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1.  Effect of Folic Acid and Betaine Supplementation on Flow-Mediated Dilation: A Randomized, Controlled Study in Healthy Volunteers 
PLoS Clinical Trials  2006;1(2):e10.
We investigated whether lowering of fasting homocysteine concentrations, either with folic acid or with betaine supplementation, differentially affects vascular function, a surrogate marker for risk of cardiovascular disease, in healthy volunteers. As yet, it remains uncertain whether a high concentration of homocysteine itself or whether a low folate status—its main determinant—is involved in the pathogenesis of cardiovascular disease. To shed light on this issue, we performed this study.
This was a randomized, placebo-controlled, double-blind, crossover study.
The study was performed at Wageningen University in Wageningen, the Netherlands.
Participants were 39 apparently healthy men and women, aged 50–70 y.
Participants ingested 0.8 mg/d of folic acid, 6 g/d of betaine, and placebo for 6 wk each, with 6-wk washout in between.
Outcome Measures:
At the end of each supplementation period, plasma homocysteine concentrations and flow-mediated dilation (FMD) of the brachial artery were measured in duplicate.
Folic acid supplementation lowered fasting homocysteine by 20% (−2.0 μmol/l, 95% confidence interval [CI]: −2.3; −1.6), and betaine supplementation lowered fasting plasma homocysteine by 12% (−1.2 μmol/l; −1.6; −0.8) relative to placebo. Mean (± SD) FMD after placebo supplementation was 2.8 (± 1.8) FMD%. Supplementation with betaine or folic acid did not affect FMD relative to placebo; differences relative to placebo were −0.4 FMD% (95%CI, −1.2; 0.4) and −0.1 FMD% (−0.9; 0.7), respectively.
Folic acid and betaine supplementation both did not improve vascular function in healthy volunteers, despite evident homocysteine lowering. This is in agreement with other studies in healthy participants, the majority of which also fail to find improved vascular function upon folic acid treatment. However, homocysteine or folate might of course affect cardiovascular disease risk through other mechanisms.
Editorial Commentary
Background: Evidence from observational studies indicates a link between high concentrations of homocysteine (an amino acid) in the blood and increased risk of cardiovascular disease. However, the basis for the link between homocysteine concentrations and cardiovascular disease risk is not clear. Supplementing the diet with B-vitamins lowers homocysteine levels, and large-scale trials are underway that will determine whether B-vitamin supplementation has an effect on cardiovascular outcomes, such as heart attacks and strokes. These trials also involve administration of folic acid as well as other B-vitamins. It is not obvious, however, whether the effects of B-vitamin supplementation arise as a result of homocysteine lowering or via some other biochemical pathway.
What this trial shows: Olthof and colleagues aimed to further understand the effects of homocysteine lowering by randomizing 40 healthy volunteer participants to receive either folic acid supplementation; placebo; or betaine, a nutrient that lowers homocysteine levels via a different biochemical pathway than folic acid. Each participant in the trial received each supplement for 6 wk, with a 6-wk washout period before the next supplement was given. The researchers then used a technique called flow-mediated dilation (FMD) to measure functioning of the main artery of the upper arm, as a surrogate for cardiovascular disease risk. In this trial, both folic acid and betaine supplementation significantly lowered homocysteine levels over the 6-wk supplementation period. However, both forms of supplementation failed to result in any significant change in functioning of the artery, as measured using FMD.
Strengths and limitations: In this trial 40 participants were recruited, and 39 were followed up to trial completion. A crossover design was used, with each participant receiving each supplement and a placebo in sequence. This method enabled a smaller number of participants to be used to answer the question of interest, as compared to parallel-group designs. The majority of participants in the trial were followed up. However, the trial's outcomes are surrogates for cardiovascular disease risk, measured over fairly short time periods, and no clinical outcomes were examined.
Contribution to the evidence: This trial adds to the evidence on the effects of nutrient supplementation on surrogate outcomes for cardiovascular disease risk. The results show that over a 6-wk study period, these surrogate outcomes are not affected by either folic acid or betaine supplementation.
PMCID: PMC1488898  PMID: 16871332
2.  Acute Effect of Folic Acid, Betaine, and Serine Supplements on Flow-Mediated Dilation after Methionine Loading: A Randomized Trial 
PLoS Clinical Trials  2006;1(1):e4.
We investigated whether reducing post-methionine homocysteine concentrations via various treatments other than folic acid affects vascular function, as measured through flow-mediated dilation (FMD) of the brachial artery. High fasting and post-methionine homocysteine concentrations are associated with cardiovascular disease risk, but homocysteine might be a surrogate marker for low folate status.
This was a randomized, placebo-controlled, double-blind, crossover study.
The study took place at Wageningen University in Wageningen in the Netherlands.
Participants were 39 apparently healthy men and women, aged 50–70 y.
Participants ingested 10 mg of folic acid, 3 g of betaine, 5 g of serine, and placebo together with an oral methionine load. Each supplement was tested on two different days.
Outcome Measures:
On each of the eight treatment days, plasma homocysteine concentrations and FMD were measured before (t = 0 h, fasting) and 6 h (t = 6 h) after methionine loading.
The mean (± SD) fasting homocysteine concentrations averaged over the eight test days were 9.6 ± 2.1 μmol/l. Mean fasting FMD was 3.1 ± 2.4 FMD%. A methionine load with placebo increased homocysteine concentrations by 17.2 ± 9.3 μmol/l at 6 h after loading, similar to the increase following methionine loading with folic acid. A methionine load together with betaine and with serine increased homocysteine by 10.4 ± 2.8 μmol/l (p < 0.001 relative to placebo) and by 12.1 ± 8.2 μmol/l (p < 0.001 relative to placebo), respectively. Methionine loading with placebo did not affect FMD, and neither did methionine loading with folic acid, betaine, or serine; differences relative to placebo were +0.7 FMD% (95%CI, −0.6; 1.9), +0.2 FMD% (−1.0; 1.3), and +0.3 FMD% (−0.8; 1.4), respectively.
Experimentally induced acute changes in homocysteine concentrations did not affect FMD in healthy volunteers. This implies that potential adverse effects of high homocysteine concentrations on the cardiovascular system are not mediated through vascular function. However, homocysteine or folate may affect cardiovascular disease risk through other mechanisms.
Editorial Commentary
Background: It is already known from observational studies that people with high concentrations of homocysteine (an amino acid) in the blood are at increased risk of disease involving the heart and blood vessels, known as cardiovascular disease. Some randomized trials have also shown that lowering homocysteine levels decreases the risk of cardiovascular disease, but not all trials show this. The mechanisms linking homocysteine levels and cardiovascular disease are not well understood. Olthof and colleagues wanted to explore further the mechanisms linking homocysteine levels and the risk of cardiovascular disease. The investigators did a trial in healthy volunteers in which homocysteine concentrations were experimentally raised, then lowered, over short periods of time. The volunteers took a methionine supplement (an amino acid that is converted to homocysteine in the body) to raise homocysteine levels and either betaine (a dietary nutrient), serine (an amino acid), or folic acid (a B vitamin) to lower them. During the trial, the researchers then looked at how well the volunteers' arm arteries functioned, as a surrogate for measuring cardiovascular disease risk.
What this trial shows: The investigators found that functioning of the volunteers' arteries, as measured through flow-mediated dilation (FMD), was not affected by the changes in homocysteine levels that were brought about in the experiment.
Strengths and limitations: Although the number of participants analyzed in the trial was small (n = 39), it was large enough to adequately test the researchers' hypothesis. The vast majority of participants received the experimental treatment, with only one participant dropping out of the study. However, the observations were made over a short period of time, within 6 h of the experimental treatments being given. The investigators did not look at clinical outcomes, such as heart disease or stroke. Therefore, this trial does not provide evidence on whether altered homocysteine levels cause, or could be manipulated to prevent, such clinical outcomes.
