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Protein and Amino Acids, 1999

Pp. 169-216. Washington, D.C.

National Academy Press

9
Inherent Difficulties in Defining Amino Acid Requirements

D. Joe Millward1

INTRODUCTION

To address the role of protein and amino acids in performance, this chapter is based on the premise that it is an inherently difficult problem to define the dietary requirements of human adults for indispensable amino acids and to assess the nutritional value (protein quality) of different food protein sources to provide for those needs. There are three major reasons for this difficulty. The first is adaptation, that is, a variable metabolic demand for amino acids set by the habitual intake. Thus, the extent to which any intake appears to be adequate will depend on the completeness of adaptation to that intake. The second is methodology, with no entirely satisfactory practical nitrogen or amino acid balance method or other measure of dietary adequacy. The third is lack of quantifiable, unequivocal metabolic indicators of adequacy that can validate

1  

D. Joe Millward, Centre for Nutrition and Food Safety, School of Biological Sciences, University of Surrey, Guildford GU2 5XH United Kingdom.



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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Protein and Amino Acids, 1999 Pp. 169-216. Washington, D.C. National Academy Press 9 Inherent Difficulties in Defining Amino Acid Requirements D. Joe Millward1 INTRODUCTION To address the role of protein and amino acids in performance, this chapter is based on the premise that it is an inherently difficult problem to define the dietary requirements of human adults for indispensable amino acids and to assess the nutritional value (protein quality) of different food protein sources to provide for those needs. There are three major reasons for this difficulty. The first is adaptation, that is, a variable metabolic demand for amino acids set by the habitual intake. Thus, the extent to which any intake appears to be adequate will depend on the completeness of adaptation to that intake. The second is methodology, with no entirely satisfactory practical nitrogen or amino acid balance method or other measure of dietary adequacy. The third is lack of quantifiable, unequivocal metabolic indicators of adequacy that can validate 1   D. Joe Millward, Centre for Nutrition and Food Safety, School of Biological Sciences, University of Surrey, Guildford GU2 5XH United Kingdom.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance balance measurements. The questions posed in this review cannot currently be answered because of the absence of studies of outcome in terms of physical performance in long-term, controlled feeding trials. BACKGROUND TO THE CURRENT CONTROVERSY In 1985, the Food and Agriculture Organization (FAO) report on protein and energy requirements was published (FAO/WHO/UNU, 1985), a feature of which was the recommendation that protein quality should be evaluated by the PDCAAS method (protein-digestibility corrected amine acid score), making use of age-specific amine acid scoring patterns. Because the indispensable amine acid (IAA) requirement values used to calculate the scoring patterns fell markedly with age, from over 50 percent of total protein requirement in infants to only 16 percent in adults (see Table 9-1), the quality of any protein would now be assessed as higher when used for adults than for children. Furthermore the low requirement level of IAA in adults meant that all natural diets and food proteins would be adequate. Thus, apart from digestibility, protein quality ceased to be an issue in the nutrition of adults. Considerable disquiet arose about the 1985 report. Young (1986) argued that the adult IAA requirement values were seriously flawed because of the way Rose (1957) conducted his nitrogen (N) balance studies (mainly excess energy and no account for miscellaneous N losses). Millward and Rivers (1988) reviewed the subject, paying particular attention to the adaptive changes in amine acid oxidation that can occur and that will influence requirement values. They argued that the marked fall with age in the requirement values was mainly a reflection of the methodologies used in their assessment. Thus, the infant values were largely patterned on the composition of breast milk, while the adult values, measured in balance studies with excess nonessential nitrogen and low levels of indispensable amine acid, would have identified minimum requirement values. They concluded that IAA requirements are complex, include an adaptive component, and can only be defined under specific artificial conditions that would allow definition of a minimum value and that ''current estimates of adult requirements may be close to this level." To identify which IAA might be rate limiting for the obligatory N losses (ONL), they calculated the obligatory oxidative amine acid losses (OOL) as estimates of the losses of tissue IAAs that would give rise to the ONL, as discussed in detail below. Young et al. (1989) then published a paper entitled "A Theoretical Basis for Increasing Current Estimates of the Amine Acid Requirements in Adult Man with Experimental Support." This paper reproduced the table of eel values from Millward and Rivers (1988). After making some small adjustments in lysine, threenine, and valine values derived from their stable isotope studies and increasing all values assuming a 70 percent efficiency of utilization, Young and colleagues proposed that this pattern, the "MIT" (Massachusetts Institute of

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance TABLE 9-1 Protein and Indispensable Amino Acid Requirements (mg/kg/d) and Obligatory Indispensable Amino Acid Oxidative Losses*           Obligatory oxidative losses†   Infants (3-4 too) Children (2 y) Schoolboys (10-12y) Adults Value Requirement multiple Protein             Growth 625 187 1 06 —     Maintenance 750 738 681 600     Total 1,375 925 787 600 338 0.56 Amino acids             Histidine 28 (20) (20) 8-12 11.5 1.05 Isoleucine 70 31 30 10 16.2 1.62 Leucine 161 73 45 14 27.4 1.96 Lysine 103 64 60 12 30.1 2.51 TSA 58 27 27 13 13.5 1.01 TAA 125 69 27 14 27 1.93 Threonine 87 37 35 7 15.5 2.21 Tryptophan 17 12.5 4 3.5 4.0 1.14 Valine 93 38 33 10 16.9 1.69 Total 742 372 281 94 162 1.72 % Protein requirement 54 40 36 16 48   NOTE: TSA, total sulfur amino acids; TAA, total aromatic amino acids. * Protein requirements arc mean values † These are the rates of oxidative loss of IAAs predicted to occur based on the assumption that the ONL, (54 mg N/kg/d) derive from the oxidation of amino acids liberated from body protein (amino acid composition as beef), and the composition of the free amino acid pool does not change. SOURCE: Adapted from FAO/WWHO/UNU, 1985.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Technology) scoring pattern, should be used as the basis for protein quality evaluation in adults and in children given the similarity between their pattern and that of the FAO preschool child pattern. In 1989, FAO/WHO convened a meeting to consider protein quality evaluation and to endorse the PDCAAS method recommended by FAO in 1985. However the report (FAO/WHO, 1991) rejected both the 1985 adult and older school child IAA requirement values as flawed, was unable to identify any other appropriate adult scoring pattern, and proposed that the scoring pattern for the preschool child be utilized for older children and for adults as a strict interim measure. It was argued that (a) the preschool child data were reliable, (b) in the absence of any other data, some pattern was needed for older children and adults, and (c) the slow growth of children compared with adults means that a major change in the requirement pattern with age was unlikely. Although Young and colleagues broadly agreed with this conclusion in that the MIT and preschool patterns were similar, Millward (1994) argued that the report was flawed. In fact, the data that formed the basis of the preschool child pattern had never been published and were not available for scrutiny except for some "typical" data for lysine published in a book review (Pineda et al., 1981). The data, which were derived from study of preschool children who had recovered from protein energy malnutrition (PEM), show that the N balances were so large in the children studied that they would have been exhibiting catchup growth as far as lean tissue was concerned (growth rates and N retentions of 3 times the expected values). This growth would markedly increase the need for indispensable amine acids compared with that of normal preschool children, older children, and especially adults. Millward (1994) also argued against acceptance of the MIT scoring pattern on the grounds that amine acid requirements for maintenance cannot be predicted from the amine acid composition of body proteins. Fuller and Garlick (1994) have reviewed the controversy, and while they did not endorse the MIT pattern, they did conclude that the FAO values were likely to be underestimates, having failed to include miscellaneous N losses in the original balance studies. They reported adjusted higher values, taking into account estimated miscellaneous N losses. They also raised concern that the tracer studies may also suffer from an underestimate of losses and that neither N nor tracer studies are inherently better than the other. These issues are considered in more detail below. Waterlow (1996) made a detailed analysis of the tracer studies and came to conclusions similar to Fuller and Garlick, that is, they did not accept the theoretical basis of the MIT pattern but recognized that the 13C leucine studies do point to a higher leucine requirement than does the FAO value. The issue was considered at an international meeting of an expert group in London in 1994. However, contrary to what was published (Clugston et al., 1996), the MIT pattern was not endorsed at this meeting since, as subsequently reported by Millward and Waterlow (1996), the published statement "a large majority of the group accepted as an interim operational

