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Apple Cider Vinegar Modulates Serum Lipid Profile, Erythrocyte,
Kidney, and Liver Membrane Oxidative Stress in Ovariectomized
Mice Fed High Cholesterol
Mustafa Nazırog
˘lu •Mustafa Gu
¨ler •
Cemil O
¨zgu
¨l•Gu
¨ndu
¨zalp Saydam •
Mustafa Ku
¨c¸u
¨kayaz •Ercan So
¨zbir
Received: 8 March 2014 / Accepted: 13 May 2014 / Published online: 4 June 2014
ÓSpringer Science+Business Media New York 2014
Abstract The purpose of this study was to investigate the
potentially beneficial effects of apple cider vinegar (ACV)
supplementation on serum triglycerides, total cholesterol,
liver and kidney membrane lipid peroxidation, and anti-
oxidant levels in ovariectomized (OVX) mice fed high
cholesterol. Four groups of ten female mice were treated as
follows: Group I received no treatment and was used as
control. Group II was OVX mice. Group III received ACV
intragastrically (0.6 % of feed), and group IV was OVX
and was treated with ACV as described for group III. The
treatment was continued for 28 days, during which the
mice were fed a high-cholesterol diet. The lipid peroxida-
tion levels in erythrocyte, liver and kidney, triglycerides,
total, and VLDL cholesterol levels in serum were higher in
the OVX group than in groups III and IV. The levels of
vitamin E in liver, the kidney and erythrocyte glutathione
peroxidase (GSH-Px), and erythrocyte-reduced glutathione
(GSH) were decreased in group II. The GSH-Px, vitamin
C, E, and b-carotene, and the erythrocyte GSH and GSH-
Px values were higher in kidney of groups III and IV, but in
liver the vitamin E and b-carotene concentrations were
decreased. In conclusion, ACV induced a protective effect
against erythrocyte, kidney, and liver oxidative injury, and
lowered the serum lipid levels in mice fed high cholesterol,
suggesting that it possesses oxidative stress scavenging
effects, inhibits lipid peroxidation, and increases the levels
of antioxidant enzymes and vitamin.
Keywords Oxidative stress Glutathione peroxidase
Ovariectomize Hypercholesterolemia Antioxidant
vitamins Liver
Abbreviations
ACV Apple cider vinegar
GSH Glutathione
GSH-Px Glutathione peroxidase
HDL High-density lipoprotein
i.p. Intraperitoneal
LDL Low-density lipoprotein
OVX Ovariectomized
ROS Reactive oxygen species
VLDL Very low-density lipoprotein
Introduction
Diseases of liver and kidney abnormalities constitute a
serious health problem in menopause (Poli 1993). These
diseases are usually treated with drugs, dietary or metabolic
changes, and vaccines. Liver disease in menopause also
induces lipid profile abnormalities because of its important
role in lipid metabolism (Luotola et al. 1986; Ulas¸ and Cay
2011). In this regard, finding new, safe, and effective food
supplements has become an important line of research.
Oxidative stress is the result of an imbalance between
the rates of free radical production and elimination via
endogenous antioxidant mechanisms such as the enzymes
glutathione peroxidase (GSH-Px) and catalase as well as
M. Nazırog
˘lu (&)M. Ku
¨c¸u
¨kayaz E. So
¨zbir
Department of Biophysics, Medical Faculty, Suleyman Demirel
University, Isparta, Turkey
e-mail: mustafanaziroglu@sdu.edu.tr
M. Gu
¨ler G. Saydam
Suleyman Demirel University, Isparta, Turkey
C. O
¨zgu
¨l
Regenerative and Restorative Medical Research Center,
Istanbul Medipol University, Istanbul, Turkey
123
J Membrane Biol (2014) 247:667–673
DOI 10.1007/s00232-014-9685-5
low molecular weight reductants like a-tocopherol, gluta-
thione (GSH), and ascorbate (Kovacic and Somanathan
2008). This imbalance can be initiated by numerous factors
including acidosis, transition metals, nitric oxide, LDL-
oxidation, and uncouplers of mitochondrial electron
transport (Nazırog
˘lu and Bransch 2006; Espino et al. 2012).
