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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

(introductory text...)

KG Dewy1, G Beaton2, C Fjeld3, B Lönnerdal1 and P Reeds4 (with input from KH Brown1, MJ Heinig1, E Ziegler5, NCR Räihä6 and IEM Axelsson6)

1 Department of Nutrition and Program in International Nutrition, University of California, Davis, CA 95616-8669;
2 Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1 A8;
3 Section of Nutritional and Health-Related Environmental Studies, International Atomic Energy Agency, PO Box 100, A1400 Vienna; 4 USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine 1100 Bates Street, Houston, TX 77030; 5 Division of Nutrition, Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242; 6 Department of Pediatrics, University of Lund, Malmo General Hospital, S-214 01 Malmo, Sweden

Descriptors: protein, amino acids, nutrition, nitrogen, catch-up growth, diet quality

1. Introduction

There are several approaches for estimating the protein requirements of infants and children. During early infancy, the intake of breastfed infants has been used as a model, under the assumption that protein requirements are satisfied by human milk alone. Alternatively, a factorial model can be used to calculate requirements, or a direct experimental approach can be taken whereby key outcomes are measured while subjects are fed varying levels of protein. Finally, an approach which has been called 'operational', based on protein-energy ratio, has been proposed (Waterlow, 1990). This variety of approaches has created a serious dilemma, as the estimates obtained in the past using these alternative models have differed considerably, partly because of confusion over which aspects of the actual distribution of protein requirements they were describing (Beaton, 1994). Furthermore, all the above approaches conceptualize requirements in terms of total protein, whereas recently there has been an increased focus on estimating requirements in terms of the needs for individual amino acids (Scrimshaw and Schürch 1991). Although there has been heated debate on this latter issue, and some data relevant to adults, there is a paucity of data for infants and children.

Another major dilemma is posed by the question: protein requirements for what? Historically, a satisfactory growth rate during infancy and childhood has been the 'litmus test' for the adequacy of protein intake. However, in recent years there has been greater attention to the need for assessing functional outcomes such as immune function and behavioral development. It is not clear whether the rate of growth is an adequate proxy for these other outcomes. Under conditions of nutrient deficiency, for example, it may be that growth falters only after other aspects of function, such as behavioral development, are compromised. Basing requirements on adequate growth might therefore underestimate the true need for optimal function. On the other hand, reports of differences in growth rates between breastfed and formula-fed infants (e.g. Dewey et al, 1992) have raised the issue of whether maximal growth is synonymous with optimal growth. It has been suggested that excessive protein intake may jeopardize certain physiological functions (see section 2.3). Therefore, it is theoretically possible that a protein intake that maximizes growth may be disadvantageous in other respects.

Note: assistance in preparing this report does not imply that all of the contributors agree with all of the conclusions and recommendations.
Correspondence: KG Dewey.

The task set out for this position paper - to evaluate whether the protein requirements for infants and children described in the 1985 FAO/WHO/UNU report on Energy and Protein Requirements should be revised - is thus very complex. Rather than attempting to cover all the issues in depth, the objective of this paper is to critically review the basis for the 1985 recommendations, to suggest which sections should be revised, and to identify topics requiring further research. The paper first reviews the protein needs of normal infants and children, followed by sections on protein requirements in situations of catch-up growth or in association with infections, methods for assessing the protein quality of weaning diets, and future research needs. The special needs of low birthweight infants are not covered.

It is useful to begin by clarifying the terminology that will be used in this paper. The literature on nutrient requirements in general and protein or amino acid requirements in particular is plagued by the fact that the term 'requirement' is used very loosely and is often confused with the notion of a recommended dietary allowance. In keeping with the 1985 report, this paper will use the word 'requirement' when discussing the true biological need for protein or amino acids (the lowest intake that will maintain functional needs of the individual). As recognized in the 1985 report, there is a distribution of such requirements among seemingly similar individuals, so the mean requirement is usually taken as the starting point. The 'safe level of intake' is defined as the amount that will meet or exceed the requirements of practically all individuals in a population, which is generally calculated as the mean requirement + 2 s.d. of the requirement. In the case of protein, this refers to a quantity of high-quality (or 'reference' protein). With mixed diets, the 'safe level' will need to be adjusted for digestibility and amino acid composition of the foods consumed in order to arrive at recommended intakes for a specific population. Understanding the distinction between biological requirements and recommended intakes is critical, particularly for infants whose total diet may be prescribed based on recommended allowances. When assessing the observed mean intake of a group, it should be kept in mind that this should be somewhat higher than the 'safe level' of intake. This is because the group mean intake required to ensure adequacy for all individuals must take into account both the distribution of requirements among individuals and the distribution of intakes among individuals (and the correlation between intake and requirement), whereas the conventional calculation of a 'safe level' considers only the distribution of requirements.

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.

3. Protein requirements of children and adolescents

3.1. Basis of the 1985 recommendations

In the 1985 report, the factorial approach was used to estimate protein requirements of children and adolescents. For maintenance nitrogen requirements, values were interpolated based on the two 'anchor' points of 120 mg N/kg/d at 1 year and 100 mg N/kg/d at 20 years of age. The coefficient of variation was assumed to be 12.5%, similar to that observed in adult balance studies. For growth, the expected nitrogen accretion was based on data from Fomon et al (1982) up to age 10; after age 10 it is unclear what reference values were used. The same assumptions as used for infants were included in the calculations: (a) the efficiency of conversion from dietary protein to body protein was assumed to be 70%, (b) the coefficient of variation for growth was taken as 35% and (c) an additional 50% was added to the growth increment to allow for day-today variation in growth rate.

It is worthwhile to reevaluate the 1985 estimates, given that questions could be raised about all the above assumptions. With regard to the efficiency of protein utilization for growth and the CV for growth, revisions in the above assumptions are not likely to have much of an impact on requirement estimates after infancy. This is because protein needs for growth are a small percentage of total protein needs after the first year of life, and the magnitude of changes caused by minor revisions in those assumptions (e.g. using an efficiency of 90% instead of 70%) is thus small. The assumptions that are most likely to influence requirement estimates for children and adolescents are the maintenance nitrogen requirement (and its CV) and the 50% growth increment. The lack of justification for the latter adjustment was discussed in section 2.2.4. In the next section, the maintenance needs of children and adolescents are re-examined, and in the subsequent section, revised estimates are presented.

3.2. Maintenance nitrogen requirements/nitrogen balance studies

In the 1985 report, the maintenance nitrogen requirements for children and adolescents depended heavily on the estimated value of 120 mg N/kg/d at 1 year of age. As explained in section 2.2.2 above, only one study was cited to support this estimate, which was based on infants fed very low amounts of energy. Section 2.2.2 presents an argument for considering a lower value, such as 90 mg N/kg/d. If this were accepted, then the interpolation of values from 1 to 20 years would clearly be very different.

Several short-term balance studies in preschool children were described in Table 30 of the 1985 report (Town et al, 1981a; Intengan et al, 1981; Egaña et al, 1983; Iyengar et al, 1979). In all but one of these (which had a sample size of only 3 with adequate energy intake: Iyengar et al, 1979), the average maintenance requirement (corrected for digestibility) listed in Table 30 was £ 100 mg N/kg/d. One study not cited in the 1985 report measured obligatory nitrogen losses in five preschool children 17 to 31 months of age given a protein-free diet for 7-9 days (Town and Viteri, 1981a). Total obligatory nitrogen losses were 59 mg N/kg/d, assuming 5 mg N/kg/d for integumental nitrogen losses. Assuming that nitrogen intake to meet maintenance needs is 130-145% of inevitable nitrogen losses (FAO/ WHO/UNU, 1985; Young et al, 1989), the maintenance requirement would be 77-86 mg N/kg/d. These studies thus provide support for the idea that maintenance nitrogen requirements at this age may be closer to 90-100 mg N/kg/d than to the previously assumed value of 120 mg N/kg/d. No new balance studies (since the 1985 report) for this age range were located.

