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1 From the Department of Physiology and Nutrition Research Center, St John’s Medical College, Bangalore, India (AVK, TR, RK, MV, DC, and VGV), and the Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA (MMR, AE-K, SB, and VRY).
2 Antoine El-Khoury is deceased. 3 Supported by the Nestlé Foundation, Switzerland, and by NIH grants RR88, DK 42101, and P-30-DK-40561. 4 Address reprint requests to AV Kurpad, Department of Physiology and Nutrition Research Center, St John’s Medical College, Bangalore, India. E-mail: a.kurpad{at}divnut.net. 5 Address correspondence to VR Young, Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA 02139.
ABSTRACT
Background: The mean lysine requirement of healthy Indian subjects was estimated from short-term experimental diet periods to be 29 mg · kg-1 · d-1, which is higher than the 1985 FAO/WHO/UNU upper requirement of 12 mg · kg-1 · d-1.
Objective: Our objective was to confirm our proposed requirement of 29 mg · kg-1 · d-1 by extending the diet period to 21 d and by using 4 test lysine intakes (12, 20, 28, and 36 mg · kg-1 · d-1) and a 24-h indicator amino acid oxidation and balance approach.
Design: During two 21-d diet periods, 18 healthy Indian men were randomly assigned to receive 12 and 28 or 20 and 36 mg lysine · kg-1 · d-1 as part of an L-amino acid diet. At 1800 on days 6 and 20, [13C]leucine was infused over 24 h to assess leucine oxidation and daily leucine balance at each test intake.
Results: Leucine oxidation, balance, and flux did not differ significantly between days 7 and 21. Twenty-four–hour leucine oxidation was lower at lysine intakes of 28 and 36 mg than at 12 and 20 mg. Leucine balances at lysine intakes of 12 and 20 mg were negative and significantly less than equilibrium (P < 0.01) and lower (P < 0.02) than balances at 28 and 36 mg lysine. Two-phase regression analysis indicated a breakpoint at 31 mg lysine · kg-1 · d-1 in the relation between lysine intake and 24-h leucine oxidation and balance.
Conclusions: Full adaptation to a low lysine intake occurs within 7 d. The previously proposed tentative mean lysine requirement for Western subjects of 30 mg · kg-1 · d-1 is confirmed for healthy Indian adults.
Key Words: Indian adults • lysine requirements • 24-h indicator amino acid oxidation • 24-h balance • amino acid oxidation • adaptation • [13C]leucine
INTRODUCTION
There is an expanding body of evidence justifying the recommendation of the proposed, revised indispensable amino acid requirement (1), which is 2–3 times the current international recommendations (2). Support for a revised mean lysine requirement of 30 mg · kg-1 · d-1 (1, 3–5) comes from a series of 24-h [13C]lysine tracer studies in Western subjects (6, 7) and 24-h indicator (leucine) amino acid oxidation (24-h IAAO) and balance (24-h IAAB) studies in Indian subjects (8, 9). These data were generated by giving subjects experimental L-amino acid diets for 1 wk before the tracer studies began. This estimated requirement is entirely consistent with results from other studies that used a short-term IAAO technique (10, 11), from recalculations of earlier nitrogen balance data (12–14), and from studies of postprandial [13C]leucine metabolism (15, 16).
The earlier Indian studies measured dietary lysine adequacy with the use of the 24-h IAAO and IAAB techniques (oxidation and balance) with leucine as the tracer (8, 9). In one of these studies (8), the leucine intake was 40 mg · kg-1 · d-1, whereas in the other study (9) the leucine intake was 107 mg · kg-1 · d-1. These leucine intakes were chosen to represent "requirement" and "generous" leucine intakes, respectively. At the lower, or requirement, leucine intake, the subjects were in zero leucine balance at a lysine intake of 28 mg · kg-1 · d-1, suggesting lysine adequacy at this intake. However, at a higher, generous intake of leucine in the second study (9), the subjects were in distinct positive leucine balance at lysine intakes of either 28 or 36 mg · kg-1 · d-1. Although the pattern of 24-h leucine oxidation (which decreased as lysine intakes increased, to a nadir from 28 to 36 mg lysine · kg-1 · d-1) and 24-h leucine balance (which increased as lysine intakes increased, with a breakpoint and then a constant balance at an intake of 29 mg · kg-1 · d-1) suggest a mean lysine requirement of 29 mg · kg-1 · d-1, the finding of a distinct positive daily leucine balance at higher test lysine intakes was somewhat unexpected for these well-nourished Indian subjects. These findings differed from those of our previous studies with generous intakes of leucine in Western subjects studied at the Massachusetts Institute of Technology (MIT) (17, 18), where leucine balance approximated an equilibrium value. Therefore, the first aim of the present study was to extend and confirm our previous conclusion that leucine (the indicator amino acid) equilibrium is achieved at and above a lysine intake of 29 mg · kg-1 · d-1, but in the present case for subjects receiving a leucine intake of 50 mg · kg-1 · d-1, which is more similar to the proposed requirement (5).
