CLINICAL INVESTIGATION

Folic Acid Supplementation for

Stroke Prevention in Patients
With Cardiovascular Disease
Tao Tian, MD, PhD, Kun-Qi Yang, MD, Jin-Gang Cui, MD, PhD,
Lan-Lan Zhou, BN and Xian-Liang Zhou, MD, PhD

ABSTRACT

Background: Controversy remains regarding the efficacy of folic acid supplementation in reducing the risk of stroke. This
study aimed to evaluate the effect of folic acid supplementation on stroke prevention in patients with cardiovascular disease
(CVD).
Materials and Methods: We searched the PubMed, EMBASE and Cochrane Library databases through October 2016 to
identify randomized clinical trials of folic acid supplementation to prevent stroke in patients with CVD. Relative risks (RRs) with
95% CIs were used to examine the association between folic acid supplementation and the risk of stroke with a fixed-effect
model. Stratified analyses were performed according to modifiers that may affect the efficacy of folic acid supplementation.
Results: Eleven studies with a total of 65,790 participants were included. Folic acid supplementation was associated with a
significant benefit in reducing the risk of stroke in patients with CVD (RR ¼ 0.90; 95% CI: 0.84-0.97; P ¼ 0.005). In the
stratified analysis, greater beneficial effects were observed in participants with a decrease in homocysteine concentrations of
25% or greater (RR ¼ 0.85; 95% CI: 0.74-0.97; P ¼ 0.03), those with a daily folate dose of less than 2 mg (RR ¼ 0.78; 95%
CI: 0.68-0.89; P ¼ 0.01), and populations in regions with no or partly fortified grain (RR ¼ 0.87; 95% CI: 0.81-0.94;
P ¼ 0.04).
Conclusions: Our meta-analysis demonstrated that folic acid supplementation is effective in stroke prevention in patients
with CVD.
Key Indexing Terms: Folic acid; Homocysteine; Stroke; Cardiovascular disease; Meta-analysis. [Am J Med Sci 2017;354
(4):379–387.]

INTRODUCTION and stroke could be reduced by 11% and 19%,

respectively.4

C
ardiovascular disease (CVD), a severe disease
burden in both developed and developing coun-
tries, will be one of the most prominent global
public health challenges in the 21st century.1 Stroke is
one of the leading causes of death in the world and
threatens the lives of those diagnosed with CVD,2 thus
stroke prevention is of great importance, particularly in
patients with CVD. Hyperhomocysteinemia has been
identified as a modifiable, independent risk factor for
CVD,3-5 and is associated with an increased risk of
stroke. Considerable experimental evidence has been
accumulated to support the role of homocysteine in
promoting atherosclerosis, including inducing oxidative
stress,6 enhancing inflammatory responses,7,8 and facil-
itating endothelial dysfunction.9 Since 1976, a series of
case-control studies have provided epidemiologic evi-
dence that elevated homocysteine concentrations are
associated with cardiovascular events.10-13 Folic acid
and B vitamins play important roles in regulating homo-
cysteine metabolism, and it is suggested that folic acid
and vitamin B6 and B12 supplementation could reduce
plasma homocysteine levels.14,15 A meta-analysis of
observational studies showed that, with a 25% lower
homocysteine level, the risk of ischemic heart disease
In the past 2 decades, studies regarding folic acid
supplementation for preventing stroke were widely per-
formed. A population-based cohort study showed that,
after 2-year implementation for mandatory folic acid
fortification of grain products in the United States and
Canada, the mortality rate from stroke improved overall
and in nearly all population strata, while there was no
improvement in mortality from stroke in England and
Wales where folic acid fortification was not required
during the same period.16 Moreover, the HOPE2 study
and the SU.FOL.OM3 study showed a 24% and a 41%
reduction in the risk of stroke by folic acid supplemen-
tation, respectively.17,18
However, the efficacy of folic acid supplementa-
tion in stroke prevention is still a matter of debate.
Several randomized controlled trials (RCTs) with folic
acid therapy reported null results.19-22 A recent meta-
analysis indicated that B vitamin supplementation was
not associated with a lower risk of stroke.23 Further-
more, another meta-analysis that analyzed individual
participant data of 8 randomized trials concluded that
dietary supplementation with folic acid had no signifi-
cant benefits within 5 years on cardiovascular events,

Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. 379
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Tian et al
including stroke.24 Conversely, Wang et al25 reported
that folic acid supplementation was effective in stroke
prevention. In addition, in a recent meta-analysis by Ji et
al,26 the significant benefits of B vitamin supplementa-
tion on stroke were also observed, especially in subjects
with 4130 mm Hg systolic blood pressure and with
lower antiplatelet use.
It is of note that these studies analyzed data from
participants with different pre-existing conditions,
including CVD, end-stage renal disease, esophageal
dysplasia, and colorectal adenomas, whereas the effi-
cacy of folic acid supplementation for stroke prevention
in CVD population has not been sufficiently analyzed.
Therefore, with the recent completion of several random-
ized trials,18-20 we decided to perform an updated
meta-analysis, focusing on stroke as the disease
endpoint in patients with CVD in relation to folic acid
supplementation.
MATERIALS AND METHODS
Search Strategy
We searched PubMed, the Cochrane Library, and
EMBASE databases up to October 2016 for randomized
clinical trials that presented the effects of folic acid
supplementation on stroke prevention in patients with
CVD. The proceedings from the American Heart Asso-
ciation and American College of Cardiology were man-
ually retrieved. In addition, the reference lists of the
identified studies and relevant review articles were also
searched. No language restrictions were used. The
following terms were used in the search: “homocys-
teine,” “folic acid,” “folate,” “vitamin B12” and “vitamin
B6” crossed with the terms “cardiovascular disease,”
“myocardial infarct,” “myocardial ischemia,” “coronary
heart disease,” “angina,” “heart attack,” “stroke,” “cer-
ebrovascular disease,” “cerebrovascular attack,” “brain
attack,” “brain infarct,” “brain hemorrhage” and “intra-
cranial hemorrhage.” The search was limited to studies
in human adults.
Inclusion and Exclusion Criteria
Design of the included study had to meet the
following criteria: (1) an RCT, with duration of interven-
tion of at least 1 year; (2) the enrolled participants were
at risk of or had established CVD; (3) the intervention
group received folic acid with or without vitamin B12 or
vitamin B6, and the comparison group received placebo,
usual care or low-dose B vitamins and (4) the number of
events for stroke that occurred during the trial was
reported by intervention and control groups. Privacy
rights had been observed for patients in these studies.
Our study was approved by the Ethics Committee of
Fuwai Hospital.
The exclusion criteria were as follows: (1) studies
with patients with end-stage renal disease; (2) the
control group received another active therapy that the
active treatment group did not receive and (3) a lack of
380
adequate details of study methodology or results from
the article or study investigators.
Data Extraction and Quality Assessment
Two investigators (T.T. and K.-Q.Y.) independently
selected suitable trials and extracted data from and
assessed the quality of included trials. Discrepancies
were resolved by discussion with a third reviewer
(L.-L.Z.) and by referencing the original report. For each
study, the following information was extracted: name of
the study, first author, year of publication, sample size,
mean age, percentage of male participants, homocys-
teine levels at baseline and at the end of the study,
baseline folic acid levels, vitamin B6 and B12 levels,
daily folic acid and vitamin B6 and B12 doses, duration
of follow-up, whether there was folic acid fortification,
regions where the studies conducted and stroke events.
The quality of the studies was assessed according to
the criteria proposed by Juni et al.27 The criteria included
generation of a random sequence, concealment of a
treatment allocation schedule, whether the groups were
similar at baseline, blinding of patients and caregivers,
blinding of outcome assessment, percentage of patients
lost to follow-up, and whether all patients were treated
as assigned.
Statistical Analysis
Statistical analyses were performed with Review
Manager (RevMan version 5.0; The Nordic Cochrane
Centre, The Cochrane Collaboration, Copenhagen, Den-
mark) and Stata Statistical Software, Release 12 (Stata-
Corp LP, College Station, TX). Relative risks (RRs) with
95% CIs were used to assess the association between
folic acid supplementation and the risk of stroke. And we
considered the hazard ratio as RR in the studies.
Heterogeneity between trials was assessed using the
chi-squared test and I2 statistics. Heterogeneity was
considered significant when the P value of chi square
statistics was o0.05. We regarded an I2 of o40% as
minimal heterogeneity, 40%-75% as modest and 475%
as considerable.28 We planned to pool data across trials
according to the fixed-effects model based on Mantel-
Haenszel methods if considerable heterogeneity, P o
0.05, or I2 4 75% was not present.29,30 We also
compared results obtained from a fixed-effects model
with those obtained from a random-effects model to
evaluate the influence of small-study effects on the
results.28 Planned stratified analyses were performed
based on a decrease in homocysteine levels, interven-
tion regimen, daily dose of folic acid and vitamin B12,
baseline level of vitamin B12, prior folic acid grain
fortification, history of stroke, and duration of interven-
tion. Meta-regression analysis was performed to identify
the relation between a reduction in homocysteine con-
centrations and the RR of stroke. We also conducted
sensitivity analysis by excluding studies that provided
unclear methodology or failed to adopt a blind method.
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 Folic Acid for Stroke Prevention