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance pattern that [that was] proposed by Young et al.," had in fact emerged during postmeeting editing. The current views of Young and colleagues are described in the chapter following this one. What follows is an account of this author's current understanding of the debate. Much of the argument made here has been reported previously in publications by the author and in correspondence relating to publications by Young and colleagues (Millward and Rivers, 1988, 1989; Millward, 1990, 1991, 1992, 1993, 1994; Millward et al., 1989, 1990; Millward and Pacy, 1995). METABOLIC BACKGROUND One cause of potential confusion within this debate is lack of consistency in terminology. To avoid that here, protein and amino acid requirements will be discussed in terms of metabolic demand, dietary requirement, and dietary allowances. Metabolic demand (MD) is determined by the nature and extent of those metabolic pathways that consume amino acids. The dietary requirement is the amount of protein and/or its constituent amino acids that must be supplied in the diet to satisfy the metabolic demand, usually greater than the MD because of less-than-perfect protein utilization. Dietary Reference Values (U.K. terminology) or Recommended Dietary Allowances (U.S. terminology) are a range of intakes derived from estimates of individual requirements that are designed to meet the dietary requirements of the population and that take into account the variability among individuals in that dietary requirement. This chapter focuses on the MD and dietary requirement. Obligatory Metabolic Demand The MD for dietary protein is to provide amino acid precursors for the synthesis of tissue proteins and a range of nonprotein products. Although most proteins are in a dynamic state of constant turnover, little metabolic demand for amino acids is generated by this avenue because of amino acid recycling. "Wear and tear" as a driver of MD is not an appropriate biological analogy. Only net protein synthesis contributes to MD. This growth aspect of MD is straightforward in that the qualitative nature of MD is determined by the amino acid pattern of tissue protein deposited. This pattern is usually assumed to be influenced only by the changes in body composition occurring during growth and is not generally assumed to vary with the diet within cells and tissues. However, as discussed by Fuller and Garlick (1994), some evidence exists for changes in amino acid content of tissues during growth on amino acid-limiting diets. Nonprotein products of amino acid precursors include nucleic acids, and a range of smaller molecules such as creatine, taurine, glutathione, hormones

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance (e.g., catecholamines and thyroxine), neurotransmitters (serotonin, dopamine), and nitric oxide, a key regulator of blood flow and other physiological processes. In human nutrition, growth occurs very slowly after the first few months of life. Net protein synthesis contributes a small and decreasing component of MD during pre- and immediate post-adolescence. In the adult, it comprises only that associated with continuing growth of skin and hair and the synthesis of those gastric secretions (e.g., threonine-rich mucus glycoproteins) that pass into the colon to be utilized for bacterial metabolism. Thus, apart from these small components, humans are normally at nitrogen or amine acid equilibrium, with MD reflecting mainly nonprotein pathways of amine acid metabolism and catabolism associated with maintenance of normal function and composition. In the traditional nutritional terminology of human growth and maintenance, growth needs are low at all ages after early infancy with maintenance dominating the MD. The task, then, is to define the amounts and amine acid pattern of the maintenance requirement. Obligatory Metabolic Demands and Obligatory Oxidative Losses The diverse obligatory maintenance MDs for amine acids represent an important, but small, intrinsic part of MD, the magnitude of which is the main subject of current debate. Table 9-1 shows the requirement values for IAAs and for protein as reported by the FAO (FAO/WHO/UNU, 1985). The feature of these values that has been at the heart of the controversy is that the IAA requirement as a proportion of the protein requirement falls markedly with age, from 54 percent in infants to 16 percent in adults. Millward and Rivers (1988) argued that some information could be obtained from the magnitude of the ONL. In subjects fed a protein-free but otherwise nutritionally adequate diet, N losses fall to a stable and reproducible low level after 7 to 14 days. Subjects lose body protein at a constant daily rate, about 54 mg/kg/day, which is equivalent to 0.34 g of protein/kg/day (FAO/WHO/UNU, 1985). These ONLs are assumed to represent nitrogen end products of amine acids derived from body protein and utilized for the obligatory metabolic demand (OMD) that is tacitly assumed to be the same in subjects consuming a protein-free diet as in subjects consuming a normal diet. The ONL is a function of body weight and, when normalized to "metabolic body size" (kg0.75), varies little with age (FAO/WHO/UNU, 1985). Millward and Rivers (1988) reported a simple calculation of the eels. These are the oxidation rates of amine acids that give rise to the ONL and they are equal to the amounts of amine acids in the protein equivalent of the ONL. It was assumed that tissue protein composition could be approximated by that of muscle and the values for beef muscle listed by FAO (FAO/WHO/UNU, 1985) were used. This was only a first approximation calculation, since some of the ONL occurs as protein per se (skin, hair, some fecal nitrogen, and secretions), and protein is lost from several tissues in addition to skeletal muscle. They made

