QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kurpad, A. V. Articles by Gnanou, J. V. Search for Related Content PubMed PubMed Citation Articles by Kurpad, A. V. Articles by Gnanou, J. V.

---

Branched-Chain Amino Acids: Metabolism, Physiological Function, and Application: Session III

Branched-Chain Amino Acid Requirements in Healthy Adult Human Subjects^1–4,

Anura V. Kurpad^*,5, Meredith M. Regan, Tony Raj^* and Justin V. Gnanou^*

^* Institute of Population Health and Clinical Research, St. John's National Academy of Health Sciences, Bangalore, India, and Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA 02139–4307

⁵ To whom correspondence should be addressed. E-mail: a.kurpad{at}iphcr.res.in.

ABSTRACT

There is now an expanding body of evidence to recommend, in the case of adult humans, the use of revised indispensable amino acid requirement values; these are 2 to 3 times higher than the current international recommendations. The earlier methodologies for determining amino acid requirements, based on nitrogen balance, were criticized because of their design and the associated high energy intakes. The 1985 World Health Organization/Food & Agriculture Organization/United Nations University requirement for leucine has been demonstrated to be too low by short- and long-term (24-h) tracer-derived estimates of leucine oxidation and balance. The best values for leucine requirements come from 24-h direct amino acid oxidation (DAAO) and direct amino acid balance (DAAB) studies. Finally, we also collated all available data from studies on fed-state leucine oxidation with an adequate dietary adaptation period to assess the inflection on the leucine oxidation–leucine intake curve. The mean requirements for leucine, valine, and isoleucine are likely to be 40, 17–25, and 19 mg · kg^–1 · d^–1, respectively. This adds up to a total of 84 mg · kg^–1 · d^–1, which is much lower than the lowest estimate of the total BCAA requirement of 110 mg · kg^–1 · d^–1 made by the short-term indicator amino acid oxidation (IAAO) method, which determined the BCAA requirement from the pattern of oxidation of an indicator amino acid (phenylyalanine) at different levels of BCAA intake. An additional estimate of the leucine requirement was also made by a meta-analysis of all available 24-h DAAO/DAAB data from different studies. This resulted in a higher value for the leucine requirement than that obtained by the specific studies that utilized the 24-h DAAO/DAAB approach; however, even adding this value to the total BCAA requirement does not account for the difference in the total BCAA requirement estimates and the summed individual BCAA estimates.

KEY WORDS: • BCAA requirement • direct amino acid oxidation • direct amino acid balance • indicator amino acid oxidation • indicator amino acid balance

There is now an expanding body of evidence to recommend, in the case of adult humans, the use of revised indispensable amino acid (IAA)⁶ requirement values (1); these are 2 to 3 times higher than the current international recommendations (2). The earlier methodologies for determining amino acid requirements based on nitrogen balance were criticized because of their design and the associated high energy intake (3). A further problem was related to the exclusion of the miscellaneous nitrogen losses when calculating nitrogen balance (see below). Young et al. (4) proposed a new set of amino acid requirement values calculated from estimates of obligatory nitrogen loss (ONL), based on the pattern of IAA in tissue protein mobilized to provide for the ONL. The use of tracer methodologies in the next phase of studies (e.g., 5,6) also suggested that the daily requirement for BCAAs was greater than the 1985 World Health Organization (WHO)/Food & Agricultural Organization (FAO)/United Nations University (UNU) value (2). Particularly for leucine, the short-term tracer balance findings were then confirmed in longer term 24-h tracer balance studies in western subjects (7,8) and Indian subjects (9), where leucine equilibrium was achieved at a leucine intake of 40 mg · kg^–1 · d^–1, but not at an intake of 14 mg · kg^–1 · d^–1. Estimates of the total BCAA requirement have also been made by a tracer technique used in an indicator amino acid oxidation (IAAO) paradigm (10), as described below, and these estimates of the BCAA requirement, when portioned into the constituent amino acids, also suggest higher requirements than those in the 1985 WHO/FAO/UNU report. A recently convened Expert Committee of the WHO/FAO/UNU (11) has also reviewed the evidence available from recent tracer studies and concluded that BCAA requirements are higher than set out in the earlier 1985 WHO/FAO/UNU report, as discussed below.

THE REQUIREMENT FOR LEUCINE

From the earlier nitrogen balance studies (12) and studies in women (13), it was concluded that the mean requirement for leucine approximated 10 mg · kg^–1 · d^–1 or less (Table 1). An important difficulty in accurate measurement of nitrogen balance is determining the integumental and other minor or unmeasured routes (miscellaneous) of nitrogen loss; these were not measured in the earlier nitrogen balance studies. A reanalysis of nitrogen balance studies in adults, using a correction factor of 0.5 g of nitrogen as the daily integumental loss, increased the derived leucine requirement values severalfold (14); however, a similar reassessment, using an assumed +0.3-g nitrogen daily miscellaneous loss gave generally lower values for amino acid requirements; for leucine, this was 26 mg · kg^–1 · d^–1 (15). It is not possible to state the magnitude of the miscellaneous nitrogen loss with any confidence; therefore, the nitrogen balance method does not provide a result for the leucine requirement with any certainty. The nitrogen balance is also highly sensitive to changes in energy intake and, at high energy intake, could account for an underestimation of the requirement. These problems have been reviewed earlier with reference to the earlier nitrogen balance studies (3,4,15,16). An alternative theoretical paradigm of IAA requirements was proposed in 1989 (4) based on an estimate of the minimal obligatory nitrogen loss (ONL) and the intake of IAA necessary to balance this loss, predicted from the composition of mixed body proteins, with the assumption that the efficiency of absorption of the IAA was 70%. Based on these considerations, the leucine requirement was calculated to be nearly 40 mg · kg^–1 · d^–1 (Table 1; 17,18).

