Clinical Science (2004) 107, 477–484 (Printed in Great Britain) 477

B-group vitamin supplementation mitigates

oxidative damage after acute ischaemic stroke

Rajesh ULLEGADDI∗ , Hilary J. POWERS† and Salah E. GARIBALLA∗

∗
Sheffield Institute for Nutritional Studies on Ageing, The University of Sheffield, Northern General Hospital, Sheffield S5 7AU,
U.K., and †Human Nutrition Unit, The University of Sheffield, Northern General Hospital, Sheffield S5 7AU, U.K.

A B S T R A C T

Evidence shows that there is a rapid increase in the production of markers of oxidative damage
immediately following acute stroke and that endogenous antioxidant defences are rapidly depleted,
thus permitting further tissue damage. Several studies point to an antioxidant effect of B-group
vitamins and a pro-oxidant effect of elevated plasma tHcy (total homocysteine). In the present
study, we assessed whether supplementary B-group vitamins during this critical period will enhance
antioxidant capacity and mitigate oxidative damage. Forty-eight patients with acute ischaemic
stroke within 12 h of symptom onset were assigned to receive daily oral supplements of B-group
vitamins comprising 5 mg of folate, 5 mg of vitamin B2 , 50 mg of vitamin B6 and 0.4 mg of vitamin
B12 (n = 24) or no supplements (n = 24) for 14 days. The treatment group and controls were
matched for stroke subtype and age. Blood samples were obtained before intervention and also at 7
and 14 days post-recruitment for measurement of the following biomarkers: red cell folate (whole
blood folate corrected with haematocrit), erythrocyte glutathione reductase activity coefficient
(EGRAC; measure of vitamin B2 status), plasma pyridoxal phosphate (vitamin B6 status), plasma
vitamin B12 , plasma α-tocopherol, plasma ascorbic acid, plasma TAOC (total antioxidant capacity),
plasma MDA (malondialdehyde), plasma tHcy and CRP (C-reactive protein). Supplementation
for 14 days with B-group vitamins significantly increased the plasma concentrations of pyridoxal
phosphate and red blood cell folate and improved a measure of B2 status compared with the
control group (P < 0.05). Plasma tHcy decreased in both groups albeit less in the control group, but
differences in cumulative changes were not significant. There was, however, a decrease in plasma
MDA concentration in the treatment group, in contrast with the increase seen in the control
group and these differences were significant (P = 0.05). CRP concentration, a marker of tissue
inflammation, was significantly lower in the treatment group compared with controls (P < 0.05). In
conclusion, B-group vitamin supplementation immediately post-infarct may have antioxidant and
anti-inflammatory effects in stroke disease independent of a homocysteine-lowering effect.

INTRODUCTION among Western people living in their own homes [2].

Stroke is the third most common cause of death in most
Western populations after coronary heart disease and
cancer [1]. The majority of strokes are not fatal, and the
major burden is long-term disability. It is therefore
the most important single cause of severe disability
Stroke in the developing world is less well docu-
mented. A statement from the Asia–Pacific Consensus
Forum on Stroke Management predicts that ‘In the next
30 years or so the burden of stroke will grow most
in developing countries rather than in the developed
world’ [3].

Key words: B-group vitamin, C-reactive protein, homocysteine, ischaemia, stroke, malondialdehyde, total antioxidant capacity.
Abbreviations: CRP, C-reactive protein; CV, coefficient of variation; EGRAC, erythrocyte glutathione reductase activity coefficient;
LACI, lacunar infarct; LDL, low-density lipoprotein; MDA, malondialdehyde; PACI, partial anterior circulation infarct; POCI,
posterior circulation infarct; TACI, total anterior circulation infarct; TAOC, total antioxidant capacity; tHcy, total homocysteine.
Correspondence: Dr Salah E. Gariballa (email s.e.gariballa@sheffield.ac.uk).


