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1 From the Laboratory of Human Nutrition, School of Science and Clinical Research Center, Massachusetts Institute of Technology, Cambridge, MA, and Shriners Burns Hospital, Boston.
2 Supported by NIH grants DK15856, DK42101, and RR00088 and grants-in-aid from the Shriners Hospitals for Children.
3 Address reprint requests to VR Young, Laboratory of Human Nutrition, Room E17–434, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: vryoung{at}mit.edu.
ABSTRACT
Background: We previously studied methionine kinetics and oxidation with the tracer L-[1-13C, methyl-2H3]methionine.
Objectives: We sought to explore methionine-cysteine interrelations in adults by using L-[1-13C]cysteine under different dietary conditions.
Design: In experiment 1, 12 adults consumed a protein-free diet for 6 d. On day 7, methionine (n = 6) or cysteine (n = 6) oxidation rates were measured during an 8-h continuous infusion of L-[1-13C, methyl-2H3]methionine or L-[1-13C]cysteine, respectively. In experiment 2, 6 young men consumed 3 diets for 6 d each before a tracer study on day 7 with L-[1-13C]cysteine. The amounts (in mg•kg-1•d-1) of methionine and cysteine, respectively, were: high-methionine (HM) diet, 13 and 0; low-methionine (LM) diet, 6.5 and 0; and methionine-plus-cystine (MC) diet, 6.5 and 5.6. Cysteine flux and oxidation rates were determined and sulfur amino acid (SAA, methionine plus cysteine) balances were estimated.
Results: In experiment 1, rates of methionine and cysteine oxidation were similar to losses predicted from obligatory nitrogen losses. In experiment 2, SAA balance was less negative when subjects consumed the HM diet than the LM and MC diets (interaction, P = 0.034), largely because of a difference in fed-state balance (HM compared with LM, P < 0.01; HM compared with MC, P < 0.05). There was no evidence of a sparing effect of dietary cystine on the methionine requirement.
Conclusion: These studies support use of [1-13C]cysteine for studying whole-body SAA oxidation and conclusions that maintenance of SAA balance is best achieved by supplying methionine at approximately the FAO/WHO/UNU recommendations for total SAA intake (13 mg•kg-1•d-1).
Key Words: Cysteine oxidation • cysteine kinetics • sulfur amino acid metabolism • obligatory oxidative losses • obligatory nitrogen losses • young adults • methionine • cystine
INTRODUCTION
We have investigated the relations between dietary methionine and cystine in young adults (1–3) and elderly individuals (4) by using L-[1-13C, methyl-2H3]methionine and [3,3-2H2]cysteine as tracers. From estimates of methionine transsulfuration (oxidation), we have concluded that the mean requirement for dietary methionine, in the absence of dietary cystine, is 13 mg•kg-1•d-1 (5). This compares with the upper requirement of 13 mg•kg-1•d-1 for total sulfur amino acids (SAAs, methionine plus cystine) proposed by FAO/WHO/UNU (6). Furthermore, we have not detected, with these tracer techniques, any major sparing effect of dietary cystine on the methionine intake needed to balance methionine oxidation (1, 2).
To expand our investigations of SAA kinetics in healthy adults, we conducted studies to quantify whole-body cysteine turnover and oxidation. We believed that if the results for cysteine oxidation were found to be consistent with those predicted from the findings in young adults for methionine oxidation, this would provide additional support for the conclusions drawn previously (2, 3).
During the first study (experiment 1), 2 groups of healthy adult subjects consumed a protein-free but otherwise adequate diet for 6 d. We then determined the rate of either methionine or cysteine oxidation. In the second study (experiment 2), we explored the effects of 3 diets (providing adequate methionine without cystine or low methionine with or without cystine) on cysteine kinetics and oxidation.
SUBJECTS AND METHODS
Subjects
A total of 12 healthy volunteers (10 males and 2 females) participated in this investigation. In experiment 1, the subjects were divided into 2 groups. Group A included 4 males and 2 females (72.6 ± 12.7 kg body weight, 174.9 ± 11.1 cm height, 23.3 ± 3.5 y of age) and group B included 6 males (68.9 ± 6.9 kg body weight, 173.1 ± 5.4 cm height, 22.8 ± 2.2 y of age). Group B also participated in experiment 2. The subjects were all from the Massachusetts Institute of Technology (MIT) student community and were screened for health by medical history and physical examination. In addition, blood and urine samples were collected for a biochemical and clinical screening that was carried out in the Clinical Research Center laboratories.
