Methylenetetrahydrofolate reductase mutation (677C → T) negatively influences plasma homocysteine response to marginal folate intake in elderly women

Methylenetetrahydrofolate Reductase Mutation (677C 8 T) Negatively Influences Plasma Homocysteine Response to Marginal Folate Intake in Elderly Women Gail P.A. Kauwell, Chad E. Wilsky, James J. Cerda, Kelli Herrlinger-Garcia, Alan D. Hutson, Douglas W. Theriaque, Anita Boddie, Gail C. Rampersaud, and Lynn B. Bailey Individuals who are homozygous for the methylenetetrahydrofolate reductase (MTHFR) 677C 8 T mutation have depressed serum folate (SF) and elevated plasma total homocysteine (tHcy) concentrations, which may affect folate requirements and increase the risk for coronary artery disease. A controlled metabolic study (14 weeks) using a depletion/repletion protocol was performed in women (aged 60 to 85 years, N ⴝ 33) to provide age-specific data on the effects of the MTHFR mutation on SF and tHcy status. Subjects consumed a moderately folate-deplete diet (118 ␮g/d) for 7 weeks, followed by 7 weeks of folate repletion with 200 or 415 ␮g/d provided as two different treatments. Following folate depletion, the mean SF concentration was lower for homozygous (P ⴝ .017) versus heterozygous subjects. Homozygotes for the 677C 8 T mutation showed a higher (P ⴝ .015) percent increase in plasma tHcy (44%) than heterozygous (20%) or normal (15%) subjects. At week 7, the mean plasma tHcy concentration was higher in homozygous subjects (12.5 ⴞ 5.3 ␮mol/L, mean ⴞ SD) versus the heterozygous (10.8 ⴞ 3.8 ␮mol/L, P ⴝ .008) or normal (11.3 ⴞ 2.7 ␮mol/L, P ⴝ .001) genotype groups. Following folate repletion, plasma tHcy concentrations were not different between genotype groups, despite a higher (P F .016) SF concentration in subjects with the homozygous genotype. These data suggest that older women who are homozygous for the MTHFR 677C 8 T mutation may be at risk for greater elevations in plasma tHcy in response to moderately low folate intake as compared with individuals with the normal or heterozygous genotypes. Copyright r 2000 by W.B. Saunders Company

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COMMON GENETIC polymorphism results from a 677C = T substitution in the gene encoding methylenetetrahydrofolate reductase (MTHFR), the enzyme that catalyzes the reduction of 5,10-methylenetetrahydrofolate (5,10-methylene-THF) to 5-methyltetrahydrofolate (5-methyl-THF), the methyl donor for methionine synthesis from homocysteine.1 Metabolic abnormalities associated with the 677C = T MTHFR mutation include changes in one-carbon derivatives and homocysteine metabolism.1 This mutation is considered the most common genetic abnormality resulting in hyperhomocysteinemia.2 Approximately 12% of the Caucasian population is homozygous for the 677C = T MTHFR gene mutation,3 while African-Americans exhibit a very low incidence of the homozygous genotype.4 Previous studies indicated that individuals who are homozygous (TT) for the 677C = T MTHFR mutation have elevated plasma total homocysteine (tHcy) concentrations compared with individuals with either the heterozygous (CT) or normal (CC) genotype.3,5,6 Low serum folate (SF) concentrations have

From the Food Science and Human Nutrition Department, College of Agricultural and Life Sciences, Department of Gastroenterology and Nutrition, College of Medicine, Division of Biostatistics, Department of Statistics, and General Clinical Research Center, University of Florida, Gainesville, FL; and Rexall Sundown, Boca Raton, FL. Submitted December 10, 1999; accepted April 5, 2000. Supported in part by Florida Department of Citrus Grant No. 95044 and National Institutes of Health Clinical Research Center Grant No. RR0082. Florida Agricultural Experiment Station, Journal Series No. R-07476. Presented in part at the Experimental Biology ’99 Meeting, April 19, 1999, Washington, DC and published in abstract form (FASEB J 13:A228, 1999). Address reprint requests to Gail P.A. Kauwell, PhD, Food Science and Human Nutrition Department, University of Florida, Box 110370, Gainesville, FL 32611-0370. Copyright r 2000 by W.B. Saunders Company 0026-0495/00/4911-0002$10.00/0 doi:10.1053/meta.2000.16555 1440

