| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Original Research Communications |
1 From the General Clinical Research Center, Department of Medicine, Fletcher Allen Healthcare and College of Medicine, and the Department of Nutrition and Food Sciences, College of Agriculture and Life Sciences, University of Vermont, Burlington.
2 Supported by the American Heart Association (9797850S) and the National Institutes of Health (NIH RR00109 and NIH AG00599).
3 Address reprint requests to NK Fukagawa, University of Vermont College of Medicine, Given Building, Room C-207, Burlington, VT 05405-0068. E-mail: nfukagaw{at}zoo.uvm.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Objective: This study was designed to determine whether sex-related differences exist in methionine cycle kinetics, which may account for the reportedly lower fasting homocysteine concentrations in premenopausal women.
Design: Eleven healthy young men and 11 premenopausal women without cardiac risk factors were studied by using stable-isotope-labeled L-[methyl-2H3,1-13C]methionine and L-[methyl- 2H3]leucine. After 3 h of tracer infusion, 100 mg unlabeled L-methionine/kg body wt was ingested. Blood and breath samples were obtained at timed intervals. Fat-free mass was estimated by dual-energy X-ray absorptiometry and muscle mass by urinary creatinine excretion.
Results: No significant sex-related differences were found in fasting homocysteine concentrations, responses to the oral methionine load, or rates of methionine flux based on carboxyl or methyl labels. However, women had significantly higher remethylation rates than did men (P < 0.005) and a tendency toward higher transmethylation (P < 0.10). Whereas adjustment of remethylation rates for fat-free mass tended to attenuate the sex-related effect (P = 0.08), adjustment for muscle mass did not (P < 0.04). In contrast, significant sex-related differences in leucine flux (P < 0.02) were eliminated after adjustment for either fat-free mass or muscle mass.
Conclusion: Reported differences between men and women in homocysteine concentrations may be partially explained by differences in rates of homocysteine remethylation.
Key Words: Homocysteine • sex • amino acids • kinetics • sulfur amino acids • methionine • cysteine • premenopausal women • remethylation
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Homocysteine, a sulfur-containing amino acid formed during the metabolism of the essential amino acid methionine, is metabolized by 1 of 2 pathways: remethylation or transsulfuration (Figure 1
). In remethylation, homocysteine is salvaged by the acquisition of a methyl group from N5-methyl-tetrahydrofolate in a vitamin B-12–dependent pathway or from betaine in a pathway occurring primarily in the liver (10). When excess methionine is present or cysteine synthesis is required, homocysteine enters the transsulfuration pathway, in which it condenses with serine to form cystathionine. Cystathionine can subsequently be hydrolyzed to form cysteine which, in turn, can be incorporated into glutathione or further metabolized to sulfate and excreted in urine (11, 12). An early study by Mudd and Poole (13) suggested that rates through the methionine cycle differ between men and women because of different demands for labile methyl groups. Mudd and Poole suggested that the difference between men and women may be related to the stoichiometric formation of homocysteine in connection with creatine or creatinine synthesis in proportion to muscle mass (which would therefore be higher in men than in women). It was also believed that more rapid cycling in women resulted in a greater proportion of homocysteine being diverted to cystathionine (14). Blom et al (7) further suggested that a higher rate of methionine transamination in premenopausal women may contribute to lower homocysteine concentrations and hence protect against vascular disease.
|
|
SUBJECTS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and no cardiac risk factors. The studies were initiated in late 1997, just before the start of mandatory fortification of cereal grain products with folic acid in the United States. All volunteers were carefully informed of the nature, purpose, and possible risks of the study before they gave their written consent to participate. The Committees on Human Research and Medical Sciences at the University of Vermont approved the protocol and consent form.
|
CO2) were made at intervals throughout the study. At 180 min, 0.1 g (0.7 mmol) unlabeled methionine/kg body wt (Sigma Chemical Co, St Louis) was administered orally in 200 mL Crystal Lite (Kraft Foods Inc, White Plains, NY).
Isotopes
Infusates of the tracers were prepared from sterile powders of high chemical purity (99%), high optical purity, and high isotopic enrichment. The dual tracer of methionine, L-[methyl-2H3,1-13C]methionine [99% atom percent excess (APE)], and the cysteine tracer, L-[3,3-2H2]cysteine•HCl (98% APE), were purchased from Cambridge Isotopes Inc (Woburn, MA). L-[methyl-2H3]Leucine (99% APE) and [13C]bicarbonate (99% APE) were obtained from Tracer Technologies Inc (Somerville, MA). The methionine, cysteine, and leucine tracer priming doses were equivalent to 
1 times the amount of tracer infused each hour.