Publication bias was evaluated using funnel plots and
the Begg and Egger tests.
RESULTS
Literature Search
Initially, 802 references were yielded by retrieving the
databases PubMed, the Cochrane Library and EMBASE,
of which 780 were excluded based on screening titles
and abstracts (Figure 1). The remaining 22 studies with
potential eligibility were obtained and evaluated in detail.
Eleven of these studies were excluded for the following
reasons: 4 studies were sub-analyses, 2 studies for
rationales and designs, 4 studies did not report stroke
as an endpoint and the follow-up duration of 1 study
was less than 1 year. Finally, 11 RCTs met the inclusion
criteria and were included in the analysis.
Study Characteristics
Details of the characteristics of the included trials are
shown in Table 1. Eleven RCTs were included in our final
analysis, consisting of 65,790 participants. All of the
individuals who were enrolled in these 11 trials suffered
from CVD. The number of participants in the trials
ranged from 283-20,702, and more men were enrolled
in 9 of the 11 studies. These trials compared the stroke
events (1 of the endpoints) between folic acid supple-
mentation groups (with or without combination with
other B vitamins, including B6 and B12) and either
placebo or usual care groups. The dosage of folic acid
in the intervention groups ranged from 0.5-5.0 mg/day.
The duration of the trials’ follow-up period ranged from
12-87 months. These 11 trials were performed in differ-
ent regions, including regions with and without folic acid
fortification, and partly fortified regions (i.e., both fortified
and nonfortified).
1,360 stroke events in 32,250 participants (4.2%) in the
intervention group, and 1,466 in 30,582 participants
(4.8%) in the control group. No significant heterogeneity
was found in the overall analysis (I 2 ¼ 36%, P ¼ 0.12).
Five studies either provided unclear information about
methodology or failed to adopt blind design.21,31-34
When sensitivity analyses removed any 1 of these
5 studies, they showed that the RR and 95% CI did
not substantially change.
At the end of the intervention, a variable degree of
reduction in homocysteine concentrations among the
trials was found. The net reduction in homocysteine
concentrations ranged from 1.6-3.8 μmol/L, whereas the
relative reduction ranged from 13%-29% (Table 3).
When stratified by the rate of reduction in homocysteine,
the subgroup with a higher degree of lowering of
homocysteine (Z25%) showed a greater reduction in
the RR of stroke (RR ¼ 0.85; 95% CI: 0.74-0.97; P ¼
0.03; Table 4). Combined intervention of B vitamins did
not seem to be associated with greater reduction in the
RR of stroke. When stratified by treatment regimen (folic
acid alone versus folic acid with B vitamins), the RR for
the trials with folic acid and B vitamins was 0.91 (95%
CI: 0.82-1.00, P ¼ 0.13). Despite the homocysteine-
lowering effect of folic acid, there was no indication that
a higher dosage of folic acid was better. When stratified
by daily folic acid dose (o2 versus Z2 mg), the RR for
the trials with a lower dosage exhibited significance
(0.78; 95% CI: 0.68-0.89; P ¼ 0.01). Subgroup analyses
based on daily vitamin B12 dosage (o0.5 versus
Z0.5 mg) and baseline vitamin B12 levels (o300 versus
Z300 pmol/L) revealed that these factors were not
associated with a lower risk of stroke. We observed a
benefit in the subgroup in which trials that were

Study Quality
The methodological quality of the included 11 trials is
shown in Table 2. All of these trials were randomized, but 4
studies did not reveal the method of generating a random-
ized sequence,21,31-33 and 2 studies did not describe the
method of concealing allocation.33,34 Significant differen-
ces were not observed in baseline characteristics between
intervention and control groups in each study. Among the
trials, 2 were open label,33,34 whereas the others were a
double-blind design. Blinding of outcome assessors was
reported by all 11 studies. All participants in each study
were treated as assigned. The percentage of patients lost
to follow-up in each study was o10%.

