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T Polymorphism and by Dietary Folate Restriction in Young Women1,2,3
,**
* Food Science & Human Nutrition Department, Institute of Food and Agricultural Sciences,
Division of Endocrinology and Metabolism, Department of Medicine,
** Department of Biochemistry and Molecular Biology, and
Department of Statistics, College of Medicine, University of Florida, Gainesville, FL 32611-0370;
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA;
Vitamin Metabolism, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;
# Department of Biochemistry, Vanderbilt University, and
Department of Veterans Affairs Medical Center, Nashville, TN 37232
4To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T polymorphism on the kinetics of homocysteine metabolism are unclear. We measured the effects of dietary folate restriction on the kinetics of homocysteine remethylation and synthesis in healthy women (20–30 y old) with the MTHFR 677 C/C or T/T genotypes (n = 9/genotype) using i.v. primed, constant infusions of [13C5]methionine, [3-13C]serine, and [2H3]leucine before and after 7 wk of dietary folate restriction (115 µg dietary folate equivalents/d). Dietary folate restriction significantly reduced folate status (
65% reduction in serum folate) in both genotypes. Total remethylation flux was not affected by dietary folate restriction, the MTHFR 677CT polymorphism, or their combination. However, the percentage of remethylation from serine was reduced 15% (P = 0.031) by folate restriction in C/C subjects. Further, homocysteine synthesis rates of T/T subjects and folate-restricted C/C subjects were twice that of C/C subjects at baseline. In conclusion, elevated homocysteine synthesis is a cause of mild hyperhomocysteinemia in women with marginal folate status, particularly those with the MTHFR 677 T/T genotype.
KEY WORDS: • methionine • one-carbon metabolism • S-adenosylmethionine • S-adenosylhomocysteine • women
Elevated plasma total homocysteine concentration is an independent risk factor for vascular diseases (1). The strong inverse relation between plasma total homocysteine concentration and folate status (2) makes dietary folate intake one of the most potent and easily modifiable determinants of plasma total homocysteine concentration. Methylenetetrahydrofolate reductase (MTHFR)5 produces 5-methyltetrahydrofolate (5-CH3THF), the major substrate for homocysteine remethylation (RM). The MTHFR 677CT polymorphism, which causes an alanine-to-valine substitution at position 222 of the mature enzyme, reduces MTHFR activity in vitro (3,4). The presence of 5-CH3THF and other folates protects against loss of activity by maintaining the affinity of the enzyme for its flavin cofactor (5). As a result, the MTHFR 677 T/T genotype increases susceptibility to hyperhomocysteinemia by increasing the sensitivity of MTHFR activity to low folate status (6,7). This polymorphism also alters the partitioning of one-carbon units in favor of formylated forms of folate (8), and accumulation of these forms of folate in RBCs correlates positively with plasma total homocysteine concentration (9).
The concentrations of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) are key regulators of, and are themselves regulated by several reactions involved in homocysteine metabolism (10). Plasma AdoHcy concentration might be a more sensitive indicator of perturbed one-carbon metabolism than plasma total homocysteine concentration (11). We recently reported that marginal folate status and the MTHFR 677CT polymorphism did not alter the concentrations of AdoMet and AdoHcy in plasma of young women (9), but the effects of folate status and the MTHFR 677CT polymorphism on the relations among the activities of methionine cycle enzymes and the concentrations of methionine cycle metabolites in humans are unknown.
Previous investigations addressed how factors such as dietary amino acid intake (12,13), age (14), gender (15), and pathologic conditions (16,17) affect the kinetics of homocysteine metabolism in humans. However, only one investigation of the effects of folate status on the kinetics of human homocysteine metabolism was reported (18). Those data suggest that the rate of homocysteine synthesis is a more important determinant of plasma total homocysteine concentration than RM in individuals with marginal folate status. In contrast, severe folate restriction reduced RM in a human intestinal cell culture model (19).
We developed a multitracer approach for simultaneous measurement of total and folate-dependent RM that also permits estimation of homocysteine synthesis rates in humans (20). This approach is used here to test the hypothesis that the MTHFR 677CT polymorphism modulates the effects of folate status on the kinetics of homocysteine metabolism.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Human subjects.