Blood and expired air samples
The blood sampling lines were kept open with a slow drip of sterile physiologic saline solution. As described previously (15–17), collection of arterial blood samples might have been more desirable, but to minimize the degree of invasiveness of these studies, arterialized venous blood samples were taken by using a hand-warming device. Expired breath samples for 13CO2 analysis were collected and stored in 20-mL evacuated tubes until analyzed. CO2 was measured by indirect calorimetry (DeltaTrac Metabolic Monitor; Sensor Medics Corp, Yorba Linda, CA).
Biochemical analyses
Plasma amino acid concentrations were measured by HPLC (Waters Chromatography, Milford, MA) with the phenylisothiocyanate derivative, a reversed-phase Nova-Pak C18 column (Waters Chromatography), and ultraviolet-visible detection at 254 nm (PicoTag; Waters Chromatography). Plasma homocysteine concentrations were measured after reduction with tributylphosphine (Sigma) and derivatization with the thiol-specific fluorogenic reagent, 4-fluoro-7-sulfobenzoflurazan, ammonium salt (SBD-F; Wako, Kyoto, Japan). The derivatives were separated by reversed-phase HPLC with use of a 150 x 4.6-mm column packed with Adsorbosphere C18 (5 µm; Alltech Inc, Deerfield, IL) and equipped with a guard column as described previously (18). Fluorescence intensity was measured with excitation at 385 nm and emission at 515 nm by using a scanning fluorescence detector (model 474; Waters Chromatography). All HPLC results were analyzed by using MILLENNIUM HPLC software (Waters Chromatography).
Measurement of isotope enrichments
Plasma enrichments of [1-13C]methionine, [methyl-2H3,1-13C] methionine, [3,3-2H2]cysteine, and [2H3]leucine/
-ketoisocaproate were determined from 200 mL plasma as described previously by using the N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (Pierce, Rockford, IL) derivatives of the amino acids (15–17, 19). Ethanethiol was included in the derivatization mixture to convert cystine to cysteine and to serve as an antioxidant, which greatly increased the yield of the cysteine derivatized. Cysteine bound to protein and dipeptides was not recovered in this assay because the ethanethiol was added after the free amino acids had been extracted from plasma. The cysteine isotope enrichments reflect, therefore, the combined free cysteine and cystine in plasma.
Plasma enrichments were measured on a 5890 series II gas chromatograph coupled to an HP 5988A mass spectrometer (Hewlett-Packard, Palo Alto, CA) as described previously (15–17, 19). All values are expressed as mole fractions above baseline after mass isotopomer deconvolution (20). 13CO2 production rates (13CO2) were computed from CO2 multiplied by the mole fractional enrichment at isotopic plateau in breath adjusted for 70% recovery of 13C during the fasted state (21).
The model for methionine metabolism (with Qc and Qm used when referring specifically to measurements of methionine flux with the carboxyl and methyl tracers, respectively) and the assumptions applied were described previously (15–17, 19). Briefly, whole-body methionine-methyl flux rates (Qm) and whole-body methionine-carboxyl flux rates (Qc) were calculated from the isotope infusion rate and enrichment and the plasma plateau enrichments of methionine at m + 1 (carboxyl label) and m + 4 (methyl and carboxyl label). To account for the dilution of label within the intracellular pool from intracellular protein breakdown, relative to that measured in plasma, a 20% adjustment was made to the m + 4 methionine species, as described previously (15–17, 19). This factor permits determination of rates of methionine oxidation that are consistent with predicted rates, based on the assumption that the volunteers are in approximate body methionine balance. Admittedly, this assumed value requires further validation, which is currently underway (22).
The following 2 equations relate methionine flux at steady state to the individual components:
 |
 |
where I is dietary intake, Bmet is the appearance in plasma of methionine via tissue protein breakdown, RM is the appearance of methionine from homocysteine remethylation, Smet is the disappearance of methionine from plasma via nonoxidative catabolism (assumed to be protein synthesis), TM is methionine that undergoes transmethylation, and TS is methionine oxidation (which we assumed to be equivalent to transsulfuration). We assume that the transamination pathway of methionine metabolism is not significant. However, if it were significant, then the 13CO2 from the [13C]carboxyl-labeled tracer would arise via this S-adenosylmethionine–independent pathway and methionine oxidation could not be attributed entirely to transsulfuration (15, 16, 19).
