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1 From the Department of Physiology and Division of Nutrition (AVK and SV) and the Department of Biochemistry (JG), St John’s Medical College, Bangalore, India, and the Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge (MMR and VRY).
2 Supported by the Nestle Research Foundation, Lausanne, Switzerland, and NIH grant DK42101. 3 Reprints not available. Address correspondence to AV Kurpad, Institute of Population Health and Clinical Research, St John’s Medical College, St John’s National Academy of Health Sciences, Bangalore, India. E-mail: a.kurpad{at}divnut.net.
ABSTRACT
Background: Earlier studies of the requirement for total sulfur amino acids (SAAs; methionine in the absence of cystine) in healthy, well-nourished Indians indicated a value of 15 mg · kg–1 · d–1, but it is unknown whether this estimate is applicable to chronically undernourished subjects.
Objective: We assessed the total SAA requirement in otherwise clinically healthy, young, chronically undernourished adult Indians by using 7 test methionine intakes (3, 6, 9, 13, 18, 21, and 24 mg · kg–1 · d–1), without cystine, and by using both the 24-h indicator amino acid oxidation (24-h IAAO) and the 24-h indicator amino acid balance (24-h IAAB) methods.
Design: Twenty-one men were studied during each of 3 randomly assigned 7-d diet periods supplying methionine intakes (diet devoid of cystine) above and below the putative total 1985 FAO/WHO/UNU SAA requirement of 13 mg · kg–1 · d–1. Twenty-four–hour IAAO and IAAB were measured on day 7 by use of a 24-h [13C]leucine tracer infusion. The breakpoint in the relation between these values and methionine intake was determined.
Results: Two-phase linear regression of daily leucine oxidation or the daily leucine balance against methionine intake estimated a breakpoint in the response curve at a methionine intake of 16 mg · kg–1 · d–1 (95% Fiellers CI: 13, 22 mg · kg–1 · d–1).
Conclusions: On the basis of the 24-h IAAO-IAAB approach, a mean total SAA requirement of 16 mg · kg–1 · d–1 is proposed for undernourished Indian adults. This is not significantly different from that determined in Western and Indian well-nourished adults.
Key Words: Indian undernourished adults • methionine requirement • sulfur amino acid requirement • 24-h indicator amino acid oxidation • 24-h indicator amino acid balance
INTRODUCTION
The daily methionine requirement in the absence of dietary cystine (ie, the total sulfur amino acid requirement) was determined to be 15 mg · kg–1 · d–1 in well-nourished, healthy Indian men by use of the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) methods with leucine as the indicator amino acid (1). This value is similar to that in the 1985 FAO/WHO/UNU report (2), which was based on nitrogen balance studies (3), and is similar to the requirement measured by use of direct amino acid oxidation and balance methods (4, 5), even though some doubts exists about the exact identification of the precursor pool for irreversible methionine oxidation (6). This value is also similar to the total sulfur amino acid requirement measured by use of a short-term IAAO technique (7) and the value predicted from the obligatory amino acid loss method (8).
However, there are concerns that estimates of amino acid requirements obtained in healthy, well-nourished US or Indian subjects may not be representative of global methionine requirement patterns, particularly in populations living under environmental stressors such as chronic or subclinical infection, parasitic infestation, or pollution. In an earlier investigation of chronically undernourished men who lived in poor or unsanitary environments, we found that the daily lysine requirement was increased by 50% (9). Part of this increased lysine requirement was due to the presence of intestinal parasites, as shown by a subsequent study of similar subjects after they were treated for parasites (10). It is possible that subclinical infections or trauma increase the requirement for indispensable amino acids (11). This is particularly possible with the sulfur-containing amino acids, because of the important role of glutathione in the body’s response to infection (12).
The present study was designed to assess the daily methionine requirement in the absence of cystine (ie, the total sulfur amino acid requirement) in otherwise healthy, young adult, chronically undernourished Indian males. We used a 7-d dietary adaptation period and the 24-h IAAO and IAAB approach with [13C]leucine as the indicator amino acid.
