Vol. 104, Issue 20 | October 17, 2012
Advance Access publication October 12, 2012
1562 Articles | JNCI
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effect of combined Folic Acid, Vitamin B6, and Vitamin B12 on
Yiqing Song, JoAnn E. Manson, I-Min Lee, Nancy R. Cook, Ligi Paul, Jacob Selhub, Edward Giovannucci, Shumin M. Zhang
Manuscript received February 10, 2012; revised July 25, 2012; accepted July 26, 2012.
Correspondence to: Shumin M. Zhang, MD, ScD, Division of Preventive Medicine, Brigham and Women’s Hospital, 900 Commonwealth Avenue East,
Boston, MA 02215 (e-mail: firstname.lastname@example.org).
Background Folic acid, vitamin B6, and vitamin B12 act in concert in the one-carbon metabolism and may protect against
colorectal neoplasia. We examined the effect of combined B-vitamin treatment on the occurrence of colorectal
Methods The Women’s Antioxidant and Folic Acid Cardiovascular Study was a randomized, double-blind, placebo-con-
trolled trial of 5442 female health professionals at high risk for cardiovascular disease from April 1998 through
July 2005. Participants were randomly assigned to receive a combination pill of folic acid (2.5 mg), vitamin B6
(50 mg), and vitamin B12 (1 mg) or placebo. This study included 1470 participants who were followed up for as
long as 9.2 years and underwent an endoscopy at any point during follow-up. We estimated relative risks using a
generalized linear model with a natural logarithm link function and Poisson distributed errors. All statistical tests
Results The risk of colorectal adenoma was similar among participants receiving treatment (24.3%, 180 of 741 partici-
pants) vs placebo (24.0%, 175 of 729 participants) (multivariable adjusted relative risk = 1.00, 95% confidence
interval = 0.83 to 1.20). Treatment was not associated with the risk of adenoma when data were analyzed by sub-
site, size, stage, and the number of adenomas. There was no statistically significant effect modification by alcohol
intake, history of cancer or adenoma, or baseline plasma levels or intakes of folate, vitamin B6, or vitamin B12.
Conclusion Our results indicate no statistically significant effect of combined folic acid, vitamin B6, and vitamin B12 treatment
on colorectal adenoma among women at high risk for cardiovascular disease.
J Natl Cancer Inst 2012;104:1562–1575
Folate, vitamin B6, and vitamin B12 are essential cofactors that play
important roles in one-carbon metabolism, which is required for
the maintenance of intracellular DNA synthesis and methylation.
Data from both in vitro and animal studies have suggested a pro-
tective effect of B vitamins against colorectal carcinogenesis (1,2),
although the results have been somewhat mixed and the complex
mechanisms have not yet been fully elucidated. Observational evi-
dence, though not entirely consistent, has suggested that blood
levels of folate or vitamin B6 are inversely related to the risk of
colorectal neoplasia. Some, but not all prospective studies have
suggested a 20%–40% reduction in the risk of colorectal cancer or
adenoma in those with the highest intake of folate compared with
those with the lowest intake (1–3). A recent meta-analysis of nine
prospective studies showed a 10% reduction in colorectal cancer in
individuals with the highest vitamin B6 intake compared with those
with the lowest intake (4).
However, most randomized trials have focused on folic acid
supplementation alone and found neither beneficial nor harmful
effects of folic acid supplementation (0.5–5 mg/d for as long as
3 years) on recurrence of colorectal adenomas (1,2,5,6). Mandatory
fortification of cereals and grain products with folic acid in the
United States beginning in the year 1998 has improved folate
status in the general population (7,8), but there has been concern
about limited protective effects or a potential cancer-promoting
effect of folic acid treatment alone in a folic acid–fortified
population. Because folic acid, vitamin B6, and vitamin B12 act in
concert to affect the pathways of one-carbon metabolism (1,2),
combined folic acid, vitamin B6, and vitamin B12 supplements
may have greater promise for preventing colorectal adenoma, the
precursor of invasive cancer, than any single B-vitamin supplement.
