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1 From the Division of Nutrition (AVK, SV, and AL) and the Biochemistry Laboratory (JG), Institute of Population Health and Clinical Research, St John’s National Academy of Health Sciences, Bangalore, India, and the Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA (MMR and VRY)
2 Dedicated to the memory of Vernon R Young. 3 Supported by the Nestlé Research Foundation, Lausanne, Switzerland, and NIH grant DK42101. 4 Reprints not available. Address correspondence to AV Kurpad, St John’s Medical College, Department of Physiology and Division of Nutrition, Sarjapur Road, Bangalore 560034, India. E-mail: a.kurpad{at}divnut.net.
ABSTRACT
Background: The 1985 FAO/WHO/UNU requirement for methionine in healthy adults consuming a cystine-free diet is 13 mg · kg–1 · d–1. It is unclear whether this daily requirement is influenced by dietary cystine.
Objective: We assessed the effect of 2 intakes of cystine (5 and 12 mg · kg–1 · d–1) on methionine requirements in well-nourished Indian men by using 7 test methionine intakes (3, 6, 9, 13, 18, 21 and 24 mg · kg–1 · d–1) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) methods. We combined these data with those from an experiment with zero cystine intake and in which the exact same method was used.
Design: Two studies were performed in which a diet containing either 5 or 12 mg cystine · kg–1 · d–1 was fed to 21 well-nourished Indian men over three 7-d periods. The 24-h IAAO and 24-h IAAB values were measured on day 7 with the use of a 24-h intravenous [13C]leucine tracer infusion. The breakpoints in the relation between these values and methionine intake in each study were assessed by two-phase linear regression.
Results: Breakpoints in the response curve were obtained at methionine intakes of 20 (95% Fiellers CI: 17, 26) and 10 (95% Fiellers CI: 8, 16) mg · kg–1 · d–1 with cystine intakes of 5 and 12 mg · kg–1 · d–1 intakes, respectively, which suggested a sparing effect of cystine. Although the 5- and 12-mg cystine breakpoints differed from one another, they did not differ significantly from that estimated previously with 0 mg cystine.
Conclusion: Cystine may spare the methionine requirement in healthy men, although the amount of sparing is difficult to quantify.
Key Words: Indians • methionine requirement • cystine intake • methionine sparing • sulfur amino acid requirement • 24-h indicator amino acid oxidation • 24-h indicator amino acid balance
INTRODUCTION
The daily requirement for total sulfur amino acids (TSAAs; methionine plus cystine) has important implications in the evaluation of and planning for the nutritional adequacy of diets based principally on vegetable protein sources. For healthy adults, the 1985 FAO/WHO/UNU Expert Consultation (1) set the upper daily dietary requirement for TSAA at 13 mg · kg–1 · d–1, which is based on nitrogen balance studies (2–4). More recent short-term, direct, or indicator amino acid–based tracer measurements (5–11) suggest that the mean methionine requirement is similar to this value. An unresolved issue is the sparing effect of dietary cystine on the methionine or TSAA requirement.
The percentage of the TSAA requirement of the young chick (12), cat (13), pig (14), rat (15, 16), and young dog (17, 18) that can be met with cystine approximates 50–60%. There are some similar but less complete and more variable nitrogen balance data available for human subjects (3). According to Rose and Wixom (19), a major portion (80–89%) of the dietary need for TSAAs in adult humans could be met with cystine. The current estimates, derived from nitrogen balance studies (1), are based largely on results obtained with a racemic mixture used by Rose (20) in which D-methionine appeared to be as effective as L-methionine. In later studies by others, D-methionine was shown to be less efficiently utilized in humans than was the L-isomer (3). This raises a question about the security of the original methionine requirement estimate made by Rose and Wixom (19, 20) and the validity of the apparently high-sparing effects, noted above, on the dietary need for methionine.
