Criteria and Significance of Dietary Protein Sources in Humans

Transcription

1 Criteria and Significance of Dietary Protein Sources in Humans Nitrogen and Amino Acid Requirements: The Massachusetts Institute of Technology Amino Acid Requirement Pattern 1,2 Vernon R. Young 3 and Sudhir Borgonha Laboratory of Human Nutrition, School of Science and Clinical Research Center, Massachusetts Institute of Technology, Cambridge, MA ABSTRACT We review the current international recommendations concerning the protein (nitrogen) and amino acid requirements of healthy individuals, from infancy to the later years of adult life and describe the changes in the recommendations for protein that have been made, since those issued in 1985 by Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU), by the International Dietary Energy Consultative Group. The current international requirements for the specific indispensable amino acids are critiqued briefly, and the rationale and basis for the proposed Massachusetts Institute of Technology (MIT) amino acid requirement pattern are presented. The evidence is then summarized that supports its use in practical considerations of protein nutrition. It is suggested that this MIT amino acid requirement pattern provides the best current estimates of the minimum physiological requirements for the indispensable amino acids in children and adults. It is further concluded that it would be difficult to argue for the continued use of the amino acid requirement values proposed by FAO/WHO/UNU in 1985 in the planning and assessment of dietary protein intakes for population groups worldwide. The MIT amino acid requirement pattern supports and strengthens the relevance of dietary protein quality as an important factor in human protein and amino acid nutrition. J. Nutr. 130: 1841S 1849S, KEY WORDS: infants children adults factorial method protein quality protein requirements The major focus of this workshop is on the capacity, and comparative assessment, of food proteins to efficiently meet the nitrogen and amino acid requirements of humans. Although food protein sources serve as a vehicle for the mineral elements, vitamins and other functionally active compounds, potentially with a relatively high bioavailability, especially if the foods are of animal origin, this is not a topic for consideration here. Rather, the major postulate behind the discussion here is that the principal determinant of the nutritional quality of a food protein and its comparative value in reference to other food proteins is the concentration, per unit of protein (nitrogen), of the available indispensable amino acids. Hence, we review briefly the human requirements for nitrogen and for 1 Presented at the symposium Criteria and Significance of Dietary Protein Sources in Humans, held in San Francisco, CA, on October 4, The symposium was sponsored by the National Dairy Council; International Dairy Federation; United Kingdom Dairy Association; Dairy Farmers of Canada; Davisco Foods International, Inc.; New Zealand Milk; CAMPINA MELKUNIE, Zaltbommel, The Netherlands; Land O Lakes; and CERIN. Published as a supplement to The Journal of Nutrition. Guest editors for this publication were Gregory D. Miller, National Dairy Council, Rosemont, IL, and Daniel Tome, Institut National Agronomique, Paris, France. 2 Supported by National Institutes of Health Grants RR88, DK15856, DK42101, and P-30-DK and by the Nestlé Research Foundation, Lausanne, Switzerland, and Global Cereal Fortification Initiative, Tokyo, Japan. 3 To whom correspondence should be addressed. the indispensable amino acids. This may help to set the stage for an assessment of the Criteria and Significance of Dietary Protein Sources in Humans during the course of the workshop. Nitrogen (protein) requirements The requirement for dietary protein consists of two components: (1) total nitrogen, to serve the needs for synthesis of the nutritionally dispensable (nonessential) and conditionally indispensable amino acids, as well as for other physiologically important nitrogen-containing compounds, and (2) the nutritionally indispensable (essential) amino acids (IAA) 4 that cannot be made by human tissues at rates commensurate with metabolic needs and so must be supplied via an exogenous source (diet or parenteral formulation). With respect to the requirements for total nitrogen (protein), the current international recommendations are now broadly accepted (FAO/WHO/UNU 1985) (Table 1). Some refinements in these recommendations have been proposed 4 Abbreviations used: CV, coefficient of variation; FAO, Food and Agriculture Organization; IAA, indispensable amino acids; IDECG, International Dietary Energy Consultative Group; MIT, Massachusetts Institute of Technology; UN, United Nations; UNU, United Nations University; WHO, World Health Organization /00 $ American Society for Nutritional Sciences. 1841S

2 1842S SUPPLEMENT TABLE FAO/WHO/UNU safe protein intakes for selected age groups and physiological states1 Group Age Safe protein level y g kg 1 day 1 Infants Children Adolescent (girls) (boys) 0.97 Young Adults Elderly Women 0.75 Pregnant 2nd Trimester 6 g daily 3rd Trimester 11 g daily Lactating 0 6 mo 16 g daily 6 12 mo 11 g daily 1 Summarized from FAO/WHO/UNU (1985). Values are for proteins such as provided by hen s egg, cow s milk, meat, fish. more recently by the International Dietary Energy Consultative Group (IDECG) for the protein needs of infants and young children (Dewey et al. 1996). Because there are some differences between these 1996 values and those proposed in the 1985 United Nations (UN) report, it is worth summarizing the basis for and the magnitude of the differences. Some subsequent comments about the protein needs in elderly persons are also in order. The major bases for the 1985 UN protein requirement values in infants, children and adolescents are summarized in Table 2: (1) for infants 6 mo old, the recommendation is derived from data on breast milk protein intake, and (2) for infants 6 mo old and for children and adolescents, a modified factorial approach was used in which i) the maintenance requirement at 6 mo was taken to be 120 mg N kg 1 d 1, declining to 103 mg N kg 1 d 1 by age 18, ii) a growth component was estimated and to which a 50% addition was made to provide a margin of safety because growth rates vary from day to day and because protein is not stored in times of a relative excess in intake, iii) the fractional efficiency of nitrogen utilization from high quality protein sources for both maintenance and growth was taken to be 0.7 and iv) the coefficients of variation (CV) for maintenance and growth TABLE 2 Protein requirements of infants, children, adolescents: Summary of essentials of the approach (FAO/WHO/UNU 1985) 1. Infants from birth to 6 months. Based on intake data 2. Children from 6 months onward. Modified factorial method (a) Maintenance (M): N balance, 120 mg Assume 12.5% CV (b) Requirement for growth (i) Mean N increment 50% to account for intra-individual variation in growth (ii) Assume protein used with 0.7 efficiency (iii) Allowance for inter-individual variation in growth (G) taken to be a CV of 37% for growth, therefore: CV total MxCV m 2 GxCV g 2 / M G TABLE 3 Protein requirements in adults, elderly and women: Summary of essentials of the approach 1. Young men: Short term N balance (mean 0.63/Kg) Long term N balance ( 0.58 g/kg) Mean: 0.6 g: CV 12.5% Hence, safe level % 0.75 g/kg/day 2. Young women: Concluded to be the same as for men 3. Elderly: Not lower that 0.75 g/kg/day 4. Pregnancy: Based on average 925 g protein increment 30% with 0.70 efficiency factor: Therefore, 1.2, 6.1 and 10.7 g additional protein during 1st, 2nd, 3rd trimesters 5. Lactation: From protein secreted: 0.7 efficiency factor CV of 12.5% (birth wt.); 16 g daily (0 6 mo); 12 g daily (6 12 mo) were taken to be 12.5 and 35%, respectively, for purposes