also been reported in individuals with the TT genotype compared with the CT or CC genotypes.5-7 Several studies have demonstrated a positive association between the TT genotype and coronary artery disease,8-12 although not all studies support this association,13-17 in particular the meta-analysis by Brattstrom et al.3 Higher folate requirements have been suggested for individuals with the MTHFR mutation in order to regulate the plasma tHcy concentration18-20 and potentially reduce the risk of vascular disease.21 When establishing the Dietary Reference Intakes for folate, the Institute of Medicine22 considered the possibility that individuals who are homozygous for the 677C = T MTHFR mutation may have a different folate requirement. The data were considered insufficient for definitive conclusions regarding the influence of the MTHFR genotype on folate status, since the reported studies were all observational in nature.22 There have been no controlled dietary intake studies in which the metabolic response to folate intake is compared between different MTHFR genotypes. The present study is the first to investigate the folate status response to a controlled dietary folate intake and to evaluate the effect of the MTHFR genotype on the folate and tHcy response to changes in folate intake. SUBJECTS AND METHODS

Study Subjects and Design The study (14 weeks) was designed to estimate the adequacy of dietary folate intake in elderly women with folate status data reported separately.23 The current report includes the relative response of the folate status by MTHFR genotype. The study was approved by the University of Florida Institutional Review Board and informed consent was obtained from all participants. Exclusion criteria were as follows: a history of chronic disease as determined by a medical diagnosis of cardiovascular disease, cancer, diabetes, renal disease, malabsorptive disorders, and/or hypertension; use of tobacco products; chronic alcohol consumption; abnormal blood chemistry profile or blood cell count; low SF and/or red blood cell folate concentration; elevated fasting plasma tHcy concentration; body weight greater than 120% of ideal; use of Metabolism, Vol 49, No 11 (November), 2000: pp 1440-1443

LOW FOLATE AND MTHFR MUTATION INCREASE tHcy

estrogen replacement therapy, steroids, and/or anti-folate medications; or vegetarianism. The study design included a 7-week folate depletion period followed by a 7-week folate repletion period. Thirty-three healthy female subjects (60 to 85 years) completed the depletion phase (7 weeks) and 30 completed the entire 14-week study. Subjects consumed breakfast and dinner at the University of Florida General Clinical Research Center located in Shands Hospital. Lunch and an evening snack were provided for subjects to consume at home or work. Compliance to the study protocol was ensured by personal monitoring by the research team and weekly SF analyses. During the first 7 weeks (depletion phase), all subjects consumed a folate-restricted diet (1,895 kcal, 13% protein, 22% fat, and 65% carbohydrate) providing approximately 118 ⫾ 25 µg folate per day (mean ⫾ SD). Following the depletion phase, subjects were randomly assigned to receive either 200 or 415 µg folate per day during the 7-week repletion period. Treatment consisted of the folaterestricted diet supplemented with either folic acid or a combination of folic acid and endogenous food folate from orange juice. A 5-day cycle menu consisting of conventional foods was used throughout the study and is similar to that reported previously.24 To minimize naturally occurring folate in the diet, food items such as chicken, rice, and vegetables were boiled 3 times and the cooking water was discarded after each boiling. The folate composition of the menu was analyzed using a modification of the tri-enzyme extraction method25 and analysis by the microplate adaptation of the Lactobacillus casei microbiologic assay.26,27 Vitamins and minerals not provided in sufficient quantity by the diet were provided in custom-manufactured folic acid–free vitaminmineral supplements (Tishcon, Westbury, NY) to meet 100% of the 1989 recommended dietary allowance. A daily iron supplement (18 mg/d) was provided to all subjects (General Nutrition Center, Pittsburgh, PA). The body weight was monitored on a weekly basis, and the total kilocalories were adjusted to correct for changes ⫾ 5% of baseline weight.

Sample Collection and Processing Baseline and weekly fasting venous blood samples were collected in EDTA tubes and serum separator (SST) gel and clot activator tubes (Vacutainer; Becton Dickinson, Rutherford, NJ). Plasma and leukocytes were collected after centrifugation of EDTA blood (2,000 ⫻ g for 30 minutes at 4°C). Serum was collected after centrifugation of SST blood (650 ⫻ g for 15 minutes at 21°C). SF concentrations were determined using an adaptation of the L. casei microbiologic assay.26,27 Plasma tHcy concentrations were determined using a modified high-performance liquid chromatography method.28 Baseline blood samples were used to perform MTHFR genotyping, and the DNA for genotyping was extracted from leukocytes using a commercially available DNA preparation kit (QIAamp blood kit; Qiagen, Santa Clarita, CA). The MTHFR genotype determination was accomplished using a modification of the polymerase chain reaction and HinfI restriction enzyme digestion procedure.29 The primers used generated a 198–base pair (bp) fragment. If the mutation was present, the HinfI restriction enzyme digested the 198-bp fragment into 175-bp and 23-bp fragments that were identified by gel electrophoresis.

