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The requirements estimates in the 1985 FAO/WHO/UNU (1) report were taken directly from the 1973 FAO/WHO report (2). These were based on the nitrogen balance studies by Rose (3) in men, on similar studies by various investigators in women as summarized by Irwin & Hegsted (4), and on both sets of data having been re-analysed by regression of loge intakes on balance by Hegsted (5). As discussed in section 4, since 1985 concerns have been expressed about the derived values, and all now agree that they were certainly too low. While the chosen requirement estimates in the 1973/1985 reports were conservative, being based on the highest values available (in each case, the values from Rose, 3), all reviewers of these studies (e.g. 6-8) have concluded that they are unsatisfactory for a variety of reasons, especially in the studies reported by Rose. The most serious problem in all studies was that no allowance was made for miscellaneous losses, so the values selected in the 1973/1985 reports were certainly too low. A set of higher values calculated with a realistic value for the miscellaneous losses have been reported by Fuller & Garlick (7) and by Millward (8). Also Rand & Young (9) have re-analysed one set of values relating to the lysine requirements. These recalculated higher values represent the best estimates from nitrogen balance studies.
These nitrogen balance estimates are, however, in many cases lower than newer estimates made in stable isotope studies. As argued in section 4, these stable isotope methods involve a number of assumptions in their interpretation and there is, as yet, no complete consensus as to their relative merits. Those studies which involve prior dietary adaptation are logistically difficult and laborious, so only a limited number of intakes can be studied. Thus the estimates obtained are approximate and probably represent conservative overestimates, rather than underestimates, of the true values. Those stable isotope approaches that have not involved prior dietary adaptation have covered a wide range of intakes, but concern has been expressed about the interpretation of the results of such studies. In some cases, stable isotope approaches have been used to estimate the lysine requirement from measurements of the relative utilization of wheat compared with milk protein. The values obtained in these studies nevertheless depend on several assumptions and require cautious interpretation.
Among the stable isotope studies, the Consultation agreed that the most reliable approach involved measurements over an entire 24-hour period representative of a normal day with [sup.13]C tracers which can be reliably interpreted in terms of calculation of oxidation rates after some adaptation to the intakes. Recognizing that this limits the database to [[sup.13]C]leucine studies of lysine, leucine and threonine, all other published studies were considered as sources of variable quality which need to be interpreted cautiously, and judgments were made on final values for recommendations. All of the methods are based on a physiological response to graded intakes of the test amino acid. To best define the requirement level of the test amino acid, a range of intakes must be used, but the logistics of conducting 24-hour tracer balance studies means that in many cases fewer intakes were tested than would have been desirable.
Theoretical predictions of the requirement pattern have also been published based on the obligatory oxidative losses, i.e. the pattern of tissue protein and the magnitude of the obligatory nitrogen losses, although as discussed in section 4, the theoretical basis of such predictions has been questioned, and this approach has not received general support. All agree, however, that these values are a likely guide to the magnitude of one rate-limiting amino acid which determines the magnitude of the obligatory oxidative losses--probably methionine.
Finally, it must be recognized that these new values have not been validated in any entirely satisfactory way, i.e. in long-term studies at the requirement intakes with measurement of body weight, body composition and well-being. Indeed such studies, based on nutritionally complete real food containing the requirement pattern and adequate dispensable nitrogen, would be difficult to design. Only in the case of lysine have attempts been made with studies of the nutritional adequacy of wheat-based diets, which are generally agreed to be lysine-limited. However, while these studies provide useful information on the adequacy of one intake level, they do not enable a requirement intake to be defined.
8.1 Requirements for indispensable amino acids
The indispensable amino acids are leucine, isoleucine, valine, lysine, threonine, tryptophan, methionine, phenylalanine and histidine. Histidine is considered to be an indispensable amino acid because of the detrimental effects on haemoglobin concentrations that have been observed (10) when individuals are fed histidine-free diets.
The requirement for lysine has received most attention given its nutritional importance as the likely limiting amino acid in cereals, especially wheat.