of calculating the total CV and a recommended safe protein intake level. Similarly, in Table 3, the basis is presented on which the protein requirements in adult men and women, including elderly persons, are estimated. Results from both short-term and long-term nitrogen balance studies were used to set a mean requirement of 0.6 g high quality protein kg 1 d 1 for men. From this, a safe protein intake level of 0.75 g protein kg 1 d 1 was set for men, women and elderly persons. Additional estimates, as also indicated in this table, were made to arrive at the recommendations for pregnant and lactating women. Infants and children. As mentioned, the IDECG group (Dewey et al. 1996) reevaluated the bases used to derive the 1985 UN recommendations for infants, and this expert group revised the 1985 UN estimates of intakes of breast-fed infants. The revision took into account new data for estimates of 1) the intake of breast milk; 2) the content of protein nitrogen and nonprotein nitrogen; 3) the efficiency of retention of nonprotein N, which the IDECG group took to be 46 61%, compared with an assumed value of 100% used by the UN; and 4) body weights of breast-fed rather than of bottle-fed babies. The 1996 IDECG protein intake estimates for breast-fed infants are shown in Table 4, and they are compared with the 1985 FAO/WHO/UNU values; the 1996 values are 10 26% lower than the 1985 values, depending on age. For infants 6 mo old and for young children, the IDECG group used the factorial approach but with some important differences from that followed by the UN in 1985; these were 1) the 1985 UN maintenance requirement of 120 N kg 1 d 1 was revised downward to 90 N kg 1 d 1, 2) the 50% TABLE IDECG revised estimates of protein intake by breast-fed infants, 0 6 mo (males) Age mo 1985 FAO/WHO/UNU1 Crude protein g/kg/day 1996 IDECG Adjusted protein From Table 29: FAO/WHO/UNU (1985). 2 Milk protein concentration plus 46 61% of the NPN (protein 6.25 N). Taken from Dewey et al. (1996).

3 MIT AMINO ACID REQUIREMENT PATTERN 1843S TABLE 5 Safe level of protein intake for infants and children: Comparison of 1985 FAO/WHO/UNU and 1996 IDECG1 Age 1985 FAO/WHO/UNU 1996 IDECG2 y Values are g protein/kg/day. 2 Calculated and taken from Dewey et al. (1996). addition to account for day-to-day variation in growth was discarded and 3) new estimates were made of the CV for interindividual variability in requirements for infants. However, the IDECG group retained the 70% (0.7 fraction) retention efficiency value. A comparison of the safe levels of protein intake for selected groups of older infants and children as proposed in 1985 by the UN and in 1996 by the IDECG is given in Table 5. The revised estimates are 25 30% lower than those made in the 1985 FAO/WHO/UNU report. It remains to be seen whether the new IDECG recommendations will be used in a future UN revision of current international (FAO/WHO/UNU) (Table 1). Adults and elderly persons. The 1985 FAO/WHO/UNU protein requirements and recommendations for dietary protein intakes in adults have not yet been revisited by an international expert group. However, in view of the rapidly growing number of elderly subjects in the developed, as well as developing, regions of the world (World Health Organization 1998), it is important that increased attention also be given to the nitrogen and amino acid needs of this sector of the population. The UN group (FAO/WHO/UNU 1985) concluded that the safe intake of protein should not be lower than 0.7 g kg 1 d 1 for older adults and elderly persons. However, there has been limited additional study on the protein requirements of elderly persons since that recommendation was made. Thus, Campbell and others (Campbell et al. 1994, Campbell and Evans 1996) have more recently proposed a higher safe protein intake of g kg 1 d 1 based on their own investigations and a reassessment of the literature. In addition, in their review of the published literature, Millward and Roberts (1996) concluded that it has not been demonstrated unequivocally there is an increase with progressive adult age in the mean protein requirement. Indeed, the group at the University of Surrey concluded from 13 C-leucine tracer studies that the apparent protein requirement is lower in elderly persons on the basis of body weight as well as fat free mass (Fereday et al. 1997b, Millward et al. 1997). However, the experimental approach used by these investigators has its inherent limitations, and in particular, their study subjects had not been adjusted to a standard diet before their of the tracer studies. Thus, the nutritional requirement implications of the metabolic studies by Millward, Fereday and their colleagues remain somewhat uncertain. Some years ago, Young et al. (1982) proposed that because of the increased morbidity rate and disease burden in elderly persons, a rational and sound recommendation would be in the region of 1 g good quality protein kg 1 d 1 for this age group. There seems little reason not to follow this recommendation, based on the foregoing. Clearly, further research into the protein requirements in this age group would be highly desirable, although it is may be clear from this overview that the nitrogen requirements at various stages in the life of generally healthy individuals are not currently a topic that arouses strong debate or major controversy. This differentiates it from that of the requirements for IAA, which we consider later. However, before doing so, it is worth noting briefly, with reference to nitrogen requirements, that there is much current research interest on the metabolism of and possibly conditional needs for a number of the so-called dispensable amino acids. These include studies on glutamine, arginine, cyst(e)ine, tyrosine, glycine and proline, which we do not review here. In addition, new research on the in vivo metabolism of glutamic acid (see Reeds 2000), which plays a pivotal role in the transactions and economy of body nitrogen (Young and Ajami, 2000), coupled with the possibility that nonamino nitrogen sources may not be effectively used for the net synthesis of -amino N (see, e.g., Young et al. 2000), has raised again the question of the importance of the qualitative nature of the so-called nonspecific nitrogen component of the total nitrogen requirement. It appears to us that a distinct possibility exists that a preformed source of -amino nitrogen, possibly preferably as glutamic acid, is a necessary component of an optimal dietary protein (nitrogen) intake, in addition to that of the IAA. If this is so, this adds a new perspective to our metabolic understanding and the nutritional significance of the total protein requirement in humans. Requirements for indispensable amino acids Problem/controversy. We (Young 1991 and 1999, Young and Marchini 1990, Young et al. 1987) have reviewed, on a number of occasions, the state-of-the-art with respect to the definition and determination of the quantitative needs for the specific IAA. Our starting point in the present overview is the 1985 FAO/WHO/UNU recommendations for four age groups; these are shown in Table 6. These values indicate that the requirements, per unit body weight, decline substantially between infancy and adulthood, with the requirement for the total of the IAA falling from 714 mg kg 1 d 1 in 3- to 4-mo-old infants to 84 mg kg 1 d 1 in the adult. When expressed per unit of safe protein intake (less histidine), the Amino acid TABLE FAO/WHO/UNU1 estimates of amino acid requirements at different ages Infants Children School boys (3 4 months) (2 years) (10 12 years) Adults mg/kg per d Histidine 28?? [8 12] Isoleucine Leucine Lysine Methionine & Cystine Phenylalanine & Tyrosine Threonine Tryptophan Valine Total Total per g protein FAO/WHO/UNU (1985). 2 Total mg per g crude protein. Taken from Table 38 in FAO/WHO/ UNU (1985), and based on all amino acids minus histidine.