Subjects were healthy, nonpregnant, nonsmoking 20- to 30-y-old women who did not use oral contraceptives or other medications that might interfere with folate metabolism and who agreed to abstain from alcohol consumption during the study period. Subjects were selected for either the C/C or T/T genotypes of the MTHFR 677CT polymorphism, as determined by a PCR/restriction fragment length polymorphism procedure (21). At the time of screening, plasma concentrations of folate (>7 nmol/L), vitamin B-12 (>150 pmol/L), pyridoxal 5'-phosphate (PLP > 30 nmol/L), and homocysteine (<15 µmol/L) were normal in all subjects. A medical history questionnaire, physical examination, and clinical blood chemistry screening were used to confirm general health, including renal function. Informed consent was obtained from all subjects. This protocol was approved by the University of Florida Institutional Review Board and the General Clinical Research Center (GCRC) Scientific Advisory Committee. Of the 23 subjects who enrolled in the study (11 C/C and 12 T/T), 18 (9 C/C and 9 T/T) completed the entire protocol.
Folate-restricted diet. All meals were prepared by the Bionutrition Unit of the GCRC. Nutritionally adequate meals of controlled protein content were consumed by subjects for 3 d before the first infusion. On the next day, subjects began consuming the folate-deficient diet (115 ± 20 µg dietary folate equivalents/d) for 5 (n = 1 C/C and 1 T/T) or 7 (n = 8 C/C and 8 T/T) wk, including weekends and holidays. The folate status of the 2 subjects who followed the diet for 5 wk was similar to that of the subjects of their genotype after the full 7 wk of folate restriction. The 5-wk restriction period for these 2 subjects was necessitated by scheduling constraints. All subjects consumed breakfast in the GCRC, were given a take-out lunch to eat at their convenience, and returned to the GCRC to consume their evening meal. With the exception of dietary folate, potential vitamin and mineral inadequacies of the study diet (i.e., dietary intake less than the Recommended Dietary Allowance) were compensated for by custom supplements administered daily to the subjects. Compliance with the dietary regimen was monitored by measuring serum folate concentrations at weekly intervals throughout the study (22,23). A more detailed description of the diet was published (24).
Analytical methods. Folate concentrations in serum and RBCs were measured using the Lactobacillus casei microbial assay in 96-well plates (22,23). The concentrations of folate and vitamin B-12 in plasma were measured by a commercial competitive radiobinding assay (radioassay) method (Quantaphase II B12/Folate radiobinding assay, Bio-Rad). Plasma PLP and plasma total homocysteine concentration were measured by reverse-phase HPLC with fluorescence detection (25,26).
RBC total folates and the distribution of folate forms within RBCs were measured for at least one time point (either baseline or after folate restriction) for 7 C/C subjects and 8 T/T subjects. Data were available both at baseline and after folate restriction in 5 C/C subjects and 7 T/T subjects. Individual folate species and their percentage distributions were measured in isolated, washed RBCs by HPLC and electrochemical detection (HPLC-EC) (27). RBC folate data were normalized to hemoglobin content, which was measured by a commercially available kit (Hemoglobin Reagent Kit, Pointe Scientific). Concentrations of AdoMet and AdoHcy in plasma were measured as the isoindole-derivatives by HPLC with fluorescence detection as previously described (9,28).
Infusion protocol.
Subjects were admitted to the GCRC on the evening before each infusion protocol and consumed no food between 2030 h and initiation of the infusion. On the morning of the infusion, a heparin lock was established in one vein of each arm, one for blood collection and one for the tracer infusion. Blood samples were taken before infusion to measure concentrations of homocysteine, vitamin B-12, and vitamin B-6 in plasma, folate in plasma, serum, and RBCs, and for measurement of background isotopic enrichment of amino acids. Infusions were initiated at 0830 h with a 5-min, 20-mL priming dose that delivered 9.26, 1.62, and 1.87 µmol/kg of [3-13C]serine, [13C5]methionine, and [2H3]leucine, respectively. The 9-h constant infusion followed immediately after the priming dose, and delivered 20 mL/h of infusion solution containing 9.26, 1.62, and 1.87 µmol/kg of [3-13C]serine, [13C5]methionine, and [2H3]leucine, respectively. Heparinized blood samples were taken at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7.5, and 9 h after the infusion. Blood samples were stored on ice until isolation of plasma by centrifugation (1500 x g for 10 min at 4°C). Aliquots of plasma were stored at –80°C until analysis. Subjects were kept from a catabolic state during the infusion by hourly consumption of a protein-free formulation containing carbohydrate (70% of energy) and fat (30% of energy) that provided one twenty-fourth of the subject’s daily energy requirement per serving [5.23 kJ/(kg · h)].
