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T Polymorphism and Dietary Folate Restriction Affect Plasma One-Carbon Metabolites and Red Blood Cell Folate Concentrations and Distribution in Women1,2
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;
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
3To 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 interact to affect methionine-cycle metabolite concentrations is uncertain. We evaluated the effects of dietary folate restriction on relations among folate status indices and plasma concentrations of methionine cycle metabolites in women with the MTHFR 677 C/C and T/T genotypes. Healthy, normohomocysteinemic women (n = 18; 20–30 y old) of adequate B vitamin status, and equally divided according to MTHFR 677CT genotype (9 C/C and 9 T/T) were recruited. Folate status indices and methionine cycle metabolites were measured in blood samples collected at baseline and after 7 wk of dietary folate restriction (115 µg dietary folate equivalents/d). Significant negative correlations between plasma total homocysteine concentrations and total or 5-methyl folate concentrations (P = 0.041 and 0.023, respectively) in RBCs were found only in T/T subjects. Formylated folates were detected in RBCs of T/T subjects only, and their abundance was predictive of plasma total homocysteine concentration despite no significant alteration by folate restriction. Plasma concentrations of S-adenosylmethionine and S-adenosylhomocysteine were not significantly affected by dietary folate restriction and the MTHFR 677 T/T genotype. In conclusion, plasma total homocysteine concentrations in subjects with the MTHFR 677 T/T genotype were inversely related to 5-methyl folate concentrations and directly related to formylated folate concentrations in RBCs, even though the latter were not significantly affected by moderate folate restriction.
KEY WORDS: • homocysteine • methionine • S-adenosylmethionine • S-adenosylhomocysteine • women
Although homocysteine is a key intermediate in sulfur amino acid metabolism, elevated plasma total homocysteine concentration is an independent risk factor for vascular diseases (1–3). Many factors affect plasma total homocysteine concentration in humans, including the nutritional status of folate, riboflavin, and vitamins B-6 and B-12 (4). Of these nutrients, folate is the most important determinant of plasma total homocysteine concentration. Inverse relations exist between plasma total homocysteine concentration and folate status or dietary folate intake (5). Plasma total homocysteine concentrations are elevated in folate deficiency (6) or during dietary folate-restriction (7), whereas folate supplementation (8) and folate fortification of foods (9) reduce plasma total homocysteine concentration.
Genetic factors that affect folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR)4 677CT polymorphism, also can influence plasma total homocysteine concentration (10). The MTHFR 677CT polymorphism causes an alanine-to-valine substitution at position 222 of the enzyme (11) and reduces its activity in vitro (11) and ex vivo (12,13). The loss of enzyme activity appears to be due to increased dissociation of the essential flavin cofactor (14). The presence of 5-methyltetrahydrofolate (5-CH3THF) and other folates protects against loss of FAD in a dose-dependent manner (14). Homozygosity for the MTHFR 677CT polymorphism is not a significant determinant of plasma total homocysteine concentration when folate status in high, but does increase sensitivity to the effects of low folate status on plasma total homocysteine concentration (10,15). Another consequence of this polymorphism is altered partitioning of one-carbon units, which is reflected in the accumulation of formylated forms of folate in RBCs (16). The relation between folate status and accumulation of formylated folates has not been examined.
The ratio of the intracellular concentrations of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) is thought to be a key regulator of AdoMet-dependent methyltransferase activity (17), but the significance of plasma concentrations of AdoMet and AdoHcy for evaluating methionine cycle metabolism is unclear. Plasma AdoHcy concentration might be a more sensitive indicator of perturbed methionine cycle metabolism than plasma total homocysteine concentration (18,19). However, it is unknown whether folate status or the MTHFR 677CT polymorphism influences plasma concentrations of AdoMet and AdoHcy.
Few investigations have addressed the difference between MTHFR 677 genotypes in responses of methionine cycle metabolites in plasma to dietary folate restriction. In this report, we test the hypothesis that the MTHFR 677CT polymorphism interacts with folate status or dietary folate restriction in their effects on the concentrations of methionine cycle metabolites (AdoMet, AdoHcy, and homocysteine) in plasma and on folate distribution in RBCs.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T polymorphism and folate status on homocysteine remethylation kinetics using stable isotope-labeled amino acid tracers. Some data were used in a previous analysis of the effects of dietary folate restriction and the MTHFR 677CT polymorphism on the rate of change of folate status and plasma total homocysteine concentration (20).
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 variants 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 (9.7–36.2 nmol/L), vitamin B-12 (151–625 pmol/L), pyridoxal 5'-phosphate (PLP; 33.4–117 nmol/L), and homocysteine (6.5–13 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. Subjects consumed a 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. The folate status of the 2 subjects who followed the diet for 5 wk was similar to that of 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. Subjects consumed breakfast on the GCRC and were given a take-out lunch to eat at their convenience. Subjects returned to the GCRC to consume their evening meal. Nutrient inadequacies of the study diets (other than folate) were compensated for by custom vitamin and mineral supplements administered each morning 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 (20).