Statistical Methods One-way ANOVA was used to test for differences in age, weight, vitamin status, and plasma tHcy between genotype groups at baseline. Differences between the mean and percent changes in plasma tHcy concentrations were determined by ANOVA. Analysis of covariance (ANCOVA) was used to account for subject variability upon entry into the study and to evaluate group differences in SF and plasma tHcy concentrations, adjusting for either baseline or week 7 values at weeks 7 and 14, respectively. The magnitude of the difference between each group was evaluated using the least square (LS) mean at the average

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covariate value. Bonferroni correction was used for multiple pairwise comparisons within each ANOVA and ANCOVA, and any of the three possible pairwise comparisons between genotype groups were considered significant at an ␣ level of .05/3, or .017. All other differences were considered significant at a P level of .05 or less. Statistics were computed using SAS version 6.12 (SAS Institute, Cary, NC). RESULTS

Data relative to the two folate repletion intakes (ie, 200 and 415 µg/d) were pooled during analysis to provide more definitive comparisons between genotypes. Randomization resulted in 5 of the 7 homozygous subjects being placed in the 415-µg/d folate treatment group. The genotype distribution for the 677C = T MTHFR mutation was 21% TT (n ⫽ 7), 46% CT (n ⫽ 15), and 33% CC (n ⫽ 11). There were no significant differences in age (P ⫽ .51) or weight (P ⫽ .66) between genotype groups. Mean SF and plasma tHcy concentrations for the MTHFR genotypes at baseline and weeks 7 and 14 are presented in Table 1. At baseline, mean SF levels were not significantly different (P ⫽ .53) between the genotype groups. At week 7, the mean SF concentration was significantly lower for TT subjects versus CT subjects (P ⫽ .017), but not significantly different versus CC subjects. Following 7 weeks of folate repletion, the mean SF level was significantly higher for TT subjects versus CC (P ⫽ .014) or CT (P ⫽ .016) subjects. Plasma tHcy concentrations were not different (P ⫽ .47) at baseline for the three genotype groups. In response to folate depletion, TT subjects had a 44% increase in plasma tHcy, which was significantly greater (P ⫽ .015) than the change in plasma tHcy for CC (15% increase) or CT (20%) subjects (Fig 1). At week 7, the mean plasma tHcy concentration was significantly higher for TT subjects versus CC (P ⫽ .001) or CT (P ⫽ .008) subjects. Differences in mean plasma tHcy concentrations were not detected (P ⫽ .12) between the three genotypes following folate repletion (ie, week 14). DISCUSSION

The present study is the first to investigate the influence of the MTHFR polymorphism on the response to controlled dietary folate intake. At baseline, mean SF concentrations were above normal for all genotypes and there were no differences in mean SF or plasma tHcy concentrations between genotypes. These data are consistent with the observation of no association between genotype and fasting tHcy in subjects with plasma folate levels at or above the median (15.4 nmol/L),19 and data from a meta-analysis concluding that plasma tHcy concentrations are not different between genotypes when folate concentrations are above the median.3 In response to the moderately low folate diet, subjects who were homozygous for the 677C = T mutation had a more dramatic increase (44%) in plasma tHcy than subjects who were either heterozygous (20% increase) or normal (15% increase). Postdepletion, subjects with the TT genotype showed a significantly higher mean plasma tHcy concentration than CC or CT subjects and a significantly lower mean SF concentration than CT subjects. Elevated tHcy concentrations in individuals with the TT genotype have been observed when SF concentrations are below the geometric mean,5 median, or in the lowest quartile.3 The results of the current

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Table 1. SF and Plasma tHcy Concentrations by MTHFR Genotype at Baseline and Weeks 7 and 14 Genotype Parameter

SF (nmol/L) Baseline Mean, unadjusted LS mean Week 7 Mean, unadjusted LS mean Week 14 Mean, unadjusted LS mean Plasma tHcy (µmol/L) Baseline Mean, unadjusted LS mean Week 7 Mean, unadjusted LS mean Week 14 Mean, unadjusted LS mean

