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Laboratory of Human Nutrition, MIT, Cambridge, MA, and * Division of Nutrition, St. John’s Medical College, Bangalore, India
1To whom correspondence should be addressed. E-mail: vryoung{at}mit.edu.
The importance of obtaining reliable estimations of the quantitative requirements for the indispensable amino acids, especially of lysine, in human protein nutrition has been articulated (1 ,2 ). However, there has been a great deal of discussion and also disagreement about the methods used for determining the estimated requirements (3 –7 ). Tracer-based experimental approaches have gained preference over the use of earlier nitrogen balance techniques, which provided the basis for the FAO/WHO (8 ) and FAO/WHO/United Nations University (UNU) (9 ) international recommendations for amino acid requirements and dietary amino acid requirement patterns. Nevertheless, Millward and co-workers (10 ) recently criticized earlier tracer-based estimations of the lysine requirement in healthy adults, including those involving the 24-h direct (11 ,12 ) and indicator amino acid oxidation (IAAO) methods (13 ,14 ) as well as those based on the short-term, fed-state IAAO technique (15 ,16 ). Hence, these investigators (10 ) determined an "apparent requirement" for lysine in subjects adapted to their usual diets. The determination was based on the oxidation of 13C-leucine, using a 13C-leucine tracer model that involved a single meal of wheat gluten protein, over a 6-h postprandial period. At the end of 6 h, leucine oxidation and plasma concentrations had returned to their premeal values, suggesting that the absorption and initial metabolism of the meal protein was complete.
The postprandial utilization (efficiency of utilization; PPU) of the nitrogen (N) intake was calculated as the ratio of the N utilized in protein deposition (measured as the cumulative difference between the postmeal leucine oxidation rate and the premeal oxidation rate, with a conversion to an N retention value based on an assumed body tissue protein leucine:N ratio) and the N intake supplied by the meal. Thus, the N utilized in this case is assumed to be the equivalent of "leucine intake less the meal-dependent leucine oxidation." Also, assuming that the utilization of wheat gluten is limited by its lysine content, the requirement for lysine was then calculated from the ratio of an assumed average requirement for good quality protein [0.6 g/(kg · d)] and the PPU, with the lysine content of the largely wheat gluten protein meal being taken to be 18.7 mg/g wheat protein. On the basis of this approach, Millward et al. (10 ) concluded that the daily lysine requirement for healthy subjects, adapted to their usual and presumably adequate diet, is 18.3 mg/(kg · d). This interpretation of the data raises two critical issues: 1) the derivation of this so-called "apparent" requirement value; and 2) whether it is appropriate for the authors to conclude that their study "indicates an average lysine requirement of 18.3 mg lysine per day... ." In doing so, they link this estimate to the amino acid requirement values in United Nations reports (8 ,9 ), used for the evaluation of food protein quality, diet adequacy and for the planning of food supplies and diets.
We examine here the assumptions and approach used by Millward et al. (10
) and show how difficult it is to draw, from their model, any firm conclusions about the physiologic requirements for lysine in healthy adults. Thus, with respect to the first issue above, there are problems with both the reasoning and the specific assumptions made by Millward et al. (10
) in applying their PPU approach. It is impossible to estimate exactly how much of the leucine oxidized during the fed period is due specifically to exogenous and endogenous sources of leucine. Therefore, it is not possible to measure precisely the PPUnitrogen for the N present in the test meal. Thus, for example, the assumption might be made that essentially all of the leucine oxidized in the fed state is derived from the exogenous leucine (wheat protein). In this case, a recalculated PPU would be 0.2, and the lysine "requirement" would be 
58 mg/(kg · d). However, this is an unlikely metabolic condition because wheat gluten N is retained with a higher efficiency (17
) than this calculation would imply. Hence, it is likely that the "apparent lysine requirement" as determined by the present PPU approach would be less than this relatively high, upper estimate.