GC-MS analysis of amino acid isotopic enrichments. Plasma free amino acids were isolated and derivatized as previously described (20). N-Heptafluorobutyryl-n-propyl ester derivatives were dried and solubilized in ethyl acetate and stored at –20°C until analysis. Isotopic enrichment was determined by negative chemical ionization/MS using a Finnigan-Thermoquest Voyager GC-MS and a 30-m poly (5% diphenyl/95%dimethylsiloxane) fused silica capillary column (Equity 5; Supelco) (20). Isotopic enrichments are expressed as molar ratios of labeled:nonlabeled isotopomers after correction for the natural abundance of stable isotopes (29).
Kinetic analyses. The [13C5]methionine and [3-13C]serine tracer paradigms are illustrated in Figure 1. Briefly, one 13C-atom is lost when [13C5]methionine is utilized in the methionine cycle for AdoMet-dependent methyltransferase reactions. Subsequent RM of the resulting [13C4]homocysteine with unlabeled 13C methyl groups generates [13C4]methionine. Total RM is calculated from the plasma plateau enrichments of these 3 species using equations modified from previous tracer models (29,30). Use of the 13C-labeled carbon of the [3-13C]serine tracer for RM via methionine synthase generates [13C1]methionine. Folate-dependent RM from serine is calculated from the plateau enrichments of these 2 species using equations adapted from a radiotracer model used in studies of rat liver (31).
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Results were analyzed statistically using SigmaStat 3.0 (SPSS). Plasma total homocysteine concentrations were transformed (log10) before analyses to normalize distributions. Differences between genotypes at baseline were assessed by 2-sample t test. The paired t test was used to detect differences within genotypes due to dietary folate restriction. The effects of folate restriction on all measures (changes from baseline) were compared between genotypes by a 2-sample t test. Data are reported as means or slopes and SE, and differences were considered significant at P < 0.05. Some data were used in previous analyses of the effects of dietary folate restriction and the MTHFR 677CT polymorphism on the plasma concentrations of metabolites related to one-carbon metabolism (9) and on the rate of change of folate status and plasma total homocysteine concentration (24).
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isotopic enrichment of the infused tracers rapidly reached a plateau in the plasma compartment (0.5–1 h) with no effect of genotype or dietary folate on the shape of the enrichment curves (online supplemental Fig. 1). The plateau enrichments of the [13C5]methionine tracer (7%), remethylated [13C4]methionine (2–2.5%), the [3-13C]serine tracer (6–8%), [13C1]methionine remethylated from serine (>1%), and the [2H3]leucine tracer (2%) did not differ among groups. Mean [13C4]homocysteine plateau enrichments (6–7%) did not differ among groups, and reached 90–100% of the enrichment of plasma [13C5]methionine (online supplemental Fig. 2). No enrichment of [13C1]cysteine in plasma was detected (data not shown). These results are similar to our previous investigations (20).
Estimates of QM and QC and total RM did not differ significantly between the calculation methods of Storch et al. (29) and MacCoss et al. (30). Results are reported as calculated from the latter model unless stated otherwise. QM, QC, and RM did not differ among the groups (Fig. 2, Table 1). Similar to our previous report (20), RM accounted for 30% of QM in all groups. The fluxes of leucine and serine (Qleu and Qser, respectively) did not differ among the groups, nor did the Qser/Qleu and Qmet/Qleu ratios (online supplemental Table 1).