Longer-term balance studies (3-6 months) have also been conducted in preschool children (Town and Viteri, 1981b,c; Begum et al, 1970), and were described in the 1985 report (Table 35). In all these studies, the level of protein provided (1.07-1.40 g/kg/d when corrected for digestibility, equivalent to 171-224 mgN/kg/d) was judged to be adequate or more than adequate to support expected growth rates. There is a lack of information regarding long-term nitrogen balance of children with protein intakes closer to the estimated requirement.

One factor that has generally not been considered in the longer-term balance studies is the adequacy of micronutrient intake. This is particularly important when diets relying extensively on plant sources are utilized, given the low bioavailability of some nutrients (e.g. iron and zinc) from such foods. Incorrect estimates of protein requirements may result if growth is limited by nutrients other than protein during balance studies.

There is much less information available for older children and adolescents than for preschool children. The 1985 report cites one study of adolescent males (Prothro et al, 1973) as showing that an intake of 100120 mg N/kg/d from a mixed diet (with considerable animal protein) was necessary to produce consistently positive nitrogen balances. Since the 1985 report, two short-term balance studies have been published (Gattas et al, 1990, 1992) both conducted in Chile and using a mixed, predominantly vegetable diet. In one, the mean intake to achieve balance (allowing 8 mg N/kg/d for unmeasured losses and 10 mg N/kg/d for growth) among males 8-10 y of age was 150 mg N/kg/d (Gattas et al, 1990), not correcting for digestibility. A very similar value was obtained for males 12-14y of age: 147 mg N/kg/d (not correcting for digestibility), allowing 8 mg N/kg/d for unmeasured losses and 19 mg N/kg/d for growth (Gattas et al, 1992). If corrected for digestibility, the estimates would be approximately 125 mg N/ kg/d. In the latter study, it is difficult to evaluate the validity of the allowance for growth (19 mg N/kg/d), as no growth data were presented.

The two studies conducted in Chile estimated 'safe' levels of protein intake assuming a CV of 12.5%. The resulting estimates for intake from a mixed diet (not correcting for digestibility) were 1.2 g protein/kg/d for boys 8-10 y and 1.15 g protein/kg/d for boys 12-14y. However, when calculated using the actual CVs observed in the balance studies (5% and 9%, respectively), the 'safe' levels were 1.03 and 1.09 g/kg/d, respectively.

3.3. Revised estimates of protein requirements for children and adolescents

Based on the information described above, Tables 19 and 20 were developed assuming a maintenance requirement of 100 mg N/kg/d (the same as for adults), and without the 50% augmentation for intra-individual variation in growth used in the 1985 report. Both tables use the same assumptions as the 1985 report for the efficiency of conversion of dietary protein to body protein, the CV for growth, and the CV for maintenance (12%)

Table 19 Revised estimates for Table 33 in the 1985 FAO/WHO/UNU report on Energy and Protein Requirements (children)

Age (y)

Maintenance (mg N/kg/d)

Growtha
(mg N/kg/d)

Total requirement (mg N/kg/d)

Total + 2 s.d.b (mg N/kg/d)

1-1.5

100

27

127

160

1.5-2

100

21

121

151

2-3

100

19

119

148

3-4

100

16

116

144

4-5

100

14

114

141

5-6

100

11

111

138

6 7

100

11

111

138

7-8

100

11

111

138

8-9

100

11

111

138

9-10

100

11

111

138

a Assumes 70% efficiency of utilization, as in 1985 report.
b Using CVs shown in Table 33 of the 1985 report.

Table 20 Revised estimates for Table 34 in the 1985 FAO/WHO/UNU report on Energy and Protein Requirements (adolescents)

Age (y)

Maintenance (mg N/kg/d)

Growtha (mg N/kg/d)

Total requirement (mg N/kg/d)

Total + 2 s.d.b (mg N/kg/d:)

Girls

10-11

100

13

113

140

11-12

100

11

111

138

12-13

100

10

110

136

13-14

100

9

109

135

14-15

100

6

106

131

15-16

100

5

105

130

16-17

100

1

101

125

17-18

100

0

100

124

Boys

10-11

100

11

111

138

11-12

100

11

111

138

12-13

100

14

114

141

13-14

100

11

111

138

14-15

100

11

111

138

15-16

100

9

109

135

16-17

100

7

107

133

17-18

100

5

105

130

a Assumes 70% efficiency of utilization, as in 1985 report.
b Using CVs shown in Table 33 of the 1985 report.

Among preschool children, estimated requirements from balance studies using milk-based or mixed diets with considerable animal protein range from 98 to 117 mg N/kg/d (Town et al, 1981a; Intengan et al, 1981; Egana et al, 1983). These are consistent with the values in Table 19, which are about 17-20% lower than the 1985 values for this age range. Among older children and adolescents, estimated requirements from balance studies are 100-120 mg N/kg/d for males 13-17 y in the US (Prothro et al, 1973) and about 125 mg N/kg/d for males 8-10 and 12-14 y in Chile (Gattas et al, 1990, 1992). The estimate from the US study is consistent with Table 20, but the estimate from the latter two studies is higher than estimated needs in Tables 19 and 20. Thus, the few data available suggest that the 1985 values are overestimates for preschool children but may be appropriate for older children and adolescents. It should be noted that most balance studies include only boys; there are almost no data on requirements of girls, which theoretically could be quite different during adolescence.

3.4. Amino acid requirements of children and adolescents

In the 1985 report, the amino acid requirements of preschool children were based on experimental studies in which the amount of each essential amino acid provided was varied one at a time (Pineda et al, 1981; Torun et al, 1981b). The 1991 Expert Consultation on Protein Quality Evaluation (FAO/WHO Expert Consultation, 1991) accepted these values, although it has been pointed out (Millward et al, 1992) that the original data have not been published in full. In addition, the applicability of these estimates (obtained from six previously malnourished children) to other populations is debatable (Millward et al, 1992). Nevertheless, the data for preschoolers are considered the best available to date. One of the co-investigators contributing these data (Town, 1989) has pointed out, however, that the published values should be considered as safe levels rather than minimal requirements.

The amino acid requirements for older children published in the 1985 report were considered inappropriate by the 1991 Consultation (FAO/WHO Expert Consultation, 1991) due to problems in the original balance studies on which they were based. In the 1985 report, amino acid requirements (per kg) were predicted to decrease considerably with age, but the biological plausibility of this 'trend' has since been questioned. Millward et al (1992) suggest that the age-related decline in estimated amino acid needs is merely an artifact of the different dietary designs of the original balance studies. Newer data suggest that amino acid needs of adults may have been underestimated; based on these data, Young and colleagues (Pellett and Young, 1988) proposed a new amino acid pattern to be used for all ages except during infancy. However, the 1991 Consultation considered that more data were required before a new pattern could be adopted. Nonetheless, it was recognized that after the first year of life, amino acid needs per unit body weight should not differ greatly by age because growth is such a small proportion of total needs. Therefore, the 1991 Consultation recommended that until additional information is available, the amino acid pattern for preschool children adopted in the 1985 report should be used to estimate requirements for all age groups except infants.

3.5. Recommendations for revision of the 1985 report

(1) The factorial model used to calculate protein requirements for children and adolescents should not use estimates of maintenance requirements that are interpolated from the 'anchor point' of 120 mg N/kg/d at 12 months, given that the latter value is questionable (see section 2). Rather, a value of 100 mg N/kg/d (at all ages) seems more appropriate, based on data from balance studies of children and adolescents (see section 3.2). When updated information on maintenance needs of adults is available, this should be used to help judge whether the estimate of 100 mg N/kg/d is sufficient for older children and adolescents.