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance the assumption that of the individual amino acids that comprise the obligatory MD (which, on a protein-free diet, is in effect fueled by tissue protein), one amino acid would be rate limiting, with the highest ratio of obligatory MD to OOL. All other amino acids with a lower ratio would be present in excess in the OOL but would be nevertheless oxidized because they could not be returned on their own to the tissue protein pool. They argued that if protein turnover is tightly regulated, allowing just enough of the rate-limiting amino acid to be withdrawn from tissue protein to provide for its MD, the OOL of this amino acid should be a reasonable guide to its requirement. For all others, the values for the OOL should be greater than the maintenance requirements. Use of the Obligative Oxidative Loss Pattern to Predict a Requirement Pattern The actual listed values of the OOL were adopted by Young et al. (1989) as the basis of the MIT scoring pattern, something that was contrary to what Millward and Rivers (1988) intended. In effect, this defined a maintenance pattern with the same composition as tissue protein, a novel assumption given the widespread assumption of different amino acid patterns for maintenance and growth. Millward and Rivers (1988) assumed that the pattern of the obligatory MD is different from that of tissue protein, so that there would be a rate-limiting amino acid that "drives" the ONL. The identification of this driver can be done by reference to an actual requirement pattern. Thus, they compared the values of OOL with the 1985 FAO/WHO/UNU requirement values such that if the FAO values were accurate, the values would be similar for one amino acid. In fact, while the OOL for most amino acids was greater than the FAO requirement, with lysine, threonine, leucine, and the aromatic amino acids being particularly in excess (2-2.5 times the requirement), the OOL of the total sulfur amino acids (TSAs) was quite close to the FAO requirement values; this latter observation showed that the TSAs are rate determining for the mobilization of tissue protein to provide for obligatory MD. The possibility that the TSAs are the rate-limiting amino acids that drive the ONL in humans was attractive on the basis of animal studies. Providing the rate-limiting amino acid to the protein-free diet fed during measurement of ONL should result in a fall in N excretion to the rate determined by the demand for the second limiting amino acid. Studies in dogs (Allison et al., 1947) and rats (Yoshida, 1983) have shown that supplementation by S amino acids reduces N excretion. Animal Data for the Pattern of the Obligatory Metabolic Demand Animal data clearly indicate that the amino acid pattern of the obligatory maintenance MD differs from that of tissue protein, which necessarily repre-

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance sents the pattern for the growth requirement. However, this view is not accepted by Young (e.g., Young and El-Khoury, 1995), who, having used the OOL pattern to derive the MIT pattern, assumed maintenance requirements to be broadly similar to tissue protein. Before reviewing the animal data, their relevance to human nutrition needs to be addressed. Young and El-Khoury (1995) have discussed the relevance of the high-quality nitrogen balance data obtained in the young pig (Fuller et al., 1989). They argued that any comparison of human requirement with that of the young pig is invalid because (a) relative amounts of maintenance and growth vary too much between the species (maintenance = < 10 percent total [growth + maintenance] requirement in the young pig and < 5 percent total in the rat), and (b) the efficiency of dietary protein utilization in the young pig at maintenance is much higher (100 percent) compared with human values (assumed to be 70 percent). In fact, the first point is irrelevant when human-animal comparisons are limited to discussions of either maintenance or growth needs specifically, and the second point is irrelevant when what is considered is MD rather than dietary requirement. Young also argues that data from adult pigs (boars) support the proposition that the pattern of tissue protein is similar to the maintenance pattern. Yet he acknowledges the data to be poor and at variance with most other animal data. Finally, Young and El-Khoury (1995) argue that maintenance patterns derived from rapidly growing animals held at maintenance by food restriction are unphysiological and, consequently, may be an unreliable guide to the human obligatory IAA MD. To date there has yet to be a claim that human IAA bid can be accurately predicted from animal values, only that consideration of the animal data as a whole, including both growing and adult data, may provide useful general information. There appear to be few major differences between mammalian species with regard to the fundamentals of amine acid and protein metabolism. With obvious exceptions (e.g., arginine requirements for growing cats and growing and adult dogs, a taurine requirement for the kittens, and a high-maintenance amine acid requirement in avian species for feather growth), interspecies comparisons appear to be legitimate since robust animal data should provide general principles about the nature of human needs. Two kinds of studies are pertinent. The first is deletion studies, in which individual amine acids are removed from the diet and the extent of the negative balance is monitored. If the maintenance requirement patterns corresponded exactly to the patterns of tissue protein, then there should be a similar negative balance on removal of each IAA. If not, then negative balance will occur in proportion to the ratio of obligatory MD to tissue content of each amine acid. Only one report exists for the adult rat (Said and Hegsted, 1970), a high-quality study based on measured changes in body water. Gahl et al. (1991) reported N balance data for young rats, while Fuller et al. (1989) studied 41 kg pigs, with N balance; data from the latter study are widely recognized as the most robust data for the pig.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Table 9-2 shows the relative losses normalized for the response to a protein-free diet. The first, second, and third limiting amino acids are threonine, TSA, and isoleucine for the growing rat; threonine, isoleucine, and tryptophan for the adult rat; and TSA, threonine, and tryptophan for the pig. The most highly conserved, least-limiting amino acids are lysine and leucine in the rat and all three branched-chain amino acids (BCAAs) and lysine in the pig. Supplementation with each limiting amino acid allows the slope of the balance curve to be established and the consequent requirement values to be determined; these are shown in Table 9-3 compared with carcass protein content. For ease of comparison, these patterns have been normalized for threonine. Leucine and lysine are the two most abundant amino acids in carcass proteins and in the growth requirement patterns for both rat and pig; in the maintenance requirement patterns, the most abundant amino acids are threonine and TSA in the pig; threonine, isoleucine, valine, and TSA in both adult and growing rats. TABLE 9-2 Responses (Negative Balance) to Deletion of Individual Indispensable Amino Acids or a Protein-Free Diet Amino acid Growing pig* Growing rat† Adult rat‡‡ Histidine nd 0.31 0.55 Isoleucine 0.24 0.94 0.90 Leucine 0.21 0.38 0.36 Lysine 0.33 0.25 0.30 TSA 0.93 1.09 0.51 TAA 0.34 0.06 0.47 Threonine 0.68 1.19 0.92 Tryptophan 0.53 0.75 0.80 Valine 0.23 0.88 0.71 All (protein free) 1.00 1.00 1.00 NOTE: TAA, total aromatic amino acids; TSA, total sulfur amino acid; nd, not determined. * Fuller et hi. (1989). † Gahl et al. (1991). ‡ Said and Hegsted (1970).

