Abstract
Background: High homocysteine concentrations may be neurotoxic and contribute to cognitive decline in older persons. Objective: The objective was to examine the effect of supplementation with folic acid, vitamin B-12, and vitamin B-6 on cognitive change in women with cardiovascular disease (CVD) or CVD risk factors. Design: The Women's Antioxidant and Folic Acid Cardiovascular Study is a randomized placebo-controlled trial designed to test the effect of a combination of B vitamins (2.5 mg folic acid/d, 50 mg vitamin B-6/d, and 1 mg vitamin B-12/d) on secondary prevention of CVD. Female health professionals aged ͧ40 y (n ҃ 5442) with CVD or ͧ3 coronary risk factors in 1998 (after folic acid fortification began in the United States) were randomly assigned to treatment. Shortly after randomization (mean: 1.2 y), a substudy of cognitive function was initiated among 2009 participants aged ͧ65 y. Telephone cognitive function testing was administered up to 4 times over 5.4 y with 5 tests of general cognition, verbal memory, and category fluency. Repeated-measures analyses were conducted, and the primary outcome was a global composite score averaging all test results. Results: Mean cognitive change from baseline did not differ between the B vitamin and placebo groups (difference in change in global score: 0.03; 95% CI: Ҁ0.03, 0.08; P ҃ 0.30). However, supplementation appeared to preserve cognition among women with a low baseline dietary intake of B vitamins. Conclusions: Combined B vitamin supplementation did not delay cognitive decline among women with CVD or CVD risk factors. The possible cognitive benefits of supplementation among women with a low dietary intake of B vitamins warrant further study.
Abstract
Am J Clin Nutr 2008;88:1602-10.
INTRODUCTION
Substantial research implicates vascular factors in both cognitive decline and dementia-including cognitive outcomes not traditionally associated with vascular health, such as general cognition, episodic memory, and Alzheimer dementia (AD) (1) . Thus, identifying methods to prevent cognitive decline and dementia in this high-risk group with vascular disease is important.
INTRODUCTION
Addressing homocysteine concentrations may be promising. High concentrations of homocysteine have been shown to be neurotoxic: homocysteine raises intracellular amyloid ␤ concentrations (believed to be one of the earliest pathologic features in AD) (2, 3) , increases plaque deposition (3), sensitizes hippocampal neurons to the toxic effects of amyloid ␤ (4, 5), and lowers spatial learning in animal models (3, 6) .
INTRODUCTION
Thus, we hypothesize that homocysteine lowering via supplementation with folic acid, vitamin B-6, and vitamin B-12 may be an effective intervention against cognitive impairment in a highrisk group with vascular disease or major risk factors. Two previous studies reported no cognitive benefits of supplementation with B vitamins in this high-risk population, yet they have been of relatively short duration (3 y) (7, 8) .
INTRODUCTION
Therefore, in a randomized placebo-controlled trial of 2009 women with 6.6 y of treatment and 5.4 y of follow-up for cognitive function we examined the effect of supplementation with a combination of B vitamins on cognitive function in older women with cardiovascular disease (CVD) or vascular risk factors.