(2) As suggested for the infancy requirements (see section 2.6), the 50% augmentation in the growth component to allow for day-to day variability should be abandoned. If any adjustment is considered necessary to allow for spurts in growth (particularly during adolescence), it should be included in the CV component for growth when calculating safe levels, not added onto the mean protein increment for growth.

(3) The factorial model incorporating the above revisions (Tables 19 and 20) should be considered a good approximation of requirements for preschool children, as it yields estimates that are consistent with short-term balance studies. These estimates are 17-20% lower than the 1985 values. However, limited data on older children and adolescents suggest that the 1985 values may be appropriate for that age range. More information is needed to determine whether the new factorial model for older children and adolescents should be modified.

(4) The recommendations of the 1991 Consultation (FAO/WHO Expert Consultation, 1991) regarding amino acid requirements (see section 3.4) should be adopted until additional data are available, but further attention needs to be paid to the distinction between requirement vs safe level of intake.

4. Protein needs during catch-up growth

4.1. Basis of recommendations in the 1985 report

In the 1985 report (p. 143), protein requirements for catch-up growth were considered separately according to whether children were low in weight-for-height (wasted) or low in height-for-age (stunted). In the case of wasting, protein needs were estimated to be 0.23 g per gram of tissue deposited (assuming that 16% of tissue deposited is protein, and that the efficiency of conversion of dietary protein to tissue protein is 70%: 0.16/ 0.70 = 0.23). However, this estimate was not used in a factorial model to calculate total requirements. In the case of stunted children, examples of how protein needs might be calculated based on the 'safe' levels of intake for normal children were illustrated (Table 52 in the 1985 report). The approach proposed was to estimate average requirement for catch-up by using the 'safe level' for a child of the same height-age (not chronological age) and at the median weight for height. The 1985 report defended this approach on the basis that it provided estimates that were consistent with observed rates of catch-up growth.

4.2. Protein and energy needs for rapid catch-up growth

In the 1985 report, an example was given for catch-up growth of an undernourished child with a weight deficit of 3.3-3.8 kg and a height-age of 12 months, provided with a protein intake of 1.37 g/kg/d (p. 198). It was calculated that with this level of intake, satisfactory catch-up would require 5-6 months. However, for hospitalized malnourished children, such a long duration of recovery is impractical. Furthermore, it is likely that during this period there would be illnesses causing additional catabolic losses. Hastening catch-up growth would be advantageous, if it could be achieved by practical nutritional measures.

To test the hypothesis that catch-up growth could be more rapid without compromising body composition or other indices of nutritional recovery, Fjeld et al (1989a) refed marasmic children using diets formulated to meet the theoretical requirements for energy, protein and micronutrients. Children were randomly assigned to dietary treatments that permitted either moderate (4-6 g/kg/d) or rapid (12-16g/kg/d) rates of weight gain. The composition of total weight gained and final body composition were similar in the two groups, indicating that appropriate nutritional therapy can reduce the time required for catch-up growth while restoring reference levels of body composition.

In the above study, the rate of weight gain in the rapid gain group (12-16g/kg/d) was well within the range of growth velocity observed in other studies in hospital settings (up to 20 g/kg/d: Waterlow, 1992a). This rate of growth is far above the average growth velocity for a normal young child (approximately 1.3 g/kg/d at 6-12 months). Such a high rate of catch-up growth cannot be expected with dietary interventions in community settings, in which few children would be likely to have more than mild to moderate growth impairment (compared with the severe cases treated in a clinical setting). However, a report from The Gambia describes rates of catch-up growth of up to eight times the average daily growth rate calculated from the normal annual growth increment (Rowland et al, 1977).

In the 1985 report (p. 147), the percentage increase in protein requirements for children to grow at twice the 'normal' growth rate was presented as an example of what might be applicable in the community. Although there is clearly a need for more information on rates of catch-up growth in non-hospital settings, the 'twice normal' example given in the 1985 report is almost certainly well below the potential, given appropriate dietary interventions.

For a stunted child with normal body composition, the amount of protein required for rapid catch-up growth can be estimated based on the gain in lean tissue associated with normal growth. In such cases, protein makes up approximately 16-17% of weight gain. Nitrogen balance data from recovering malnourished children (Waterlow, 1992a; Fjeld et al, 1989a) and reference values for normal children at 9-12 months of age (Fomon et al, 1982) are consistent with this estimate. For severely wasted children, catch-up growth will likely include a higher proportion of fat tissue, and therefore a proportionately lower amount of lean tissue. Table 21 provides estimates of the protein and energy needs at various rates of weight gain under conditions of either normal growth (i.e. applicable to the stunted child), or a high rate of fat deposition (i.e. applicable to the severely wasted child). In the latter case, the estimates of fat and lean tissue gain were taken from those expected of a normal infant at 2-3 months of age, when fat deposition is rapid (Fomon et al, 1982); the assumed percentage of fat tissue (43%) is consistent with data for recovering marasmic children in Peru (Fjeld et al, 1989a). In both parts of Table 21, the maintenance protein need was assumed to be 0.63 g/kg/d (90 mg N/ kg/d). The efficiency of conversion of dietary protein to tissue protein was estimated at 70%, to be consistent with the factorial models presented in previous sections. It is possible, however, that recovering malnourished children are more efficient at protein deposition. Table 21 presents energy needs based on two levels of 'maintenance' energy expenditure (i.e. including BMR and activity but not growth): 80 and 90 kcal/kg/d. The latter value is typical of normal infants at 9-12 months of age (see the position paper by Butte), but may be higher than would be expected of malnourished infants if they are less active. The former value (80 kcal/kg/d) is similar to the average energy expenditure of preschool children (see the position paper by Torun et al) and to energy expenditure for maintenance and activity of recovering malnourished children in Peru (Fjeld et al, 1989b). The table also presents the ratios of requirements expressed as percentage of energy from protein (P: E) in each situation.

Several points are noteworthy with regard to the estimates in Table 21:

(1) At the higher rates of catch-up growth (10-20g/kg/ d), the amount of protein needed ranges from 2.0 to 5.4 g/kg/d, depending on the composition of weight gain. The P: E ratio is much higher for children expected to have a normal composition of weight gain (up to 15% dietary protein) than for those expected to have a high rate of fat deposition (up to 7% dietary protein). Because most wasted children are also stunted, it is probably prudent to err on the high side when calculating the P: E ratio for refeeding, especially given the fact that the P: E ratios in Table 21 are based on average estimated requirements, and are therefore not analogous to 'safe levels'.

(2) Energy needs are higher for wasted children than for non-wasted children, because of the higher energy cost of depositing fat tissue. At the higher rates of weight gain, energy needs range from 113 to 156 kcal/kg/d for nonwasted children and from 140 to 210 kcal/kg/d for wasted children. The duration of such feeding regimes should be determined on a case-by-case basis; if an initially wasted child is fed a very high energy diet for too long, he or she may become obese.