A second possible limitation of our earlier studies of lysine requirements (6–9) is that relatively short-term (ie, 1 wk) diets were used. Previously, we conducted an experiment (19) in which [13C]leucine balances were estimated in adult Western subjects after 1 or 3 wk consumption of an L-amino acid diet based on the 1985 FAO/WHO/UNU (2), the MIT (1), or a hen’s egg protein amino acid pattern. The subjects did not achieve body leucine equilibrium with the 1985 FAO/WHO/UNU pattern, even at the end of the 3-wk experimental period. However, there are no data available about this aspect of adaptation for subjects given a limiting intake of lysine but with an otherwise constant pattern of indispensable amino acid intake. Therefore, the second aim of the present study was to compare 24-h leucine oxidation and 24-h leucine balance at the end of the first and third weeks of experimental diets supplying different lysine intakes.
SUBJECTS AND METHODS
Subjects
Eighteen young men recruited from the student population of St John’s Medical College (Bangalore, India) participated in this experiment. The physical characteristics of the subjects on day 1 of the experiment are given in Table 1. All subjects were in good health as determined by medical history, physical examination, blood cell count, routine blood biochemical profile, and urinalysis. The subjects’ habitual intake of lysine was estimated to be < 60 mg · kg-1 · d-1 on the basis of a 3-d weighed dietary intake record. Subjects who smoked cigarettes, consumed 5 alcoholic drinks/wk, or drank > 6 cups of caffeinated beverages/d were excluded from participation. The purpose of the study and the potential risks involved were explained to each subject. Written consent was obtained from each subject. The Human Ethical Approval Committee of St John’s Medical College approved the research protocol, which was endorsed by the MIT Committee on the Use of Humans as Experimental Subjects.
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TABLE 1 . Characteristics of healthy Indian men studied for their lysine requirements1
Anthropometric measurements
Anthropometric and skinfold-thickness measurements were made on day 0. Subjects were weighed while wearing minimal clothing with the use of a digital scale (Soehnle-Waagen GmbH & Co, Murrhardt, Germany) that had a precision of 0.1 kg. All weights were measured twice and the means were expressed to the nearest 0.1 kg. The heights of shoeless subjects were recorded with the use of a vertically mobile scale (Holtain Ltd, Crymych, United Kingdom) and were expressed to the nearest centimeter. Skinfold-thickness measurements of the biceps, triceps, subscapula, and suprailium were made in duplicate while the subjects were in a standing position, and the mean of each was used for additional calculations. All skinfold-thickness measurements were standardized (20) and carried out to the nearest 0.2 mm with the use of skinfold calipers (Holtain Ltd). The logarithm of the sum of the 4 skinfold thicknesses was used in age- and sex-specific equations (21) to obtain an estimate of body density, from which the percentage body fat was determined (22).
Diet and experimental design
Each subject was studied during 2 separate 21-d diet periods, during which time they consumed a weight-maintaining diet based on an L-amino acid mixture (Table 2). The tracer (intravenous [13C]leucine) experiments were carried out on 2 separate occasions during each 21-d diet period. The first tracer experiment was carried out after a 6-d diet period and the second on day 20 of the experimental diet period. Daily energy intakes were designed to maintain body weight, and the energy requirement was calculated to be 1.6 x basal metabolic rate during the days of feeding and 1.35 x basal metabolic rate on days 7 and 21 (days of the tracer infusion). The subjects were encouraged to maintain their customary levels of physical activity but were asked to refrain from excessive or competitive exercise. The major energy supply was given in the form of a sugar-oil formula and as protein-free, wheat starch cookies (Table 3). Nonprotein energy was provided as fat (43% of energy) and carbohydrate (56% of energy). The main sources of carbohydrate were beet sugar and wheat starch, to achieve a relatively low 13C content in the diet and a steady background in breath 13CO2 enrichment over the 24-h period. Breath 13CO2 enrichments obtained during the leucine tracer studies were corrected to account for the small changes in background 13CO2 output that would have occurred without the [13C]leucine tracer (9).