There is no input from remethylation in the case of carboxyl flux (Qc; Equation 2) because the carboxyl label is conserved within a turn of the transmethylation-remethylation cycle. Remethylation and transmethylation are derived from Equations 1 and 2 where RM = Qm - Qc and TM = RM + TS. Methionine disappearance via nonoxidative metabolism is taken to be an index of the rate of protein synthesis (Smet). Methionine appearance from protein breakdown (Bmet) also can be determined from a rearrangement of Equation 2. Thus, Bmet = Qc - I and Smet = Qc - TS. In the present study, the oral methionine load did not include a tracer and therefore splanchnic extraction could not be calculated.
Cysteine flux (Qcys) can be estimated in 2 ways, as described previously (15–17). The first, or predictive, estimate of cysteine flux can be derived from an assessment of the rates of appearance in plasma of cysteine via protein breakdown (Bcys = Bmet x 1.4) and disappearance from plasma via protein synthesis (Scys = Smet x 1.4). In the postabsorptive state we assume that these rates can be estimated by dividing the corresponding methionine flux values by the molar ratio of cysteine to methionine in average proteins in human tissues, taken to be 1.4 (19), and assuming that there are no other sources of plasma cysteine. Qcys can then be estimated, in theory, because values can be obtained for all 3 cysteine inputs: from diet (Icys), TS, and Bcys such that Qcys = inputs = Icys + Bcys + TS.
The second estimate of cysteine flux is made directly from the plasma cysteine isotopic enrichment values. Here flux is calculated by using the standard steady state equation as in our earlier tracer studies (15–17). To make a detailed comparison between these 2 estimates of cysteine flux, it also is necessary to attempt to account for the fact that the intracellular enrichment of labeled cysteine, as in the case of methionine, is probably lower than that measured in the plasma compartment. For our purpose, we propose that the additional dilution within the intracellular pools, relative to the labeling of cysteine measured in plasma, is 20% as used for methionine (15–17).
Calculations
Muscle mass was estimated from the average of three 24-h urine collections for creatinine excretion by using the following equation (23):
 |
Amino acid kinetics were calculated as described above. Creatinine clearance was calculated from urine volume and creatinine concentrations in serum and urine.
Statistics
Results are reported as means ± SDs unless otherwise noted. Comparisons between groups were made by using Student's two-sample t tests with equal variances and analysis of covariance (ANCOVA) with sex as the grouping variable and fat-free mass or muscle mass as covariates. Multivariate regression analysis was also used to examine the relations between the multiple independent factors and dependent variables. Changes in amino acid concentrations over time and in response to the oral methionine load were compared with the use of repeated-measures analysis of variance. All analyses were carried out with BMDP (SPSS Inc, Chicago) or SAS (SAS Institute Inc, Cary, NC).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The men were heavier and taller than the women and had higher body mass indexes. Fat-free mass, muscle mass, and creatinine clearance were also greater in the men than in the women.
Isotopic enrichments and rates of CO2 during the basal period are summarized in Table 2. Basal rates of methionine and leucine turnover are summarized in Table 3 and Figure 2. Qc and Qm did not differ significantly between men and women. In contrast, plasma leucine flux estimated by using -ketoisocaproate enrichments (QKIC) was significantly higher in men than in women. When adjusted for fat-free mass or muscle mass by ANCOVA, the sex-related difference in QKIC disappeared. As shown in Figure 2 and Table 3, the rate of remethylation was significantly higher in women than in men and there was a trend toward a slightly higher rate of transmethylation in women than in men. Whereas adjustment of remethylation for fat-free mass by ANCOVA attenuated the effect of sex (P = 0.08), adjustment for muscle mass had a minimal effect (P < 0.04). This suggests that differences between men and women in remethylation were partially related to differences in fat-free mass but not muscle mass.
|
|
|
Fasting methionine concentrations were also not significantly different between men (35 ± 12 µmol/L) and women (33 ± 13 µmol/L). The oral methionine load resulted in a prompt rise in plasma methionine concentrations in all subjects (Figure 3). The magnitude of the response in methionine concentrations did not differ significantly between men and women. Plasma methionine concentrations peaked 60 min after the oral load and had not returned to baseline at the end of the study. In contrast with methionine, homocysteine concentrations rose gradually over the 5 h of observation after the oral methionine load (Figure 4). However, as with methionine, there was no significant difference in the response over time between men and women.
|
|
. Fasting plasma glycine and serine concentrations were significantly higher in women than in men. In the last 90 min of observation after the oral methionine load, when the concentrations of most amino acids reached a new plateau, plasma concentrations of glycine were reduced in both men and women but the change was more dramatic in women (Figure 5). The responses of plasma serine and glutamate were the opposite in men and women. Postload increases in leucine and cysteine were not significantly different between men and women.