The major roles of GSH-Px are to catalyze the reduction
of hydrogen peroxide to water (Kovacic and Somanathan
2008) and the removal of organic hydroperoxides (Nazır-
og
˘lu 2009). GSH is a peptide involved in maintaining
oxidant homeostasis and in the cellular detoxification of
reactive oxygen species (ROS) in tissues including brain,
kidney, and liver (Halliwell 2006). Vitamin E is the most
important lipid antioxidant in cells. In addition to its role as
free radical scavenger, vitamin C also transforms vitamin E
to its active form (Frei et al. 1989).
It has been reported that a number of phenolic compound
extracted from fruit can protect against oxidative stress-
induced liver and kidney injury because of their antioxidant
properties (Yang et al. 2010;Nazırog
˘lu et al. 2011a,b). This
has promoteda substantial increase in the use of supplemental
dietary phenols and alternative therapy to treat menopause-
induced liver and kidney diseases in women and animals in
favor of hormone replacement treatment (Nazırog
˘lu et al.
2004a,b;Kireevetal.2010;Nazırog
˘lu et al. 2011a,b).
Apple cider vinegar is widely used in salad dressings,
marinades, vinaigrettes, food preservatives, chutneys, and
other common foods. The main classes of phenols in apples
and ACV are flavonoids and polyphenolic compounds (Denis
et al. 2013). A number of studies have shown that these foods
have a variety of pharmacological functions, including anti-
oxidant (Yang et al. 2010; Denis et al. 2013), antidiabetic
(Shishehbor et al. 2008), and cholesterol lowering (Budak
et al. 2011) properties, without adverse effects. However,
information on the effects of ACV on antioxidant systems is
very limited. For example, there are no scientific reports on the
use of ACV in menopausal women or ovariectomized animals
as a potential source of natural antioxidant phenols.
It is known, however, that the deficiency of ovarian hor-
mones promotes the generation of ROS, resulting in oxidative
stress, cell damage, or death (Budak et al. 2011). The bene-
ficial effects of estrogen include reducing total- and low-
density cholesterol (LDL) through enhanced LDL receptor
binding and clearance (Noh et al. 1999; Sanchez-Rodriguez
et al. 2011). Additionally, by reducing hepatic lipase activity,
estrogen promotes the formation of larger, less atherogenic
LDL (Nazırog
˘lu et al. 2004a,b; Sanchez-Rodriguez et al.
2011). Hence, the use of ACV instead of cholesterol lowering
drug therapy in menopause may improve oxidative stress-
induced lipid profile liver and kidney function in ovariecto-
mized mice as a model of postmenopausal women.
The aim of the current study was to investigate protec-
tive effects of ACV on ovariectomy-induced erythrocyte,
liver, and kidney oxidative damage as well as lipid profile
changes in mice fed high cholesterol and its free radical
scavenging activity in vivo.
Materials and Methods
Animals
Forty female Swiss mice weighing 36–40 g were used for the
experimental procedures. Twenty of them remained intact as
control and ACV groups, and the ovaries were removed in
the remaining 20 animals in order to constitute groups II and
IV. The mice were housed in individual plastic cages with
bedding. Standard food and tap water were available
ad libitum for the duration of the experiments unless other-
wise noted. The temperature was maintained at 22 ±2°C.
A 12/12 h light/dark cycle was maintained, unless otherwise
noted. The ACV was provided as a gift from the Agricultural
Faculty of Suleyman Demirel University and it was diluted
with water so that it would provide 0.6 % of the animals’
daily diet (Budak et al. 2011). This concentration was found
to be innocuous to mice in a pre-study in which various
concentrations of ACV were tested. Hypercholesterolemia
was induced by daily gavage administration of 1 ml/100 g
body weight of a cocktail containing 100 g cholesterol, 30 g
propylthiouracil, and 100 g cholic acid in 1 liter peanut oil
(Vogel and Vogel 1997).
The mice were maintained and used in accordance with
the Animal Welfare Act for the Care and Use of Laboratory
Animals of Suleyman Demirel University (SDU), and the
local ethical committee of the Medical Faculty, SDU,
approved the experimental protocol designed for this study
(Protocol Number; 2009: 27-08).
Experimental Groups
The mice were randomly divided into four groups of ten
that were treated for 28 days, as follows:
Group I was used as control and was fed a cholesterol-
rich feed (5 % cholesterol). The only treatment was intra-
gastrical application of physiological saline, as placebo
(Dilek et al. 2010).