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TABLE 2 . Composition of the amino acid mixtures used to supply 4 daily lysine intakes
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TABLE 3 . Composition of the experimental diet used to supply adequate energy with 1 of 4 test lysine intakes
The subjects were randomly assigned to receive 2 experimental diets providing either 12 and 28 or 20 and 36 mg lysine · kg-1 · d-1; the order of intake was also randomized. The leucine content of all diets was 50 mg · kg-1 · d-1 (Table 2). The subjects were terminated from the experimental protocol at the end of the second 24-h tracer study (day 21), and the second diet period began within 4–6 wk. During this interval, the subjects consumed a diet of their choice. The L-amino acids were obtained from Ajinomoto USA, Inc (Washington, DC).
All other nutrients consumed during the experimental period were provided in adequate amounts (Table 3). A choline supplement of 500 mg was given daily, and dietary fiber was provided in the form of 20 g psyllium husk (Sat-Isabgol; Charak Pharmaceuticals Ltd, Gujarat, India) when requested by a subject. The total daily food intake was consumed as 3 isoenergetic, isonitrogenous meals (at 0800, 1300, and 2000). Each morning, body weight was measured and vital signs were monitored. All of the subjects’ meals were consumed in the kitchen of the Nutrition Research Center, under supervision of the dietary staff.
Twenty-four–hour tracer-infusion protocol
The primed tracer-infusion protocol was conducted in all subjects according to a standard 24-h design (9). After the subjects consumed their last meal of the day at 1500, the tracer administration began at 1800 on days 6 and 20 and ended at 1800 on days 7 and 21, respectively. Subjects received 10 small isoenergetic, isonitrogenous meals at hourly intervals beginning at 0600 and ending at 1500; together these meals provided the complete 24-h dietary intake for that day). Indirect calorimetry was performed on alternate hours and blood samples were half-hourly for measurement of [13C]-ketoisocaproic acid (KIC) enrichment. Throughout the 24-h study, the subjects remained in bed, in a reclining position, except during sleep when they lay supine. Thus, the 24-h study was divided into two 12-h metabolic periods (fasted and fed); additional details were provided previously (8, 9, 23).
Collection and analysis of breath samples
Three baseline breath samples were collected 30, 15, and 5 min before the 24-h tracer infusion started and then at consecutive half-hourly intervals throughout the 24-h study. Breath gas was collected in a specially designed bag that permitted the removal of dead space air and was transferred into three 10-mL nonsilicon-coated glass tubes (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) with a thin needle (PrecisionGlide, 24G; Becton Dickinson) that was attached to the bag by means of a 3-way tap. When the breath-sample collection coincided with the hourly meals, the breath sample was collected first. The samples were stored at room temperature until isotope ratio mass spectrometry (Europa Scientific, Crewe, United Kingdom) was used to analyze the ratio of 13CO2 to 12CO2 as previously described (8). The increase in breath enrichment after the isotope administration was expressed as atom percent excess (APE). The APE was calculated as the arithmetic difference between the enrichment of each breath sample and the predose baseline breath sample.
Collection and analysis of blood and urine samples
Blood samples were collected at 30-min intervals between 0000 and 2400 of the tracer-infusion period, except between 0600 and 1200, at which times hourly samples were collected because this was the time period when the subjects were sleeping. Three baseline samples were collected 30, 15, and 5 min before administration of the [13C]leucine tracer. Blood samples (5 mL/sample) were collected through a 20-gauge, 5-cm catheter placed into a superficial vein of the dorsal hand or wrist on the nondominant side. The catheter was introduced in an antiflow position to facilitate blood collection while the hand was in a custom-made warming box that was maintained at 65 °C for 15 min before withdrawal of each sample to achieve arterialization of venous blood. The arterialization of the blood sample was checked earlier by measuring hemoglobin saturation; saturation was > 90%. The patency of the vein was maintained by slow infusion of isotonic sodium chloride. Blood samples were drawn into 5-mL syringes and transferred into anticoagulant tubes and centrifuged for 15 min at 1200 x g in a refrigerated centrifuge (4 °C). The plasma was removed and the samples were stored at -80 °C until analyzed in our laboratory for the isotopic enrichment of KIC, according to procedures described previously (9, 17). The isotopic abundance of plasma [13C]KIC was considered to represent enrichment of the intracellular leucine pool (24) that was undergoing leucine oxidation. We validated this assumption previously (17).