|
|
. Values calculated by using plasma cysteine enrichments were not significantly different from those predicted from methionine Qc and transsulfuration. When predicted from leucine flux (QKIC), Qcys was similar to the values obtained from the correction that assumes that intracellular [2H]cysteine was 0.8 of plasma [2H]cysteine enrichment. Because plasma cysteine enrichments remained at steady state throughout the study, an estimate of Qcys was made by using the enrichments obtained during the last 90 min of the study. There was no significant difference between basal Qcys and Qcys at the end of the study for both men and women. Values of Qcys at the end of the study were 29.7 ± 5.8 and 37.4 ± 7.3 µmol•kg-1•h-1, using uncorrected and corrected enrichments, respectively, for men and 31.9 ± 6.2 and 40.2 ± 7.7 µmol•kg-1•h-1 for women. Hence, the only significant difference in basal flux through the pathways of the methionine cycle was in remethylation. Non–steady state plasma methionine concentrations and low m + 1 enrichments of labeled methionine precluded calculation of postload methionine fluxes.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To our knowledge, the present study is the first to report and quantify differences in the rates of remethylation of homocysteine between men and women. Despite similar fasting homocysteine concentrations and similar changes after the oral methionine load, rates of remethylation and transmethylation were higher in women than in men. If men have higher requirements for labile methyl groups as proposed by Mudd and Poole (13), this alone cannot explain the slightly higher fasting homocysteine concentrations and the similar rise in homocysteine in men after equivalent doses of methionine. Our finding of higher rates of remethylation could be due to sex-related differences in the methionine synthase (5-methyltetrahydrofolate–homocysteine S-methyltransferase) or the betaine–homocysteine S-methyltransferase pathway, or both. To our knowledge, the influence of sex on these pathways has not been investigated nor have sex-related differences in the activity of the enzymes involved been implicated. Unfortunately, the present study was not designed to delineate the contribution of each of the remethylation pathways to the total estimate of remethylation.
Many of the previous reports of lower homocysteine concentrations in premenopausal women than men were made before methods were developed for measuring total plasma homocysteine (the sum of all homocysteinyl moities whether in sulfhydryl or disulfide forms, free or protein-bound) (6, 7, 14). On the basis of those findings it was proposed that a uniquely efficient methionine metabolism protects premenopausal women against vascular disease (6). Despite the development of methods for measuring total plasma homocysteine, this important issue has received little attention. Using measures of total plasma homocysteine, both Andersson et al (9) and Silberberg et al (24, 25) found slightly lower homocysteine concentrations in women when all ages were combined because older women tended to have higher homocysteine concentrations. When volunteers were grouped by age, however, no significant differences in fasting homocysteine concentrations were found between men and women (9, 24). Moreover, in the latter study (24), sex-related differences were found in the response to a methionine load (ie, greater rise in women). Hence, when interpreting the literature, careful attention must be paid to both age and sex distributions before definitive statements are made. Furthermore, recent fortification of cereal grain products with folic acid in the United States may influence the plasma homocysteine concentrations observed and the sex-related differences if food intake patterns vary significantly between men and women. In the present study, however, diet composition did not differ significantly between men and women.
The lack of a significant difference between men and women in the change in homocysteine concentrations after the oral methionine load in this study suggests efficient handling of the metabolic pertubation in both sexes. This is consistent with the results of some studies (9, 39, 40), but not others (6, 8, 14, 24). A tracer was not included in the oral methionine load and, hence, splanchnic extraction could not be estimated in the present study. The almost identical rise in plasma methionine concentrations suggests that differences in splanchnic extraction were not likely to be responsible, however.
Despite our inability to detect significant differences between men and women in fasting methionine and homocysteine concentrations, sex-related differences in fasting glycine, leucine, and serine concentrations were reported previously (41). Of interest are the differential responses of these amino acids to the oral methionine load. The decline in glycine, serine, and glutamate concentrations was much greater in women than in men. In fact, both serine and glutamate increased in men 4 h after the methionine load. This suggests that men and women differ in their utilization of certain amino acids for other metabolic pathways. For example, glycine is important in the synthesis of nucleotides, creatine, and glutathione, and serine is a necessary component in the transsulfuration pathway, ie, the condensation with homocysteine to form cystathionine.