Contribution to the evidence: This trial adds information on the short-term effects of changes in homocysteine levels.
PMCID: PMC1488894  PMID: 16871326
3.  Effect of Homocysteine-Lowering Treatment With Folic Acid and B Vitamins on Risk of Type 2 Diabetes in Women 
Diabetes  2009;58(8):1921-1928.
Homocysteinemia may play an etiologic role in the pathogenesis of type 2 diabetes by promoting oxidative stress, systemic inflammation, and endothelial dysfunction. We investigated whether homocysteine-lowering treatment by B vitamin supplementation prevents the risk of type 2 diabetes.
The Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS), a randomized, double-blind, placebo-controlled trial of 5,442 female health professionals aged ≥40 years with a history of cardiovascular disease (CVD) or three or more CVD risk factors, included 4,252 women free of diabetes at baseline. Participants were randomly assigned to either an active treatment group (daily intake of a combination pill of 2.5 mg folic acid, 50 mg vitamin B6, and 1 mg vitamin B12) or to the placebo group.
During a median follow-up of 7.3 years, 504 women had an incident diagnosis of type 2 diabetes. Overall, there was no significant difference between the active treatment group and the placebo group in diabetes risk (relative risk 0.94 [95% CI 0.79–1.11]; P = 0.46), despite significant lowering of homocysteine levels. Also, there was no evidence for effect modifications by baseline intakes of dietary folate, vitamin B6, and vitamin B12. In a sensitivity analysis, the null result remained for women compliant with their study pills (0.92 [0.76–1.10]; P = 0.36).
Lowering homocysteine levels by daily supplementation with folic acid and vitamins B6 and B12 did not reduce the risk of developing type 2 diabetes among women at high risk for CVD.
PMCID: PMC2712772  PMID: 19491213
4.  Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials 
BMJ : British Medical Journal  1998;316(7135):894-898.
Objective: To determine the size of reduction in homocysteine concentrations produced by dietary supplementation with folic acid and with vitamins B-12 or B-6.
Design: Meta-analysis of randomised controlled trials that assessed the effects of folic acid based supplements on blood homocysteine concentrations. Multivariate regression analysis was used to determine the effects on homocysteine concentrations of different doses of folic acid and of the addition of vitamin B-12 or B-6.
Subjects: Individual data on 1114 people included in 12 trials.
Findings: The proportional and absolute reductions in blood homocysteine produced by folic acid supplements were greater at higher pretreatment blood homocysteine concentrations (P<0.001) and at lower pretreatment blood folate concentrations (P<0.001). After standardisation to pretreatment blood concentrations of homocysteine of 12 μmol/l and of folate of 12 nmol/l (approximate average concentrations for Western populations), dietary folic acid reduced blood homocysteine concentrations by 25% (95% confidence interval 23% to 28%; P<0.001), with similar effects in the range of 0.5-5 mg folic acid daily. Vitamin B-12 (mean 0.5 mg daily) produced an additional 7% (3% to 10%) reduction in blood homocysteine. Vitamin B-6 (mean 16.5 mg daily) did not have a significant additional effect.
Conclusions: Typically in Western populations, daily supplementation with both 0.5-5 mg folic acid and about 0.5 mg vitamin B-12 would be expected to reduce blood homocysteine concentrations by about a quarter to a third (for example, from about 12 μmol/l to 8-9 μmol/l). Large scale randomised trials of such regimens in high risk populations are now needed to determine whether lowering blood homocysteine concentrations reduces the risk of vascular disease.
Key messages Higher blood homocysteine concentrations seem to be associated with higher risks of occlusive vascular disease and with lower blood concentrations of folate and vitamins B-12 and B-6 Proportional and absolute reductions in blood homocysteine concentrations with folic acid supplements are greater at higher pretreatment blood homocysteine concentrations and at lower pretreatment blood folate concentrations In typical Western populations, supplementation with both 0.5-5 mg daily folic acid and about 0.5 mg daily vitamin B-12 should reduce blood homocysteine concentrations by about a quarter to a third Large scale randomised trials of such regimens in people at high risk are now needed to determine whether lowering blood homocysteine concentrations reduces the risk of vascular disease
PMCID: PMC28491  PMID: 9569395
5.  Hyperhomocysteinemia: An emerging risk factor for cardiovascular disease 
There is considerable epidemiological evidence, which confirms the importance of plasma homocysteine as a powerful predictor of future risk of coronary heart disease and other complications of atherosclerosis. Treatment of hyperhomocysteinemia varies with the underlying cause. However, an inexpensive vitamin supplementation with folic acid, vitamin B12 and vitamin B 6 is generally effective in reducing homocysteine concentrations. Several randomised, controlled trials evaluating the effects of folic acid based supplements on homocysteine concentrations have been conducted over the last decade. In most patients, folic acid alone, and in combination of vitamin B12 and B6, has been shown to reduce homocysteine concentrations within four to six weeks after the initiation of therapy (34).
However, no study has yet demonstrated that lowering of homocysteine by vitamin supplementation decreases the cardiovascular morbidity or mortality. Avoidance of excessive meat intake and increased consumption of fresh vegetables and fruits is a dietary measure, which has many health benefits, including a potential to reduce elevated homocysteine levels. The other reasonable approach is to determine levels of fasting homocysteine in high risk patients and it may be advisable to increase their intake of vitamin fortified foods and/or to suggest the daily use of supplemental vitamins. Several large scale randomised trials like Heart Outcomes Prevention Evaluation (HOPE-2) Study, Mcmaster University, Canada, Study of the Effectiveness of Additional Reductions in Cholesterol and Homocysteine (SERCH), Clinical Trial Service Unit, Oxford, U.K, Cambridge Heart Antioxidant Study (CHAOS-2) University of Cambridge, U.K, Bergen Vitamin Study, University of Bergen Norway, Women's Antioxidant and Cardiovascular Disease Study (WACS) Harvard Medical School, U.S.A, Prevention with a combined inhibitor and folate in Coronary Heart Disease (PACIFIC) study, University of Sydney, Australia, and many others are ongoing to assess the effect of homocysteine—lowering by vitamin supplementation on risk of vascular disease.
PMCID: PMC3453741  PMID: 23105365
6.  The role of Homocysteine as a predictor for coronary heart disease 
Background and objective
There is an ongoing debate on the role of the cytotoxic aminoacid homocysteine as a causal risk factor for the development of coronary heart disease. Results from multiple case control-studies demonstrate, that there is a strong association between high plasma levels of homoysteine and prevalent coronary heart disease, independent of other classic risk factors. Furthermore, results from interventional studies point out that elevated plasma levels of homocysteine may effectively be lowered by the intake of folic acid and B vitamins. In order to use this information for the construction of a new preventive strategy against coronary heart disease, more information is needed: first, whether homocysteine actually is a causal risk factor with relevant predictive properties and, second, whether by lowering elevated homocysteine plasma concentrations cardiac morbidity can be reduced. Currently in Germany the determination of homocysteine plasma levels is reimbursed for by statutory health insurance in patients with manifest coronary heart disease and in patients at high risk for coronary heart disease but not for screening purposes in asymptomatic low risk populations.
Against this background the following assessment sets out to answer four questions:
Is an elevated homocysteine plasma concentration a strong, consistent and independent (of other classic risk factors) predictor for coronary heart disease?Does a therapeutic lowering of elevated homoysteine plasma levels reduce the risk of developing coronary events?What is the cost-effectiveness relationship of homocysteine testing for preventive purposes?Are there morally, socially or legally relevant aspects that should be considered when implementing a preventive strategy as outlined above?
