Mol Cell Biochem (2014) 391:211–216
DOI 10.1007/s11010-014-2004-8
Vitamin E does not regress hypercholesterolemia-induced
oxidative stress in heart
Kailash Prasad • Erick D. McNair •
Gudrun Caspar-Bell • A. Mabood Qureshi
Received: 21 November 2013 / Accepted: 21 February 2014 / Published online: 6 March 2014
Ó Springer Science+Business Media New York 2014
Abstract Vitamin E suppresses the hypercholesterolemia-
induced cardiac oxidative stress. The objectives were to
investigate: if vitamin E regresses the hypercholesterolemia-
induced oxidative stress in hearts and if regression is asso-
ciated with decreases in the antioxidant reserve. The rabbits
were assigned to 4 groups: I, regular diet (2-months); II,
0.25 % cholesterol diet (2-months); III, 0.25 % cholesterol
diet (2-months) followed by regular diet (2-months); and IV,
0.25 % cholesterol diet (2-months) followed by regular diet
with vitamin E (2-months). Blood samples were collected
before and at the end of protocol for the measurement of total
cholesterol (TC). Hearts were removed at the end of the
protocol under anesthesia for the assessment of oxidative
stress parameters, malondialdehyde (MDA), and tissue
chemiluminescent (CL) activity. High cholesterol diet
increased the serum levels of TC, and regular diet with or
without vitamin E reduced the TC levels to a similar extent.
The MDA content of the heart in groups I, II, III, and IV were
0.074 ± 0.015, 0.234 ± 0.016, 0.183 ± 0.028 and
0.169 ± 0.016 nmol/mg protein, respectively. Regular diet
following high cholesterol diet reduced the MDA levels
(0.234 ± 0.016 vs. 0.183 ± 0.028 nmol/mg protein but
K. Prasad (&)
Department of Physiology, College of Medicine, University of
Saskatchewan, 105 Wiggins Road, Saskatoon, SK S7N 5E5,
Canada
e-mail: k.prasad@usask.ca
E. D. McNair
A. Mabood Qureshi
Department of Pathology and Laboratory Medicine, University
of Saskatchewan and Royal University Hospital, Saskatoon,
SK, Canada
G. Caspar-Bell
Department of Medicine, University of Saskatchewan and Royal
University Hospital, Saskatoon, SK, Canada
vitamin E did not reduce the MDA levels. The cardiac-CL
activities were similar in groups’ I, II, and III
(30.11 ± 0.7 9 106, 32.9 ± 1.43, and 37.92 ± 8.35 9 106
RLU/mg protein). The activity decreased in group IV, sug-
gesting that vitamin E increased the antioxidant reserve
while lowering serum cholesterol did not increase antioxi-
dant reserve. In conclusion, hypercholesterolemia increases
cardiac oxidative stress and regular diet regresses hyperch-
olesterolemia-induced oxidative stress but vitamin E does
not further regress hypercholesterolemia-induced cardiac
oxidative stress. Vitamin E reduces oxidative stress in the
heart tissue in spite of a decrease in CL activity (increase in
antioxidant reserve).
Keywords Hypercholesterolemic cardiac oxidative
stress
Malondialdehyde
Chemiluminescent activity
(CL)
Vitamin E
Regression
Introduction
Hypercholesterolemia increases the levels of lipid peroxi-
dation product, malondialdehyde (MDA), a measure of
oxidative stress in rabbit [1] rats [2] and Apo B-100
transgenic mice [3]. The increases in MDA could be due to
increases in the production of reactive oxygen species and/
or decreases in antioxidants. Hypercholesterolemia
increases the levels of ROS through various mechanisms
described by Prasad and Kalra [4], and Prasad et al. [5]. A
high cholesterol diet increases cardiac superoxide anion
generation and nicotinamide adenine dinucleotide phos-
phate (NADPH)-oxidase expression [3]. It also increases
the production of superoxide anion from endothelial cells
[6], and polymorphonuclear leukocytes [7]. Increases in the
levels of ROS could also be due to decreases in the levels
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of antioxidants, enzymatic [superoxide dismutase (SOD),
catalase, glutathione peroxidase (GSH-Px)], and non-
enzymatic [reduced glutathione (GSH), vitamins E, and C].