The studies and their respective consent forms were approved by the MIT Committee on the Use of Humans as Experimental Subjects and the Advisory Committee of the MIT Clinical Research Center. Informed consent was obtained from the volunteers and they were paid for their participation in the study.
Experimental designs
Experiment 1
Twelve healthy subjects (10 male, 2 female) were given a protein-free diet for 6 d. On the morning of day 7, the subjects underwent an 8-h tracer study consisting of an initial 3-h fast followed immediately thereafter by a 5-h fed period. The tracers used were either L-[1-13C, methyl-2H3]methionine and L-[3,3-2H2]cysteine (n = 6, group A) or L-[1-13C]cysteine (n = 6, group B).
Experiment 2
Six subjects (all males) participated in this experiment, which consisted of 3 separate 7-d diet periods. During the first 6 d of each period, subjects adjusted to the different dietary intakes of methionine and cystine. The 3 diets were: 1) high methionine (HM), which provided methionine (13 mg•kg-1•d-1) with no cystine; 2) low methionine (LM), which provided methionine (5 mg•kg-1•d-1) with no cystine; and 3) methionine plus cystine (MC), which provided methionine (5 mg•kg-1•d-1) with cystine (6.5 mg•kg-1•d-1). On day 7 of each period, an 8-h tracer study similar to the one performed in experiment 1 was conducted. Each subject received the 3 diets in random order. Between the different diet periods there were intervals of 10–20 d, during which subjects consumed free-choice diets. One subject dropped out of the study for personal reasons after he had completed one of the diet periods (diet HM).
Diets
Subjects received 3 isoenergetic, isonitrogenous meals on each of the 6 d preceding every 8-h tracer experiment. Daily energy intake was constant; the diet provided between 170 and 190 kJ/kg (41–45 kcal/kg) for these subjects. The dietary energy was mainly derived from lipid and carbohydrate sources provided in the form of protein-free wheat-starch and butter cookies and a sherbet-based drink (Table 1). Nitrogen (160 mg•kg-1•d-1) was supplied as an L-amino acid mixture (amino acids were obtained from Ajinomoto USA, Inc, Teaneck, NJ) (Table 2). The amino acid mixture was similar to that used previously (2); it supplied indispensable amino acids in amounts that we have suggested are sufficient to meet the mean requirement in healthy young adults (7) but that are considerably higher than the requirements proposed by FAO/WHO/UNU (6). Dispensable (nonessential) amino acids were adjusted to maintain a constant total nitrogen content for the 3 diets. Beet sucrose and flavoring agents (Vivonex flavor packets; Norwich Eaton Pharmaceuticals, Norwich, NY) were added to improve the taste of the amino acid mixture. Beet sucrose was used to avoid changes in the background 13C-isotope enrichment of the expired carbon dioxide between the fasting and fed states.
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TABLE 1.. Composition of protein-free and of L-amino acid–based diets varying in methionine and cystine contents that were given to young adults for 6 d
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TABLE 2.. Composition of L-amino acid mixtures used to supply various methionine and cystine intakes1
Vitamins and minerals were supplied in daily supplements designed to meet or exceed recommended allowances or the safe and adequate intakes, as described previously (8). Choline supplements providing 500 mg/d were also given to subjects.
Tracer studies
Experiment 1
On the morning of day 7 of the protein-free-diet period, subjects were admitted to the infusion room of the MIT Clinical Research Center after they had fasted for 12 h overnight. As described in greater detail previously (2), an indwelling catheter was inserted in retrograde direction into a dorsal hand or low forearm vein and a second catheter was inserted into the antecubital vein of the same arm. The hand was then placed in a heating box for purposes of sampling arterialized venous blood. After blood and breath samples were collected from subjects to measure background isotopic enrichments, subjects received intravenous priming doses of [13C]bicarbonate (0.8 µmol/kg; MassTrace, Woburn, MA) and either L-[1-13C, methyl-2H3]methionine (2.0 µmol/kg; MassTrace) and L-[3,3–2H2]cysteine (1.5 µmol/kg; CIL, Andover, MA) in group A or L-[1-13C]cysteine (1.5 µmol/kg; MassTrace) in group B. Then the labeled methionine (2.0 µmol•kg-1•h-1) and [2H2]cysteine or [13C]cysteine (both 1.5 µmol•kg-1•h-1) were infused continuously throughout the 8-h experiment in groups A and B, respectively.
