FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS	ESN: FAO/WHO/UNU EPR/81/Inf. 8 September 1981
	WORLD HEALTH ORGANIZATION
	THE UNITED NATIONS UNIVERSITY

INFORMATION PAPER No. 8

Joint FAO/WHO/UNU Expert Consultation on
Energy and Protein Requirements

Rome, 5 to 17 October 1981

WHOLE BODY AMINO ACID TURNOVER WITH ¹³C TRAVERS

A NEW APPROACH FOR ESTIMATION OF HUMAN AMINO ACID REQUIREMENTS

by

Carol Meredith, Dennis M. Bier, Michael M. Meguid
Dwight E. Matthews, Zhimei Wen, and Vernon R. Young

Massachusetts Institute of Technology
Cambridge, Massachusetts
and
Washington University School of Medicine
St. Louis, Missouri

INTRODUCTION

The normal intake of foods and, thus, of the essential nutrients required for maintenance of body functions may be disrupted as a consequence of disease or trauma. Hence, it is often necessary to resort to the use of one of many available formulations and various therapeutic techniques, including tube-feeding and intravenous alimentation, in order to supply the patient with the nutrients necessary to prevent development of malnutrition, reverse nutritional deficiencies and to promote effective response to therapy and recovery. In the context of the protein and amino acid nutrition of patients and the appropriate design of protein and amino acid mixtures intended for either oral or intravenous administration, it is important to have an adequate knowledge of the requirements for total nitrogen and for specific indispensable (essential) amino acids. This information is necessary to assess both the basal requirements and the quantitative impact of pathophysiologic states on the metabolism and needs for these nutrients.

Here we will examine current knowledge about the amino acid requirements of healthy adult subjects. In doing so we will take this opportunity to discuss some of our recent studies that have been undertaken to exploit modern views of body protein and amino acid metabolism and the application of stable isotope techniques for investigating whole body amino acid kinetics. Our purpose has been to attempt the development of a new approach for improving estimates of the quantitative requirements for specific amino acids in human subjects.

CURRENT ESTIMATES OF AMINO ACID REQUIREMENTS FOR ADULTS

In their excellent review of parenteral nutrition, Shenkin and Wretlind (22) emphasized the importance of the recommendations made, in 1957, by Rose (21) concerning the requirements for individual essential amino acids. These recommendations have served as a major basis for comparing amino acid requirements of infants, children and adults and for assessing possible differences in the requirements for maintenance as compared with those necessary for effective body protein repletion (e.g. Table 1). In view of the significance of the data shown in Table 1 for the evaluation of indispensable amino acids in human nutrition, it is instructive to review briefly the methods and approaches used to quantify the essential amino acid requirements in adults. Also it is necessary to raise some problems concerned with interpretation of the nutritional significance of these data and the similar values for adult amino acid requirements as derived from more recent investigations, the latter summarized by Irwin and Hegsted (12).

We have discussed this area in a recent review (30) and so the major points will be summarized here, together with an update of our observations and thoughts concerning the determination of amino acid requirements by novel methods.