C 2004 The Biochemical Society
478 R. Ullegaddi, H. J. Powers and S. E. Gariballa
There is strong indirect evidence that free radical pro-
duction is an important mechanism of brain injury after
exposure to ischaemia and reperfusion [4]. Lipid perox-
idation with accumulation of both conjugated dienes and
thiobarbiturate-reactive material is consistently found
when cerebral ischaemia is followed by reperfusion [4].
Recent studies in humans have reported an association
between plasma concentration of antioxidants, markers
of oxidative damage and early clinical outcome after acute
ischaemic stroke [5,6].
Elevated levels of plasma tHcy (total homocysteine)
and deficiencies in folate or vitamin B6 or B12 cofactors
have been found in atherosclerotic patients, including
strokes [7]. Several studies suggest that tHcy may have a
pro-oxidant effect and a role in the production of reactive
oxygen species that results in oxidative damage to arterial
endothelial cells [8,9]. Through cofactor roles in homo-
cysteine metabolism, increased intakes of B-vitamins
have homocysteine-lowering effects [9]. The effect is
strongest for folate, but vitamins B6 , B12 and B2 have
all been shown to be independently predictive of plasma
homocysteine. There is also recent evidence suggesting an
acute antioxidant effect of folate independent of its effect
on homocysteine [10,11]. Riboflavin may also have pro-
tective effects independent of homocysteine-lowering;
this vitamin may protect tissues from ischaemia/reper-
fusion injury, probably through an antioxidant effect [12].
We hypothesize that there is a rapid increase in the pro-
duction of markers of oxidative damage following acute
stroke due to the reperfusion event following ischaemia.
Supplementary B-group vitamins during this critical
period will enhance antioxidant capacity and mitigate
oxidative damage. The aim of the present study was,
therefore, to test the effect of supplementary B-group
vitamins during this critical period on plasma markers of
antioxidant capacity, oxidative damage and plasma tHcy
concentration.
METHODS
Subjects
For a 12-month period, all patients admitted to a Uni-
versity Teaching Hospital with a diagnosis of ischaemic
stroke according to the World Health Organization
criteria were identified prospectively. The study was ap-
proved by the Local Health Research Ethics Committee.
After providing informed written consent, 48 patients
with acute ischaemic stroke admitted within 12 h of
symptom onset (excluding those with cerebral or sub-
arachnoid haemorrhage on CT head scan) were randomly
assigned to receive oral supplements of B-group vitamins
comprising 5 mg of folate, 5 mg of vitamin B2 , 50 mg of
vitamin B6 and 0.4 mg of vitamin B12 (n = 24) daily, or no
supplement (n = 24), for 14 days. All patients, however,
received standard medical treatment for acute stroke. No

C 2004 The Biochemical Society
subject received thrombolytic therapy. Stroke patients
with active gastrointestinal disease, severe medical
or psychiatric illness, serum creatinine concentration
> 150 µmol/l, history of gout or acute renal failure,
supplemental vitamins or inability or refusal to give con-
sent were excluded. The treatment group and controls
were matched for age and stroke subtype. The class-
ification of stroke subtypes was based on clinically
identifiable subtypes of cerebral infarction as described
by Bamford et al. [13]. This classification has important
prognostic implications and is useful for planning stroke
treatment trials. The classification is as follows: large
anterior circulation infarcts with both cortical and sub-
cortical involvement [TACI (total anterior circulation
infarcts)]; more restricted and predominantly cortical in-
farcts [PACI (partial anterior circulation infarcts)];
infarcts associated with the vertebro-basilar arterial
territory [POCI (posterior circulation infarcts)]; and
infarcts confined to the territory of the deep perforating
arteries [LACI (lacunar infarcts)].
Protocol
Non-fasting venous blood was obtained before treatment
(within 12 h of stroke onset) and at day 7 and day 14.
Blood was collected in appropriate tubes, the haematocrit
measured and an aliquot of whole blood stored at − 70 ◦ C
with ascorbic acid for the measurement of red blood
cell folate. Following separation from red blood cells,
plasma was stored, with stabilizer where appropriate, at
− 70 ◦ C for analysis within 12 weeks. Red blood cells
were washed, haemolysed and stored at − 70 ◦ C for the
later measurement of vitamin B2 status. All analyses were
performed blind to the identity of the sample. Plasma B12
was measured by competitive immunoassay using direct
chemiluminescence on an ADVIA Centaur system (kit
reference 09544818; Bayer, Newbury, Berkshire, U.K.).
The inter-batch CV (coefficient of variation) was
7.0 %. The automated Ion Capture Assay system (IMX
folate assay; Abbott Laboratories, Abbott Park, IL,
U.S.A.) was utilized to determine the total folate con-
centration of whole blood, and had an inter-batch CV
of 6.1 %. Simultaneously measured haematocrit values
were then used to calculate red cell folate concentra-
tions. Riboflavin status was assessed as the EGRAC
(erythrocyte glutathione reductase activity coefficient),
using the Cobas BioAutoanalyser (Roche Diagnostics,
Indianapolis, IN, U.S.A.) [14], with an inter-batch CV
of 3.4 %. Ratios above 1.4 were considered to reflect
biochemical deficiency of riboflavin. Pyridoxine (vitamin
B6 ) was measured as plasma pyridoxal phosphate by
HPLC [15]; this assay had an inter-batch CV of 8.0 %.
Plasma tHcy was determined by an automated fluor-
escence polarization immunoassay (IMX Hcy assay;
Abbot Laboratories) with an inter-batch CV of 5.4 %.
Plasma α-tocopherol was measured by HPLC [16],
and plasma total ascorbic acid was by a fluorescence assay

