Yoshiaki Somekawa, M.D., a Kimiko Kobayashi, M.D., b Shigeo Tomura, M.D., Ph.D., c Takeshi Aso, M.D., Ph.D., d and Hideo Hamaguchi, M.D., Ph.D.

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1 FERTILITY AND STERILITY VOL. 77, NO. 3, MARCH 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Effects of hormone replacement therapy and methylenetetrahydrofolate reductase polymorphism on plasma folate and homocysteine levels in postmenopausal Japanese women Yoshiaki Somekawa, M.D., a Kimiko Kobayashi, M.D., b Shigeo Tomura, M.D., Ph.D., c Takeshi Aso, M.D., Ph.D., d and Hideo Hamaguchi, M.D., Ph.D. b Toride Kyodo General Hospital, Toride, and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan Received May 21, 2001; revised and accepted August 21, Presented in part at the 11th World Congress of Endocrinology, Sydney, New South Wales, Australia, October 30, Reprint requests: Yoshiaki Somekawa, M.D., Toride Kyodo General Hospital, Hongo Toride, Ibaraki, Japan (FAX: ; pv2t-sigi@asahinet.or.jp). a Department of Obstetrics and Gynecology, Toride Kyodo General Hospital. b Institute of Basic Medical Sciences, University of Tsukuba. c Institute of Community Medicine, University of Tsukuba. d Department of Obstetrics and Gynecology, Tokyo Medical and Dental University, Tokyo, Japan /02/$22.00 PII S (01) Objective: To evaluate the relationships among the methylenetetrahydrofolate reductase (MTHFR) polymorphism, plasma folate, total homocysteine (Hcy) levels, lipids, and the reduction of Hcy levels resulting from hormone replacement therapy (HRT). Design: Clinical study. Setting: Outpatient department of obstetrics and gynecology in a general hospital. Patient(s): Two hundred seventeen postmenopausal Japanese women. Intervention(s): Of the 217 women, 172 patients were under continuous treatment with oral conjugated equine estrogen and medroxyprogesteron acetate. Main Outcome Measure(s): Fasting Hcy, folate, methionine, lipids, and apolipoproteins were measured before and after 3 months of HRT. Result(s): The plasma Hcy concentration was significantly higher in the low folate than in the high-folate group only in patients with the homozygous (T/T) mutant. Plasma Hcy concentrations were significantly correlated with age (R 0.64, P.02) or years since menopause (R 0.73, P.02) only in the low-folate group with T/T. The plasma Hcy concentration decreased significantly in all genotypes after 3 months of HRT, but the levels of serum folate and methionine remained unchanged. Conclusion(s): The MTHFR polymorphism was associated with a higher Hcy concentration, and this association was related to the serum folate level. Hormone replacement therapy reduced the plasma Hcy concentration independently of the MTHFR polymorphism. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Methylenetetrahydrofolate reductase polymorphism, folate, homocysteine Recent studies have shown that moderately elevated blood levels of homocysteine (Hcy) are associated with cardiovascular disease (1). Hcy is a sulfur amino acid whose metabolism is at the intersection of two metabolic pathways: remethylation and transsulfuration (2). In the remethylation pathway, methylenetetrahydrofolate reductase (MTHFR) catalyzes 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate, the principal circulating form of folate and methyl donor for the remethylation of Hcy to methionine. Kang et al. reported a thermolabile form of MTHFR that is associated with elevated Hcy levels and an increased risk of coronary heart disease (3). Frosst et al. demonstrated that thermolabile MTHFR is caused by an alanine-to-valine missense mutation (4). Furthermore, folate supplementation has been reported to normalize hyperhomocysteinemia in patients with thermolabile MTHFR, which might have a potential as a preventive therapy for coronary heart disease (5, 6). The effects of estrogen on plasma Hcy remain unclear. Plasma Hcy levels tend to increase after menopause and a correlation between this increase and the reduction of serum 481