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15%; P = 0.031; Table 1) in C/C subjects by folate restriction. The percentage of RM from serine was not reduced by folate restriction in T/T subjects. The fractional synthesis rate of methionine from homocysteine did not differ significantly among groups, although the value in the C/C group at baseline (10.4 ± 1.2%/d) was 20% greater (P = 0.083) than in the C/C restricted (7.5 ± 1.0%/d). The percentage QSer for RM also did not differ among the groups. Although there were no significant differences in FSRHcy due to folate restriction and the T/T genotype (Fig. 3B), when plasma total homocysteine concentrations (Fig. 3A) were taken into account, the values for ASRHcy were twice as large in T/T subjects and in C/C subjects after folate-restriction compared with C/C subjects at baseline (t test and paired t test, respectively; P < 0.05; Fig. 3C) when using EpHcy M+4 to estimate intracellular [13C5]methionine enrichment (i.e., precursor enrichment). Similar results were obtained using EpMet M+5 as the precursor enrichment (data not shown), although the difference between C/C subjects at baseline and after folate restriction was not significant (paired t test; P = 0.09).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T polymorphism have focused mainly on the direct effect that each might have on the availability of 5-CH3THF for use in RM. However, this approach does not account for the possibility that reduced RM might ultimately restrict methionine-cycle flux. Also, betaine-homocysteine methyltransferase activity in liver and kidney could compensate, at least in part, for the loss of methionine synthase activity (33,34). Furthermore, focusing solely on RM ignores the regulatory role of 5-CH3THF on transmethylation flux (35,36).
Three findings of this study are particularly noteworthy. First, neither the MTHFR 677CT polymorphism nor dietary folate restriction significantly altered total RM despite modestly reducing folate-dependent RM. Second, homocysteine synthesis doubled in C/C subjects after folate restriction. Third, folate-dependent RM and homocysteine synthesis in T/T subjects at baseline were characteristic of those rates in folate-restricted subjects of both genotypes. These results imply that a one-carbon donor(s) other than serine (e.g., betaine) can be used to compensate for impaired folate-dependent RM, that increased homocysteine synthesis through transmethylation contributes to elevated plasma total homocysteine concentration in moderate folate restriction, and that the MTHFR 677CT polymorphism alters homocysteine metabolism even when folate status is within the range currently deemed adequate.
The modest reduction in the percentage of RM from serine caused by dietary folate restriction in the C/C genotype is consistent with the current view that methionine synthase activity is sensitive to the concentration of its substrate, 5-CH3THF. Because total RM was not reduced, these data also are consistent with the use of one-carbon donors other than serine to compensate for impaired folate-dependent RM. The small magnitude of these changes in the rates of folate-dependent RM resulting from dietary folate restriction might be explained in part by a simultaneous reduction in the size of the intracellular 5-formyltetrahydrofolate pool. Both 5-formyl and 5-methyl forms of tetrahydrofolate are inhibitors of serine hydroxymethyltransferase, and 5-formyltetrahydrofolate was shown to regulate folate-dependent pathways in a cultured cell model (37). Reduction of the intracellular pool due to folate restriction might release serine hydroxymethyltransferase from inhibition and partially counter the effects of reduced THF availability on this pathway.
The magnitude of the stimulatory effects of moderate dietary folate restriction and the MTHFR 677CT polymorphism on homocysteine synthesis was unexpected. Elevated homocysteine synthesis (i.e., increased transmethylation) might be explained in large part by increased glycine N-methyltransferase activity. This enzyme is subject to reciprocal regulation by AdoMet (positive cooperativity) and 5-CH3THF (allosteric inhibition) and is only weakly inhibited by AdoHcy compared with other AdoMet-dependent methyltransferases (38). A reduction of 5-CH3THF concentration caused by dietary folate restriction or the MTHFR 677 CT polymorphism might release glycine N-methyltransferase from inhibition and increase the production of AdoHcy (and homocysteine) from AdoMet, as was observed in rats (35). Analysis of plasma sarcosine, the product of the glycine N-methyltransferase reaction that is reported to accumulate in plasma of folate or vitamin B-12 deficient humans (39), will improve our understanding of the role of this pathway in hyperhomocysteinemia.
Whether reduced RM or increased synthesis causes the initial increase of plasma total homocysteine concentration probably depends on which enzyme (methionine synthase or glycine N-methyltransferase, respectively) is more sensitive to changes in 5-CH3THF production. The strong correlation between plasma total homocysteine concentration and measures of homocysteine synthesis reported here suggests that glycine N-methyltransferase activity is perturbed first.