View larger version (11K): [in this window] [in a new window]   FIGURE 1  Different patterns of responses of tracer oxidation or balance to graded intakes of test amino acid. The lines represent kinetic responses to graded intakes of test amino acid. The point of inflection of each line represents the requirement for the test amino acid. Both IAAB and DAAB would be expected to plateau at a zero balance. For IAAO, either whole-body indicator amino acid oxidation, or a surrogate in the form of the proportion of tracer oxidized, can be used. DAAO, short-term or 24-h direct amino acid oxidation; DAAB, 24-h direct amino acid balance; IAAO, short-term fed-state or 24-h indicator amino acid balance; IAAB, 24-h indicator amino acid balance.

Earlier tracer studies involving the short-term DAAB technique suggested that the mean requirement fell in the range of 20–40 mg · kg^–1 · d^–1 (5; Table 1) and these data, when reanalyzed through a breakpoint analysis, yielded a value of 25 mg · kg^–1 · d^–1 (23). The 24-h DAAB tracer studies by El-Khoury (7,8) confirmed that the WHO/FAO/UNU (2) requirement of 14 mg · kg^–1 · d^–1 leucine was too low and their results gave a requirement value of 38 mg · kg^–1 · d^–1, thereby also supporting the prediction of a leucine requirement of 40 mg · kg^–1 · d^–1. More recently, Kurpad et al. (9,17), using four test intake levels of leucine and the 24-h DAAB approach, have estimated the mean leucine requirement to be 37 and 40 mg · kg^–1 · d^–1 in well-nourished and undernourished Indian subjects, respectively (Table 1).

Another approach that has been used to measure the leucine requirement is the IAAO technique (24; Fig. 1). Thus, the requirement for an IAA (e.g., lysine) is determined from the pattern or rate of change in oxidation of another (indicator) amino acid (e.g., ¹³C-phenylalanine). This method, which is deployed in the fed state over short periods of time, also uses the measurement of a surrogate for whole-body IAAO by measuring the proportion of tracer oxidized (F¹³CO₂). There are a number of advantages to this short-term IAAO approach, including 1) the possibility of carrying out a relatively large number of relatively short-term tracer studies within the same subject; 2) problems arising from changes in pool sizes and kinetics that might affect the behavior of a direct tracer are largely avoided when an indicator amino acid tracer is used; and 3) there is no a priori reason to determine the actual rate of IAAO because the pattern of release of the ¹³C label in expired air can be assessed through a breakpoint analysis on the intake–oxidation response curve. This pattern of ¹³C appearance, which represents the proportion of tracer oxidized (F¹³CO₂), should, in theory, parallel that for the absolute oxidation rate of the indicator according to the concept on which this approach is based. Based on the determination of the total BCAA requirement by the short-term IAAO technique, Riazi et al. (10) derived a leucine requirement value of 47–55 mg · kg^–1 · d^–1 based on the proportion of leucine in an egg protein–based amino acid mix that was fed to the study subjects. The range of requirement values in this study was because different values within this range were derived depending on which outcome measure of indicator amino acid equilibrium was used (F¹³CO₂, phenylalanine oxidation, or balance). These values could have been overestimated by 10% (18) and therefore the range of values of leucine requirement derived from the short-term IAAO measurement of the total BCAA requirement is probably from 42–50 mg · kg^–1 · d^–1. These are still higher than estimates from the 24-h DAAO/DAAB studies and, as has been pointed out above, one criticism of the latter studies is that they did not assess leucine balance at leucine intake above the putative requirement value of 40 mg · kg^–1 · d^–1. An argument against this is that, in an indicator amino acid balance (IAAB) paradigm, the breakpoint should ideally occur at a zero leucine balance and therefore the regression estimate of the zero balance intercept should be identical to the breakpoint estimate.