In 1988, we developed a tracer model for the measurement of methionine oxidation using the direct amino acid oxidation (DAAO) technique (10). At a methionine-free intake, dietary cystine decreased methionine oxidation (21), but our subsequent DAAO studies suggest that the sparing action of cystine on methionine metabolism is small or not detectable when the methionine intake is within the mid submaintenance range or at 0.6–1.0% of the dietary protein intake (6, 8, 22). A methionine-sparing effect of cystine was observed in the elderly when fed submaintenance methionine intakes (7). In addition, the authors of a recent study, using the DAAO method to evaluate transmethylation and transsulfuration rates with differing intakes of methionine and cystine (in the range of normal diets), suggested that their results were consistent with a significant sparing effect of dietary cystine on methionine oxidation rates and, therefore, on requirements in healthy young adults (23). However, because of the design of this study (23), it is not possible to determine whether the observed reduction in methionine oxidation was the result of added dietary cystine or whether it was due solely to a lower methionine intake. Also, a short-term fed-state indicator amino acid oxidation (IAAO) method has shown the methionine requirement to be 4 mg · kg–1 · d–1 in the presence of a relatively large amount (21 mg · kg–1 · d–1) of dietary cystine (24). However, the level of dietary cystine supplied, relative to methionine, was well outside a normal dietary proportion and so the nutritional significance of these studies remains uncertain.
Therefore, the current study was designed to assess methionine-cystine relations in healthy young Indian men with the use of a 7-d dietary adaptation period and the 24-h IAAO and indicator amino acid balance (IAAB) approach, with [13C]leucine as the indicator amino acid. The approach avoids some of the problems associated with the nitrogen balance method (25) as well as those related to the determination of the isotopic enrichment of the precursor pool of methionine for estimating its oxidation rate (26) with the DAAO approach. We compared the findings from the current study with those reported previously in an experiment in which methionine was given in the absence of dietary cystine but in which the experimental factors and subjects were similar (11). Our original experimental plan was to make such comparisons.
SUBJECTS AND METHODS
Subjects and anthropometry
Methionine requirement experiments were performed at intakes of 5 and 12 mg cystine · kg–1 · d–1 with 21 healthy men participating in each experiment. The subjects were weighed to the nearest 0.1 kg, and their height was measured to the nearest 0.1 cm. The logarithm of the sum of 4 skinfold thicknesses (biceps, triceps, subscapular, and suprailiac) was used in age- and sex-specific equations (27) to obtain an estimate of body density, from which percentage body fat and fat-free mass (FFM) were determined (28) (Table 1). The purpose of the study and the potential risks involved were explained to each subject, and the Human Ethical Review Board of St John’s Medical College approved the research protocol.
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TABLE 1. Characteristics of well-nourished Indian men studied for their methionine requirements1
Diet and experimental design
In each experiment, groups of 9 subjects each were randomly assigned to 3 separate 7-d experimental diet periods when they received a weight-maintaining diet based on an L-amino acid mixture, as previously described (29–31). The test quantities of methionine during the respective diet periods were chosen from 7 designated methionine intakes: 3, 6, 9, 13, 18, 21, and 24 mg · kg–1 · d–1 (n = 9 per intake) (Tables 2 and 3), which replicated our previous study of methionine requirements with a cystine-free diet (11). The quantities of methionine and cystine in the diet were adjusted on an equal molar basis with glycine. This involved an approximate 8% reduction in the intake of glycine between the lowest and highest intakes of dietary methionine. The 3 methionine intakes that each subject was given were distributed around a putative requirement intake of 13 mg · kg–1 · d–1. The subjects received their daily dietary intake as 3 isoenergetic, isonitrogenous meals (at 0800, 1300, and 2000), except on day 6 and 7 (see below).