(a) N Balance Studies

Much of the data available on the amino acid requirements of adults are based on results obtained from metabolic N-balance studies. Although useful information can be gained from N-balance determinations, it is important to appreciate that there are a number of significant limitations to this approach, in order to further assess the significance of the published data. These limitations include: (a) Body N equilibrium does not necessarily reflect an adequate state of organ protein metabolism or of nutritional status because it does not reveal alterations in the intensity, quality and/or distribution of tissue and organ protein metabolism. (b) In addition to the cumulative, positive errors involved in determination of N balance from measurement of the intake and rates of N output via feces and urine, it is difficult to quantify all of the N losses from the body. However, unless all of these losses are measured, erroneous estimates may be made of the absolute requirement level for an amino acid. It is noteworthy that most of the N balance estimates used in arriving at amino acid requirements have involved measurements of only the N intake and of urinary and fecal N losses. The problem created by this approach can be illustrated in reference to the data of Rose et al. (19) on the tryptophan requirement in adult men. As indicated in Table 2, these investigators concluded from apparent N balance estimations that 150 mg L-tryptophan would be sufficient to meet the physiological requirement for this amino acid. However, if an allowance of about 5 mg N kg^-1 day^-1 had been made to account for integumental and other minor losses (4) before computing the N balance data, it is clear for the subject shown here that even 250 mg L-tryptophan daily would not have been sufficient. A more extensive evaluation of this problem has been undertaken by Hegsted (9) who re-analyzed, using regression techniques, the published N balance data used to estimate the amino requirements in women. Based on this analysis it was found that there was a marked difference, almost five-fold for threonine and the sulfur amino acids, between the estimated minimum requirement for individual amino acids when determined from apparent N balance data as compared with that derived when an allowance of 0.5 g N daily for unmeasured nitrogen losses was included in the estimation of body nitrogen balance. In our studies on lysine requirements of healthy adult men, we found that when unmeasured nitrogen losses, estimated at 5 mg/kg.day, were included in the balance equation, the mean lysine requirement for N equilibrium increased from 12 mg/kg.day to 17 mg/kg.day. This estimate is higher than currently accepted values (4); however, Fujita et al. (5) measured nitrogen balances in young men consuming graded amounts of a lysine-deficient protein (wheat gluten) and from their findings suggested that the requirement for lysine is about 24 mg/kg.day. On the basis of nitrogen balance studies alone, therefore, it seems likely that the published values for requirements may represent significant underestimates of the actual need for indispensable amino acids in adults.

Even if all routes of N loss are considered in estimating N balance during short experimental diet periods, this technique does not necessarily provide an adequate, sole basis for estimating the minimum requirements for specific amino acids in the adult. The reasons for this, discussed by us previously (29) include: (a) there are significant confounding effects on N balance due to the influence of dietary energy level (3, 7, 11) and these have not been adequately considered in relation to current estimates of the amino acid requirements in adult humans. For example, in the studies by Rose (21) the experimental diets routinely supplied high energy intakes (about 55 kcal kg^-1day^-1). Although Rose (21) stated that debate on the possible influence of high energy intakes was irrelevant to the evaluation of minimum requirements for amino acids we do not accept this view. For example, Rose and Wixom (20) carried out a study to determine the minimum nitrogen requirement using a diet supplying all indispensable amino acids at a “safe” level, or double the minimum requirement found for a subject with the highest need (21). In this study (Table 3) one of their volunteer subjects gained about 5 kg during the course of the experiment and another increased body weight by about 2 kg. Clearly, this indicates that energy intakes were excessive for the test subjects. This casts further doubt about the reliability of estimates of the minimum requirement values, because the high energy intakes would tend to lead to an underestimation of the actual requirements where energy intakes were sufficient to just maintain energy balance (e.g. see ref. 11). (b) interpretation of the metabolic and nutritional significance of N is difficult. Hegsted (10) has pointed out that in many studies with increasing nitrogen intakes above requirement levels, N balance in adults becomes progressively more positive and that unrealistic positive balances are obtained over long periods of time. Furthermore at low nitrogen intakes that seem adequate because they support nitrogen equilibrium during brief experimental periods, abnormalities in some functional parameters begin to appear at these N intakes in some subjects over longer periods of time (8). Therefore, it is not easy to determine whether N balance data reflect precisely the status of body protein nutriture or whether this approach provides a satisfactory means for establishing requirement values.

For these reasons, and because of the conclusion by Weller et al. (27) that the one, or more, of the values for the requirements for indispensable amino acids as established by Rose (21) is more than one-third below the actual need in healthy young men, the nutritional value of current estimates for amino acid requirements in adults remains highly uncertain.