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance TABLE 9-3 Amino Acid Composition and Requirement Patterns   Growing pig*   Growing rat†   Adult rats‡   Body G M Body G M G M Histidine 0.74 — 0.65 0.34 0.14 0.41 0.48 — Isoleucine 0.92 0.92 0.30 0.81 1.07 0.56 1.08 1.03 Leucine 1.88 1.66 0.43 1.67 1.61 0.38 1.35 0.94 Lysine 1.87 1.45 0.68 1.58 1.23 0.33 1.76 0.74 TSA 0.74 0.76 0.92 0.87 1 .40 0.63 0.98 0.97 TAA 1.89 1.80 0.70 1.67 1.25 0.20 1.41 1.15 Threonine 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Tryptophan nd 0.26 0.21 0.26 0.16 0.06 0.22 0.22 Valine 1.25 1.12 0.38 1.03 1.27 0.66 1.10 1.03 NOTE: G, growth; M, maintenance; TSA, total sulfur amino acids; TAA, total aromatic amino acids; nd, not determined. * Fuller et al. (1969). † Bencvenga et al. (1994). ‡ Said and Hegsted (1970). The major implication of these animal data is that there are marked differences between the MD for maintenance and for growth. It is clear that in the growing rat and pig and the adult rat, leucine and lysine exhibit the biggest difference between growth and maintenance patterns, these two amine acids being most abundant for growth and among the least abundant for maintenance. The practical consequence of this, as pointed out by Hegsted (1973), is that the balance-intake curve is extremely shallow for leucine and lysine both in the sub-maintenance and growth range. This means that small differences in balance result in large differences in maintenance intakes so that measurement of a requirement value for maintenance is very difficult and depends on the exact criterion for adequacy. The several early reports of rats maintaining body weight for 6-month periods on very low lysine diets (e.g., zein [Osborne and Mendel, 1916] or even lysine-free diets [Bender, 1961]) are probably explained by coprophagy, given the clear evidence of a metabolic need for lysine in terms of the rapid onset of symptoms on a lysine-free diet in humans (Rose, 1957). However, no evidence exists for anything other than a low metabolic need for this amine acid. A second type of study which is pertinant is the work of Yoshida (1983) who has done most to explore the concept that rate-limiting amine acids at maintenance differ from those that rate-limit growth. Having established that in

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance adult rats fed a protein-free diet, the most rate-limiting amino acids were threonine and TSA, he also showed that in adult rats fed limiting amounts of rice or wheat diets, the limiting amino acids were threonine and the sulfur amino acids. When these two amino acids were added to the cereal diets, they restored nitrogen balance and transformed body weight loss to growth (See Figure 9-1). This may explain why attempts to show in human adult supplementation trials that lysine is the limiting amino acid in wheat were so disappointing (Scrimshaw et al., 1973). Although the nature of the relative metabolic need for individual amino acids is by no means clear, Fuller's work with the pig points to ileal amino acid losses as a partial explanation, accounting for some 60 percent of pig amino acid maintenance requirements (Wang and Fuller, 1989). Table 9-4 compares ileal losses of the pig and humans. These data show that in each case, threonine is the largest component, and while the patterns differ somewhat, the absolute values are much lower in humans than in the pig. Thus, despite discussion of the pig as an inappropriate model for humans, to the extent that ileal losses comprise a component of obligatory ME), these data point to a lower MD in humans than in the pig. To summarize research on obligatory maintenance MD, a consistent and extensive body of animal data shows the maintenance pattern to differ from the growth pattern, with lower levels of lysine and leucine in the maintenance pattern. As a result, the rate-limiting amino acids in dietary proteins for maintenance may differ from those for growth, the example being that lysine limits wheat for growth but not for maintenance. FIGURE 9-1 Adult rats were fed limiting amounts of a wheat diet that did not allow balance and were supplemented with a mixture of indispensable amino acids that did allow balance. By removing individual amino acids, the limiting amino acids were identified as threonine and the sulfur amino acids. Lysine was not needed to improve balance. Note: threo, threonine; met, methionine; lieu, isoleucine; val, valine; N, nitrogen; IAA, indispensable amino acids. Source: Adapted from Yoshida (1983).