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TABLE 2. Composition of the amino acid mixtures used to supply 7 methionine intakes per day (cystine intake: 5 mg · kg–1 · d–1)
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TABLE 3. Composition of the amino acid mixtures used to supply 7 methionine intakes per day (cystine intake: 12 mg · kg–1 · d–1)
24-h Tracer-infusion protocol and sample collection
The primed 24-h intravenous [13C]leucine approach was used with the protocol of indirect calorimetry and blood and breath sampling as previously described (29-31). Briefly, 1-[13C]leucine (99.3 atom%; MassTrace, Woburn, MA) was given as a primed, constant intravenous infusion at a known rate of 2.8 µmol · kg–1 · h–1 (the prime was 4.2 µmol/kg) into an antecubital vein. The bicarbonate pool was primed with 0.8 µmol/kg [13C]sodium bicarbonate (99.9 atom%; MassTrace, Woburn, MA). The tracer administration began at 1700 on day 6, with subjects having consumed their last meal of that day at 1500, and lasted until 1800 on day 7. Therefore, the tracer infusion was given for 25 h, although only the data from the last 24 h were used in calculation daily leucine oxidation and balance. On the day of the infusion study, the subjects received, at hourly intervals, 10 isoenergetic, isonitrogenous small meals beginning at 0600 on day 7, lasting until and including 1500 h (which together were equivalent to the 24-h dietary intake for that day). A similar feeding pattern was imposed on the subjects on day 6 as well, so that the feeding pattern on the infusion day was not suddenly different from the pattern on the previous day.
The analyses of breath for 13CO2 enrichment by isotope ratio mass spectrometry (Europe Scientific Ltd, Crewe, United Kingdom) and blood samples for 13C enrichments of plasma -ketoisocaproic by gas chromatography–mass spectrometry (Varian, Palo Alto, CA) were as previously described (29).
Leucine oxidation and balance calculations
Leucine oxidation (µmol · kg–1 · 0.30 min–1) was computed for consecutive half-hourly intervals as the ratio of the 13CO2 production rate (µmol · kg–1 · 0.30 min–1) and the plasma [13C]ketoisocaproate enrichment (mole percent excess) at that time. Leucine balance (mg · kg–1 · d–1) was computed as the difference between the leucine input (dietary leucine + intravenous tracer) minus the leucine output (sum of leucine oxidation at half-hourly intervals).
Statistical methods and data evaluation
Data are presented as means ± SDs. The weight change and metabolic variables were analyzed by using mixed-models analysis of variance (ANOVA). The model for weight change over the 6-d experimental diet periods included a factor for diet period. The models for 12-h leucine oxidation and flux included diet period, metabolic phase (fasted compared with fed), methionine intake, and the intake by metabolic phase interaction. Model contrasts were used to make pairwise comparisons of interest, as appropriate, based on the significance of the interaction and main effects. The model for 24-h IAAB (leucine) included diet period and methionine intake; comparisons versus zero balance were made by using the model. A two-sided P value of 0.05 indicated significance for all tests of interaction and main effects; P values for pairwise comparisons were adjusted by using Tukey’s method.
Data from the 3 studies [the present study at 5 and 12 mg · kg–1 · d–1 and from a previous study (11) with cystine at 0 mg · kg–1 · d–1] were combined to compare the effects of methionine intake on 24-h IAAB and IAAO and on 12-h fed IAAO (leucine) across cystine intakes. The mixed-models ANOVA included factors for methionine intake, cystine intake, and the methionine-by-cystine intake interaction; model contrasts were used to make comparisons of interest, as appropriate, based on the significance of the interaction and main effects, and P values were adjusted by using Tukey’s method.
