Protective Effect of Squalene on Endogenous Antioxidant Vitamins in Experimentally Induced Myocardial Infarction in Rats
In the present study an attempt has been made to assess the cardioprotective effect of squalene on isoprenaline-induced myocardial infarction in male albino rats with respect to changes in the levels of endogenous antioxidant vitamins in heart tissue. Levels of endogenous antioxidants such as ascorbic acid, α-tocopherol and endogenous squalene content in heart tissue were determined. Significant (p<0.001) reduction was observed in the levels of ascorbic acid, α-tocopherol and endogenous squalene content in the heart tissue of isoprenaline administered rats compared to normal control rats. It is worth noting that, the prior administration of squalene at 2% level along with feed for 45 days significantly (p<0.001) reduced the isoprenaline-induced decline in the levels of these vitamins and restored the membrane bound squalene content at near normal. The results of the present study indicates that the cardioprotective effect of squalene might be ascribable to its antioxidant property thereby sharing the responsibility of these antioxidant vitamins in counteraction of free radicals generated during isoprenaline-induced oxidative stress.
Myocardial infarction is the death of part of the heart muscle due to sudden
loss of blood supply, which is typically caused by complete blockage of coronary
arteries by blood clot (Qureshi et al., 2002).
Myocardial injury is irreversible in nature and most of the drugs available
are effective only for the prevention of spreading or dispersal of necrotic
damage to the adjacent cells. Hence, it is important to find drugs capable of
protecting myocardial cells from necrotic damage by strengthening the cardiac
cell membrane. Since the major abnormalities noticed in myocardial infarction
are lipidemia, peroxidation and loss of plasma membrane integrity (Rodriguez
et al., 2005), the drug should possess antilipidemic, antiperoxidative
and membrane stabilizing properties. Also, it should be devoid of any adverse
Squalene, an isoprenoid molecule present in shark liver oil in higher quantities,
has been reported to possess antilipidemic, antioxidant and membrane stabilizing
properties (Qureshi et al., 1996; Ko
et al., 2002; Ivashkevich et al., 1981).
A phase I trial in adult males given 869 mg of squalene daily for 20 weeks to
study the cholesterol lowering effect of squalene showed that oral squalene
is safe and tolerable (Chan et al., 1996). The
preventive effect of squalene on mineral status (Farvin
et al., 2005), lipid metabolism (Farvin et
al., 2006), proteins and glycoprotein components in experimentally induced
myocardial infarction in rats (Farvin et al., 2007),
has already been explored. In the present study, an attempt has been made to
assess the protective effect of squalene on endogenous non-enzymatic antioxidants
in isoprenaline induced myocardial infarction in rats, a well established animal
model for studying the effects of many drugs on the process of myocardial infarction.
MATERIALS AND METHODS
Isoprenaline (isoproterenol; L-(3,4-dihydroxyphenyl)-isopropylaminoethanol
hydrochloride), Ascorbic acid, α-tocopherol and squalene standards were
purchased from M/s. Sigma Chemical Company, St. Louis. MO, USA in 2003 to 2006.
Squalene (Specific gravity: 0.853; Refractive index: 1.493; Saponification Value:
30; Iodine value: 344; Boiling point: 240-245°C and 95% purity) was prepared
from the shark liver oil of Centrophorus sp. caught in the Andaman waters
(Farvin et al., 2004). All the other chemicals
used were of analytical grade.
Male Wistar strain albino rats, weighing 100-120 g were selected for the
study. The animals were housed individually in polyurethane cages under hygienic
and standard environmental conditions (28±2°C, humidity 60-70%, 12
h light/dark cycle). The animals were allowed free access to food (M/s Sai Feeds,
Bangalore, India) and water. The experiment was carried out as per the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA), New Delhi, India and approved by the Institutional Animal Ethics Committee
Seven days after acclimatization, the animals were divided into four groups
of 6 rats each. Group I and III animals were fed on commercial feed with added
coconut oil at 2% level for 45 days and group II and IV animals were fed on
commercial feed with added squalene at 2% level for a period of 45 days. After
45 days feeding, the group III and IV animals were intraperitoneally (i.p.)
injected with isoprenaline (11 mg (dissolved in physiological saline) 100 g-1
b.wt. day-1 for 2 days) for the induction of myocardial infarction.