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Maclean, W.C., G.L. De Romana, R.P., Placko, and G.G. Graham. 1981. Protein quality and digestibility of sorghum in pre school children: Balance studies and plasma free amine acids. J. Nutr. 111:1928-1936. Marchini, J.S., J. Cortiella, T. Hiramatsu, T.E. Chapman, and V.R. Young. 1993. Requirements for indispensable amine acids in adult humans: Longer-term amine acid kinetic study with support for the adequacy of the Massachusetts Institute of Technology amine acid requirement pattern. Am. J. Clin. Nutr. 58:670-683. Meguid, M.M., D.E. Matthews, D.M. Bier, C.N. Meredith, J.S. Soeldner, and V.R. Young 1986a. Leucine kinetics at graded leucine intakes in young men. Am. J. Clin. Nutr. 43:770-780. Meguid, M.M., D.E. Matthews, D.M. Bier, C.N. Meredith, and V.R. Young. 1986b. Valine kinetics at graded valine intakes in young men. Am. J. Clin. Nutr. 43:781-786. Meredith, C., D.M. Bier, M.M. Meguid, D.E. Matthews, Z. Wen, and V.R. Young. 1982. Whole body amine acid turnover with 13C tracers: A new approach for estimation of human amine acid requirements. Pp. 42-59 in Clinical Nutrition '81, R.I.C. Wesdorp and P.B. Soeters, eds. Edinburgh and London: Churchill Livingstone. Meredith, C.N., Z-M. Wen, D.M. Bier, D.E. Matthews, and V.R. Young. 1986. Lysine kinetics at graded lysine levels in young men. Am. J. Clin. Nutr. 43:787-794. Millward, D.J. 1990. Amine acid requirements in adult man. Am. J. Clin. Nutr. 51:492-493. Millward, D.J. 1992. The metabolic basis of the amine acid requirement. Pp. 31-57 in Protein-Energy Interactions I/D/E/C/G, N.W. Scrimshaw and B. Schurch, eds. Lausanne, Switzerland: Nestle Foundation. Millward, D.J. 1993. Stable-isotope-tracer studies of amine acid balance and human indispensable amine acid requirements Am. J. Clin. Nutr. 57(1):81-86. Millward, D.J. 1994. Can we define indispensable amine acid requirements and assess protein quality in adults? J. Nutr. 124:1509-1516. Millward, D.J., and J.P.W. Rivers. 1988. The nutritional role of indispensible amine acids and the metabolic basis for their requirements. Eur. J. Clin. Nutr. 42:367-393. Millward, D.J., and J.P. Rivers. 1989. The need for indispensable amine acids: The concept of the anabolic drive. Diab. Metab. Rev. 5(2):191-211. Millward, D.J., and P.J. Pacy. 1995. Postprandial protein utilization and protein quality assessment in man. Clin. Sci. 88:597-606. Millward, D.J., and J.C. Waterlow. 1996. Letter to the editor. Eur. J. Clin. Nutr. 50:832-833. Millward, D.J., and S.R. Roberts. 1996. Protein requirement of older individuals . Nutr. Res. Rev. 9:67-88. Millward, D.J., A.A. Jackson, G. Price, and J.P.W. Rivers. 1989. Human amine acid and protein requirements: Current dilemmas and uncertainties Nutr. Res. Revs. 2:109-132. Millward, D.J., G.M. Price, P.J.H. Pacy, and D. Halliday. 1990. Maintenance protein requirements: the need for conceptual revaluation. Proc. Nutr. Sec. 49:473-487. Millward, D.J., G. Price, P.J.H. Pacy, and D. Halliday. 1991. Whole body protein and amino acid turnover in man: What can we measure with confidence? Proc. Nutr. See. 50:197-216. Millward, D.J., J.L. Bowtell, P. Pacy, and M.J. Rennie. 1994. Physical activity, protein metabolism and protein requirements. Proc. Nutr. Sec. 53(1):223-240. Millward, D.J., A. Fereday, N. Gibson, and P.J. Pacy. 1996. Postprandial protein metabolism. Ballieres Clin. Endocrinol. Metabol. 10(4):533-549. Munro, H.N. 1964a. General aspects of the regulation of protein metabolism by diet and hormones. Pp. 381-481 in Mammalian Protein Metabolism, Vol. I., Munro H.N. and J.B. Allison, eds. New York: Academic Press. Osborne, T.B., and L.B. Mendel. 1916. The amino-acid minimum for maintenance and growth, as exemplified by further experiments with lysine and tryptophan. J. Biol. Chem. 25:1-8.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Pacy, P.J., G.M. Price, D. Halliday, M.R. Quevedo, and D.J. Millward. 1994. Nitrogen homeostasis in man: The diurnal responses of protein synthesis and degradation and amino acid oxidation to diets with increasing protein intakes. Clin. Sci. 86(1):103-116. Pelletier, V., L. Marks, D.A. Wagner, P.A. Hoerr, and V.R. Young. 1991a. Branched-chain amino acid interactions with reference to amino acid requirements in adult men: Valine metabolism at different leucine intakes. Am. J. Clin. Nutr. 54:395-401. Pelletier, V., L. Marks, D.A. Wagner, P.A. Hoerr, and V.P. Young. 1991b. Branched-chain amino acid interactions with reference to amino acid requirements in adult men: Leucine metabolism at different valine and isoleucine intakes. Am. J. Clin. Nutr. 54:402-407. Pineda, O., B. Torun, F.E. Viteri, and G. Arroyave. 1981. Protein quality in relation to estimates of essential amino acids requirements. Pp. 131-139 in Protein Quality in Humans: Assessment and In Vitro Estimation, C.E. Bodwell, ed. Westport, Conn.: AVI Publishing Company Inc. Price, G.M., D. Halliday, P.J. Pacy, M.R. Quevedo, and D.J. Millward. 1994. Nitrogen homeostasis in man: 1. Influence of protein intake on the amplitude of diurnal cycling of body nitrogen. Clin. Sci. 86:91-102. Quevedo, M.R., G.M. Price, D. Halliday, P.J. Pacy, and D.J. Millward. 1994. Nitrogen homeostasis in man: 3. Diurnal changes in nitrogen excretion, leucine oxidation and whole body leucine kinetics during a reduction from a high to a moderate protein intake. Clin. Sci. 86:155-193. Reddy, V. 1971. Lysine supplementation of wheat and nitrogen retention in children. Am. J. Clin. Nutr. 24:1246-1249. Reeds, P.J, and P.R. Becket. 1996. Protein and amino acids. Pp. 67-86 in Present Knowledge in Nutrition, 7th ed., E.E. Ziegler and L.J. Filer, eds. Washington, D.C.: ILSI Press. Rose, W.C. 1957. The amino acid requirements of adult man. Nutr. Abstr. Rev. 27(3):631-647. Rowland, I.R, T. Granli, O.C. Bockman, P.E. Key, and P.C. Massey. 1991. Endogenous N nitrosation in man assessed by measurement of apparent total N nitroso compounds in faeces. Carcinogenesis 12:1359-1401. Said, A.K., and D.M. Hegsted. 1970. Response of adult rats to low dietary levels of essential amino acids. J. Nutr. 100:1363-1376. Scrimshaw, N.S, Y. Taylor, and V.R. Young. 1973. Lysine supplementation of wheat gluten at adequate and restricted energy intakes in young men. Am. J. Clin. Nutr. 26:965-972. Soares, M.J., L.S. Piers, P.S. Shetty, S. Robinson, A.A. Jackson, and J.C. Waterlow. 1991. Basal metabolic rate, body composition and whole body protein turnover in Indian men with differing nutritional status . Clin. Sci. Colch. 81(3):419-425. Stephen, A.M., and J.H. Cummings. 1979. The influence of dietary fibre on fecal nitrogen excretion in man. Proc. Nutr. Soc. 38:141A. Thompson, G.N., P.J.H. Pacy, H. Merritt, G.C. Ford, M.A. Read, K.N. Cheng, and D. Halliday. 1989. Rapid measurement of whole body and forearm protein turnover using a [2H5]phenylalanine model. Am. t. Physiol. 256:E631-E639. Tolbert, B, and J.H. Watts. 1963. Phenylalanine requirements of women consuming a minimal tyrosine diet and the sparing effect of tyrosine on the phenylalanine requirement. J. Nut. 80, 111-117. Wallace, W.M. 1959. Nitrogen content of the body and its relation to retention and loss of nitrogen. Fed. Proc. 18:1125-1130. Wang, T.C, and M.F. Fuller. 1989. The optimum dietary amino acid pattern for growing pigs. 1. Experiments by amino acid deletion. Br. J. Nutr. 62(1):77-89. Waterlow, J.C. 1996. The requirements of adult man for indispensable amino acids . Eur. J. Clin. Nutr. 50 (suppl. 1):S151-176.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Waterlow, J.C., P.J. Garlick, and D.J. Millward. 1978. Protein Turnover in Mammalian Tissues and the Whole Body. Amsterdam: North Holland Elsevier. Weller, L.A., D.H. Calloway, S. Margen. 1971. Nitrogen balance of men fed amine acid mixtures based on Rose's requirements, egg white protein, and serum free amine acid patterns. J. Nutr. 101(11):1499-1507. Yoshida, A. 1983. Specificity of amine acids for the nutritional evaluation of proteins. Pp. 163-182 in Proceedings of the International Association of Cereal Chemists Symposium on Amine Acid Composition and Biological Value of Cereal Proteins, R. Lasztity and M. Hidvegi, eds. Budapest: Akademiai Kiado. Young, V.R. 1986. Nutritional balance studies: Indicators of human requirements or adaptive mechanisms. J. Nutr. 116:700-703. Young, V.R., and A. E. El-Khoury. 1995a. Can amine acid requirements for nutritional maintenance in adult humans be approximated from the amine acid composition of body mixed proteins? Proc. Natl. Acad. Sci. USA 921:300-304. Young, V.R., and J.S. Marchini. 1990. Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amine acids with reference to nutritional adaptation in humans Am. J. Clin. Nutr. 51:270-289. Young, V.R., Y.S.M. Taylor, W.R. Rand, and N.S. Scrimshaw. 1973. Protein requirements of man: efficiency of egg protein utilization at maintenance and sub-maintenance levels in young men. J. Nutr. 103:1164-1174. Young, V.R., L. Fajardo, E. Murray, W.M. Rand, and N.S. Scrimshaw. 1975. Protein requirements of man: Comparative nitrogen balance response within the submaintenance-to-maintenance range of intakes of wheat and beef proteins. J. Nutr. 105:534-544. Young, V.R., M. Puig, E. Queiroz, N.S. Scrimshaw, and W.M. Rand. 1984. Evaluation of the protein quality of an isolated soy protein in young men: Relative nitrogen requirements and effect of methionine supplementation. Am. J. Clin. Nutr. 39:16-24. Young, V.R., D.M. Bier, and P.L. Pellet. 1989. A theoretical basis for increasing current estimates of the amine acid requirements in adult man with experimental support Am. J. Clin. Nutr. 50:80-92. Zello, G.A., P.B. Pencharz, and R.O. Ball. 1990. Phenylalanine flux, oxidation and conversion to tyrosine in humans studied with 13C phenylalanine. Am. L Physiol. 259:E835-E843. Zello, G.A., P.B. Pencharz, and R.O. Ball. 1992. Lysine requirement in young adult males. Am. J. Physiol. 264:E677-E685. Zhao, X-H., Z-M. Wen, C.N. Meredith, D.E. Matthews, D.M. Bier, and V.R. Young. 1986. Threonine kinetics at graded threonine intakes in young men. Am. J. Clin. Nutr. 43:795-802. DISCUSSION ROBERT NESHEIM: Thank you. Are there questions? Yes, sir, John. JOHN VANDERVEEN: I am interested in the reality of the current situation. At the present time, our troops are getting plenty of protein in amounts far beyond this whole discussion. But the issue of adaptation raises the issue of survival. If, indeed, higher levels of protein intake change our metabolism to the point where additional adaptation, if you will, occurs in our needs, are we