During the first 3 h of each tracer study, the subjects continued to fast. Throughout the next 5 h (fed phase), every 30 min they received small, isoenergetic meals, each of which supplied one–twenty-fourth of the daily intake.
Experiment 2
The tracer protocol followed on day 7 of each of the 3 diet periods was essentially the same as for experiment 1, except that we gave only [1-13C]cysteine in place of the specific methionine and cysteine tracers used in experiment 1 for groups A and B. After an intravenous priming dose (1.5 µmol/kg), it was infused at a constant rate of 1.5 µmol•kg-1•h-1.
Blood and expired air samples
Blood and breath samples were collected every 15 min during the last hour of each metabolic phase (fasting and fed states). Blood was collected in chilled tubes with heparin and was then immediately centrifuged (15 min at 1200 x g at 4 °C); the plasma was stored at -20°C until analyzed. Breath samples were collected as described previously (9) and were stored at room temperature until analyzed by isotope ratio mass spectrometry (MAT Delta E; Finnigan, Bremen, Germany). Total carbon dioxide production and total oxygen utilization were measured by indirect calorimetry (DeltaTrak; Datex, Yorba Linda, CA) twice during each phase over a period of 30 min for each.
Sample analysis
We have previously described in detail the treatment of blood and expired air samples for determination of isotopic enrichment, measurement of total 13CO2 production, and analysis of plasma free methionine and cysteine (1–3, 10). Briefly, N-methyl-N-(tert-butyl-dimethylsilyl) trifluoracetamide (Pierce Chemical Co, Rockford, IL) was used to form the tert-butyl-dimethylsilyl derivative of these amino acids. Ethanethiol was also used in the derivatization mixture to convert cystine to cysteine and to serve as an antioxidant. Also, note that the cysteine bound to protein and dipeptides would not be recovered in this assay because the ethanethiol was added after the free amino acids had been extracted from the plasma. Therefore, the cysteine isotope enrichments reflect the combined free cysteine and cystine in plasma (ie, total free plasma cysteine). This point has been discussed in greater detail in our previous papers on cysteine kinetics (1, 4).
Isotopic enrichments were measured by using a gas chromatograph and mass spectrometer (HP 5890 Series II and Hewlett Packard 5988A, respectively; Hewlett Packard, Palo Alto, CA). Methionine, [1-13C]methionine, and [1-13C, methyl-2H3]methionine were monitored at m/z 320, 321, and 324, respectively. Cysteine, [1-13C]cysteine, and [3,3-2H2]cysteine were monitored at m/z 406, 407, and 408, respectively. The isotopic enrichment of the experimental samples was determined by multivariate spectral deconvolution (11) by using the observed abundances of known tracer and tracee combinations from 0 to 0.1 mol ratio as standards. The validation standards were analyzed before and after each set of unknowns to adjust for variations in instrument response. In this study, the tert-butyldimethylsilyl derivatization approach afforded an average accuracy error and intersample precision of <7% for each. All plasma enrichment values reported here are expressed as a molar ratio (%) above baseline (11).
Whole-body kinetics
Methionine
Methionine carboxyl (Qc) and methyl (Qm) flux rates, when specifically referring to measurements with the [13C]carboxyl and [2H3]methyl tracers, respectively, were calculated as described previously (10). Briefly, Qc and Qm were calculated as follows:
RESULTS
The isotopic enrichments of plasma methionine, cysteine, or both and of 13C in expired air during the fasting and fed periods reached a relatively steady state, as shown in Figure 1 for experiment 1 and in Figure 2 for experiment 2. Group mean values for the fasting and fed periods in experiments 1 and 2 are summarized in Tables 3 and 4, respectively.