We estimated a breakpoint for the relations between methionine intake and leucine oxidation and balance. A two-phase linear regression model was fit to the 24-h oxidation data (IAAO) to estimate at what methionine intake (mg · kg–1 · d–1) the oxidation no longer decreased with increasing dietary methionine. A mixed-models ANOVA regression model estimated the intercept and slope of one line segment and the intercept of the second line segment, and the slope of the second line segment was restricted to zero. From a biological standpoint, the slope of the second line should be zero, but we first tested whether the slope was significantly different from zero before implementing the restriction. The model was constrained such that the 2 line segments intersected at the unknown breakpoint. The breakpoint parameter was estimated as –1 times the ratio of the difference between intercepts divided by the difference between slopes (32). The 95% CI for the breakpoint was calculated by using Fieller’s theorem. The analysis was repeated by using daily IAAB (leucine) data to determine when balance no longer increased with increasing dietary methionine. The analysis was implemented with the use of PROC NLMIXED (SAS Institute Inc, Cary, NC), which accounted for multiple measurements on each subject. The analysis was implemented for each experiment with cystine intakes of 5 and 12 mg · kg–1 · d–1, and the results from a previous study (11) with a cystine intake of 0 mg · kg–1 · d–1 are presented for comparison. The breakpoint estimates and the estimated values above the breakpoint (plateau values) were compared between quantities of cystine intake by considering the overlap in the 95% CIs; 2 estimates are considered significantly different when the bounds of each interval do not cover the other point estimate. As in our previous study, we also assessed whether other statistical models fitted the relation between methionine intake and leucine oxidation and balance better; the models and procedure follow, as described in our previous study (11).
RESULTS
5 mg Cystine supplementation
Anthropometry
The subjects’ anthropometric measures were similar to those in subjects from studies in our previous series (11, 29–31; Table 1). During the 6-d experimental diet periods, subjects experienced a small but statistically significant (P < 0.001) weight loss of 0.27 ± 0.21 kg on average across diet periods. There was no significant difference in weight loss between diet periods (P = 0.62).
Leucine oxidation
There was a significant interaction between methionine intake and metabolic phase (P < 0.0001), which indicated that the effects of methionine intake on 12-h leucine oxidation differed between metabolic phases. During fasting, leucine oxidation was significantly higher at the 3 mg · kg–1 · d–1 intake than at each other intake (each P < 0.05); leucine oxidation did not differ significantly among the other intakes. During feeding, leucine oxidation was significantly higher at the 3, 6, 9, and 13 mg · kg–1 · d–1 intakes than at the 18, 21, and 24 mg · kg–1 · d–1 intakes (each P < 0.05); leucine oxidation did not differ significantly among the 3, 6, 9, and 13 mg · kg–1 · d–1 intakes, except for the 6 compared with the 13 mg · kg–1 · d–1 intakes (P < 0.05), nor did it differ significantly among the 18, 21, and 24 mg · kg–1 · d–1 intakes.
Leucine balance
With respect to leucine balance, the results were essentially the same whether expressed as an absolute balance or as a percentage of leucine intake. Daily leucine balance was affected by methionine intake (P < 0.0001) and was significantly different from zero balance at the 3, 6, 9, and 13 mg · kg–1 · d–1 intakes (each P < 0.01) (Table 4). Leucine balance was significantly lower at the 3 mg · kg–1 · d–1 intake than at the 9, 13, 18, 21, and 24 mg · kg–1 · d–1 intakes; at the 6 mg · kg–1 · d–1 intake than at the 13, 18, 21, and 24 mg · kg–1 · d–1 intakes; at the 9 mg · kg–1 · d–1 intake than at the 18, 21, and 24 mg · kg–1 · d–1 intakes; and at the 13 mg · kg–1 · d–1 intake than at the 21 and 24 mg · kg–1 · d–1 intakes (each P 0.05). Leucine balance was not significantly different among the 18, 21, and 24 mg · kg–1 · d–1 intakes.
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TABLE 4. Leucine oxidation and flux at 7 daily methionine intakes in well-nourished Indian men (cystine intake: 5 mg · kg–1 · d–1)1
Leucine flux
There was a significant interaction between intake and metabolic phase (P = 0.015), which indicated that the effects of methionine intake on 12-h leucine flux differed between metabolic phases (Table 4). During fasting, 12-h leucine flux differed significantly between the 3 and the 24 mg · kg–1 · d–1 intakes; the 6 and the 9 mg · kg–1 · d–1 intakes; the 9 and the 18, 21, and 24 mg · kg–1 · d–1 intakes; and the 18 and the 24 mg · kg–1 · d–1 intakes (each P < 0.05). During the fed state, 12-h leucine flux differed significantly between the 3 and the 13, 21, and 24 mg · kg–1 · d–1 intakes and between the 9 and the 13, 21, and 24 mg · kg–1 · d–1 intakes (P < 0.05).