Leucine requirement from a meta-analysis of 24-h leucine balance studies

We attempted a meta-analysis of the leucine requirement by 24-h DAAO/DAAB evidence by collecting data on those studies in which there was a defined 1-wk period of experimental dietary intake in human adult subjects with adequate levels of energy and nitrogen intake, and in which there were adequate levels of intake of all amino acids (adequacy defined by the requirement pattern that was accepted by the recent WHO/FAO/UNU Expert Committee on Protein and Amino Acid Requirements; 11). Therefore, the 24-h leucine balance data were collated from all of the 24-h leucine balance studies conducted in the US (7,8) and India (9,25–29) on well-nourished healthy young adult male subjects. A total of eight studies were therefore identified that had the requisite 24-h leucine balance data at otherwise adequate energy, IAA, and protein intakes. In these studies, leucine balance was measured at different intakes; collating these yielded leucine balance data at leucine intakes of 14, 22, 30, 40, 50, 90, and 105 mg · kg^–1 · d^–1 (Fig. 2, filled circles). Because the study at the highest leucine intake (26; Fig. 2, open square) yielded balances that were far too positive for probable reasons discussed below, the data from this study were deleted from the database from which the breakpoint on the leucine balance–leucine intake curve was calculated. The breakpoint estimate for the leucine requirement was determined to be 47.6 mg · kg^–1 · d^–1 with lower and upper 95% CI of 42.2 and 55.5 mg · kg^–1 · d^–1, respectively. This breakpoint occurred at a slightly positive balance of 1.7 mg · kg^–1 · d^–1, whereas the zero intercept value (leucine intake value at which the leucine balance was zero on the ascending slope line) was 44.4 mg · kg^–1 · d^–1. It is not clear as to which estimate (breakpoint or zero balance) of the leucine requirement is correct. Ideally, they would be expected to be the same and the small positive balance at the breakpoint may have been due to technical reasons; it is possible that the calculation of leucine balance from intake and oxidation is not completely accurate because the tracer-based estimate of oxidation does not capture all the leucine loss from the body. For example, there may be some intestinal loss of metabolic leucine, which would not be captured by the tracer model. In contrast, if the positive balances were due to subjects accreting protein, it is unlikely that this small anabolic gain over a week could be assessed by anthropometric measures such as body weight or even body composition. The subjects in these studies did not gain weight and, in the Indian studies, actually lost 0.2–0.3 kg at the end of a week on the experimental diet. This was attributed to the loss of body glycogen and associated water through the measurement of substrate balances (17).

View larger version (13K): [in this window] [in a new window]   FIGURE 2  Twenty-four-hour leucine balance in adult human subjects measured from 13C-leucine-derived estimates at different daily leucine intakes. Data are mean ± SD 24-h leucine balances from 24-h studies in western and Indian subjects. Leucine intake was calculated as the sum of dietary leucine plus 24-h tracer. (Data taken from Refs. 7–9, 17, 25–29.) More than one leucine intake was assessed in each study and some leucine intakes were repeated in different studies; collating these gave leucine balance data at leucine intakes of 14, 22, 30, 40, 50, 90, and 105 mg · kg–1 · d–1, with a total of 11 observations from seven studies for well-nourished subjects and four observations from one study in undernourished subjects. In the undernourished subjects, leucine balance was measured at four intakes of 14, 22, 30, and 40 mg · kg–1 · d–1. Filled circles, 24-h balances in well-nourished, healthy subjects (7–9,25–29). Open triangles, 24-h balance data in chronically undernourished subjects (17). Open square, 24-h balance data in one study on well-nourished subjects; these data not included in breakpoint analysis (26). Solid line, breakpoint estimation from well-nourished data alone (7–9,25,27–29). Dotted line, breakpoint estimation from well-nourished and undernourished data (7–9,17,25,27–29).

Interestingly, adding the leucine balance data of undernourished Indian subjects (17) into the database (Fig 2, open triangles) and then estimating the breakpoint gave a higher estimate for the leucine requirement than when only the data on the well-nourished subjects were subjected to breakpoint analysis. The breakpoint for the leucine requirement occurred at a leucine intake of 50.2 mg · kg^–1 · d^–1, with 95% CI of 43.1 and 58.1 mg · kg^–1 · d^–1. The zero balance intercept also occurred at a higher value of 45.6 mg · kg^–1 · d^–1, and this was higher than the leucine requirement of 39 mg · kg^–1 · d^–1 (95% CI = 32.56 mg · kg^–1 · d^–1) measured in chronically undernourished Indian subjects by the 24-h DAAB method, using a zero balance intercept instead of a breakpoint (17). Two points are noteworthy here: first, the slope of the 24-h leucine balance response to graded leucine intakes was shallower with the combined dataset because the mean balances tended to be less negative in the undernourished subjects, particularly at lower leucine intakes, and it is possible that the shallower slope of the leucine balance response influenced the breakpoint and zero intercept analysis of the data, such that a higher estimate was obtained. Second, the higher leucine requirement estimate with the mixed undernourished and well-nourished database is reminiscent of a higher lysine requirement in undernourished subjects measured by the 24-h IAAO/IAAB technique (31), although, in this case, it should be emphasized that the estimate was from a mixed group. Certainly, the undernourished subjects could accrete protein under ideal dietary (and activity) conditions as experienced during the dietary adaptation period, where adequate energy, nitrogen, and micronutrients were provided because their muscle mass (as a proportion of body weight) was lower than that of well-nourished subjects (17).