automated for the Cobas BioAutoanalyser [17], giving
inter-batch CVs of 4.3 and 9.0 % respectively. TAOC
(total antioxidant capacity) was measured as the ability
of plasma to inhibit free-radical-induced oxidation in an
assay automated for the Cobas BioAutoanalyser, with
an inter-batch CV of 5.0 % [18]. Serum total cholesterol
concentrations, measured by a cholesterol oxidase assay
using the Synchron LX System (kit reference 467825;
Beckman, Fullerton, CA, U.S.A.), were used to adjust
α-tocopherol values. The inter-batch CV of this assay
was 6.0 %. Plasma MDA (malondialdehyde), a marker of
lipid peroxidation, was also measured by HPLC using
fluorescence detection [19], and this had an inter-batch
CV of 9.0 %. CRP (C-reactive protein) concentration, a
marker of acute inflammation in stroke [20] and other
illnesses (normal range, < 6 mg/l), was measured by
a modified latex-enhanced immuno-turbidimetric assay
(Synchron LX System; kit reference 465131; Beckman).
Plasma uric acid concentration was measured by an
enzymic uricase fluorescence method (normal range, 160–
400 µmol/l) on the Synchron LX System (kit reference
442785; Beckman). The inter-batch CVs for these assays
were 6.5 % and 5.0 % respectively.
All patients had demographic and medical data col-
lected at baseline, including history of hypertension,
smoking, alcohol and drug intake, diabetes mellitus
and cardiovascular diseases. Disability at baseline was
assessed using the Barthel score on a 100-point scale
[21]. The Barthel scores 10 functions on a scale 0 (fully
dependent) to 100 (independent).
Allocation of treatment
The method of minimization [22] is an accepted form of
randomization and was chosen because it minimizes any
differences and provides treatment groups very similar for
major prognostic variables. The effect of this procedure
was that the groups were similar with regard to the
chosen variables of interest. In this trial, clinical stroke
subtype (TACI, PACI, LACI and POCI) and age (< 75
and
75 years) were used for minimization.
Sample size calculation
A previous study [23] has shown that daily supplement-
ation with B-group vitamins (5 mg of folate, 50 mg of
vitamin B6 and 0.4 mg of vitamin B12 ) to patients with
recurrent venous thrombosis (19 on supplements com-
pared with 15 on placebo) reduced plasma tHcy concen-
trations by up to 33 %. A sample size of 48 ischaemic
stroke patients (24 treatment group + 24 controls) would
therefore allow the detection of a 30 % difference between
groups in plasma tHcy with 80 % power and type 1 error
probability of  0.05.
Data analysis
Statistical analyses were performed with SPSS software,
version 11.0 (SPSS Inc., Chicago, IL, U.S.A.). Descriptive
Effect of B-group vitamin supplementation following acute ischaemic stroke 479
tests (median and inter-quartile range) were used to
describe the baseline characteristics of the subjects.
Kruskal–Wallis and Mann–Whitney U tests were used to
test between-group differences where appropriate. The
Friedman test was used to test within-group differences.
Forward stepwise multiple regression analysis was per-
formed to determine the predictive importance of baseline
variables (age, stroke subtype, drugs, chronic disease and
CRP) for plasma TAOC, MDA and tHcy. Adjusted
and change in R2 values were then used to determine
the extent to which the TAOC, MDA and tHcy concen-
trations could be explained by individual antioxidants
(vitamin C, vitamin E and uric acid) and B-group vita-
mins, but also other clinical variables included in the
model.
RESULTS
Table 1 shows baseline characteristics of the treatment and
control groups on entry into the study. The two groups
were comparable with respect to clinical stroke subtype,
chronic disease, drug and alcohol intake and time between
stroke onset and randomization. Although there were
some differences between the two groups in age, rate
of atrial fibrillation, disability, blood glucose and serum
creatinine, these differences did not reach statistical
significance, except for blood glucose (P < 0.05). Vitamin
supplements were prescribed in the drug cards of the
patients, and compliance for surviving patients was 100 %
with no treatment failure or withdrawal.
The concentrations of plasma vitamins B6 and B12 , red-
cell folate and EGRAC (vitamin B2 ) values are shown in
Table 2. Supplementation of B-group vitamin for 14 days
significantly increased concentrations of plasma vitamin
B6 and red cell folate and reduced EGRAC (inversely
associated with riboflavin status) in the intervention
group compared with controls. Concentrations of plasma
vitamin B12 increased significantly in both groups over
2 weeks (P < 0.05 for within-group differences), but there
was no significant difference between groups.
Table 3 shows plasma concentrations of ascorbic acid,
α-tocopherol, TAOC and MDA during the study period.
Within-group and between-group differences in plasma
ascorbic acid, α-tocopherol and TAOC were not signi-
ficant. Although plasma TAOC concentrations decreased
in both groups, albeit less so in the treatment group
over the study period, cumulative differences between the
two groups were not statistically significant. However,
the reduction in plasma MDA concentration seen in the
treatment group was in contrast with the increase seen
in the control group and these different responses were
significant. Plasma tHcy concentrations decreased more
in the treatment group than the controls, but difference in
cumulative changes between the two groups was not
significant (Table 4).