Group II (OVX), the ovaries were surgically removed.
Otherwise, the mice were treated as described for the
controls.
Group III (ACV) for which in addition to the high-
cholesterol diet, the animals were given 0.6 % apple cider
vinegar instead of a placebo.
Group IV (ACV ?OVX) in which the animals were
ovariectomized and treated with ACV, as described for
group III.
668 M. Nazırog
˘lu et al.: Ovariectomy and Hypercholesterolemia
123
Animal Model of Menopause
For removal of the ovaries, the two OVX groups of mice
were anesthetized by intraperitoneal administration of a
cocktail of ketamine hydrochloride (50 mg/kg) and xylazine
(5 mg/kg) (Kireev et al. 2010; Dilek et al. 2010). Briefly, the
surgical procedure for ovariectomy equithesin and two
4-mm incisions were made through the skin and the muscle
back walls in parallel with the bodyline. The ovaries were
then located, and a silk thread was tightly tied around the
oviduct, including the ovarian blood vessels. The wound was
sutured with a synthetic absorbable thread. Depocilin was
used to prevent of infection. The ovariectomized mice were
allowed to fully recover before beginning the study protocol.
Blood and Tissue Samples
All the mice were sacrificed after 12 h of the last ACV
administration to obtain the necessary blood and tissue sam-
ples. The blood was separated into plasma and erythrocytes by
centrifugation at 1,5009gfor 10 min at ?4°C. The eryth-
rocyte samples were washed three timesin cold isotonic saline
(0.9 %, v/w), and hemolysis was accomplished by adding a
nine-fold volume of 50 mM, pH 7.4 phosphate buffer. The
hemolyzed samples were stored at -30 °C for not more than
months pending measurement of enzymatic activity.
All preparation procedures were performed on ice. The
liver and kidney samples were washed twice with cold
saline solution, placed into glass bottles, labeled, and stored
in at -33 °C until needed. After weighing, the tissue
samples were placed on ice, cut into small pieces with
scissors and mixed with five volumes (1:5 w/v) of ice cold
50 mM, pH 7.4 Tris-HCl buffer, and then homogenized for
2 min at 5,000 rpm using a glass Teflon homogenizer
(Caliskan Cam Teknik, Ankara, Turkey). The homogenate
was immediately used for establishing the extent of lipid
peroxidation and antioxidant enzyme levels. The antioxi-
dant vitamin analyses were performed within 3 months.
Lipid Peroxidation Determination
The lipid peroxidation levels were determined by the TBA
method of Placer et al. (1966) using a UV-1800 Spectrom-
eter (Schimadzu, Kyoto, Japan). The values are expressed as
lmol/gram protein and as lmol/gram hemoglobin (Hb).
Reduced Glutathione (GSH), Glutathione
Peroxidase (GSH-Px), and Protein Assays
The GSH content in erythrocyte, kidney, and liver samples
was measured by the spectroscopic method of Sedlak and
Lindsay (1968), as described in a previous study (Nazır-
og
˘lu et al. 2004a). The GSH-Px activity in erythrocyte,
kidney, and liver was measured spectrophotometrically at
37 °C according to the method of Lawrence and Burk
(1976). The protein contents in the liver and kidney were
measured by the method of Lowry et al. (1951) with bovine
serum albumin as the standard. Drabkin’s reagent was used
in the erythrocyte for determination of Hb.
Vitamins A, C, and E and b-carotene Analyses
The levels of vitamins A and E were determined in the
kidney and liver samples by a modification of the methods
described by Desai (1984) and Suzuki and Katoh (1990)as
described in a previous study (Nazırog
˘lu et al. 2004a).
Liver and kidney samples (0.25 g) were saponified by the
addition of 0.3 ml KOH (60 % w/v in water) and 2 ml of
1 % (w/v in ethanol) ascorbic acid, followed by heating at
70 °C for 30 min. After cooling on ice, 2 ml of water and
1 ml of n-hexane were added to the samples, mixed, and
allowed to separate into phases. An aliquot of 0.5 ml of
n-hexane extract was taken, and the vitamin A concentra-
tions were measured at 325 nm. Then, reactants were
added and the absorbance value of hexane was measured in
a spectrophotometer at 535 nm. Calibration was performed
using standard solutions of all-trans retinol and a-tocoph-
erol in hexane.