(b) Alternative criteria

In addition to the limitations, discussed above, of the N balance data upon which most estimations of adult human amino acid requirements have been made, it is worthwhile to state that requirements for essential nutrients might be determined on the basis of more than one metabolic or biochemical criterion. For example, in the case of thiamine, a number of different biochemical and metabolic parameters have been used to arrive at the adult requirement for this B-complex vitamin. By determining how different degrees of dietary thiamine restriction affect metabolic and physiological parameters, it has been possible to define a gradation of deficits, beginning with what might be termed “adaptive responses to lowered nutrient intakes” and proceeding to alterations in whole body homeostasis that carry pathological consequences. Similarly, alternative methods other than body nitrogen balance, might be used to understand how the organism responds and adapts to decreasing intakes of a specific amino acid, and possibly, to facilitate estimation of requirements for these nutrients. Thus, in the case of the adult human, use of plasma free amino acid responses has been explored by us ( e.g. 25, 29) for this purpose but this approach requires further examination and refinement because the pattern of changes in plasma amino acid levels varies according to the specific amino acid under test. A valuable technique for studying the dynamic response of body protein metabolism to alterations in the intake of indispensable amino acids involves application of labeled tracers. Radioactive isotope tracer studies in growing (2, 13, 14) and adult rats (23), as well as other biochemical studies (28), have indicated that when the intake of a specific indispensable amino acid is gradually decreased, significant alterations in amino acid homeostasis coincide with appearance of detrimental changes in whole body physiological parameters such as reduced growth, in the young, or loss of weight maintenance in the adult. These findings indicate that the biochemical mechanisms responsible for maintaining the homeostasis of whole body proteins and of the specific limiting amino acid are closely integrated with the individual's requirement for these nutrients. On this basis it seems likely that when intakes of protein and/or specific amino acids range from the generous down to inadequate levels in humans, the dynamic state of the whole body metabolism of specific amino acids and/or proteins will change greatly. Therefore, recent studies involving stable isotope tracer techniques have been carried out in our laboratories (15, 16) in order to determine how kinetic parameters of amino acid and protein metabolism in adult human subjects respond to changes in the dietary supply of specific indispensable amino acids. Furthermore, we have designed our studies in a way that should aid a further definition of human protein and amino acid requirements. A brief account of the approach we have followed and some of our preliminary findings and conclusions will be discussed below.

¹³C-TRACER STUDIES IN ADULT HUMANS

(a) Overall approach

Various approaches might be followed in studies of whole body amino acid kinetics in adult humans. Our work has concentrated on use of a method of continuous isotope infusion, that has been explored extensively and described by Waterlow and his co-workers (26). We chose this approach to avoid some of the difficulties inherent in the earlier, single isotope dose approaches. The continuous infusion of tracer may either be coupled with measurement of end products of N metabolism, or with determination of the enrichment of isotope (¹⁵N or ¹³C) in plasma following administration of a labeled amino acid or precursor (26). By combining this latter approach with measurements of ¹³C in expired CO₂ (15) or ¹⁵N in urinary urea or other metabolic or excretory products the components of whole body amino acid flux can be determined. These are; incorporation of amino acids into and their release from proteins and the conversion of amino acids to intermediateor end-products of their metabolism. Specifically, our studies have utilized amino acids labeled with ¹³C on the carboxyl carbon moiety. Details of the methodology and general aspects of the protocol design have been described (15, 16).

(b) Validity and ¹³C-tracer infusion model

The problems and limitations of the stable isotope tracer model used in our studies have been discussed by Waterlow and his colleagues (26) as well as in our reports (15, 16). We recognize that the model does involve use of assumptions that may not hold under all experimental conditions or that are difficult to validate. However, as a further examination of the model, it is important to determine whether use of different isotope probes such as [¹³C]-leucine, -valine or -lysine will allow similar quantitative estimates of the dynamics of whole body protein metabolism in adult subjects under defined dietary conditions. This is important if close comparisons are to be made among the various studies that involve application of different tracers. As shown in Fig. 1, we have found that when young adult men receive diets providing a generous protein intake, estimates of rates of whole body protein synthesis and breakdown are quite comparable between experiments based on either use of [1-¹³C]-leucine, -valine or -lysine. Furthermore, with diets that are very low in either leucine, valine or lysine, use of these labels indicates quantitatively similar and markedly lower rates of whole body protein synthesis and breakdown (Fig. 2), as compared with those determined for subjects receiving nutritionally adequate diets. These observations further support use of different ¹³C-labeled amino acids and application of the continuous isotope infusion model for exploration of dynamic aspects of whole body metabolism of indispensable amino acids.

