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INTRODUCTION their isoprenoid side chain. The common nomenclature
Oxidative stress is due to an imbalance between pro- of menaquinones is MK-n, where n stands for the
14, 15, 16
oxidant molecules (reactive oxygen (ROS) and number of isoprenoid residues . Vitamin K
nitrogen species), and antioxidant defence systems functions as a cofactor for an enzymatic carboxylation
which eventually may have pathological consequences reaction, which is necessary for the functional activity
1,2,3 17,18
on the cells if not properly managed .Every system of vitamin K–dependent (VKD) proteins . Vitamin
of the body has put in place antioxidant defense K-hydroquinone, the active cofactor for gamma-
mechanisms which protection then against oxidative glutamyl carboxylase, is continuously regenerated in
19, 20
stress. Although, these defense mechanisms are the vitamin K cycle . The successive steps in this
4, 5
sometimes inadequate. cycle are: (i) the oxidation of hydroquinone to vitamin
K 2, 3-epoxide, followed by (ii) reduction to the
Testicular oxidative stress is a fundamental factor in the quinone and finally (iii) the reduction to the
6,7,8
pathophysiology of the testis . Spermatogenesis and hydroquinone. Vitamin K-hydroquinone is a potent
steroidogenesis are two important testicular processes radical scavenger of ROS species in tissue like the brain
21, 22
that lead to the production of mature sperm cells and the . In this study, we have investigated the potential
male androgen hormones respectively and these two antioxidant effect of vitamin K on testicular oxidative
9,10
processes can be disrupted by oxidative stress . stress.
Although, the testis has a wide range of antioxidant
defence systems enzymatic or non- enzymatic in nature MATERIALS AND METHODS
and sometimes additional small molecular weight Chemicals
antioxidant factors such as vitamin C and E are required Glutathione (GSH), 1-chloro-2, 4-dinitrobenzene
11,12,13 (CDNB), 5, 5'-dithio-bi's-2-nitrobenzoic acid (DTNB),
to sufficiently combat the ROS attack .
thiobarbituric acid (TBA), trichloroacetic acid (TCA),
epinephrine and hydrogen peroxide were purchased
Vitamin K is a fat-soluble vitamin that is well known for
from Sigma chemical company (London, UK).
its role in the coagulation process and it occurs in two
K2HPO4 (dipotassium hydrogen phosphate) and
natural forms: vitamin K1 (phylloquinone) and vitamin
KH 2P O4 (Potassium d ihydrogen p ho sp hate)
K2 (menaquinone), which is a group-name for a
purchased from BDH chemical company Poole,
number of related compounds differing in the length of
Vitamin K: Its Antioxidant Effect on Testicular Oxidative Stress and Male
Reproductive Hormone Level in Sprague-Dawley Rats
O, Oremosu AA Osinubi AA and BakareAA
,
Department of Anatomy, Faculty of Basic Medical, College of Medicine,
University of Lagos, Idi-Araba, Lagos State, Nigeria , PMB12003.
Corresponding Author: Sanyaolu, AO.
E-mail: arislois@yahoo.com; asanyaolu@unilag.edu.ng; +2347039869563
Sanyaolu A
A S N
Journal of Anatomical
Sciences
Email:anatomicaljournal@gmail.com
J Anat Sci 9 (1)
ABSTRACT
Oxidative stress is a common cause of testicular dysfunction that leads to disturbance in the endocrine system,
thereby affecting the production of male reproductive hormones. Evidence suggests that vitamin K exhibits
antioxidant potentials that mitigate the effect of oxidative stress in tissues like liver. The effect of vitamin K on
testicular oxidative stress was not well elucidated. In this study, various diets were formulated to investigate the
effects of a warfarin-induced vitamin K-deficient diet and different dietary forms and concentrations of vitamin K1
and K2 (menaquinone-4, MK-4) on testicular oxidative stress and serum levels of reproductive hormones in
Sprague-Dawley rats. Different vitamin K1 and MK-4 dietary levels were achieved by the addition of the pure
vitamers to a vitamin K-deficient diet. Both forms of vitamin K reduced the level of testicular malondialdehyde and
increased the activities of testicular antioxidant enzymes. The findings of this study showed that high dietary
concentrations of K2 exhibit a more antioxidant potential than K1 in the testes. The serum level of testosterone was
higher in the group receiving MK-4 compared to K1. Consistently, the warfarin-induced vitamin K-deficiency
group showed an elevated level of testicular oxidative stress and a decreased serum testosterone level.