The concentrations of b-carotene in the kidney and liver
samples were determined according to the method of Su-
zuki and Katoh (1990). 2 ml of hexane was mixed with
0.25 g tissue sample. The concentration of b-carotene in
hexane was measured spectrophotometrically at 453 nm.
The quantitative determination of ascorbic acid in the
kidney and liver samples was performed according to the
method of Jagota and Dani (1982). The absorbance of the
samples was measured spectrophotometrically at 760 nm.
Measurement of Triglyceride and Total Cholesterol
Serum triglycerides, total cholesterol, VLDL cholesterol,
and element levels were measured in an autoanalyzer
(Olympus AV 2700) at the Isparta State Hospital by using
standard laboratory techniques (Nazırog
˘lu et al. 2011a,b).
Statistical Analysis
All results were expressed as mean ±SD. Significant
values in the four groups were assessed by the unpaired
Mann–Whitney Utest. Data were analyzed using the SPSS
statistical software (v. 17.0, SPSS Inc. Chicago, Illinois,
USA). pvalues of less than 0.05 were regarded as
significant.
M. Nazırog
˘lu et al.: Ovariectomy and Hypercholesterolemia 669
123
Results
Serum Element, Total Cholesterol and Triglycerides
Levels
Serum total cholesterol, triglycerides, and VLDL choles-
terol levels are shown in Figs. 1and 2, respectively. The
total cholesterol levels as in groups I–IV were 113, 121, 79,
and 89 mg/dl, respectively. The triglycerides levels in
these groups were 111, 142, 78, and 73 mg/dl, respectively.
In the same order, the total cholesterol levels were 22, 43,
16, and 20 mg/dl, respectively. The serum triglycerides
(p\0.05), total (p\0.05) and VLDL (p\0.001) cho-
lesterol levels were significantly higher in group II relative
to controls, but significantly lower in groups III and IV
relative to groups I and II (p\0.001). The levels of
sodium, potassium, and chlorine in serum samples are
shown in Table 1. No significant changes in the levels of
these elements were detected in all study groups.
Lipid Peroxidation Results
The lipid peroxidation levels in erythrocytes, liver, and
kidney are shown in Tables 2,3, and 4, respectively. Lipid
peroxidation is considered to be one of the principal indi-
cators of OVX-induced oxidative erythrocytes, liver, and
kidney injury (Nazırog
˘lu et al. 2004a,b; Ulas¸ and Cay
2011; Sanchez-Rodriguez et al. 2011). The lipid peroxi-
dation levels in the erythrocytes (p\0.05), liver
(p\0.05), and kidney (p\0.01) were significantly higher
in group II than in the controls. However, pretreatment of
mice with ACV effectively inhibited OVX-induced lipid
peroxidation levels in l erythrocytes (p\0.05), liver
(p\0.05) and kidney (p\0.01).
GSH and GSH-Px Values in Liver and Kidney
GSH-Px is a strong antioxidant enzyme within enzymatic
ROS scavenger systems, while GSH is a non-enzymatic
thiol-containing antioxidant against OVX-induced cellular
toxicity (Nazırog
˘lu 2009; Kovacic and Somanathan 2008).
The GSH levels and GSH-Px activity in erythrocytes, liver,
and kidney are shown in Tables 2,3, and 4, respectively.
The data show that the GSH-Px activity in erythrocytes
(p\0.05) and kidney (p\0.01), and GSH levels in
erythrocytes (p\0.05) significantly decreased in group II.
However, the GSH-Px activity in erythrocytes (p\0.05)
and kidney (p\0.01), and the GSH levels in erythrocytes
(p\0.05) were higher in groups III and IV relative to
group II. The GSH level in liver and kidney and GSH-Px
activity in liver did not change in the four groups
statistically.
Antioxidant Vitamin Concentrations in Liver
and Kidney
The concentrations of b-carotene and the levels of vitamins
A, C, and E in liver and kidney are shown in Tables 3and
4, respectively. The vitamin E stores in liver were mark-
edly depleted in group II (p\0.05), and the vitamin E and
b-carotene concentrations in liver were significantly
depleted in groups III and IV (p\0.01) relative to groups
II and I.
