Prospective study of methylenetetrahydrofolate reductase (MTHFR) variant C677T and risk of all-cause and cardiovascular disease mortality among 6000 US adults.

BACKGROUND: The association between blood homocysteine concentration and the risk of cardiovascular disease (CVD) remains controversial, but few studies have examined the association between MTHFR C677T (a proxy for high homocysteine concentration) and death from CVD. OBJECTIVE: The objective was to examine associations of MTHFR C677T, a proxy for high homocysteine concentrations, with CVD mortality and with all-cause mortality in a national representative prospective cohort of the US adult population before the introduction of mandatory folic acid fortification of flour. DESIGN: We used Mendelian randomization to examine the association of MTHFR C677T with cause-specific mortality in 5925 participants by accessing the NHANES III (1991-1994) Linked Mortality File (through 2006). RESULTS: A comparison of homozygotes at baseline showed that individuals with a TT genotype had a 2.2-µmol/L higher homocysteine and a 1.4-ng/mL lower folate concentration, respectively, than did those with a CC genotype. The TT genotype frequency varied from 1.2% (95% CI: 0.7, 2.0) in non-Hispanic blacks and 11.6% (95% CI: 9.6, 14.0) in non-Hispanic whites to 19.4% (95% CI: 16.7, 22.3) in Mexican Americans. After adjustment for ethnic group and other CVD risk factors, the MTHFR C677T TT genotype was associated with significantly lower CVD mortality (HR: 0.69; 95% CI: 0.50, 0. 95) but had no significant effect on all-cause mortality (HR: 0.79; 95% CI: 0.59, 1.05). After stratification by period of follow-up, the inverse association of MTHFR with CVD mortality was significant only in the period after introduction of mandatory folic acid fortification. CONCLUSION: The inverse association of MTHFR with CVD mortality was unexpected and highlights the need for caution in interpretation of Mendelian randomization studies, which, like other observational studies, can be influenced by chance, bias, or confounding.

Genomic DNA methylation changes in response to folic acid supplementation in a population-based intervention study among women of reproductive age.

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (-14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.

MTHFR 677C->T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation.

BACKGROUND: The methylenetetrahydrofolate reductase (MTHFR) genotype is associated with modification of disease and risk of neural tube defects. Plasma and red blood cell (RBC) folate and plasma homocysteine concentrations change in response to daily intakes of folic acid supplements, but no large-scale or population-based randomized trials have examined whether the MTHFR genotype modifies the observed response. OBJECTIVE: We sought to determine whether the MTHFR 677C→T genotype modifies the response to folic acid supplementation during and 3 mo after discontinuation of supplementation. DESIGN: Northern Chinese women of childbearing age were enrolled in a 6-mo supplementation trial of different folic acid doses: 100, 400, and 4000 µg/d and 4000 µg/wk. Plasma and RBC folate and plasma homocysteine concentrations were measured at baseline; after 1, 3, and 6 mo of supplementation; and 3 mo after discontinuation of supplementation. MTHFR genotyping was performed to identify a C→T mutation at position 677 (n = 932). RESULTS: Plasma and RBC folate and homocysteine concentrations were associated with MTHFR genotype throughout the supplementation trial, regardless of folic acid dose. MTHFR TT was associated with lower folate concentrations, and the trend of TT < CC was maintained at even the highest doses. Folic acid doses of 100 µg/d or 4000 µg/wk did not reduce high homocysteine concentrations in those with the MTHFR TT genotype. CONCLUSION: MTHFR genotype was an independent predictor of plasma and RBC folate and plasma homocysteine concentrations and did not have a significant interaction with folic acid dose during supplementation. This trial was registered at clinicaltrials.gov as NCT00207558.

Folic acid source, usual intake, and folate and vitamin B-12 status in US adults: National Health and Nutrition Examination Survey (NHANES) 2003-2006.