4 1844S SUPPLEMENT TABLE 7 Some recent estimates of the amino acid requirements of infants, pre-school children and adults 3 6 Months 2 3 Year Adult Amino acid 1985 FAO 1996 IDECG 1999 Millward 1985 FAO 1999 Millward 1985 FAO 1999 Millward mg kg 1 day 1 Lysine [23 27]1 Aromatic Sulfur Valine Leucine Isoleucine Threonine Tryptophan per mg protein Lysine [38 45]1 Aromatic Sulfur Valine Leucine Isoleucine Threonine Tryptophan Values taken from Millward (2000). pattern of change with growth development is also marked in that there is a fall in the total IAA-to-protein ratio, with the value for infants being 434 mg g 1 protein 1 compared with adults, in whom the comparative value is 111 mg g 1 protein 1. The biological basis for this dramatic change in the proportion of IAA to total nitrogen is unclear to us, particularly because daily protein maintenance accounts for such a high proportion of the total amino acid requirement, even in the young. Earlier, we (Young 1991) had estimated that for the 2-y-old child, maintenance accounts for 80 90% of the total protein requirement, and Millward (1999), using a somewhat different data set, suggested a value of 85%. Therefore, it seemed likely to us, some years ago, that the picture of a greater change in the IAA requirements relative to the total protein requirements that emerges from the UN data may be a reflection of experimental approaches and their limitations for the determination of requirements rather than a reflection of true changes in the biology of human protein and amino acid metabolism with the development and achievement of maturity. We (Young 1999, Young and Marchini 1990, Young et al. 1989) discussed the various experimental limitations of the amino acid requirement values shown in Table 6, and they will not be repeated here, except to make the following points: 1) the requirements for infants are derived from a combination of the lowest intakes that were found to be adequate for all infants tested in the studies by Holt and Snyderman (1965) or those calculated by Fomon and Filer (1967) that were the lowest intakes of amino acids by infants fed a variety of formulas at levels that maintained adequate growth; 2) the values for the preschool-age child were obtained from studies carried out at the Institute of Nutrition for Central America and Panama, Guatemala, of which the data were presented in only summary form at a conference proceedings (Pineda et al. 1981, Torun et al. 1981); 3) the values for school-age children are limited to a series of careful studies carried by Nakagawa et al. (1964) in Japan; and 4) the adult values are based on the studies by Rose (1957) in men and from similar investigations by others in women (summarized by Irwin and Hegsted 1971); the adult values given in this 1985 report (Table 6) are no longer considered to be acceptable or nutritionally relevant (Working Group 1996). The amino acid requirements in infants also were reassessed by IDECG (Dewey et al. 1996) using a factorial approach. The IDECG values for 3- to 6-mo-old infants are summarized in Table 7 and compared with the 1985 FAO/WHO/UNU estimates (which were taken from the 1973 FAO/WHO report), as well as with those suggested recently by Millward (1999), which were also derived via a factorial method. There are some differences, although not substantial, between the IDECG and Millward (1999) factorial estimates, due essentially to differences in the amino acid requirements assumed for maintenance. However, both of these factorially derived estimates of the amino acid requirements (expressed per kg body weight) in 3- to 6-mo-old infants are substantially lower than those proposed in 1985 FAOWHO/UNU report. This is also true for the infant amino acid requirement values, when expressed per unit of protein, due here to the fact that the UN values are based on the amino acid composition of breast milk protein rather on experimentally derived requirement values. The requirement estimates for the preschool child are also summarized in Table 7, giving the FAO/WHO/UNU direct estimates and the factorially derived values of Millward (1999). IDECG did not reassess the amino acid requirement values for preschool- or school-age children. The differences for the preschool child between the two values summarized here and expressed per kg per day are relatively large for all of the amino acids. Finally, the factorially derived estimates of the amino acid requirement in adults made by Millward (1999) are also compared in Table 7 with the 1985 FAO/WHO/UNU values. For this age group, the values of Millward (1999) are consistently

5 MIT AMINO ACID REQUIREMENT PATTERN 1845S higher than the FAO (UN) values. The even more recent estimates of the lysine requirement made by Millward (2000), based on 13 C-leucine kinetic studies (shown in Table 7 in parentheses) are, per unit of protein, approximately two to almost three times that proposed by the UN group. Furthermore, for reasons given later, the recent lysine requirement values proposed by Millward (2000) are the important ones to be considered, whereas at the same time, they have probably been underestimated. Massachusetts Institute of Technology requirement values and pattern The amino acid requirement estimates for adults that were produced by the 1981 UN Consultation (Table 7), which were reported in 1985 (FAO/WHO/UNU 1985), have been widely used internationally, and they were based on nitrogen balance studies by Rose and others in the 1950s and 1960s (Irwin and Hegsted 1971, Rose 1957, Williams et al. 1974). Furthermore, the requirement values proposed by Millward (1999) and summarized in Table 7 are also derived from the same nitrogen balance studies, except for his 13 C-tracer based estimates of the requirements for lysine (Millward 2000). However, the validity of these earlier nitrogen balance studies and the interpretations drawn from them have been seriously criticized (Young 1991 and 1999, Young and Marchini 1990, Young et al. 1989). Therefore, in our laboratories at the Massachusetts Institute of Technology (MIT) Clinical Research Center, we have been developing and refining a new method of estimating IAA requirements based. In its latest version, the method involves an infusion for 24 h of an amino acid labeled with 13 C and measurement of 13 CO 2 output. From this information, the carbon balance can be calculated, and if the IAA of interest is given at different levels of intake, a value for the requirement can be obtained by estimating the minimal intake necessary to maintain balance. Briefly, our overall approach has involved both the application of 13 C-labeled amino acid carbon balance at varying test levels of amino acid intake (Young 1999, Young et al. 1987) and the prediction of the obligatory amino acid losses and intakes necessary to just balance these, especially for the amino acids that we have not yet studied directly using 13 C tracers (Young and El-Khoury 1995). Our working definition