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

^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/kg^0.75/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:

(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)

^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

^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

^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

^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.

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