In this large-scale, placebo-controlled randomized trial among high-risk women, we found no overall effects of a combination of folic acid, vitamin B6, and vitamin B12 on the primary outcome of total CVD events over the largest number of person-years and the longest follow-up period reported thus far (7.3 years). In subgroup analyses, there was no heterogeneity of treatment effect among those above or below the median of folate intake and among women with or without prior vascular disease. A possible interaction with randomized vitamin C and with nonrandomized angiotensin converting enzyme inhibitor treatment on the primary outcome was observed; however, due to the large number of comparisons these results could have been due to chance.
These null results for women are consistent with those previously reported in randomized trials composed primarily of men with pre-existing vascular disease3
. In the Heart Outcomes Prevention Evaluation Trial (HOPE) 25
, the same regimen of folic acid, vitamin B6
, and vitamin B12
failed to significantly lower the risk of a combined outcome of death from cardiovascular causes, MI, and stroke among 5522 patients with prior vascular disease over an average of five years. Although there was a significant reduction in the secondary end point of stroke in this trial (relative risk, 0.75; 95% confidence interval, 0.59 to 0.97), a reduction in stroke was not found with a similar B-vitamin regimen in the Vitamins Intervention for Stroke Prevention (VISP) trial13
, where stroke was the primary endpoint. The HOPE-2 trial also reported an increased risk of hospitalization for unstable angina (relative risk, 1.24; 95% confidence interval, 1.04–1.49) among those randomized to active treatment.
In the Norwegian Vitamin (NORVIT) Trial14
, a 2 × 2 factorial trial of vitamin B6
and a combination pill of folic acid/vitamin B12
among recent post MI patients, a marginally significant 22% (95% confidence interval, 0–50%; p=0.05) increased risk of recurrent MI, stroke, and sudden death was found among those assigned to the combination of folic acid/B12
and vitamin B6
over a median follow-up of 40 months. Smaller studies among patients post coronary intervention have found both decreased15
rates of restenosis among patients treated with B-vitamin regimens. In the present study, we found no evidence for a benefit on stroke or any evidence for harm regarding the primary composite endpoint or any of the individual secondary endpoints including coronary revascularization.
Concerns have been raised regarding the power of this and other current trials to adequately test the homocysteine hypothesis17, 18
; especially in countries where folic acid fortification of the food supply has taken place 19
. Observational studies suggested that fortification of grain products with 140 mcg of folic acid per 100g, which began in 1996 and became mandatory in the United States and Canada by 1998, significantly reduced mean plasma homocysteine concentrations among middle-aged individuals, and decreased the prevalence of high homocysteine levels (>13 μmol per liter) from 29.8% to 18.7% 20
. Based upon these data, it has been estimated that additional B-vitamin supplementation in such fortified population would only lower homocysteine by about 10%21
. In our trial, we observed a greater but still somewhat modest lowering of homocysteine (18.5% reduction). Although, we observed almost complete elimination of low folic acid concentrations (<7 ng per milliliter) in the placebo group after fortification, there was no apparent reduction over time on homocysteine concentrations or on the prevalence of elevated homocysteine levels (15+ μmol per liter) in this population. Although homocysteine levels were unchanged in the placebo group, folic acid fortification likely prevented further elevations in homocysteine levels that would have otherwise taken place due to the aging of the population.
Initially, epidemiologic studies, which were primarily retrospective and cross-sectional, suggested that reducing plasma homocysteine by 5 μmol/L would decrease vascular risk by one third22
. However, a more recent meta-analysis of prospective observational studies suggested that risk reductions associated with homocysteine lowering would be much more modest1
. In these studies, a 25% lower homocysteine (approximately 3 μmol per liter) was associated with 11% reduction in coronary heart disease risk and a 19% lower stroke risk1
. The expected reductions in cardiovascular events may have been even lower in our trial where the reduction in homocysteine levels was only 18.5% (2.3 μmol per liter) among compliant patients. Although our trial was not initially powered to detect such modest reductions in cardiovascular events, the 95% confidence intervals for the primary endpoint excludes with reasonable certainty reductions as low as a 10% in the combined endpoint of total cardiovascular events. However, such modest plausible reductions in the individual secondary endpoints of stroke, MI, and cardiovascular death cannot be excluded even in a trial of this size.
There are several caveats and/or limitations to this study, which warrant consideration. First, the study was conducted in a population of health professionals, who were at a relatively low risk of folate deficiency. Women were allowed to take the RDA of folic acid and B-vitamins and were also exposed to folic acid fortified grain products during the course of the trial. Although the blood study suggests that a significant proportion of the women were folate deficient at the beginning of the trial, this was virtually eliminated over the course of study. Therefore, we cannot rule out the possibility that this same regimen may have resulted in an even greater reduction in homocysteine levels in a more folate deficient population, which might have translated into an observable benefit on cardiovascular events. Alternatively, the optimal dose of these vitamins may actually be lower than that tested in this and other trials, and the potential for harm at higher doses has been raised by other studies14
. Also, since homocysteine levels were only measured in 5% of the sample, we were unable to determine whether women with high homocysteine levels at baseline may have benefited to a greater extent both with respect to homocysteine lowering and cardiovascular events.
Although this trial was the first to include a significant number of participants without prior cardiovascular disease (n=1950), power is still insufficient to exclude moderate treatment effects in primary prevention. Also, morbidity and mortality follow-up rates in this high-risk population were lower than in other trials of primary prevention utilizing similar methodology23
; and it is thus plausible that a larger primary prevention population with higher rates of follow-up might have demonstrated a benefit. However, since the lost to follow-up rates did not differ between the two treatment groups in our study; this is unlikely to account for the null findings observed. These remaining issues, along with the hypotheses regarding possible novel drug interactions with vitamin C and angiotensin enzyme converting inhibitors raised by our subgroup analyses, warrant further investigation in future studies.
In summary, in the WAFACS trial, a combination pill of 2.5 mg of folic acid, 50 mg of vitamin B6
, and 1 mg vitamin B12
had no beneficial or adverse effects on a combined outcome of total major cardiovascular events in a high-risk population of women with prior cardiovascular disease or three or more coronary risk factors over 7.3 years of follow-up. Background folic acid fortification in the food supply, although not associated with homocysteine lowering over the long-term follow-up, may still have contributed to these null findings in this population, which was at low risk for folic acid deficiency. Our results are consistent with prior randomized trials performed primarily among men with established vascular disease3
, and do not support the use of folic acid and B-vitamin supplements as preventative interventions for CVD in these high-risk fortified populations.