Phosphatidylethanolamine N-methyltransferase (PEMT) might also contribute to elevated homocysteine synthesis in response to folate restriction or the MTHFR 677CT polymorphism. This enzyme generates 3 homocysteine molecules in the synthesis of phosphatidylcholine from phosphatidylethanolamine. Noga and colleagues (40) recently demonstrated that mice lacking PEMT have 50% less homocysteine in plasma due to 50% less homocysteine secreted from the liver. Further, transfection of rat hepatoma cells with PEMT yields greater secretion of homocysteine into extracellular fluid. If a significant amount of hepatic choline is used to supply betaine for homocysteine remethylation to help compensate for reduced 5-CH3THF availability, then less choline would be available for phosphatidylcholine production. Greater production of phosphatidylcholine by PEMT would be required, which would cause increased homocysteine synthesis under the low-folate conditions of our protocol.
These findings challenge the role of RM in the hyperhomocysteinemia that occurs during the initial decline in 5-CH3THF production caused by folate deficiency. However, these data might still fit into the proposed model of impaired RM in hyperhomocysteinemia if the contributions of homocysteine transport and the tissue-specificity of one-carbon cycle enzyme expression are considered. Hyperhomocysteinemia might be the product of increased export of homocysteine from peripheral tissues that cannot adequately metabolize homocysteine when 5-CH3THF production is insufficient because they are completely dependent on folate-dependent RM. For example, homocysteine is exported from cultured human intestinal epithelial cells (19). Hyperhomocysteinemia might reflect in part increased homocysteine transit from the periphery to those organs possessing the transsulfuration pathway (liver, kidney, intestine, and pancreas) and betaine homocysteine S-methyltransferase (BHMT) expression (liver and kidney). This model could account for the negative vascular consequences of mild hyperhomocysteinemia even if total RM is unimpaired.
It is worth noting that we were unable to detect [13C1]cysteine, which would be produced when [13C1]serine is used as a substrate by cystathionine ß-synthase. Although this might be related to insufficient sensitivity of our analytical measurements, the most likely explanation for this observation is that there is little transsulfuration flux under the experimental conditions. Specifically, the subjects began the study after an overnight fast and consumed negligible dietary protein during the 9-h protocol; these conditions combined to minimize the availability of S-adensylmethionine for inhibition of MTHFR and activation of cystathionine ß-synthase, thus limiting transsulfuration flux.
In conclusion, total RM did not significantly change in response to moderate dietary folate restriction or the MTHFR 677CT polymorphism despite modestly reduced folate-dependent RM. Under these conditions, increased homocysteine synthesis probably contributes more to elevated plasma total homocysteine concentration than the decline in folate-dependent RM. Rates of homocysteine synthesis and folate-dependent RM for subjects with the MTHFR 677 T/T genotype suggest that homocysteine metabolism, particularly homocysteine synthesis, requires higher folate status in that genotype. Further study is required to determine the mechanism(s) responsible for increased homocysteine synthesis, to measure directly the contribution of betaine to RM when 5-CH3THF production declines, and to determine what level of folate deficiency is required to impair total RM.
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FOOTNOTES |
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T polymorphism on homocysteine synthesis in healthy young females. FASEB J. 17: A308 (abs.)]. 
2 Supported by National Institutes of Health grants DK56274 (J.F.G.), DK42033 (B.S.), DK15289 (C.W.), and GCRC M01-RR00082, U.S. Department of Agriculture-National Research Initiative grants 00–35200-9113 (J.F.G.) and 00–35200-9102 (L.B.B.), and the Department of Veterans Affairs Medical Center (C.W.). This paper is Florida Agricultural Experiment Station Journal Series No. R-10264.
3 Supplemental Figures 1–2 and Table 1 are available with the online posting of this paper at www.nutrition.org.
5 Abbreviations used: AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; 5-CH3THF, 5-methyltetrahydrofolate; GCRC, General Clinical Research Center; HPLC-EC, HPLC and electrochemical detection; MTHFR, methylenetetrahydrofolate reductase; PEMT, phosphatidylethanolamine N-methyltransferase; PLP, pyridoxal 5'-phosphate; RM, remethylation.
Manuscript received 29 November 2004. Initial review completed 30 December 2004. Revision accepted 22 January 2005.
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