12 mg Cystine supplementation
Anthropometry
The subjects’ anthropometric measures were similar to those in subjects from the 5 mg cystine study referred to above (Table 1). During the 6-d experimental diet periods, these subjects also experienced a small but statistically significant (P < 0.001) weight loss of 0.26 ± 0.30 kg on average across diet periods. There was no significant difference in weight loss between diet periods (P = 0.52).
Leucine oxidation
With respect to leucine oxidation (Table 5), there was no significant interaction between methionine intake and metabolic phase, a significant main effect of methionine intake (P < 0.001) and no significant main effect of metabolic phase, which indicated that the effects of methionine intake on 12-h leucine oxidation were similar across metabolic phases and that there were no fasted compared with fed differences. Without regard to metabolic phase, leucine oxidation was significantly higher at the 3 mg · kg–1 · d–1 intake than at each of the other intakes (each P < 0.05); no significant differences were found between these latter intakes.
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TABLE 5. Leucine oxidation and flux at 7 daily methionine intakes in well-nourished Indian men (cystine intake: 12 mg · kg–1 · d–1)1
Leucine balance
With respect to leucine balance (Table 5), the results were essentially the same whether expressed as an absolute balance or as a percentage of leucine intake. Daily leucine balance was affected by methionine intake (P < 0.0001) and was significantly different from zero balance at the 3, 6, and 9 mg · kg–1 · d–1 intakes (each P < 0.01). Leucine balance was significantly lower at the 3 mg · kg–1 · d–1 intake than at each other intake (P < 0.05 compared with the 6 mg · kg–1 · d–1 intake and P < 0.01 otherwise) and was significantly lower at the 6 mg · kg–1 · d–1 intake than at the 24 mg · kg–1 · d–1 intake (P < 0.05) but otherwise was not significantly different among the intakes.
Leucine flux
For leucine flux (Table 5), there was no significant interaction between intake and metabolic phase, but significant main effects of intake (P < 0.001) and metabolic phase (P = 0.02) were observed. These findings indicate that the effects of methionine intake on 12-h leucine flux were similar across metabolic phases and that fasted compared with fed differences were similar across methionine intakes. Without regard to metabolic phase, leucine flux differed significantly between the 3 and the 6 mg · kg–1 · d–1 intakes; the 6 and the 9, 13, 18, and 21 mg · kg–1 · d–1 intakes; the 9 and the 13 mg · kg–1 · d–1 intakes; and the 13 and the 18 and 24 mg · kg–1 · d–1 intakes (each P < 0.05).
Comparison of leucine indexes: 24-h oxidation and balance and 12-h fed state oxidation at intakes of 0, 5, and 12 mg cystine
Data for the 5 and 12 mg cystine intakes are from the current study, whereas data for the 0 mg cystine intake are from a previous study (11). For each of the 3 leucine indexes, there was a significant interaction between methionine and cystine intakes (each P < 0.01), which indicated that the effect of methionine intake on the leucine index differed between the cystine intakes (Table 6). Overall, for each leucine index, there were significant differences in the index between cystine intakes at intakes of 3, 6, 9, and 21 mg methionine · kg–1 · d–1 (each P < 0.05), but there were no significant differences at intakes of 13, 18, and 24 mg methionine · kg–1 · d–1 (Table 6). Overall, the results suggest that there were differences in leucine indexes for different quantities of cystine intake at low methionine intakes (3, 6, and 9 mg) but, generally, not at higher intakes (13–24 mg).