In order to answer the first question, a systematic overview of prospective studies and metaanalyses of prospective studies is undertaken. Studies are included that analyse the association of homocysteine plasma levels with future cardiac events in probands without pre-existing coronary heart disease or in population-based samples. To answer the second question, a systematic overview of the literature is prepared, including randomised controlled trials and systematic reviews of randomised controlled trials that determine the effectiveness of homocysteine lowering therapy for the prevention of cardiac events. To answer the third question, economic evaluations of homocysteine testing for preventive purposes are analysed. Methodological quality of all materials is assessed by widely accepted instruments, evidence was summarized qualitatively.
For the first question eleven systematic reviews and 33 single studies (prospective cohort studies and nested case control studies) are available. Among the studies there is profound heterogeneity concercing study populations, classification of exposure (homocysteine measurements, units to express “elevation”), outcome definition and measurement, as well as controlling for confounding (qualitatively and quantitatively). Taking these heterogeneities into consideration, metaanalysis of single patient data with controlling for multiple confounders seems to be the only adequate method of summarizing the results of single studies. The only available analysis of this type shows, that in otherwise healthy people homocysteine plasma levels are only a very weak predictor of future cardiac events. The predictive value of the classical risk factors is much stronger. Among the studies that actively exclude patients with pre-existing coronary heart disease, there are no reports of an association between elevated homocysteine plasma levels and future cardiac events.
Eleven randomized controlled trials (ten of them reported in one systematic review) are analysed in order to answer the second question. All trials include high risk populations for the development of (further) cardiac events. These studies also present with marked clinical heterogeneity: primarily concerning the average homocysteine plasma levels at baseline, type and mode of outcome measurement and as study duration. Except for one, none of the trials shows a risk reduction for cardiac events by lowering homocysteine plasma levels with folate or B vitamins. These results also hold for predefined subgroups with markedly elevated homocysteine plasma levels.
In order to answer the third questions, three economic evaluations (modelling studies) of homocysteine testing are available. All economic models are based on the assumption that lowering homocysteine plasma levels results in risk reduction for cardiac events. Since this assumption is falsified by the results of the interventional studies cited above, there is no evidence left to answer the third question.
Morally, socially or legally relevant aspects of homocysteine assessment are currently not being discussed in the scientific literature.
Discussion and conclusion
Many currently available pieces of evidence contradict a causal role of homocysteine in the pathogenesis of coronary heart disease. Arguing with the Bradford-Hill criteria at least the criterion of time-sequence (that exposure has to happen before the outcome is measured), the criterion of a strong and consistent association and the criterion of reversibility are not fulfilled. Therefore, homocysteine may, if at all, play a role as a risk indicator but not as risk factor.
Furthermore, currently available evidence does not imply that for the prevention of coronary heart disease, knowledge of homocysteine plasma levels provides any information that supersedes the information gathered from the examination of classical risk factors. So, currently for the indication of prevention, there is no evidence that homocysteine testing provides any benefit. Against this background there is also no basis for cost-effectiveness calculations.
Further basic research should clarify the discrepant results of case control studies and prospective studies. Maybe there is a third parameter (confounder) associated with homocysteine metabolism as well with coronary heart disease. Further epidemiological research could elucidate the role of elevated homocysteine plasma levels as a risk indicator or prognostic indicator in patients with pre-existing coronary heart disease taking into consideration the classical risk factors.
PMCID: PMC3011327  PMID: 21289945
7.  Antiplatelet therapy and the effects of B vitamins in patients with previous stroke or transient ischaemic attack: a post-hoc subanalysis of VITATOPS, a randomised, placebo-controlled trial 
Lancet Neurology  2012;11(6):512-520.
Previous studies have suggested that any benefits of folic acid-based therapy to lower serum homocysteine in prevention of cardiovascular events might be offset by concomitant use of antiplatelet therapy. We aimed to establish whether there is an interaction between antiplatelet therapy and the effects of folic acid-based homocysteine-lowering therapy on major vascular events in patients with stroke or transient ischaemic attack enrolled in the vitamins to prevent stroke (VITATOPS) trial.
In the VITATOPS trial, 8164 patients with recent stroke or transient ischaemic attack were randomly allocated to double-blind treatment with one tablet daily of placebo or B vitamins (2 mg folic acid, 25 mg vitamin B6, and 500 μg vitamin B12) and followed up for a median 3·4 years (IQR 2·0–5·5) for the primary composite outcome of stroke, myocardial infarction, or death from vascular causes. In our post-hoc analysis of the interaction between antiplatelet therapy and the effects of treatment with B vitamins on the primary outcome, we used Cox proportional hazards regression before and after adjusting for imbalances in baseline prognostic factors in participants who were and were not taking antiplatelet drugs at baseline and in participants assigned to receive B vitamins or placebo. We also assessed the interaction in different subgroups of patients and different secondary outcomes. The VITATOPS trial is registered with, number NCT00097669, and Current Controlled Trials, number ISRCTN74743444.
At baseline, 6609 patients were taking antiplatelet therapy and 1463 were not. Patients not receiving antiplatelet therapy were more likely to be younger, east Asian, and disabled, to have a haemorrhagic stroke or cardioembolic ischaemic stroke, and to have a history of hypertension or atrial fibrillation. They were less likely to be smokers and to have a history of peripheral artery disease, hypercholesterolaemia, diabetes, ischaemic heart disease, and a revascularisation procedure. Of the participants taking antiplatelet drugs at baseline, B vitamins had no significant effect on the primary outcome (488 patients in the B-vitamins group [15%] vs 519 in the placebo group [16%]; hazard ratio [HR] 0·94, 95% CI 0·83–1·07). By contrast, of the participants not taking antiplatelet drugs at baseline, B vitamins had a significant effect on the primary outcome (123 in the B-vitamins group [17%] vs 153 in the placebo group [21%]; HR 0·76, 0·60–0·96). The interaction between antiplatelet therapy and the effect of B vitamins on the primary outcome was significant after adjusting for imbalance in the baseline variables (adjusted p for interaction=0·0204).
Our findings support the hypothesis that antiplatelet therapy modifies the potential benefits of lowering homocysteine with B-vitamin supplementation in the secondary prevention of major vascular events. If validated, B vitamins might have a role in the prevention of ischaemic events in high-risk individuals with an allergy, intolerance, or lack of indication for antiplatelet therapy.
Australia National Health and Medical Research Council, UK Medical Research Council, Singapore Biomedical Research Council, and Singapore National Medical Research Council.
PMCID: PMC3361667  PMID: 22554931
8.  Status of B-Vitamins and Homocysteine in Diabetic Retinopathy: Association with Vitamin-B12 Deficiency and Hyperhomocysteinemia 
PLoS ONE  2011;6(11):e26747.
Diabetic retinopathy (DR) is a common cause of blindness. Although many studies have indicated an association between homocysteine and DR, the results so far have been equivocal. Amongst the many determinants of homocysteine, B-vitamin status was shown to be a major confounding factor, yet very little is known about its relationship to DR. In the present study, we, therefore, investigated the status of B-vitamins and homocysteine in DR. A cross-sectional case–control study was conducted with 100 normal control (CN) subjects and 300 subjects with type-2 diabetes (T2D). Of the 300 subjects with T2D, 200 had retinopathy (DR) and 100 did not (DNR). After a complete ophthalmic examination including fundus fluorescein angiography, the clinical profile and the blood levels of all B-vitamins and homocysteine were analyzed. While mean plasma homocysteine levels were found to be higher in T2D patients compared with CN subjects, homocysteine levels were particularly high in the DR group. There were no group differences in the blood levels of vitamins B1 and B2. Although the plasma vitamin-B6 and folic acid levels were significantly lower in the DNR and DR groups compared with the CN group, there were no significant differences between the diabetes groups. Interestingly, plasma vitamin-B12 levels were found to be significantly lower in the diabetes groups compared with the CN group; further, the levels were significantly lower in the DR group compared with the DNR group. Higher homocysteine levels were significantly associated with lower vitamin-B12 and folic acid but not with other B-vitamins. Additionally, hyperhomocysteinemia and vitamin-B12 deficiency did not seem to be related to subjects' age, body mass index, or duration of diabetes. These results thus suggest a possible association between vitamin-B12 deficiency and hyperhomocysteinemia in DR. Further, the data indicate that vitamin-B12 deficiency could be an independent risk factor for DR.