Chemiluminescent activity of tissue is a measure of anti-
oxidant reserve [8]. ROS act on membrane phospholipids
to produce lipid peroxides MDA [9]. ROS have been
implicated in the pathophysiology of the heart failure [10],
ischemic heart disease [11], and ischemia/reperfusion
injury [8]. Vitamin E, a nonenzymatic antioxidant, [12]
suppresses [1] and slows the progression [13] of hyperch-
olesterolemia-induced oxidative stress in the heart. To be
of potential clinical benefit in patients with hypercholes-
terolemia-induced oxidative stress, the drug should be able
to regress the oxidative stress. However, it is not known if
vitamin E regresses established hypercholesterolemia-
induced cardiac oxidative stress. Agents that suppress the
development of atherosclerosis do not necessarily regress
the atherosclerosis. For example, probucol which sup-
presses atherosclerosis [5] and slows the progression of
atherosclerosis [14] does not regress atherosclerosis [14,
15]. Calcium antagonists suppress but do not regress ath-
erosclerosis [16]. Nifedipine has no effect on progression
or regression of atherosclerosis but prevents the develop-
ment of atherosclerosis [17]. However, Thiery et al. [18]
reported that niphedipine regresses atherosclerosis. Amlo-
dipine, another calcium antagonist regresses atherosclero-
sis. It appears that calcium antagonists have variable
effects. The main objectives of the present study were to
investigate if: (a) vitamin E regresses the hypercholester-
olemia-induced oxidative stress in the heart and
(b) regression is associated with alterations in serum total
cholesterol (TC), and increased antioxidant reserve (car-
diac chemiluminescent (CL) activity). An investigation
was, therefore, made of the effects of vitamin E on the
regression of oxidative stress and serum TC in high cho-
lesterol fed rabbits. Oxidative stress parameters were
assessed by measuring cardiac MDA and antioxidant
reserve (CL activity) .
Methods
The studies were conducted on New Zealand white rabbits
weighing between 1.2 and 1.5 kg. After 1 week of adap-
tation on regular rabbit chow diet, the rabbits were
assigned to one of four groups: Group I, (n = 6) regular
rabbit chow diet for 2 months; Group II, (n = 6), 0.25 %
cholesterol diet for 2-months; Group III, (n = 5), 0.25 %
cholesterol diet for 2-months followed by a regular diet for
an additional 2-months; and Group IV (n = 5), 0.25 %
cholesterol diet for 2-months followed by regular diet with
vitamin E (40 mg/kg body weight/day) for an additional
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Mol Cell Biochem (2014) 391:211–216
2 months. The measured amount of vitamin E was laced on
a small piece of lettuce and fed orally. The person in charge
stayed there till the lettuce was eaten by the rabbits to
confirm that vitamin E was ingested. Vitamin E levels were
not measured in the blood. The Department of Agriculture
and Biosource Engineering, University of Saskatchewan
prepared the 0.25 % cholesterol diets, using regular rabbit
chow diet purchased from Purina. Food and water were
provided ad libitum. The rabbits were housed in individual
cages at 20 °C under a 12-h light/12-h dark cycle. The
Ethics Committee of the University of Saskatchewan
approved the experimental protocol, and the animal care
was conducted according to the approved standards for
Laboratory Animal Care. Fasting blood samples from the
ear marginal artery were collected before, and at the end of
the protocol for the measurement of TC. At the end of the
protocol, the rabbits were anesthetized with pentobarbital
sodium (40 mg/kg intravenously), and hearts were
removed to measure their MDA content and CL activity.
Serum TC
Serum TC was measured on an automated Beckman Syn-
chron LX20 Clinical System Analyzer.
