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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
Background: Elevated fasting homocysteine concentrations are considered a risk factor for vascular disease. Homocysteine, which is produced by the transmethylation of methionine, can be either remethylated back to methionine or metabolized via transsulfuration to cystathionine. It has been speculated that the lower risk of vascular disease among premenopausal women may be related to lower homocysteine concentrations in women than in men.
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
A link between elevated plasma homocysteine concentrations and cardiovascular disease has been suspected since 1969 (1). Several investigators have since reported a significant relation between elevated plasma homocysteine concentrations and atherosclerosis in the coronary, cerebral, and peripheral vasculature (2–5). Because several studies showed that women tend to have lower fasting homocysteine concentrations than men do, it was speculated that the lower risk of vascular disease among premenopausal women may be related to lower homocysteine concentrations (6–9).
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.
FIGURE 1. . A schematic depiction of the methionine cycle, the major components of which are transmethylation (TM), remethylation (RM), transsulfuration (TS), and methionine entry into or release from body proteins. In TM, methionine transfers its methyl group via S-adenosylmethionine to various methyl group acceptors, yielding S-adenosylhomocysteine, which is hydrolyzed to form homocysteine. Homocysteine can be remethylated to methionine, accepting a methyl group from 5-methyl-tetrahydrofolate (5-me-THF) or betaine, or undergo TS to yield cysteine and -ketobutyrate. Vitamin B-6, vitamin B-12, and folate are key cofactors in the methionine cycle.
We previously investigated kinetic aspects of methionine metabolism in humans (15–17), but found no significant differences between men and women. However, the number of subjects studied was small, most of the women were postmenopausal, and homocysteine concentrations were not measured. Therefore, we designed the present study to determine whether differences in methionine cycle kinetics between men and women exist, which might then account for the reportedly lower fasting homocysteine concentrations in premenopausal women than in men.
SUBJECTS AND METHODS
Subjects
Eleven men and 11 premenopausal women in good health as determined by a history, a physical examination, and routine blood and urine tests were studied at the General Clinical Research Center (GCRC) of the University of Vermont and Fletcher Allen Health Care. Women were studied in the follicular phase of their menstrual cycle and none were taking oral contraceptives. All participants were screened for the use of vitamin supplements and had normal blood concentrations of vitamin B-12, vitamin B-6, and folate (Table 1) 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.
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TABLE 1.. Subject characteristics1
Experimental protocol
Volunteers were admitted to the GCRC 6 d before the study for dietary stabilization. The volunteers consumed a standard diet providing 1 g protein•kg-1•d-1 and energy estimated to maintain body weight. For measurement of creatinine excretion, urine was collected on days 4–6 of the diet. Body composition was estimated by using dual-energy X-ray absorptiometry (Lunar DPX-L, version 1.3Y; Lunar Radiation, Madison, WI). On day 7, after a 12-h overnight fast, 2 intravenous lines were inserted: 1 for the infusion of isotope tracers into an antecubital vein and 1 for blood sampling, retrograde into an ipsilateral hand vein. After baseline samples of blood and breath were collected, a primed, constant infusion of stable-isotope-labeled methionine, cysteine, and leucine tracers was begun, which lasted for 480 min. A priming dose of [13C]bicarbonate (1.7 µmol/kg) was also given to enrich the bicarbonate pool. For measurement of substrate concentrations and isotopic enrichments, samples of blood and expired breath were collected at intervals between 120 and 180 min of the isotope infusion period (basal) and then every 30 min until the end of the study. Five 20–30-min measurements of the rate of carbon dioxide production (
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.
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 (
The following 2 equations relate methionine flux at steady state to the individual components:
RESULTS
Subject characteristics are summarized in Table 1. 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
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TABLE 2.. Basal plasma isotopic enrichments and carbon dioxide production (
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TABLE 3.. Basal rates of methionine cycle kinetics1
FIGURE 2. . Mean (±SEM) rates of carboxyl (Qc) and methyl (Qm) turnover of labeled methionine and of leucine flux based on -ketoisocaproate enrichments (QKIC) and rates of remethylation (RM), transsulfuration (TS), and transmethylation (TM) of methionine in men and women.
Despite the difference in remethylation between men and women, fasting plasma homocysteine concentrations did not differ significantly between men (7.1 ± 2.1 µmol/L) and women (6.5 ± 1.2 µmol/L), although there was a trend toward lower values in women. Multivariate regression analysis with fasting homocysteine concentration as the dependent variable and creatinine clearance, fat-free mass, muscle mass, remethylation, or transmethylation as independent variables revealed that the sex-related difference in fasting homocysteine concentrations approached significance at the P < 0.08 level. A larger sample size may have brought out the differences to a greater extent, as shown previously (8, 24, 25).
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.
FIGURE 3. . Mean (±SEM) plasma methionine concentrations before (-30 to 180 min) and after an oral methionine load (0.1 g/kg) administered at 180 min in men (•) and women ().
FIGURE 4. . Mean (±SEM) plasma homocysteine concentrations before (-30 to 180 min) and after an oral methionine load administered at 180 min in men (•) and women ().
Fasting concentrations of selected amino acids are summarized in Table 4. 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.
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TABLE 4.. Fasting amino acid concentrations1
FIGURE 5. . Mean (±SEM) percentage change in plasma concentrations of selected amino acids from the basal period to the last 90 min of the study.
Basal Qcys values calculated by using several approaches are shown in Table 5. 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.
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TABLE 5.. Basal cysteine flux as measured by different approaches1
DISCUSSION
Recent reports of differences between men and women in the incidence and severity of coronary artery disease underscore the importance of understanding potential sex-related differences in risk factors for atherosclerosis (26, 27). Although a high plasma homocysteine concentration has been deemed an independent risk factor for vascular disease (2–5, 28–30), the underlying mechanism for the pathogenic response is still unclear (2, 31). A recent review concluded that high total homocysteine itself may not be deleterious but instead provokes vascular occlusion under conditions predisposing to cardiovascular disease (32). Hence, although large studies reported lower homocysteine concentrations in women than in men, the effect of homocysteine on vascular disease is confounded by other risk factors such as smoking, age, vitamin status, and obesity (33–35). A rise in homocysteine concentrations with menopause was described (36, 37), and the influence of sex steroids was implicated. Additionally, Giltay et al (38) found that the hormones given to transsexuals appear to influence homocysteine concentrations.
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
We are grateful to the volunteers for their participation and to the staff of the GCRC for their assistance in the conduct of the studies.
REFERENCES