Main Analysis
After pooling all 11 RCTs, folic acid supplementation
(with or without other B vitamins) significantly reduced
the risk of stroke by 10% compared with the control
group (RR = 0.90, 95% CI: 0.84-0.97, P = 0.005;
I2 = 32%, P = 0.15; Figure 2). There was a total of FIGURE 1. Flow diagram of study selection.

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 382

Tian et al
TABLE 1. Characteristics of study participants and study design.

Percentage Percentage Active treatment

of of lipid- Folic
Study Age Antiplatelet lowering acid Vitamin B12 Vitamin B6 Duration Grain
(year) Number (year) Sex (male) use drugs use (mg) (mg) (mg) Control (months) fortification Countries
VISP (2004)31 3,680 66.3 2,301 (63%) NR NR 2.5 0.4 25 20 μg folic acid, 24 Yes USA, Canada
6 μg vit B 12, and
200 μg vit B6 Scotland
FOLARDA (2004)33 283 59 197 (70%) 98 NR 5.0 – – Usual care 12 No Netherlands
Goes (2005)34 593 65.2 462 (78%) NR 100 0.5 – – Usual care 42 No Netherlands
HOPE2 (2006)17 5,522 68.9 3,963 (72%) 80 60 2.5 1 50 Placebo 60 Partially Canada, USA,
Brazil,
western
Europe,
Slovakia
and Italy
NORVIT (2006)32 3,749 63.2 2,815 (74%) 90 82 0.8 0.4 40 Placebo 40 No Norway
WAFACS (2008)22 5,442 62.8 0 51 34 2.5 1 50 Placebo 87 Yes USA
WENBIT (2008)21 3,090 61.7 2,458 (79%) 90 88 0.8 0.4 40 Placebo 38 No Norway
SEARCH (2010)19 12,064 64.2 10,012 (83%) 91 100 2 1 – Placebo 80 No UK
Su.Fol.Om3 (2010)18 2,501 60.7 1,987 (79%) 93 85 0.56 0.02 3 Placebo 56 No France
THE AMERICAN JOURNAL

VITATOPS (2010)20 8,164 62.6 5,218 (64%) NR NR 2 0.5 25 Placebo 41 No 20 countries

from 4
continents
CSPPT (2015)48 20,702 60.0 8,497 (41%) 2.9 0.8 0.8 þ 10 mg – – 10 mg enalapril 54 No China
VOLUME 354

enalapril

NR, not reported.

OF THE
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 Folic Acid for Stroke Prevention

TABLE 2. Quality assessment of studies included in this meta-analysis.

Patients selection Blinding methods Attrition

Adequate Similar Blinding
generation Adequate group at of Blinding Percentage
of concealment the start patients of of patients Intention-
allocation of allocation of the and outcome lost to to-treat
Study (year) sequences sequences study caregivers assessors follow-up analysis

VISP (2004)31 Unclear Yes Yes Double Yes 7.2 Yes

FOLARDA (2004)33 Unclear Unclear Yes Open Yes 8.1 Yes
Goes (2005)34 Yes Unclear Yes Open Yes 0 Yes
HOPE2 (2006)17 Yes Yes Yes Double Yes 0.7 Yes
NORVIT (2006)32 Unclear Yes Yes Double Yes 0 Yes
WAFACS (2008)22 Yes Yes Yes Double Yes 7.4 Yes
WENBIT (2008)21 Unclear Yes Yes Double Yes 0.1 Yes
SEARCH (2010)19 Yes Yes Yes Double Yes 1 Yes
Su.Fol.Om3 (2010)18 Yes Yes Yes Double Yes 0.7 Yes
VITATOPS (2010)20 Yes Yes Yes Double Yes 8.6 Yes
CSSPT (2015)48 Yes Yes Yes Double Yes 0.3 Yes