Table 21 Protein and energy needs for catch-up growth at different rates of weight gain



Normal composition of weight gaina

High rate rate of fat depositionb



EEc = 80

EEc = 90


EEc = 80

EEc = 90

Rate of gaind (g/kg/d)

Proteine (g/kg/d)

Energyf (kcal/kg/d)

P/E (%)g

Energyf (kcal/kg/d)

P/E (%)g

Proteine (g/kg/d)

Energyf (kcal/kg/d)

P/E (%)g

Energyf (kcal/kg/d)

P/E (%)g

1

0.87

83

4.2

93

3.7

0.77

86

3.6

96

3.2

2

1.11

87

5.1

97

4.6

0.91

92

4.0

102

3.6

5

1.83

97

7.5

107

6.8

1.33

110

4.8

120

4.4

10

3.03

113

10.7

123

9.9

2.03

140

5.8

150

5.4

20

5.43

146

14.9

156

13.9

3.43

200

6.9

210

6.5

a 17% protein, 9% fat; assume energy cost of growth = 3.3 kcal/g (based on 5.65 kcal/g protein and 9.25 kcal/g fat, with efficiencies of synthesis of 42% and 85%, respectively (Roberts and Young, 1988): 0.17g protein × 5.65 kcal/g/0.42 = 2.3 kcal; 0.09g fat × 9.25 kcal/g/0.85 = 1.0 kcal); protein needs for growth = protein need/efficiency = 0.17/0.7 = 0.24 g/kg/d.
b 10% protein, 43% fat; assume energy cost of growth = 6.0 kcal/g (based on 5.65 kcal/g protein and 9.25 kcal/g fat, with efficiencies of synthesis of 42% and 85%, respectively (Roberts and Young, 1988): 0.10 g protein × 5.65 kcal/g/0.42 = 1.3 kcal; 0.43 g fat × 9.25 kcal/g/0.85 = 4.7 kcal); protein needs for growth = protein need/efficiency = 0.10/0.7 = 0.14g/kg/d.
c EE = energy expenditure for maintenance and activity.
d In normal children, average rates of weight gain are about 1.3 g kg/d at 6-12 months, 0.8 g/kg/d at 12-18 months and O.5 g/kg/d at 18-24 months.
e Assume maintenance needs for protein of 0.63 g/kg/d.
f Metabolizable energy intake.
g Ratio of requirements expressed as percentage of energy from protein. This differs from the concept of a 'sale level' of P: E as used in Section 2.4.

Waterlow (1992a) noted that requirements based only on gains in tissue protein do not account for restoration of proteins that may be depleted in malnourished children, such as serum albumin. However, he calculated that the amount of protein required for this is only about 5% of the tissue protein deficit, which is too small to strongly affect the estimates in Table 21. Waterlow (1992a) also illustrated the impact of illnesses during the period of recovery on protein needs for catch-up growth, using the assumptions that each day of infection produces a deficit of 30 g in weight gain and that the extra protein is consumed only during the days when the child is well. For a recovery period of 100 days, protein needs for catch-up increase by about 15-25% when the prevalence of infections is 20-30%. The impact of infections is discussed in more detail in section 5.

For a stunted child with normal body composition, the rate of catch-up growth may be limited by the amount of time needed to recover in height. It has been observed that when recovering malnourished children reach a normal weight for height, their appetite and rate of weight gain usually diminish, even though they may still be stunted. Thus, in these situations it would be rare to observe rates of weight gain as high as the upper end of the range in Table 21 (20 g/kg/d). A more realistic example is shown in Table 22, which provides estimates of the protein and energy needs for a child whose initial height-for-age is three standard deviations below the reference median and who reaches normal height-forage within 6 months. Weight gain in this case is expected to be 1.63 g/kg/d. At this rate, energy needs are only about 4% higher than normal, whereas protein needs are 37% greater than the requirement for a normal child. Table 22 is meant to provide only an example of the needs for catch-up in height, and is not intended to be used prescriptively.

Table 22 Example of protein and energy needs for catch-up in height


N

S

Height (cm):

24 months

85.6

76.0

30 months

90.4

90 4

Weight (kg):

24 months

12.3

10.0

30 months

13.5

13.5

Height gain (cm/month)

0.8

2.4

Weight gain:

g/d

6.6

19.1

g/kg/d (at mean wt))

0.51

1.63

(kcal/kg/d)

Energy cost of wt gain @ 3.3 kcal/g

1.68

5.38

TEE

80

80

Total energy need

82

85

(g kg/d)

Protein gain @ 17% of wt. Gain

0.087

0.277

Protein cost @ 70% efficiency

0.12

0.40

Protein need for maintenance

0.63

0.63

Total protein need

0.75

1.03

(%)

P: E ratio

3.7

4.8

N = normal boy, 2 years of age, initial Z-score in height = 0.
S = stunted boy, 2 years of age, initial Z-score in height = - 3, but weight-for-height normal.
Assuming full catch-up in height in 6 months (with normal body composition)

4.3. Evidence from nutrition intervention studies

Results of nutrition interventions in developing countries appear to provide support for the value of relatively high protein intakes for malnourished children (Kabir et al, 1992, 1993; Fjeld et al, 1989a; Malcolm, 1970; Jackson et al, 1990). Unfortunately, most studies have not controlled for the levels of micronutrients provided by the supplementary foods given, so it is difficult to determine whether the observed improvement in growth can be attributed to extra protein or to higher intakes of other nutrients. For example, Kabir et al (1992, 1993) demonstrated that catch-up growth can be accelerated in children 2-4 y of age recovering from malnutrition secondary to shigellosis. Accelerated linear growth was achieved by supplementing the standard recovery diet (lentils, banana, bread, rice, oil, milk powder, sugar) with egg, milk and chicken. The standard diet provided 7.5% of dietary energy as protein, whereas the experimental diet provided 15% of energy as protein, as well as increased levels of other nutrients. The linear growth rate in control children was comparable with the NCHS reference; the rate in the experimental group was significantly higher. In the study of malnourished Peruvian children by Fjeld et al (1989a), height gain in the control children fed the standard recovery diet (approximately 125 kcal/kg/d, 8% energy as protein, micronutrient intake supplemented by level of energy intake) was comparable to the NCHS reference based on height-age, whereas height gain in the children fed the experimental diet (approximately 165 kcal/kg/d, 11 % energy as protein, micronutrient intake supplemented by level of energy intake) was significantly greater than the NCHS reference based on height-age. In several studies, children fed control diets (considered adequate for children not experiencing catch-up growth, but having less protein and other nutrients than the experimental diets) tended to deposit fat tissue at the expense of lean tissue (Malcolm, 1970; MacLean and Graham, 1980; Jackson et al, 1990; Fjeld et al, 1989), which supports the idea that restoration of appropriate body composition requires nutrient intakes different from those needed at lower rates of weight gain.

The mechanism by which dietary treatment stimulates linear growth probably involves increased synthesis of insulin-like growth factor I (IGF-I) and IGF-binding proteins (Kabir et al, 1992; Kabir et al, 1993). The above studies suggest that dietary interventions can stimulate the anabolic drive during convalescence from malnutrition, which implies that nutrient requirements during this period should be calculated on the basis of the maximal rate of growth achievable without compromising optimal body composition or function. It is important to note that the increased needs for catch-up growth include not only protein and energy but nearly all other nutrients as well.

At present there is some disagreement about whether complete catch-up growth in height of stunted children is achievable after the first 2-3 years of life (Uauy & Alvear et al, 1992; Waterlow, 1992a; Martorell et al, 1994; Golden, 1994). If there is a 'critical period' for linear catch-up growth, the benefits of increased nutrient intake to support accelerated growth may not be demonstrable in older children.

4.4. Recommendations for revision of the 1985 report

Requirements for catch-up growth should include a factor for maintenance and a factor for growth. The latter should be presented for various rates of weight gain, as illustrated in Table 21. The values used for maintenance nitrogen needs and the efficiency of conversion from dietary protein to body protein should be consistent with those chosen for the protein requirement tables for infants and children in general.