2 estradiol levels suggests that estrogen lowers the plasma Hcy concentration (7, 8). Estrogen lowers the levels of vitamins required for remethylation (folate and vitamin B12) and transsulfuration (vitamin B 6 ) of Hcy, however, which might be expected to raise plasma Hcy concentrations (9, 10). Estrogen replacement therapy (ERT) and hormone replacement therapy (HRT) have not shown consistent effects on plasma Hcy concentrations. For example, some studies showed that HRT reduced plasma Hcy concentrations (8, 11, 12), while another showed no such effect of HRT (13). According to some latest research reports, plasma Hcy concentration is weakly correlated with serum lipids including serum total cholesterol (TC), high-density lipoprotein (HDL) cholesterol and triglyceride (TG) (14, 15), and folate supplement was found to reduce the plasma Hcy concentration, TC, low-density lipoprotein (LDL)-cholesterol, and TG in end-stage renal disease (16). The purpose of this study was to determine the relationships among the MTHFR polymorphism, plasma folate, and plasma total Hcy concentrations, as well as the effects of estrogen and HRT on Hcy levels. Plasma Hcy, folate, and lipid concentrations were also followed to determine the extent to which folate and Hcy modified these cardiovascular risk factors. MATERIALS AND METHODS Patients The authors assessed the MTHFR polymorphism among 217 postmenopausal Japanese women aged 44 to 80 years (mean age years). In all cases, more than 6 months had elapsed since the last menstrual bleeding, the serum estradiol level was lower than 20 pg/ml (73.42 pmol/ L), and the serum follicle-stimulating hormone (FSH) level was more than 50 IU/dL. Those who exercised regularly, smoked or drank heavily (consuming over 30 cigarettes or drinking over 80 g of alcohol per day for longer than 5 years), or were clinically diagnosed with liver disease, ischemic heart disease, diabetes, renal disease, or metabolic or other endocrine diseases that could influence lipid metabolism were excluded. None of the women were receiving medications that could affect lipid metabolism. The study was approved by the institutional review of board of the hospital and was performed in accordance with the Declaration of Helsinki. Each subject gave her written informed consent for participation in the study before participation. Variables in the backgrounds of the patients such as age, height, weight, body mass index (BMI), and age at menopause were measured. MTHFR Genotype and Homocysteine Venous blood samples were collected from 217 postmenopausal women. DNA was isolated from the leukocytes by the phenol extraction method, and subjected to the polymerase chain reaction. The amplified fragments were digested with HinfI restriction digestion, which can recognize C to T substitution. This single nucleotide substitution corresponds to a conversion of alanine to valine in the MTHFR encoding region. If the mutation is present, HinfI digests the 198-bp fragment into a 175-bp and a 23-bp fragment. The fragments were resolved by electrophoresis in polyacrylamide gels and stained with ethidium bromide. Genotypes were expressed as C/C for homozygous normal, C/T for heterozygous, and T/T for homozygous mutant. Plasma Hcy levels were assayed by high-performance liquid chromatography (HPLC) (17). Plasma folate levels were measured microbiologically (18), and plasma methionine levels were assayed by HPLC (19). Lipids and Apolipoproteins Analysis The values of fasting serum TC, TG, HDL-cholesterol, LDL-cholesterol, apolipoprotein (Apo) A1, Apo B, and Apo E were measured to evaluate the risk of atherosclerosis. After an overnight fast, blood was collected from each patient for the estimation of lipids and lipoproteins. TC and TG levels were measured using enzymatic colorimetric methods (Ono Pharmaceutical, Osaka, Japan) and the enzymatic method (Shino-test, Tokyo, Japan). HDL-cholesterol levels were measured by a direct method (20) using commercially available kits (Kyowa Medix, Tokyo, Japan) on a Hitachi chemical analyzer. LDL-cholesterol levels were calculated with Friedewald s equation (21). Apo AI, Apo B, and Apo E were measured by an immunoturbidimetric method using commercially available kits (Daiichi Pure Chemical, Tokyo, Japan). The intraassay coefficients of variation (CVs) for TC, TG, HDL-cholesterol, Apo AI, Apo B, and Apo E, were 0.7%, 1.9%, 0.7%, 2.5%, 4.0%, and 2.3%, respectively. Hormone Replacement Therapy (HRT) Of these 217 women, 172 women with severe menopausal symptoms, diagnosed as osteoporosis according to the criteria [patients with low lumbar bone mineral density (BMD) (less than 70% of young adult