In addition to the 24-h tracer studies, we also attempted an assessment of the breakpoint on the 12-h fed leucine oxidation vs. leucine intake curve derived from several short- and long-term studies. To do so, we collated data from 24-h (as above) and short-term fed-state leucine oxidation studies (5,32–37). In the short-term studies, the reported leucine oxidation was extrapolated to a 12-h value and the leucine intake was taken as the total from the diet and the projected 24-h tracer intake. We chose to use the 24-h tracer intake because this would allow for the 24-h studies (9,25–29) and the short-term studies to be used in the same dataset. Where necessary, these studies were corrected for the use of the enrichment of plasma leucine as the precursor pool instead of keto-isocaproic acid (5,35,36) by the use of a factor of 0.8 (19). A total of 16 short- and long-term studies employing different leucine intake levels in each study were therefore identified for this analysis, for a total of 47 observations. The data from the studies are presented in Figure 3; it was not possible to achieve a breakpoint estimate because these did not converge and the relation was best described by a straight-line equation. However, in Figure 3 it might be seen that, from an intake of 40 mg · kg^–1 · d^–1, there was a more obvious increase in leucine oxidation.

View larger version (15K): [in this window] [in a new window]   FIGURE 3  Twelve-hour fed-state leucine oxidation rates at different leucine intakes. Data are mean ± SD. Fed-state leucine oxidation in short-term (2–5 h) and long-term (24 h) studies. (Data taken from references as noted in text and represent mean values taken at different intakes from Refs. 5,7–9,25–29,32–37.) A total of 48 observations were made from 16 studies. Oxidation data from all short-term studies were extrapolated to 12-h values. Leucine intake was calculated as the sum of dietary (12-h fed) leucine plus 24-h tracer. No breakpoint evaluation was possible. The straight line describing the relation had the equation: 12-h fed-state leucine oxidation (mg · kg–1 · d–1) = 0.5. * Daily leucine intake (mg · kg–1 · d–1) + 45.

The requirement for valine

The earlier nitrogen balance studies on the valine needs in healthy adults (38,39) suggested requirements in the region of 10 mg · kg^–1 · d^–1 for valine (Table 2; 40); therefore, the 1985 WHO/FAO/UNU (2) daily requirement for valine was set at 10 mg · kg^–1 · d^–1. As referred to earlier, the nitrogen balance technique has several disadvantages and reanalyses of the nitrogen balance data by Hegsted (14) and Millward (15) gave valine requirement estimates of 17 and 14 mg · kg^–1 · d^–1, respectively. Based on the ONL model (see above), Young et al. (4) suggested a value of 24 mg · kg^–1 · d^–1 for the daily valine requirement. Because the BCAAs share common enzymes in their oxidative pathways, the valine requirement can also be theoretically estimated by an assumed proportionality with the leucine requirement based on the amino acid composition of body protein (41); such a procedure yields a value of 26 mg · kg^–1 · d^–1 when a value of 40 mg · kg^–1 · d^–1 is used for the leucine requirement (9,18). The response of plasma valine concentrations to different levels of valine intake has also suggested a requirement value of 20 mg · kg^–1· d^–1 (6).

The disadvantages of the IAAO method as usually conducted are that it has been based essentially on a short-term fed-state model without any significant prior adaptation of the subjects to the test diets. To circumvent some of the limitations of the short-term IAAO technique, a 24-h IAAO/IAAB approach has been developed (25; Fig 1) and applied in ¹³C-leucine tracer studies of the lysine, threonine, and methionine requirement of adult Indian subjects (25–29) and in studies of the threonine requirement in US adults (42). The approach is similar in concept to that of the IAAO technique, but is based on a 24-h IAAO–daily balance protocol using leucine (for which the kinetics are well described) as the indicator amino acid. It has the advantage of providing a direct estimate of 24-h IAAO and IAAB. The disadvantage of the 24-h IAAO and IAAB approaches relates to the complexity of the 24-h tracer study and possibly the rather stringent demands and restraints that it places on the experimental subject.

A recent 24-h IAAO/IAAB study, using phenylalanine as the indicator amino acid, in well-nourished young adult Indians determined the valine requirement to be 17 mg · kg^–1 · d^–1, with 95% CI of 12 to 28 mg · kg^–1 · d^–1 (40). This study assessed phenylalanine balances at a zero tyrosine intake, using a protocol that had earlier obtained fairly neutral phenylalanine balances at an intake of 39 mg · kg^–1 · d^–1 phenylalanine on a tyrosine-free diet (43). Because the labeled tyrosine formed from the hydroxylation of labeled phenylalanine tracer is partitioned into oxidation and protein synthesis, this partitioning could be variable depending on the needs of protein synthesis. In this respect, the provision of a tyrosine-free diet does not reduce the conversion of phenylalanine into tyrosine and could add an additional variability to determination of the breakpoint estimate from estimates of phenylalanine oxidation. It is not possible to predict whether this would have changed the estimate of the valine requirement in this study, but it can also be assumed that the net phenylalanine oxidation and balance under a defined and constant set of conditions would effectively serve as an indicator of valine equilibrium. In addition, this study also measured the valine requirement by the short-term IAAO tracer technique by measuring the fraction of tracer oxidized, similar to the method used in the short-term IAAO method. Using this latter approach gave a numerically higher requirement value of 20 mg · kg^–1 · d^–1, with 95% CI of 12 to >35 mg · kg^–1 · d^–1. Given the large CIs, it is quite likely that an expensive study involving a very large number of subjects would be required to actually determine whether there was a significant difference between the 24-h and the short-term methods.

