What is apparent is not always real: lessons from lysine requirement studies in adult humans.

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 C-leucine, using a C-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 consum1 To whom correspondence should be addressed. E-mail: vryoung@mit.edu. 2 Abbreviatons: EAR, estimated average requirement; IAAB, indicator amino acid balance; IAAO, indicator amino acid oxidation; PPU, postprandial protein utilization; UNU, United Nations University.