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TABLE 6. Comparison of leucine indexes at 7 daily methionine intakes, when 0, 5, or 12 mg cystine · kg–1 · d–1 was fed1
Comparison of breakpoint estimates of the methionine requirement and the leucine equilibrium values from the present study with estimates from a cystine-free diet
Data for the 5 and 12 mg cystine intakes are from the present study, whereas data for the 0 mg cystine intake are from a previous study (11). The estimated methionine requirements were 15, 20, and 10 mg · kg–1 · d–1 for cystine intakes of 0, 5, and 12 mg · kg–1 · d–1, respectively (Table 7). The estimated requirements at 5 and 12 mg · kg–1 · d–1 differed significantly from each other but did not differ significantly from the estimated requirement for the group receiving methionine without cystine. The individual data for leucine oxidation and leucine balance for all 3 cystine intake groups are presented in Figures 1 and 2. Note that in the previous study (11) the relation between daily leucine balance or oxidation and methionine intake was adequatelydescribed by either a breakpoint model (with second slope constrained to zero) or a sigmoid model (11); in the present studies, there was no evidence that a sigmoid model provided an improved fit as compared with the breakpoint model. Because the only model that fit the data well in all 3 experiments was the 2-phase linear regression model (with the second slope constrained to zero), we used this model to look for differences in the methionine requirement when different quantities of dietary cystine were provided. This also allowed for consistency in model application across all 3 data sets and was justified because there were no significant differences between this model and the sigmoid fit in all cases.
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TABLE 7. Two-phase regression analysis of the relation between 24-h leucine oxidation or balance and methionine intake at different cystine intakes
FIGURE 1.. Relation between 24-h leucine oxidation and methionine intake at cystine intakes of 0 mg · kg–1 · d–1 [A; from previous study (11)], 5 mg · kg–1 · d–1 (B), and 12 mg · kg–1 · d–1 (C). The observed (•) and mean (horizontal bars) values are plotted, and the fitted breakpoint model summarized in Table 7 is overlaid as a dashed line in each panel.
FIGURE 2.. Relation between 24-h leucine balance and methionine intake at cystine intakes of 0 mg · kg–1 · d–1 [A; from previous study (11)], 5 mg · kg–1 · d–1 (B), and 12 mg · kg–1 · d–1 (C). The observed (•) and mean (horizontal bars) values are plotted, and the fitted breakpoint model summarized in Table 7 is overlaid as a dashed line in each panel.
Finally, the estimated leucine balances obtained at methionine intakes that were above the estimated breakpoint values (Table 7) were also compared to assess whether there was an effect of the cystine intake on leucine equilibrium when the methionine intake was adequate. These estimated values did not differ significantly from each other; thus, there does not appear to be a consistent statistical relation between level of cystine intake and the plateau leucine balance.
DISCUSSION
For adult US males, the 50th centile for the dietary cystine-to-methionine ratio is 0.6 (34). There are no comparable values for healthy adult Indians living in the Bangalore area, but the ratios for soy, chickpea, and polished rice are 1.1, 1.1, and 0.7, respectively. Hence, the dietary quantities of cystine tested in these experiments, relative to the requirement for TSAAs (methionine in the absence of cystine), bear a reasonable similarity for diets (33) and plant food proteins (34).