Preparation of supernatant
The hearts were removed, cleaned of adventitial tissue and
atria, and immersed in cold Hank’s balanced salt solution
(HBSS). The supernatant from cardiac tissue was prepared
by the previously described method [10]. In short, 1.5 g of
tissue was washed in cold saline and cut into small pieces.
The tissue was immersed in 10 volumes (W/V) of KCl–
phosphate buffer (pH 7.4) containing 140 mM KCl and
10 mM phosphate buffer and homogenized with a polytron
homogenizer (PT-10 Brinkman Instrument, Rexdale, ON,
Canada) at a setting of 5 for two periods of 10 s each at
0–4 °C. The homogenate was centrifuged at 4009g in a
Beckman model J2-21 refrigerated centrifuge (4 °C) for
2-min. The supernatant was pipetted and used for the
measurement of MDA and CL activity.
MDA measurement
The levels of MDA in the supernatant were measured as
thiobarbituric acid reactive substances (TBARS) using an
earlier described method [19, 20]. TBARS were extracted
in a mixture of butanol: pyridine (15:1) that was separated
by centrifugation. The fluorescence intensity of butanol–
pyridine solution was measured at 553 nm with an exci-
tation at 513 nm. The MDA contents of the cardiac tissue
were expressed as nmol/mg protein.
Mol Cell Biochem (2014) 391:211–216 213

Measurement of CL activity

The CL activity is a measure of antioxidant reserve. An

increase in tissue CL indicates a decrease in the antioxidant
reserve of tissue and vice versa. Antioxidant reserve of tissue
is the amount of antioxidant present in the tissue at the time of
exposure of the tissue to oxidants. CL activity of the cardiac
tissue was measured as previously described [5, 8]. In short,
tissue supernatant (0.8 mL) was added to a counting vial
containing 0.4 mL of 2 9 10-4 M luminol, placed in an
AutoLumat LB953 luminometer (EG&G Berthold, Berthold
GmbH & Co. KG. Bad, Wildbad, Germany) at 37 °C and
incubated for 5 min. The reaction was initiated by adding
0.1 ml of 200 mM tert-butyl hydroperoxide. The chemilu-
minescence was monitored every 6 s for a period of 10 min.
The unit of CL is a relative light unit (RLU). The tissue CL
was expressed as RLU/mg protein.

Protein measurement
The protein content of the supernatant samples was mea-
sured by the Biuret method [21].
Statistical analysis
The results were expressed as mean ± SE. An unpaired
student’s ‘‘t’’ test was used to compare data between
groups. A p value of \0.05 was considered significant.
Fig. 1 The MDA content of cardiac tissue of the four experimental
groups. Group I, control (regular diet) for 2 months; Group II, 0.25 %
cholesterol diet for 2-months; Group III, 0.25 % cholesterol diet for
2-months followed by a regular diet for an additional 2-months; and
Group IV, 0.25 % cholesterol diet for 2-months followed by regular
diet plus vitamin E for an additional 2 months. Results are expressed
as mean ± SE. *p \ 0.05, group I versus other groups.  p \ 0.05,
groups II versus groups III or IV
MDA

The MDA content of the cardiac tissue from the four

Results
groups are summarized in Figure 1. The MDA content of
the cardiac tissue in group I was 0.074 ± 0.015 nmol/mg
Serum TC
protein. The content increased to 3.16-, 2.47- and 2.28-
folds in groups II, III, and IV, respectively, compared to
The basal levels of serum TC were 2.04 ± 0.23, 1.57 ± 0.27,
group I. The MDA content of cardiac tissue in group III
1.39 ± 0.12, and 1.75 ± 0.19 mmol/L, respectively, in
and group IV, respectively, was 21.83 and 27.84 % lower
groups I, II, III, and IV, and they were not significantly dif-
than those in group II. The values in groups III and IV were
ferent from each other except the values in group III were
similar (0.183 ± 0.028 vs. 0.169 ± 0.016 nmol/mg pro-
lower than those in group I. At month-2 the levels were lower
tein) but, higher than those in group I. These data suggest
in group I (1.29 ± 0.07 mmol/L) but higher in group II
that hypercholesterolemia increased the MDA content of
(38.91 ± 3.72 mmol/L), group III (31.40 ± 5.10 mmol/L),
the heart and that the removal of a high cholesterol diet
and group IV (36.94 ± 3.06 mmol/L) compared to their
after a hypercholesterolemic diet reduced the MDA content
respective initial levels. The levels at month-2 were similar in
of the cardiac tissue. Reduction of the MDA levels in the
groups II, III, and IV. The values at month-4 decreased sig-
heart was similar to the reduction in the serum cholesterol
nificantly in group III (6.08 ± 4.13 mmol/L) and group IV
with or without vitamin E. These data suggest that vitamin
(4.89 ± 1.0 mmol/L) compared to the values at month 2 in
E did not regress cardiac oxidative stress-induced by
the respective groups. These values were not significantly
hypercholesterolemia.