5. protein needs associated with infection

5.1. Basis of recommendations in the 1985 report

The 1985 report pointed out that young children in disadvantaged populations experience frequent infections, which can have a significant impact on protein requirements. However, because there were few quantitative estimates of the increased need for protein during infection, this section of the 1985 report was brief. Information from one long-term nitrogen balance study of six children 8-12 months of age in Thailand (Tontisirin et al, 1984) was used to estimate the order of magnitude of increases that might be needed. In that study, measurements were made under relatively controlled conditions, but the children were exposed to some degree of infection and parasitism. The average intake of 1.35 g/kg/d was considered to meet their protein requirements, but was not sufficient to be considered a 'safe level'. It was suggested that if 1.35 g/kg/d could be considered the requirement level under those conditions, the safe level would be 1.69 g/kg/d (1.35 × 1.25), which was 14% higher than the requirement calculated by the factorial method for children of that age. In a later section, a 20% increment in protein intake for children 12-18 months exposed to infection was suggested as an approximation. It was pointed out that children younger than this would need a larger increase and older children less of an increase. The conclusion was that until more information was available, more detailed calculations were unwarranted. It was recommended that field studies be conducted to test assumptions about protein requirements under actual environmental conditions.

5.2. Metabolic changes accompanying infection: implications regarding protein and amino acid requirements

Ideally, the calculation of the likely impact of infection on nutrient requirements should take into account the metabolic changes that occur during and after an illness. Severe infection obviously requires immediate clinical intervention and while febrile illness has a major metabolic impact, supporting the infected individual's nutritional needs is not a substitute for the appropriate therapeutic measures. Of greater concern at the population level are the circumstances of infants and children in environments in which persistent immune activation, not necessarily manifested as overt infection, may be prevalent. Solomons et al (1993) have suggested that such continuous 'low level' activation of the host defense mechanisms diverts nutrients away from growth and towards the needs of the immune system. They cite studies showing that chickens raised in sanitary environments grow more rapidly and utilize their diet more efficiently than those housed under less advantageous conditions even though these animals do not display any clinically identifiable infections (Libby and Schaible, 1955; Hill et al, 1952; Lillie et al, 1952).

Although the nutrient requirements of severely infected individuals should not, perhaps, be the model when we seek to define estimates of nutrient requirements for populations, the changes that accompany more severe illness can provide insights into the ways in which less severe, but continuous, immune activation might impact nutrient requirements. With regard to protein and amino acid nutrition, the key factors are (1) synthesis of new proteins associated with host defense, (2) muscle protein loss, (3) metabolic diversion of nutrients during the active infection and (4) impaired intestinal absorption with some infections. All these responses occur in concert, largely initiated by the increase in cytokine levels consequent upon immune activation and the increased secretion of the so-called stress hormones: cortisol, epinephrine and glucagon.

5.2.1. Synthesis of immune-response proteins. Host defense depends on the ability to support the proliferation of the cells of the immune system, the synthesis of the positive acute-phase proteins and factors involved in the complement system, and the maintenance of peroxidative defenses by increased synthesis of glutathione and other free radical scavenging molecules (such as nitric oxide) derived from amino acids. It is important to bear in mind that the new protein synthesis associated with the activation of the body's defense mechanisms is highly specific and therefore may demand specific nutrients. Little quantitative information on the scale of these 'anabolic' responses is available. Waterlow (1991) has calculated that at the height of a 'typical' infection the circulating positive acute-phase reactant proteins may rise by a total of 1.2 g/kg. This is a substantial proportion of whole body protein synthesis. In well-nourished individuals it seems reasonable that the substrates available for these changes are adequate, but the inability of undernourished individuals to mount an adequate response is well established. The suppression of immune activity by undernutrition may persist even when immediate nutritional deficits have been replenished. For example, Doherty et al (1993) have shown that in infants the response of C-reactive protein and serum amyloid A to vaccination with diphtheria-pertussis-tetanus vaccine was impaired for some time after an episode of severe protein energy malnutrition, despite nutritional rehabilitation. In this case a specific role of protein deficiency cannot be established because of the likelihood of multiple micronutrient deficiencies accompanying an episode of severe protein energy malnutrition.

5.2.2. Catabolism of skeletal muscle. A uniform finding in a wide variety of traumatic conditions, including injury, is a loss of muscle protein. This may be particularly marked in undernourished individuals (Nessan et al, 1974). The muscle protein mobilization is greater (Garlick et al, 1980) than would be expected from the reduction in intake that almost invariably accompanies infection, and it appears to be actively regulated by the combination of catabolic cytokines and hormones that also initiate a number of the other metabolic responses to infection (Beisel, 1975). It is presumed that muscle protein mobilization is directed towards supplying amino acids for other processes. These could include the provision of glutamine, glycine and cysteine for glutathione synthesis, the provision of essential amino acids for cellular proliferation and new secretory protein synthesis and the possibility that amino acids assume a greater importance as energy sources during active infection. A recent study of the amino acid composition of the major acute phase proteins (Reeds et al, 1994) has revealed that, as a group, they have unusually high concentrations of phenylalanine and tryptophan. It has been hypothesized that this necessitates excessive muscle protein mobilization. The mobilization of muscle protein is not, in and of itself, a contributor to increased amino acid requirements, but depletion of muscle protein is an indication that these requirements have increased beyond those being supplied by the diet of the infected individual. The replenishment of the depleted muscle mass is an important factor in the nutritional requirements for the recovery period.

5.2.3. Metabolic diversion of nutrients - specific effects on amino acid needs. Overt infection is characteristically associated with substantial changes in circulating and tissue amino acid concentrations. The large majority of the amino acids show an early and rapid decline in concentration (Beisel, 1975; Wannemacher, 1977). The decline in plasma amino acid concentrations occurs early in the illness. In a number of studies of experimental infection (Feigen and Dangerfield, 1967; Feigen et al, 1967, 1968; Wannemacher et al, 1972), the decline in circulating amino acids preceded the onset of fever. In general it is believed that these changes are a reflection of increased hepatic utilization which, in this context, includes both acute-phase protein synthesis and gluco-neogenesis (Beisel, 1975).

The general decline in amino acid concentrations is not uniform. Research in the 1970s with Diplococcus pneumoniae infection (Wannemacher et al, 1971; Wannemacher, 1977) showed early reductions in the branched chain amino acids, threonine, tyrosine, proline and arginine. Subsequent studies (Souba et al, 1990) have also established consistent reductions in circulating glutamine concentrations in a variety of traumatic circumstances. These changes persist as long as the infection persists. It has generally been presumed that the particularly marked changes in the branched chain amino acids and glutamine are related to their metabolic interrelationships, their involvement in muscle energy metabolism and interorgan nitrogen transport and hence that the alterations are primarily associated with derangements in energy metabolism. By contrast, the concentrations of phenylalanine, tryptophan and glycine increase during infection (Wannemacher, 1977). The underlying reason for these changes remains obscure, as the release of these amino acids from skeletal muscle is not excessive (Bostian et al, 1976) and infection is associated with accelerated hepatic uptake, at least of phenylalanine. The changes are particularly paradoxical given that a case can be made for a specific increase in phenylalanine and tryptophan requirements during infection (Reeds et al, 1994).

Recent work has also suggested an increase in proline requirements under traumatic conditions. Jaksic et al (1987) have shown that the ability of humans to synthesize this 'non-essential' amino acid is limited; in burn injury, humans move into a profound negative proline balance (Jaksic et al, 1991).

5.2.4. Implications regarding protein and amino acid requirements. The above sections illustrate that overt infection not only alters protein requirements in general but probably has effects on the needs for some specific amino acids, notably the aromatic amino acids as well as cysteine, glutamine, arginine and proline However, the quantitative impact of infection still requires investigation, particularly with regard to persistent subclinical infections.

For children, one approach that has been taken is to estimate the amount of protein needed to restore the growth deficit caused by infections. However, this ignores the unique alterations of amino acid metabolism that accompany infection.