PMCID: PMC3206053  PMID: 22069468
9.  Folic Acid: A Marker of Endothelial Function in Type 2 Diabetes? 
Endothelial dysfunction is a common feature of type 2 diabetes. Recent studies suggest that the B-vitamin folic acid exerts direct beneficial effects on endothelial function, beyond the well known homocysteine lowering effects. Therefore, folic acid might represent a novel “biomarker” of endothelial function. We sought to determine whether plasma levels of folic acid determine endothelial-dependent vasodilation in patients with type 2 diabetes.
Forearm arterial blood flow (FABF) was measured at baseline and during intrabrachial infusion of the endothelial-dependent vasodilator acetylcholine (15 μg/min) and the endothelial-independent vasodilator sodium nitroprusside (2 μg/min) in 26 type 2 diabetic patients (age 56.5 ± 0.9 years, means ± SEM) with no history of cardiovascular disease.
FABF ratio (ie, the ratio between the infused and control forearm FABF) significantly increased during acetylcholine (1.10 ± 0.04 vs 1.52 ± 0.07, p < 0.001) and sodium nitroprusside (1.12 ± 0.11 vs 1.62 ± 0.06, p < 0.001) infusions. After correcting for age, gender, diabetes duration, smoking, hypertension, body mass index, microalbuminuria, glycated hemoglobin, low-density lipoprotein cholesterol, and homocysteine, multiple regression analysis showed that plasma folic acid concentration was the only independent determinant (p = 0.037, R2 = 0.22) of acetylcholine-mediated, but not sodium nitroprusside-mediated, vasodilatation.
Folic acid plasma concentrations determine endothelium-mediated vasodilatation in patients with type 2 diabetes. These results support the hypothesis of a direct effect of folic acid on endothelial function and the rationale for interventions aimed at increasing folic acid levels to reduce cardiovascular risk.
PMCID: PMC1993928  PMID: 17319100
folic acid; homocysteine; endothelium; type 2 diabetes
10.  Effect of Homocysteine-Lowering Nutrients on Blood Lipids: Results from Four Randomised, Placebo-Controlled Studies in Healthy Humans 
PLoS Medicine  2005;2(5):e135.
Betaine (trimethylglycine) lowers plasma homocysteine, a possible risk factor for cardiovascular disease. However, studies in renal patients and in obese individuals who are on a weight-loss diet suggest that betaine supplementation raises blood cholesterol; data in healthy individuals are lacking. Such an effect on cholesterol would counteract any favourable effect on homocysteine. We therefore investigated the effect of betaine, of its precursor choline in the form of phosphatidylcholine, and of the classical homocysteine-lowering vitamin folic acid on blood lipid concentrations in healthy humans.
Methods and Findings
We measured blood lipids in four placebo-controlled, randomised intervention studies that examined the effect of betaine (three studies, n = 151), folic acid (two studies, n = 75), and phosphatidylcholine (one study, n = 26) on plasma homocysteine concentrations. We combined blood lipid data from the individual studies and calculated a weighted mean change in blood lipid concentrations relative to placebo. Betaine supplementation (6 g/d) for 6 wk increased blood LDL cholesterol concentrations by 0.36 mmol/l (95% confidence interval: 0.25–0.46), and triacylglycerol concentrations by 0.14 mmol/l (0.04–0.23) relative to placebo. The ratio of total to HDL cholesterol increased by 0.23 (0.14–0.32). Concentrations of HDL cholesterol were not affected. Doses of betaine lower than 6 g/d also raised LDL cholesterol, but these changes were not statistically significant. Further, the effect of betaine on LDL cholesterol was already evident after 2 wk of intervention. Phosphatidylcholine supplementation (providing approximately 2.6 g/d of choline) for 2 wk increased triacylglycerol concentrations by 0.14 mmol/l (0.06–0.21), but did not affect cholesterol concentrations. Folic acid supplementation (0.8 mg/d) had no effect on lipid concentrations.
Betaine supplementation increased blood LDL cholesterol and triacylglycerol concentrations in healthy humans, which agrees with the limited previous data. The adverse effects on blood lipids may undo the potential benefits for cardiovascular health of betaine supplementation through homocysteine lowering. In our study phosphatidylcholine supplementation slightly increased triacylglycerol concentrations in healthy humans. Previous studies of phosphatidylcholine and blood lipids showed no clear effect. Thus the effect of phosphatidylcholine supplementation on blood lipids remains inconclusive, but is probably not large.
Folic acid supplementation does not seem to affect blood lipids and therefore remains the preferred treatment for lowering of blood homocysteine concentrations.
Lowering homocysteine might reduce the risk for heart disease. Betaine seems to have an adverse effect on blood lipids. This would make it less suitable than folic acid, which does not affect blood lipids.
PMCID: PMC1140947  PMID: 15916468
11.  Folic Acid, Vitamin B6, and Vitamin B12 in Combination and Age-related Macular Degeneration in a Randomized Trial of Women 
Archives of internal medicine  2009;169(4):335-341.
Observational epidemiologic studies indicate a direct association between homocysteine concentration in the blood and risk of age-related macular degeneration (AMD), but randomized trial data to examine the effect of homocysteine-lowering in AMD are lacking.
To examine incidence of AMD in a trial of folic acid/vitamin B6/vitamin B12.
Randomized, double-masked, placebo-controlled trial.
5,442 female health professionals aged 40 years or older with preexisting cardiovascular disease (CVD) or 3 or more CVD risk factors. A total of 5,205 of these women did not have a diagnosis of AMD at baseline and were included in this analysis.
Participants were randomly assigned to receive a combination of folic acid (2.5 mg/d), vitamin B6 (50 mg/d), and vitamin B12 (1 mg/d), or placebo.
Main Outcome Measures
Total AMD, defined as a self-report documented by medical record evidence of an initial diagnosis after randomization, and visually-significant AMD, defined as confirmed incident AMD with visual acuity of 20/30 or worse attributable to this condition.
After an average of 7.3 years of treatment and follow-up, there were 55 cases of AMD in the folic acid/B6/B12 group and 82 in the placebo group (relative risk [RR], 0.66; 95% confidence interval [CI], 0.47–0.93; p=0.02). For visually-significant AMD, there were 26 cases in the folic acid/B6/B12 group and 44 in the placebo group (RR, 0.59; 95% CI, 0.36–0.95; p=0.03).
These randomized trial data from a large cohort of women at high risk of CVD indicate that daily supplementation with folic acid/B6/B12 may reduce the risk of AMD.
PMCID: PMC2648137  PMID: 19237716
12.  Vitamin Status as a Determinant of Serum Homocysteine Concentration in Type 2 Diabetic Retinopathy 
Journal of Diabetes Research  2014;2014:807209.