CC

CT

TT

40.7 ⫾ 28.4 (n ⫽ 11) NA

44.5 ⫾ 22.7 (n ⫽ 15) NA

56.3 ⫾ 33.9 (n ⫽ 7) NA

13.3 ⫾ 7.4 (n ⫽ 11) 14.4

14.7 ⫾ 7.5 (n ⫽ 15) 15.0

12.1 ⫾ 5.4* (n ⫽ 6) 9.1

20.3 ⫾ 14.0 (n ⫽ 10) 20.7

22.0 ⫾ 12.0 (n ⫽ 13) 21.4

29.8 ⫾ 9.8† (n ⫽ 7) 30.8

10.0 ⫾ 2.8 (n ⫽ 11) NA

9.0 ⫾ 2.9 (n ⫽ 15) NA

8.5 ⫾ 2.9 (n ⫽ 7) NA

11.3 ⫾ 2.7 (n ⫽ 11) 10.4

10.8 ⫾ 3.8 (n ⫽ 15) 11.1

12.5 ⫾ 5.3‡ (n ⫽ 7) 13.3

10.4 ⫾ 2.9 (n ⫽ 10) 10.7

10.9 ⫾ 4.0 (n ⫽ 13) 11.1

10.1 ⫾ 2.1 (n ⫽ 7) 9.3

NOTE. Unadjusted values are the mean ⫾ SD. The LS mean represents the average covariate value (ANCOVA). Divide by 2.266 to convert SF from nmol/L to ng/mL. Abbreviations: CC, normal; CT, heterozygous; TT, homozygous; NA, not applicable (baseline values not adjusted). *Significantly different v CT (P ⫽ .017, ANCOVA). †Significantly different v CT (P ⫽ .016) or CC (P ⫽ .014) (ANCOVA). ‡Significantly different v CT (P ⫽ .008) or CC (P ⫽ .001) (ANCOVA).

study suggest that elderly women who are homozygous for the MTHFR mutation may be at greater risk for an increase in plasma tHcy in response to moderately low folate intake. At week 14, subjects who were homozygous for the MTHFR 677C = T mutation showed significantly higher mean SF concentrations than CT or CC subjects, although there was no significant difference in plasma tHcy levels between the genotype groups. In contrast, Nelen et al30 reported that women with the homozygous genotype had significantly lower median SF concentrations after 2 months of FA supplementation (500 µg/d) compared with women with the normal or heterozygous geno-

type. However, the current study included two levels of folate repletion (ie, 200 and 415 µg/d) and the results may be confounded by the fact that 5 of the 7 homozygous subjects were randomized to the folate 415-µg/d folate treatment group. In response to folate repletion, a folate intake of 415 µg/d seemed to have the most pronounced effect on plasma tHcy levels in subjects with the TT genotype (3.4-µmol/L decrease) compared with genotypes CC (1.5-µmol/L decrease) and CT (0.8-µmol/L decrease). These data agree with the finding of Malinow et al5 that subjects with the TT genotype had larger decreases in plasma tHcy after 1 or 2 mg folic acid supplemen-

Fig 1. Mean percent increase in plasma total homocysteine at week 7 (postdepletion) compared with baseline for the 3 genotype groups. a v b, P ⴝ .015 by ANOVA.

LOW FOLATE AND MTHFR MUTATION INCREASE tHcy

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tation for a 3-week period, whereas smaller decreases in tHcy were observed in subjects with the CC genotype. Nelen et al30 reported that women (aged 22 to 41 years) with the TT genotype had the greatest decrease in fasting tHcy (41%), compared with a 26% decrease for the CC and CT genotypes, after supplementation with 0.5 mg/d folic acid for 4 weeks. These data indicate that compared with the CC or CT genotypes, individuals who are homozygous for the MTHFR 677C = T mutation may be more responsive to a folate intake of 415 µg/d with respect to the reduction in the plasma tHcy concentration. Due to the small

sample size of the TT group, comparisons were not made between genotypes in response to repletion with 200 µg/d folate. The current study suggests that elderly women who are homozygous for the MTHFR 677C = T mutation are at greater risk for an increase in plasma tHcy in response to low folate intake, which may predispose this group to an increased risk for vascular disease. These data demonstrate the potential impact of inadequate folate intake on homocysteine metabolism in the large segment of the population who are homozygous for this common genetic mutation.

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