different from each other and from their respective initial
values at month-0. These data suggest that a high cholesterol
diet increases the serum levels of TC and that removal of high CL activity
cholesterol reduced the high cholesterol diet-induced increase
in serum cholesterol to normal levels. The data also suggest The CL activity of cardiac tissue from the four experi-
that vitamin E did not lower serum levels of TC. mental groups is summarized in Fig. 2. The cardiac-CL

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Fig. 2 The CL activity of the cardiac tissue from the four experi-
mental groups. Group I, (n = 6) regular rabbit chow diet for 2
months; Group II, (n = 6), 0.25 % cholesterol diet for 2-months;
Group III, (n = 5), 0.25 % cholesterol diet for 2-months followed by
a regular diet for an additional 2-months; and Group IV (n = 5),
0.25 % cholesterol diet for 2-months followed by regular diet with
vitamin E (40 mg/kg body weight/day) for an additional 2 months.
Results are expressed as mean ± SE.  p \ 0.05, group II versus
group IV
activity in group I was 30.11 ± 0.7 9 106 RLU/mg pro-
tein. The values in groups II, III, and IV were not signifi-
cantly different from group I. However, the values in group
IV were 16.82 % lower than those in group II. The data
suggest that hypercholesterolemia did not affect the anti-
oxidant reserve of the cardiac tissue. However, vitamin E
increased the antioxidant reserve in cardiac tissue follow-
ing a high cholesterol diet.
Discussion
In this study, we have shown that hypercholesterolemia
increases the oxidative stress in the myocardium. Lowering
of serum levels of TC in hypercholesterolemic rabbits by
regular diet decreased the cardiac oxidative stress but
vitamin E did not further regress oxidative stress.
A high cholesterol diet in the present study increased the
serum levels of TC similarly to previous the studies [4, 5,
22, 23]. Regular diet with or without vitamin E for 2
months following the high cholesterol diet reduced the
serum levels of TC to a similar extent. Lowering of serum
TC with a regular diet following high cholesterol-induced
hypercholesterolemia has also been reported earlier [24–
26]. This study suggests that vitamin E does not lower
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Mol Cell Biochem (2014) 391:211–216
serum TC. These findings are consistent with the findings
of previous investigators [1, 4, 13, 27].
In the present study, cardiac tissue MDA levels were ele-
vated in rabbits on a high cholesterol diet which is consistent
with other studies [1–3, 13]. Regular diet following a high
cholesterol diet reduced the cardiac MDA which could be due
to lowering of serum cholesterol. The reduction in serum
cholesterol could reduce the levels of ROS. Hypercholester-
olemia is known to increase ROS levels through various
mechanisms [3–7]. Lower cholesterol levels may also
increase the levels of antioxidants. It has been reported that
hypercholesterolemia reduces the SOD activity, but has no
effect on catalase activity of cardiac tissue in rabbits [1].