Pretreatment of mice with ACV effectively increased
the levels of vitamins C (p\0.05), E (p\0.05), and
b-carotene (p\0.01) in kidney. The vitamin A concen-
trations in liver and kidney did not significantly change in
any of the four groups.
Discussion
It was observed that the lipid peroxidation values for
erythrocytes, liver, and kidney and the serum triglycerides,
total and VLDL cholesterol concentrations were increased
and that the GSH-Px activity and level of GSH were
decreased in ovariectomized mice, proving that the proto-
col worked as an experimental menopause model. Sup-
plementation with ACV resulted in an increase of the
Fig. 1 Effects of apple cider vinegar (ACV) on total cholesterol and
triglyceride levels in serum of ovariectomized (OVX) mice
(mean ±SD and n=10).
a
p\0.05 and
b
p\0.001 as compared
with group control.
c
p\0.001 as compared with OVX group
Fig. 2 Effects of apple cider vinegar (ACV) on VLDL cholesterol
levels in serum of ovariectomized (OVX) mice (mean ±SD and
n=10).
a
p\0.001 p\0.001 versus control.
b
p\0.001 versus
OVX group
670 M. Nazırog
˘lu et al.: Ovariectomy and Hypercholesterolemia
123
antioxidants GSH and GSH-Px and a decrease of the tis-
sues lipid peroxidation and serum lipid profiles.
Lipid peroxidation has been implicated in the patho-
genesis of OVX-induced oxidative injury due to meno-
pause-induced estrogen deficiency, which results in cell
membrane damage. Sexual hormones play an important
role in the progression of liver diseases. The lipid
peroxidation level was higher in the OVX mice, revealing a
deficient antioxidant defense system. This observation
confirms previous studies showing that OVX led to an
increase of lipid peroxidation and decrease of GSH-Px
activity and antioxidant vitamin levels (Poli et al. 1993;
Nazırog
˘lu et al. 2011a,b; Nazırog
˘lu et al. 2004a,b; Dilek
et al. 2010; Lucas et al. 2006).
Table 1 Effects of apple cider
vinegar (ACV) supplementation
on serum sodium, potassium,
and chloride levels in
ovariectomized (OVX) mice
(mean ±SD)
Parameters Control (n=10) OVX (n=10) ACV (n=10) OVX ?ACV (n=10)
Sodium (mg/dl) 163.2 ±24.8 145.2 ±9.2 142.5 ±7.4 155.5 ±21.2
Potassium (mg/dl) 4.60 ±0.89 4.91 ±0.81 4.70 ±0.40 4.68 ±0.59
Chloride (mg/dl) 124.8 ±16.7 110.2 ±4.9 107.0 ±7.8 113.3 ±14.8
Table 2 Effects of apple cider vinegar (ACV) supplementation on erythrocytes lipid peroxidation (LP), reduced glutathione (GSH), and
glutathione peroxidase levels in ovariectomized (OVX) mice (mean ±SD)
Parameters Control (n=10) OVX (n=10) ACV (n=10) OVX ?ACV (n=10)
GSH-Px (IU/g Hb) 13.00 ±1.49 10.90 ±3.52
a
12.80 ±3.07
b
12.90 ±2.90
b
GSH (lmol/g Hb) 10.8 ±1.28 9.37 ±1.55 10.40 ±1.73 10.20 ±1.51
LP (lmol/g Hb) 11.80 ±1.44 14.30 ±1.31
a
12.00 ±1.85
b
11.70 ±1.42
b
a
p\0.05 versus control group
b
p\0.05 versus OVX group
Table 3 Effects of apple cider vinegar (ACV) supplementation on liver lipid peroxidation (LP), reduced glutathione (GSH), glutathione
peroxidase (GSH-Px), vitamin A, C, E and b-carotene levels in ovariectomized (OVX) mice. (mean ±SD)
Parameters Control (n=10) OVX (n=10) ACV (n=10) OVX ?ACV (n=10)
