d)

2 (mg/d)

800 ml milk (mg/d)

Total (mg/kg/d)a

Lysine

134

264

267

398

401

482

63

Aromatic

132

246

248

378

380

564

60

Sulfur

68

101

102

169

170

244

27

Valine

89

151

153

240

242

346

38

Leucine

142

195

198

337

340

707

54

Isoleucine

65

139

139

204

204

360

32

Threonine

78

132

134

210

212

299

34

Tryptophan

34

38

379

72

71

170

11

Glycine

307

ND

ND



150


Arginine

237

ND

ND



244


Alanine

225

ND

ND



238


Total

1890

4725

4274

6515

6164

6800

1014

Essential

742

1266

1278

2008

2020

3196

320

Nonessential

1148

3459

2996

4607

4144

3604


a Mean of the two methods of calculation.
Maintenance protein method 1 = 6.3 × 120 × 6.25 = 4725 mg protein; maintenance protein method 2 = (6.3)0.75 × 172 × 6.25 = 4274 mg protein; total maintenance essential amino acids = 4274 × 0.3 = 1288 mg.

The final values for total need (growth + maintenance, in mg/d) are then compared with the quantities of amino acids provided by 800 ml of milk from a well nourished woman. It is important to note that (a) the estimates of need do not include any adjustment for digestibility or efficiency of utilization (i.e. it is assumed that milk amino acids are 100% bioavailable) and (b) it is assumed that full milk production is achieved within the first month post partum.

The results of this exercise suggest that at no age do the needs (mg/d) for essential amino acids as a group exceed the average amount provided by human milk. (Note that this is not the ideal comparison, as the estimated needs in Tables 16-18 are analogous to requirements, whereas the average amounts provided by human milk should be more than 2 s.d. above mean requirements in order to meet the needs of virtually all infants; without data on the variability of amino acid requirements, the more appropriate comparison cannot be made). However, it should be noted that if the Rose 'safe' estimate of sulfur amino acid needs for maintenance had been used, total sulfur amino acids would have been in short supply by 3-6 months. The results also show that, even without ascribing specific maintenance needs for specific amino acids, the amounts of the so-called 'non-essential' amino acids (particularly glycine) supplied by milk are lower than estimated needs at all three ages. This indicates that the breastfed infant must utilize essential amino acids for synthesis of non-essential amino acids. The average estimated 'surplus' of essential amino acids is more than enough to meet the average need for non-essential amino acids at each of the ages examined, but again, this comparison does not take into account variability in amino acid requirements or intake.

This assessment of amino acid needs does not account for the use of glycine in creatine synthesis or of cysteine in taurine synthesis. It could be argued that the supply of these two end-products of glycine and cysteine metabolism in breast milk is crucial both to the economy of these amino acids and, more importantly, to the functional development of the infant. The apparently higher need of the formula fed infant for protein (i.e. the apparently lower efficiency of protein utilization of these infants) might be related to the lack of creatine and taurine in many infant formulas.

This exercise has demonstrated that, although the pattern of amino acids required by infants differs from that of human milk, the average amounts provided to breastfed infants are generally greater than estimated needs. The needs for essential amino acids listed in Table 18 (mg/kg/d) for the 3-6 months infant are much lower than the average requirement values listed in the 1985 report (p. 65) for infants at 3-4 months. This is because the values in the 1985 report were based on the intakes of cow's milk formula or human milk that were observed to support satisfactory growth, whereas the values in Table 18 were calculated using a factorial approach. Interestingly, the values in Table 18 are very similar to the 1985 estimated requirements for preschool children (per kg), which were determined using experimental diets in which one essential amino acid at a time was partially replaced in the diet by glycine at five different levels. Torun (1989) points out that the values published for preschoolers should actually be considered safe levels rather than requirements, which would place the preschool requirements somewhat below the estimated needs of infants (per kg). This makes sense, given the difference in the proportion of total protein needed for growth at 3-6 months (31%) vs 2 y (approximately 15%).