Our earlier DAAO studies (6, 8, 22) failed to show marked or consistent sparing by cystine of the methionine requirement, which otherwise would have been expected from earlier nitrogen balance studies (3, 19). It should be noted, however, that use of declining methionine oxidation rates as the outcome indicator of the effect of cystine on methionine sparing is complicated by an independent and strong effect of methionine intake alone on methionine oxidation rates. Fed-state methionine oxidation rates, measured with the same technique used in several studies of young adults who received a cystine-free diet at different intakes of methionine (5–8, 21–23), are shown in Figure 3. There is a strong and linear association between the methionine intake over a range of 0 and 24 mg · kg–1 · d–1 (r = 0.99, P < 0.0001) and the methionine oxidation rate. This analysis suggests that DAAO studies attempting to assess methionine sparing by replacing dietary methionine with cystine would a priori need to take this effect of methionine intake per se into consideration. The DAAO study by Hiramatsu et al (6) did meet this provision, and it showed that there were no significant differences in short-term, fed-state methionine oxidation when cystine was supplemented in the diet over a range of 0–20 mg · kg–1 · d–1, with a constant methionine intake of 6.5 mg · kg–1 · d–1. DiBuono et al (23) also studied methionine sparing by cystine supplementation at adequate and submaintenance intakes of methionine; on the basis of a reduced methionine oxidation rate with cystine supplementation, they suggested that this was indicative of a methionine-sparing effect. Although this study showed that the rate of methionine oxidation and the transsulfuration to transmethylation ratio (an index of the rate at which newly formed homocysteine molecules are diverted to form cystathionine) decreased when a high dietary methionine intake (24 mg · kg–1 · d–1) was partially replaced with different amounts of cystine, it could be determined whether the decline in methionine oxidation was due to the cystine supplementation or to the decrease in methionine intake. In addition, there was no significant difference in the methionine oxidation rate between diets that provided 13 and 11 mg · kg–1 · d–1 or 5 and 19 mg · kg–1 · d–1 of methionine and cystine, respectively. Together with data from other studies that assessed the rate of methionine oxidation with different quantities of cystine intake, Figure 3 shows that, in general, there is a considerable overlap between rates of methionine oxidation for cystine-supplemented and unsupplemented diets, and it further illustrates the difficulty in separating the effect of a decreased methionine intake from that of cystine supplementation on methionine oxidation in experiments in which methionine intake varies.
FIGURE 3.. Mean (±SD) methionine oxidation rates in the fed state at different methionine and cystine intakes in healthy adults. •: Methionine oxidation at different intakes of methionine with no cystine supplementation. The regression line refers to these data (r = 0.99, P < 0.0001). Data abstracted from references 5, 6, 8, 10, 21, 22, and 23. : Methionine oxidation at different methionine intakes supplemented by 20 mg cystine · kg–1 · d–1. Data abstracted from references 6, 21, and 23. : Methionine oxidation at different methionine intakes supplemented by 10 mg cystine · kg–1 · d–1. Data abstracted from references 6 and 23. : Methionine oxidation at different methionine intakes supplemented by 5 mg cystine · kg–1 · d–1. Data abstracted from references 6 and 22.
The 24-h IAAO and IAAB approaches (35) used in the present study are suited for defining the methionine-sparing effect of cystine in an integrated, whole-body approach that avoids the problems peculiar to the DAAO technique. The comparison of the breakpoint on the indicator 24-h balance –methionine intake curve at different quantities of cystine supplementation provides an elegant way to assess the nutritionally relevant effect of cystine supplementation on the methionine requirement.
When the breakpoints were compared between the 2 dietary groups in the current study (5 and 12 mg cystine, respectively, and various methionine intakes), they were significantly different (P < 0.05). This finding suggested that dietary cystine within the range tested affects the requirement for methionine and indicates a sparing effect of cystine. However, because of the variability in estimated methionine requirements from one set of experiments to another, we could not conclude that the 5- or 12-mg breakpoint estimates differed from the estimate of the 0-mg cystine experiment. In addition, the 5-mg cystine estimate and CI are not consistent with the 15 mg · kg–1 · d–1 methionine requirement, whereas the estimate obtained from the 12-mg cystine study was consistent with a requirement of 14–15 mg · kg–1 · d–1 determined from our earlier study (11). A requirement of 15 mg methionine · kg–1 · d–1 in the absence of dietary cystine appears to be reasonably well established on the basis of our own 24-h IAAO studies (11); the short-term, fed, IAAO study of Di Buono et al (9), and a reanalysis (36) of the nitrogen balance data of Reynolds et al (37). Hence, it is reasonable to conclude that although dietary cystine might spare the requirement for methionine, our studies suggest that the sparing is probably <30% of the requirement for methionine, estimated in the absence of dietary cystine, and this value agrees with our earlier conclusions (6, 8, 22) that there was neither a marked nor a consistent sparing of the methionine requirement by dietary cystine.