The valine requirement in the 24-h IAAO/IAAB study was similar to that suggested from earlier DAAO studies at different valine intakes (6). However, this estimate was lower than that predicted from the total BCAA requirement by the short-term IAAO technique (10), or by the obligatory amino acid losses (OAAL) prediction (4). It is not possible to state with any certainty what the reason for this discrepancy is. One possibility is that the short-term IAAO study (10) determined the total BCAA requirement and from that predicted the valine requirement based on a similar proportionality in the amino acid composition of the fed protein. A similar higher valine requirement value is predicted from the OAAL method based on the same principle (4), and it may be that the prediction of the valine requirement based on an assumed proportionality of the BCAA in body protein is unwarranted. A second possibility is that the short-term IAAO technique is conducted on relatively unadapted subjects compared with the 24-h IAAB technique. Whereas studies on pigs suggest that the 1-d experimental dietary period in the short-term IAAO technique may be adequate for adaptation to the diet (44), it is still not clear whether the cause for the difference between these two estimates of the valine requirement is related to the adaptation period to the experimental diet. For example, if a dietary effect of the habitual diet persisted into the experimental period, then this would, in the simplest interpretation, push the requirement estimate upward, assuming that the habitual dietary intake had generous amounts of the test amino acid. A third possibility is the composition of the intake of the other BCAAs during the adaptation period of the 24-h IAAO/IAAB study. In this case, the leucine intake was held at the maintenance requirement value of 40 mg · kg^–1 · d^–1. This is not a safe intake that would meet the needs of 95% of the population and it is conceivable that this intake may have been suboptimal for some of the subjects in this study and hence might have been responsible for a lower valine requirement. However, as discussed below, it has been shown that varying leucine intake in the range supplied by normal diets (40 to 80 mg · kg^–1 · d^–1) had no effect on valine oxidation (45) and hence it is possible that the concern about the level of leucine intake is unwarranted.

Overall, the valine requirement measured by the IAAB technique (which might be considered to be the best estimate available) of 17 mg · kg^–1 · d^–1 is lower than what is expected from proportionality with the leucine requirement, based on the composition of mixed-body proteins. It is also lower than the indirect estimate obtained from total BCAA requirement estimates by the IAAO method (10,18).

THE REQUIREMENT FOR ISOLEUCINE

Based on recalculated data from nitrogen balance studies (12, 46; Table 3), it is possible that the mean requirement for isoleucine may be in the region of 20–30 mg · kg^–1 · d^–1. The prediction of the isoleucine requirement from the ONL gives a value of 23 mg · kg^–1 · d^–1 (4). No tracer studies have been conducted to assess the requirement for isoleucine. The isoleucine requirement can also be predicted from the leucine requirement if it can be assumed that this requirement for isoleucine is in proportion to its relative concentration in body protein compared with that for leucine. Based on the composition of mixed-body protein, the isoleucine requirement, when the leucine requirement is 40 mg · kg^–1 · d^–1, is 19 mg · kg^–1 · d^–1.

Because BCAAs share common membrane transport systems (47,48) and metabolic enzymes (49,50), it is possible that these amino acids can influence each other's kinetics and requirements. For example, in short-term animal experiments, a high (5-fold) leucine intake has been shown to result in an increased valine oxidation, while also increasing the free level of leucine in the plasma and depressing the free levels of the other BCAAs in plasma and tissue amino acid pools (51). However, with excess isoleucine, whereas the plasma isoleucine levels increased dramatically, there was no change in plasma leucine or valine levels (51). Similar findings have been observed in humans, with leucine intake having an effect on plasma concentrations of valine and isoleucine, but with valine and isoleucine intake having little effect on the plasma leucine concentration (52,53) or oxidation (54). Shwenk et al. (55) observed that, whereas isoleucine infusion did not have any effect on leucine metabolism, the infusion of leucine stimulated leucine oxidation and decreased plasma concentrations of other amino acids.

However, whereas these effects of leucine on BCAA metabolism may change their requirement level, it has been shown that varying leucine intake in the range supplied by normal diets (40 to 80 mg · kg^–1 · d^–1) had no effect on valine oxidation (45); in addition, the variation of valine intake did not have any effect as expected from the earlier experiments. This is corroborated by comparing the findings of the recent 24-h IAAB study on valine intake (40) with a requirement level (40 mg · kg^–1 · d^–1) of leucine intake with an earlier short-term DAAO study (6) in the presence of generous amounts (egg protein–based amino acid mixture) of leucine. In these two experiments based on different principles, the valine requirement estimate was very similar even though the amount of leucine supplied in the diet varied 2-fold. Therefore, it appears that there may be no regulatory effect of dietary leucine on valine catabolism within the physiological range of leucine intake.