It could be argued that the essential amino acid 'requirements' for infants listed in the 1985 report should be revised downward to be more consistent with calculated needs. However, because there are other functions for amino acids besides their role in protein metabolism (Reeds and Hutchens, 1994), and we do not yet fully understand the functional implications of the amino acids in human milk, it is safer to continue to recommend that a pattern similar to that of human milk be taken as the 'required' pastern.

2.6. Recommendations for revision of the 1985 report

(1) Updated estimates of the protein intake of breastfed infants, such as those shown in Table 1, should be included. At this time, the assumption that at least 46% of the non-protein nitrogen in human milk is utilizable appears to be justified. The estimates of intake in Table 1 are 10-26% lower than those in the 1985 report (see section 2.1.4).

(2) If the model of the breastfed infant is used to estimate requirements in the first 6 months, the approach taken should not assume that mean intake equals mean requirement. The epidemiological, probability approach suggests that the nitrogen requirement is less than 170 mg/kg/d at 3-4 months (Table 9), whereas mean intake is 231 mg/kg/d (see section 2.2.8).

(3) Several aspects of the factorial model for estimating protein requirements of infants should be reconsidered:

(a) The estimate of maintenance requirement in the 1985 report (120 mg N/kg/d) is probably too high. The evidence suggests that a value of 90 mg N/kg/d is more realistic (see section 2.2.2).

(b) The use of a 50% augmentation to the protein needs for growth, to account for day-to-day variability, should be abandoned. If any adjustment is considered necessary to allow for spurts in growth, it should be based on data regarding the true biological needs for rapid deposition of tissue. The adjustment factor should be included in the CV component for growth (i.e. in calculating safe levels), not added on to the mean protein increment for growth (see section 2.2.4).

(c) In the factorial model presented (Table 6), 70% was chosen as a conservative estimate of efficiency of utilization of dietary protein for growth, based on the slopes of nitrogen balance equations for infants. However, the factor of 70% may be an underestimate, especially for rapidly-growing infants and those with intakes close to estimated requirements. In considering this assumption, the definition of 'efficiency' should be clearly stated. Empirical data based on infants fed human milk or formula must indicate how the 'protein' content of the milk was calculated, as 'efficiency' based on intake calculated as total N × 6.25 will obviously be lower than if intake is calculated adjusting for the presumed bioavailability of the protein and non-protein nitrogen fractions.

(d) The factorial model presented in Table 6 is generally consistent with the estimates obtained using the model of the breasfed infant, though there may still be some overestimation of requirements (see section 2.2.8). A relatively conservative approach was taken in choosing the assumptions for Table 6, so as to provide estimates that would be appropriate for a range of ages and feeding modes. At this time there is insufficient evidence to determine whether protein requirements of non-breastfed infants are higher than those for breastfed infants (regardless of growth rate) {see section 2.3). More research on the efficiency of utilization of infant formulas is needed to resolve this question.

(e) In arriving at estimates of safe levels of intake, the CV component for growth should use data for intervals greater than one month (e.g. 3-month increments). Otherwise, the safe levels calculated for older infants would be considerably further from the mean requirement than would be the case for younger infants (see section 2.2.6). When more data are available regarding protein needs during growth spurts, this information should be considered when calculating the CV for growth.

(4) The operational approach to estimating the adequacy of protein intake, based on P: E ratios, can be used to derive diets which will provide safe levels of intake when infants are no longer exclusively breastfed, as described in section 2.4.

(5) In estimating metabolic needs for amino acids of infants, it should not be assumed that the pattern of amino acids in human milk :is the same as the pattern of requirements. Nonetheless, to provide a generous margin of safety with regard to essential amino acids, the pattern in human milk should be acceptable for estimating dietary allowances in most cases. Thus, the estimates listed in the 1985 report need not be revised at this time, but the rationale for their adoption should be clarified. However, more information is needed to determine the appropriate amino acid composition of infant formulas; because of differential rates of utilization of proteins in human milk vs infant formulas, it cannot be assumed that a formula that mimics the amino acid composition of human milk will be optimal for the non-breastfed infant.