Another series of observations, also made recently in our laboratories that underscore the potential of determining the quantitative aspects of whole body amino acid metabolism with ¹³C-tracers as a new approach for estimation of human amino acid requirements, are depicted in Figs. 3 and 4. For example, on the basis of a large series of studies, we have found that there is an apparent linear relationship between the adequacy of protein or amino acid intake and magnitude of body N balance, as determined during short diet periods, and the extent of net body protein synthesis during the time of the day when protein-containing meals are consumed (defined as the fed state). This relationship is found under conditions where diets supplying widely varying intakes of high quality egg protein (Fig. 3) as well as for diets providing a constant and adequate intake of total nitrogen but differing in the concentration of a specific indispensable amino acid (Fig. 4).

Finally, on the basis of these data, it is apparent that an intake of protein sufficient to maintain body protein nutriture during relatively long experimental periods, namely 0.8 g egg protein.kg^-1.day^-1, there is a net protein synthesis, approximating 23 mg.kg^-1.h^-1 when subjects are in the fed state (Fig. 3). A net protein synthesis is to be anticipated because the post-absorptive state is characterized by an excess of body protein breakdown over synthesis (e.g. 6, 16), and thus the fed state must support an anabolic status if there is to be a compensation for the net loss of body protein that occurs during that period of the day when food is not consumed. This estimate of the degree of net protein synthesis at an adequate, but not too generous, a level of protein intake will be referred to again in the section below.

From these various findings we have concluded that the continuous isotope infusion model, using ¹³C-labels, provides consistent estimates of whole body amino acid kinetics and protein turnover. Therefore, we have thought it worthwhile to further exploit this approach for increasing our understanding of the relationships between dietary factors and body N balance, particularly in reference to determination of amino acid requirements for maintenance of body protein homeostasis in healthy adults.

(c) Studies designed to determine amino acid requirements

In addition to the encouraging findings obtained in our initial human studies, the published evidence based on studies in young rats has revealed that the rate of oxidation of a number of the indispensable amino acids remains low and relatively constant for intakes that are less than sufficient for supporting maximal rates of net body protein synthesis (or growth). This rate increases linearly when intakes are progressively raised above this requirement level (e.g. 1) (Fig. 5). A similar response, although perhaps not as well differentiated, also occurs in adult rats (Fig. 6) (23). Thus, if adult human subjects show similar patterns of change in the dynamics of whole body amino acid and N metabolism with graded intakes of specific amino acids, distinct alterations in amino acid oxidation rates and/or in whole body protein turnover might occur when the dietary intake level of a given indispensable amino acid changes in the region approximating the minimal physiological need. If so, it should be possible to determine the requirement for the amino acid, using the tracer techniques outlined above. In addition, determination of the intake level of a specific amino acid necessary to support a given net protein synthesis rate might provide another criterion for estimation of the physiological requirement for the amino acid.

A series of experiments currently under way in our laboratories have allowed us to evaluate these possibilities. Thus, our results for the rate of whole body lysine oxidation in young men receiving an amino acid diet supplying graded intakes of lysine are depicted in Fig. 7 and in Fig. 8 results for whole body leucine kinetics in young men receiving graded leucine intakes are shown. These data suggest that a marked change in the dynamics of whole body leucine metabolism, specifically in this case the rate of incorporation into body proteins, occurs at an intake of about 28 mg.kg^-1day^-1 (Fig. 8). For lysine the changes in the oxidation response curve occur at about 35 mg.kg^-1day^-1 (Fig. 7). In contrast to these “breakpoint” type of responses to graded intakes of either leucine or lysine, we did not find this same pattern for the response of whole body valine kinetics to graded intakes of this amino acid, as shown in Figs. 9 and 10 for valine oxidation and incorporation into body proteins, respectively.