of the requirement for an IAA in a healthy individual is that minimal intake level which represents a single point on a dose-response curve and that is sufficient to maintain a specific criterion of nutritional adequacy (such as growth performance, body composition, body amino acid balance, or measure of organ [liver, muscle] or system [immuno/defense, nervous] function). For practical reasons we have chosen to use body amino acid balance, as determined by the difference between the intake of the test amino acid (e.g., leucine or lysine) and the whole body oxidation of that amino acid, as measured by the appearance of the 13 C label of the amino acid in expired carbon dioxide. Noninvasive measures of specific organ or system function have not yet been sufficiently explored or developed for use in estimating the requirements for specific IAA. From our initial series of 13 C-tracer studies, we estimated the requirements in adults for leucine, lysine, threonine and methionine (without dietary cysteine) to be 30 40, 30, 15 and 13 mg kg 1 d 1, respectively (Young et al. 1989). Except for the sulfur amino acid requirement, these values are far higher than the 1985 FAO/WHO/UNU values for adults shown in Table 7. In addition, we attempted to predict the requirements for those IAA that we had not yet studied with TABLE 8 Lysine oxidation and balance, plasma 13C-lysine precursor, for subjects given low and intermediate lysine levels1 Variable Low (n 5) Intermediate (n 6) Lysine intake (daily) Diet Tracer TOTAL Lysine oxidation: 12 h fast h fed ratio fast/fed h h lysine balance ,5 1 Taken from El-Khoury et al. (2000). 2 Values are mg/kg 1 for the period (Mean SD). 3 For low group: vs. zero: P For intermediate group: vs. zero: P 0.1 (NS). 5 Low vs. intermediate; P (one-tailed t test). the aid of tracer techniques. This prediction was based on assumed obligatory oxidative amino acid losses (Young and El-Khoury 1995), and we have recently validated these estimates (Raguso et al. 1999). Our earlier 13 C-tracer studies provided an initial basis for setting tentative, new requirement values for adults. These studies involve relatively short-term tracer infusion protocols lasting for 3 8 hours. It was then considered important to assess the 24-h kinetics and balance of the test amino acids, so we have undertaken a major series of 24-h tracer studies, beginning with leucine as the test amino acid (El-Khoury et al. 1994a, 1994b and 1995). This permitted us to validate our technique and to provide a stronger basis for follow-up and interpretation of 24-h studies with other amino acids. Thus, more recently, we obtained extensive data on the requirements for the aromatic amino acids (Basile-Filho et al and 1998, Sánchez et al and 1996) and for lysine (El-Khoury et al and 2000). In Table 8 a summary of rates of lysine oxidation and estimates of balance are given for two test intakes of lysine: a low intake of 15.5 mg kg 1 d 1 [ 30% higher than the UN upper requirement value (FAO/WHO/UNU 1985) of 12 mg kg 1 d 1 ] and an intermediate intake of 29.1 mg kg 1 d 1 [similar to the tentative new MIT requirement of 30 mg kg 1 d 1 (El-Khoury et al. 2000)]. Balance was significantly negative at the low intake, but body lysine equilibrium was achieved at the tentative MIT requirement intake level. A point worth noting from the data shown in Table 8 is that the daily rate of oxidation was estimated to be almost identical for the two test lysine intake levels. Studies in sheep (Brooks et al. 1973) and rats (Bergner et al. 1978, Brooks et al. 1972) have shown that at intakes approximating a requirement intake of lysine and below the rate of lysine, oxidation is essentially constant. When this physiological requirement intake level is exceeded, the oxidation increases with further increases in amino acid intake. Hence, our findings in healthy adults are consistent with the view that the two test lysine intakes studied here fall in the range approximating a minimum physiological requirement level and below. The preceding point is further substantiated when the present oxidation data are evaluated against our previous data obtained at a generous lysine intake (77 mg kg 1 d 1 ) (El-Khoury et al. 1998). At this latter intake, the rate of lysine

6 1846S SUPPLEMENT oxidation was determined to be minimally 70 mg kg 1 d 1, although in reality it was more likely to approximate the intake. Thus, when the dietary intake is reduced to a level of 30 mg kg 1 d 1, oxidation declined almost quantitatively, but with a further intake reduction below this level, the rate of oxidation remains essentially constant. Hence, when these two lysine tracer studies (El-Khoury et al and 2000) are viewed together, it is apparent that the minimum physiological requirement approximates (or is possibly higher than) the intermediate lysine studied in our recent investigation. Other lines of evidence support our proposal that the lysine requirement is 30 mg kg 1 d 1 ; these have been discussed and reviewed (Young and El-Khoury 1996) and now include the results of our (Rand and Young 1999) recent analysis of the earlier nitrogen balance data of Jones et al. (1956) and the findings in the studies by the Toronto group using the indicator amino acid oxidation technique for determining amino acid requirements. Thus, on the basis of the oxidation of L-[ 13 C]phenylalanine, as the indicator amino acid, Zello et al. (1993) concluded that the lysine requirement of adult males is three times greater than the World Health recommendation of 12 mg/kg/d. Subsequent studies by this group (Duncan et al. 1996) confirmed their previous estimate of 40 mg kg 1 d 1, which is somewhat higher than our new tentative estimate of 30 mg kg 1 d 1. Furthermore, Millward and colleagues (Fereday et al. 1995and 1997a, Millward 2000) applied a 13 C-leucine tracer model of postprandial protein utilization to evaluate the efficiency of protein retention when small or large meals supplying either cow s milk proteins or wheat protein were the major nitrogen components of these meals. Based on the 13 C-tracer data, they (Millward 2000) estimated that the mean lysine requirement in healthy adults is mg kg 1 d 1, a figure that is close to our proposed value of 30 mg kg 1 d 1. The recent data of Millward are very similar to those we had predicted a few years ago (Young and El-Khoury 1996). Also, in 24-h 13 C-leucine indicator amino acid oxidation/balance studies carried out in Bangalore, India, in healthy adult Indian subjects, Kurpad et al. (1998) provided evidence to further support the 30 mg kg 1 d 1 lysine requirement figure. This has been confirmed in a more extensive study carried out at the Indian center, and the results obtained are now being TABLE 9 Lysine content of wheat flour in comparison with other foods or amino acid requirement patterns Food or requirement pattern Lysine content mg/g protein Whole wheat flour1 24 Wheat flour (70% 80% extraction rate)1 20 Wheat bran 16 Animal protein Legumes FAO/WHO/UNU pattern Adults 16 School children 44 Pre-school children FAO/WHO pattern3 58 MIT pattern 50 1 Sikka et al. (1975). 