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480 R. Ullegaddi, H. J. Powers and S. E. Gariballa

Table 1 Baseline characteristics of the treatment and in the multivariate model, only stroke subtype showed
control stroke groups significant correlation with TAOC at day 7 (Table 5).
Values are medians (inter-quartile range), or numbers (%). ∗ P < 0.05 compared Most of the variance in MDA was explained by age
with control. AF, atrial fibrillation; TIA, transient ischaemic attack. at baseline and α-tocopherol at day 7, but tHcy showed
significant positive correlation with MDA at baseline and
Treatment group Control group
day 14 (Table 6).
n 24 24 We also analysed the effects of B-group vitamins, age,
Age (years) 77 (68–81) 79 (73–84) stroke subtype, tissue inflammation (CRP), drugs and
Sex (males) 15 (63) 15 (63) alcohol on plasma tHcy during the study period using a
Stroke sub-type (n) multivariate model. Folate and age were significant pre-
TACI 6 (25) 6 (25) dictors of tHcy during the study period (Table 7).
PACI 8 (33) 8 (33) Figure 1 shows the CRP profile over a period of
LACI 6 (25) 6 (25) 3 months following the stroke. CRP concentrations were
POCI 4 (17) 4 (17) significantly lower in the treatment group compared with
Smoking history (n) controls (P < 0.05). There were no statistically significant
Never smoked 10 (42) 12 (50) differences in the rate of infective complications (pneu-
Ex-smoker 11 (46) 8 (33) monia, urinary tract infection and septicaemia) or re-
Current smoker 3 (12) 4 (17) infarction between the treatment group and controls
Alcohol intake > 21 units/week (n) 1 (4) 1 (4) during the first 2 weeks post-randomization [two (8 %)
Drugs/patient† (n) 1.9 1.5 and no patients in the treatment group had infective
Previous stroke/TIA (n) 8 (33) 10 (42) complications during the first and second weeks post-
AF on ECG (n) 3 (12) 8 (33) randomization respectively, compared with three (13 %)
Hypertension (n) 8 (33) 9 (38) and one (4 %) in the control group; two [8 %] stroke
Ischaemic heart disease (n) 8 (33) 6 (25) patients in the treatment group had recurrent cerebral
Diabetes mellitus (n) 1 (4) 1 (4) infarction compared with two (8 %) controls].
CRP (mg/l) 6 (6–12.5) 6 (6–9)
Blood glucose (mmol/l) 5.7 (5.3–7.0)∗ 6.7 (5.8–7.6)
Serum creatinine (µmol/l) 88 (71–117) 99 (69–142)
DISCUSSION
Barthel score 40 (16–59) 28 (20–65) Supplementation with B-group vitamins within 12 h
Time from onset of stroke to 8.0 (5.0–12.8) 9.5 (5.0–12.0) post-infarct after ischaemic stroke reduced lipid perox-
randomization (h) idation products (MDA) after 2 weeks. This occurred
† Diuretics, angiotensin-converting-enzyme (ACE) inhibitors, β-blockers, calcium channel in association with a predicted increase in the plasma
blockers, statins, aspirin, warfarin and clopidogrel. concentrations of certain B vitamins. Although tHcy de-
creased more in the treatment group compared with con-
trols, overall there was no significant difference between
In a multivariate analysis, plasma uric acid and red the two groups. Plasma CRP, a marker of tissue
cell folate explained most of the variance in TAOC du- inflammation, was also lower over 90 days in those
ring the study period. Of the other variables included subjects who received B-group vitamin supplements

Table 2 Changes in concentrations of B-group vitamins over the study period in the treatment and control stroke groups
Values are medians (inter-quartile range), or median percentage change over 2 weeks. ∗ P values are for the differences in cumulative changes between groups over
2 weeks.