SUBJECTS AND METHODS
In 1995-1996, the Women's Antioxidant Cardiovascular Study began as a 2 ҂ 2 ҂ 2 randomized placebo-controlled trial of 3 antioxidants (vitamin E, vitamin C, and ␤-carotene) for the secondary prevention of CVD among 8171 women (9) . In April 1998, a fourth arm including folic acid (2.5 mg/d), vitamin B-6 (50 mg), and vitamin B-12 (1 mg/d) was added, and the Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS) was initiated among 5442 women; thus, the 5442 women were assigned to 16 treatment assignments that were combinations of the 3 antioxidants and 1 B vitamin supplement (10) . Folic acid, vitamin B-6, vitamin B-12, and placebos were provided by the BASF Corporation (Mount Olive, NJ). Eligible women were female health professionals aged ͧ40 y with CVD or ͧ3 coronary risk factors. CVD included myocardial infarction, stroke, revascularization procedures (percutaneous transluminal angioplasty, coronary artery bypass graft, carotid endarterectomy, and peripheral artery surgery), and symptomatic angina pectoris or transient cerebral ischemia. Risk factors included current tobacco use, hypertension, high cholesterol, diabetes, parental history of premature myocardial infarction, or obesity [body mass index ͧ 30 (inkg/m 2 )]. For assessment of baseline diet, including intake of B vitamins, a semiquantitative food-frequency questionnaire (FFQ) was administered; this FFQ was developed and validated in a similar cohort of women (11, 12) . In a 3-mo run-in phase to assess compliance, women received placebo caplets. Women who reported good compliance; who had no history of cancer, active liver disease, chronic kidney failure, or use of anticoagulants; and who expressed willingness to forego the use of out-of-study vitamin supplements (an exception was made for vitamin supplements, including multivitamins that provided only up to the Recommended Dietary Allowances; any supplements that exceeded Recommended Dietary Allowances were not permitted) were randomly assigned to treatment.
SUBJECTS AND METHODS
Every 12 mo, the women were sent a year's supply of monthly calendar packs containing active agents or placebo. Women completed mailed questionnaires annually to update information on compliance, side effects, health and lifestyle characteristics, and the occurrence of clinical endpoints. They were followed through the scheduled end (31 July 2005); the overall follow-up of the cohort for mortality and morbidity exceeded 98% of total potential person-years of follow-up (10) . The average compliance during follow-up was 83% and did not differ significantly between the 2 groups (10). All trial participants provided written informed consent; the trial was approved by the institutional review board of Brigham and Women's Hospital (Boston, MA) and was monitored by an external data and safety monitoring board.
SUBJECTS AND METHODS
The results of the primary trial of B vitamin supplementation were reported previously (10) . Briefly, supplementation with B vitamins was not found to protect against recurrent CVD, and no major adverse effects of treatment were reported (10) .
Cognitive cohort
From December 1998 to July 2000, a mean of 1.2 y after B vitamin randomization, a substudy of cognitive function was initiated among active WAFACS participants aged ͧ65 y (n ҃ 2164). Of these, 155 women (7%) were unreachable, had declined participation, or had died; thus, 2009 (93%) women completed the initial telephone cognitive assessment (Figure 1) . Participation in the initial cognitive interview was virtually identical by treatment group.
Cognitive cohort
Participants received 3 follow-up cognitive assessments approximately every 2 y. A high level of follow-up was maintained in both the treatment and placebo groups ( Figure 1 ): 94% completed at least one follow-up assessment, and 83% completed ͧ3 of 4 assessments. In the fourth assessment, 24% of participants were not contacted for their assessment because only a short interval had passed between their third interview and the end of the trial in July 2005. Follow-up rates were nearly identical across treatment groups at each assessment.
Cognitive function assessment
We assessed cognitive function using a telephone cognitive battery ( Table 1) with 5 tests measuring general cognition, verbal memory, and category fluency. For general cognition, we used the Telephone Interview of Cognitive Status (TICS) (13)-a telephone adaptation of the Mini-Mental State Examination. For verbal memory, we administered the delayed recall of the TICS 10-word list and the immediate and delayed recalls of the East Boston Memory Test (14) in which a short paragraph is read and 12 key elements must be repeated immediately and 15 min later. Finally, for category fluency (used to measure executive retrieval functions) (15) , women were asked to name as many animals as possible in 1 min. PLACEBO The primary prespecified outcome was a global composite score averaging all 5 cognitive tests using z scores. Because verbal memory is strongly associated with the risk of AD (16), our key secondary outcome was a verbal memory composite score, calculated by averaging 4 measures of verbal memory (the immediate and delayed recalls of both the East Boston Memory Test and the 10-word list). To calculate the composite scores for participants who did not complete all tests (only 0.5% of participants for the global composite score and 0.5% for the verbal memory score), we used the mean of the z scores of the tests that were completed.