Reported values are shown in Table 16. The Consultation's estimate of the requirement for lysine (30 mg/kg per day) is derived from tracer studies using the 24-hour indicator amino acid method (11, 12), which are considered to provide the best stable isotope data currently available. However, even with these studies the possibility of a value intermediate between 22 mg and 30 mg cannot be ruled out, in line with the most recent of these studies. Intakes of either 30 mg/kg per day or 45 mg/kg per day maintained similar, slightly positive [[sup.13]C]leucine balances in Indian subjects with low body mass index fed their habitual diet after treatment for intestinal parasites (13). Studies with the indicator amino acid oxidation method, with [[sup.13]C]phenylalanine in the fed state only and without prior adaptation to the diet (10, 14-16), have generally indicated higher values (35-45 mg/kg per day) for reasons that are not understood. Two of these latter studies (10, 16) allow a comparison between females and males using the same tracer model. Also, [[sup.13]C]lysine oxidation studies in the fed state only or over 24 hours have yielded values of 20-30 mg/kg per day (17-19).
The 1973/1985 FAO/WHO values for lysine derived from a value of 800 mg/day (3) expressed as 12 mg/kg per day, the highest value of a number of studies on men and women (20-22). The most extensive single nitrogen balance study is that on young women (21), although even in this study intakes are either below (<13 mg/kg per day) or above (>23 mg/kg per day) the predicted requirement value. Recalculation after curve fitting indicates values ranging from 13 mg/kg per day to 36 mg/kg per day according to the fitting model and value chosen for miscellaneous nitrogen loss (5, 7-8). The best estimate from these re-analyses of this nitrogen balance study is about 17 mg/kg per day (95% CI = 14-27 mg/kg per day) based on the current estimate of miscellaneous loss, 5 mg/kg per day (23).
Indirect estimates of the lysine requirement have been reported from [[sup.13]C] leucine balance studies in normal adults, which measure leucine retention from wheat and the efficiency of wheat protein utilization, i.e. postprandial protein utilization. The most secure value, i.e. 23 mg/kg per day, derives from assessment during a small repeated meal protocol at two levels of protein intake, where postprandial protein utilization should reflect only protein utilization (24). A slightly lower value, 18 mg/kg per day, has been reported from calculations of postprandial protein utilization using a large single meal protocol, where the model assumptions are different. This is less secure, since postprandial protein utilization is a measure of the utilization of the whole meal, with leucine balance influenced by energy intake as well as protein (25).
Finally, two long-term nitrogen-balance studies in male college students fed wheat-based diets provide support for a value between 17 mg/kg per day and 30 mg/kg per day. In these studies, diets providing either 0.94 g protein/kg per day (26) or 0.51 to 0.73 g protein/kg per day (27) showed maintenance of nitrogen balance, body weight and fitness with lysine intakes over 2 months of 18 mg/kg per day, or 20-30 mg lysine/kg per day. Although the authors of each of these reports state that the subjects were in energy balance, concern has been expressed over whether the high energy intakes associated with high levels of physical activity could have influenced the outcome (9).
Leucine is the most abundant amino acid in tissue and food proteins but specific demands for non-protein functions have not been identified. Reported requirements are shown in Table 17. The Consultation's estimate of the requirement for leucine (39 mg/kg per day) derives from 24-hour [[sup.13]C]leucine balance studies which indicated leucine requirements of 37 mg/kg per day and 40 mg/kg per day (28, 29). A limitation of these studies is that the highest test dose of leucine fed was 40 mg/kg per day, and thus did not exclude the possibility that the requirement was higher than 40 mg/kg per day. Conversely, others have argued that these values may be overestimates because of the way the tracer and meal leucine intakes were distributed between the fed and fasted state, although a study to investigate this possibility did not identify any design problem (30) and previous 24-hour [13C]leucine balance studies indicated similar values ([approximately equal to] 40 mg/kg per day; 31, 32). Earlier fed-stateonly …