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 was measured as the semicarbazone-derivative by reverse-phase HPLC with fluorescence detection (24). Plasma total homocysteine was measured as the SBDF-derivative by reverse-phase HPLC with fluorescence detection (25).
RBC total folate, the distribution of folate forms within RBCs, and the polyglutamate chain lengths of RBC folates 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 and 7 T/T subjects. RBC folates, folate distribution, and polyglutamate chain length were measured by a method that combines affinity chromatography with HPLC and electrochemical detection (HPLC-EC) (26). RBC folate data were normalized to hemoglobin content, which was measured by a commercially available kit (No. 525, Sigma).
Concentrations of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) in plasma were measured as the isoindole-derivatives by HPLC with fluorescence detection (27). Briefly, deproteinized plasma extracts were partially purified by reverse-phase HPLC, the eluates were desalted on solid phase extraction columns, and the fractions containing AdoMet and AdoHcy were derivatized in a reaction involving napthalenedialdehyde and cyanide. The isoindole derivatives of AdoMet and AdoHcy were analyzed in separate injections by reverse-phase HPLC and quantified with fluorescence detection. Intra-assay CV, which were calculated from a single sample measured 6 times on 1 d, were 4.2% for AdoMet and 12.9% for AdoHcy. Interassay CV for a single sample measured on 12 separate days were 7.1% for AdoMet and 12.0% for AdoHcy.
Differences in numeric values between genotypes were assessed by 2-sample t tests. Plasma total homocysteine concentrations and the AdoMet:AdoHcy ratio values were transformed (log10) before analyses to minimize the effects of outliers. The Mann-Whitney rank-sum test (nonparametric t test) was used for intergenotype comparison of the percentage of RBC folates found in the formyl form because these data could not be normalized. Fisher’s exact test was used for intergenotype comparison of the proportion of RBC samples that contained formylated folates. Within genotypes, the paired t test was used to detect differences between means, and the sign test (binary outcome) was used to detect differences in proportions due to dietary folate restriction. The effects of folate restriction on all measures (changes from baseline) were compared between genotypes by the same methods used for baseline comparisons. Linear regression was used to estimate how concentrations of methionine cycle metabolites in plasma changed with folate status (28). Data are reported as means or slopes with SE. Differences were considered significant at P < 0.05, two-sided.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T genotype groups did not differ. B vitamin status of subjects at baseline and after dietary folate restriction is summarized in Table 1 (n = 9 for most analyses). For HPLC-EC measurements of RBC folates and mean polyglutamate chain length of RBC folates, data were available both before and after folate restriction for 5 C/C subjects and 7 T/T subjects. Only these paired data were used to assess quantitative changes of RBC total folates, RBC 5-CH3THF, percentage of RBC folates in the formylated form, and polyglutamate chain length of RBC folates. Data were available for 2 additional C/C subjects and 1 additional T/T subject at baseline only, and these data were included in reporting the fraction of subjects whose RBCs contained detectable levels of formylated folates (i.e., n = 7 for C/C genotype and n = 8 for T/T genotype at baseline, and n = 5 for C/C genotype and n = 7 for T/T genotype after folate restriction). At baseline, all measures of blood folate concentrations were greater (31–47%) in subjects with the C/C genotype than subjects with the T/T genotype. Formylated folates were detected in RBCs from 5 of 7 T/T subjects and accounted for a mean of 
10% of total RBC folates (range 0–40.7%). Formylated folates were not detected in the RBCs of C/C subjects. The polyglutamate chain length of RBC folates did not differ between genotypes. Vitamin B-12 status also was greater in C/C subjects at baseline (37%), but vitamin B-6 status (i.e., plasma PLP) did not differ significantly between genotypes. Plasma total homocysteine was 39% greater in T/T subjects at baseline (Table 2). No differences in plasma AdoMet or AdoHcy were detected between genotypes at baseline.
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Linear regression was performed on data from subjects within MTHFR 677 C/C and T/T genotypes after folate restriction as an exploratory assessment of the dependence of the concentrations of methionine cycle metabolites in plasma on indices of vitamin status. Plasma total homocysteine data and the AdoMet:AdoHcy ratio were transformed (log10) before analyses to minimize the effect of outliers. The dependence of plasma total homocysteine concentration on folate status after folate restriction is summarized in Table 3. Slopes from linear regression analyses of the dependence of plasma total homocysteine concentration on RBC total folates and RBC 5-CH3THF measured by HPLC-EC were significantly negative (P < 0.05) in the T/T genotype only. The percentage of RBC folates detected in the formyl form was directly proportional to plasma total homocysteine concentration in T/T subjects (Table 3, P < 0.001). No significant relations between vitamin status and the plasma concentrations of AdoMet, AdoHcy, or the AdoMet:AdoHcy ratio were detected (data not shown). Further, no correlations between plasma total homocysteine concentration and AdoMet, AdoHcy, or the AdoMet:AdoHcy ratio were detected (data not shown).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The lower folate status of T/T subjects compared with C/C subjects at baseline is not uncommon (20,29), and might have accounted for the difference in plasma total homocysteine concentration concentrations at that time point (Table 1). However, no subjects were initially folate deficient, and the effects of dietary folate restriction on indices of folate status did not differ between genotypes. Plasma vitamin B-12 concentrations were also lower in T/T subjects at baseline, but no subjects were vitamin B-12 deficient before or after dietary folate restriction, and the gap between the genotypes narrowed during that time period.
