CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 1, February 2012
AFRICA
19
and handling were in accordance with guidelines provided by the
experimental animal laboratory and approved by the Animal
Care Committee of the Tabriz University of Medical Sciences.
The rabbits were equally divided (
n
=
9) into four groups:
group 1 rabbits were fed a regular diet, group 2 were fed a diet
containing 2% cholesterol, group 3 had a regular diet plus 5 mg/
kg/day oral amlodipine, and group 4 had a diet with 2% choles-
terol plus amlodipine 5 mg/kg/day, for eight weeks. Cholesterol
powder and amlodipine powder were provided from Merck and
Aria companies, respectively. Cholesterol powder was mixed into
the feed. Amlodipine was dissolved in distilled water and was
given with a special gavage tube at 09:00 daily for eight weeks.
The study protocol was designed in accordance with the
Guidelines for the Care and Use of LaboratoryAnimals
published
by the US National Institutes of Health (NIH Publication, No.
86-23, revised 1996) and approved by the Ethics Committee for
the Use of Animals in Research at Tabriz University of Medical
Sciences.
At the end of the experiments, all animals were fasted for
eight hours and then anesthetised by injecting ketamine (25 mg/
kg, intravenously) and sodium pentobarbital (20 mg/kg, intra-
venously) via the ear vein. Blood samples were drawn from the
inferior vena cava and were stored in tubes for determination of
serum lipid profiles and blood oxidative stress.
After decapitation, the heart was quickly removed, washed
in ice-cold saline and the atria and great blood vessels were
trimmed away. The ventricles were weighed and quickly frozen
in liquid nitrogen.
For analysis of oxidative stress, cardiac and blood homogen-
ates were prepared at 0–4ºC as described by Rothermel
et al
.
14
In brief, 50 mg of ventricle muscle were homogenised on ice in
1 ml of ice-cold lyses buffer (10 mM NaCl, 1.5 mM MgCl
2
, 20
mM HEPES, 20% glycerol, 0.1% Triton X-100, 1 mM dithi-
othreitol, pH 7.4). The homogenates were centrifuged at 4 500
g
for 1 min at 4°C (Avanti J 25 USA). The supernatant containing
the cytoplasmic protein fraction was collected and a protease
inhibitor cocktail (104 mM AEBSF, 0.08 mM aprotinin, 2 mM
leupeptin, 4 mM bestatin, 1.5 mM pepstatin A, and 1.4 mM
E-64) (P8340, Sigma-Aldrich, St Louis, MO) was added, and it
was stored at –80ºC until use. Protein concentration of the super-
natant was estimated using the Bradford technique.
15
Blood samples were drawn from the inferior vena cava and
stored in tubes for an hour. The serum was prepared and used
for the determination of serum lipid profiles, including total
cholesterol and triglycerides. These were determined by enzy-
matic methods using an automatic analyser (Abbott, Alcyon
300
Falcor, USA).
Lipid peroxidation was analysed by measuring thiobarbituric
acid-reactive substances (TBARs) in the homogenates, as previ-
ously described by Draper and Hadley.
16
Briefly, the samples were
mixed with 1 ml 10% trichloroacetic acid (TCA) and 1 ml 0.67%
thiobarbituric acid. The samples were heated in a boiling water
bath for 15 min, and butanol (2:1 v:v) was added to the solu-
tion. After centrifugation at 800
g
for 5 min (Avanti J 25 USA),
TBAR levels were determined from the absorbance at 535 nm.
SOD activity was determined using a RANSOD kit (Randox
labs. Crumlin, UK), according to Delmas-Beauvieux
et al
.
17
SOD
activity was measured in the supernatant on a spectrophotometer
(Stat Fax, 2100, Awareness, USA) at 505 nm. In this method,
xanthin and xanthin oxidase were used to generate superoxide
radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-
phenyl tetrazolium chloride (ITN) to form a red formazan dye.
Concentrations of substrates were 0.05 mmol/l for xanthin and
0.025 mmol/l for INT. SOD activity was measured by the degree
of inhibition of this reaction. To assay the mitochondrial SOD
(mtSOD) activity in the heart, the cytosolic SOD was inhibited
with 1 mm KCN.
Glutathione peroxidase activity was determined using a
RANSEL kit (Randox labs Crumlin, UK) according to the
method of Paglia and Valentine.
18
GPX catalyses the oxidation of
glutathione (at a concentration of 4 mmol/l) by cumene hydro-
peroxide. In the presence of glutathione reductase (at a concen-
tration
≥
0.5 units/l) and 0.28 mmol/l of NADPH, the oxidised
glutathione is immediately converted to the reduced form with
a concomitant oxidation of NADPH to NAD
+
. The decrease in
absorbance at 340 nm was measured using a spectrophotometer.
Statistical analysis
All determinations were performed at least in duplicate. Data
were expressed as mean
±
SEM and were analysed by a one-
way ANOVA using a standard computerised statistical program,
SPSS13.0 for windows software (SPSS INC, Chicago, IL, USA).
When a significant
p
-value was obtained, the Tukey
post hoc
test
was used to determine the differences between groups. A level of
p
<
0.05 was selected to indicate statistical significance.
Results
Our results clearly demonstrate that eight weeks on a 2% high-
cholesterol diet significantly increased serum levels of total
cholesterol (TC), low-density lipoprotein cholesterol (LDL-C),
high-density lipoprotein cholesterol (HDL-C) and triglycerides
(TG). These observations indicate that an atherogenic diet
induced hypercholesterolaemia in our
in vitro
model. Although
amlodipine treatment tended to enhance HDL-C:LDL-C and
HDL-C:TC ratios in this group, these effects were not statistical-
ly significant. The significant increase observed in plasma levels
of HDL-C and decrease in LDL-C, TG and TC is considered the
main effect of amlodipine treatment on serum lipid profiles in
the rabbits fed a regular diet (Table 1).
Lipid peroxidation
In the blood samples, the level of TBARs increased in the rabbits
on the 2% cholesterol diet (group 2) and those on 2% choles-
terol plus amlodipine (group 4), and decreased in the group on
a regular diet plus amlodipine (group 3) (
p
<
0.05). In addition,
in those on a regular diet plus amlodipine (group 3) and those on
2% cholesterol plus amlodipine (group 4), the level of TBARs
was less than in the group on a diet of 2% cholesterol (group 2)
(Fig. 1). Lipid peroxidation levels in all the heart samples showed
a similar trend to that of the blood samples, except in the rabbits
on a regular diet plus amlodipine (group 3), where the levels of
TBARs did not diminish compared to the control group (Fig. 1).
Antioxidant enzymes
Fig. 2 shows that the activity of total SOD (tSOD) in the blood
samples decreased in the rabbits on the 2% cholesterol diet
(group 2) (
p
<
0.05), and markedly increased in those on a regu-
lar diet plus amlodipine (group 3) and those on 2% cholesterol