Group Day Vitamin B12 (pmol/l) Vitamin B6 (nmol/l) Red cell folate (nmol/l) Vitamin B2 EGRAC

Treatment (n = 24) 0 302 (207–417) 23.1 (15.1–37.1) 481 (327–720) 1.33 (1.23–1.61)
7 482 (307–706) 212.4 (143.2–295.7) 603 (413–910) 1.15 (1.11–1.24)
14 404 (293–697) 222.4 (121.1–330.9) 616 (388–902) 1.17 (1.12–1.27)
Percentage change over 2 weeks 46 778 21 9
Control (n = 24) 0 345 (268–481) 31.7 (16.8–45.2) 606 (426–720) 1.28 (1.19–1.37)
7 417 (305–737) 35.5 (22.9–53.1) 590 (403–757) 1.24 (1.18–1.35)
14 452 (270–733) 32.6 (19.5–48.9) 546 (410–676) 1.27 (1.18–1.49)
Percentage change over 2 weeks 16 14 3 2
∗
P value 0.64 < 0.01 < 0.01 < 0.01


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 Effect of B-group vitamin supplementation following acute ischaemic stroke 481

Table 3 Change in plasma ascorbic acid, α-tocopherol, TAOC and MDA over the study period in the treatment and control
stroke groups
Values are medians (inter-quartile range), or median percentage change over 2 weeks. ∗ P values are for the differences in cumulative changes between groups over
2 weeks.

Group Day Ascorbic acid (µmol/l) Adjusted α-tocopherol (µmol/l) TAOC (mmol/l) MDA (µmol/l)

Treatment (n = 24) 0 39.7 (18.2–55.4) 4.68 (3.51–5.62) 1.30 (1.16–1.38) 0.60 (0.49–0.76)
7 28.6 (14.8–49.8) 4.3 (3.76–5.19) 1.25 (1.14–1.38) 0.57 (0.48–0.71)
14 28.0 (22.4–48.1) 4.31 (3.39–5.72) 1.26 (1.17–1.40) 0.53 (0.37–0.77)
Percentage change over 2 weeks 20 6 2 5
Control (n = 24) 0 33.6 (21.3–63.8) 4.40 (3.77–4.98) 1.29 (1.19–1.38) 0.58 (0.49–0.71)
7 30.2 (19.3–46.2) 4.42 (3.72–5.89) 1.24 (1.17–1.41) 0.68 (0.49–0.88)
14 33.6 (19.0–50.4) 4.98 (3.57–5.90) 1.21 (1.07–1.43) 0.70 (0.52–0.95)
Percentage change over 2 weeks 12 5 7 8
∗
P value 0.76 0.22 0.42 0.05

Table 4 Change in plasma tHcy over the study period in the variance in MDA was explained by age, vitamin E
the treatment and control stroke groups and tHcy. Folate status and age were the most significant
Values are medians (inter-quartile range), median change or median percentage independent predictors of tHcy.
change over 2 weeks. Many recent studies have reported an association
between plasma tHcy concentrations and risk of stroke
Group Day tHcy (µmol/l)
independent of other prognostic indicators [7]. Mech-
Treatment (n = 24) 0 16.73 (14.44–19.67) anisms whereby elevated plasma tHcy increases risk of
7 15.28 (12.02–22.19) stroke are not fully understood, but several studies point
14 14.46 (11.90–23.67) to a pro-oxidant effect. High homocysteine levels may
Change in tHcy lead to endothelial damage, affect platelet function and
Day 0–7 − 1.63 (− 4.59–+ 0.05) coagulation factors, and promote LDL (low-density lipo-
Day 0–14 − 2.30 (− 3.52–+ 0.92) protein) oxidation [8,9,24]. Hyperhomocysteinaemia has
Percentage change over 2 weeks 12 been suggested to play a role in the production of re-
active oxygen species, resulting in oxidative damage to ar-
Control (n = 24) 0 16.48 (11.97–24.06)
terial endothelial cells [9,25]. The homocysteine-lowering
7 13.21 (11.26–19.42)
activity of B-group vitamins may therefore be viewed
14 12.82 (10.82–15.56)
as potentially antioxidant in nature. However, folate can
Change in tHcy
evidently behave as a free-radical scavenger, which might
Day 0–7 − 0.91 (− 4.10–+ 1.43)
explain reported beneficial effects of high-dose folate
Day 0–14 − 0.70 (− 3.95–+ 2.66)
on the vasculature [26]. Nakano et al. [10] have shown
Percentage change over 2 weeks 4
recently that folate has a direct protective antioxidant
effect on LDL oxidation in vitro, and others have shown
for 14 days compared with controls. Plasma TAOC protective effects on the endothelium that appear to be
concentrations decreased in both groups and cumulative independent of homocysteine-lowering [11].
differences between the two groups were not statistically Elevated plasma CRP concentration during the acute
significant. Plasma TAOC was significantly accounted phase or recovery in stroke is independently predictive of
for by serum uric acid and red cell folate status. Most of poor long-term outcome [27,28]. We found a significant
Table 5 Multiple regression analysis for treatment and control stroke groups with TAOC as the dependent variable
P < 0.05 is significant.