2 Pellett and Young (1990). 3 FAO/WHO (1991). TABLE 10 Lysine content of whole wheat flour in relation to an estimate of protein quality, using different amino acid scoring patterns Amino acid pattern Amino acid score 1985 FAO/WHO/UNU for adults1 100 (L) 1991 FAO/WHO2 41 (L) Young et al. (MIT pattern)3 48 (L) 1985 FAO/WHO/UNU pre-school child1 41 (L) 1 FAO/WHO/UNU (1985). 2 FAO/WHO (1991). 3 Young et al. (1989). L lysine, first limiting amino acid, not corrected for digestibility. summarized and prepared for publication (Kurpad, A. V., Raj, T., El-Khoury, A. E., et al., unpublished data). Finally, the proposed MIT lysine requirement value is supported by nitrogen balance data on the nutritional quality of whole wheat proteins. Thus, the lysine content of wheat (Sikka et al. 1975) is summarized in Table 9, together with the lysine content of a number of international amino acid scoring patterns. In addition, the usual concentration of lysine in most animal proteins and legumes (Pellett and Young 1990) and that for the MIT requirement pattern are also given for comparison in Table 9. Therefore, if an amino acid score ([amino acid content in the food protein/amino acid content in the reference amino acid requirement pattern] 100) is calculated for wheat four, it would be 100 when the FAO/WHO/ UNU (1985) amino acid requirement pattern for the adult is used as the reference pattern (Table 10). This means that the nutritional value of wheat is predicted to be equal to that of high quality animal protein foods, such as milk, egg or meat. On the other hand, for scoring purposes, the FAO/WHO/ UNU (1985) preschool amino acid pattern (or the FAO/ WHO pattern 1991) predicts a relative nutritional quality of 41% (lysine is limiting); and with the MIT pattern, the score predicts a slightly higher value of 48%. In each case, lysine is determined to be the most limiting amino acid. These foregoing and lower predictive estimates of the nutritional quality of wheat proteins in adults are consistent with the results of nitrogen balance experiments in healthy adults carried out at MIT some years ago (Young et al. 1975). The nitrogen balance response to graded intakes of test dietary protein in healthy adults, expressed as relative protein value (RPV [N balance slope with wheat/n balance slope with TABLE 11 Biological assessment of the nutritional quality of whole wheat proteins in young adults1 Measure of quality Experimental value Predicted from amino acid values 1985 FAO/ WHO/UNU2 MIT pattern3 Relative protein value Relative nitrogen requirement Expressed in comparison with beef protein as reference protein. From Young et al. (1975). 2 FAO/WHO/UNU (1985). 3 Young et al. (1989).

7 MIT AMINO ACID REQUIREMENT PATTERN 1847S TABLE 12 Some recent adult amino acid requirement estimates Amino acid 1985 FAO 1999 Millward MIT mg kg 1 day 1 Lysine [23 27]1 30 Aromatic Sulfur Valine Leucine Isoleucine Threonine Tryptophan mg g protein 1 Lysine [38 45] 50 Aromatic Sulfur Valine Leucine Isoleucine Threonine Tryptophan Values taken from Millwood (2000). reference protein] 100), was found to be 54 for whole wheat protein, using beef protein as a reference. Expressed as relative nitrogen requirement (RNR 1/[amount of wheat protein to achieve nitrogen balance in 97.5% of population amount of beef protein] 100), the response was 56 (Table 11). The MIT amino acid requirement pattern predicted a value of 48. Hence, there is very good agreement between these experimentally derived (nitrogen balance) and predicted (from amino acid score) estimates of the nutritional quality of whole wheat proteins when the MIT lysine requirement value is used to generate the reference amino acid scoring pattern. In contrast, use of the 1985 FAO/WHO/UNU adult amino acid pattern gives an invalid prediction of the nutritional value of wheat protein. Again, therefore, these observations support the conclusion that the 1985 FAO/WHO/UNU lysine requirement value of 12 mg kg 1 d 1 for the adult should be discarded. They also provide additional justification for the tentative working value of 30 mg kg 1 d 1 proposed above (or 50 mg lysine/g protein). From the foregoing, the proposed MIT lysine and other IAA requirement values seem to offer the current best estimates of the minimum physiological requirements for these amino acids in healthy adults. Our tentative requirement values are given in Table 12 and compared with those proposed by FAO/WHO/UNU (1985) and by Millward (1999 and 2000). The nutritionally relevant amino acids, from a public health nutritional standpoint, are, first, lysine, followed by the sulfur amino acids, threonine and perhaps tryptophan (FAO/WHO/UNU 1985). It may be seen that there is evidently relatively good agreement between the MIT values and those proposed recently by Millward (1999 and 2000) for these amino acids in healthy adults (Table 12). In contrast to the availability of some, although variable, experimentally based estimates of the requirements for total protein (nitrogen) in elderly subjects, there is an almost complete absence of experimental data on the requirements for the IAA in this age group. At the present, the requirements are assumed to be essentially the same as those in young adults (Young 1992). We (Fukagawa et al. 1998), however, studied methionine/cyst(e)ine relationships in elderly subjects, and our initial data suggest that elderly persons may adjust less well to a reduction in the dietary intake of the sulfur amino acids than do young adults. There is, clearly, a need to establish with great confidence whether the amino acid requirement values for healthy young adults apply as well to the healthy elderly population. Importance of revised requirement values The question might first be raised as to whether the MIT amino acid requirement pattern for healthy adults differs in any fundamental way from that proposed by FAO/WHO/ UNU in 1985 for the preschool-age group with respect to the application to older children and adults (FAO/WHO 1991). It can be seen from Table 13 that the amino acid requirement pattern (expressed per unit of protein) for the school-age child has higher lysine and threonine values than for the MIT pattern but similar values for the other amino acids. The higher FAO/WHO/UNU lysine value has been suggested by Millward (1994) to be due to the possibility that the preschool-age children who participated in the amino acid re- TABLE 13 Some amino acid requirement (scoring) patterns for pre-school children and adults Pre-school Adult Amino acid 1985 FAO/WHO/UNU1 Millward FAO/WHO/UNU1 Millward MIT3 Isoleucine Leucine Lysine (38 45)4 50 Total SAA Total AAA Threonine Tryptophan Valine TOTAL From Table 38; FAO/WHO/UNU (1985). 2 From Table 3; Millward (1999). 3 From Table 12; Young and El-Khoury (1995). 4 Values taken from Millwood (2000).