We investigated the association of serum homocysteine levels and vitamin status with type 2 diabetic retinopathy. This study included 65 patients with and 75 patients without diabetic retinopathy. Patients with diabetic retinopathy had significantly higher serum homocysteine levels (P < 0.001), higher prevalence of hyperhomocysteinemia (P < 0.001), lower serum folic acid (P < 0.001), and vitamin B12 (P = 0.014) levels than those without diabetic retinopathy. Regression analysis revealed that homocysteine was an independent risk factor for diabetic retinopathy and there was a threshold in its serum level (13.7 μmol/L), above which the risk of diabetic retinopathy greatly increases (OR = 1.66, P = 0.001). Folic acid was associated with decreased odds for diabetic retinopathy (OR = 0.73, P < 0.001). There was a threshold in serum vitamin B12 level (248.4 pg/mL), below which serum homocysteine concentration significantly increases with decreasing serum vitamin B12 (P = 0.003). Our findings suggest that hyperhomocysteinemia is an independent risk factor for the development and progression of diabetic retinopathy. Decreased serum levels of folic acid and vitamin B12, through raising serum homocysteine concentrations, may also affect the diabetic retinopathy risk.
PMCID: PMC4071945  PMID: 25006590
13.  Homocysteine and Coronary Heart Disease: Meta-analysis of MTHFR Case-Control Studies, Avoiding Publication Bias 
PLoS Medicine  2012;9(2):e1001177.
Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.
Moderately elevated blood levels of homocysteine are weakly correlated with coronary heart disease (CHD) risk, but causality remains uncertain. When folate levels are low, the TT genotype of the common C677T polymorphism (rs1801133) of the methylene tetrahydrofolate reductase gene (MTHFR) appreciably increases homocysteine levels, so “Mendelian randomization” studies using this variant as an instrumental variable could help test causality.
Methods and Findings
Nineteen unpublished datasets were obtained (total 48,175 CHD cases and 67,961 controls) in which multiple genetic variants had been measured, including MTHFR C677T. These datasets did not include measurements of blood homocysteine, but homocysteine levels would be expected to be about 20% higher with TT than with CC genotype in the populations studied. In meta-analyses of these unpublished datasets, the case-control CHD odds ratio (OR) and 95% CI comparing TT versus CC homozygotes was 1.02 (0.98–1.07; p = 0.28) overall, and 1.01 (0.95–1.07) in unsupplemented low-folate populations. By contrast, in a slightly updated meta-analysis of the 86 published studies (28,617 CHD cases and 41,857 controls), the OR was 1.15 (1.09–1.21), significantly discrepant (p = 0.001) with the OR in the unpublished datasets. Within the meta-analysis of published studies, the OR was 1.12 (1.04–1.21) in the 14 larger studies (those with variance of log OR<0.05; total 13,119 cases) and 1.18 (1.09–1.28) in the 72 smaller ones (total 15,498 cases).
The CI for the overall result from large unpublished datasets shows lifelong moderate homocysteine elevation has little or no effect on CHD. The discrepant overall result from previously published studies reflects publication bias or methodological problems.
Please see later in the article for the Editors' Summary
Editors' Summary
Coronary heart disease (CHD) is the leading cause of death among adults in developed countries. With age, fatty deposits (atherosclerotic plaques) coat the walls of the coronary arteries, the blood vessels that supply the heart with oxygen and nutrients. The resultant restriction of the heart's blood supply causes shortness of breath, angina (chest pains that are usually relieved by rest), and sometimes fatal heart attacks. Many established risk factors for CHD, including smoking, physical inactivity, being overweight, and eating a fat-rich diet, can be modified by lifestyle changes. Another possible modifiable risk factor for CHD is a high blood level of the amino acid homocysteine. Methylene tetrahydofolate reductase, which is encoded by the MTHFR gene, uses folate to break down and remove homocysteine so fortification of cereals with folate can reduce population homocysteine blood levels. Pooled results from prospective observational studies that have looked for an association between homocysteine levels and later development of CHD suggest that the reduction in homocysteine levels that can be achieved by folate supplementation is associated with an 11% lower CHD risk.
Why Was This Study Done?
Prospective observational studies cannot prove that high homocysteine levels cause CHD because of confounding, the potential presence of other unknown shared characteristics that really cause CHD. However, an approach called “Mendelian randomization” can test whether high blood homocysteine causes CHD. A common genetic variant of the MTHFR gene—the C677T polymorphism—reduces MTHFR efficiency so TT homozygotes (individuals in whom both copies of the MTHFR gene have the nucleotide thymine at position 677; the human genome contains two copies of most genes) have 25% higher blood homocysteine levels than CC homozygotes. In meta-analyses (statistical pooling of the results of several studies) of published Mendelian randomized studies, TT homozygotes have a higher CHD risk than CC homozygotes. Because gene variants are inherited randomly, they are not subject to confounding, so this result suggests that high blood homocysteine causes CHD. But what if only Mendelian randomization studies that found an association have been published? Such publication bias would affect this aggregate result. Here, the researchers investigate the association of the MTHFR C677T polymorphism with CHD in unpublished datasets that have analyzed this polymorphism incidentally during other genetic studies.
What Did the Researchers Do and Find?
The researchers obtained 19 unpublished datasets that contained data on the MTHFR C677T polymorphism in thousands of people with and without CHD. Meta-analysis of these datasets indicates that the excess CHD risk in TT homozygotes compared to CC homozygotes was 2% (much lower than predicted from the prospective observational studies), a nonsignificant difference (that is, it could have occurred by chance). When the probable folate status of the study populations (based on when national folic acid fortification legislation came into effect) was taken into account, there was still no evidence that TT homozygotes had an excess CHD risk. By contrast, in an updated meta-analysis of 86 published studies of the association of the polymorphism with CHD, the excess CHD risk in TT homozygotes compared to CC homozygotes was 15%. Finally, in a meta-analysis of randomized trials on the use of vitamin B supplements for homocysteine reduction, folate supplementation had no significant effect on the 5-year incidence of CHD.
What Do These Findings Mean?
These analyses of unpublished datasets are consistent with lifelong moderate elevation of homocysteine levels having no significant effect on CHD risk. In other words, these findings indicate that circulating homocysteine levels within the normal range are not causally related to CHD risk. The meta-analysis of the randomized trials of folate supplementation also supports this conclusion. So why is there a discrepancy between these findings and those of meta-analyses of published Mendelian randomization studies? The discrepancy is too large to be dismissed as a chance finding, suggest the researchers, but could be the result of publication bias—some studies might have been prioritized for publication because of the positive nature of their results whereas the unpublished datasets used in this study would not have been affected by any failure to publish null results. Overall, these findings reveal a serious example of publication bias and argue against the use of folate supplements as a means of reducing CHD risk.
Additional Information
Please access these Web sites via the online version of this summary at
The American Heart Association provides information about CHD and tips on keeping the heart healthy; it also provides information on homocysteine, folic acid, and CHD, general information on supplements and heart health, and personal stories about CHD
The UK National Health Service Choices website provides information about CHD, including personal stories about CHD
Information is available from the British Heart Foundation on heart disease and keeping the heart healthy
The US National Heart Lung and Blood Institute also provides information on CHD (in English and Spanish)
MedlinePlus provides links to many other sources of information on CHD (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC3283559  PMID: 22363213
14.  Effect of Folic Acid and B-Vitamins on Risk of Cardiovascular Events and Total Mortality among Women at High Risk for Cardiovascular Disease: A Randomized Trial 
Recent randomized trials among patients with pre-existing cardiovascular disease (CVD) have failed to support benefits of B-vitamin supplementation on cardiovascular risk. Observational data suggest benefits may be greater among women, who have been underrepresented in published randomized trials.
To test whether a combination of folic acid, vitamin B6, and vitamin B12 lowers risk of CVD among high-risk women with and without CVD.