Control animals (Group I and II) were i.p. injected with physiological saline
alone for 2 days. At the end of the experimental period, i.e., 24 h after last
injection of isoprenaline, the experimental animals were sacrificed. The heart
tissue was excised immediately and washed with chilled physiological saline.
The heart tissue homogenates prepared in ice cold 0.1 M Tris-HCl buffer, pH
7.2 was used for the biochemical analysis.
Determination of Ascorbic Acid (Vitamin C)
Ascorbic acid (Vitamin C) in the heart tissue was determined by the method
of Roe and Kuether (1942). In brief : 1.0 mL of heart
homogenate was taken into test tubes containing 1.0 mL 10% ice cold trichloro
acetic acid (TCA) and centrifuged at 3000 rpm for 20 min. The 0.5 mL of supernatant
was taken into another test tube, to this 2.0 mL 5% TCA and 0.1 mL DTC reagent
(0.4 g thio urea, 0.05 g CuSO4, 3 g 2,4 dinitrophenyl hydrazine (DNPH)
dissolved in 100 mL of 9 N H2SO4) were added and incubated
for 3 h at 37°C. Cooled to room temperature and 0.75 mL ice cold 65% H2SO4
was added. After 30 min absorbance was read at 520 nm by using Shimadzu- UV
spectrometer. A blank was carried out using 1.0 mL of distilled water. The standards
of different concentrations were also treated similarly.
Vitamin E (α-Tochopherol)
α-Tochopherol (Vitamin E) was determined in the heart tissue by the
method of Baker et al. (1980). In brief: 200
mg heart sample was homogenized with 2.0 mL of ethyl alcohol and centrifuged
at 3000 rpm for 10 min. One milliliter of the supernatant was taken into test
tubes containing 1 mL of 2% epinephrine and incubate at 70°C for 2 min.
To this 0.3 mL of saturated KOH was added and kept in a water bath at 70°C
for 2 min. After cooling in ice bath, 1 mL distilled water and 4.0 mL hexane
was added and centrifuged at 2800 rpm for 10 min. Three milliliter of the upper
hexane layer was taken into a test tube and evaporated to dry. To the residues
and standards (0.1-0.5 mL), 3.0 mL ethyl alcohol, 0.2 mL bathophenanthroline,
0.2 mL FeCl3 and 0.2 mL H3PO4 was added. Mixed
well and absorbance was read at 550 nm by using Shimadzu-UV spectrometer.
Determination Endogenous Squalene Content
Endogenous squalene content of the heart tissue was estimated by a modified
method of Liu et al. (1976) using GC-MS (Thermo
trace GC ultra) equipped with Elite 220 capillary column (30 mx0.54 mm dia)
and a flame ionization detector. The carrier gas used was nitrogen with a flow
rate of 0.8 mL min-1. Initial temperature was set at 220°C and
was increased 3°C min-1 until the temperature of 260°C was
reached. Injector and detector temperature was kept at 260 and 275°C, respectively.
Squalene separated was identified by comparison of retention time with those
obtained by standard. Measurement of peak areas and data processing were carried
out by Thermo chrom cord software.
Mean values of data obtained were calculated and the results were expressed
as Mean±SD of 6 animals. One way Analysis of Variance (ANOVA) was carried
out and the statistical comparisons among the groups were performed with Bonferroni
multiple comparison test by using a statistical package program Graphpad prism
4 (Graphpad Software Inc., San Diego, USA). A p-value <0.05 was considered
as statistically significant.
RESULTS AND DISCUSSION
In the present study, a significant (p<0.001) decline was observed in the
content of non-enzymic antioxidants such as ascorbic acid and α-tocopherol
in the heart tissue of Group III isoprenaline-administered rats as compared
to Group I control rats (Fig. 1a, b). Which concurs with an
earlier reported study (Pinelli et al., 2004).