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance putting soldiers at risk to survive starvation or low protein intake for a period of time on rations? D. JOE MILLWARD: I think it is a very complicated question. I think that it is quite possible that we are putting soldiers at risk. Certainly, the view that Chittenden expressed was that high protein intakes represent a metabolic load that is hard to deal with. Now, Mike Rennie will present data on physical activity and intakes and deal with this problem to a certain extent. But, in my view, the real point about the human response to protein intake is that we adapt our metabolic demand according to how much we eat. The point about any individuals who stuff themselves with very high-protein diets are two things. Firstly, as soon as they stop taking in that high protein diet, they lose lean tissue very, very rapidly. So the soldier who suddenly finds himself without food for a few days on a background of a high protein diet will lose more lean body mass than somebody who has been on a lower intake. Secondly, the response to exercise, I think, can be shown to induce a bigger loss when one's background intake is higher. Now, there are things about infection and all of that which complicate the issue and that I do not think we have an answer to. ROBERT NESHEIM: Yes? G. RICHARD JANSEN: Joe, I guess, having been recruited to work on the ill-fated lysine program at Dupont 40 years ago, this is like dèja vu all over again. I remember a 1960 conference in Chicago at the Federation Meetings discussing protein reserves. If you forget the turnover, it was the flux data. It is the same argument basically. But it seems to me that your position as you have published it is more agnostic than atheistic to Vernon's dogma here. And it seems to me you have not really addressed his argument that there is an advantage in terms of metabolic control to that increased oxidation. And, in fact, in your slide there you said, number one, that we basically cannot determine the indispensable requirements because we do not have enough data. However, you say protein scoring is invalid. It seems to me that those two statements cannot be made together. If we cannot define it, we cannot determine what is valid or invalid yet. D. JOE MILLWARD: Okay. The point about protein scoring is that it is a procedure that was adopted for quality assessment in the rat because it was shown to work. In other words, scoring correlated with net protein utilization in the growing rat. The reason that it correlates is simply because for the growing