Millward and co-workers (10
) recognized that the rate of endogenous protein breakdown and, in turn, of endogenous leucine oxidation declines with ingestion of a protein-containing meal. We reviewed our data (unpublished) generated on leucine kinetics in well-nourished, Indian subjects given a protein intake of 1 g/(kg · d), with differing levels of leucine intake [50 and 107 mg/(kg · d); n = 60] and with all other indispensable amino acids given in adequate amounts. Our analysis indicates a 29% reduction in protein breakdown with the feeding of small frequent meals. Studies by other investigators, using the same kinetic model and tracer, have reported similar results. Melville et al. (18
) observed a 65% reduction in protein breakdown postprandially in subjects receiving 1.5 g protein/(kg · d). Motil et al. (19
,20
) reported 29 and 49% decreases in postprandial protein breakdown in adults consuming 0.6 and 1.3 g protein/(kg · d), respectively. In another short-term leucine kinetic study of normal Indian adults receiving 1.1 g protein/(kg · d), the postprandial protein breakdown was reduced by 36% (21
). In children fed 4 g protein/(kg · d), the postprandial protein breakdown was reduced by 34% (22
). Hence, a reasonable assumption is that there is a 35% reduction in protein breakdown with ingestion of a complete meal. If so, then this would lead to a higher calculated value for the postmeal-dependent excess leucine oxidation [by 7 mg/(kg · 6 h)] and, thus, a lower PPU for wheat nitrogen, or 0.45, according to our calculation. This recalculated PPU, along with an estimated average requirement (EAR) for protein of 0.6 g/(kg · d) would then give a requirement for wheat protein of 1.32 g/(kg · d), and an apparent requirement for lysine of 25 mg/(kg · d). In this calculation, the intake of tracer leucine [3 mg/(kg · d)] is ignored, as was also done by Millward et al. (10
). Further support for this recalculation comes from a study of postprandial milk protein oxidation by Gaudichon et al. (23
), who found that 15–20% of the protein-based amino acid intake was oxidized, leading to a PPUnitrogen for milk protein of 0.85. This compares with the higher estimation of the PPUnitrogen for milk of 0.93 made by Millward et al. (10
) and in part probably indicates the approximate error involved in estimating the meal-derived N utilization by this method.
A further assumption made by Millward et al. (10
) is that the bicarbonate recovery factor was constant for the last 5 h of the fed state. This factor was based on data, reported in an abstract, from an hourly, small meal feeding experiment (24
), rather than from an experiment that mimicked the single large meal protocol used by Millward et al. (10
). Direct experiments at MIT on the recovery of bicarbonate using a large meal feeding paradigm have shown that the recovery of bicarbonate increases from 75% in the fasted state to 90% immediately after the meal begins, and then declines gradually such that it is near premeal level 3 h after beginning the meal (25
). If this reduction in the recovery of bicarbonate also applies to the study by Millward et al. (10
), this would tend to raise the estimate of lysine requirement; a reduction in the mean recovery of bicarbonate over 6 h, from the assumed value used of 0.9 to that of 0.85 or 0.8, would lead to an increase in the apparent requirement for lysine of 2–3 mg/(kg · d). Similarly, if the tracer given during the postabsorptive state was oxidized rather than retained, this would result in a further increase in the estimated rate of meal-dependent oxidation and thus the estimate of requirement. These and a number of other problems related to 13C isotopic measurements of amino acid oxidation rates were reviewed by Millward (26
) but they were not critically considered in the evaluation of their recent experiment (10
). However, we (27
) recently determined, in the case of a leucine tracer given during the postabsorptive period, that the tracer is not oxidized and is thus presumably retained in the free amino acid pool with its later utilization for protein synthesis or oxidation when a mixture of meal-derived amino acids is absorbed.
The EAR for protein used in the calculations by Millward et al. (10 ) for estimating the lysine requirement is 0.6 protein/(kg · d). However, a recent analysis of a large body of published N balance data leads to an EAR of 0.66 g/kg for good quality protein (28 ). If this new figure is used to estimate the lysine requirement in the example above, it would further increase the apparent lysine requirement value (Table 1 ), although only slightly.