Design, Setting, and Participants
Within an ongoing randomized trial of antioxidant vitamins, 5,442 female US health professionals 42 years of age or older, with either a history of CVD or three or more coronary risk factors were randomized to a combination pill containing folic acid, vitamin B6, and vitamin B12 or a matching placebo and were followed for 7.3 years from April, 1998 until July, 2006.
2.5 mg of folic acid, 50 mg of vitamin B6, and 1 mg vitamin B12
Main Outcome Measures
A composite outcome of myocardial infarction (MI), stroke, coronary revascularization, or CVD mortality.
Compared to placebo, active treatment with the combination pill did not decrease the risk of the primary combined endpoint (226.9 per 10,000 person-years versus 219.2 per 10,000 person-years; relative risk =1.03; 95 percent confidence interval, 0.90–1.19, P=0.65), or any of the secondary outcomes including MI (34.5 per 10,000 person-years versus 39.5 per 10,000 person-years; relative risk =0.87; 95 percent confidence interval, 0.63–1.22), stroke (41.9 per 10,000 person-years versus 36.8 per 10,000 person-years in placebo; relative risk =1.14; 95 percent confidence interval, 0.82–1.57), and CVD mortality (50.3 per 10,000 person-years versus 49.6 per 10,000 person-years; relative risk =1.01; 95 percent confidence interval, 0.76–1.35). In a blood sub-study, geometric mean plasma homocysteine level was decreased by 18.5% (95% CI, 12.5–24.1; P<0.001) in the active arm (n=150) over that observed in the placebo arm (n=150) for a difference of 2.27 μmol per liter (95% CI, 1.54 –2.96).
Over the longest follow-up recorded thus far, a combination of folic acid/vitamin B6/vitamin B12 did not reduce a combined endpoint of total cardiovascular events among high-risk women despite significant homocysteine lowering.
Trial Registration Identifier: NCT00000541
PMCID: PMC2684623  PMID: 18460663
15.  Efficacy of homocysteine lowering therapy with folic acid in stroke prevention: a meta-analysis 
Background and Purpose
Although lower serum homocysteine concentration is associated with a reduced risk of stroke in epidemiologic studies, randomized controlled trials (RCTs) have yielded mixed findings regarding the effect of therapeutic homocysteine lowering on stroke prevention. We performed a meta-analysis of RCTs to assess the efficacy of folic acid supplementation in the prevention of stroke.
Salient trials were identified by formal literature search. Relative risk (RR) with 95% confidence interval (CI) was used as a measure of the association between folic acid supplementation and risk of stroke, pooling data across trials using a fixed-effects model.
The search identified 13 RCTs of folic acid therapy to reduce homocysteine, enrolling 39,005 participants, in which stroke was reported as an outcome measure. Across all trials, folic acid supplementation was associated with a trend toward mild benefit that did not reach statistical significance in reducing the risk of stroke (RR 0.93, 95% CI 0.85-1.03; p=0.16). The RR for non-secondary prevention trials was 0.89 (95% CI 0.79-0.99; p=0.03). In stratified analyses, a greater beneficial effect was seen in the trials testing combination therapy of folic acid plus vitamins B6 and B12 (RR 0.83, 0.71-0.97; p=0.02) and in the trials which disproportionately enrolled male patients (men/women > 2, RR 0.84, 0.74-0.94; p=0.003).
Folic acid supplementation did not demonstrate a major effect in averting stroke. However, potential mild benefits in primary stroke prevention, especially when folate is combined with B vitamins and in male patients, merit further investigation.
PMCID: PMC2909661  PMID: 20413740
Homocysteine; Folic Acid; Stroke; Prevention; Meta-Analysis
16.  Effects of Common Anti-epileptic Drugs on the Serum Levels of Homocysteine and Folic Acid 
Elevated total plasma homocysteine has been established as an independent risk factor for CVD. A strong relationship between plasma homocysteine levels and mortality has been reported in patients with CAD. Interference with folate and homocysteine metabolism by some drugs, may lead to increased plasma homocysteine levels. The object of the study was to examine the effect of AEDs on the serum concentrations of folic acid.
A total of 22, older than 18-year-old, epileptic patients, admitted in the Neurology Clinic, who were treated with AED at least for one year were selected. Twenty-two sex- and age-range-matched controls were enrolled in the study. Concentrations of total homocysteine and folic acid in the serum were measured in a fasted status. Demographic and medicine information was collected via a questionnaire. Data were analyzed by spss16 software.
Mean of serum Hcy concentration in the patients was significantly higher compared to that in the controls (p = 0.04). Serum folic acid had a nonsignificant negative correlation with the dose of drug used (p = 0.2). Serum homocysteine was not significantly correlated with the dose and duration of drug consumption (p values were 0.4, 0.24, respectively). Serum homocysteine was not significantly correlated with the kind of drug (p = 0.4), but folic acid concentration was significantly lower in the monotherapy group than in the poly therapy group (p = 0.02).
Homocysteine (Hcy) was not different between the epileptic and nonepileptic groups, although the means of the serum folic acid were similar. Possible mechanisms by which AEDs could cause hyper-homocysteinemia might be through the dysfunction of homocysteine metabolism, the acceleration of vitamin metabolism, and the interference in the metabolism of folic acid coenzymes.
PMCID: PMC3399294  PMID: 22826764
Antiepileptic drug; Folic acid; Homocysteine
High homocysteine levels may be neurotoxic and contribute to cognitive decline in older persons.
Examine the effect of supplementation with folic acid, vitamin B12 and vitamin B6 on cognitive change among women with cardiovascular disease (CVD) or CVD risk factors.
The Women's Antioxidant and Folic Acid Cardiovascular Study is a randomized, placebo-controlled trial to test a combination of B vitamins (folic acid 2.5 mg, vitamin B6 50 mg, and vitamin B12 1 mg, daily) for secondary prevention of CVD. Randomization took place among 5,442 female health professionals, 40+ years, with CVD or at least three coronary risk factors in 1998 (after folic acid fortification began in the US). Shortly after randomization (mean=1.2 years), a cognitive function substudy was initiated among 2009 participants aged 65+ years. Telephone cognitive function testing was administered up to four times over 5.4 years with 5 tests of general cognition, verbal memory and category fluency. Repeated measures analyses were conducted. The primary outcome was a global composite score averaging all tests.
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 −0.03, 0.08; p=0.30). However, supplementation appeared to confer benefits in preserving cognition among women with low baseline dietary intake of B vitamins.
Combined B vitamin supplementation did not delay cognitive decline among women with CVD or CVD risk factors. Possible cognitive benefits of supplementation among women with low dietary intake of B vitamins warrant further study.
PMCID: PMC3470481  PMID: 19064521
18.  Effect of lipid-lowering and anti-hypertensive drugs on plasma homocysteine levels 
Elevated plasma concentrations of homocysteine, a sulfur-containing amino acid, are a risk factor for coronary, cerebral and peripheral artery disease. Next to other factors, drugs used for the prevention or treatment of cardiovascular disease may modulate plasma homocysteine levels. Thus, a drug induced homocysteine increase may counteract the desired cardioprotective effect. The aim is to summarize the current knowledge on the effect of two important classes of drugs, lipid-lowering drugs and anti-hypertensive drugs, on homocysteine metabolism. Among the lipid-lowering drugs, especially the fibric acid derivatives, which are used for treatment of hypertriglyceridemia and low HDL-cholesterol, are associated with an increase of homocysteine by 20%–50%. This increase can be reduced, but not totally avoided by the addition of folic acid, vitamin B12 and B6 to fibrates. HMG-CoA reductase inhibitors (statins) do not influence homocysteine concentrations substantially. The effects of nicotinic acid and n3-fatty acids on the homocysteine concentrations are less clear, more studies are necessary to clarify their influence on homocysteine. Antihypertensive drugs have also been studied with respect to homocysteine metabolism. A homocysteine increase has been shown after treatment with hydrochlorothiazide, a lowering was observed after treatment with ß-blockers, but no effect with ACE-inhibitors. The clinical significance of the homocysteine elevation by fibrates and thiazides is not clear. However, individual patients use these drugs for long time, indicating that even moderate increases may be important.