8 1848S SUPPLEMENT TABLE 14 Some amino acid scores for two major protein sources, alone and in combination1 Amino acid requirement pattern Milk Wheat Milk/wheat (40/60) Lysine SAA Lysine SAA Lysine SAA 1985 FAO/WHO/UNU Pre-School Adult Millward Millward Millward MIT Not corrected for digestibility. 2 Using a lysine requirement estimate of 38 mg g protein 1. 3 Using a lysine requirement estimate of 45 mg g protein 1. 4 Young and El-Khoury (1995). quirement studies at the Institute of Nutrition for Central America and Panama had not fully recovered from earlier protein-energy malnutrition. However, it can equally well be argued that this is not an adequate explanation, because rapid growth during recovery might be expected to be associated with a higher efficiency of lysine (or other IAA) utilization and retention than would be the case in the fully replete state. Nevertheless, we consider that a requirement value of 50 mg lysine/g protein should be adequate to support the needs for growth in healthy preschool-age children. Thus, the 1996 IDECG group (Dewey et al. 1996) proposed a mean lysine requirement of 63 mg kg 1 d 1 for 3- to 6-mo-old infants; when expressed in relation to the IDECG proposed mean protein requirement of 1.06 protein kg 1 d 1 for the 3- to 4-mo-old age group, this would give a mean value of 59 mg lysine kg 1 d 1 for a young infant who is gaining 56 mg N kg 1 d 1. The comparable rate gain in the 2-y-old child might be 13 mg n kg 1 d 1, so this would support our view that a lysine requirement of 50 mg/g protein should be sufficient to meet the needs of the preschool-age child. A similar argument can be made for the adequacy of the threonine content in the MIT amino acid requirement pattern, with respect to its application to the preschool-age group. The foregoing leads us to recommend use of the proposed MIT amino acid requirement pattern in considerations of food/diet protein quality for all ages above 1 2 y. If this recommendation is accepted, then we can briefly consider its implications for estimates of the quality of two major food protein sources: wheat and milk. In Table 14 we present the amino acid scores (for lysine and the sulfur amino acids) of wheat and milk and a combination of the two sources when the estimates are based on different reference amino acid requirement patterns. This table shows that when the 1985 FAO/WHO/UNU amino acid requirement pattern for adults is used, the nutritional qualities of wheat and milk are both very high and equal to each other, when each are consumed as a sole source of dietary protein. On the other hand, the more recent estimates of Millward (1999 and 2000) of the lysine requirement in adults and that in the MIT amino acid requirement pattern lead to the conclusion that wheat has a protein nutritional value much lower than that of milk and that when milk is added to wheat in the proportion of 40% of milk protein nitrogen and the remainder from wheat, a high quality protein mixture is obtained. From the foregoing discussion, it can be concluded that the MIT requirement pattern makes dietary protein quality an important factor in practical considerations of human amino acid nutrition. The pattern is supported by data from a variety of metabolic studies, and it provides a rationale for drawing nutritional distinctions among different food protein sources. Acceptance of the earlier FAO/WHO/UNU (1985) recommendations would make the question of dietary protein quality totally unimportant, except perhaps for the infant and young child. On the basis of the totality of the evidence, this latter position would be hard to justify. We reviewed the current international recommendations concerning the nitrogen and amino acid requirements of healthy individuals from infancy to the later years of adult life. Changes in the recommendations for protein that have been made, since those issued in 1985 by FAO/WHO/UNU, by the IDECG are described. The current international requirements for the specific IAA are critiqued briefly, and the rationale and basis for the MIT amino acid requirement pattern are presented. The evidence is then summarized that supports its use in practical considerations of human protein nutrition. It is proposed that this MIT pattern provides the current best estimates of the minimum physiological requirements for IAA in children and adults. It is further concluded that it would be difficult to argue for the continued use of the amino acid requirement values proposed by FAO/WHO/UNU in 1985 in the planning and assessment of dietary protein intakes for population groups worldwide. LITERATURE CITED Basile, A., Beaumier, L., El-Khoury, A., Kenneway, M., Gleason, R. E. & Young, V. R. (1998) Twenty-four hour L-[1-13 C]tyrosine and L-[3 3-2 H 2 ] phenylalanine oral tracer studies at generous, intermediate and low phenylalanine intakes to estimate aromatic amino acid requirements in adults. Am. J. Clin. Nutr. 67: Basile-Filho, A., El-Khoury, A. E., Beaumier, L., Wang, S. Y. & Young, V. R. (1997) Continuous twenty-four hour L-[1-13 C]phenylalanine and L- [3,3-2 H 2 ]tyrosine oral tracer studies at an intermediate phenylalanine intake to estimate requirements in adults. Am. J. Clin. Nutr. 65: Bergner, H., Simon, O. & Adams, K. (1978) Determination of lysine requirement in growing rats based on catabolism rate of 14 C- and 15 N-labeled lysine. Arch. Tierernahrung 28: Brookes, I. M., Owens, F. N., Brown, R. E. & Garrigus, U. S. (1972) Influence of amino acid level in the diet upon amino acid oxidation by the rat. J. Nutr. 102: Brookes, I. M., Owens, F. N., Brown, R. E. & Garrigus, U. S. (1973) Amino acid oxidation and plasma amino acid levels in sheep with abomasal infusion of graded amounts of lysine. J. Anim. Sci. 36: Campbell, W. W., Crim, M. C., Dallal, G. E., Young, V. R. & Evans, W. J. (1994) Increased protein requirements in elderly people: new data and retrospective reassessments. Am. J. Clin. Nutr. 60:

9 MIT AMINO ACID REQUIREMENT PATTERN 1849S Campbell, W. W. & Evans, W. J. (1996) Protein requirements of elderly people. Eur. J. Clin. Nutr. 50(suppl 1): S180 S185. Dewey, K. G., Beaton, G., Fjeld, C., Lonnerdal, B. & Reeds, P. (with input from Brown, K. H., Heinig, M. J., Ziegler, E., Räihä, N.C.R. & Axelsson I.E.M.) (1996) Protein requirements of infants and children. Eur. J. Clin. Nutr. 50(suppl 1): S119 S150. Duncan, A. M., Ball, R. O., Pencharz, P. B. (1996) Lysine requirement of adult males is not affected by decreasing dietary protein. Am. J. Clin. Nutr. 64: El-Khoury, A. E., Basile, A., Beaumier, L., Wang, S. Y., Al-Amiri, H. A., Selvaraj, A., Wong, S., Atkinson, A., Ajami, A. & Young, V. R. (1998) Twenty-four hour intravenous and oral tracer studies with L-[1-13 C]-2-aminoadipic acid and L-[1-13 C]lysine as tracers at generous nitrogen and lysine intakes in healthy adults. Am. J. Clin. Nutr El-Khoury, A. E., Fukagawa, N. K., Sánchez, M., Tsay, R. H., Gleason, R. E., Chapman, T. E. & Young, V. R. (1994a) Validation of the tracer-balance concept with reference to leucine: 24h intravenous tracer studies with L-[1-13 C]leucine and [ 15 N]urea. Am. J. Clin. Nutr. 59: El-Khoury, A. E., Fukagawa, N. K., Sánchez, M., Tsay, R. H., Gleason, R. E., Chapman, T. E. & Young, V. R. (1994b) The 24h pattern and rate of leucine oxidation, with particular reference to tracer estimates of leucine requirements in healthy adults. Am. J. Clin. Nutr. 59: El-Khoury, A. E., Pereira, P.C.M., Borgonha, S., Basile-Filho A., Beaumier, L., Wang, S. Y., Metges, C. C., Ajami, A. M. & Young, V. R. (2000) Twenty-four hour oral tracer studies with L-[1-13 C]lysine at a low (15 mg kg 1 day 1 ) and intermediate (29 mg kg -1 day -1 )lysine intake in healthy adults. Am. J. Clin. Nutr. (in press). El-Khoury, A. E., Sánchez, M., Fukagawa, N. K., Gleason, R. E., Tsay, R. H. & Young, V. R. (1995) The 24h kinetics of leucine oxidation in healthy adults receiving a generous leucine intake via three discrete meals. Am. J. Clin. Nutr. 62: FAO/WHO (1991) Protein Quality Evaluation: Report of a Joint FAO/WHO Expert Consultation. Paper No. 51. Food and Agriculture Organization of the United Nations, Rome. FAO/WHO/UNU (1985) Energy and Protein Requirements: Report of an FAO/ WHO/UNU Expert Consultation. WHO Tech. Rept. Ser. No World Health Organization, Geneva. Fereday, A., Gibson, N., Cox, M., Halliday, D., Pacy, P. D. & Millward, D. J. (1995) Postprandial protein utilization in normal adults, II: milk protein measured in the non-steady state. Proc. Nutr. Sci. 54: 63A (abs.). Fereday, A., Gibson, N., Cox, M., Halliday, D., Pacy, P. D. & Millward, D. J. (1997a) Postprandial protein utilization of wheat protein from a single meal in normal adults. Proc. Nutr. Soc. 56: 90A (abs.). Fereday, A., Gibson, N. R., Cox, M., Pacy, P. J. & Millward, D. J. (1997b) Protein requirements and ageing: metabolic demand and efficiency of utilization. Br. J. Nutr. 77: Fomon, S. J. & Filer, L. T., Jr. (1967) Amino acid requirements for normal growth. In: Amino Acid Metabolism and Genetic Variation (Nyhan, W. L., ed.), pp McGraw-Hill, New York. Fukagawa, N. K., Yu, Y.-M. & Young, V. R. (1998) Methionine-cysteine kinetics at different intakes of methionine and cystine in elderly men and women. Am. J Clin. Nutr. 68: Holt, L. E., Jr. & Snyderman, S. E. (1965) Protein and amino acid requirements of infants and children. Nutr. Abstr. Rev. 37: Irwin, M. I. & Hegsted, D. M. (1971) A conspectus of research on amino acid requirements of man. J. Nutr. 101: Jones, E. M., Bauman, C. A. & Reynolds, M. S. (1956) Nitrogen balances of women maintained on various levels of lysine. J. Nutr. 60: Kurpad, A. V., El-Khoury, A. E., Beaumier, L., Srivatsa, A., Kuriyan, R., Raj, T., Borgonha, S., Ajami, A. M. & Young, V. R. (1998) An initial assessment, using 24 hour 13 C-leucine kinetics, of the lysine requirement of healthy adult Indian subject. Am. J. Clin. Nutr. 67: Millward, D. J. (1999) The nutritional value of plant-based diets in relation to human amino acid and protein requirements. Proc. Nutr. Soc. 58: Millward, D. J. (2000) Post-prandial protein utilization: implications for clinical nutrition. In: Proteins, Peptides and Amino Acids in Enteral Nutrition. Proceedings of 3rd Nestlé Nutrition Workshop, Stockholm, Sweden, Sept. 9 10, 1999 (in press). Millward, D. J. (1994) Can we define indispensable amino acid requirements and assess protein quality in adults? J. Nutr 124: Millward, D. J., Fereday, A., Gibson, N. & Pacy, P. J. (1997) Aging, protein requirements, and protein turnover. Am. J. Clin. Nutr. 66: Millward, D. J. & Roberts, S. B. (1996) Protein requirements of older individuals. Nutr. Res. Rev. 