SUBJECTS AND METHODS
Subjects and anthropometry
Twenty-one adult male undernourished subjects participated in this experiment. They were judged to be undernourished on the basis of having a body mass index (in kg/m2) < 18.5 (13). Anthropometric and body-composition variables (Table 1) were measured as described earlier (1). The health status of the subjects was determined by medical history, physical examination, analysis of blood for cell counts and erythrocyte sedimentation rate, routine blood biochemical profile, urine analysis, and blood serology for hepatitis and HIV. 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 undernourished Indian men studied for their methionine requirements1
Diet and experimental design
Groups of 9 subjects each were randomly assigned to 3 separate 6-d experimental diet periods, during which the subjects received a weight-maintaining diet based on an L-amino acid mixture, as described previously (1, 14-16). The daily test methionine intakes during the respective diet periods were chosen from 7 designated intakes: 3, 6, 9, 13, 18, 21, and 24 mg · kg–1 · d–1 (Table 2). 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 days 6 and 7 (see below). For each subject, energy intake was titrated against energy expenditure, which was estimated by performing time and motion studies on each subject during his stay in the metabolic ward. The observed physical activity level was 1.5–1.6. This energy expenditure level was similar to the subjects’ habitual daily energy expenditure, which we had measured earlier by use of physical activity diaries. For these subjects, daily energy intake was 35 kcal/kg.
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TABLE 2. Composition of amino acid mixtures used to supply 7 different methionine intakes
Before the first of the experimental studies in each subject, the subject was treated for intestinal parasites as described earlier (10), and the efficacy of the treatment was verified by fecal examination for 3 consecutive days after the treatment. The anti-parasite treatment was given only at the start of the first experimental period, and the subjects were verified to be parasite free at the beginning of each subsequent experimental diet period.
Twenty-four–hour 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 (1, 14-16). On day 6, the subjects consumed their last meal of the day at 1500; tracer administration began at 1700 and lasted until 1800 on day 7. Therefore, the tracer was infused for 25 h, although only the data from the last 24 h were used to calculate daily leucine oxidation and balance. On day 7, the subjects received, at hourly intervals, 10 isoenergetic, isonitrogenous small meals beginning at 0600 and lasting until and including 1500 (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, so that the feeding pattern on the infusion day was not suddenly different from the pattern on the previous day. The analyses of breath and blood for 13CO2 enrichment and for 13C enrichments of plasma -ketoisocaproic and the calculations for leucine oxidation and balance were as previously described (1, 14-17).
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. 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 x metabolic phase interaction. Model contrasts were used to make pairwise comparisons of interest, as appropriate based on the significance of the interaction and the main effects. The model for 24-h IAAB (leucine) included diet period and methionine intake; comparisons with 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 of pairwise comparisons were adjusted by using Tukey’s method. The data were analyzed by using SAS version 8.2 (SAS Institute Inc, Cary, NC).
We also 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) leucine oxidation no longer decreased with increasing dietary methionine. A mixed-models analysis of variance 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. 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 the intercepts divided by the difference between the slopes (18). 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 by using PROC NLMIXED, which accounted for multiple measurements on each subject.
RESULTS
Anthropometry
The subjects’ anthropometric characteristics were comparable with those of chronically undernourished subjects in our previous studies (9, 10). During the 6-d experimental diet periods, the subjects experienced a small but significant (P = 0.02) weight loss of –0.10 ± 0.18 kg on average across diet periods. There was no significant difference in weight loss between diet periods (P = 0.15).
Leucine oxidation
The relation between leucine oxidation and methionine intake is depicted in Figure 1. There was no significant interaction between methionine intake and metabolic (fed or fast) phase (P = 0.08) and a significant main effect only of methionine intake (P < 0.0001). This finding indicates that the effects of methionine intake on leucine oxidation were not significantly different across both metabolic phases and not significantly different between metabolic phases (Table 3).
FIGURE 1.. Relation between daily methionine intake and 24-h leucine oxidation and 24-h leucine balance. Individual data are plotted together with the mean value (horizontal line in each column of data) and the two-phase linear regression (dashed line).
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TABLE 3. Summary of leucine oxidation and flux at 7 methionine intakes in undernourished Indian men1
Leucine oxidation was significantly higher at the 3-mg · kg–1 · d–1 intake than at the 9-, 13-, 18-, 21-, and 24-mg · kg–1 · d–1 intakes (each P < 0.05) and was significantly higher at the 6-mg · kg–1 · d–1 intake than at the 13-, 18-, 21-, and 24-mg · kg–1 · d–1 intakes (each P < 0.05). Leucine oxidation was not significantly different between the 3- and 6-mg · kg–1 · d–1 intakes nor between the 6- and 9-mg · kg–1 · d–1 intakes; it also was not significantly different between the 9-, 13-, 18-, 21-, and 24-mg · kg–1 · d–1 intakes, except that leucine oxidation was significantly higher at the 9- than at the 21-mg · kg–1 · d–1 intake (P < 0.05).
