Biodrugs 2006; 20 (5): 271-273

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The Pro-Oxidant Activity of High-Dose Vitamin

E Supplements in Vivo
Philip Pearson,1 Sarah A. Lewis,1 John Britton,2 Ian S. Young3 and Andrew Fogarty2
1 Division of Respiratory Medicine, University of Nottingham, Nottingham City Hospital, Nottingham, UK
2 Division of Epidemiology and Public Health, University of Nottingham, Nottingham City Hospital, Nottingham, UK
3 Queen’s University Belfast, Welcome Research Laboratories, Royal Victoria Hospital, Belfast, UK

Abstract Background: A recent meta-analysis has demonstrated that the regular administration of high-dose vitamin E
supplements may be associated with increased mortality. The biological mechanism for this effect is uncertain.
Methods: A ferrous oxidation xylenol assay was used to assess plasma oxidation activity levels in samples from
a randomized, placebo-controlled, 6-week trial of daily vitamin E supplementation in adults with asthma
(n = 72).
Results and conclusion: A 27% increase in plasma oxidation activity levels was observed in patients receiving
vitamin E. We demonstrate a pro-oxidant effect of high-dose vitamin E supplementation that may explain the
increase in mortality observed in intervention studies using this nutrient.

In the US, the dietary antioxidant vitamin E (α-tocopherol) is
taken daily by 11% of adults,[1] while 37% of adults reported
taking it in the previous month.[2] A recent meta-analysis of the
dose-response relationship between vitamin E supplementation
and total mortality demonstrated an increase in mortality with
high-dose vitamin E supplementation (defined as >400IU vitamin
E per day).[3] A more detailed dose-response analysis suggested
that this increased mortality may be observed with as little as
150IU vitamin E per day. Long-term vitamin E supplementation
was also associated with higher rates of heart failure and hospital-
izations for heart failure.[4] No single mechanism has been pro-
posed to explain these observations, although it is noted that
high-dose vitamin E may have pro-oxidant effects in in vitro
models.[5] We have used data from a randomized control trial of
the potential therapeutic effect of vitamin E supplementation in
asthma to investigate the effect of vitamin E upon a marker of lipid
peroxidation in vivo, ferrous oxidation xylenol (FOX).
Methods
We recruited 72 participants aged 18–60 years who had a
current doctor’s diagnosis of asthma and were taking regular
inhaled corticosteroids. As part of a study of vitamin E’s effect on
asthma reported in more detail elsewhere,[6] participants were
randomized to receive either two capsules of vitamin E (250mg
natural vitamin E [d-α-tocopherol] in soya bean oil [R P Scherer
Ltd, Swindon, UK], equivalent to 372IU vitamin E per capsule)
[n = 35] or two identical placebo capsules (gelatina base) [n = 36]
per day for 6 weeks. The baseline characteristics of the study
population are reported in the original paper[6] and patients in the
two limbs of the study were comparable in terms of age and sex. A
baseline blood sample was requested at entry into the study and a
second sample was requested at the end of the study after 6 weeks
of supplements. Lipid peroxidation was assessed in the baseline
and 6-week samples using the FOX assay.[7,8]
Statistical Analysis
FOX measurements were log transformed to achieve normality;
FOX values were then compared between the vitamin E and
placebo groups at 6 weeks by analysis of covariance with adjust-
ment for baseline FOX level. Our primary analysis was by inten-
tion to treat, presuming no change from baseline for those who did
not provide a FOX measurement at 6 weeks. We also carried out a
per-protocol analysis, excluding those who withdrew and also
those who changed asthma medication during the course of the
treatment period. Statistical analysis was carried out with SPSS
version 12 (SPSS Inc., Chicago, IL, USA).
272
Results
Of the 72 randomized subjects, 71 (35 randomized to vitamin E
and 36 randomized to placebo) provided a baseline FOX sample
and were eligible for inclusion in the present intention-to-treat
analysis. A total of 68 patients completed the supplementation
period of the study and provided FOX measurements at 6 weeks;
the remaining three (two allocated to vitamin E, one to placebo)
did not provide a serum sample for FOX analysis at 6 weeks and
were, therefore, allocated a FOX value at 6 weeks equivalent to
their baseline value for intention-to-treat analysis. Sixty two par-
ticipants provided paired samples with no change in their asthma
medication and were included in the per-protocol analysis.
The treatment and placebo groups were similar at baseline in
terms of age, lung function, bronchial reactivity, asthma symp-
toms, vitamin E intake, and plasma vitamin E. Baseline FOX
levels were also similar at baseline (geometric mean 0.86 μmol/L
and 0.88 μmol/L for vitamin E and placebo groups, respectively).
In the intention-to-treat analysis, FOX values at 6 weeks were
significantly greater for those receiving vitamin E (geometric
mean 0.97μmol/L) than for those receiving placebo (geometric
mean 0.77 μmol/L); geometric mean for the ratio vitamin
E : placebo adjusted for baseline was 1.27 (95% CI 1.05, 1.54;
p = 0.02) equating to a 27% increase of FOX with vitamin E
compared with placebo. The results of the per-protocol analysis
were virtually identical.
Discussion
We have demonstrated that 6 weeks of supplementation with
natural vitamin E 745IU has a pro-oxidant activity as assessed by
the FOX assay, a marker of initial fatty acid oxidation.[7]
It is unlikely that the increase in plasma oxidation activity is
confounded by selective withdrawals from the study as the find-