9: Nakagawa, I., Takahashi, T., Suzuki, T. & Kobayashi, K. (1964) Amino acid requirements of children: nitrogen balance at the minimal level of essential amino acids. J. Nutr. 83: Pellett, P. L. & Young, V. R. (1990) Role of meat as a source of protein and essential amino acids in human protein nutrition. In: Meat and Health: Advances in Meat Research, Vol. 6 (Pearson, A. M. & Dutson, T. R., eds.), pp Elsevier Science Publishing, New York. Pineda, O., Torun, B., Viteri, F. E. & Arroyave, G. (1981) Protein quality in relation to estimates of essential amino acid requirements. In: Protein Quality in Humans: Assessment and In Vitro Estimation (Bodwell, C. E., Adkins, J. S. & Hopkins, D. T., eds.), pp AVI Publishing, Westport, CT. Raguso, C., Pereira, P. & Young, V. R. (1999) A tracer investigation of the obligatory oxidative amino acid losses in healthy, young adults. Am. J. Clin. Nutr. 70: Rand, W. M. & Young, V. R. (1999) Statistical analysis of N balance data with reference to the lysine requirement in adults. J. Nutr. 129: Reeds, P. J. (2000) Dispensable and indispensable amino acids for humans. J. Nutr. 130: 1835S 1840S. Rose, W. C. (1957) The amino acid requirements of adult man. Nutr. Abstr. Rev Sánchez, M., El-Khoury, A. E., Castillo, L., Chapman, T. E., Basile, A., Beaumier, L. & Young, V. R. (1996) Twenty-four hour intravenous and oral tracer studies with L-[1-13 C]phenylalanine and L-[3,3-2 H 2 ]tyrosine at a tyrosine-free generous phenylalanine intake in adults. Am. J. Clin. Nutr. 63: Sánchez, M., El-Khoury, A. E., Castillo, L., Chapman, T. E. & Young, V. R. (1995) Phenylalanine and tyrosine kinetics in young men throughout a continuous 24h period, at low phenylalanine intake. Am. J. Clin. Nutr. 61: Sikka, K. C., Johari, R. P., Duggan, S. K., Ahuja, V. P. & Austin, A. (1975) Comparative nutritive value and amino acid content of different extractions of wheat. Agri. Food Chem. 23: Torun, B., Pineda, O., Viteri, F. E. & Arroyave, G. (1981) Use of amino acid composition data to predict protein nutritive value for children with specific reference to new estimates of their essential maino acid requriements. In: Protein Quality in Humans: Assessment and In Vitro Estimations (Bodwell, C. E., Adkins, J. S. & Hopkins, D. T., eds.), pp AVI Publishing, Westport, CT. Williams, W. H., Harper, A. E., Hegsted, D. M., Arroyave, G. & Holt, L. E., Jr. (1974) Nitrogen and amino acid requirements. In: Improvement of Protein Nutriture (Committee on Amino Acids/Food and Nutrition Board/National Research Council, eds.), pp. l National Academy of Sciences, Washington, D.C. Working Group. (1996) Clugston, G., Dewey, K. C., Fjeld, C., Millward, J., Reeds, P., Scrimshaw, N. S., Tontisirin, K., Waterlow, J. C. & Young, V. R. Report of a working group on protein and amino acid requirements. Eur. J. Clin. Nutr. 50(suppl 1): S193 S195. World Health Organization (1998) World Health Report: Executive Summary: Life in 21st Century A Vision for All. World Health Organization, Geneva ( Young, V. R. (1991) Nutrient interactions with reference to amino acid and protein metabolism in non-ruminants: particular emphasis on protein-energy relations in man. Zeit. Ernahrungswissenschaft 30: Young, V.R. (1992) Macronutrient needs in the elderly. Nutr. Rev. 50: Young, V. R. (1999) Amino acid flux and requirements: counterpoint: tentative estimates are feasible and necessary. In: The Role of Protein and Amino Acids in Sustaining and Enhancing Performance (Committee on Military Nutrition/ Food and Nutrition Board/Institute of Medicine, eds.), pp National Academy Press, Washington, D.C. Young, V. R. & Ajami, A. M. (2000) Glutamate: an amino acid of particular distinction. J. Nutr. 130: 892S 900S. Young, V. R., Bier, D. M. & Pellett, P. L. (1989) A theoretical basis for increasing current amino acid requirements in adult men, with experimental support. Am. J. Clin. Nutr. 50: Young, V. R. & El-Khoury, A. E. (1995) Can amino acid requirements for nutritional maintenance in adult humans be approximated from the amino acid composition of body mixed proteins? Proc. Natl. Acad. Sci. U.S.A. 92: Young, V. R. & El-Khoury, A. E. (1996) Human amino acid requirements: a re-evaluation. Food Nutr. Bull. 17: Young, V. R., El-Khoury, A. E., Raguso, C. A., Forslund, A. H. & Hambraeus, L. (2000) Urea production, hydrolysis and leucine oxidation change linearly over widely varying protein intakes in healthy adults. J. Nutr. 130: Young, V. R., Fajardo, L., Murray, E., Rand, W. M. & Scrimshaw, N. S. (1975) Protein requirements of man: comparative nitrogen balance response within the submaintenance-to-maintenance range of intake of wheat and beef proteins. J. Nutr. 105: Young, V. R., Gersovitz, M. & Munro, H. N. (1982) Human aging: protein and amino acid metabolism and implications for protein and amino acid requirements. In: Nutritional Approaches to Aging Research (Moment, G. B., ed.), pp , CRC Press, Boca Raton, FL. Young, V. R. & Marchini, J. S. (1990) Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amino acids, with reference to nutritional adaptation in humans. Am. J Clin. Nutr. 51: Zello, G. A., Pencharz, P. B. & Ball, R. O. (1993) The dietary lysine requirement of adult males determined by the oxidation of an indicator amino acid L-[1-13 C]phenylalanine. Am. J. Physiol. 264: E677 E685.