Cognitive function assessment
The telephone cognitive interviews were administered by trained interviewers. Our telephone cognitive test battery had high reliability and validity. In a test-retest reliability study of the TICS, administered twice 31 d apart, we found a correlation of 0.7 (P 0.001) among 35 high-functioning, educated women. In a validation study of our telephone instrument, 61 women who had completed an extensive in-person interview were administered our brief telephone-administered assessment; we found a correlation of 0.81 comparing the overall performance on those 2 measures, which indicated the high validity of our telephone method. In addition, among 88 older female health professionals, cognitive impairment as determined by our telephone assessment was strongly associated with dementia diagnosis after 3 y; poor performance in the TICS and in verbal memory were both associated with significant 8-and 12-fold increases in dementia, respectively.
Statistical analysis
Characteristics at baseline between randomized groups were compared by using Wilcoxon's rank-sum tests for continuous variables and chi-square tests for proportions. To analyze differences in patterns of change in cognition between the active and placebo groups, we selected an approach that addressed the nonlinearity of the data. Because of a "learning effect," test scores generally improved with time, particularly at the second assessment; however, this common, well-recognized phenomenon in cognitive function studies (17) does not prohibit detection of overall differences between treatment groups. To address the nonlinearity of scores over time, general linear models of response profiles were fitted, with time modeled with indicator variables for assessment, rather than with linear variables (18); this approach imposes minimal structure on outcome trends over time, yet still permits valid estimation of effects in nonlinear data. 4 455 (323-700) 455 (319-685) 0.84 Total vitamin B-6 intake from diet and supplements (mg/d) 4 2.6 (1.9-3.9) 2.7 (1.9-4.01) 0.34 Total vitamin B-12 intake from diet and supplements (g/d) 4 7.3 (4.8-11.1) 7.4 (4.7-11.1) 0.76 Total alcohol intake (g/d) 4 0 (0-2.9) 0 (0-3. First, we evaluated mean performance at each cognitive assessment by treatment assignment using repeated-measures analysis of means, accounting for correlations between assessments:
Statistical analysis
where Y ij is 1 of the 4 cognitive outcomes for group i (active or placebo) at time j (1) (2) (3) (4) . Second, mean cognitive change from baseline over the second through fourth assessments by treatment assignment was our main analytic measure and it was modeled as follows:
Statistical analysis
where Y ij Ҁ Y i1 is the change from baseline for 1 of the 4 cognitive outcomes for group i (active or placebo) at time j (2) (3) (4) . Because the risk factors for cognitive decline were distributed similarly between the active and placebo groups, the main analyses did not adjust for other factors. All models were fitted by maximum likelihood, incorporating the longitudinal correlation within study subjects with the use of unstructured covariance structures, and treatment assignment was modeled as fixed effects; for statistical testing, we used Wald tests (18) . For all statistical analyses, Proc Mixed in SAS (SAS release 9.1; SAS Institute Inc, Cary, NC) was used. We examined effect modification by key risk factors (all assessed before randomization) for cognitive decline-such as age, baseline cognitive performance, education, the presence of either prevalent CVD events or prevalent CVD risk factors, and incident CVD during the trial-and by factors that may influence B vitamin metabolism, such as alcohol drinking, cigarette smoking, multivitamin use, and dietary intake of B vitamins. Tests of effect modification were performed by evaluating interaction terms in the models of mean change in cognition from baseline. We examined the influence of noncompliance by repeating the main analyses after excluding women who were taking less than one-third of their assigned study medications.
Statistical analysis
To consider the antioxidant arms, in models adjusting for antioxidant assignments, results did not change materially from the main analysis; thus, we did not include them as covariates in any models for the main analyses. Effect modification by assignment to antioxidant agents was also not observed.
Statistical analysis
Finally, to assess the impact of B vitamin supplementation on the risk of cognitive impairment, we fitted logistic regression models, defining the outcome as the worst 10% of the distribution of change from the initial to the final cognitive assessment. In these models, we adjusted for the follow-up time between the first and last assessments.