PMCID: PMC1994037  PMID: 17583180
homocysteine; fibrates; diuretics; cardiovascular disease
19.  B Vitamins and Antioxidants Intake is Negatively Correlated with Risk of Stroke in Iran 
International Journal of Preventive Medicine  2013;4(Suppl 2):S284-S289.
Stroke is a leading cause of death in developed countries. However, current therapeutic strategies for stroke have been largely unsuccessful. Several studies have reported important benefits on reducing the risk of stroke and improving the post-stroke-associated functional declines in patients who ate foods rich in micronutrients, including B vitamins. Folic acid, vitamin B6, and vitamin B12 are all cofactors in homocysteine metabolism. Growing interest has been paid to hyperhomocyste inemia as a risk factor for stroke. Experimental studies suggest that oxidative stress plays an important role in the pathogenesis of ischemic cerebral injury, and higher intake of antioxidants has been associated with a lower risk of stroke in large population studies. The aim of this study was to examine whether the dietary intake of B vitamins and antioxidants in patients with stroke were comparatively worse than those in patients without stroke.
In this case control study, 69 stroke patients (46 male, age = 56 ± 18 years and 23 female, age = 52 ± 7 years) admitted to Azzahra hospital between April 2009 and May 2010 were matched for age and sex with 60 patients (30 male and 30 female) from the same hospital who were not affected with acute cerebrovascular diseases and did not have a history of stroke. Dietary intake was assessed with a validated self-administered food frequency questionnaire (FFQ). FFQ was collected conducting face-to-face interview with one of the patients’ close relatives. Food intakes, translated into nutrient data, were compared between the two groups and with the recommended values.
Intake of folic acid in men with stroke and vitamin B12 in women with stroke was significantly lower than that in the patients without stroke (P < 0.05), but there was no significant difference between the two groups in the level of antioxidant consumption in women and men (P > 0.05).
Our findings suggest that increased folic acid, vitamin B12, and vitamin E, C intake may be associated with decreased risk of stroke.
PMCID: PMC3678233  PMID: 23776739
Dietary quality; folic acid; stroke; vitamin B6; vitamin B12
20.  High-Dose B-Vitamin Supplementation and Progression of Subclinical Atherosclerosis: A Randomized Controlled Trial 
Background and Purpose
Although plasma total homocysteine (tHcy) levels are associated with cardiovascular disease (CVD), it remains unclear whether homocysteine is a cause or a marker of atherosclerotic vascular disease. We determined whether reduction of tHcy levels with B-vitamin supplementation reduces subclinical atherosclerosis progression.
In this double-blind clinical trial, 506 participants 40–89 years of age with an initial tHcy >8.5 μmol/L without diabetes and CVD were randomized to high-dose B-vitamin supplementation (folic acid 5 mg + vitamin B12 0.4 mg + vitamin B6 50 mg) or matching placebo for 3.1 years. Subclinical atherosclerosis progression across 3 vascular beds was assessed using high-resolution B-mode ultrasonography to measure carotid artery intima-media thickness (primary outcome) and multidetector spiral computed tomography to measure aortic and coronary artery calcium (secondary outcome).
Although the overall carotid artery intima-media thickness progression rate was lower with B-vitamin supplementation than with placebo, statistically significant between-group differences were not found (p=0.31). However, among subjects with baseline tHcy≥9.1 μmol/L, those randomized to B-vitamin supplementation had a statistically significant lower average rate of carotid artery intima-media thickness progression compared with placebo (p=0.02); among subjects with a baseline tHcy <9.1 μmol/L there was no significant treatment effect (p-value for treatment interaction=0.02). B-vitamin supplementation had no effect on progression of aortic or coronary artery calcification overall or within subgroups.
High-dose B-vitamin supplementation significantly reduces progression of early stage subclinical atherosclerosis (carotid artery intima-media thickness) in well-nourished healthy B-vitamin “replete” individuals at low-risk for CVD with a fasting tHcy >9.1 μmol/L.
PMCID: PMC2701290  PMID: 19118243
Atherosclerosis; Computed tomography; Folate; Homocysteine; Intima-media thickness; Randomized controlled trials; Vitamin B12
21.  The effect of long-term homocysteine-lowering on carotid intima-media thickness and flow-mediated vasodilation in stroke patients: a randomized controlled trial and meta-analysis 
Experimental and epidemiological evidence suggests that homocysteine (tHcy) may be a causal risk factor for atherosclerosis. B-vitamin supplements reduce tHcy and improve endothelial function in short term trials, but the long-term effects of the treatment on vascular structure and function are unknown.
We conducted a sub-study of VITATOPS, a randomised, double-blind, placebo-controlled intervention trial designed to test the efficacy of long term B-vitamin supplementation (folic acid 2 mg, vitamin B6 25 mg and vitamin B12 0.5 mg) in the prevention of vascular events in patients with a history of stroke. We measured carotid intima-medial thickness (CIMT) and flow-mediated dilation (FMD) at least two years after randomisation in 162 VITATOPS participants. We also conducted a systematic review and meta-analysis of studies designed to test the effect of B-vitamin treatment on CIMT and FMD.
After a mean treatment period of 3.9 ± 0.9 years, the vitamin-treated group had a significantly lower mean plasma homocysteine concentration than the placebo-treated group (7.9 μmol/L, 95% CI 7.5 to 8.4 versus 11.8 μmol/L, 95% CI 10.9 to 12.8, p < 0.001). Post-treatment CIMT (0.84 ± 0.17 mm vitamins versus 0.83 ± 0.18 mm placebo, p = 0.74) and FMD (median of 4.0%, IQR 0.9 to 7.2 vitamins versus 3.0%, IQR 0.6 to 6.6 placebo, p = 0.48) did not differ significantly between groups. A meta-analysis of published randomised data, including those from the current study, suggested that B-vitamin supplements should reduce CIMT (-0.10 mm, 95% CI -0.20 to -0.01 mm) and increase FMD (1.4%, 95% CI 0.7 to 2.1%). However, the improvement in endothelial function associated with homocysteine-lowering treatment was significant in short-term studies but not in longer trials.
Although short-term treatment with B-vitamins is associated with increased FMD, long-term homocysteine-lowering did not significantly improve FMD or CIMT in people with a history of stroke.
Trial Registration
Clinical Trial Registration URL:
Trial Registration number: 12605000005651
PMCID: PMC2559819  PMID: 18803866
22.  Homocysteine-Lowering by B Vitamins Slows the Rate of Accelerated Brain Atrophy in Mild Cognitive Impairment: A Randomized Controlled Trial 
PLoS ONE  2010;5(9):e12244.
An increased rate of brain atrophy is often observed in older subjects, in particular those who suffer from cognitive decline. Homocysteine is a risk factor for brain atrophy, cognitive impairment and dementia. Plasma concentrations of homocysteine can be lowered by dietary administration of B vitamins.
To determine whether supplementation with B vitamins that lower levels of plasma total homocysteine can slow the rate of brain atrophy in subjects with mild cognitive impairment in a randomised controlled trial (VITACOG, ISRCTN 94410159).