Another approach is to estimate nutrient losses during infection and calculate the amount needed from the diet to recover those losses during convalescence. Scrimshaw (1992) has utilized this approach in a recent review of the effect of infection on nutritional status. He cites data from Powanda (1977) as indicating that the average additional loss of protein during infection in adults is about 0.6 g/kg/d; losses are higher (0.9 g/kg/d) for diseases associated with diarrhea or dysentery. Some infections, such as typhoid fever, are associated with even higher losses, up to 1.2 g/kg/d. Scrimshaw estimated that it generally takes two to three times longer to replete than to deplete an individual. Thus, the amount of extra protein required to recover losses due to infection would be one-half to one-third of the daily loss during infection, provided that this augmentation was available for the total duration of the convalescent period. Taking the mean daily loss of 0.6 g/kg/d, and assuming that this estimate also applies to children, the protein increment during convalescence would be 0.2-0.3 g/kg/d (0.3-0.5 g/kg/d for diarrhea). This represents about 20-30% of the normal protein requirement (3050% in the case of diarrhea), depending on the age of the child.

The duration of the period needed to recover nitrogen losses will depend on the severity and duration of the illness. In the 1985 report it was recommended that children be fed according to their appetite, which seems the only practical way to proceed. However, if appetite is impaired by illness (even after the acute phase) the time needed for repleting nutrient losses will be longer. It should also be emphasized that for many children in disadvantaged populations, the needs for catch-up growth may be superimposed on the extra needs during the period of recovery from infection. The total need will obviously depend on the desired rate of catch-up growth, as explained in the previous section.

While calculation of nitrogen losses during infection can provide an overall estimate of total protein needs, this approach does not stipulate the optimal amino acid balance that would restore those losses most efficiently. Given the disproportionate needs for certain amino acids during infection, merely providing more of the same mix of amino acids as consumed when healthy may be less effective than a mixture specifically designed for the recovery period. One approach to this is to measure flux rates, rates of efflux from skeletal muscle, and rates of influx into acute-phase proteins of amino acids in response to specific infections and in varying states of initial nutritional status. This kind of investigation can be done in children using stable isotopes; a multi-site study of this nature is currently underway as part of a Coordinated Research Programme by the Section of Nutritional and Health-Related Environmental Studies, International Atomic Energy Agency and several co-funders. When the results are available, it may be possible to make more precise estimates of amino acid requirements associated with infection.

Whatever the approach taken, it is important to note that, just as for catch-up growth, the extra nutrient needs associated with infections include not just protein and amino acids but energy and other nutrients as well.

5.3. Recommendations for revision of the 1985 report

Until further data are available, a reasonable estimate of protein needs following infection is a 20-30% increase in total protein (30-50% in the case of diarrhea) during a recovery period that is two to three times longer than the duration of the illness. In the case of persistent diarrhea with accompanying anorexia, the desired increase may be very difficult to achieve. When children have diarrhea for 20-30% of the time, following the above recommendation will essentially result in a permanent increase in the protein level of the diet provided (until they reach an age when diarrhea is less prevalent).

6. Assessment of protein quality of weaning diets

The procedures for making adjustments for protein digestibility and quality were described thoroughly in the 1985 report and in a subsequent Joint FAO/WHO expert report (Joint FAO/WHO Expert Consultation on Protein Quality Evaluation, 1991) and do not need to be reiterated here. Use of the protein digestibility corrected amino acid score is considered the most appropriate method for evaluating protein quality. This method obviously depends heavily on the estimated amino acid requirements at each age. For infants, both the 1985 report and the 1991 Consultation recommended that the amino acid composition of human milk be accepted as the suggested pattern of requirement. However, as the discussion in section 2.5 indicates, the essential amino acid content of human milk is generally above 'requirements' based on calculated needs for growth and maintenance. Thus, using the human milk amino acid pattern (mg/g protein) as the basis for evaluating weaning foods may underestimate their protein quality (depending on which amino acids are potentially limiting in the food being evaluated).

For children, the 1991 Consultation (FAO/WHO Expert Consultation, 1991) recommended that the amino acid scoring pattern for preschool children (see section 3.4) be used for all age groups (except infants). Until more information is available on amino acid requirements, this recommendation appears to be justified. However, because that scoring pattern was based on only one set of studies in a small sample of children, replication of the findings is essential. The lack of information on sulfur amino acid needs is particularly unfortunate as these amino acids, together with Lysine, may be marginal in vegetable protein sources if Rose's (1957) 'safe' rather than 'minimal' level is closer to true needs (see section 2.5). Even so, data from Guatemala (Scrimshaw et al, 1961; Torun and Viteri, 1981b; Torun et al, 1984) suggest that predominantly vegetable multi mixes can provide an adequate source of amino acids for children beyond the age of one year.

Aside from protein quality, any evaluation of weaning foods must also consider the content and bioavailability of other nutrients, particularly trace elements, and the potential presence of 'antinutritional' factors such as plant lectins.

7. Future research needs

To better understand protein requirements of infants and children, more information is needed on a variety of topics. The following is a partial list of research priorities:

(1) There is a need for studies of protein requirements that include functional outcome measures (both short term and long-term), not just growth rate or nitrogen balance. The question 'protein requirements for what?' has not been adequately addressed. Such studies should include not only the potential consequences of marginal intakes, but also the long-term impact of moderately high protein intakes (such as later kidney function in formula-fed infants).

(2) Information on the efficiency of utilization of dietary protein at various ages and in children with different growth rates is needed. In particular, the efficiency of protein utilization in formula-fed vs breastfed infants should be studied to determine whether protein requirements differ by feeding mode in early infancy. Additional data on the utilization of non-protein nitrogen in human milk and infant formulas would also be useful.

(3) It is essential to determine the degree of intra-individual day-to-day variation in growth at various ages, and the nutrient needs (not just protein) required to permit the child to deposit new tissue rapidly during growth 'spurts'. Limited information (Lampl et al, 1992) suggests that spurts in linear growth may be of short duration (24-48 h), but it is not known whether the magnitude of growth during such episodes or the frequency of episodes can be affected by protein intake. Both animal models and human studies would be useful in answering these questions.

(4) Updated information on body composition during infancy and childhood, particularly the percentage of weight gain that is fat-free body mass, is important for accurate estimates of protein requirements using the factorial method.

(5) Better data on maintenance nitrogen requirements of infants, children and adolescents are needed, although ethical issues may limit the types of studies that can be carried out. Studies in children who were not previously malnourished, and data on the protein needs of female adolescents should be a high priority. In addition, long-term balance studies at protein intakes close to estimated requirements would provide key evidence to support or reject the adequacy of the estimates based on short-term balance studies.

(6) Amino acid requirements require further study at all ages, including children. Such research should consider not only amino acid needs for protein metabolism, but the other functions of amino acids as well. Requirements for the sulfur amino acids in particular require investigation. These, ironically are the amino acids for which calculated 'needs' are lower in the MIT pattern (see section 2.5) than in Rose's estimates. Yet data from rats, pigs and chicks suggest a high requirement for cysteine (as a proportion of total amino acid needs) at protein intakes close to maintenance levels, and recent data from the Rowett Research Institute (Fuller, personal communication) suggest that a significant proportion of this need appears to be devoted to the synthesis of taurine.

(7) To determine protein and amino acid needs for catch-up growth, data are needed on the maximal rate of growth that can be achieved without compromising body composition or function. The rates of catch-up growth achievable in non-hospital settings should be documented.

(8) In disadvantaged populations, community studies evaluating the impact of nutrition interventions that provide extra protein, controlling for the intakes of energy and other nutrients, would be particularly useful in determining whether protein is a key limiting nutrient for such children.