performed in regions without fortified grain were pooled
(RR ¼ 0.87; 95% CI: 0.81-0.94; P ¼ 0.04; Table 4).
Nevertheless, any benefit in reducing stroke risk was not
observed in the subgroups based on history of stroke
and duration of intervention.
Publication Bias
Funnel plots of the studies suggested no significant
asymmetry in the meta-analysis of the effect of
homocysteine-lowering treatment on the risk of stroke
(Figure 3). A funnel plot of the RR of stroke (Begg’s test,
P ¼ 0.697; Egger’s test, P ¼ 0.727) showed that there
was no strong evidence of publication selection bias.
DISCUSSION
Our meta-analysis, which included 11 RCTs with
65,790 individuals, showed a significant benefit in the
effect of folic acid supplementation on stroke preven-
tion. In contrast to other published meta-analyses, we
primarily focused on stroke prevention in individuals at
risk of or with established CVD, and RCTs involving
end-stage renal disease, esophageal dysplasia and
colorectal adenomas were excluded. The benefitial
effect of folic acid supplementation on stroke prevention
was modified by the degree of decrease in homocys-
teine concentrations, daily folic acid dosage and folate
fortification.
Folic acid supplementation could reduce plasma
homocysteine concentrations, and we found a 14%
reduction in the RR of stroke related to a decrease in
homocysteine concentrations of 25% or greater.
Although there was no significant reduction in the RR
of stroke with a decrease in homocystine concentrations
of less than 25%. This finding is concordant with the
results of a meta-analysis of prospective studies, which
showed that a reduction of 19% in the RR of stroke was
associated with a 25% lower homocysteine level.4
Whereas, changes in homocysteine levels in the
included RCTs were variable, and we failed to describe

FIGURE 2. Meta-analysis of the efficacy of homocysteine-lowering intervention with folic acid on stroke prevention in patients with
cardiovascular disease.

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Tian et al

TABLE 3. Relative risk of stroke and change in homocysteine concentration.

Net decrease Stroke events/total patients

in homocysteine Change in
Study (year) (μmol/L) homocysteine (%) Intervention Control RR (95% CI)

VISP (2004)31
2.3 17.2 152/1,827 148/1,853 1.04 [0.84-1.29]
FOLARDA (2004)33 NR NR 1/140 0/143 3.06 [0.13-74.58]
Goes (2005)34 2.6 21.5 8/300 12/293 0.65 [0.27-1.57]
HOPE2 (2006)17 3.2 26.2 111/2,758 147/2,764 0.76 [0.59-0.96]
NORVIT (2006)32 3.8 29.0 49/1,872 27/943 0.91 [0.58-1.45]
WAFACS (2008)22 1.6 13.0 79/2,721 69/2,721 1.14 [0.83-1.57]
WENBIT (2008)21 2.8 25.9 28/1,540 19/779 0.75 [0.42-1.33]
SEARCH (2010)19 3.3 24.4 269/6,033 265/6,031 1.01 [0.86-1.20]
Su.Fol.Om3 (2010)18 3.5 27.3 21/622 36/626 0.59 [0.35-0.99]
VITATOPS (2010)20 3.8 27.2 360/4,089 388/4,075 0.92 [0.81-1.06]
CSSPT (2015)48 NR NR 282/10,348 355/10,354 0.79 [0.68-0.93]
NR, not reported.