Summary

The mean requirement for leucine, valine, and isoleucine is likely to be 40, 17–20, and 19 mg · kg^–1 · d^–1, respectively. This adds up to a total of 79 mg · kg^–1 · d^–1, which is much lower than the lowest estimate of the total BCAA requirement made by the short-term IAAO method (10,18). The estimate of the leucine requirement by meta-analysis gave a higher value for the leucine requirement than that obtained by the 24-h DAAO/DAAB approach, but even adding this value to the total BCAA requirement does not account for the difference in the total BCAA requirement estimates and the summed individual BCAA estimates. An unexplored variable that may account for this difference is the length of the adaptation period for which the experimental diet is consumed because this differs by 5 d between the short-term IAAO and the 24-h IAAO/IAAB or 24-h DAAO/DAAB methods. Within the range of normal dietary intake, it is also unlikely that each of the BCAAs exerts a significant influence on the others in terms of their requirements.

FOOTNOTES

¹ Published in a supplement to The Journal of Nutrition. Presented at the conference "Symposium on Branched-Chain Amino Acids" held May 23–24, 2005, in Versailles, France. The conference was sponsored by Ajinomoto USA, Inc. The organizing committee for the symposium and guest editors for the supplement were Luc Cynober, Robert A. Harris, Dennis M. Bier, John O. Holloszy, Sidney M. Morris, Jr., and Yoshiharu Shimomura. Guest editor disclosure: L. Cynober, R. A. Harris, D. M. Bier, J. O. Holloszy, S. M. Morris, Y. Shimomura: expenses for travel to BCAA meeting paid by Ajinomoto USA; D. M. Bier: consults for Ajinomoto USA; S. M. Morris: received compensation from Ajinomoto USA for organizing BCAA conference.

² Supported by the Nestle Research Foundation, Lausanne, Switzerland, and by the National Institutes of Health (grant no. DK 42101).

³ This paper is dedicated to the memory of Vernon R. Young.

⁴ Author Disclosure: No relationships to disclose.

⁶ Abbreviations used: CI, confidence interval; DAAB, direct amino acid balance; DAAO, direct amino acid oxidation; IAA, indispensable amino acids; IAAB, indicator amino acid balance; IAAO, indicator amino acid oxidation; OAAL, obligatory amino acid losses; ONL, obligatory nitrogen loss.

LITERATURE CITED

1. Young VR, Borgonha S. Nitrogen and amino acid requirements; the Massachusetts Institute of Technology amino acid requirement pattern. J Nutr. 2000;130:1841S–9S.

2. World Health Organization (1985) FAO/WHO/UNU Expert Consultation. Energy and protein requirements. Geneva: WHO. [WHO Technical Report No 724, 264 pages].

3. Young VR, Marchini JS. Mechanisms and nutritional significance of metabolic responses to altered adaptation in humans. Am J Clin Nutr. 1990;51:270–89.

4. Young VR, Bier DM, Pellett PL. A theoretical basis for increasing current estimates of the amino acid requirements in adult men with experimental support. Am J Clin Nutr. 1989;50:80–92.

5. Meguid MM, Matthews DE, Bier DM, Meredith CN, Soeldner JS, Young VR. Leucine kinetics at graded leucine intakes. Am J Clin Nutr. 1986;43:770–80.

6. Meguid MM, Matthews DE, Bier DM, Meredith CN, Young VR. Valine kinetics at graded valine intakes in young men. Am J Clin Nutr. 1986;43:781–6.

7. El-Khoury AE, Fukagawa NK, Sanchez M, Tsay RH, Gleason RE, Chapman TE, Young VR. Validation of the tracer-balance concept with reference to leucine: 24-h intravenous tracer studies with L-[1-¹³C]leucine and [¹⁵N-¹⁵N]urea. Am J Clin Nutr. 1994;59:1000–11.

8. El-Khoury AE, Fukagawa NK, Sanchez M, Tsay RH, Gleason RE, Chapman TE, Young VR. The 24h pattern and rate of leucine oxidation, with particular reference to tracer estimates of leucine requirements in healthy adults. Am J Clin Nutr. 1994;59:1012–20.

9. Kurpad AV, Raj T, El-Khoury AE, Kuriyan R, Maruthy K, Borgohna S, Chandukudlu D, Regan MM, Young VR. The daily requirement for, and splanchnic uptake of, leucine in healthy adult Indian subjects. Am J Clin Nutr. 2001;74:747–55.

10. Riazi R, Wykes LJ, Ball RO, Pencharz PB. The total branched-chain amino acid requirement in young healthy adult men determined by indicator amino acid oxidation by use of L-[1-¹³C]phenylalanine. J Nutr. 2003;133:1383–9.

11. Working Groups FAO. http://www.fao.org/es/ESN/nutrition/groups.htm (accessed: 22 July 2005).

12. Rose WC, Eades CH, Jr, Coon MJ. The amino acid requirements of man. XII. The leucine and isoleucine requirements. J Biol Chem. 1955;216:225–34.

13. Leverton RM, Ellison J, Johnson N, Pazar J, Schmidt F, Geschwerder D. The quantitative amino acid requirements of young women. I. Leucine. J. Nutr. 1956;58:355–65.