mean) or one or more nontraumatic vertebral fractures and lumbar BMD less than 80% of young adult mean] proposed by the Japanese Society of Bone Metabolism in 1996 (22), or showing unfavorable lipid profiles [serum TC levels over 220 mg/dl or LDLcholesterol levels over 140 mg/dl (23)] were treated by oral conjugated equine estrogen (0.625 mg/day) and medroxyprogesteron acetate (2.5 mg/day) continuously with the agreement to be treated with HRT. Levels of Hcy, folate, methionine, lipids, and lipoproteins were measured before and after 3 months of HRT. All patients in this study were advised to maintain a normal diet not to be changed throughout the duration of the study. 482 Somekawa et al. Homocysteine, HRT, and MTHFR polymorphism Vol. 77, No. 3, March 2002

3 TABLE 1 Baseline clinical characteristics in MTHFR genotypes. C/C (N 96) C/T (N 91) T/T (N 30) P value a Age (y) NS Height (m) NS Weight (kg) NS BMI NS Age of menopause (y) NS Years since menopause (y) NS Systolic blood pressure (mmhg) NS Diastolic blood pressure (mmhg) NS Plasma homocysteine (nmol/ml) b.05 Plasma folate (nmol/l) NS Note: Values are means SEs. BMI body mass index; NS not significant. a P value by one-way factorial analysis of variance. b P.05 by Scheffe s F vs. C/C genotype. Somekawa. Homocysteine, HRT, and MTHFR polymorphism. Fertil Steril Statistical Analysis Results are given as the mean standard error (SE). Data analysis was performed using a Stat View 5.0 software package (SAS Institute Inc., Cary, NC). Baseline parameters were compared among the groups by the Kruskal Wallis test. One-way factorial analysis of variance (ANOVA) and Scheffe s F as a post hoc test were used for the evaluation of differences among three stratified groups according to the serum folate level in each MTHFR genotype. ANOVA for repeated measures was used to evaluate the significance of any changes in the plasma Hcy, methionine, folate, and serum lipids. Linear regression analysis was used to determine correlation among age, years since menopause, and plasma Hcy levels. Linear regression analysis was also used to determine whether plasma Hcy or folate concentrations are correlated with lipids. Differences with P.05 were defined as statistically significant. RESULTS The frequencies of the three genotypes were 44% for homozygous normal (C/C), 42% for heterozygous (C/T) and 14% for homozygous mutant (T/T); and the allele frequency of the substitution was 35%, which is consistent with the values previously reported in Japan (24, 25). Background variables such as age, height, weight, BMI, and age at menopause did not exhibit significant differences among the genotypes. The women with the T/T genotype had higher levels of Hcy concentration ( nmol/ml) than those with the C/C genotype ( nmol/ml). Those with the C/C or C/T genotype had higher plasma folate concentrations than those with the T/T genotype, but these differences were not statistically significant ( nmol/l and nmol/l vs nmol/l) (Table 1). Mean folate levels in these Japanese subjects were higher than those reported in Western communities (1, 6). The effects of folate on the relation between Hcy and genotype were examined by stratifying each genotype into three groups by the plasma folate level (low-folate group, 13.6 nmol/l; moderate-folate group, nmol/l; high-folate group, 20.4 nmol/l). The plasma Hcy concentration was significantly higher in the low-folate group than in the high-folate group only in the patients with the T/T genotype, and the Hcy concentrations did not vary by folate level in those with the C/C or C/T genotype (Table 2). A statistically significant negative correlation between Hcy and folate was found only for those with the T/T genotype. The effect of folate on the relation between Hcy concentrations and age or years since menopause was further examined. Plasma Hcy concentrations were significantly correlated with age (R 0.64, P.02) or years since menopause (R 0.73, P.02) only in the low-folate group TABLE 2 Relationships among plasma homocysteine levels and folate levels according to MTHFR genotype. MTHFR genotype Low-folate group ( 13.6 nmol/l) Moderate-folate group ( nmol/l) High-folate group ( 20.4 nmol/l) P value a C/C (14) (38) (44) NS C/T (21) (33) (37) NS T/T b (8) (11) (11).05 Note: Values in parentheses are numbers of patients. NS not significant. a P value by one-way factorial analysis of variance. b P.05 by Scheffe s F vs. high-folate group. Somekawa. Homocysteine, HRT, and MTHFR polymorphism. Fertil Steril FERTILITY & STERILITY 483

