See corresponding editorial on page 253.
Effects of vitamins C and E and b-carotene on the risk of type 2
diabetes in women at high risk of cardiovascular disease: a randomized
Yiqing Song, Nancy R Cook, Christine M Albert, Martin Van Denburgh, and JoAnn E Manson
Background: Vitamin C, vitamin E, and b-carotene are major anti-
oxidants and as such may protect against the development of type 2
diabetes via reduction of oxidative stress.
Objective: The purpose of this study was to investigate the long-
term effects of supplementation with vitamin C, vitamin E, and
b-carotene for primary prevention of type 2 diabetes.
Design: In the Women’s Antioxidant Cardiovascular Study, a ran-
domized trial that occurred between 1995 and 2005, 8171 female
health professionals aged ?40 y with either a history of cardiovas-
cular disease (CVD) or ?3 CVD risk factors were randomly as-
signed to receive vitamin C (ascorbic acid, 500 mg every day),
vitamin E (RRR-a-tocopherol acetate, 600 IU every other day),
b-carotene (50 mg every other day), or their respective placebos.
Results: During a median follow-up of 9.2 y, a total of 895 incident
cases occurred among 6574 women who were free of diabetes at
baseline. There was a trend toward a modest reduction in diabetes
risk in women assigned to receive vitamin C compared with those
assigned to receive placebo [relative risk (RR): 0.89; 95% CI: 0.78,
1.02; P = 0.09], whereas a trend for a slight elevation in diabetes
risk was observed for vitamin E treatment (RR: 1.13; 95% CI: 0.99,
1.29; P = 0.07). However, neither of these effects reached statistical
significance. No significant effect was observed for b-carotene treat-
ment (RR: 0.97; 95% CI: 0.85, 1.11; P = 0.68).
Conclusion: Our randomized trial data showed no significant over-
all effects of vitamin C, vitamin E, and b-carotene on risk of de-
veloping type 2 diabetes in women at high risk of CVD. This trial
was registered at clinicaltrials.gov as NCT00000541.
Am J Clin
Oxidative stress, which is characterized by excessive pro-
duction of reactive oxygen species and reduction of antioxidant
defense capacity, has been implicated in the pathogenesis of type
2 diabetes and its complications (1–4). Evidence from basic
research and observational studies has suggested that oxidative
stress elicits systemic inflammation (3), promotes endothelial
dysfunction (5), impairs pancreatic b cell insulin secretion (2, 6),
and interferes with glucose disposal in peripheral tissues (2, 3),
thereby accelerating the development and progression of type 2
Vitamin C (ascorbic acid), vitamin E (a-tocopherol), and
b-carotene are considered important antioxidants in humans. In
vitro studies suggest that they protect against free radical–mediated
damage by reducing free oxygen radicals and replenishing an-
tioxidant reserves (7–9). In experimental animal models, anti-
oxidant administration has been shown to delay the onset of
diabetes (10, 11). It has thus been hypothesized that supple-
mentation with antioxidants may help prevent the development
of type 2 diabetes in humans.
Observational epidemiologic studies have shown significant
inverse correlations between antioxidant concentrations and
several biomarkers of insulin resistance or glucose intolerance in
healthy individuals (12). Concentrations of antioxidants in the
blood, such as vitamins C (12–14) and E (15, 16) and b-carotene
(15–17), were also significantly lower in individuals with type 2
diabetes than in nondiabetic control subjects. Evidence from
most previous prospective cohort studies generally supports an
inverse association between incidence of type 2 diabetes and
dietary, serum, or plasma concentrations of vitamins C (18) and
E (19–21) and b-carotene (21, 22) in nondiabetic individuals.
Some (23–25), but not all (26–28), of the short-term randomized
trials in patients with type 2 diabetes also showed the beneficial
effects of oral supplementation of vitamin C or E at high doses
on risk factors linked to insulin resistance and diabetes, in-
cluding oxidative stress, blood pressure (23), lipid metabolism
(24), endothelial function (25), and insulin-mediated glucose
disposal (24). However, few large trials with long treatment
1From the Division of Preventive Medicine (YS, NRC, CMA, MVD, and
JEM) and Cardiology Division (CMA), Brigham and Women’s Hospital,
Harvard Medical School, Boston, MA, and the Department of Epidemiology
(NRC and JEM), Harvard School of Public Health, Boston, MA.
2Supported by investigator-initiated grant HL46959 from the National
Heart, Lung, and Blood Institute. YS is supported by a grant (K01-
DK078846) from the National Institute of Diabetes and Digestive and Kid-
ney Diseases, National Institutes of Health. Vitamin E and its placebo were
supplied by Cognis Corporation (LaGrange, IL). All of the other agents and
their placebos were supplied by BASF Corporation (Mount Olive, NJ). Pill
packaging was provided by Cognis and BASF. Neither Cognis nor BASF
provided any input into the design and conduct of the study; collection,
management, analysis, and interpretation of the data; and preparation, re-
view, or approval of the manuscript.