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance rat the requirement is quite simple and straight-forward. It has to do with the requirement for tissue deposition. What I am saying is that for adult humans, our requirement is more complicated because it is variable. We have this fixed requirement that may well be very low. And, in fact, FAO took the view that you use the minimum requirement value and you score protein with that. If you do that, then all proteins in the world are nutritionally adequate. Now, they are only adequate if you can actually adapt to them successfully. So for a population with a habitual protein intake that is relatively high, the question is what is adequate for that population. That is what is very difficult to define, because ultimately one has to test it against that group and against the extent to which they are adapted to their intakes. If you add the problem that there may well be other components of intake that are more important than balance, like performance, like the immune response, like any of these things, and we talked about the anabolic drive on growth and development, then it becomes even more difficult to define nutritional adequacy against this single pattern of individual amine acids. That is the point that I am trying to make. G. RICHARD JANSEN: I assumed that. But I think, at the same time, I do not think that you addressed at all Young's argument that there is an advantage in terms of metabolic flux and control to that increased protein turnover, which then would make the argument that he has made, that the pattern should be what it is and, therefore, it would be valid. I understand the rat gross data. I have run a lot of those experiments; but you are not addressing the argument he is making. D. JOE MILLWARD: Well, I did address it partly. That argument is made basically in the Young and Marchini paper, which he quotes in his abstract, where they show that whole body protein synthesis rates plummet dramatically as the intake of essential amine acids plummet. I think that data are wrong, to be quite honest. I think they are artifactual. There are other data where the same diet is fed, for example, with decreasing lysine intake, but the protein turnover is measured with another amine acid, namely phenylalanine, and there is no change in the phenylalanine flux over the whole range of intake. So I think there is a technical problem with the observation that higher intakes are associated with higher rates of protein turnover. Because I do not think there are higher rates of protein turnover with higher intakes. I do not think they actually change.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance SUSAN HUDSON: I have a question. We have argued about this for years. But is there any way to make the steady isotope labeling data more precise, that is, those you say are flawed? Is there any kind of information that could be gathered that would, say for leucine, make the data better? D. JOE MILLWARD: I think that if Vernon were here, what he would say is that over the years, the studies have gotten better. There has been a better understanding of what was going on. The way the calculations have been done has been improved. The studies that Vernon is doing at the moment in India where he looking at lysine intakes and he is using leucine balances are probably as good as you can actually get. So we do await the outcome of those studies. The difficulty is that many of the data that have been used in the past to support the MIT scoring pattern, in my view, simply are unusable because they are seriously flawed. So we are still waiting for hard data that support a better requirement pattern. Now, Denny [Bier] was a co-author of those papers, so I am sure he will want to respond. ROBERT NESHEIM: Defray? DENNIS BIER: Well, first, I think that Joe has probably presented the clearest and the fairest view of this subject that I have heard in the last 10 years. I would like to see it come out as clearly as it was presented here today. I would agree with you on this whole business of requirements, that is, the adaptive component and how it affects those requirements. I think it is precisely in that slide you showed us of the obligatory losses. I think that, unfortunately, that is really what we would like to know. That is what the military would like to know in the circumstances that they are addressing. How do we get at that? I think we are trying to address a problem about intake and requirements in the face of a body that is filled with amino acids, as you pointed out in your remark about the utilization and recycling. It becomes very difficult, I think, to solve the problems in that context when really what we are looking for is some change from the baseline. In addition, the balance studies really do not tell us in any way, of course, what happened to what was in the body at that time and whether or not there has been an advantage or disadvantage to what it took to get the balance in the context of these sort of intakes. So adding functional measurements, adding things like whole body nitrogens or whole body potassium, something that really lets us know over the long term what happened to the body proteins, would be very useful. Now, to go back to the models themselves, my position on this, and I have stated it many times, is that the models are not very good. But the original data that went into some of these calculations (I am not going to talk about scoring

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance patterns because I have no idea what they even mean), but, the original data that went into the calculations were based not on any complicated model but simply on the amount of C13 recovered outside of the body during the course of the tracer study. Admittedly, that has its own set of problems. But, if anything, most of those problems entail underestimation of loss rather than overestimation. If you just take those numbers, they were those net losses that generated the initial part of this. So, admittedly, they were done at MIT with young, healthy adult men, adapted longterm, and using a different protein technique for a maximum of three weeks. I have no idea, frankly, whether that applies to Northern Thais who eat rice or adapt to a different type of protein for the rest of their lives. K. SREEKUMARAN NAIR: I want to echo what was said. This is a complex problem. My concern is we are going to try to solve this problem by a very simplistic approach. What we need is a long-term study on the effects of different proteins on performance changes. They are not easy to perform. D. JOE MILLWARD: I think we have tried many times to erect very complicated models to understand amine acid and protein metabolism. I have always taken the view that, if you cannot actually measure the total amount of body protein that is there and whether it is going up or whether it is going down, any other information is basically irrelevant. You have got to start with good data about what is the state of the overall system, i.e. the lean body mass. And, while that is a simplistic approach, it is a necessary precondition to answering any of the other questions. DENNIS BIER: But the balance method does not allow that—the only way to do that is with the total body nitrogen. D. JOE MILLWARD: Exactly. I think that we need much more data on whole body nitrogen. NANCY BUTTE: Joe, the committee has been asked to address a very straightforward question. Is the military RDA for protein, which is one hundred grams per men and 80 for women, appropriate? Given the current state of knowledge with the techniques we have, would you recommend that they even attempt to revise that right now?

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance D. JOE MILLWARD: Well, there is certainly, in my mind, no evidence to suggest that it is inadequate. The only issue is whether that level is too much. NANCY BUTTE: And what would you advise them if you were asked to advise them at this time? Do you think the recommendations are too high? D. JOE MILLWARD: Well, I think it is. To be quite honest, I do not think we have yet done the right studies. I mean, if you work on the basis of this very significant series of studies that were done over three or four years on Army recruits and young athletes, then you would have to conclude that the current requirements are too high and that soldiers may well perform better on a lower intake. Mike [Rennie] is going to present data this afternoon that suggest that for physical activity, there may well be benefits of lower protein intakes. ROBERT WOLFE: I would just like to follow up on Dr. Jansen's point. I do not know that it can be so readily dismissed that a higher protein intake affects protein turnover, because even your own anecdotal comment, that if you eat a higher protein intake and then stop the protein intake, you will lose protein faster. The only way that could occur would be if you have a higher protein turnover; the higher protein intake caused the higher protein turnover. The only way you would lose it more rapidly would be if it were turning over more rapidly before you stopped. But, furthermore, there are some studies that have done what you have suggested and avoided the problem of using the same amino acid tracer. Because we performed a study a number of years ago, albeit not in normal subjects, but in severely burned patients in a crossover study in which they were given different levels of protein, and three different essential amino acids were used, and all three tracers showed parallel increases in whole body protein turnover when the protein intake was increased, although none indicated an improvement in balance. So I think the fact that the same amino acid tracer as the test compound has been used with different levels of protein intake may not be valid, and that it is a jump to go from that observation to the conclusion that there is no effect of protein intake on protein turnover. D. JOE MILLWARD: Let me say that it was not an anecdotal statement. We have published a series of studies where we used a multiple tracer approach. We used N15, we used C13 leucine, we used deuterated phenylalanine to address the question of whether the level of protein intake affects the overall rate of protein turnover. The outcome of that experiment was that from an intake of .3 grams per kilogram, up to an intake of 2.5 grams per kilogram over three weeks, there was no effect on the overall replacement rate. However, those subjects who

