|Food and Nutrition Bulletin Volume 17, Number 3, 1996 (UNU Food and Nutrition Bulletin, 1996, 104 pages)|
|Protein and amino acid requirements|
|Human amino acid requirements: A re-evaluation|
Lysine is the first limiting amino acid in most cereal-based diets characteristic of populations in certain areas of the developing world , and it is likely that the protein nutritional quality of these diets could be improved by increasing the supply and availability of lysine Therefore, it is highly important to resolve the uncertainties concerning the minimum physiological requirement for this indispensable amino acid. Twenty-four-hour C-labelled amino acid tracer studies comparable to those described above for leucine and phenylalanine, using both C-lysine and C-leucine tracers and estimations of their body amino acid balances as criteria for evaluating the lysine requirements of healthy adults, are now under way in our laboratories. However, they have not yet reached the point at which we are able to confirm our proposed tentative requirement estimate of 30 mg/kg/day (table 2) a value significantly higher than the FAO/WHO/UNU  figure of 12 mg/kg/day. Nevertheless, it is worthwhile overviewing the status of our current knowledge about the minimum requirements for lysine in healthy adults, particularly because it is consistent with the tentative requirement value that we propose.
Various criteria and methods have been used to estimate the lysine requirement in healthy adults. The nitrogen balance studies carried out by Rose et al.  and those by later investigators in men and women [10, 19], including our own nitrogen balance experiments [38, 39], are in general similar (table 5). These give estimates of the mean minimal lysine requirement on the order of 9 to 12 mg/kg/day. It might be noted that the FAO/WHO/UNU  value of 12 mg/kg/day was taken from the 1973 FAO/WHO report , and was derived from the upper range of the individual requirements found in men and women. Our own data on nitrogen balance are not inconsistent with these values, except that we estimated a nitrogen balance derived requirement figure to be somewhat higher, namely 17 mg/kg/day, when the nitrogen balance data included an estimate of miscellaneous and unmeasured nitrogen losses [37, 38]. These similar findings are not unexpected, because it might be anticipated that nitrogen balance studies carried out in different laboratories should give comparable estimates of the minimum needs for dietary lysine However, the apparent consistency in the data in no way validates the technique or the derived estimates of the requirements for specific amino acids. Indeed, we have illustrated previously why we believe some of the classical balance experiments, and those by Rose et al.  in particular, would lead to erroneous requirement estimates .
TABLE 5: Estimates of the lysine requirement in healthy adults
|Rose et al. ||9 |
|N balance (mean; n = 5) |
safe intake (double minimum)
|FAO/WHO/UNU ||12||N balance; upper level for group|
|MIT studies to date |
[37, 38. and this article]
|~ 12 |
> 15 < 80
(17 with miscellaneous) |
13C-lysine; short-term fed
plasma amino acid responses
diurnal retention (after Millward et al. )
current 24-h 13C kinetics (incomplete)
|Zello et al. ||37||indicator amino acid oxidation|
|Duncan et al. ||40||indicator amino acid oxidation|
On the basis of earlier short-term C-lysine tracer infusion studies, we estimated the mean lysine requirement as approximately 30 mg/kg/day ; our factorial prediction gave a somewhat higher value of 42 mg/kg/day . Furthermore, preliminary results from our recent 24-hour C-lysine kinetic studies indicate that the minimal requirement for lysine is likely to be much greater than the FAD/WHO/UNU value . However, we have not completed a sufficient number of these C-lysine studies to establish what minimal level of lysine intake might be sufficient to maintain body lysine balance or protein nutritional status over the long term.
A group in Toronto has used the so-called indicator amino acid oxidation technique for determining amino acid requirements [39, 40]. This approach is based on the concept that the incorporation of an indispensable amino acid into proteins or its oxidation is dependent on the level of intake of the most limiting amino acid (see
Using the oxidation of L-[13C]phenylalanine  as the indicator amino acid, Zello et al.  concluded that "the lysine requirement of adult males is three times greater than the World Health Organization recommendation of 12 mg/kg/day." Recent studies by this group  have confirmed their previous estimate of 40 mg/kg/day, which is consistent with, although somewhat higher than, our new tentative estimate of 30 mg/kg/day.
Two additional lines of evidence can be used in support of our view that the lysine requirement value for adults is considerably higher than that derived from nitrogen balance studies and, in consequence, the recommended values proposed earlier by national  and international [9, 11] expert groups. The first is in reference to the response of plasma free lysine concentrations to changes in the level of lysine intake. The reason for exploring this aspect of lysine metabolism is that plasma amino acid concentrations are known to be influenced by the level and adequacy of the amino acid intake, with particularly marked changes occurring for the limiting dietary amino acid [44, 45]. Indeed, plasma amino acid changes have been used as a basis for identifying the limiting amino acids in food proteins and for quantifying protein nutritional quality [45-47]. In our previous studies, we found that amino acid-containing meals produce an increase in the plasma concentration of the non-limiting indispensable amino acids in the diet, as compared with the concentrations found during the fasting (post-absorptive) state. In the case of the "limiting" amino acid, meal ingestion was associated with an increased concentration of the amino acid in plasma only when the meal provided an adequate supply of this amino acid. Below a given dietary level, meal ingestion reduced the concentration of the limiting amino acid below that of the fasting blood values. Hence, the intake level at which a changeover in the prandial/post-absorptive plasma amino acid response occurs may well correspond closely to the minimum physiological requirement. Thus, with lysine as the "limiting," or test, amino acid, the observed plasma lysine response was as shown in figure 2 (see
The second additional line of evidence is based on considerations of the diurnal cycle of feeding and fasting, resulting in gains and losses of body proteins throughout the day in subjects who are in overall nitrogen balance. This area of protein and amino acid metabolism has been explored [49, 50], with the suggestion that diurnal cycling has important implications for understanding the metabolic basis of the protein requirement . A recent study examined experimental work on nitrogen and amino acid homeostasis in relation to the determination and evaluation of post-prandial protein utilization (PPU) in the human adult . This investigation represents a conceptual development that is quite valuable, in our opinion, and may also be used to further assess and develop an approximation of the minimum lysine requirement in adult subjects.
PPU is defined as protein deposition plus post-absorptive losses/intake . For milk as the test dietary protein source, a value of 0.86 was obtained from metabolic studies . Further, Millward and Pacy  have estimated from the results of our studies with amino acid-based diets  that when subjects receive the FAO/WHO/UNU  amino acid requirement pattern, the PPU is 0.53. This compares with higher values of 0.74 and 0.70 for amino acid-based diets supplying the MIT and egg patterns, respectively. Again, these calculations imply that the FAO/WHO/UNU  amino acid requirement values are inferior to those represented by the new, tentative MIT amino acid requirement pattern for adults.
We can apply the concepts that Millward and his colleagues have developed, concerning the magnitude of the post-prandial retention of protein necessary to balance subsequent post-absorptive losses, to estimate a minimum requirement level of lysine. This estimate is based on the following line of reasoning. First, it is accepted that, for the purposes of this argument, the minimum needs for total protein in adults can be estimated, as illustrated in figure 3 (see
There is a cohesive though limited body of data indicating that the mean requirement value for lysine in adults is probably in the region of 30 mg/kg/day (table 5). When expressed per unit of the mean protein requirement, the lysine requirement pattern would be 50 mg per gram of protein. Again, this far exceeds the FAO/WHO/UNU  value of 16 mg lysine per gram of protein. The question now arises, therefore, as to whether there are additional experimental data to support this new tentative requirement value of 50 mg lysine per gram of protein.