Leucine oxidation and balance
Leucine oxidation was computed for consecutive half-hourly intervals to improve the accuracy of the 24-h leucine oxidation value, as previously described (8, 9). The 24-h leucine balance (input - measured output) was computed as follows:
RESULTS
Anthropometry
The mean (± SD) anthropometric indexes of the subjects are summarized in Table 1. The mean body mass index (BMI; in kg/m2) was 20.77 ± 1.64, and the mean percentage body fat was 19.01 ± 4.00%. The subjects in this study were very similar to those in our previous 24-h tracer studies (8, 9). The mean weight of the subjects at the start of the study was 59.7 ± 6.1 kg. There was a clinically small, but significant loss of weight over the 21-d period. The 3-way interaction was not significant, nor was the interaction between lysine intake and tracer-infusion day, suggesting that the trends in weight over time do not differ by lysine intake. However, the interaction between diet period and tracer-infusion day was significant (P = 0.02), suggesting that trends in weight over time differed by diet period, with subjects losing slightly more weight in their second diet period (estimated mean ± SE weight losses: 0.69 ± 0.19 and 1.02 ± 0.19 kg for the first and second 21-d experimental periods, respectively).
Leucine oxidation and balance
Data for the primary variables measured, including carbon dioxide output, breath 13CO2 enrichment, 13CO2 production, and plasma [13C]KIC enrichment at each test lysine intake, were similar to those published previously (8, 9) and thus are not shown here. The data for leucine oxidation, balance, and flux on the infusion days are summarized in Table 4 (for day 7) and in Table 5 (for day 21). As indicated below, statistical analyses are presented for the combined data.
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TABLE 4 . Summary of leucine oxidation, balance, and flux at 4 lysine intakes in healthy Indian men on day 7 of the 21-day diet period1
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TABLE 5 . Summary of leucine oxidation, balance, and flux at 4 lysine intakes in healthy Indian men on day 21 of the 21-d diet period1
There were no significant interactions with, or main effects of, tracer-infusion day on leucine oxidation; therefore, the final statistical model included lysine intake, metabolic period, and the interaction (P = 0.023). The mean (± SD) values calculated from the model without regard to tracer-infusion day are summarized in Table 6. Leucine oxidation was higher during the fed period than during the fasting period; the differences were significant at the 2 lowest lysine intakes (P < 0.01) but not at the 2 highest lysine intakes. Leucine oxidation differed with intake; the effect was stronger during feeding (P < 0.01) than during fasting (P < 0.05). The daily leucine oxidation rate was lower with the 28- and 36-mg intakes than with the 12- and 20-mg intakes (all P < 0.05). Daily leucine oxidation was not significantly different between the 12- and 20-mg intakes or between the 28- and 36-mg intakes.
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TABLE 6 . Summary of leucine oxidation, balance, and flux at 4 lysine intakes in healthy Indian men, without regard to tracer-infusion day1
The results for leucine balance were essentially the same as those for leucine oxidation, whether expressed as absolute values or as a percentage of the leucine intake. There was no significant interaction with, or main effect of, tracer-infusion day. There was a significant effect of intake on balance (P < 0.001). Without regard to tracer-infusion day (Table 6), balance did not differ significantly between the 12- and 20-mg intakes or between the 28- and 36-mg intakes. The balances at lysine intakes of 12 and 20 mg each differed (P < 0.02) from the lysine intakes of 28 and 36 mg. Leucine balances were significantly different (P < 0.01) from zero balance or equilibrium at the 12- and 20-mg lysine intakes but were not significantly different from zero at the 28- and 36-mg lysine intakes.