Because remethylation remained significantly different between the sexes in the present study after adjustment for muscle mass, it is not likely that differences in creatine or creatinine synthesis account for the higher homocysteine concentrations in men as suggested by some (13, 38). Formation of N-methylglycine is reportedly a major catabolic route for the methyl portion of methionine when methionine and S-adenosylmethionine are present in excess (42). We did not measure this metabolite but N-methylglycine synthesis could account for some of the decline in glycine concentrations. As suggested by Blom et al (7), sex-related differences in the transamination pathway may exist, although it is uncertain whether this pathway is quantitatively important (19).
The differential responses of plasma glutamate and serine are difficult to explain. Glutamate is an important component of glutathione, and serine is necessary for condensation with homocysteine and the eventual synthesis of cysteine, the rate-limiting amino acid in the synthesis of glutathione. Although our estimate of transsulfuration did not differ significantly between men and women, it is possible that women utilized serine to a greater extent than men did and hence the slightly higher plasma cysteine concentrations in women at the end of the study. The methionine load did not significantly affect cysteine flux, but this may have been due to buffering by the glutathione pool as we suggested previously (15, 43).
Together with the trend toward a higher transmethylation rate, the present data suggest that women may have a more brisk response through the methionine cycle than men. The present study had >80% power to detect differences in remethylation but further studies are needed to elucidate the underlying mechanisms of the sex-related differences. A logical initial focus would be the differences in sex steroids. Understanding the effects of estrogen, progesterone, or androgens on the transcription, translation, or activity of the multiple enzymes involved in the methionine cycle may provide other avenues for intervention to minimize risks for cardiovascular disease. Furthermore, the nutritional implications of sex-related differences in methionine metabolism may affect specific amino acid requirements or utilization.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. Atkinson, J. Elmslie, M. Lever, S. T Chambers, and P. M George Dietary and supplementary betaine: acute effects on plasma betaine and homocysteine concentrations under standard and postmethionine load conditions in healthy male subjects Am. J. Clinical Nutrition, March 1, 2008; 87(3): 577 - 585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S H. Mudd, J. T Brosnan, M. E Brosnan, R. L Jacobs, S. P Stabler, R. H Allen, D. E Vance, and C. Wagner Methyl balance and transmethylation fluxes in humans Am. J. Clinical Nutrition, January 1, 2007; 85(1): 19 - 25. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M Stead, J. T Brosnan, M. E Brosnan, D. E Vance, and R. L Jacobs Is it time to reevaluate methyl balance in humans? Am. J. Clinical Nutrition, January 1, 2006; 83(1): 5 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Davis, E. P. Quinlivan, K. P. Shelnutt, H. Ghandour, A. Capdevila, B. S. Coats, C. Wagner, B. Shane, J. Selhub, L. B. Bailey, et al. Homocysteine Synthesis Is Elevated but Total Remethylation Is Unchanged by the Methylenetetrahydrofolate Reductase 677C->T Polymorphism and by Dietary Folate Restriction in Young Women J. Nutr., May 1, 2005; 135(5): 1045 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R Davis, J. B Scheer, E. P Quinlivan, B. S Coats, P. W Stacpoole, and J. F Gregory III Dietary vitamin B-6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men Am. J. Clinical Nutrition, March 1, 2005; 81(3): 648 - 655. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Davis, P. W. Stacpoole, J. Williamson, L. S. Kick, E. P. Quinlivan, B. S. Coats, B. Shane, L. B. Bailey, and J. F. Gregory III Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor Am J Physiol Endocrinol Metab, February 1, 2004; 286(2): E272 - E279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Joshi, S. Rao, A. Golwilkar, M. Patwardhan, and R. Bhonde Fish Oil Supplementation of Rats during Pregnancy Reduces Adult Disease Risks in Their Offspring J. Nutr., October 1, 2003; 133(10): 3170 - 3174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Kim, J. M. Ordovas, J. Selhub, and H. Campos B Vitamins and Plasma Homocysteine Concentrations in an Urban and Rural Area of Costa Rica J. Am. Coll. Nutr., June 1, 2003; 22(3): 224 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H Glew, M. Williams, C. A Conn, S. M Cadena, M. Crossey, S. N Okolo, and D. J VanderJagt Cardiovascular disease risk factors and diet of Fulani pastoralists of northern Nigeria Am. J. Clinical Nutrition, December 1, 2001; 74(6): 730 - 736. [Abstract] [Full Text]
|
|
|
|
|
|
M. J. MacCoss, N. K. Fukagawa, and D. E. Matthews Measurement of intracellular sulfur amino acid metabolism in humans Am J Physiol Endocrinol Metab, June 1, 2001; 280(6): E947 - E955. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
).