FIGURE 1. . Breath 13CO2 enrichment () [panels A and B; atom percent excess (APE) x 1000] and enrichment in plasma (mol ratio %) of [1-13C, methyl-2H3]methionine (•), [1-13C]methionine (), and [3,3-2H2]cysteine () (group A, panel A) or of [1-13C]cysteine () (group B, panel B) during fasting and fed periods in volunteers receiving a protein-free diet (experiment 1). ± SD; n = 6.
FIGURE 2. . Breath 13CO2 enrichment () [atom percent excess (APE) x 1000] and enrichment in plasma (mol ratio %) of [13C]cysteine () during fasting and fed periods in volunteers receiving the high-methionine diet (n = 6; panel A), low-methionine diet (n = 5; panel B), or methionine plus cysteine diet (n = 5; panel C) in experiment 2. Data are means ± SD.
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TABLE 3.. Plasma methionine and cysteine enrichments, breath 13CO2 enrichment, and total carbon dioxide production (
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TABLE 4.. Cysteine kinetics in young adults with different intakes of methionine and cystine (experiment 2)1
Experiment 1: protein-free diet
On the basis of the enrichments of plasma methionine and cysteine and the 13CO2 output (Table 3), we estimated the fluxes of methionine and cysteine as well as methionine and cysteine oxidation. These results are summarized in Table 5.
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TABLE 5.. Methionine and cysteine kinetics in young adults consuming a protein-free diet (experiment 1)1
For group A, the carboxyl methionine flux (Qc) in the fasting state was 14 µmol•kg-1•h-1 and was significantly higher than that in the fed state (P < 0.05, paired t test). Cysteine flux was 37 and 28 µmol•kg-1•h-1 in the fasting and fed states, respectively (P < 0.01, ANOVA). In group B, the cysteine flux (Q[13C]cys) was 48 and 34 µmol•kg-1•h-1 for the fasting and fed states, respectively (P < 0.01, ANOVA). These values were higher (P < 0.01, ANOVA) than those obtained in group A with the deuterium tracer. The fasting-state methionine flux, under this condition of a protein-free diet, reflects the breakdown of body protein. Hence, we can calculate that this is equivalent to 3 g protein•kg-1•d-1. The cysteine flux reflects cysteine entering the pool from protein turnover, its formation from methionine (a relatively small fraction, 4–5%, of the cysteine flux), and the turnover of circulating glutathione, as shown previously (22). Therefore, we cannot use these cysteine fluxes to compute protein turnover.
For the protein-free diet condition, methionine oxidation (extrapolated to 24 h) was 41 µmol•kg-1•d-1 and cysteine oxidation was 125 µmol•kg-1•d-1. These values compare well with predicted oxidation rates of 40 and 110 µmol•kg-1•d-1, respectively.
Experiment 2: methionine and cystine intakes
A summary of the main data used to assess the kinetic status of plasma [13C]cysteine metabolism is given in Table 6 for the 3 experimental diets. The output of expired carbon dioxide was not different among the 3 diet groups and increased (P < 0.001, ANOVA) with the feeding of small meals. The enrichment of plasma cysteine was also higher in the fed than in the fasting state (P < 0.01, ANOVA) across all diet groups.
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TABLE 6.. Rates of cysteine oxidation for fasting and fed periods and daily for 6 subjects receiving different intakes of methionine and cysteine (experiment 2)1
Plasma cysteine flux was consistently reduced with feeding (P < 0.01) across all diets and was higher for the high-cystine (MC) diet (P < 0.05; Table 5). In comparison with the diets adequate in methionine (HM) and high in cysteine (MC), the daily rate of cysteine oxidation was lowest when the diet low in methionine and free of cystine (LM) was given (P < 0.001 and P < 0.05, respectively) (Tables 5 and 6).
We calculated the daily total SAAB by using cysteine oxidation as an index of the combined methionine and cysteine sulfur loss from the SAA pool. The input was the sum of total SAA intake via diet and tracer and the output was the cysteine oxidation. These balance values are summarized in Table 7. For diet HM, the calculated daily balance was less negative than for diets LM and MC (interaction, P = 0.034). This was essentially due to the higher positive balance achieved in the fed state for diet HM (42.2 µmol•kg-1•12 h-1) than for diet LM (9.0 µmol•kg-1•12 h-1, P < 0.01) and diet MC (13.9 µmol•kg-1•12 h-1, P < 0.05). There were no significant differences in balance between diet HM and diets LM and MC for the fasting state (-43.6 compared with -34.5 and -61.6 µmol•kg-1•12 h-1, respectively). Finally, there was no indication of a sparing effect of dietary cystine on overall SAAB for the amount of dietary methionine intake evaluated in this experiment.