Combined B-vitamin supplementation also helps address the
concern about potentially masking vitamin B12 deficiency by folic
acid supplementation alone in adults. Because 28%–35% of the
US population report the use of dietary supplements that contain
folic acid and vitamins B6 and B12 (9,10), definitive data from large
randomized trials with long treatment duration are warranted to
evaluate the potential yet unproven benefits or risks of combined
folic acid, vitamin B6, and vitamin B12 supplementation.
In the Women’s Antioxidant and Folic Acid Cardiovascular
Study (WAFACS), a large cardiovascular disease (CVD) prevention
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JNCI | Articles 1563
trial conducted during the folic acid fortification era, we specifically
examined the effect of daily supplementation with folic acid, vita-
min B6, and vitamin B12 on the occurrence of colorectal adenoma
among participants followed up for as long as 9.2 years (7.3 years
of active treatment and 1.9 years of postintervention follow-up).
Study Design and Population
The WAFACS was a randomized, double-blind, placebo-controlled
trial evaluating the effects of combined folic acid, vitamin B6, and
vitamin B12 in the secondary prevention of CVD among 5442
high-risk women with either a history of CVD or at least three
cardiovascular risk factors (http://clinicaltrials.gov, identifier:
NCT00000541) (11–14). The trial was approved by the institu-
tional review board of Brigham and Women’s Hospital, and all
participants provided written informed consent. An external inde-
pendent data and safety monitoring board monitored the safety of
the participants and the overall quality and scientific integrity of
Details of the WAFACS trial design and the main results have
been reported previously (11–14). Briefly, from June 1995 through
October 1996, 8171 female health professionals in the Women’s
Antioxidant Cardiovascular Study (the parent trial of the WAFACS)
were randomly assigned to receive vitamin C (500 mg/d), vitamin E
(600 IU every other day), and β-carotene (50 mg every other day)
vs respective matching placebos (11–14). Women were eligible
for the Women’s Antioxidant Cardiovascular Study if they were at
least 40 years of age, were postmenopausal, or had no intention of
becoming pregnant, and had a self-reported history of CVD or had
at least three traditional cardiac risk factors (12).
In April 1998, 5442 of these women provided consent and were
additionally randomly assigned in a factorial design to receive a
daily combination pill containing folic acid (2.5 mg), vitamin B6
(pyridoxine hydrochloride, 50 mg), and vitamin B12 (cyanocobala-
min, 1 mg) or a matching placebo to form the WAFACS trial popu-
lation (11,12,14). Our study included 1470 WAFACS participants
who underwent at least one endoscopy after random assignment to
treatment and were followed up through June 30, 2007 (Figure 1).
Following random assignment and annually thereafter, participants
were mailed monthly calendar packs containing active agents or
placebos, along with questionnaires on adherence, use of nonstudy
supplements, and occurrence of major illnesses or adverse events.
At baseline, 98.8% of the participants also answered a semiquan-
titative food-frequency questionnaire to assess dietary nutrient
intakes (11–14). At the scheduled end of the trial (July 31, 2005),
morbidity and mortality follow-up was 92.6% complete. If assessed
in terms of person-time, mortality and morbidity information
was complete for 98.9% and 98.0% of person-years of follow-up,
respectively (11,13,14). The average adherence, defined as taking at
least two-thirds of the study pills over the course of follow-up, was
83% for both the active and placebo groups.
Women in the WAFACS provided a baseline blood sample in
the year 1996 (75.4% of the participants), before folic acid fortifi-
cation in the year 1998 (7,8). Randomly selected from participants
who were adherent with study medications, 300 (150 women in
the active treatment group and 150 women in the placebo group)
provided a blood sample at the end of randomized treatment. As
reported previously (11,14), median plasma folate (8.8 vs 8.9 ng/
mL, P = .94) and homocysteine levels (12.1 vs 12.5 µmol/L, P =
.96) were similar between the active treatment group and the pla-
cebo group at baseline. At the end of trial, the median plasma folate
level in the active group was statistically significantly higher (38.9
vs 15.4 ng/mL, P < .001) and homocysteine was statistically sig-
nificantly lower (9.8 vs 11.8 µmol/L, P < .001) compared with the
Ascertainment of Colorectal Adenoma
Participants in WAFACS who reported a recent endoscopy (colo-
noscopy and/or sigmoidoscopy) or a diagnosis of colon polyps on
the trial questionnaires were mailed a medical release form to get
permission to obtain and review relevant medical records from the
treating physician and/or hospital. An endpoints committee of phy-
sicians who were blinded to the participants’ treatment assignment
reviewed the medical records that included endoscopy and histo-
pathologic reports for final confirmation of a reported colorectal
adenoma diagnosis and/or endoscopy procedure through June 30,
2007. Additional details of polyp characteristics and indications for
endoscopy procedures were also extracted from medical records.