Similar to previous reports involving human RBCs and immortalized lymphocytes derived from humans, only the MTHFR 677 T/T genotype was associated with an accumulation of formylated folates (16,29,30). Also, one study found no detectable difference in formylated folate accumulation in RBCs between T/T subjects with high vs. low RBC folate concentrations (29). Data from the present investigation demonstrate for the first time that dietary folate restriction does not significantly change the percentage of folates found in the formyl form within individuals (Table 1). Consistent with a previous report (16), formylated folates were not detectable in RBCs from some (2 of 8) of T/T subjects, even after dietary folate restriction. Further, accumulation of formylated folate was highly variable in those subjects that accumulated this folate species (2–41%). Together, these results suggest that the MTHFR 677 CT genotype might not be the only genetic factor affecting formylated folate production, deposition, and/or retention in erythrocytes.
Consistent with previous reports (16,30), the mean glutamate chain length of RBC folates was remarkably consistent between MTHFR C/C and T/T genotypes (Table 1). From the current investigation, we can add that glutamate chain length changed little in response to dietary folate restriction in either genotype and did not significantly correlate with folate status within the range associated with this study.
Previous studies reported differences in the effects of folate status or dietary folate intake on plasma total homocysteine concentration between the MTHFR 677 C/C and T/T genotypes (12,13,15,20). The significant correlations between plasma total homocysteine concentration and RBC folate concentrations measured by HPLC-EC only in TT subjects in this study (Table 3) are consistent with those reports (15). These data also support the viewpoint that RBC folate concentration is a better predictor of intracellular folate status than is plasma or serum folate (31). A significant inverse correlation between the percentage of formylated folates in RBCs and plasma total homocysteine concentration within the T/T genotype is consistent with the hypothesis that the MTHFR 677 CT polymorphism diverts folates away from production of 5-CH3folate (which is required for folate-dependent homocysteine remethylation) and toward formylated folates.
Concentrations of AdoMet and AdoHcy in plasma were shown to correlate with intracellular AdoMet and AdoHcy concentrations in lymphocytes (19), and are considered to be important markers of methionine cycle metabolism (18,19). The present study is the first to examine the effects of the MTHFR 677CT polymorphism and a controlled dietary folate restriction protocol on the plasma concentrations of AdoMet and AdoHcy. No significant relations were found between the concentrations of AdoMet and AdoHcy in plasma, or their ratio (AdoMet:AdoHcy) with MTHFR 677 genotypes, dietary folate intake (Tables 1, and 2), or folate status. These results, combined with the fact that plasma total homocysteine concentration was significantly dependent on folate status, do not support the notion that plasma AdoMet and AdoHcy concentrations are more sensitive markers than plasma total homocysteine concentration of the effects of folate status on methionine cycle metabolism, at least in otherwise healthy young women with marginal folate status.
The concentrations of plasma folate measured by the radioassay were, on average, less than half of the value of serum folate measured by the microbial assay (Table 1). These results are consistent with those from an interlaboratory comparison study that directly addressed this issue (32). The factors responsible for large differences in response between the radioassay and microbial assay for measurement of serum or plasma folate remain to be determined. Despite the difference in absolute values obtained by the 2 assays, a 60–70% reduction in plasma or serum folate due to dietary folate restriction was measured for both genotypes with each assay, which indicates that either assay might suffice for determining relative changes of folate status.
In conclusion, we confirmed that the concentrations of folates in RBCs are more closely associated with plasma total homocysteine concentration than are serum or plasma folate measurements in the MTHFR 677 T/T genotype. We also confirmed that although RBC formylated folates are accumulated only by individuals with the T/T genotype, not all individuals with the T/T genotype accumulate this form of the vitamin. Further, dietary folate restriction does not significantly affect the percentage of RBC folates produced in this form within individuals. Plasma concentrations of AdoMet and AdoHcy are insensitive to short-term dietary folate restriction and to folate status within the range from high to low normal, and are not affected by MTHFR 677 genotype. Whether these conclusions can be extended to conditions of more pronounced folate deficiency is a question that should be addressed in future studies.
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FOOTNOTES |
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2 Supported by National Institutes of Health grants DK56274 (J.F.G.), 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-10234.
4 Abbreviations used: AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; CC, methylenetetrahydrofolate reductase 677 CC genotype; 5-CH3THF, 5-methyltetrahydrofolate; GCRC, General Clinical Research Center; HPLC-EC, HPLC and electrochemical detection; MTHFR, methylenetetrahydrofolate reductase; PLP, pyridoxal 5'-phosphate; TT, methylenetetrahydrofolate reductase 677 TT genotype.
Manuscript received 29 November 2004. Initial review completed 13 January 2005. Revision accepted 22 January 2005.
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