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TABLE 7.. Sulfur amino acid balance for fasting and fed periods and daily for 6 subjects receiving different intakes of methionine and cysteine (experiment 2)1
DISCUSSION
The main objective of this investigation was to explore the whole-body kinetics of cysteine by using [1-13C]cysteine as a tracer, with particular reference to our earlier conclusions that dietary cystine has little detectable sparing effect on the methionine requirement in healthy young adults (1, 2); in contrast, there may be a small sparing effect of cystine in elderly subjects (4). Furthermore, we wished to confirm our earlier theory (5) that a daily intake of methionine that was about half of the total SAA requirement proposed in the 1985 FAO/WHO/UNU report, when either consumed as the sole source of SAAs or with a significant cystine intake, would not be sufficient to maintain body methionine balance, as estimated by the 13C-tracer balance approach (1, 2).
Because there are no published 13C-tracer estimates of whole-body cysteine oxidation in healthy adults, we conducted experiment 2 to assess whether our approach for estimating whole-body cysteine oxidation (at various SAA intakes) would yield reasonable values. For experiment 1, our objectives were to estimate the rate of cysteine oxidation while subjects consumed a protein-free diet and to compare this with the predicted total loss (oxidation) of this SAA that could be derived from the amount of obligatory nitrogen loss (54 mg N•kg-1•d-1) and an assumed composition of mixed body proteins (6, 21).
Thus, we predict that while consuming a protein-free diet, the population mean total endogenous SAA loss would be 110 µmol•kg-1•d-1. By using the rate of cysteine oxidation that we measured during the 8-h tracer study and extrapolating it to a 24-h day, we estimated a mean value of 125 µmol•kg-1•d-1 for whole-body cysteine oxidation. Hence, there is relatively good agreement between these different estimates. On this basis, it appears that our approach for determination of whole-body cysteine oxidation is satisfactory. Had we included an 80% correction factor, as for methionine, then the rate of cysteine oxidation would have been 50% higher than the prediction. Considering this, taken together with our calculations of body balance in experiment 2 (Table 7), it seems that a correction of this magnitude is not appropriate for estimating whole-body cysteine oxidation.
We can also conclude from Table 4 that the measured rate of methionine oxidation was quite close to that predicted from obligatory nitrogen losses. The extrapolated 24-h rate of methionine oxidation was 41 µmol•kg-1•d-1, whereas the predicted loss is 40 µmol•kg-1•d-1.
The agreement between the measured and predicted methionine or total SAA (methionine plus cysteine) losses implies that we have not substantially overestimated their rates of oxidation by using the 13C-tracer technique. However, we may have underestimated the rates of endogenous methionine and cysteine oxidation because these comparisons were made under conditions of a significant input of tracer during the 8-h infusion period. In the case of cysteine, this amounted to an input of 13 µmol/kg over the tracer period, whereas for methionine the input was 16 µmol/kg. However, whether these amounts should be used to correct the daily estimates of methionine and cysteine oxidation is unclear. It seems likely that there was retention of tracer methionine and cysteine and possibly even a sparing effect of cysteine on methionine oxidation during this protein-free, SAA-free diet condition. This situation is reminiscent of the response in rats, in terms of growth or nitrogen balance, when a protein-free diet was supplemented with methionine (23).
In experiment 1, we obtained different estimates for cysteine flux in the 2 groups; results based on the deuterium tracer were lower than those obtained with the 13C tracer. The reason for this finding is unclear, because both estimates fall within the range of values that we obtained previously with the deuterium tracer (1, 4). We do not believe that our findings were due to an analytic problem or necessarily to an isotope effect of the kind we reported for deuterated phenylalanine (24) and that others noted for various 2H-labeled compounds (25–27). However, to rule out or accept this latter possibility, it would be desirable to explore simultaneously the effects of bolus doses of the 2 cysteine tracers in healthy adults under conditions similar to our investigations reported here. Our current conclusions, however, are not influenced by this particular issue.