RESULTS

Primary analyses
The average time from randomization to the initial cognitive assessment was 1.2 y (range: 0.7-1.7 y), and from randomization to the last assessment was 6.6 y (range: 6.3-6.9 y). Compliance was comparable (83%) in the 2 treatment groups (10). Demographic and health characteristics at randomization were similar between treatment and placebo groups (Table 1) .
Primary analyses
At the first cognitive assessment, cognitive performance did not differ by treatment ( Table 2) ; the mean difference in the global composite score between the vitamin B and placebo groups was 0.01 (95% CI: Ҁ0.05, 0.07). At the final cognitive assessment (after a mean 6.6 y of treatment), there was no difference observed between the treatment and placebo groups (mean difference: 0.05; 95% CI: Ҁ0.04, 0.14). Similarly, for our secondary endpoint of verbal memory, the groups did not differ at any of the cognitive assessments: for example, at the final assessment, the mean difference in performance was 0.07 (95% CI: Ҁ0.03, 0.16). The treatment group also did not show better performance in either the TICS or category fluency.
Primary analyses
When we evaluated the mean change in cognitive performance from the initial through the final assessments, we observed similar results ( Table 3) , with no significant differences by treatment assignment. The mean difference in cognitive decline over time between the treatment and placebo groups was 0.03 standard units per year (95% CI: Ҁ0.03, 0.08) for the global score, 0.03 (95% CI: Ҁ0.03, 0.09) for the verbal memory score, 0.16 points (95% CI; Ҁ0.11, 0.44) for the TICS, and Ҁ0.13 points (95% CI: Ҁ0.51, 0.25) for category fluency.
Primary analyses
The B vitamin group did not differ from the placebo group in the odds of substantial cognitive decline from the first through the final assessment. Compared with the placebo group, the relative risk (RR) for the B vitamin group was 1.04 (95% CI: 0.69, 1.56) in the global score (defined as 0.8 point decline), 1.08 (95% CI: 0.72, 1.63) for the verbal memory score (defined as 1.0 point decline), and 0.82 (95% CI: 0.55, 1.22) for the TICS. For category fluency there was some suggestion that supplementation was associated with an increased risk of decline in fluency (RR: 1.51; 95% CI: 0.97, 2.35; P ҃ 0.07).
Subgroup analyses
We investigated whether the effect of B vitamin supplementation differed by various characteristics ( Table 4 ). For the primary outcome of global score, we observed no statistically significant effect modification. However, we observed a general trend across the outcomes, where B vitamin supplementation conferred cognitive benefits among a subset of women with low total intakes of B vitamins. We defined the cutoff for a "low" intake of vitamin B-6 as 1.9 mg/d and for folate as 279 g/d; these cutoffs were based on the intakes that were found to be significantly associated with elevated homocysteine (ͧ13 mol/L) in the Framingham Study (19) . For vitamin B-12 , there were no such low dietary intake threshold levels; thus, we used the Reference Dietary Intake of 2.4 g for older persons (20) . Finally, we created strata for women with either a low intake for ͧ1 of the 3 B vitamins or with adequate intakes of all 3 B vitamins.
Subgroup analyses
Although there were no significant interactions with dietary intake of individual B vitamins for the global composite score, we noted a possible benefit of combined B vitamin supplementation on TICS in those with low dietary intakes of folate (mean difference in change from baseline: ҃ 1.24 points; 95% CI: 0.47, 2.00), vitamin B-6 (mean difference in change:҃ 0.56 points; 95% CI: 0.03, 1.10), or vitamin B-12 (mean difference in change: 1.35 points; 95% CI: 0.16, 2.54), but not among those with adequate intakes of these vitamins; the P for interaction was significant for folate (P ҃ 0.002). For category fluency, we observed similar effect modifications, particularly with dietary intake of vitamin B-12 (P ҃ 0.02): in those with a low vitamin B-12 intake, the mean difference in change was 1.62 (95% CI: 0.11, 3.13). When we stratified women by those with a low intake of at least one B vitamin and those with an adequate intake of all B vitamins, we observed that B vitamin supplementation significantly slowed the rate of decline in TICS by 0.74 units (95% CI: 0.23, 1.25), but not for those with adequate intakes (P for interaction ҃ 0.01).