Many issues need to be raised with respect to the current study and our interpretation of the results. First, the nitrogen content of the amino acid mixture given to the subjects was kept constant at each level of methionine and cystine intake by altering the glycine content. Although glycine is involved in several parts of the transmethylation and transsulfuration pathway of methionine metabolism (methyl acceptor, glutathione synthesis), it is unlikely that this manipulation of the glycine intake would have altered our findings. Endogenous glycine synthesis would be expected to be maintained at the adequate and constant nitrogen intake provided during the dietary periods and to be unaffected by a change of <10% in the total glycine intake between the lowest and highest methionine intakes (38, 39). Furthermore, the range of glycine intake supplied by the experimental diets was somewhat higher than would be present in diets based predominantly on cereal, legumes, or animal proteins. Hence, it appears unlikely that changing the glycine content of the amino acid mixtures to maintain isonitrogenous intakes would have compromised our findings or their interpretation.
Second, the desirable length of the dietary adaptation period, which in our case was 6 d, needs resolution, as the Toronto group emphasized (36). Third, the mode of feeding, ie, small equal and frequent meals that were similar in our studies and those by DiBuono et al (9, 23, 24), might influence quantitative aspects of methionine-cystine relations. We have shown, although to a small extent, that the utilization of leucine differs between a frequent small meal design and one involving 3 discrete meals (40, 41). This is a topic for further investigation. Finally, our interpretation is based, in part, on a comparison with an earlier study that limited the extent to which definitive conclusions could be made. However, the earlier study was of a similar design, used the same techniques as used in the current study, had results that were supported by those of Di Buono et al (9).
The findings of the current study and our previous studies (6, 8, 22) need also to be reconciled with those from in vitro studies, which have shown that there was a significant effect of the cystine intake on the lowering of liver cystathionine synthase (EC 4.2.1.22) activity, which suggests that cystine supplementation can spare methionine (42, 43). It is, however, difficult to extrapolate results of isolated organ and in vitro studies to an integrated whole-body response (44, 45). Furthermore, studies in young, growing animals (12–18) have clearly shown a quantitatively significant cystine sparing of the methionine requirement. The difference between these studies and ours in adult human subjects may have been a consequence of the relatively high growth rates of the animals or to the fact that fur or feathers increase the metabolic need for cystine. It is also of interest that Webel and Baker (46) showed that cystine is the first limiting amino acid for utilization of endogenous amino acids in chicks fed a protein-free diet.
In summary, the current investigation examined 24-h [13C]leucine tracer indicator kinetics in well-nourished Indian subjects in response to 7 test intakes of methionine that included the 1985 FAO/WHO/UNU (1) SAA requirement of 13 mg · kg–1 · d–1 along with a cystine supplement of 5 or 12 mg · kg–1 · d–1. Our findings suggest that, although there is a sparing of the methionine requirement by cystine, it is difficult to quantify for intakes that, relative to methionine, are within normal dietary ranges or similar to those from major plant-food protein sources. The data from the current study add to the earlier tracer-derived balance data that we generated using the diet-adapted 24-h IAAO and 24-h IAAB paradigm to quantify adult amino acid requirements (11, 29, 30, 31, 35) in South Asian (Indian) and North American subjects (47). This pattern of amino acid requirements (for leucine, lysine, threonine, and methionine) is similar to the amino acid pattern recommended for adults by a recently convened FAO/WHO/UNU Expert Consultation on Protein and Amino Acid Requirements (48).
ACKNOWLEDGMENTS
AVK was involved in the study design, data collection, sample and data analyses, and writing of the manuscript. SV and AL were involved in the data collection and analysis. JG was involved in the data collection and sample analysis. VRY and MMR were involved in the study design, data analysis, and interpretation and writing of the manuscript. The authors had no conflicts of interest.
REFERENCES