Leucine balance
The relation between leucine balance and methionine intake is depicted in Figure 1. With respect to leucine balance, the results were essentially the same whether expressed as absolute balance or as a percentage of leucine intake (Table 3). 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–1intakes. Leucine balance was significantly lower at the 3- and 6-mg · kg–1 · d–1 intakes than at the 9-, 13-, 18-, 21-, and 24-mg · kg–1 · d–1 intakes (each P < 0.05). Leucine balance was not significantly different between the 3- and 6-mg · kg–1 · d–1 intakes, nor between the 9-, 13-, 18-, 21-, and 24-mg · kg–1 · d–1 intakes, except that leucine balance was significantly lower at the 9- than at the 21-mg · kg–1 · d–1 intake (P < 0.05).
Leucine flux
There was no significant interaction between methionine intake and metabolic phase (P = 0.60) and a significant effect of metabolic phase only on 12-h leucine flux (P = 0.03). This finding indicates that leucine flux was higher, on average across methionine intakes, during fasting than during feeding and was unaffected by methionine intake (Table 3).
Breakpoint analysis
Two-phase regression analysis of the relation between methionine intake and 24-h leucine oxidation and balance (Figure 1) yielded a breakpoint at a methionine intake of 16 mg · kg–1 · d–1 (Table 4). The 95% CIs and the equation of the 2 lines are also given in Table 4. As in our previous study (1) on the methionine requirement of well-nourished subjects, we fitted alternative models such as a linear, quadratic, and a sigmoid curve to the data to assess whether these gave better fits. We found that a sigmoid curve fitted the data as well as the 2 lines in the breakpoint method (–2 log likelihood of 276.3 and 277.0 in the sigmoid and breakpoint methods, respectively), whereas the other curves represented oversimplified models. More importantly, both the sigmoid and the breakpoint methods gave similar estimates of the daily methionine requirement.
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TABLE 4. Two-phase regression analysis of the relation between methionine intake and leucine oxidation and balance
DISCUSSION
The results of the present study confirm that the requirement for total sulfur amino acids (methionine in the absence of cystine) in chronically undernourished men is not less than that in well-nourished subjects, which is a finding similar to the results of our investigations of leucine and lysine requirements in chronically undernourished subjects (1, 9, 10, 19). These requirements are similar to the requirements recommended for adults by a recently convened FAO/WHO/UNU Expert Consultation on Protein and Amino Acid Requirements (20). It is recognized that a majority of the world’s population live in developing countries, with low food resources, and in unsanitary and polluted environments. This makes the assessment of amino acid requirements outside the framework of clinically normal, healthy, affluent, well-nourished subjects very important, both in terms of possible adaptive survival mechanisms and in terms of the increased nutritional cost of dealing with environmental stressors.
The first position that may be taken on the amino acid requirement in chronic undernutrition resulting from low food intakes is that there may be adaptive reductions in the requirement for amino acids when habitual diets are low in protein or amino acids (21). However, the alternate position is that there might be increased amino acid requirements as the result of factors such as the chronic but subclinical immunostimulation (22) or intestinal parasitic infestation (10, 23) that appear to be more prevalent in subjects living in urban slums. Pollution may also play a role in increasing specific protein synthesis, as shown by the increased plasma concentration of cell adhesion molecules in slum-dwelling individuals (24). The actual situation may be more complex owing to the interaction between these 2 positions, because malnutrition can attenuate the acute phase response to infection (25, 26), and interestingly, increased concentrations of acute phase proteins are hypothesized to predispose to chronic disease such as syndrome X in Asians (27). The requirement for sulfur amino acids cannot therefore be easily predicted on the basis of either an adaptive or a stress response and requires actual study.
The sulfur-containing amino acids are specifically important in terms of glutathione and the response to infection. The role of glutathione in the immune response to infection is well known (28-30) and its concentrations have been shown to be dependent on cysteine availability in healthy individuals (31), in protein-energy malnutrition (32), and in HIV infection (33). Indeed, cysteine supplementation improves the glutathione synthesis rate in malnourished children (34). Cysteine sulfur in turn is derived from methionine through the transsulfuration pathway and is important for the synthesis of glutathione (35). Therefore, the measurement of the total sulfur amino acid requirement, or of methionine in the absence of dietary cystine intake, is an important starting point in the assessment of sulfur amino acid requirements under differing pathophysiologic conditions. In summary, the present investigation of 24-h [13C]leucine tracer kinetics in chronically undernourished but otherwise healthy Indian men indicates that the international mean requirement for total sulfur amino acids should be close to the value of 15 mg · kg–1 · d–1.
ACKNOWLEDGMENTS
AVK was involved in study design, data collection, sample and data analysis, and writing of the manuscript. SV was involved in data collection and analysis. JG was involved in data collection and sample analyses. VRY and MMR were involved in study design, data analysis, and writing of the manuscript. The authors had no conflicts of interest.
REFERENCES