4 TABLE 3 Changes of plasma homocysteine, folate, and methionine levels after hormone replacement therapy for 3 months according to MTHFR genotype. MTHFR genotype Baseline with the T/T genotype. This correlation was not significant in the moderate or high-folate group with the T/T genotype, or in any folate concentration groups with the C/C genotype or C/T genotype. No statistically significant differences occurred in serum TC, TG, LDL-cholesterol, HDL-cholesterol, Apo AI, Apo B, or Apo E levels between patients in different folate concentration groups among the three genotypes. Linear regression analysis revealed that plasma Hcy concentrations and plasma folate concentrations were not statistically significantly associated with serum TC, TG, LDL-cholesterol, HDL-cholesterol, Apo AI, Apo B, or Apo E. Plasma Hcy concentrations decreased significantly in all genotypes after 3 months of HRT, but no statistically significant changes occurred in the concentrations of plasma folate or methionine at the same time point (Table 3). Plasma Hcy concentrations were decreased 5.9% in the C/C genotype, 4.7% in the C/T genotype, and 8.3% in the T/T genotype. Reduction rate of plasma Hcy after HRT for 3 months tended to be higher in the patients with the T/T genotype than the C/C or C/T genotype, but these differences were not statistically significant among the three groups. DISCUSSION After HRT for 3 months P value Homocysteine (nmol/ml) C/C (n 76) a.05 C/T (n 71) a.05 T/T (n 23) a.05 Folate (nmol/l) C/C (n 76) NS C/T (n 71) NS T/T (n 23) NS Methionine ( mol/dl) C/C (n 19) NS C/T (n 18) NS T/T (n 6) NS Note: Values are means SEs. HRT hormone replacement therapy; NS not significant. a P.05 by analysis of variance for repeated measures vs. baseline. Somekawa. Homocysteine, HRT, and MTHFR polymorphism. Fertil Steril The effects of HRT to reduce plasma Hcy concentrations observed in the present study are in accord with those reported (8, 11, 12), although one study showed no such effect (13). One explanation for this discrepancy is that the subjects who showed no change in Hcy level after HRT had normal baseline Hcy concentrations, while those who showed reduction in Hcy level after HRT had primarily elevated Hcy concentrations as in this study. These normal levels of Hcy may not be affected by HRT. This study has yielded three intriguing results. First, individuals heterozygous or homozygous for mutations with an alanine-to-valine substitution have reduced enzyme activity and thermolability, resulting in elevated plasma Hcy caused by suboptimal intake of folate. It has been hypothesized that the region linked with the MTHFR polymorphism is involved in folate binding and that the enzyme may be stabilized in the presence of sufficient levels of folate (4). Therefore, the combination of the genetic defect and inadequate folate intake may cause elevated Hcy concentrations, and this elevation of Hcy could be corrected with folic acid supplements. This prompts the following question: What is the adequate folate level to compensate for this genetic defect? Folate levels are considerably high in Japan due to differences in nutritional intake from the Western community. The World Health Organization (WHO) has proposed a lower limit of 13.6 nmol/l for folate concentration (26). This study employed the cut off folate level of the low group according to WHO criteria in this study. Pancharuniti et al. have reported that a folate concentration below 12.5 nmol/l increases the Hcy concentration (27). In our study, thermolabile MTHFR in the T/T genotype was associated with a higher Hcy level than that in the homozygous normal (C/C) genotype with a folate level below 13.6 nmol/l. Thus, a plasma folate level above 13.6 nmol/l may be necessary to compensate for this genetic defect. Second, plasma Hcy concentrations are higher in postmenopausal women than in premenopausal women, and plasma fasting Hcy concentrations are reduced by E2-dydrogesterone therapy in postmenopausal women (7, 8). These studies suggest a relation between plasma Hcy concentration and serum estrogen status. Moreover, in the present study, age, and