(9) The impact of infection on amino acid and protein needs requires further investigation. The impact of acute-phase protein synthesis on whole body protein economy and requirements is of particular interest. Data are needed to determine whether chronic stimulation of the immune system diverts essential nutrients from whole body growth in undernourished children, and, secondly, the dietary needs to prevent or compensate for this outcome. Several outcome variables would be useful: (a) the ratio of protein synthesis to protein breakdown under conditions of immunostimulation and refeeding; (b) the rate of oxidative loss of dietarily indispensable amino acids and (c) the rates of acute-phase protein synthesis, of transport protein synthesis, and the interactions between these and whole body protein metabolism. In studies of amino acid requirements in infected children, identification of the infective agent(s) should be attempted and concentrations of hormones known to mediate amino acid/protein metabolism during infection should be determined because these indices may facilitate predictions of the impact of infection in other populations.

Acknowledgements - Data for the pooled analysis of growth of breastfed infants came from seven studies conducted by the following investigators: KG Dewey, MJ Heinig, LA Nommsen and B Lonnerdal (USA); NF Krebs, CJ Reidinger and KM Hambidge (USA); KF Michaelsen and G Samuelson (Denmark); LA Persson and G Samuelson (Sweden); L Salmenpera, J Perheentupa and MA Siimes (Finland); AA Paul, TJ Cole, EA Ahmed and RG Whitehead (UK); and D Yeung (Canada). The analysis was funded by a grant from Wellstart International through the US Agency for International Development (AID), Bureau for Research and Development, Office of Health (Cooperative Agreement No. DPE-5966-A-00 1045-00) to the University of California, Davis. The contribution of Peter Reeds to this paper has been funded in part with federal funds from the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement number 58-6250-1-003. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement from the US Government. We gratefully acknowledge comments on the initial draft from JC Waterlow and SJ Fomon.

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Discussion

In all discussions on protein requirements it is assumed that the needs for energy and micronutrients are met. In the breast-fed infant this must indeed be the case, and it is therefore appropriate that the definition of the protein requirements of infants should be based on the 'biological reality' of the breast-fed baby. Discussion centered initially on some of the components of the factorial estimates put forward by Dewey in her paper

Efficiency of utilization of nitrogen for maintenance and for growth

Two options were initially proposed for the table of factorial estimates (Table 6): 70% and 90%. It was agreed that 90% is too high, at least for infants over 4 months of age, and that 70% is more appropriate. Higher efficiencies have indeed been found in children who have reached reference weight-for-height after recovering from malnutrition, but this is not a normal situation, as these children may still be depleted of protein (Scrimshaw).

The question was considered, whether the utilization of protein in infant formulae is less good than in breast milk. Although there are inevitably differences in composition, the evidence is that there are no major differences in efficiency: slope of N balance studies with formula is about 0.74 (Reeds). One factor that may affect the utilization of N is the much higher content of NPN, particularly of urea, in breast milk. Dewey had proposed a value of 17% for the utilization of urea-N, derived from the work of Donovan et al (1990) and of Fomon et al (1987) (see also Darling et al (1993)). There are difficulties in the interpretation of these isotopic measurements; several participants (Young, Millward, Waterlow) considered that the figure of 17% for urea, leading to a value of 46% for total NPN, is too low. It was argued (Reeds) that in breastfed children there may be direct benefit from the utilization of urea-N because breast milk tends to be low in non-essential amino acids. Other components of the NPN glutathione, creatine, taurine and nucleotides (mainly pyrimidines) -may be used with 100% efficiency because they spare the metabolism of their precursor amino acids. It was suggested that a range of utilization of total NPN (4661%) be used for the estimates of protein intake of breastfed infants.

Allowance for irregular (saltatory) growth

The arbitrary figure of 50% by which the growth requirement was multiplied in the 1985 report was considered unsatisfactory by most participants. The alternative proposed in the paper is to use the observed CV of weight gain over 3-month intervals in the calculation of 'safe levels', but not in determining mean requirement. The CV over 1-month intervals would have been far too large. However, according to the validation analysis (Section 2.2.8), the CV of weight gain does not have a large influence on the calculated percentage of breastfed infants with 'inadequate' intakes. Moreover, since the physiological concern is with day-to-day variations, a correction derived from these longer intervals is also arbitrary. It may be, however, that there is an automatic adjustment of intake: when the baby needs more milk, the mother is able to produce it (Butte). To translate this in terms of requirement, it would be necessary to know the day-to-day variation in breast milk intake.

The point was also made that, in the 1985 report, growth of the infant was considered entirely in terms of weight gain, and no account was taken of a possible specific requirement for growth in length, through deposition of protein at the ends of the long bones. This requirement may be quantitatively small but qualitatively important, since long bone growth is a major stimulus for skeletal muscle growth (Millward) (see also below, under 'catch-up').

Amino acid requirements of the infant

There is remarkably little information on the amino acid composition of human milk in different populations (Reeds). All the emphasis has been on the amino acids in the NPN fraction. However, since the amino acid composition of most milks is dominated by two proteins, b-casein and a-lactalbumin, there is little room for significant changes (Reeds). Traditionally, it has been assumed that if an infant is growing adequately on a certain amount of breast milk or its equivalent, then the amino acid requirements will be met; but the actual intakes may be much greater than the minimum requirements.

In fact we do not have enough information about the SDs of amino acid intakes or requirements to enable us to arrive at a safe level. Young raised the question of how, for example, the specific value for the Lysine requirement had been calculated. Reeds explained that there is no problem about the growth component, which is based on the Lysine content of tissue protein. The protein requirement for maintenance can be calculated in various ways, but the results are not very different. Two assumptions were then made: first, that the total requirement for indispensable amino acids (IAAs) is 30% of the maintenance protein requirement; secondly, that the pattern of IAA requirements is the MIT pattern derived by Young and coworkers (see JCW's paper). The results of these calculations, which assume 100% availability, are shown in Tables 16-18. It is important to emphasize that these are not tables of requirements. The object of the exercise is to show that, with normal milk intake, regardless of the method of calculation, the IAA content of the milk more than covers requirements, even with a fairly large standard deviation. It is interesting also that the values in Table 18 are quite similar to those obtained by Torun for the safe levels of IAA intakes by pre-school children, using a completely different approach.

The values in the tables are compared with the amino acids in 800 ml of breast milk. The point was raised that this figure may over-estimate the intakes in developing countries, but both Prentice and Dewey have found that they are very similar to intakes in industrialized countries.

Older infants and children; the factorial vs operational (P/E) approach

It was agreed that the factorial approach for estimating the needs of older children is satisfactory. It is logical to use the adult figure of 100 mg N/kg/d for maintenance; both recent and earlier studies on children give similar results. However, it should not be implied that maintenance in a child is physiologically the same as in an adult (Young). It may be quite artificial to separate the maintenance and growth requirements.

Waterlow suggested that an alternative to the factorial approach is to look at the P/E ratios in the diets of children who are no longer exclusively breast-fed, starting from the assumption that the P/E ratio in breast milk represents a safe level (Section 2.4). This is quite different from the approach in the 1985 report, based on the ratio of requirements for protein and energy. The P/E ratio in breast milk is about eight; as growth falls off the safe level of P/E will decrease to about six. In Whitehead's studies in The Gambia and Uganda, P/E ratios were measured in the diets of individual children (Whitehead et al, 1977). In The Gambia, where diets are cereal-based, the ratios were very similar in the diets of all children, with an average of about eight. In Uganda there was a much wider scatter, and about 10% of children had diets with a ratio of five or less, which indicated a risk of protein deficiency. It is interesting that, at least at that time, kwashiorkor was commoner in Uganda than in The Gambia.

This approach does not require any assumptions about the extent to which protein and energy requirements are correlated. It was agreed that the P/E ratio provides a good way of looking at the adequacy of intakes, but it cannot be used to define requirements. Since, after infancy, protein requirements per kg are more or less fixed, while energy requirements per kg fall, the safe P/E ratio for adults is higher than that for children. It was pointed out that there are very few family diets around the world which provide less than 10% protein calories (Scrimshaw). However, although this may be so for adults, it is not necessarily so for children (Waterlow).