3Address reprint requests and correspondence to Y Song, Division of
Preventive Medicine, Brigham and Women’s Hospital, 900 Commonwealth
Avenue East, Boston, MA 02215. E-mail: firstname.lastname@example.org.
Received January 15, 2009. Accepted for publication April 21, 2009.
First published online June 2, 2009; doi: 10.3945/ajcn.2009.27491.
Am J Clin Nutr 2009;90:429–37. Printed in USA. ? 2009 American Society for Nutrition
duration have addressed the primary prevention of type 2 di-
abetes. Two previous randomized clinical trials provided no
evidence for significant effects of vitamin E or b-carotene sup-
plementation on the incidence of type 2 diabetes in apparently
healthy women (29) and men (30), respectively. To our knowl-
edge, there are no previous trials examining the efficacy of
vitamin C in preventing type 2 diabetes. It thus remains un-
answered whether vitamins E and C and b-carotene, indepen-
dently or in combination, protect against the development of
type 2 diabetes.
To address this question, we investigated whether long-term
supplementation of vitamins C and E and b-carotene reduces the
incidence of type 2 diabetes in the Women’s Antioxidant Car-
diovascular Study (WACS), a randomized, double-blind, placebo-
controlled trial conducted over a duration of ’9.4 y.
SUBJECTS AND METHODS
The WACS is a randomized, double-blind, placebo-controlled
trial evaluating the effects of vitamin C [500 mg/d synthetic
vitamin C (ascorbic acid); provided by BASF Corporation
(Mount Olive, NJ], vitamin E [600 IU vitamin E (RRR-a-
tocopherol acetate) every other day; provided by Cognis Cor-
poration (La Grange, IL)], and b-carotene (50 mg Lurotin every
other day; provided by BASF Corporation) in the secondary
prevention of important vascular events in a 2 · 2 · 2 factorial
design among women at high risk (either a history of vascular
disease or ?3 cardiovascular risk factors). Details of the design
have been reported previously (31, 32). Briefly, a total of 8171
female health professionals, who were willing, eligible, and
compliant during a 12-wk run-in period, were entered into the
trial from June 1995 through October 1996. They were ?40 y
old, postmenopausal or had no intention of becoming pregnant,
and had either a self-reported history of cardiovascular disease
(CVD), ie, a history of myocardial infarction, stroke, re-
vascularization procedure, angina pectoris, or transient cerebral
ischemia, or ?3 cardiac risk factors. These cardiac risk factors
were self-reported diagnosis of hypertension, high cholesterol
concentration, or diabetes mellitus; parental history of pre-
mature myocardial infarction (MI) before age 60 y; obesity
[body mass index (BMI; in kg/m2) ?30]; and current cigarette
smoking. Women were excluded if they had a self-reported
history of cancer (excluding nonmelanoma skin cancer) within
the past 10 y, had any serious non-CVD illness, or were cur-
rently using warfarin or other anticoagulants. Potential partic-
ipants also had to be willing to forgo individual supplements of
vitamins A, C, and E and b-carotene at amounts beyond the US
Recommended Dietary Allowance during the trial. The trial was
approved by the institutional review board of the Brigham and
Women’s Hospital (Boston, MA) and was monitored by an ex-
ternal data and safety monitoring board. From the 8171 women
enrolled in the WACS trial, we excluded those with prevalent
diabetes at baseline (n = 1597), leaving 6574 nondiabetic
women who were randomly assigned to 8 groups for the present
analyses (Figure 1).
Study treatment and follow-up
for the first year and then annually the women were sent monthly
calendar packs containing active agents or placebos along with
A semiquantitative food-frequency questionnaire at baseline was
used to assess dietary nutrient intake (31, 32). Compliance was
assessed through self-report and was defined as taking at least
two-thirds of study pills. As previously reported (32), mean
compliance over follow-up was ’73% for all active and placebo
agents. In 1999 blood samples were obtained from 30 local
participants to evaluate biomarkers for compliance. Blood con-
centrations were elevated in each active group compared with
the placebo group [ascorbic acid: 1.9 compared with 1.3 mg/dL
(P = 0.007); vitamin E: 20.2 compared with 12.2 lg/mL (P =
0.007); and b-carotene: 54.4 compared with 19.5 lg/mL (P =
0.003)]. Information was also obtained on outside supplements
of study medications for ?4 d/mo (“drop-ins”). Outside use of
vitamin C and vitamin E supplements was ,15% and outside
FIGURE 1. Flow diagram illustrating diabetes outcomes in the randomly assigned treatment components of the Women’s Antioxidant Cardiovascular
Study (WACS). A total of 1597 participants who had a diagnosis of type 2 diabetes at baseline were excluded in the analysis.
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ANTIOXIDANT SUPPLEMENTATION AND DIABETES IN WOMEN