The leucine fluxes did not differ significantly between days 7 and 21, and so the final statistical model included lysine intake, metabolic period, and the interaction (P = 0.004). Hence, without regard to tracer-infusion day (Table 6), the difference between intakes depended on whether the flux was determined for fasting or feeding. The effect was stronger during fasting (P < 0.01) than during feeding (P < 0.10). The 24-h leucine fluxes differed between the 20- and 36-mg intakes (P < 0.01), but did not differ significantly between the 12- and 28-mg intakes. A summary of the leucine oxidation, balance, and flux data without regard to tracer-infusion day is shown in Table 6. Mean (± SD) values were calculated from a mixed-models ANOVA to adjust for the correlation between observations on the days of the tracer infusion (days 7 and 21).
Breakpoint analysis
We fit two-phase linear regression models to the 12-h fed leucine oxidation, 24-h leucine oxidation, and 24-h leucine balance data by using the data from days 7 and 21 separately and combined; these results are summarized in Table 7. The breakpoint estimated from each of the 3 variables approximates a lysine intake of 31 mg · kg-1 · d-1. The 95% CIs are also given in Table 7.
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TABLE 7 . Two-phase regression analysis of the relation between lysine intake, leucine oxidation, and leucine balance
Three points can be made on the basis of the data in Table 7. First, the breakpoint occurred consistently at a lysine intake of 31 mg · kg-1 · d-1 for the 24-h oxidation and daily leucine balance data. Second, the daily rate of leucine oxidation beyond the breakpoint, assuming a zero slope, was essentially the same as the daily leucine intake, consistent with the calculated balance data for the 28- and 36-mg lysine intakes. Third, there was a tendency for the breakpoint to be somewhat lower when based on the 12-h fed state rate of leucine oxidation than when based on the 24-h rate of leucine oxidation.
Muscle function tests
The MVC of the forearm flexors was tested in 8 subjects at lysine intakes of 12 and 28 mg · kg-1 · d-1 and in 7 subjects at lysine intakes of 20 and 36 mg · kg-1 · d-1. There was no effect of intake on the mean MVC, measured between the beginning and the end of the 3-wk feeding period. The mean MVC decreased by 1.9 ± 6.8 kg at a lysine intake of 12 mg · kg-1 · d-1. The same subjects showed no negative trend at a lysine intake of 28 mg · kg-1 · d-1; the mean difference in MVC was 0.7 ± 3.3 kg. At a lysine intake of 20 mg · kg-1 · d-1, the mean difference in MVC between the subjects was 0.7 ± 5.4 kg, whereas at an intake of 36 mg · kg-1 · d-1 the difference was 3.1 ± 4.8 kg. None of these changes were significantly different from zero.
DISCUSSION
The present finding of a breakpoint at 31 mg · kg-1 · d-1 in the relation between lysine intake and 24-h leucine oxidation and lysine intake and 24-h leucine balance supports the results of our previous studies (8, 9) and strengthens our conclusion that the mean lysine requirement of healthy adults from Western countries and South Asia is 30 mg · kg-1 · d-1 (7, 9). We used the 24-h IAAO and 24-h IAAB techniques to determine the estimated lysine requirement; these techniques are considered to be the reference methods for estimating amino acid requirements. Furthermore, we showed here that a 7-d experimental diet period is entirely sufficient for the purpose of determining minimum physiologic requirements in healthy adults because essentially identical results were obtained at the end of a 3-wk dietary period. This observation is important because it was proposed by Millward et al (14) that the adaptive component of the metabolic demand for amino acids (and, therefore, for amino acid intakes to balance losses) complicates the determination of minimum physiologic amino acid requirement values. Specifically, Millward et al suggest that the time required for complete adaptation "...would appear to involve weeks and possibly months."
The data from the present study on the relation of 24-h leucine oxidation rates to changes in lysine intakes, together with data from our recent study on leucine oxidation rates at low threonine intakes (28) and data on the rate of adjustment in nitrogen excretion after changes in nitrogen intake from previous studies (29–32), are consistent with a view that contrasts with that of Millward et al (14). In other words, the adaptive diet-related oxidation of the so-called metabolic demand for amino acids responds quantitatively to an alteration in the intake of specific amino acids and reaches a new steady state within 5 d, or earlier. Our observations also contrast with those made by Millward and colleagues (33), from which it appears that their view about the adjustment of the adaptive demand for amino acids may have developed. As we noted previously (34), the results obtained by Quevedo et al (33), indicating a relatively slow rate of change in nitrogen and leucine metabolism to a reduction in protein intake, may have been confounded by the relatively low and possibly inadequate energy intakes of their subjects. Another possible difference between their experiment (33) and ours is that, in our experiment, only the lysine intake varied; the intake of nitrogen and of all the other indispensable amino acids was kept constant.