BACKGROUND: US adults have access to multiple sources of folic acid. The contribution of these sources to usual intakes above the tolerable upper intake level (UL) (1000 microg/d) and to folate and vitamin B-12 status is unknown. OBJECTIVE: The objective was to estimate usual folic acid intake above the UL and adjusted serum and red blood cell folate, vitamin B-12, methylmalonic acid, and homocysteine concentrations among US adults by 3 major folic acid intake sources-enriched cereal-grain products (ECGP), ready-to-eat cereals (RTE), and supplements (SUP)-categorized into 4 mutually exclusive consumption groups. DESIGN: We used data from the National Health and Nutrition Examination Survey (NHANES) 2003-2006 (n = 8258). RESULTS: Overall, 2.7% (95% CI: 1.9%, 3.5%) of adults consumed more than the UL of folic acid. The proportions of those who consumed folic acid from ECGP only, ECGP+RTE, ECGP+SUP, and ECGP+RTE+SUP were 42%, 18%, 25%, and 15%, respectively. Of 60% of adults who did not consume supplements containing folic acid (ECGP only and ECGP+RTE), 0% had intakes that exceeded the UL. Of 34% and 6% of adults who consumed supplements with an average of < or = 400 and >400 microg folic acid/d, <1% and 47.8% (95% CI: 39.6%, 56.0%), respectively, had intakes that exceeded the UL. Consumption of RTE and/or supplements with folic acid was associated with higher folate and vitamin B-12 and lower homocysteine concentrations, and consumption of supplements with vitamin B-12 was associated with lower methylmalonic acid concentrations (P < 0.001). CONCLUSION: At current fortification levels, US adults who do not consume supplements or who consume an average of < or =400 microg folic acid/d from supplements are unlikely to exceed the UL in intake for folic acid.

Folate status and homocysteine response to folic acid doses and withdrawal among young Chinese women in a large-scale randomized double-blind trial.

BACKGROUND: There are no large randomized trials of the effect of folic acid dosing regimens on blood folate and homocysteine concentrations. OBJECTIVE: We aimed to evaluate the changes in folate and homocysteine concentrations in response to different folic acid doses and to withdrawal in young women not exposed to other sources of folic acid. DESIGN: Women (n = 1108) were randomly assigned to 1 of 6 intervention groups for which daily intakes of folic acid for 6 mo were 100 microg 1 time/d, 25 microg 4 times/d, 400 microg 1 time/d, 100 microg 4 times/d, 4000 microg 1 time/d, or 4000 microg 1 time/wk. Plasma and red blood cell folate and homocysteine concentrations were measured at baseline; at 1, 3, and 6 mo; and 3 mo after the discontinuation of folic acid. RESULTS: Folate and homocysteine concentrations were not different at baseline between the groups who had the same daily intake of folic acid as a single dose or multiple doses (P = 0.058). Plasma folate concentrations plateaued at 3 mo with 108% (95% CI: 97.7%, 120%), 259% (95% CI: 240%, 279%), 460% (95% CI: 417%, 503%), and 142% (95% CI: 123%, 162%) observed increases for the folic acid groups receiving 100, 400, and 4000 microg/d and 4000 microg/wk, respectively. The rate of reduction in folate concentrations during the 3 mo after cessation of folic acid was dose-dependent-higher intakes were associated with faster reductions. CONCLUSIONS: Changes in folate and homocysteine concentrations were unaffected by different dosing schedules. After folic acid cessation, blood folate declined rapidly, which indicated that the intervention-enhanced folate status was rapidly diminished.

Prevalence and effects of gene-gene and gene-nutrient interactions on serum folate and serum total homocysteine concentrations in the United States: findings from the third National Health and Nutrition Examination Survey DNA Bank.

BACKGROUND: Abnormalities of folate and homocysteine metabolism are associated with a number of pediatric and adult disorders. Folate intake and genetic polymorphisms encoding folate-metabolizing enzymes influence blood folate and homocysteine concentrations, but the effects and interactions of these factors have not been studied on a population-wide basis. OBJECTIVE: The objective was to assess the prevalence of these genetic polymorphisms and their relation to serum folate and homocysteine concentrations. DESIGN: DNA samples from 6793 participants in the third National Health and Nutrition Examination Survey (NHANES III) during 1991-1994 were genotyped for polymorphisms of genes coding for folate pathway enzymes 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T and 1298A-->C, methionine synthase reductase (MTRR) 66A-->G, and cystathionine-beta-synthase 844ins68. The influence of these genetic variants on serum folate and homocysteine concentrations was analyzed by age, sex, and folate intake in 3 race-ethnicity groups. RESULTS: For all race-ethnicity groups, serum folate and homocysteine concentrations were significantly related to the MTHFR 677C-->T genotype but not to the other polymorphisms. Persons with the MTHFR 677 TT genotype had a 22.1% (95% CI: 14.6%, 28.9%) lower serum folate and a 25.7% (95% CI: 18.6%, 33.2%) higher homocysteine concentration than did persons with the CC genotype. Moderate daily folic acid intake (mean: 150 microg/d; 95% CI: 138, 162) significantly reduced the difference in mean homocysteine concentrations between those with the MTHFR 677 CC and TT genotypes. We found a significant interaction between MTHFR 677C-->T and MTRR 66A-->G on serum homocysteine concentrations among non-Hispanic whites. CONCLUSIONS: The MTHFR 677C-->T polymorphism was associated with significant differences in serum folate and homocysteine concentrations in the US population before folic acid fortification. The effect of MTHFR 677C-->T on homocysteine concentrations was reduced by moderate daily folic acid intake.