a linear relation between lowering of homocysteine
levels and stroke prevention.
The intervention regimen of folic acid is regarded as
a possible modifying factor to modify the intervention
effect on stroke prevention. A meta-analysis showed
that the addition of vitamin B12 to folate provided a
further reduction of 7% in homocysteine concentra-
tions.14 And previous studies have suggested the supe-
riority of combined intervention of B vitamins (i.e.,
supplementation of folic acid and vitamins B6 and
B12) in reducing the risk of stroke.35,36 Discordant with
these findings, our analysis showed no association
between intervention regimen and stroke prevention.
Nevertheless, there is an interesting story concerning
the daily folate dose, that is, lower daily folate dose
could bring about a better outcome. Our analysis
showed that a daily folate dose of less than 2 mg had
a significant effect on reducing stroke risk, whereas
most other studies only suggested a benefit in lowering
homocysteine levels but there was no improvement in
the risk of stroke related to a particular folate dose. A
possible explanation for the effectiveness of low-dose
folic acid is that low-dose folic acid may have been
capable of improving vascular function to its maximum.
Shirodaria et al37 showed that the recommended
daily allowance for folic acid (400 μg/day) can induce
a similar increment in vascular endothelium 5-
methyltetrahydrofolate levels as high-dose folic acid
(5 mg/day), despite high-dose folate leading to higher
plasma 5-methyltetrahydrofolate levels. This recom-
mended daily allowance for folic acid is sufficient for
providing the greatest benefit on vascular function,
including improvement in endothelial function, intracel-
lular redox state, and elastic properties of large
vessels.37 A negative effect of high-dose folic acid
netralizing the benefit might be another possible inter-
pretation. Methylation, which is an important step in
the metabolism of homocysteine involving folate, is
enhanced with high-dose folate administration.38 Hyper-
methylation of arginine and several proatherogenic
genes can elevate levels of asymmetrical dimethylargi-
nine and increase expression of proatherogenic
molecules,39,40 respectively, leading to deterioration of
clinical outcomes.
The same as folic acid, vitamin B12 plays an
important role in modulating the metabolism of homo-
cysteine as well.36 There is evidence that vitamin B12
deficiency could limit the ability of intervention to reduce
elevated homocysteine levels.41 In an efficacy analysis
of the Vitamin Intervention for Stroke Prevention trial
(VISP), which showed no benefit in reducing stroke with
the combined intervention of B vitamins, Spence et al42
found a significant reduction in the risk of stroke when
patients with vitamin B12 deficiency and renal impair-
ment were excluded. In our analysis, we did not find
higher daily vitamin B12 dosage and higher baseline
vitamin B12 concentrations were associated with a lower
stroke risk, which may attribute to individuals with
vitamin B12 deficiency enrolled in the RCTs. A further
analysis based on individual participant data from RCTs
may be needed to clarify the role of vitamin B12 in
modifying the effect of folic acid supplementation on
stroke prevention.
Several lines of evidence support the concept that
grain fortification with folate is an important modi-
fier.25,26,43 Our analysis showed a significant reduction
in homocysteine concentrations and the risk of stroke in
regions without folate fortification. This finding is likely
attributable to the higher sensitivity to folic acid in
populations in countries where grain is not fortified.
Several genetic studies have shown that the MTHFR
677 C-T polymorphism is related to the elevated
homocysteine levels and increases the risk of stroke.44-46
A recent meta-analysis suggested the latent role of folic
acid status in modulating the relevance between MTHFR
677 C-T polymorphism and the risk of stroke, that is, low

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TABLE 4. Effect of folic acid supplementation on the risk of stroke in prespecified subgroups.

Stroke events/total patients

Subgroups Intervention group Control group Relative risk (95% CI) I2heterogeneity Pdifference

Homocysteine lowering
o25% 508/10,881 494/10,898 1.03 [0.91-1.16] 0 0.03
Z25% 569/10,881 617/9,187 0.85 [0.74-0.97] 11
Intervention regimen
FA only 290/10,648 367/10,647 0.79 [0.68-0.92] 0 0.13
FA þ B vitamins 755/13,725 834/13,761 0.91 [0.82-1.00] 41
Daily folic acid dose
o2 mg 388/14,682 449/12,995 0.78 [0.68-0.89] 0 0.01
Z2 mg 971/17,428 1017/17,444 0.96 [0.88-1.04] 35
Daily Vit B12 dose
o0.5 mg 250/5,861 230/4,201 0.93 [0.78-1.10] 34 0.90
Z0.5 mg 827/15,901 881/15,884 0.94 [0.86-1.03] 35
Baseline Vit B12 level
o300 pmol/L 580/17,303 668/17,304 0.87 [0.78-0.97] 58 0.84
Z300 pmol/L 520/8,719 562/7,782 0.88 [0.79-0.99] 0
Grain fortification
Yes 231/4,548 217/4,574 1.07 [0.90-1.29] 0 0.04
No 1,128/27,562 1,249/25,865 0.87 [0.81-0.94] 21
History of stroke
Yes 512/5,916 536/5,928 0.96 [0.85-1.07] 0 0.21
No 847/26,194 930/24,511 0.87 [0.79-0.95] 35
Duration of intervention
o48 months 598/9,768 594/8,086 0.94 [0.84-1.05] 0 0.32
Z48 months 762/22,482 872/22,496 0.87 [0.79-0.96] 63
FA, folic acid.