We could not determine the specific nature of the body weight loss of our subjects—particularly whether and to what extent the loss reflected a change in intestinal bulk, a shift in body water compartments, or differences in energy substrate utilization—because we did not make precise estimates of body composition. Although the weight changes did not appear to affect the leucine kinetic data, as summarized above, we examined the respiratory gas exchange data obtained during the 24-h tracer infusions on days 7 and 21 to assess the rates of fat and carbohydrate oxidation. Leucine oxidation was used as a surrogate for protein oxidation, based on a leucine content in mixed body protein of 8% (35). The heat equivalents of body protein and fat (in the fasting state) and of the amino acid mixture and other components of the diet (for the fed state) were calculated according to Livesey and Elia (36) and incorporated into these authors’ equations for fat and carbohydrate oxidation. A mixed-models ANOVA of the substrate oxidation data showed that lysine intake had no effect on 24-h fat or carbohydrate oxidation (g · kg-1 · d-1). Therefore, the average fat and carbohydrate oxidations across all intakes were assessed depending on the day (day 7 or 21) or the diet period (first or second diet period). The mean 24-h fat oxidation rates were 0.81 ± 0.33 and 1.18 ± 0.48 g · kg-1 · d-1on days 7 and 21, respectively, during the first diet period and were 0.71 ± 0.29 and 0.72 ± 0.30 g · kg-1 · d-1 on days 7 and 21, respectively, during the second diet period. The mean 24-h carbohydrate oxidation rates were 3.91 ± 0.74 and 3.02 ± 1.10 g · kg-1 · d-1 on days 7 and 21, respectively, during the first diet period and were 4.07 ± 0.75 and 4.21 ± 0.77 g · kg-1 · d-1 on days 7 and 21, respectively, during the second diet period. Mixed-models ANOVA showed a significantly higher (P < 0.001) rate of fat oxidation on day 21 during the first period than during the second period and a significantly higher (P < 0.001) rate of carbohydrate oxidation on day 21 during the second diet period than during the first diet period. Thus, the fasting and 24-h rates of fat oxidation on day 21 were lower during the second than during the first diet period. This suggests that increased fat mobilization and oxidation were not the cause of the greater weight loss during the second than during the first diet period. On the other hand, both the fasting and 24-h carbohydrate oxidation rates were higher on day 21 of the second diet period than on day 21 of the first diet period. There were no significant differences between the first and second diet periods on day 7. Assuming that the higher rate of carbohydrate oxidation in the fasting state occurred only in the last 14 d of the second diet period, then the total extra carbohydrate oxidized relative to that on day 7 amounted to 100 g for a person weighing 60 kg. Assuming further that the extra carbohydrate oxidation relative to day 7 came from the utilization of stored glycogen, together with the fact that glycogen deposition is associated with water at a ratio of 1 to 3 or of 1 to 4 (37), this difference in carbohydrate oxidation conceivably could account for a major proportion of the differences in weight changes observed in our subjects. It is unclear, however, why the subjects oxidized relatively more carbohydrate during the second diet period, because the dietary and other experimental conditions were essentially identical in both periods.
In addition to the 24-h IAAO and 24-h IAAB techniques used in the present study, a short-term IAAO technique has been used to determine requirements for lysine (10, 11), tryptophan (38), threonine (39), and the aromatic amino acids (40) in healthy subjects in the fed state. With this approach, the lysine requirement was found to be 35–45 mg · kg-1 · d-1 (10, 11), which is reasonably consistent with the present findings.