Colorectal polyps were first categorized by types (adenomatous,
hyperplastic) and anatomic location within the large bowel. For
adenomatous polyps, polyp characteristics were captured by the
number, size, histologic type (tubular, tubulovillous, villous), and
grade of dysplasia (low, high). Advanced histology neoplasms were
defined as tubulovillous or villous adenomas, high-grade dysplasia
(carcinoma in situ), or invasive cancer (15). Polyps were also classi-
fied into small (<1 cm in diameter) and large (≥1 cm). An advanced
adenoma was defined as a lesion that had either a large size (≥1 cm)
or advanced histology. A total of 1470 participants who received at
least one endoscopy during April 1998 through June 30, 2007 were
included in this analysis, and of them, 355 had confirmed colorectal
In this study, baseline plasma levels of folate, vitamin B6, vitamin B12,
homocysteine, cysteine, and cysteinylglycine were assayed at the Jean
Mayer US Department of Agriculture Human Nutrition Research
Center on Aging at Tufts University. Plasma folate and vitamin B12
were determined by the IMMULITE 1000 immunoassay system
(Diagnostic Products Corporation, Los Angeles, CA). Plasma levels
of vitamin B6 were determined by an enzymatic procedure using
radioactive tyrosine and the apoenzyme tyrosine decarboxylase (16).
Plasma homocysteine, cysteine, and cysteinylglycine were measured
using high-performance liquid chromatography, with fluorescence
detection (17). The mean coefficients of variation for 22 replicate
quality control plasma samples were 7.5% for folate, 15.9% for
vitamin B6, 6.2% for vitamin B12, 8.3% for homocysteine, 9.7% for
cysteine, and 9.0% for cysteinylglycine.
Baseline characteristics were compared using Wilcoxon rank-sum
test for continuous variables and Mantel–Haenszel χ2 statistics for
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Vol. 104, Issue 20 | October 17, 2012
1574 Articles | JNCI
It seems unlikely that the doses of folic acid, vitamin B6, and vita-
min B12 in our study were insufficient to test the study hypothesis.
However, the improved folate status in the general population from
the mandatory folic acid fortification (7,8) might have limited the
ability of our study to detect an effect. In the WAFACS population
(11), the prefortification prevalences of women having inadequate
levels of folate (<7 ng/mL) were 34.7% and 32.7% in the placebo
and treatment group, respectively. After the termination of the trial,
only 1.33% had folate levels below 7 ng/mL in the placebo group and
no one in the active treatment group had levels this low. Mandatory
folic acid fortification was associated with an almost 30% elevation
in plasma folate levels over a 7-year period (in the placebo group),
but the relative increase in folate levels was greater in the active
treatment group (69%), in which 49.3% of participants had folate
levels greater than 40 ng/mL compared with 4.7% in the placebo
group. Furthermore, a modest but statistically significantly greater
decrease of homocysteine levels was observed in the active treatment
group vs the placebo group (18.5% reduction). The statistically sig-
nificant elevation in plasma folate levels and statistically significant
decrease in homocysteine levels by combined folic acid, vitamin B6,
and vitamin B12 supplementation were not associated with the over-
all risk of total colorectal adenoma and advanced colorectal lesions
in our study, although we cannot exclude the possibility of a modest
effect that might have existed with longer follow-up or if only those
with true B-vitamin deficiency were enrolled and treated.
Alcohol is a known folate “antagonist” that affects dietary methyl
supply. Alcohol intake has been observed to modify the effect of
dietary folate or folic acid supplementation on adenoma risk (1,2).
However, we did not observe a statistically significant decrease in
the risk of adenoma associated with combined folic acid, vitamin
B6, and vitamin B12 treatment in those with higher alcohol intake.