The results obtained in experiment 2 are in accordance with our previous tracer experiments (1, 2) in healthy adults, which failed to reveal a sparing effect of dietary cystine on the methionine requirement under the experimental conditions tested. Furthermore, our findings indicate that at a methionine intake substantially below the 1985 FAO/WHO/UNU (6) requirement for methionine plus cystine, body SAAB cannot be achieved. As shown in Table 6, the addition of an extra 40 µmol dietary cysteine (given as cystine) per day to the LM diet increased cysteine oxidation by a somewhat greater extent than expected; the mean difference in daily oxidation between diets LM and MC amounted to 62 µmol. Although all subjects had higher rates of cysteine oxidation, there was wide interindividual variation in response to cystine supplementation. However, it is evident from this experiment that we failed to observe a significant sparing of the methionine requirement. This is consistent with our previous methionine-tracer studies (1, 2, 4). Nevertheless, this observation does not refute the elegant biochemical findings reported by Finkelstein (28, 29) on the extent and mechanism of methionine sparing by cystine in rats, and, as has been pointed out in a recent editorial by Finkelstein (30) in reference to our study in elderly subjects (4). It merely serves to emphasize, again in agreement with Finkelstein (30), the complexity of tissue and interorgan methionine metabolism as well as the interactions between numerous dietary variables and the integrated response of the whole body.
In addition, for further comparison among our series of studies, methionine balance when consuming the LM diet can be estimated from the values for SAAB given in Table 7. Thus, we found that the mean negative total SAAB was -25 µmol•kg-1•d-1. If the molar proportion of methionine to total SAAs (methionine plus cysteine) in body proteins is assumed to be 0.37 (120/326), then the methionine balance would be -25 x 0.37, or -9 µmol•kg-1•d-1. This estimate of methionine balance is similar to that reported by Hiramatsu et al (1), or -8 µmol•kg-1•d-1, for comparable methionine and cysteine intakes. However, it is less negative than the methionine balance estimated by Raguso et al (2). A possible reason for this latter difference is that the present study and that of Hiramatsu et al (1) used intravenous tracers, whereas an oral route of tracer administration was used in the study by Raguso et al (2).
However, it is also important to note that if the balance values had been determined from an estimate of the cysteine oxidation rate with an assumed 80% correction, as discussed above, all diets would have resulted in apparent negative balances, although the pattern of differences would have been the same. For the HM diet, the balance would be –33 µmol•kg-1•d-1 compared with –1.4 µmol•kg-1•d-1 (Table 7), with the latter being consistent with previous tracer methionine studies at adequate methionine intakes (1, 3, 5). Furthermore, there is no particular reason to expect that the plasma-to-intracellular enrichment ratio would be the same for the methionine and cysteine tracers because different essential amino acid tracers yield different estimates of whole-body protein turnover in the same individual (31). Also, in a study involving a 48-h constant intravenous infusion of several labeled amino acids, Reeds et al (32) found that the ratio of the equilibrium isotopic enrichment in VLDL apolipoprotein B-100 to that in plasma free amino acid differed significantly among amino acids in both the fed and postabsorptive states; the ratios were 0.7 and 0.94 for leucine and 0.81 and 1.05 for phenylalanine, respectively. We believe, therefore, that the calculations used here to determine rates of cysteine oxidation are entirely reasonable, consistent with the protein-free data, and consequently acceptable for the present purpose.
In conclusion, the present amino acid tracer studies support the use of L-[1-13C]cysteine as a probe of whole-body SAA oxidation and support the possible adequacy of the 1985 FAO/WHO/UNU (6) mean requirement for total SAAs (methionine and cysteine) of 13 mg•kg-1•d-1, with the qualification that the requirement does not seem to be met by a substantially lower intake of methionine that is supplemented with a generous amount of cystine. Thus, our experiments failed to expose a major sparing effect of dietary cystine on the minimum requirement for methionine. Our data suggest that, to meet the SAA requirements of healthy adults, it would be prudent to 1) supply methionine at an intake that approaches, if not equals, the FAO/WHO/UNU requirement for total SAAs and 2) simultaneously supply a reasonable, although as yet undefined, amount of cystine because it might be used more effectively than methionine to maintain cysteine and glutathione homeostasis (33).
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