Day 0 Day 7 Day 14

Variable/independent predictor R2 change P value R2 change P value R2 change P value

α-Tocopherol 0.000 0.882 0.024 0.191 0.010 0.446

Ascorbic acid 0.004 0.572 0.002 0.706 0.017 0.314
Red cell folate 0.024 0.163 0.064 0.031 0.072 0.033
Uric acid 0.312 0.000 0.143 0.001 0.209 0.000
Stroke type 0.014 0.193 0.064 0.016 0.007 0.448


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482 R. Ullegaddi, H. J. Powers and S. E. Gariballa

Table 6 Multiple regression analysis for treatment and control stroke groups with MDA as the dependent variable
P < 0.05 is significant.

Day 0 Day 7 Day 14

Variable/independent predictor R2 change P value R2 change P value R2 change P value

α-Tocopherol 0.002 0.714 0.064 0.031 0.040 0.116

Ascorbic acid 0.010 0.346 0.006 0.521 0.030 0.179
tHcy 0.103 0.002 0.021 0.213 0.140 0.002
Red cell folate 0.028 0.096 0.000 0.926 0.006 0.524
Age 0.071 0.006 0.015 0.296 0.022 0.202

Table 7 Multiple regression analysis for treatment and control stroke groups with tHcy as the dependent variable
P < 0.05 is significant.

Day 0 Day 7 Day 14

Variable/independent predictor R2 change P value R2 change P value R2 change P value

Age 0.087 0.004 0.135 0.001 0.113 0.009

Red cell folate 0.160 0.000 0.140 0.000 0.072 0.031
EGRAC 0.007 0.366 0.022 0.136 0.007 0.507
Vitamin B6 0.032 0.051 0.004 0.520 0.031 0.145
Vitamin B12 0.011 0.254 0.007 0.415 0.009 0.434

effect of the B-group vitamins as a result of their anti-

Figure 1 Median CRP concentration over 3 months in the
treatment and control groups
Values [medians (inter-quartile range)] for the CRP levels in the treatment and
control group are: treatment group (n = 24), 6 (6–12) on day 0, 7 (6–14) on
day 7, 6 (6–12) on day 14 and 6 (6–6) on day 90; control group (n = 24), 6
(6–9) on day 0, 23 (7–108) on day 7, 18 (6–38) on day 14 and 14 (6–72) on
day 90. P < 0.05 for the difference in cumulative change between the groups.
difference in the CRP concentrations between the stroke
patients who received B-group vitamins and the matched
controls within 3 months following the stroke. This dif-
ference could not be explained by the difference in secon-
dary complications such as infections and re-infarction
between the two groups. Although CRP has not been
directly linked to oxidative stress, recent reports suggest
that high CRP concentrations may reflect the degree of
stroke severity correlating with the degree of inflamma-
tion directly consequent to cerebral infarction, under-
lying unstable atherosclerotic lesions and/or secondary
complications of stroke at the time of sampling [20].
Could the lower CRP concentrations in the treatment
group be due to an anti-inflammatory/neuroprotective
oxidant effect, or could it simply be explained by stroke
severity and secondary complications? Although we have
adjusted for some important prognostic variables such as
the nature of cerebral infarction and secondary compli-
cations in our study, it is impossible to exclude other
sub-clinical causes for the differences in CRP concen-
trations. However, Friso et al. [29] have shown in a
population-based study that low plasma pyridoxal 5 -
phosphate status, the active form of vitamin B6 , was asso-
ciated with higher CRP concentrations independently of
homocysteine.
Supplementation induced small, but significant,
changes in certain B-group vitamins, markers of oxidative
damage and tissue inflammation. Although our sample
size was small and larger studies with clinical end points
need to be undertaken to determine the biological and
functional relevance of these changes, recent observ-
ational studies in humans have reported an association
between plasma concentration of antioxidants, markers
of oxidative damage and early functional and clinical
outcome after acute ischaemic stroke [5,6]. We are not
aware of any other studies showing an independent effect
of the other B-group vitamins used in the present study
on plasma CRP. The possible anti-inflammatory and
neuroprotective effect of B-group vitamins immediately
post-infarct will need further exploration.
We have also found that uric acid explained most of the
variance in TAOC during the study period. In a recent
study [30], we have reported reduced plasma concen-
trations of TAOC in patients with ischaemic stroke