serum estrogen levels were related to increases of plasma Hcy concentrations in women with low folate levels with thermolabile MTHFR (T/T), while no such relation is seen in women with middle to high folate levels or with the homozygous (C/C) or heterozygous normal (C/T) genotype. Brown et al. recently reported that postmenopausal women with T/T genotype plasma Hcy levels did not show a decreased response to HRT (28). This result is not consistent with our result, but their sample size of the T/T genotype, receiving HRT consisted of only five women and was not sufficient to support a definite conclusion. The present results suggest that estrogen deficiency may amplify the responsiveness of plasma folate levels to thermolabile MTHFR and that postmenopausal women with thermolabile MTHFR (T/T) are good candidates to receive HRT. Furthermore, the mechanism by which HRT reduces the plasma Hcy is unclear. HRT is reported to decrease plasma 484 Somekawa et al. Homocysteine, HRT, and MTHFR polymorphism Vol. 77, No. 3, March 2002

5 folate levels, and an increased remethylation pathway can be hypothesized as a mechanism of the reduction of Hcy. In the present study, however, plasma methionine and folate levels were unchanged, while HRT reduced plasma Hcy concentration independently of the MTHFR polymorphism. This reduction may be unrelated to the remethylation of Hcy. Plasma cystationine levels were analyzed at the same time as methionine (data not shown), but all cystationine levels were under the analytical sensitivity both at baseline and after HRT for 3 months. Thus, HRT-induced reduction of Hcy cannot be related to either the remethylation pathway or transsulfuration pathway. Another explanation for this mechanism may be related to an increase in kidney methionine synthase activity (29). A more plausible explanation may be associated with the transamination of methionine proposed by Blom et al., who showed that premenopausal women had higher methionine transamination rates than young men, suggesting that a lower plasma Hcy concentration was caused by a higher amount of methionine degradation via the transamination pathway under a high estrogen level (30). We can speculate that the plasma Hcy is reduced by this methionine degradation via the transamination pathway in a high estrogen status, but not in estrogen deficiency, resulting in hyperhomocysteinemia in low-plasma-folate women with thermolabile MTHFR (T/T). Third, is there an interaction between lipids or apolipoproteins and Hcy metabolism through HRT? Numerous data support the increased risk of cardiovascular disease in postmenopausal women, and this increased risk is closely related to the decrease in serum estradiol. HRT is cardioprotective mainly through its effects on lipid metabolism, blood vessels, or the arterial blood flow (31, 32), but it remains controversial whether HRT confers cardioprotection because it also enhances activation of the coagulation system (33). Homocysteine may be an additional parameter affected by estrogen status and may be responsible for the cardioprotective influence of estrogen status in women with MTHFR genetic defects. Recent findings have raised interest in the possible interaction between plasma Hcy or folate and serum lipids. For example, folate supplements have been shown to reduce plasma Hcy concentration, TC, LDL-cholesterol, and TG (16), and short-term supplementation with folic acid and antioxidant vitamins can reduce LDL oxidation (34). In contrast with some other studies (14, 15), the authors found no significant association among the MTHFR polymorphism, plasma Hcy concentration, folate concentrations, and serum lipids, or apolipoproteins, suggesting the prevalence of multiple cofactors among the subjects in these studies. There may be no significant interactions between plasma Hcy or folate concentrations and lipids or apolipoproteins. In conclusion, these findings indicate that the combination of MTHFR mutation and low folate levels may lead to elevations in plasma Hcy levels especially after menopause, and that folate supplementation may possibly compensate for the genetic defect. HRT reduces the plasma Hcy levels independently of the MTHFR polymorphism, but the mechanism by which HRT reduces plasma Hcy levels remains unclear. 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