Methods and Findings
Single-center, randomized, double-blind controlled trial of high-dose folic acid, vitamins B6 and B12 in 271 individuals (of 646 screened) over 70 y old with mild cognitive impairment. A subset (187) volunteered to have cranial MRI scans at the start and finish of the study. Participants were randomly assigned to two groups of equal size, one treated with folic acid (0.8 mg/d), vitamin B12 (0.5 mg/d) and vitamin B6 (20 mg/d), the other with placebo; treatment was for 24 months. The main outcome measure was the change in the rate of atrophy of the whole brain assessed by serial volumetric MRI scans.
A total of 168 participants (85 in active treatment group; 83 receiving placebo) completed the MRI section of the trial. The mean rate of brain atrophy per year was 0.76% [95% CI, 0.63–0.90] in the active treatment group and 1.08% [0.94–1.22] in the placebo group (P = 0.001). The treatment response was related to baseline homocysteine levels: the rate of atrophy in participants with homocysteine >13 µmol/L was 53% lower in the active treatment group (P = 0.001). A greater rate of atrophy was associated with a lower final cognitive test scores. There was no difference in serious adverse events according to treatment category.
Conclusions and Significance
The accelerated rate of brain atrophy in elderly with mild cognitive impairment can be slowed by treatment with homocysteine-lowering B vitamins. Sixteen percent of those over 70 y old have mild cognitive impairment and half of these develop Alzheimer's disease. Since accelerated brain atrophy is a characteristic of subjects with mild cognitive impairment who convert to Alzheimer's disease, trials are needed to see if the same treatment will delay the development of Alzheimer's disease.
Trial Registration ISRCTN94410159
PMCID: PMC2935890  PMID: 20838622
23.  Rationale, design and baseline characteristics of a large, simple, randomized trial of combined folic acid and vitamins B6 and B12 in high-risk patients: The Heart Outcomes Prevention Evaluation (HOPE)-2 trial 
Epidemiological studies suggest that mild to moderate elevation in plasma homocysteine concentration is associated with increased risk of atherothrombotic cardiovascular (CV) disease. Simple, inexpensive and nontoxic therapy with folic acid and vitamins B6 and B12 reduces plasma homocysteine levels by approximately 25% to 30% and may reduce CV events. Therefore, a large, randomized clinical trial – the Heart Outcomes Prevention Evaluation (HOPE)-2 study – is being conducted to evaluate this therapy in patients at high risk for CV events.
To evaluate whether long-term therapy with folic acid and vitamins B6 and B12 reduces the risk of major CV events in a high-risk population. The primary study outcome is the composite of death from CV causes, myocardial infarction and stroke.
A total of 5522 patients aged 55 years or older with pre-existing CV disease or with diabetes and additional risk factor(s) at 145 centres in 13 countries were randomly assigned to daily therapy with combined folic acid 2.5 mg, vitamin B6 50 mg and vitamin B12 1 mg, or to placebo. Follow-up will average five years, to be completed by the end of 2005.
The patients’ baseline characteristics confirmed their high-risk status. Baseline homocysteine levels varied between countries and regions. HOPE-2 is one of the largest trials of folate and vitamins B6 and B12 and is expected to significantly contribute to the evaluation of the role of homocysteine lowering in CV prevention.
PMCID: PMC2538982  PMID: 16450017
Atherosclerosis; Clinical trials; Coronary disease; Homocysteine; Myocardial infarction; Stroke
24.  Effect of Combined Folic Acid, Vitamin B6, and Vitamin B12 on Cancer Risk: Results from a Randomized Trial 
Folate, vitamin B6, and vitamin B12 are thought to play an important role in cancer prevention.
To evaluate the effect of combined folic acid, vitamin B6, and vitamin B12 treatment on cancer risk in women at high risk for cardiovascular disease.
Design, Setting, and Participants
In the Women’s Antioxidant and Folic Acid Cardiovascular Study, 5442 US female health professionals aged 42 years or older with preexisting cardiovascular disease or 3 or more coronary risk factors were randomly assigned to receive either a daily combination of folic acid, vitamin B6, and vitamin B12 or placebo in April 1998, and treated through July 31, 2005 for 7.3 years.
Daily supplementation of a combination of 2.5 mg of folic acid, 50 mg of vitamin B6, and 1 mg of vitamin B12 (n=2721) or placebo (n=2721).
Main Outcome Measures
Confirmed newly diagnosed total invasive cancer.
A total of 379 women developed invasive cancer (187 in the active group and 192 in the placebo group). Compared with placebo, women receiving combined folic acid, vitamin B6, and vitamin B12 had similar risk of developing total invasive cancer (101.1/10000 person-years vs 104.3/10000 person-years for the active vs placebo group; hazard ratio, 0.97; 95% confidence interval, 0.79–1.18; P=.75), breast cancer (37.8/10000 person-years vs 45.6/10000 person-years; hazard ratio, 0.83; 95% confidence interval, 0.60–1.14; P=.24), and any cancer death (24.6/10000 person-years vs 30.1/10000 person-years; hazard ratio, 0.82; 95% confidence interval, 0.56–1.21; P=.32).
Combined folic acid, vitamin B6, and vitamin B12 treatment had no significant effect on overall risk of total invasive cancer or breast cancer among women during folic acid fortification era.
Trial Registration Identifier: NCT00000541
PMCID: PMC2593624  PMID: 18984888
25.  The effect of homocysteine-lowering with B-vitamins on osteoporotic fractures in patients with cerebrovascular disease: substudy of VITATOPS, a randomised placebo-controlled trial 
BMC Geriatrics  2013;13:88.
Homocysteine has been postulated as a novel, potentially reversible risk factor for osteoporosis and related fractures. We evaluated whether homocysteine-lowering therapy with B-vitamins in patients with symptomatic cerebrovascular disease reduced the incidence of osteoporotic fractures.
VITAmins To Prevent Stroke (VITATOPS) was a prospective randomised double-blind placebo-controlled trial in which 8,164 patients with recent (within 7 months) stroke or transient ischemic attack were randomly allocated to double-blind treatment with one tablet daily of either placebo (n = 4,075) or B-vitamins (folic acid 2 mg, vitamin B6 25 mg, vitamin B12 500 μg; n = 4,089). Patients were reviewed every six months. Any osteoporotic fracture and osteoporotic hip fractures were secondary outcome events, and were reviewed by a masked adjudication committee. Analysis was by intention to treat. Logistic regression was used to identify independent predictors of fracture.
Participants had a mean age of 62.6 years (SD 12.5 years) and 64% were male, 42% of Western European descent and 75% were functionally independent (Oxford Handicap Scale of two or less). After a median duration of 2.8 years therapy and 3.4 years follow-up, there was no significant difference in the incidence of any osteoporotic fracture between participants assigned B-vitamins (67 [1.64%]) and placebo (78 [1.91%]; risk ratio [RR] 0.86, 95% confidence interval [CI] 0.62-1.18) or the incidence of hip fractures (34 [0.83%] B-vitamins vs. 36 [0.88%] placebo; RR 0.94, 95% CI 0.59-1.5). There was no significant impact of B-vitamin therapy on time to first fracture. Baseline homocysteine levels did not predict any osteoporotic fracture (p =0.43). Independent predictors of any osteoporotic fracture were female sex, age > 64 years, Western European ethnicity and use of anti-osteoporosis medication at randomization (all p < 0.01).
Once daily treatment with B-vitamins had no effect on incidence of osteoporotic fractures during a median of 3.4 years follow-up in patients with cerebrovascular disease. A modest effect of B-vitamin therapy is not excluded due to the low numbers of fracture outcome events.
Trial registration number: NCT00097669 and number: ISRCTN74743444.
PMCID: PMC3848681  PMID: 24004645
Homocysteine; B-vitamins; Osteoporosis; Fractures; Stroke

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