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Thus, the conclusion drawn by Millward et al. (10 ), that the value of 18.3 mg lysine/(kg · d) is an average apparent requirement is based on a set of questionable assumptions. As argued above and depending on the assumptions, the value might well be close to 30 mg lysine/(kg · d) (Table 1) . Millward et al. (10 ) criticized our 24-h IAAO and indicator amino acid balance (IAAB) studies of the lysine requirement (14 ) because of the estimated positive leucine balance values obtained. It might be pointed out, however, that an analysis of the pattern of the 12-h fed state and the 24-h leucine oxidation rate, as well as the daily absolute leucine balance, with changes in lysine intake all gave similar requirement estimates. Furthermore, in contrast to the positive IAAB (13C-leucine balances) obtained in our earlier studies, which we consider resulted from the high leucine intake given to subjects in that study (14 ), a recent experiment from our laboratories (30 ) using the same approach but with leucine intakes that were closer to requirement levels, provided a similar lysine requirement estimate of 30 mg/(kg · d), while simultaneously showing that the daily leucine balances were at equilibrium when lysine intakes were at or above the requirement level of lysine intake. The point about the value of the use of the pattern of change in oxidation for estimating the requirement by the indicator amino acid oxidation approach is also underscored by the recent short-term IAAO study by Kriengsinyos et al. (31 ). These investigators showed that although the absolute rate of oxidation of the 13C-phenylalanine indictor was higher when it is given orally compared with the intravenous rate, the estimated lysine requirement was virtually identical.
Finally, although our recalculations of the data of Millward et al. (1 ) provide an "apparent" lysine requirement value that is similar to the lysine requirement value obtained from our tracer studies and close to requirement estimates derived by the Toronto group (15 ,16 ) it should not be concluded that the PPU model, as applied in this investigation by Millward et al. (10 ), is necessarily suitable for measuring the physiologic requirement for lysine in healthy adults. We believe that the experiment, more specifically, provided an approximate estimate of the lysine retention following a wheat gluten-containing meal when given to healthy subjects who were fully adapted to their usual and generous intakes of both protein and lysine. The relationship between this lysine retention figure and a physiologic lysine requirement value, which we would define as the lowest continuing intake of lysine that is sufficient to maintain body lysine equilibrium, was not determined in that study nor by these investigators in their earlier, similar experiment (32 ). Indeed, we (30 ) were critical of the approach used in the latter study (32 ) as well, in which the PPU of the N intake was calculated as the slope of the line relating N balance (derived from 13C-leucine tracer balance) to N intake. However, the postprandial expansion of the free leucine pool was unaccounted for when calculating the retention of protein-bound leucine (balance). This, in turn, would have resulted in an overestimate of the efficiency of N utilization from wheat protein and an underestimate of the apparent requirement for lysine. Additionally, it is to be appreciated that the Millward model is based on a single level of protein/lysine intake with extrapolation of the findings to an "apparent" requirement intake level. It would be highly desirable to validate such an extrapolation by conducting studies at multiple intake levels of lysine.
In our opinion, these short-term protocols based on the metabolic demand and PPU concept (10
,32
) illustrate, more importantly, the ability of tissues to recycle during the prandial period the lysine released into the free lysine pool via proteolysis during the postprandial/fasting phase of amino acid metabolism. Additionally, they imply that this recycling can occur with a high degree of efficiency when a single meal deficient in lysine is consumed. It would be of considerable interest to assess the quantitative rates of leucine oxidation and utilization as in the present experiment but in subjects who were adapted to different and lower lysine intakes before the tracer study. The longer-term, steady-state, nutritional significance of a so-called "apparent" lysine requirement value of 18.3 mg/(kg · d) is not evident. In our view, the PPU model, without further validation and as applied in this (10
) and an earlier study (32
) in which the "apparent" lysine requirement was estimated to be 22 mg/(kg · d), does not provide a satisfactory basis for nor a suitable alternative to the 24-h IAAO and IAAB methods (14
,30
,33
) for establishing an adult amino acid requirement scoring pattern. Nevertheless, research efforts, such as those by Millward and his colleagues (10
,32
) and the Toronto group (31
,34
), directed toward the development of less complex validated tracer paradigms for determination of amino acid requirements are to be encouraged. In the meanwhile, it might be noted that the Institute of Medicine/Food and Nutrition Board (35
) recently proposed a value of 31 mg/(kg · d) (47 mg lysine/g protein) as the mean requirement for lysine in healthy adults. This estimate, which is 2.5 times greater than that proposed in 1985 by FAO/WHO/UNU (9
), would be appropriate to use for planning and/or assessing amino acid (lysine) intakes (36
) and for evaluation of dietary protein quality in adults (8
,35
).
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FOOTNOTES |
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Manuscript received 28 November 2002. Initial review completed 19 December 2002. Revision accepted 31 December 2002.