folic acid intake or concentrations could enhance the effect
of the MTHFR 677 C-T variant, leading to an increased
risk of stroke.47 RCTs included in our analysis were
primarily conducted in Europe and North America where
the level of folate supplementation in the population was
increasing, or grain fortification had been implemented for
several years. The China Stroke Primary Prevention Trial
(CSPPT) was the first large-scale trial of folic acid inter-
vention in low folate regions with the stratification
by MTHFR C677T genotype.48 It clearly showed that
the highest risk of stroke and the greatest benefit of folic
FIGURE 3. Funnel plot of all studies in the meta-analysis.
acid therapy were in those with the lowest baseline folate
levels.
Some criticism has been raised on the efficacy of
folic acid supplementation in the primary prevention of
stroke and with longer intervention duration. We failed to
find the benefit of folic acid supplementation in reducing
the risk of stroke in individuals without a history of stroke
and the benefit of a longer intervention duration.
However, the CSPPT trial denoted that folic acid
intervention was effective in primary prevention of
stroke. Despite the early termination of the trial owing
to a significant efficacy difference, the treatment dura-
tion of the CSPPT trial was no less than 4 years. It is of
note that the percentage of antiplatelet and lipid-
lowering drugs used are relatively high in most included
RCTs, whereas use was very low in the CSPPT trial in
which participants only had hypertension. In the post
hoc subanalysis of the HOPE2 trial and VITATOPS trial,
both Saposnik et al49 and Hankey et al50 showed a
greater benefit on the risk of stroke in individuals not
receiving antiplatelet agent. Also, a recent meta-analysis
showed that the use of statins could affect the efficacy
of folic acid supplementation as well.43 Therefore, anti-
platelet agents and lipid-lowering drugs may be 2 other
potential modifiers.
There are inherent limitations of our meta-analyses.
First, a meta-analysis, which is a study with rigorous

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Tian et al
methodological criteria and comprehensive analysis, is
incapable of adjusting for individual variables. Despite
realizing the possible interaction between antiplatelet
agents or lipid-lowering drugs and the effect of folic acid
supplementation on the risk of stroke, we could not
adjust for use of antiplatelet agents and lipid-lowering
drugs. Second, heterogeneity is an inevitable issue in
meta-analyses. Characteristics of participants, pre-
existing treatment, the intervention regimen, the duration
of intervention, clinical outcome, and other design
features are sources of heterogeneous generation. Third,
potential publication bias existed even if we adopted the
funnel plot test to assess this bias in our analysis.
CONCLUSIONS
In conclusion, our meta-analysis indicates a signifi-
cant benefit of folic acid supplementation in stroke
prevention in individuals with CVD. Furthermore, this
benefitial effect was modified by the degree of decrease
in homocysteine concentrations, daily folic acid dosage
and folate fortification. Future studies should be con-
ducted in more areas with a low folate level which are
similar to China. Also, the interaction between folic acid
supplementation and interventions modifying other car-
diovascular risk factors needs further investigation.
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From the Department of Cardiology (TT, K-QY, J-GC, X-LZ), Fuwai
Hospital, National Center for Cardiovascular Disease, Chinese Academy of
Medical Sciences, Peking Union Medical College, Beijing, China; and
Department of Hematology and Oncology (L-LZ), The Second People's
Hospital of Hefei, Hefei, Anhui, China.
The first 3 authors (TT, K-QY, and JGC) contributed equally to this
work.
Submitted March 23, 2017; accepted May 31, 2017.
The authors have no conflicts of interest to disclose.
Source of funding: This work was supported by grants from Beijing
Outstanding Talent Training Project (No. 2016000020124G066), Peking
Union Medical College Graduate Student Innovation Fund (No. 10023-
1002-1001), CAMS Innovation Fund for Medical Sciences (2016-I2M-1-
002) and the National Key Research and Development Program of China
(No. 2016YFC1300100).
Correspondence: Xian-Liang Zhou, MD, PhD, Department of Cardiology,
Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese
Academy of Medical Sciences, Peking Union Medical College, No. 167,
Beilishi Road, Beijing 100037, China (E-mail: zhouxianliang0326@hotmail.
com).
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