However, other studies have shown that hypercholesterol-
emia does not have any effect on SOD in Apo-100 transgenic
mice [3] and rats [2]. In pigs, hypercholesterolemia reduces
cardiac antioxidant enzymes (SOD, catalase, GSH-Px) and
antioxidants (vitamin E and C) [28]. Glutathione transferase
activity of cardiac tissue in rats is not affected by hypercho-
lesterolemia [2]. It appears that although the effects of
hypercholesterolemia on antioxidants are variable, it does
reduce the cardiac SOD activity in rabbits. The evidence
suggests that decreases in the oxidative stress in groups on a
regular diet following a high cholesterol diet might be due to
lower cholesterol levels. Low cholesterol levels would
decrease the production of ROS and increase antioxidants.
The low MDA levels of cardiac tissue in groups on regular
diet following a high cholesterol diet might be due to lower
cholesterol levels. Low cholesterol levels would decrease the
ROS production and increase the levels of antioxidants. The
MDA levels of cardiac tissue in groups on a regular diet with
or without vitamin E following a high cholesterol diet were
similar, suggesting that vitamin E did not lower cardiac MDA.
This is unexpected because vitamin E reduced cardiac MDA
in suppression studies1. It is possible that prior hypercholes-
terolemia has altered the antioxidant status of the cardiac
tissue and hence vitamin E is no longer effective. In this
context, it is to note that vitamin E with a regular diet fol-
lowing short-term high cholesterol diet did not lower aortic
MDA [29].
The cardiac-CL activity in groups I, II, and III were similar
but decreased in group IV in the present study. The data
suggest that hypercholesterolemia did not affect the antioxi-
dant reserve of the cardiac tissue. Similar findings have been
reported in rabbits on high cholesterol diets [1]. One would
have expected a decrease in the antioxidant reserve (increase
in CL activity) in hypercholesterolemia because hypercho-
lesterolemia decreases cardiac SOD activity in rabbits1.
However, hypercholesterolemia also increases the cardiac
GSH-Px activity in rabbits [1]. It is possible that the activities
of these two antioxidants enzymes cancel each other with no
effect on antioxidant reserve in hypercholesterolemia. Per-
haps hypercholesterolemia does not alter the vitamin E and C
Mol Cell Biochem (2014) 391:211–216
content of cardiac tissue in the rabbit. However, it has been
reported that hypercholesterolemia decreases vitamin E con-
tent of cardiac tissue in rats [2]. The lack of change in cardiac-
CL in hypercholesterolemia may be due to unaltered cardiac
antioxidants. Vitamin E in the present study reduced cardiac-
CL activity (increased antioxidant reserve). A decrease in
cardiac-CL activity with vitamin E administration has been
reported in normocholesterolemic and hypercholesterolemic
rabbits [1]. An increase in the cardiac antioxidant reserve
could be due to a reduction in oxidative stress because of
administration of vitamin E. Vitamin E reduces SOD activity
and increases GSH-Px activity of rabbit cardiac tissue [1]. In
guinea pigs, vitamin E increases the antioxidant capacity and
redox status of the myocardium [30].
The data suggest that despite, the increase in the anti-
oxidant reserve with vitamin E, the MDA levels of group
IV did not decrease compared to group III, although it was
lower than those in group II. It is possible that the reduction
of serum cholesterol by decreasing cholesterol content in
diet, following high cholesterol produces changes in the
antioxidants which are not measured by CL activity.
Another possibility for the lack of decrease in the cardiac
MDA in group IV is that the cardiac tissue is intolerant to
the effect of vitamin E after reduction of serum cholesterol
with a low cholesterol diet.
In conclusion, hypercholesterolemia induces oxidative
stress, and vitamin E does not regress hypercholester-
olemia-induced oxidative stress.
Acknowledgments This study was supported by a grant from the
Heart and Stroke Foundation of Saskatchewan and the College of
Medicine Research Fund of the University of Saskatchewan. The
technical assistance of Ms. Barbara Raney and Mr. P. K. Chatto-
padhyay is highly appreciated.
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