GSH-Px (IU/g protein) 16.90 ±1.39 17.70 ±2.06 16.50 ±3.32 16.30 ±3.52
GSH (lmol/g protein) 6.93 ±0.87 6.56 ±0.39 7.55 ±1.31 6.86 ±1.58
LP (lmol/g protein) 10.80 ±1.26 13.30 ±1.59
a
9.86 ±1.52
c
10.70 ±1.00
c
Vitamin A (lmol/g tissue) 50.16 ±0.45 53.89 ±1.84 49.84 ±1.17 53.07 ±0.95
b-carotene (lmol/g tissue) 2.62 ±0.11 2.35 ±0.09 2.06 ±0.39
b,c
1.84 ±0.17
b,d
Vitamin C (lmol/g tissue) 0.68 ±0.09 0.66 ±0.14 0.63 ±0.09 0.69 ±0.08
Vitamin E (lmol/g tissue) 9.54 ±0.56 9.10 ±0.46
a
7.62 ±1.80
b,d
6.08 ±0.96
b,d
a
p\0.05 and
b
p\0.01 versus control group
c
p\0.05 and
d
p\0.01 versus OVX group
Table 4 Effects of apple cider vinegar (ACV) supplementation on kidney lipid peroxidation (LP), reduced glutathione (GSH), glutathione
peroxidase (GSH-Px), vitamin A, C, E and b-carotene levels in ovariectomized (OVX) mice (mean ±SD)
Parameters Control (n=10) OVX (n=10) ACV (n=10) OVX ?ACV (n=10)
GSH-Px (IU/g protein) 14.70 ±0.83 12.80 ±1.21
b
15.90 ±1.12
a,e
14.88 ±1.88
c
GSH (lmol/g protein) 5.07 ±0.39 5.26 ±0.29 5.07 ±0.47 5.26 ±0.73
LP (lmol/g protein) 4.60 ±0.89 6.11 ±1.05
b
4.93 ±0.48
d
6.34 ±0.78
Vitamin A (lmol/g tissue) 10.47 ±1.97 10.37 ±1.26 12.01 ±1.38 11.13 ±1.54
b-Carotene (lmol/g tissue) 0.81 ±0.07 0.79 ±0.09 1.06 ±0.17
d
1.83 ±0.13
d
Vitamin C (lmol/g tissue) 0.35 ±0.09 0.34 ±0.09 0.50 ±0.07
c
0.50 ±0.06
c
Vitamin E (lmol/g tissue) 6.72 ±0.67 6.17 ±0.71 7.62 ±1.80
b
7.65 ±0.99
c
a
p\0.05 and
b
p\0.01 versus control group
c
p\0.05,
d
p\0.01 and
e
p\0.001 versus OVX group
M. Nazırog
˘lu et al.: Ovariectomy and Hypercholesterolemia 671
123
It is well known that menopause and removal of ovaries
induce kidney and liver oxidative injury (Ulas¸ and Cay 2011;
Barp et al. 2012). Additionally, the present study was also
designed to explore the protective effects of ACV as free
radical scavenger on OVX-induced oxidative damage to
erythrocyte, liver, and kidney tissues. Administration of ACV
improved the antioxidant defense system and decreased lipid
peroxidation in erythrocytes, kidney, and liver.
Flavonoids and phenolic agents occur widely in many
plants. Recently, the role of phenolic compound and
flavonoids in the prevention of menopause-induced oxi-
dative stress and disease has gained great interest (Yang
et al. 2010; Nazırog
˘lu et al. 2011a,b; Nikolic
´et al. 2012).
Apple cider vinegar contains high concentrations of poly-
phenols and flavonoids (Yang et al. 2010; Budak et al.
2011; Denis et al. 2013), which explains its antioxidant
properties against oxidative damage to erythrocytes and
tissues.
In mammalians, the combined actions of various cellular
antioxidants are critical for the effective detoxification of
free oxygen radicals. Among the cellular antioxidant
enzymes, GSH-Px has been extensively studied. It converts
hydrogen peroxide to water by using GSH as substrate
(Kovacic and Somanathan 2008 Nazırog
˘lu 2009 ). Ovari-
ectomized mice showed decreased antioxidant capacity in
kidney and erythrocytes, as evidenced by decreased
activity of the antioxidant enzymes, which is in agreement
with earlier apple and apple juice reports (Poli 1993;
Nazırog
˘lu et al. 2004a,b; Avci et al. 2007; Dilek et al.