The possible significance of these findings for quantifying essential amino acid requirements can be further examined by reference to intakes necessary to achieve a given level of net protein synthesis in subjects during the fed state. Thus for lysine, as shown in Fig. 11, a net protein synthesis occurs only when the intake level is above about 17 mg/kg/day, implying that intakes less than this would be insufficient to maintain body protein equilibrium for the reasons discussed earlier. Assuming, on the basis of the earlier discussion, that a net protein synthesis of about 23 mg kg^-1h^-1 should be sustained during the fed state, the necessary lysine intake would approximate 37 mg.kg^-1.day^-1. On the basis of this criterion, therefore, this represents the physiological requirement for the amino acid.

Plasma amino acid responses were also examined in our studies of whole body amino acid kinetics. By undertaking these measurements it has been possible to interpret the ¹³C-results further and to underscore our view that the current values for requirements for these test amino acids are probably too low. Our reason for measurement of plasma amino acid patterns is that they are known to be influenced by the level and adequacy of the amino acid intake and particularly that marked changes occur in the plasma concentration of the dietary limiting amino acid (29). Indeed plasma amino acid responses have been used as a basis for identifying the limiting amino acids in food proteins and for quantifying protein nutritional quality (29). We found, in our studies, that amino acid-containing meals produce an increase in the plasma concentration of the non-limiting dietary amino acids, as compared to the concentrations in plasma during the fasting state. In the case of the “limiting” amino acid, meal ingestion was associated with an increased concentration of the amino acid in p1 when meals contained an adequate supply of the amino acid. Below a specific dietary level, meal ingestion reduced the concentration of the limiting amino acid below fasting values. The intake level at which this altered plasma amino acid response occurs could be interpreted as a reflection of an inadequate intake and that this may also correspond closely with the minimal physiological requirement for the amino acid. Based on these considerations, results with lysine as the test amino acid (Fig. 12) suggest that the amino acid requirement might approximate 35 mg kg^-1.day^-1. This estimate is also considerably higher than currently accepted values (4). Similarly, measurement of the responses of plasma free leucine and valine levels to alterations in the dietary intake of these branched chain amino acids revealed a marked increase in plasma valine when the leucine intake was reduced below about 30 mg kg^-1day^-1 (Fig. 13) and a fall to a low and constant valine concentration when the dietary supply of this amino acid was restricted to levels below about 20 mg.kg^-1day^-1 (Fig. 14). These intakes of leucine and valine may also reflect levels required for maintenance of a normal physiological state and they correspond grossly to the estimates of requirement intakes based on whole body amino acid kinetics.

SUMMATION OF DATA BASE

By undertaking measurements of whole body amino acid kinetics with the aid of ¹³C-tracers and simultaneously carrying out determinations of plasma amino acid responses, as well as N balance estimates, we have begun to develop a more comprehensive picture of the status of whole body amino acid metabolism for intakes above and below that necessary for maintenance of protein nutriture in healthy adults. Furthermore these data strongly support the concept that this approach is useful for a further examination of the physiological requirements for indispensable amino acids. A summary of our multiple metabolic parameter approach for assessment of the lysine requirement is illustrated in Table 4. Interpretation of the combined data leads to the conclusion that the recommendation made by Rose (21) (see Table 1) of the lysine requirement is quite probably highly inadequate.

The number of test amino acids we have examined so far is limited and based only on a small series of experiments, each involving few subjects and studied under highly controlled conditions that are quite different from normal free-living circumstances. This criticism applies equally to the data base on which the FAO/WHO (4) and U.S. Food and Nutrition Board (18) requirements have been established. Thus, we cannot yet be precise as to the minimum physiologic requirements for amino acids in healthy young men as approached in this new way. However, based on our assumptions and interpretations of the data, we summarize in Table 5 estimates of these requirements using various criteria and compare them with those proposed in 1971 by FAO/WHO (4). From this table the FAO/WHO (4) recommendations, that were derived largely from Rose (21), appear to be too low, possibly to the extent of a factor of about 2 to 3 in the case of the amino acids we have re-examined so far. Thus it is tempting to suggest that the current view about the relative rate of change in the amounts of total protein and indispensable amino acids required for the various age groups, as shown in Fig. 15, may not be correct and that in the future this picture may require modification, as also indicated in this figure.