Subgroup analyses
When we evaluated the influence of compliance after the exclusion of participants who reported noncompliance (ie, those who took less than one-third of the assigned pills), we observed no major differences in the effect of supplementation. For example, for the global score, the mean difference in cognitive decline over time between the treatment and placebo groups (0.03 standard units; 95% CI: Ҁ0.03, 0.08) was similar to the main analysis.
DISCUSSION
In this long-term, randomized, placebo-controlled trial of 2009 older women at high risk of cognitive decline because of existing CVD or risk factors, the use of a dietary supplement containing vitamin B-6, folic acid, and vitamin B-12 conferred no cognitive benefits. However, a suggestive pattern of reduced risk of cognitive decline with supplementation was observed in women with a low dietary intake of B jessicasnyder@example.org.
DISCUSSION
Many randomized trials have examined the relation between cognitive function or dementia and folic acid, vitamin B-6, and vitamin B-12 intakes, separately or combined in a variety of older populations. (7, 8, (21) (22) (23) (24) (25) (26) (27) (28) (29) Because of heterogeneity in the doses used (eg, range: 0.4 -15 mg folic acid/d), combinations used, duration of study (range: 5 wk to 3 y), sample size (range: 7-3097), proportion of the study population with elevated homocysteine concentrations, and characteristics of the study population (eg, restricted to those with cognitive impairment or dementia or with vascular disease), it is difficult to derive a firm conclusion (30) . However, the collective results are not convincing of a beneficial effect of B vitamin supplementation on cognitive health (31) .
DISCUSSION
A post hoc power analysis, based on the mean change in cognitive function actually observed in the placebo group and its SD, showed that there was a power of ͧ80% to detect mean differences in cognitive decline between the treatment and placebo groups, which were equivalent to the mean difference in cognitive decline observed between participants who were 3.5 y apart in age. That is, we had adequate power to detect an effect of B vitamin treatment that was equivalent to delaying cognitive aging by 3.5 y in these subjects, which is a fairly modest effect. Thus, the null results could not be largely attributed to a lack of statistical power.
DISCUSSION
The present study was unique in that it provides data from a large study sample (n ҃ 2009) of older women at elevated risk because of vascular disease or risk factors and had the longest duration of treatment (6.6 y) in studies of B vitamins to date. Only 2 other studies (7, 8) of participants with vascular disease have been conducted, and these both included a relatively short period (1-2 y) of treatment. Both of these studies administered 2.5 mg folic acid in various combinations with vitamin B-6 or B-12 and, as in the present study, reported no effect of B vitamin supplementation on cognitive function. These null results for cognitive decline are consistent with results from randomized trials that also found no benefit of B vitamin supplementation concerning the secondary prevention of CVD (7, 13, (32) (33) (34) (35) (36) (37) . Although additional supplementation with B vitamins may not confer any cognitive benefits in older populations that are generally well-nourished, our data are consistent with the possibility that B vitamin supplementation may be beneficial in the small segment of the population who have low dietary intakes of folate, vitamin B-6, and vitamin B-12. Durga et al (26) also found that older persons with elevated homocysteine concentrations and low folate intakes (mean intake: 200 g/d) supplemented with folic acid (0.8 mg/d), had a slower decline over 3 y in global cognitive functioning, memory, and information processing. Nevertheless, the results in the subgroup analysis need to be interpreted with caution and need to be further confirmed in similarly large studies of long duration.