C 2004 The Biochemical Society

compared with non-stroke controls and that TAOC was
strongly correlated with serum uric acid concentrations.
Cerebral ischaemia and reperfusion is accompanied by
increased purine metabolism and uric acid formation [4];
however, the formation of uric acid from hypoxanthine,
through the activity of xanthine oxidase, is itself asso-
ciated with the production of the oxygen-centred radical
superoxide. Some investigators have shown that blocking
the activity of this enzyme may improve recovery from
cerebral ischaemia and reperfusion [31]. Further work
is needed to explore the physiological roles of uric acid
following acute ischaemic stroke.
B-group vitamins used in the present study did not
decrease plasma tHcy concentrations as evident from
other studies, although we are not aware of any trial that
has yet measured the effectiveness of B-group vitamins in
lowering tHcy immediately after acute ischaemic stroke.
Woodside et al. [32], for example, carried out a double-
blind, randomized, factorial-design, controlled trial in
which they supplemented 22 men for 8 weeks with
B-group vitamins (1 mg of folate, 7.2 mg of vitamin B6
and 0.02 mg of vitamin B12 ). They found that supp-
lementation with the B-group vitamins significantly re-
duced tHcy concentration by 27.9 % in the men with
mildly elevated homocysteine. In our present study, the
lack of change in tHcy in the supplemented group may
have been due to the shorter period of supplementation.
However, our main objective in the present study was
to evaluate the effect of B-group vitamins on markers of
antioxidant capacity and oxidative damage immediately
post-infarct after acute ischaemic stroke.
Intervention produced a very small difference in vita-
min B12 concentrations with a similar increase seen in the
control group. This lack of difference may have been due
to the relatively modest dose of vitamin B12 used, length
of treatment, tissue inflammation or differences in rate of
absorption between subjects.
In conclusion, we have demonstrated that supplement-
ation with B-group vitamins within 12 h post-infarct
after acute stroke may have antioxidant and anti-inflam-
matory effects independent of their homocysteine-
lowering effect. Although there are many ongoing
prospective controlled intervention trials using folate,
vitamin B12 and vitamin B6 as homocysteine-lowering
agents in vascular disease, there is a need to explore further
the potential antioxidant effects of B-group vitamins
and to test the potential synergistic interactions between
B-group vitamins and conventional antioxidants in acute
stroke patients immediately post-infarct.
ACKNOWLEDGMENTS
This work was supported by a grant from Sheffield
Teaching Hospital NHS Trust.
Effect of B-group vitamin supplementation following acute ischaemic stroke 483
REFERENCES
1 Warlow, C. P., Dennis, M. S., van Gijn, J. et al. (1996)
Stroke: a Practical Guide to Management, Blackwell
Science, Oxford
2 Martin, J., Meltzer, H. and Elliot, D. (1998) OPCS survey
of disability in Great Britain. Report I: the Prevalence of
Disability Among Adults, HMSO, London
3 Poungvarin, N. (1998) Stroke in the developing world
Lancet 352 (Suppl. 3), SIII19–SIII22
4 Traystman, R. J., Kirsch, J. R. and Koehler, R. C. (1991)
Oxygen radical mechanisms of brain injury following
ischemia and reperfusion. J. Appl. Physiol. 71,
1185–1195
5 Leinonen, J. S., Ahonen, J. P., Lonnrot, K. et al. (2000) Low
plasma antioxidant activity is associated with high lesion
volume and neurological impairment in stroke. Stroke 31,
33–39
6 Cherubini, A., Polidori, M. C., Bregnocchi, M. et al. (2000)
Antioxidant profile and early outcome in stroke patients.
Stroke 31, 2295–2300
7 Perry, I. J., Refsum, H., Morris, R. W., Ebrahim, S. B.,
Ueland, P. M. and Shaper, A. G. (1995) Prospective study
of serum total homocysteine concentration and risk of
stroke in middle-aged British men. Lancet 346,
1395–1398
8 Heinecke, J. W., Kawamura, M., Suzuki, L. and Chait, A.
(1993) Oxidation of low density lipoprotein by thiols:
superoxide-dependent and -independent mechanisms.
J. Lipid Res. 34, 2051–2061
9 Hankey, G. J. and Eikelboom, J. W. (1999) Homocysteine
and vascular disease. Lancet 354, 407–413
10 Nakano, E., Higgins, J. A. and Powers, H. J. (2001) Folate
protects against oxidative modification of human LDL.
Br. J. Nutr. 86, 637–639
11 Doshi, S. N., McDowell, I. F., Moat, S. J. et al. (2001)
Folate improves endothelial function in coronary artery
disease: an effect mediated by reduction of intracellular
superoxide? Arterioscler., Thromb., Vasc. Biol. 21,
1196–1202
12 Mack, C. P., Hultquist, D. E. and Shlafer, M. (1995)
Myocardial flavin reductase and riboflavin: a potential role
in decreasing reoxygenation injury. Biochem. Biophys.
Res. Commun. 212, 35–40
13 Bamford, J., Sandercock, P., Dennis, M., Burn, J. and
Warlow, C. (1991) Classification and natural history of
clinically identifiable subtypes of cerebral infarction.
Lancet 337, 1521–1526
14 Powers, H. J., Bates, C. J. and Duerden, J. M. (1983)
Effects of riboflavin deficiency in rats on some aspects
of iron metabolism. Int. J. Vitam. Nutr. Res. 53,
371–376
15 Bates, C. J., Pentieva, K. D., Prentice, A., Mansoor, M. A.
and Finch, S. (1999) Plasma pyridoxal phosphate and
pyridoxic acid and their relationship to plasma
homocysteine in a representative sample of British men
and women aged 65 years and over. Br. J. Nutr. 81,
191–201
16 Thurnham, D. I., Smith, E. and Flora, P. S. (1988)
Concurrent liquid-chromatographic assay of retinol,
α-tocopherol, β-carotene, α-carotene, lycopene, and
β-cryptoxanthin in plasma, with tocopherol acetate as
internal standard. Clin. Chem. 34, 377–381
17 Vuilleumier, J. P. and Keck, E. (1989) Fluorometric assay of
vitamin C in biological materials using a centrifugal
analyser with fluorescence attachment. J. Micronutr. Anal.
5, 25–34
18 Miller, N. J., Rice-Evans, C., Davies, M. J., Gopinathan, V.
and Milner, A. (1993) A novel method for measuring
antioxidant capacity and its application to monitoring
the antioxidant status in premature neonates. Clin. Sci. 84,
407–412
19 Wong, S. H., Knight, J. A., Hopfer, S. M., Zaharia, O.,
Leach, Jr, C. N. and Sunderman, Jr, F. W. (1987)
Lipoperoxides in plasma as measured by liquid-
chromatographic separation of malondialdehyde-
thiobarbituric acid adduct. Clin. Chem. 33, 214–220