Finally, it should be recognized that our observations deal specifically with the responses of various parameters of whole body nitrogen and amino acid metabolism in healthy well-nourished men to changes in protein and amino acid intake during relatively short experimental diet periods. Thus our findings may be indicative only of the metabolic adaptations that occur when the diet is changed from that to which our experimental subjects have long been accustomed. In this case it could be argued that it would be premature or inappropriate to use the data to establish a dietary intake that is just sufficient to maintain, over the long-term, the health and well-being of a given free-living population group. Without adequate epidemiological data to assist our interpretation, this problem cannot be resolved, and, furthermore, it is an issue common to the quantitative definition of human requirements for all known essential nutrients. Thus, we are faced with a serious dilemma but we choose to interpret our data in the form of a working hypothesis. Our purpose is to better understand the nutritional and health significance of the protein and amino acid component of human diets.

CONCLUSIONS

The minimum physiological requirements for the indispensable amino acids in adult humans have been determined largely from results of short-term N-balance determinations. The limitations of this approach have been discussed briefly in this review. We have argued that these limitations may lead to a gross underestimation of the minimum physiological requirement for maintenance of protein nutriture and that new and alternative approaches must be developed in order to establish more definitively minimum intake levels that are consistent with maintenance of adequate protein status in healthy individuals. Studies in our laboratories concerned with the quantitative determination of whole body amino acid kinetics, using ¹³C-tracer techniques, are described with the purpose of emphasizing their possible value in developing a novel method for determining requirements for essential amino acids. The results of our initial studies support the view that study of dynamic aspects of whole body protein and amino acid metabolism will aid an improved understanding of the ways by which amino acid homeostasis is achieved in human subjects under differing nutritional conditions and directly lead to more reliable estimates of nutrient requirements. Furthermore, an interpretation of our preliminary studies leads to the conclusion that the minimum physiological requirements for leucine, valine and lysine in adult humans may be about two to three times higher than the currently accepted values; the latter derived from studies in which N balance has served as the sole criterion of adequacy of amino acid intake. Additional studies on the quantitative aspects of the whole body metabolism of indispensable and dispensable (non-essential) amino acids, studied with the aid of stable isotope tracer approaches and in subjects under various dietary conditions must be carried out. This will lead to a more complete assessment of the quantitative significance of indispensable amino acids in the protein nutrition of healthy adults and those suffering from disease.

ACKNOWLEDGEMENT

We thank our colleagues, especially Dr. J.F. Burke, Massachusetts General Hospital, and the volunteer subjects who have helped us explore many exciting aspects of whole body amino acid metabolism in the human subject. The unpublished results discussed in this paper were obtained with the support of NIH grants AM15856, RR88, and GM21700 and USDA Grant 5901–0410–8–0066–0. We are grateful to the Ajinomoto Company, USA, for generously providing L-amino acids for use in our metabolic studies.

REFERENCES

1. Bergner, H., Simon, O., and Adam, K. 1978. Lysinbedarfsbestimmung bei wachsenden Ratten anhand der Katabolisierungsrate von ¹⁴C- und ¹⁵N- markiertem Lysin. Arch. Tierernahrung 28:21–29.

2. Brookes, I.M., Owens, F.N., and Garrigus, U.S. 1972. Influence of amino acid level in the diet upon amino acid oxidation by the rat. J. Nutr. 102:27–36.

3. Calloway, D.H. 1975. Nitrogen balance of men with marginal intakes of protein and energy. J. Nutr. 105:914–923.

4. FAO/WHO. 1973. Energy and protein requirements. WHO technical rept. ser. No. 522. World Health Organization, Geneva.

5. Fujita, Y., Yamamoto, T., Rikimaru, T., and Inoue, G. 1979. Effect of low protein diets on free amino acids in plasma of young men: effect of a wheat gluten diet. J. Nutr. Sci. Vitaminol. 25:427–439.

6. Garlick, P.J., Clugston, G.A., Swick, R.W., and Waterlow, J.C. 1980. Diurnal pattern of protein and energy metabolism in man. Am. J. Clin. Nutr. 33:1983–1986.