ings were observed with intention-to-treat analysis, in which we
assigned baseline values for those who did not give a second
sample for analysis. The change in plasma levels of vitamin E
(reported elsewhere[6]) confirm that adherence to the allocated
supplement was good; the p-value (0.02) for this relationship
suggests that, although a secondary analysis, it is relatively unlike-
ly that this observation occurred by chance. The FOX assays were
measured blind to vitamin E or placebo status and, hence, mea-
surement bias will not have influenced our findings. The FOX
assay measures the total plasma lipid hydroperoxides (the sum of
cholesterol, triacylglycerol, and phospholipid hydroperoxides)[9,10]
and has been used as a marker of oxidation in studies of insulin
resistance[9] and Alzheimer disease.[11] The use of a single marker
of a diffuse entity such as oxidative stress is a limitation of these
data and further investigations in this area should consider measur-
© 2006 Adis Data Information BV. All rights reserved.
Pearson et al.
ing the effect of vitamin E on a broad range of markers including
additional markers of lipid peroxidation (e.g. F2 isoprostanes, and
oxidation of proteins and DNA).
The demonstration that supplementation with vitamin E 745IU
for 6 weeks has a pro-oxidant effect, as assessed by the FOX assay,
is an important finding in the light of a recent meta-analysis of the
dose-response relationship between vitamin E supplementation
and total mortality. This demonstrated an increased mortality with
high-dose vitamin E supplementation,[3] perhaps with as little as
150 IU/day. One of the hypothesized mechanisms put forward by
Miller et al.[3] is that vitamin E supplementation has a pro-oxidant
effect. Our findings are supportive of an increase in in vivo pro-
oxidant activity with high-dose vitamin E supplementation. This is
considered biologically plausible as an explanation for the in-
creased mortality,[12] with one postulated mechanism involving the
suppression of plasma γ-tocopherol and a reduction of the as yet
poorly understood benefits of this isomer of vitamin E.[13]
We are unaware of previous placebo-controlled studies that
have studied the effect of high-dose oral vitamin E supplementa-
tion on lipid hydroperoxides as measured by the FOX assay. The
initial studies of the effect of vitamin E on low-density lipoprotein
(LDL) oxidation were promising but small, suggesting vitamin E
supplementation decreased susceptibility to LDL oxidation,[14,15]
with an effect seen in men but not post-menopausal women.[16] In a
dose-response study with a maximum daily dosage of vitamin E
2000IU using the lipid peroxidation markers of urinary 4-hydroxy-
nonenal and two urinary isoprostanes, no antioxidant effect was
seen after 8 weeks of vitamin E supplementation, although this
lack of effect may have been a consequence of the small numbers
(n = 5) who received each dose of vitamin E supplementation.[17] A
placebo-controlled study using vitamin E 500IU in 56 patients
with congestive heart failure also demonstrated no effect on mark-
ers of oxidative activity including exhaled pentane and ethane,
plasma malondialdehyde, and F2 isoprostanes after 12 weeks
supplementation.[18] In addition, no effects on plasma malondi-
aldehyde were seen in 25 patients with alcoholic hepatitis receiv-
ing vitamin E 1000IU for 1 year[19] or in hemodialysis patients
receiving vitamin E 840IU for >3 years.[20] The discrepancy be-
tween these studies and our observation of increased plasma
oxidation activity may be because of our choice of study popula-
tion, which consisted of relatively healthy adults with mild to
moderate asthma. Alternatively, the difference may be because of
our use of the FOX assay, which measures lipid hydroperoxide
moieties that are considered an initial event in the oxidative
degradation of fatty acid molecules, and the assay may be a more
sensitive outcome measure for early changes in oxidation status.[7]
The finding that high-dose natural-source vitamin E supple-
mentation has a pro-oxidant effect in vivo is analogous to previous
Biodrugs 2006; 20 (5)
The Pro-Oxidant Activity of High-Dose Vitamin E Supplements in Vivo
observations for vitamin C[21] and consistent with observations of
pro-oxidant activity of high-dose vitamin E exposures in vitro.[5,22]
This observation provides cause for concern, particularly as in-
creased oxidative activity is considered a potential contributor to
the degenerative diseases of aging such as cancer, heart disease,
cataracts, and brain dysfunction.[12] A pro-oxidant effect of high-
dose vitamin E may explain the increased mortality observed in
adults taking high-dose vitamin E supplements for >1 year,[3] the
failure of vitamin E supplementation to impact on cardiovascular
outcomes measures,[23] and possibly the increased rate of heart
failure in those at high-risk of cardiovascular events.[4] This sug-
gests that the current recommendations for the maximum vitamin
E dosage of 1000 mg/day of any form of supplementary α-
tocopherol (corresponding to synthetic vitamin E 1100I U/day or
natural vitamin E 1500 IU/day) should be reviewed in the light of
the new data.[24]
Acknowledgments
The authors contributed equally to this work. This article was funded by
Asthma UK. The authors have no conflicts of interest relevant to the publica-
tion of this article.
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Correspondence and offprints: Dr Andrew Fogarty, Division of Epidemiolo-
gy and Public Health, University of Nottingham, Nottingham City Hospi-
tal, Clinical Science Building, Hucknall Rd, Nottingham, NG5 1PB, UK.
E-mail: andrew.fogarty@nottingham.ac.uk
Biodrugs 2006; 20 (5)