2010; Nazırog
˘lu et al. 2011a,b). Treatment with ACV
prevented the reduction of the antioxidant enzyme activity
and subsequent oxidative injury to erythrocytes and kidney
(Avci et al. 2007; Ulas¸ and Cay 2011; Yuan et al. 2011).
Vitamin C has been shown to be an important antioxi-
dant, to regenerate vitamin E through redox cycling, and to
raise intracellular GSH levels (Frei et al. 1989). As such,
vitamin C plays an important role in protein -SH group
protection against oxidation. High concentrations of vita-
min C have are found in ACV (Denis et al. 2013). The
GSH-Px activity, vitamin C, vitamin E, and b-carotene
concentrations in kidney, and GSH-Px and GSH level in
erythrocytes increased in the ACV-treated groups. These
observed increases indicate an important role of ACV in
normalizing GSH-Px and antioxidant vitamin concentra-
tion in OVX mice.
The vitamin E and total cholesterol concentrations in
liver were also decreased in the ACV- and OVX ?ACV
groups. Vitamin E is an important antioxidant in biological
systems (Nazırog
˘lu 2007). Vitamin E promotes homeo-
stasis in living cells by a mechanism of incorporation into
cell membranes or by entering the cells. Liver possesses an
a-tocopherol-binding protein specific for vitamin E, facil-
itating its incorporation into cells and subsequent transfer
from liver to cells through cholesterols (Hacquebard and
Carpentier 2005; Traber 2007). The property of tocopherol
that appears to be related to most manifestations of defi-
ciency is its inhibitory effect on the auto-oxidation of
unsaturated fatty acids.
Estrogen deficiency increases generation of free oxygen
radicals, which induces oxidative stress and results in cell
damage or death (Budak et al. 2011). Beneficial effects of
estrogen in liver and blood include reducing cholesterol
through enhanced LDL receptor binding and clearance
(Noh et al. 1999; Sanchez-Rodriguez et al. 2011). Addi-
tionally, by reducing hepatic lipase activity, treatment with
this hormone promotes the formation of larger and less
atherogenic LDL cholesterol particles (Noh et al. 1999;
Nazırog
˘lu et al. 2004a,b).
In the present study, we were not able to measure all
serum lipid values such as HDL and LDL values in mice
due to limited amounts of serum. However, we observed
that OVX-induced changes of triglycerides, total and
VLDL cholesterol are modulated by ACV supplementa-
tion. Similarly, Shishehbor et al. reported that ACV
improved the serum lipid profile in normal and diabetic rats
by decreasing serum triglycerides, LDL and increasing
serum HDL-cholesterol (Shishehbor et al. 2008). It was
also recently reported that ACV decreased triglyceride and
VLDL levels in rats fed high cholesterol when compared to
animals on high-cholesterol diets without ACV supple-
mentation (Budak et al. 2011). In agreement with these
observations, total cholesterol and triglycerides were lower
in the ACV-treated mice fed high cholesterol.
In conclusion, the results presented in this study suggest
that ovariectomy is associated with an increase of serum
triglycerides, total and VLDL cholesterol and with eryth-
rocyte, liver, and kidney lipid peroxidation and reduction
of some antioxidants in tissues and cells. The administra-
tion of apple cider vinegar possesses a protective effect
against ovariectomy-induced blood, liver, and kidney oxi-
dative injury in mice fed high cholesterol and that such
protection may be due to free oxygen radical scavenging
effects, reduced lipids and lipid peroxidation, and increased
antioxidant enzyme and vitamin levels. The results in
blood, liver, and kidney may be of help to physicians and
nutritionists in the use of apple cider vinegar as supplement
in the treatment of oxidative stress-induced toxicity.
Acknowledgments MN formulated the present hypothesis and was
responsible for writing the report. MG, GS, CO
¨, MK, and ES were
responsible for analysis of the data. The authors wish thanks to Dr.
Manuel Flores-Arce (Tijuana University, Mexico) for polishing
English of the manuscript. The study was partially supported by
Scientific Turkish Scientific and Technical Research Institute
(TUBITAK-2010).
Conflict of interest The authors declare that there are no conflicts
of interest in the current study.
672 M. Nazırog
˘lu et al.: Ovariectomy and Hypercholesterolemia
123
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