7. Garza, C., Scrimshaw, N.S., and Young, V.R. 1976. Human protein requirements: the effect of variations in energy intake within the maintenance range. Am. J. Clin. Nutr. 29:280–287.

8. Garza, C., Scrimshaw, N.S., and Young, V.R. 1977. Human protein requirements: A long-term metabolic nitrogen balance study in young men to evaluate the 1973 FAO/WHO safe level of egg protein intake. J. Nutr. 107:335–352.

9. Hegsted, D.M. 1963. Variation in requirements of nutrients---amino acids. Fed. Proc. 22:1424–1430.

10. Hegsted, D.M. 1976. Balance studies. J. Nutr. 106:307–311.

11. Inoue, G., Fujita, Y. and Niiyama, Y. 1973. Studies on protein requirements of young men fed egg protein and rice protein with excess and maintenance energy intakes. J. Nutr. 103:1673–1687.

12. Irwin, M.I., and Hegsted, D.M. 1971. A conspectus of research on amino acid requirements of man. J. Nutr. 101:539–566.

13. Kang-Lee, T.A., and Harper, A.E. 1977. Effect of histidine intake and hepatic histidase activity on the metabolism of histidine in vivo. J. Nutr. 107:1427–1443.

14. Kang-Lee, T.A., and Harper, A.E. 1978. Threonine metabolism in vivo: effect of threonine intake and prior induction of threonine dehydratase in rats. J. Nutr. 108:163–175.

15. Matthews, D.E., Motil, K.J., Rohrbaugh, D.K., Burke, J.F., Young, V.R., and Bier, D.M. 1980. Measurement of leucine metabolism in man from a primed, continuous infusion of L–[1–13C] leucine. Am. J. Physiol. 238:E473–E479.

16. Motil, K.J., Matthews, D.E., Bier, D.M., Burke, J.F., Munro, H.N., and Young, V.R. 1981. Whole body leucine and lysine metabolism: response to dietary protein intake in young men. Am. J. Physiol. 240:E712–E721.

17. Munro, H.N. 1972. Amino acid requirements and metabolism and their relevance to parenteral nutrition. In: Parenteral Nutrition (ed. A.W. Wilkinson) pp. 34–67. Churchill Livingstone, London.

18. NRC. 1974. Improvement of protein nutriture. National Academy of Sciences, Washington, D.C.

19. Rose, W.C., Lambert, G.F., and Coon, M.J. 1954. The amino acid requirements of man. VII. General procedures; the tryptophan requirement. J. Biol. Chem. 211:815–

20. Rose, W.C., and Wixom, R.L. 1955. The amino acid requirements of man. XVI. The role of the nitrogen intake. J. Biol. Chem. 217:997–1004.

21. Rose, W.C. 1957. The amino acid requirements of adult man. Nutr. Abstr. Rev. 27:631–647.

22. Shenkin, A., and Wretlind, A. 1978. Parenteral nutrition. World Rev. Nutr. Diet. 28:1–111.

23. Simon, O., Adam, K., and Bergner, H. 1978, Stoffwechselorientierte Lysinbedarfsbestimmung bei ausgewachsenden Ratten anhand der Katabolisierungsrate von ¹⁴C- und ¹⁵N-markiertem Lysin. Arch. Tierernahrung 28:609–617.

24. Steffee, C.H., Wissler, R.W., Humphreys, E.M., Bendett, E.P., Woolridge, R.D., Cannon, P.R. 1950. Studies in amino acid utilization. V. The determination of minimum daily essential amino acid requirements in protein-depleted adult male albino rats. J. Nutr. 40:483–497.

25. Tontisirin, K., Young, V.R., Miller, M., and Scrimshaw, N.S. 1973. Plasma tryptophan response curve and tryptophan requirements of elderly people. J. Nutr. 103:1220–1228.

26. Waterlow, J.C., Garlick, P.J., and Millward, D.J. 1978. Protein turnover in mammalian tissues and in the whole body. pp. 805- . North Holland Publishing Co., Amsterdam.