Severe oxidative challenge results in the reduction in the levels of hydrophilic
antioxidants like vitamin C, which in turn leads to significant depletion in
lipophilic antioxidants like ubiquinol and vitamin E (Molyneux
et al., 2002), as observed in the present study. A significant (p<0.001)
reduction was also observed in the levels of endogeneous squalene content in
the heart tissue of Group III isoprenaline-administered rats as compared to
Group I rats (Fig. 2). The decline noted in the levels of
these antioxidant vitamins and endogenous squalene content in the heart tissue
indicates the severity of oxidative stress in isoprenaline-induced myocardial
infarction condition. The generation of free radicals in isoprenaline-induced
myocardial infarction is probably exceeded the free radical scavenging capability
of these non-enzymic antioxidants, resulting in deterioration of membrane integrity.
||Levels of (a) vitamin C, (b) vitamin E content in heart tissue
of control and experimental groups of rats. Group I and Group II, normal
control, rats received standard diet mixed with 2% coconut oil and 2% squalene,
respectively, for a period of 45 days; Group III and Group IV, myocardial
infarctions were induced by intraperitoneal (i.p) injection of isoprenaline
(11 mg (dissolved in physiological saline) 100 g-1 b.wt. day-1
for 2 days) after 45 days of feeding with standard diet mixed with 2% coconut
oil and 2% squalene, respectively. Results are Means±SD for 6 animals.
ap<0.001 significantly different compared with control animals,
bp<0.001 significantly different compared with squalene administered
normal rats, cp<0.001 significantly different compared with
isoprenaline-induced myocardial infracted rats
Vitamin E has a strong antioxidant capacity and has been used in several ischemia-reperfusion
studies (Singh et al., 1996; Kushi,
1999), which have demonstrated that vitamin E attenuates membrane related
morphological and biochemical alterations resulting from myocardial infarction.
Not only can the vitamin C directly quench superoxide radicals in aqueous milieu
before they can attack lipids but it can also reduce tocopheroxyl radicals and
so removes free radicals from the lipid phase (Frei et
al., 1989). As long as the concentrations of redox cycling antioxidants,
ascorbate and glutathione are maintained in the myocardium, distal antioxidant
system would not be consumed. The decrease in vitamin E in our study could be
due to the increased utilization in scavenging the oxyradicals generated or
could be due to decreased vitamin C concentration because vitamin C can regenerate
α-tocopherol by reducing α-tocopheryl radicals present on the surface
of the membranes (Freiglben and Packer, 1993).
||Levels of Endogenous squalene content (μg g-1
wet tissue) in heart tissue of normal and experimental groups of rats. Description
of the different groups are same as in Fig. 1. Results
are Means±SD for 6 animals. ap<0.001 significantly
different compared with control animals, bp<0.001 significantly
different compared with squalene administered normal rats, cp<0.001
significantly different compared with isoprenaline-induced myocardial infracted
It is worth noting that, the prior administration of squalene at 2% level along
with feed significantly (p<0.001) reduced the isoprenaline-induced decline
in the levels of these vitamins in group IV animals as compared to those of
group III isoprenaline-injected rats (Fig. 1a, b). It probably
did so by sharing the responsibility of these antioxidant vitamins in counteraction
of free radicals generated during isoprenaline-induced oxidative stress. Moreover,
Administration of squalene restored the membrane bound squalene content in the
heart tissue (Fig. 2). The unpaired electron present in the
hydroxyl radical (OH) generated during isoprenaline-induced myocardial infarction
might have been trapped for dismutation by its free radical scavenging isoprenoid
unit. Miyachi et al. (1983) reported that squalene
functions as an efficient quencher of singlet oxygen and prevents the corresponding
lipid peroxidation in human skin. Of all the other organs and tissues, heart
tissue was reported to be richest in squalene, after skin and adipose tissues
(Liu et al., 1976). Squalene occurs in the midplane
of the lipid bilayer and stabilizes the layers of cellular and subcellular membranes
through the formation of complexes with the fatty acids in the phospholipid
bilayer membranes (Haub et al., 2002). The presence
of squalene in the membrane phospholipid bilayer might have rendered the heart
muscle more stable against isoprenaline-induced oxidative injury. The antioxidative
property of squalene has been already well established (Ko
et al., 2002). Squalene consists of six 2-methyl-2-pentene units
and the electron donating property of the methyl group at the 2-position is
likely to play an important role in the quenching activity. Further more, the
methyl groups also supply hydrogen for the ene reaction (Gilbert
and Baggott, 1991), which may facilitate the quenching of singlet oxygen.
In conclusion, the results of the present study reveal that, squalene is very effective in mitigating the deleterious effect of isoprenaline-induced aberration in endogenous antioxidant vitamins in experimental rats. The overall cardio protective effect of squalene is probably related to the counteraction of free radicals by its antioxidant nature or by its membrane stabilizing action.
The financial aid from the Indian Council of Agricultural Research is greatly acknowledged.
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