Protein digestibility and quality

When breast milk is no longer the sole source of protein, the digestibility and quality of the food protein become important. In adults differences in the amounts of protein required in the diets of different populations could be entirely accounted for by differences in digestibility rather than in amino acid patterns (Rand et al, 1984). It was agreed that more information is needed on digestibility in humans, and that it is not enough to rely on conventional rat assays. These studies should be made with different diets, not just types of protein. Since there is no evidence of any difference in digestibility between adults and children, it would be easier to do these studies in adults. More attention should be paid to variations in digestibility, which may be related as much to the subject as to the food, since there is evidence that in populations accustomed to a particular diet its digestibility may improve (Scrimshaw).

Measurements of faecal N (apparent digestibility) may be satisfactory for estimating total protein requirement by nitrogen balance, but they are not appropriate for determining the availability of individual amino acids. A distinction has to be made between endogenous and exogenous faecal N. since studies with 15N have shown that at least 50% of faecal N is endogenous, with a different amino acid composition from that of the food (Gannon et al, Shulman et al, de Lange et al).

More studies are also needed on the protein quality of children's diets by balance measurements, to check estimates obtained from amino acid scores. Previous studies have been done with isolated proteins; they should now be carried out with whole foods at a low level of N intake (200 mg/kg/d) to maximize differences in quality (Waterlow).

Catch-up growth and infection

Dewey, introducing this subject, recalled that in the 1985 report it was calculated that an extra intake of 0.23 g protein was needed per gram tissue gain, based on a tissue protein content of 16% and 70% efficiency of deposition. This recommendation would apply to a stunted child; a wasted child would need to deposit more fat and therefore theoretically would need less extra protein and more energy per gram gained, leading to a different P/E ratio of the food (Table 21). In practice, however, it might be unrealistic to make such a distinction, particularly since wasting and stunting are often combined.

The main difference between the two conditions lies in the rate of weight gain, which strongly affects the required P/E ratio. Wasted children can achieve very large rates of gain, and even under field conditions may gain at several times the normal rate. The required P/E is very sensitive to the rate of weight gain (Table 21). Therefore in a wasted child the lower ratio resulting from a higher fat content of the tissue gained would be compensated by the higher ratio needed for a more rapid rate of gain. It was also pointed out (Reeds) that in early catch-up there is a preponderance of protein in the tissue gained and in the later stages a preponderance of fat. However, it would be too complicated to make allowance for this in field situations.

For catch-up in stunted children the aim is to achieve and maintain normal body composition; the rate of weight gain is then limited by the relatively smaller increase that can be achieved in the rate of linear growth. For a child of 2 years to recover 3 s.d. units of height in 6 months would be an achievable target. This would require an increase of less than 5% in energy intake but a 37% increase in protein intake (Table 22). To give more energy would only result in obesity. Studies at INCAP have shown that children getting more protein during rehabilitation showed greater growth in length, but it is not clear whether this was related to the protein or to some associated factor (Town).

It is possible that stunting results from lack of a specific amino acid (see Waterlow & Schürch 1994), so that protein quality would be important as well as quantity. There is a need to investigate the relative efficiency of different sources of protein in promoting catch-up. Lipid metabolism may also be altered by an increased intake of animal protein, so the observed effects could be combined ones, not due to protein alone. Millward pointed out that in rat experiments intakes of protein greater than those needed for maximal weight gain can stimulate long bone growth. This means that amounts of protein should be recommended even greater than those needed to exert regulatory influences in terms of the Millward-Rivers model. However, he does not agree that stunting is primarily caused by protein deficiency; more probably it results from a combination of infection and micro-nutrient deficiencies, or all three (Scrimshaw).

A point that should not be overlooked in any discussion of catch-up is the extra needs of low birth weight babies, who may gain 20 g/kg/d when appropriately fed (see Section 2.2.3).

Rehabilitation after infection is a particular example of catch-up. Unfortunately, there are no good measurements of N losses during infection in children. They are likely to be particularly high during gastroenteritis. Studies in adults have shown an average loss of 0.6 g protein kg/d. If the recovery period is two to three times as long as the depletion period (Scrimshaw), it would mean that a 3050% increase in the daily protein intake would be necessary. Calculations based on data from The Gambia and Guatemala on the average time infected and the weight loss per day infected lead to similar results. Field data from Bangladesh showed that on average it took a child about 6 weeks to regain weight after an episode of diarrhoea. In Mexico it appeared that in children aged 7 years only a small proportion of their deficit in growth could be attributed to infection. The conclusion was that catch-up from the effects of infection occurred when the food supply was adequate (Waterlow). The various studies referred to above are summarized in Waterlow (1992).

A further question that needs investigation is how far the nitrogen loss during infection involves losses of specific amino acids. Reed's studies on the composition of the acute-phase proteins suggest that this could well be the case.

Summary of issues to be resolved (as presented by Dewey):

(1) Is the breast-fed infant a suitable model?
(2) What value should be given for the utilization of NPN in human milk ?
(3) Do requirements differ for formula-fed infants?
(4) What should be taken as the requirement for maintenance? 90 mg N/kg/d in infants and 100 in older children ?
(5) What value should be used for the efficiency of utilization of protein; 70%?
(6) How should we take into account intraindividual variability of growth?
(7) How should inter-individual variability in growth be calculated and over what time interval?
(8) Should the operational approach (P/E) be used to estimate safe levels rather than factorial + 2 s.d.
(9) Should amino acid requirements for infants still be based on the composition of human milk? They may be lower.
(10) Are data on the amino acid requirements of healthy children sufficient? Probably not.
(11) How should needs for protein and amino acids after infection be calculated ?
(12) Are estimates needed for the combination of catch-up growth + recovery from infection?

References

Darling P. Wykes LJ, Clarke R. Papageorgiou A & Pendharz PB (1983): Utilization of non-protein nitrogen in whey-dominant formulae by low-birthweight infants. Clin. Sci. 84, 543-548.

de Lange CFM, Souffrant WB & Sauer WC (1990): Real ileal and amino acid digestibilities in feedstuffs for growing pigs as determined with the 15N-isotope dilution technique. J. Anim. Sci. 68, 409-418.

Donovan SM, Lönnerdal B & Atkinson SA (1990): Bioavailability of urea nitrogen for the low birthweight infants. Acta Paediatr. Scand. 79, 899905.

Fomon SJ, Matthews DE, Bier DM, Rogers RR, Edwards BB, Ziegler EE, Nelson SE (1987): Bioavailability of dietary urea nitrogen in infants. J. Pediatr. 111, 221-224.

Gannon NJ, Reeds PJ & Wong WW (1994): Endogenous nitrogen is absorbed to a greater extent than feed nitrogen from the hind gut of neonatal pigs. Proc. 15th Int. Congr. Nutr. Abstract 362, Smith Gordon, London.

Rand WM, Uauy R & Scrimshaw NS (1984): Protein-energy requirement studies in developing countries: results of international research. United Nations University: Tokyo.

Shulman RJ, Gannon M, Reeds PJ: Impact of cereal feeding on the nitrogen economy of infants. Am. 1. Clin. Nutr. (submitted)

Waterlow JC (1992): Long-term effects of infection on growth. In Protein energy malnutrition, pp 313-318. London: Edward Arnold.

Waterlow JC, Schürch B (eds.) (1994): Causes and mechanisms of linear growth retardation. Eur. J. Clin. Nutr. (Suppl. 1).

Whitehead RG, Coward WA, Lunn PG, Rutishauser IHE (1977): A comparison of the pathogenesis of PEM in Uganda and The Gambia. Trans.R. Soc. Trop. Med. Hyg. 71, 189-195.