27. Weller, L.A., Calloway, D.H., and Margen, S. 1971. Nitrogen balance of men fed amino acid mixtures based on Rose's requirements, egg white protein and serum free amino acid patterns. J. Nutr. 101:1499–1507.

28. Young, V.R., and Munro, H.N. 1973. Plasma and tissue tryptophan levels in relation to tryptophan requirements of weanling and adult rats. J. Nutr. 103:1753–1763.

29. Young, V.R., and Scrimshaw, N.S. 1978. Nutritional evaluation of proteins and protein requirements. In: Protein Resources and Technology. (eds. M. Milner, N.S. Scrimshaw, and D.I.C. Wang). pps. 136–173.

30. Young, V.R., Meguid, M., Meredith, C., Matthews, D., and Bier, D.M. 1981. Recent developments in knowledge of human amino acid requirements. In: Nitrogen Metabolism in Man. (eds. J.C. Waterlow and J.M.L. Stephen). Applied Science Publishers, London. (In press).

31. Young, V.R., Robert, J.-J., Motil, K., Matthews, D., Bier, D.M. 1981. Protein and energy intake in relation to protein turnover in man. In: Nitrogen Metabolism in Man. (eds. J.C. Waterlow and J.M.L. Stephen). Applied Science Publishers, London. (In press).

¹Partial summary of Table VI in Shenkin and Wretlind (22).
²Values based on Munro (17).
³Estimation by Shenkin and Wretlind based on studies by Steffee et al. (24) in protein-depleted rats.

¹ Abstracted from Rose et al. (19).
² Refers to L or DL mixture of the amino acid.

¹ Rose and Wixom (20).Diet provided the safe intake of eight indispensable amino acids.
² N balance (gN per day)

¹ According to Munro (17).
² Estimated upper range of individual requirements.
³ Unpublished results. See text for additional considerations.

FIGURE LEGENDS

WHOLE BODY TURNOVER (fed state) IN YOUNG MEN RECEIVING GENEROUS PROTEIN INTAKES

Fig 1

WHOLE BODY TURNOVER (fed state) IN YOUNG MEN RECEIVING AMINO ACID DEFICIENT DIETS

Fig 2

NET PROTEIN SYNTHESIS AT A “SAFE” INTAKE OF PROTEIN

Fig 3

NET PROTEIN SYNTHESIS (FED STATE) AND NITROGEN BALANCE

Fig 4

LYSINE INTAKE AND LYSINE OXIDATION IN YOUNG RATS

Calculated from : H. BERGNER et al, Arch. Tierern¨hrung 28 : 21 (1978)

Fig 5

LYSINE INTAKE AND LYSINE OXIDATION IN ADULT RATS

Calculated from : O. SIMON et al, Arch. Tierenönrung 28:609 (1978)

Fig 6

LYSINE OXIDATION RATE FOR VARIOUS DIETARY LEVELS OF LYSINE

Fig 7

[1–¹³C]-LEUCINE INCORPORATION INTO BODY PROTEINS:RELATION TO LEUCINE INTAKE IN YOUNG MEN

Fig 8

PERCENT [1-¹³C]-VALINE FLUX OXIDIZED RELATION TO VALINE INTAKE IN YOUNG MEN

Fig 9

[1–¹³C] VALINE INCORPORATION INTO BODY PROTEINS: RELATION TO VALINE INTAKE IN YOUNG MEN

Fig 10

PROTEIN SYNTHESIS MINUS BREAKDOWN IN THE FED STATE FOR VARIOUS DIETARY LEVELS OF LYSINE

Fig. 11

PLASMA LYSINE/TOTAL AMINO ACIDS RATIO FOR VARIOUS DIETARY LEVELS OF LYSINE

Fig 12

Fig 13

PLASMA VALINE AND LEUCINE CONCENTRATIONS IN RELATION TO VALINE INTAKE IN YOUNG MEN (Fed State)

Fig 14

RELATIONSHIP BETWEEN PROTEIN AND INDISPENSABLE AMINO ACID NEEDS

Fig 15