14. Hegsted DM. Variation in requirements of nutrients—amino acids. Fed Proc. 1963;22:1424–30.

15. Millward DJ. Metabolic demands for amino acids and the human dietary requirement: Millward and Rivers (1988) revisited. J Nutr. 1998;128:2563S–76S.

16. Young VR. McCollum Award Lecture. Kinetics of human amino acid metabolism: nutritional implications and some lessons. Am J Clin Nutr. 1987;46:709–25.

17. Kurpad AV, Regan MM, Raj T, Varalakshmi S, Gnanou J, Thankachan P, Young VR. Leucine requirement and splanchnic uptake of leucine in chronically undernourished adult Indian subjects. Am J Clin Nutr. 2003;77:861–7.

18. Riazi R, Rafii M, Wykes LJ, Ball RO, Pencharz PB. Valine is the first limiting branched chain amino acid in egg protein. J Nutr. 2003;133:3533–9.

19. Matthews DE, Schwarz HP, Yang RD, Motil KJ, Young VR, Bier DM. Relationship of plasma leucine and -keto isocaproate during a L 1-¹³C leucine infusion in man: a method for measuring human intracellular tracer enrichment. Metabolism. 1982;31:1105–12.

20. Harris RA, Kobayashi R, Murakami T, Shimomura Y. Regulation of branched-chain -keto acid dehydrogenase kinase expression in rat liver. J Nutr. 2001;131:841S–5S.

21. Shimomura Y, Obayashi M, Murakami T, Harris RA. Regulation of branched-chain amino acid metabolism: nutritional and hormonal regulation of activity and expression of the branched-chain alpha-keto acid dehydrogenase kinase. Curr Opin Clin Nutr Metab Care. 2001;5:419–23.

22. Kurpad AV, Regan MM, Raj T, Maruthy K, Gnanou J, Young VR. Intravenously infused ¹³C-leucine is retained in fasting healthy adult men. J Nutr. 2002;132:1906–8.

23. Pencharz PB, Ball RO. Different approaches to define individual amino acid requirements. Annu Rev Nutr. 2003;23:101–16.

24. Zello GA, Wykes LJ, Ball RO, Pencharz PB. Recent advances in methods of assessing dietary amino acid requirements for adult humans. J Nutr. 1995;125:2907–15.

25. Kurpad, AV, El-Khoury AE, Beaumier L, Srivatsa A, Kuriyan R, Raj T, Borgonha S, Ajami AM, Young VR. An initial assessment using 24 hour ¹³C-leucine kinetics, of lysine requirements of adult men. Am J Clin Nutr. 1998;67:58–66.

26. Kurpad AV, Raj T, El-Khoury AE, Beaumier L, Kuriyan R, Srivatsa A, Borgonha S, Selvaraj A, Regan MM, Young VR. Lysine requirements of healthy adult Indian subjects, measured by an indicator amino acid balance technique. Am J Clin Nutr. 2001;73:900–7.

27. Kurpad AV, Regan MM, Raj T, El-Khoury AE, Kuriyan R, Vaz M, Chandukudlu D, Venkataswamy VG, Borgohna S, Young VR. Lysine requirements of healthy adult Indian subjects receiving long-term feeding, measured with a 24-h indicator amino acid oxidation and balance technique. Am J Clin Nutr. 2002;76:404–12.

28. Kurpad AV, Raj T, Regan MM, Vasudevan J, Caszo B, Nazareth D, Young VR. Threonine requirements of healthy Indian adults, measured by a 24-h indicator amino acid oxidation and balance technique. Am J Clin Nutr. 2002;76:789–97.

29. Kurpad AV, Regan MM, Varalakshmi S, Vasudevan J, Gnanou J, Raj T, Young VR. Daily methionine requirements of healthy Indian men, measured by a 24-h indicator amino acid oxidation and balance technique. Am J Clin Nutr. 2003;77:1198–205.

30. El-Khoury AE, Sanchez M, Fukagawa NK, Gleason RE, Tsay RH, Young VR. The 24-h kinetics of leucine oxidation in healthy adults receiving a generous leucine intake via three discrete meals. Am J Clin Nutr. 1995;62:579–90.

31. Kurpad AV, Regan MM, Raj T, Vasudevan J, Kuriyan R, Gnanou J, Young VR. Lysine requirement of chronically undernourished adult Indian subjects, measured by the 24-h indicator amino acid oxidation and balance technique. Am J Clin Nutr. 2003;77:101–8.

32. Marchini JS, Cortiella J, Hiramatsu T, Chapman TE, Young VR. Requirements for indispensable amino acids in adult humans: longer term amino acid kinetic study with support for the adequacy of the Massachusetts Institute of Technology amino acid requirement pattern. Am J Clin Nutr. 1993;58:670–83.

33. Cortiella J, Matthews DE, Hoerr RA, Bier DM, Young VR. Leucine kinetics at graded intakes in young men: quantitative fate of dietary leucine. Am J Clin Nutr. 1988;48:998–1009.