Because of relatively low alcohol intake in this population, we
were only able to look at the interaction with alcohol at 5 g/d or
more and thus cannot exclude the possibility that there might be
an interaction with alcohol at higher levels. In addition, genetic
variations in enzymes involved in one-carbon metabolism may
modulate the effect of combined B-vitamin treatment on colorectal
neoplasia (25–27). Interindividual genetic variability in our study
population is an unlikely explanation for our overall null findings,
however, because genetic factors should have been comparable in
the active treatment and placebo groups because the participants
were randomly assigned to intervention.
The strengths of our study include combined folic acid, vita-
min B6, and vitamin B12 supplementation; randomized interven-
tion assignment; sufficient treatment doses; and longer duration
of treatment and follow-up. Furthermore, baseline plasma levels
of folate, vitamin B6, vitamin B12, homocysteine, cysteine, and
cysteinylglycine in this study were assayed so that effect modifica-
tion by baseline B-vitamin levels could be assessed.
Some limitations of our study also deserve consideration. First,
the use of a combination pill did not allow us to investigate the
effects of individual components or potential interactions among
them in relation to colorectal adenoma. Second, the study popula-
tion may not be representative of the participants in the parent
WAFACS study. However, we found that women included in this
analysis had similar baseline characteristics compared with the
WAFACS participants and when the randomly assigned groups
were compared. Also, the frequencies of endoscopy indications
(screening or symptoms) were similarly distributed between the
two randomly assigned groups in our study population, and there
were no statistically significant effects of combined B-vitamin sup-
plementation on colorectal adenoma risk in the analyses stratified
by indications for endoscopies. Third, confounding by extraneous
risk factors cannot be completely excluded in our post hoc analy-
sis of randomized trial data, although the baseline characteristics
seemed well balanced between the treatment and placebo groups,
as is expected in a large-scale trial with effective randomization.
Finally, our results were based on women at high risk for CVD and
may not be applicable to men or to the general population.
In conclusion, data from this study within a large, randomized,
placebo-controlled trial indicate no apparent benefit or harm of
combined folic acid, vitamin B6, and vitamin B12 supplementation on
the risk of colorectal adenoma among women. Our findings do not
support recommending B-vitamin supplementation for the preven-
tion of colorectal adenomas. Further evidence, ideally from large,
well-designed randomized controlled trials with long-term follow-up
or from large longitudinal studies, is needed to confirm our findings.
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American Cancer Society (RSG-06-263-01-CCE to SMZ); the National
Institutes of Health, Bethesda, MD (HL046959 to JEM, CA123089 to SMZ,
CA104871 to SMZ, and CA124857 to WC Willett and SMZ).
The authors would like to thank Martin Van Denburgh (Brigham and Women’s
Hospital, Harvard Medical School, Boston, MA) for his statistical analytic sup-
port and Dr Kana Wu (Harvard School of Public Health, Boston, MA) for her
statistical assistance. We acknowledge the contributions of the WAFACS Data
Safety and Monitoring Board. We also acknowledge the invaluable contributions
of AraSarkissian, Shari Bassuk, and Elaine Zaharris and other WAFACS staff, as
well as the 5442 dedicated WAFACS participants.
Vitamin E and its placebo were supplied by Cognis Corporation (LaGrange,
IL). All other agents and their placebos were supplied by BASF Corporation
(Mount Olive, NJ). Pill packaging was provided by Cognis and BASF. The
American Cancer Society, National Institutes of Health, Cognis, and BASF
did not provide any input into the design or conduct of the study; collection,
management, analysis, or interpretation of the data; or preparation, review, or
approval of the article.
Affiliations of authors: Division of Preventive Medicine (YS, JEM, I-ML, NRC,
SMZ) and Channing Laboratory (JEM, EG), Brigham and Women’s Hospital
and Harvard Medical School, Boston, MA; Departments of Epidemiology
(JEM, I-ML, NRC, EG) and Nutrition (EG), Harvard School of Public Health,
Boston, MA; and US Department of Agriculture Human Nutrition Research
Center on Aging at Tufts University, Boston, MA (LP , JS).
by guest on December 21, 2015