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LITERATURE CITED |
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13. 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-h [13C]leucine kinetics, of the lysine requirement of healthy adult Indian subjects. Am. J. Clin. Nutr. 67:58-66.[Abstract]
14. Kurpad, A. V., Raj, T., El-Khoury, A., Beaumier, L, Kuriyan, R., Srivatsa, A., Borgonha, S., Selvaraj, A., Regan, M. & Young, V. R. (2001) Lysine requirement of healthy adult Indian subjects, measured by an indicator amino acid balance technique. Am. J. Clin. Nutr. 73:900-907.
15. Zello, G. A., Pencharz, P. B. & Ball, R. O. (1993) Dietary lysine requirement of young adult males determined by oxidation of L-[1-13C]phenylalanine. Am. J. Physiol. 264:E677-E685.
16. Duncan, A. M., Ball, R. O. & Pencharz, P. B. (1996) Lysine requirements of adult males is not affected by decreasing dietary protein. Am. J. Clin. Nutr. 64:718-725.
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19. Motil, K. J., Bier, D. M., Matthews, D. W., Burke, J. F. & Young, V. R. (1981) Whole body leucine and lysine metabolism studied with [1-13C] leucine and [
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20. Motil, K. J., Matthews, D. W., Bier, D. M., Burke, J. F., Munro, H. N. & Young, V. R. (1981) Whole body leucine and lysine metabolism: response to dietary protein intake in young men. Am. J. Physiol. 240:E712-E721.
21. Macallan, D. C., McNurlan, M. A., Kurpad, A. V., DeSouza, G., Shetty, P. S., Calder, A. G. & Griffin, G. E. (1998) Whole body protein metabolism in human pulmonary tuberculosis and undernutrition: evidence for anabolic block in tuberculosis. Clin. Sci. (Lond.) 94:321-331.[Medline]
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25. El-Khoury, A. E., Sanchez, M., Fukagawa, N., Gleason, R. E., Tsay, R. H. & Young, V. R. (1995) The 24-h kinetics of leucine oxidation in healthy adults receiving a generous leucine intake via three discrete meals. Am. J. Clin. Nutr. 62:579-590.
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28. Rand, W. M., Pellett, P. L. & Young, VR (2003) Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults. Am. J. Clin. Nutr. 77:109-127.
29. Raguso, C. A., El-Khoury, A. E. & Young, V. R. (1999) Leucine kinetics in reference to the effect of the feeding mode as three discrete meals. Metabolism 48:1378-1386.[Medline]
30. Kurpad, A. V., Regan, M. M. & Raj, T. et al. (2002) Lysine requirement of healthy adult Indian subjects receiving long-term feeding, measured with a 24h indicator amino acid oxidation and balance technique. Am. J. Clin. Nutr. 76:404-412.
31. Kriengsinyos, W., Wykes, L. T., Ball, R. O. & Pencharz, P. B. (2002) Oral and intravenous tracer protocols of the indicator amino acid oxidation method provide the same estimate of the lysine requirement in healthy men. J. Nutr. 132:2251-2257.
32. Millward, D. J., Fereday, A., Gibson, N. R. & Pacy, P. J. (2000) Human adult amino acid requirements: [1-13C]leucine balance evaluation of the efficiency of utilization and apparent requirements for wheat protein and lysine compared with those for milk protein in healthy adults. Am. J. Clin. Nutr. 72:112-121.
33. Kurpad, A. V., Raj, T. & Regan, M. et al (2002) Threonine requirements of healthy Indian men, measured by a 24-h indicator amino acid oxidation and balance technique. Am. J. Clin. Nutr. 76:789-797.
34. Bross, R., Ball, R. O. & Pencharz, P. B. (1998) Development of a minimally invasive protocol for the determination of phenylalanine and lysine kinetics in humans during the fed state. J. Nutr. 128:1913-1919.
35. IOM/FNB (Institute of Medicine/Food and Nutrition Board) (2002) Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Protein and Amino Acids. A Report of the Panel on Macronutrients, Subcommittee on Upper Reference Levels of Nutrients and Interpretation and Uses of Dietary Reference Intakes and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (uncorrected prepublication version) 2002 The National Academy Press Washington, DC.
36. IOM/FNB (Institute of Medicine/Food and Nutrition Board) (2000) Dietary Reference Intakes. Applications in Dietary Assessment. A Report to the Subcommittee on Interpretation and Uses of Dietary Reference Intakes and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes 2000 The National Academy Press Washington, DC.
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