34. Hiramatsu T, Cortiella J, Marchini JS, Chapman TE, Young VR. Source and amount of dietary nonspecific nitrogen in relation to whole body leucine, phenylalanine and tyrosine kinetics in young men. Am J Clin Nutr. 1994;59:1347–55.

35. Young VR, Gucalp C, Rand WM, Matthews DE, Bier DM. Leucine kinetics during three weeks at submaintenance-maintenance intakes of leucine in men: adaptation and accommodation. Hum Nutr Clin Nutr. 1987;41:1–18.

36. Motil KJ, Bier DM, Matthews DE, Burke JF, Young VR. Whole body leucine and lysine metabolism: response to dietary protein intake in young men. Am J Physiol. 1981;240:E712–21.

37. Pelletier V, Marks L, Wagner DA, Hoerr RA, Young VR. 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. 1991;54:402–7.

38. Rose WC, Wixom RL, Lockhart HB, Lambert GF. The amino acid requirements of man. XV. The valine requirement; summary and final observations. J Biol Chem. 1955;217:987–95.

39. Leverton RM, Gram MR, Brodovsky E, Chaloupka M, Mitchell A, Johnson N. The quantitative amino acid requirements of young women. II. Valine. J. Nutr. 1956;58:83–93.

40. Kurpad AV, Regan MM, Raj T, Gnanou J, Rao V, Young VR. The daily valine requirement of healthy adult Indians determined by the 24-h indicator amino acid balance approach. Am J Clin Nutr. 2005;82:373–9.

41. Widdowson EM, Southgate DAT, Hey EN. Body composition of the fetus and infant. In: Visser HKA, editor. Nutrition and metabolism of the fetus and infant. London: Nijhoff; 1979. p. 169–77.

42. Borgonha S, Regan MM, Oh SH, Condon M, Young VR. Threonine requirement of healthy adults, derived with a 24-h indicator amino acid balance technique. Am J Clin Nutr. 2002;75:698–704.

43. Basile-Filho A, El-Khoury AE, Beaumier L, Wang SY, Young VR. Continuous 24-h L-[1-¹³C] phenylalanine and L-[3, 3-²H₂] tyrosine oral-tracer studies at an intermediate phenylalanine intake to estimate requirements in adults. Am J Clin Nutr. 1997;65:473–88.

44. Moehn S, Bertolo RFP, Pencharz PB, Ball RO. Indicator amino acid oxidation responds rapidly to changes in lysine or protein intake in growing and adult pigs. J Nutr. 2004;134:836–41.

45. Pelletier V, Marks L, Wagner DA, Hoerr RA, Young VR. Branched-chain amino acid interactions with reference to amino acid requirements in adult men: valine metabolism at different leucine intakes. Am J Clin Nutr. 1991;54:395–401.

46. Swendseid ME, Dunn MS. Amino acid requirements of young women based on nitrogen balance data. II. Studies on isoleucine and on minimum amounts of the eight essential amino acids fed simultaneously. J Nutr. 1956;58:507–17.

47. Christensen HN. Interorgan amino acid nutrition. Physiol Rev. 1982;62:1193–233.

48. Collarini EJ, Oxender DL. Mechanism of transport of amino acids. Annu Rev Nutr. 1987;7:75–90.

49. Harper AE, Miller RH, Block KP. Branched-chain amino acid metabolism. Annu Rev Nutr. 1984;4:409–54.

50. Block KP. Interactions among leucine, isoleucine and valine with special reference to branched-chain amino acid antagonism. In: Friedman M, editor. Absorption and utilization of amino acids. Boca Raton, FL: CRC Press; 1989. p. 229–44.

51. Block KP, Harper AE. Valine metabolism in vivo: effects of high dietary levels of leucine and isoleucine. Metabolism. 1984;33:559–66.

52. Swendseid ME, Villaldobos J, Figueroa WS, Drenick EJ. The effects of test doses of leucine, isoleucine or valine on plasma amino acid levels. The unique role of leucine. Am J Clin Nutr. 1965;17:317–21.

53. Eriksson S, Hagenfeldt L, Wahren J. A comparison of the effects of intravenous infusion of individual branched-chain amino acids on blood amino acid levels in man. Clin Sci. 1981;60:95–100.

54. Pelletier V, Marks L, Wagner DA, Hoerr RA, Young VR. 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. 1991;54:402–7.

55. Schwenk WF, Haymond MW. Effects of leucine, isoleucine, or threonine infusion on leucine metabolism in humans. Am J Physiol. 1987;253:E428–34.

This article has been cited by other articles:

				T. Raj, U. Dileep, M. Vaz, M. F. Fuller, and A. V. Kurpad Intestinal Microbial Contribution to Metabolic Leucine Input in Adult Men J. Nutr., November 1, 2008; 138(11): 2217 - 2221. [Abstract] [Full Text] [PDF]

				L. Cynober and R. A. Harris Symposium on Branched-Chain Amino Acids: Conference Summary J. Nutr., January 1, 2006; 136(1): 333S - 336S. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kurpad, A. V. Articles by Gnanou, J. V. Search for Related Content PubMed PubMed Citation Articles by Kurpad, A. V. Articles by Gnanou, J. V.