A third tracer approach for determining a lysine requirement has also been proposed (16); this approach is based on the measurement of the minimum requirement of wheat protein. In this case, the estimated lysine requirement is based on the assumption that the difference in the nutritional value of wheat and milk proteins is due to the limiting content of lysine in wheat proteins. Thus, the kinetics of [13C]leucine are used to measure the metabolic demand and efficiency of utilization of wheat and milk protein; the metabolic demand for protein is considered to be twice the cumulative 12-h postabsorptive losses, or the postabsorptive rate of leucine oxidation scaled to 24 h. The postprandial utilization (efficiency of utilization) of nitrogen intake is calculated as the slope of the line relating nitrogen balance (derived from [13C]leucine tracer balance) to nitrogen intake. The requirement for dietary protein is then calculated as the metabolic demand divided by the postprandial utilization. The metabolic demand and postprandial utilization for milk and wheat protein are determined separately in this manner. From the relative efficiency of utilization of wheat compared with milk, the lysine requirement as indicated from the lysine content of wheat protein, is 23 ± 2 mg · kg-1 · d-1. However, we consider this value to be an underestimate of the lowest continuing physiologic requirement for lysine, for many reasons. First, subjects in the study by Millward et al (16) were not adapted to a lower than usual or generous lysine intake before the [13C]leucine tracer study began; this lack of adaptation would lead to a much higher nutritional value of wheat than would be predicted under adapted conditions. The estimate of a higher nutritional value of wheat was possibly due, as pointed out by the authors (16), to the presence of a large free intracellular lysine pool that would have enhanced the utilization of the test meal of wheat protein. In addition, an expansion of the free leucine pool with feeding of the test levels of wheat protein was not accounted for by these investigators, and this would have also resulted in an overestimate of the efficiency of wheat protein utilization. This latter change would have also resulted in an overestimate of the efficiency of wheat protein utilization because the plasma leucine concentration rose from 59 to 91 mmol/L during the transition from a low to a high wheat-protein intake. In the milk-protein experiment, plasma leucine increased from 63 to 96 mmol/L. If, for these subjects, total body water was 60% of body weight and the extracellular water content was assumed to be 45% of total body water, then the expansion of the extracellular water leucine pool was 544 mmol for the 3-h feeding period. Hence, this would amount to an expansion of the free leucine pool size at a rate of 2.9 mmol · kg-1 · h-1 in a person weighing 63 kg. If the leucine balance estimates made by the investigators were corrected for the expansion of the leucine pool as detailed above, the postprandial utilization of nitrogen calculated from the corrected leucine balance would be 0.62. Assuming a wheat lysine content of 27 mg/g protein and a mean protein requirement of 0.6 g · kg-1 · d-1, the estimated lysine requirement would be 27 mg · kg-1 · d-1. This is a minimal correction because we assumed an expansion of only the extracellular water leucine pool, whereas it would be fully expected that the intracellular free amino acid compartment would also expand. Thus, assuming an equal expansion of the entire total body water leucine pool, the apparent lysine requirement would be 31 mg · kg-1 · d-1. We accept that this is a crude approximation, but it illustrates how much the lysine requirement might have been underestimated by not accounting for a change in the size of the free leucine pool.
All of the available measures of the physiologic adequacy of a given amino acid intake have their own limitations. These include nitrogen balance, stable isotope tracer techniques to measure specific amino acid oxidation and balances, and plasma amino acid responses (41, 42). It might be suggested that other possibly useful measures of the adequacy of a particular diet might be based on an anthropometric or performance index. However, it is difficult to assess precisely the anthropometric changes that might occur over short periods of study, as in these experiments, but it was considered worthwhile to begin looking for alternative, functional performance measures to complement the tracer indexes of amino acid adequacy, especially when they might be applied in longer-term studies. Thus, tests of forearm muscle strength have been used to assess the adequacy of protein intake in elderly persons (43) and in chronic undernutrition (25). In the present study, forearm muscle strength determined by MVC was measured by an observer who, together with the subjects, was blinded to lysine intake. There was a small negative change in MVC between the beginning and the end of the study and a small increase at the highest lysine intake. However, these differences were not significant. Thus, it is possible that the technique, which is motivational in nature, is not sensitive enough to detect the presence of small but possibly physiologically relevant changes in muscle function induced by limiting one indispensable amino acid. Nonetheless, because the technique showed a trend, we will attempt to improve on its possible utility in our future studies.
In conclusion, this study of the 24-h IAAO and 24-h IAAB techniques confirms, once again, that the current international estimate (2) of the upper lysine requirement (ie, 12 mg · kg-1 · d-1) is invalid. The results of the study also indicate that a mean requirement can be determined by using this approach with a 7-d adaptation protocol and that the mean requirement is 30 mg lysine · kg-1 · d-1 in healthy adults. This value appears to be suitable for all healthy adult populations throughout the world.
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