DISCUSSION
WAFACS began in April 1998, 3 mo after mandated grain folic acid fortification, which has led to generally increased background dietary intakes of folic acid (38) . Furthermore, our cognitive assessments began 1.2 y after randomization. At the end of the study, we evaluated plasma concentrations of folate and homocysteine in a sample of 150 participants taking B vitamins and in 150 participants taking placebo to assess whether there were still substantial differences in folate status between the 2 groups (10). Those with high plasma folate concentrations (ͧ25 ng/mL, or 56.7 nmol/L) accounted for 16.7% of the participants assigned to take placebo and 85.3% of the participants assigned to take B vitamins. However, the difference in plasma homocysteine concentrations between the groups was modest: the median was 11.8 mol/L in the placebo group and 9.8 mol/L in the treated group, which translated to an average difference of 2.0 mol/L (or 17%), which may have been insufficient for major cognitive benefits in this generally well-nourished population. It is possible, however, that of those with low dietary intakes of B vitamins at baseline, the differences in homocysteine concentrations between the active and placebo groups may have been larger; this is one possible explanation for the apparent cognitive benefits of vitamin B supplementation that we observed in this subgroup.
DISCUSSION
Other limitations of this study should be considered. Cognitive testing began at a mean of 1.2 y after randomization; thus, we were unable to evaluate changes in cognitive performance from randomization. It is possible that vitamin B supplementation may have conferred some very early cognitive changes, and, if so, we would have missed such changes when we compared performance after our first cognitive assessment 1.2 y subsequent to randomization. We believe that such an early effect, although possible, was not biologically plausible. Moreover, because we observed no absolute differences in cognitive function 1.2 y after treatment initiation and because it is likely that cognitive function was equally distributed in the 2 groups at randomization (indeed, the distribution of numerous risk factors for cognitive Table 2 .
DISCUSSION
3 Global score is a composite of the z scores from the Telephone Interview of Cognitive Status (TICS), the immediate and delayed recalls of the East Boston Memory Test, category fluency, and delayed recall of the TICS 10-word list. 4 Verbal score is a composite score of the z scores of the immediate and delayed recalls of both the TICS 10-word list and the East Boston Memory Test. Table 2 .
DISCUSSION
3 P value for interaction from testing effect modification in longitudinal linear models. 4 Defined as a low intake of ͧ1 of the 3 B vitamins: folate (279 mg/d), vitamin B-6 (1.9 mg/d), or vitamin B-12 (2.4 g/d). 5 Defined as an adequate intakes of all 3 B vitamins: folate (ͧ279 mg/d), vitamin B-6 (ͧ1.9 mg/d), and vitamin B-12 (ͧ2.4 g/d). 6 Defined as the lowest 10% of the global score (n ҃ 201), verbal score (n ҃ 201), and animal score (n ҃ 197); for the TICS score (n ҃ 199), the established cutoff of 31 was used. 7 Includes myocardial infarction, stroke, revascularization procedures (percutaneous transluminal angioplasty, coronary artery bypass graft, carotid endarterectomy, and peripheral artery surgery), and symptomatic angina pectoris or transient cerebral ischemia. 8 Includes current tobacco use, hypertension, high cholesterol, diabetes, parental history of premature myocardial infarction, or obesity (BMI ͧ 30 kg/m 2 ). 9 Includes all nonfatal myocardial infarction, nonfatal stroke, revascularization surgery, or cardiovascular death that occurred during follow-up and was updated at each cognitive assessment. impairment was comparable across treatment groups at randomization), it seems unlikely that we could have missed any meaningful changes in cognitive from the time of randomization to our initial cognitive testing. Another limitation was noncompliance, which may bias associations toward the null; however, compliance rates were generally high and similar in the treatment and placebo groups.
DISCUSSION
In conclusion, in this large-scale, long-term, randomized, placebo-controlled trial of B vitamin supplementation and cognitive decline, no cognitive benefits were observed among women at high risk of CVD. However, we observed a general pattern of cognitive benefits with supplementation among the subset of women with a low dietary intake of the B vitamins at baseline; this finding may have been due to chance, but warrants further study. Although preventing the deficiency of essential vitamins such as folate, vitamin B-6, and vitamin B-12 remains an important health concern for older persons, additional supplementation with B vitamins is unlikely to provide cognitive benefits to those who are generally well-nourished. Alternative strategies for maintaining cognitive health in high-risk older individuals need to be investigated.

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