Trends in the postfortification prevalence of spina bifida and anencephaly in the United States.

BACKGROUND: The prevalence of NTDs in the US declined significantly after mandatory folic acid fortification; however, it is not known if the prevalence of NTDs has continued to decrease in recent years relative to the period immediately following the fortification mandate. METHODS: Population-based data from 21 birth defects surveillance systems were used to examine trends in the birth prevalence of spina bifida and anencephaly during 1999-2000, 2001-2002, and 2003-2004. Prevalence data were stratified by non-Hispanic White, non-Hispanic Black, and Hispanic race or ethnicity. Prevalence ratios were calculated by dividing the birth prevalences during the later time periods (2001-2002 and 2003-2004) by the birth prevalences during 1999-2000. RESULTS: During 1999-2004, 3,311 cases of spina bifida and 2,116 cases of anencephaly were reported. Hispanic infants had the highest prevalences of NTDs for all years. For all infants, the combined birth prevalences of spina bifida and anencephaly decreased 10% from the 1999-2000 period to the 2003-2004 period. The decline in spina bifida (3%) was not significant; however the decline in anencephaly (20%) was statistically significant. CONCLUSIONS: While the prevalences of spina bifida and anencephaly in the United States have declined since folic acid fortification in the food supply began, these data suggest that reductions in the prevalence of anencephaly continued during 2001-2004 and that racial and ethnic and other disparities remain.

Race-ethnicity differences in folic acid intake in women of childbearing age in the United States after folic acid fortification: findings from the National Health and Nutrition Examination Survey, 2001-2002.

BACKGROUND: Neural tube defects are serious birth defects of the brain and spinal cord. Up to 70% of neural tube defects can be prevented by the consumption of folic acid by women before and early during pregnancy. OBJECTIVE: The objective was to examine folic acid intake in women of childbearing age in the United States. DESIGN: We analyzed nutrient intake data reported by 1685 nonpregnant women aged 15-49 y who participated in the National Health and Nutritional Examination Survey, 2001-2002. RESULTS: The adjusted geometric mean consumption of folic acid from fortified foods was 128 microg/d (95% CI: 123, 134 microg/d) in nonpregnant women. Eight percent (95% CI: 5.8%, 11.0%) of nonpregnant women reported consuming >or=400 microg folic acid/d from fortified foods. This proportion was lower among non-Hispanic black women (5.0%) than among non-Hispanic white (8.9%) or Hispanic (6.8%) women. A smaller percentage of non-Hispanic black (19.1%) and Hispanic (21%) women than of non-Hispanic white women (40.5%) consumed >or=400 microg folic acid from supplements, fortified foods, or both, in addition to food folate, as recommended by the Institute of Medicine to reduce the frequency of neural tube defects. CONCLUSIONS: Most nonpregnant women of childbearing age in the United States reported consuming less than the recommended amount of folic acid. The proportion with low daily folic acid intake was significantly higher in non-Hispanic black and Hispanic women than in non-Hispanic white women. At the present level of folic acid fortification, most women need to take a folic acid-containing dietary supplement to achieve the Institute of Medicine recommendation.

Improvement in stroke mortality in Canada and the United States, 1990 to 2002.

BACKGROUND: In the United States and Canada, folic acid fortification of enriched grain products was fully implemented by 1998. The resulting population-wide reduction in blood homocysteine concentrations might be expected to reduce stroke mortality if high homocysteine levels are an independent risk factor for stroke. METHODS AND RESULTS: In this population-based cohort study with quasi-experimental intervention, we used segmented log-linear regression to evaluate trends in stroke-related mortality before and after folic acid fortification in the United States and Canada and, as a comparison, during the same period in England and Wales, where fortification is not required. Average blood folate concentrations increased and homocysteine concentrations decreased in the United States after fortification. The ongoing decline in stroke mortality observed in the United States between 1990 and 1997 accelerated in 1998 to 2002 in nearly all population strata, with an overall change from -0.3% (95% CI, -0.7 to 0.08) to -2.9 (95% CI, -3.5 to -2.3) per year (P=0.0005). Sensitivity analyses indicate that changes in other major recognized risk factors are unlikely to account for the reduced number of stroke-related deaths in the United States. The fall in stroke mortality in Canada averaged -1.0% (95% CI, -1.4 to -0.6) per year from 1990 to 1997 and accelerated to -5.4% (95% CI, -6.0 to -4.7) per year in 1998 to 2002 (P< or =0.0001). In contrast, the decline in stroke mortality in England and Wales did not change significantly between 1990 and 2002. CONCLUSIONS: The improvement in stroke mortality observed after folic acid fortification in the United States and Canada but not in England and Wales is consistent with the hypothesis that folic acid fortification helps to reduce deaths from stroke.