C 2004 The Biochemical Society
484 R. Ullegaddi, H. J. Powers and S. E. Gariballa
20 Di Napoli, M., Papa, F. and Bocola, V. (2001) Prognostic
influence of increased C-reactive protein and fibrinogen
levels in ischemic stroke. Stroke 32, 133–138
21 (1992) Standardised assessment scales for older people. A
report of joint workshop, pp. 1–27, The Royal College of
Physicians of London and The British Geriatrics Society,
London
22 Pocock, S. J. (1988) Clinical Trials: A practical Approach,
John Wiley and Sons, Chichester
23 den Heijer, M., Brouwer, I. A., Bos, G. M. et al. (1998)
Vitamin supplementation reduces blood homocysteine
levels: a controlled trial in patients with venous thrombosis
and healthy volunteers. Arterioscler., Thromb., Vasc. Biol.
18, 356–361
24 Harker, L. A., Harlan, J. M. and Ross, R. (1983) Effect of
sulfinpyrazone on homocysteine-induced endothelial
injury and arteriosclerosis in baboons. Circ. Res. 53,
731–739
25 El Kossi, M. M. and Zakhary, M. M. (2000) Oxidative
stress in the context of acute cerebrovascular stroke.
Stroke 31, 1889–1892
26 Wilmink, H. W., Stroes, E. S., Erkelens, W. D. et al. (2000)
Influence of folic acid on postprandial endothelial
dysfunction. Arterioscler., Thromb., Vasc. Biol. 20, 185–188
Received 30 April 2004/21 July 2004; accepted 28 July 2004
Published as Immediate Publication 28 July 2004, DOI 10.1042/CS20040134

C 2004 The Biochemical Society
27 Winbeck, K., Poppert, H., Etgen, T., Conrad, B. and
Sander, D. (2002) Prognostic relevance of early serial
C-reactive protein measurements after first ischemic
stroke. Stroke 33, 2459–2464
28 Muir, K. W., Weir, C. J., Alwan, W., Squire, I. B. and Lees,
K. R. (1999) C-reactive protein and outcome after ischemic
stroke. Stroke 30, 981–985
29 Friso, S., Jacques, P. F., Wilson, P. W., Rosenberg, I. H. and
Selhub, J. (2001) Low circulating vitamin B6 is associated
with elevation of the inflammation marker C-reactive
protein independently of plasma homocysteine levels.
Circulation 103, 2788–2791
30 Gariballa, S. E., Hutchin, T. P. and Sinclair, A. J. (2002)
Antioxidant capacity after acute ischaemic stroke.
Q. J. Med. 95, 685–690
31 Patt, A., Harken, A. H., Burton, L. K. et al. (1988)
Xanthine oxidase-derived hydrogen peroxide contributes
to ischemia reperfusion-induced edema in gerbil brains.
J. Clin. Invest. 81, 1556–1562
32 Woodside, J. V., Yarnell, J. W., McMaster, D. et al. (1998)
Effect of B-group vitamins and antioxidant vitamins on
hyperhomocysteinemia: a double-blind, randomized,
factorial-design, controlled trial. Am. J. Clin. Nutr. 67,
858–866