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance were in positive balance obviously had a higher rate of synthesis than proteolysis and those on the lower intake had a lower rate of synthesis than proteolysis. But, if you calculated rate of replacement as the lower of the two processes, that rate did not change over the entire range. DENNIS BIER: Two comments, first on the last statement. If you change the protein intake, one way or another you at least have to change components of the protein turnover. That is, you have to deal with the protein that comes in. So the total protein turnover may not change, but how it is distributed has to be changed, because you have a new term in the equation that is introducing protein into the body. So the fact is that something has to happen, not to the total number [turnover] necessarily, but to how it is distributed. Our models do not really allow us to analyze that. The second comment goes back to Chittenden's study. How did he control for the training effect on this? D. JOE MILLWARD: Well, that was the basis of the problem with his initial study, because he did that study on army recruits. There was this enormous increase in strength. He concluded immediately that it was the weekly measurement over five months that trained them and that was responsible for the increase in strength. So he only controlled for it by recruiting elite athletes. He had the U.S. National Decathlon equivalent champion among his subjects. They were as highly trained as they could get. That was how he got the results that he did. But it is not a perfect study. But it is the only study that has ever seriously addressed the problem. Just because it was published ninety years ago, it is completely ignored. DENNIS BIER: Well, you know, it is not on Medline! JEFF ZACHWIEJA: There are some recent data that are germane to that issue. Others may help me out with this. I think that Bill Evans' group (when he was still at Tufts), recently published data that suggested that exercise training in older individuals, whether they had a dietary protein intake of .8 grams per kg or 1.6 grams per kg, essentially achieved the same level of muscle strength and I think those individuals who were on the lower protein intake were still in positive nitrogen balance, but not as great as those on the higher protein intake. But, in terms of performance, the increments in performance were very similar on those two types of diets. These results spoke to the issue of the body being more efficient or becoming more efficient on the lower protein intake to achieve essentially the same functional outcome.

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance ROBERT NESHEIM: I think we could have more discussion on this, and we will have more time later. HARRIS LIEBERMAN: I just have one more comment in regard to that issue of one hundred g protein per day. There is a lot of confusion about that. The really critical issue for us is not the MRDA level of protein that is recommended but the standard for operational rations. Right now, the standard operational ration must include at least a hundred grams of protein. That typically is not fully consumed. If you lower that ration content, a hundred grams or 90 grams will not be consumed; substantially less than that will be consumed. So when we think about it and talk about it, please keep in mind that what really is critical for us is what we recommend to the ration developers at Natick, the minimum level of protein they can include in a combat ration. JOHN VANDERVEEN: I have one last comment. The concern about ration design depends on how you calculate that 100 grams of protein as well. What protein quality factor do you use? Do you have a cut-off in what you consider protein? If the protein has an amino acid score that is such that you have a cutoff at 20 percent or 30 percent, or whatever, is that counted in? I think that those are issues. I guess maybe the last question I would raise is whether there is any thinking as to how long the amino acids are available in the pool so that you can take advantage of a lower-quality protein at some later time in the day or at a different time? D. JOE MILLWARD: Well, there are good data on that. There are good data on muscle biopsy studies, which I referred to in my talk, of the rate at which amino acids disappeared from the intracellular muscle pool after a meal. It is quite clear that, whereas leucine, all three BCAAs, the aromatic amino acids, and the sulfur amino acids disappear quite quickly, lysine and threonine stick around for a long time. Seven hours after an albumin meal, lysine and threonine levels are still elevated, whereas all of the branch chains have fallen below baseline levels. I think that what we know about the Kms of the catabolic pathways for lysine and threonine would indicate that the body does not turn up their oxidation quickly after a meal in the same way that it does for the BCAAs. ROBERT NESHEIM: One more question. PATRICK DUNNE: It is more of a request for background information. It is really our group that, over the years, has been doing the nutritional assessment of the ration contents. What we do not know is the amino acid composition of

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The Role of Protein and Amino Acids in Sustaining and Enhancing Performance the diets. It is way too expensive to do that. We can make some guestimates like anyone else can from the databases. We would say that the majority of our protein intake is coming from meat. But that is switching as we look at newer concepts in our rations, where we have more vegetarian items. We are getting better at providing bread to people in the field and developing shelf-staple breads. That is a changing phenomenon and may be an area where we need to gather more data. JOHN VANDERVEEN: So you are telling us that you take N values and multiply times the conversion factor and that is how you estimate ration protein content? PATRICK DUNNE. I would say that is correct, with some minor corrections using different factors for dairy versus muscle proteins. You typically perform kjeldahl nitrogen assays and that is what you get. You get approximate values for N, and that forms the database. We are trying to reconcile that database with the nutrient content of the meal, ready to eat (MRE)-15 as best we can. That is the level of knowledge. D. JOE MILLWARD: In fact, I think it is highly unlikely that protein quality is going to be an issue in the sort of rations that are provided unless they are really quite unusual. In other words, unless they are basically unsupplemented wheat. There have been a number of papers recently that have used rats to do protein quality assays of basically vegetarian diets, and these have shown that vegetable-type mixtures can have higher NPU values than those for meat and milk. So, unless these military diets were really very peculiar, then I do not think that the amine acid composition would become an issue. PATRICK DUNNE: Right. However, in the field setting, we probably have the normal level of intake of protein, lower in dairy proteins than you would have in the garrisons just because of what is shelf stable. We do look at cross-linking effects and do not think it is a problem to really reduce the quality of protein during the normal storage period, which could be three-plus years. We also have some data relative to the antioxidant issue. Until recently, when more commercial items were added to our rations, we were not feeding our troops a lot of BHA and BHT. We were feeding them lots of vitamin C and vitamin E. As we add more commercial items, you are going to see changes in macro- and micronutrients, something for the good. ROBERT NESHEIM: Thank you very much. I think it has been a very interesting morning. It is time to break for lunch.