Autosomal trisomy and maternal use of multivitamin supplements.

Recent reports suggest that women carrying certain polymorphisms of folate genes associated with suboptimal folate status might be at increased risk for having a child with Down syndrome or other autosomal trisomies, and hypothesized that maternal use of multivitamin supplements might reduce such risk. To evaluate this hypothesis, we examined data from a population-based case-control study, and contrasted cases of Down syndrome, trisomy 18, and trisomy 13, with unaffected controls. Periconceptional multivitamin use, compared to no such use, was associated with an odds ratio (OR) of 0.9 (95% confidence interval [CI], 0.6-1.3) for having a pregnancy affected by an autosomal trisomy. The OR was 0.8 (95% CI, 0.5-1.3) for Down syndrome and 1.4 (95% CI, 0.5-3.6) for trisomies 13 and 18, with little variation by maternal race or age. Periconceptional multivitamin use was not associated with a major reduction in the risk for common autosomal trisomies.

Influence of reporting error on the relation between blood folate concentrations and reported folic acid-containing dietary supplement use among reproductive-aged women in the United States.

BACKGROUND: Folic acid intake is the most important predictor of blood folate concentrations among nonpregnant women, but the reporting of folic acid-containing supplement use is subject to error. OBJECTIVE: We assessed the effect of reporting error of supplement use on blood folate concentrations. DESIGN: Data from the third National Health and Nutrition Examination Survey were analyzed. Respondents to that survey were asked twice about supplement use: ie, during the household interview, to recall use in the previous month, and during the physical examination, to recall use in the previous 24 h. To examine the effect of error reporting, we classified women (aged 15-44 y) into 5 groups according to supplement use in the previous month (nonusers, those ingesting < 400 micro g/d, and those ingesting >or= 400 micro g/d) and in the 24 h before the physical examination (yes or no). We expected nonappreciable differences in red blood cell (RBC) folate concentration by status of 24-h recall within the same category of previous-month use because RBC folate reflects long-term average consumption. We calculated covariate-adjusted means of serum and RBC folate concentrations. RESULTS: Among women who reported average daily use of >or= 400 micro g folic acid in the previous month, the adjusted mean RBC folate was 436.5 nmol/L (95% CI: 406.7, 466.3 nmol/L) in those who did not take the supplement in the previous 24 h and 519.7 nmol/L (95% CI: 496.2, 543.2 nmol/L) in those who did do so (P < 0.01). This significant difference indicates apparently erroneous reporting of supplement use in the previous month by some participants. CONCLUSION: The effect of reporting error on blood folate concentrations is important in interpreting survey results, evaluating health education campaigns, and identifying populations needing special education programs.

Method of weighted proportion of reproductive-aged women taking folic acid supplements to predict a neural tube defect rate decline.

BACKGROUND: Neural tube defect (NTD) rates can be lowered by increased consumption of folic acid (FA) by women before and during early pregnancy. The crude proportion of reproductive-aged women taking FA supplements has been used to predict a decline of the NTD rate in the general population. In this study we examine the potential error in using the crude proportion to predict NTD risk reduction, and offer an alternative method. METHODS: The crude proportion measures the number of women taking FA. It ignores the substantial variability by maternal age in the probability of giving birth. Age-specific fertility rates (ASFRs) reflect the probability that a woman in a specific age group will give birth in a given year. In this study, we show how to calculate a proportion weighted by ASFRs to predict a decline in the NTD rate, and to assess the effectiveness of FA consumption in preventing NTDs. RESULTS: Our results show that a crude proportion of 50% of women (15-49 years old) taking FA is associated with a range of 24-77% in weighted proportions. Assuming a 40% risk reduction from taking 400 microg of FA daily, the expected NTD rate decline could vary from 9.6% to 30.6%, depending on the age distribution of women taking FA. CONCLUSIONS: The ASFR-weighted proportion estimates the proportion of babies born to women taking FA, as opposed to the crude proportion of women taking FA. We recommend using the ASFR-weighted proportion to predict an NTD rate decline and measure the success of FA education campaigns. We found that when women in high-fertility age groups increased their FA consumption, the decline in the NTD rate was greater than when women in low-fertility age groups did so. Our findings suggest that the more efficient approach to NTD prevention is to focus on women with a higher probability of giving birth. For example, by focusing on <50% of women of childbearing age (20-34 years), as much as 76% of the maximum NTD rate reduction can be achieved.