Keywords: Oxidative stress; Antioxidant; Vitamin K; Testosterone; Warfarin
Journal of Anatomical Sciences 2018: Vol. 9 No. 1
28
England. Menaquinone-4 (MK-4), vitamin K1 (cat no: Determination of lipid peroxidation
V3501) and warfarin (cat no: 45706) were obtained Malondialdehyde (MDA) was determined using the
23
from Sigma- Aldrich, St. Louis, MO, USA. method of Buege and Aust . In short, 1.0 ml of testis
homogenate was added to 2.0 ml of TCA-TBA-HCl
Animals and housing conditions reagent. A blank was prepared by adding 1.0 ml of
Eight-week-old male Sprague-Dawley rats weighing distilled water to 2.0 ml of TCA-TBA-HCl reagent,
between 125g and 185g from the breeding colony of the This reagent is a mixture consisting of TBA (0.37%),
College of Medicine of the University of Lagos were TCA, 15% and 0.24 N HCl in a 1:1:1 ratio), boiled for
used. Animals were kept individually in meshed-wired 15 min and allowed to cool. The flocculent material was
bottom cages in a well-ventilated animal house under removed by centrifuging at 3000 rpm for 10 min. The
standard conditions. Each animal was allowed access to supernatant was removed, and the absorbance was read
20 g of diet per day and water ad-libitum. Animals were at 532 nm against a blank. The MDA concentration was
allowed to acclimatize for two weeks before the start of calculated using a molar extinction coefficient for
-1 -1
the experiment. All treatments and procedures were in MDATBA-complex of 1.56 × 105 M cm .
compliance with the recommendations of the ethics
committee of the College of Medicine, University of Assays for antioxidant enzymes
Lagos, Nigeria (RGEEC/49/2015). Determination of Superoxide Dismutase (SOD)
activity
Diets and Experimental design Superoxide Dismutase activity was determined by its
Vitamin K deficient diet (VKD, cat no: TD 97053) was ability to inhibit the auto-oxidation of epinephrine
obtained from Harlan-Teklad, Madison, WI, USA. The determined by the increase in absorbance at 480nm as
24
vitamin K1 low diet (VK1low) and K1 high described by Sun and Zigma . The reaction mixture (3
supplemented diet (VK1high) contained the mixture of ml) contained 2.95 ml 0.05 M sodium carbonate buffer
VK1 and VKD at a final concentration of (0.3µg and 30 pH 10.2, 0.02 ml of the testes homogenate and 0.03 ml
µg of vitamin K1/g of VKD diet, respectively). of epinephrine in 0.005 N HCL was used to initiate the
Likewise, the MK-4 low and high supplemented diets reaction. The reference cuvette contained 2.95 ml
contained the mixture of MK-4 and VKD at final buffer, 0.03 ml of substrate (epinephrine) and 0.02 ml of
concentrations of (0.3µg and 30 µg of MK-4/g of VKD water. Enzyme activity was calculated by measuring
diet) respectively. The warfarin-induced vitamin K- the change in absorbance at 480 nm for 5 min. The SOD
deficient diet (WVKD) contained a mixture of 1mg of concentration was calculated using a molar extinction
-1 -1
warfarin and 10 mg of vitamin K1 per kg of VKD. The coefficient of ∑= 4020M cm .
total of fifty rats was used in this experiment. The
experiment was divided into two phases (four weeks Catalase activity (CAT) determination
and eight weeks). Each phase utilized twenty-five Catalase activity was determined according to Sinha et
25
animals each which were randomized into five al., . It was assayed calorimetrically at 620nm and
subgroups (VK1low, MK-4low, VK1high, MK-4high, expressed as µmoles of H O consumed/min/mg
2 2
0
and WVDF) of five animals each based on the diet they protein at 25 C. The reaction mixture (1.5ml) contained
were fed. 1.0ml of 0.01M phosphate buffer (pH 7.0), 0.1ml of
testes homogenate and 0.4ml of 2M H O . The reaction
2 2
Collection and storage of samples was stopped by the addition of 2.0ml of dichromate-
Blood samples were collected from the animals via acetic acid reagent (5% potassium dichromate and
cardiac pun cture with a heparinized syr ing e glacial acetic acid were mixed in 1:3 ratio). The molar
immediately after decapitation. The blood was -1 -1
extinction coefficient of 40M cm was used for
centrifuged to prepare serum, which was stored at - calculating the concentration of the CAT activities
20ºC until testosterone was assayed. The testes were
excised, defatted and stored at -70ºC until ready to be Glu ta t hion e p er o xid as e ( G SH) acti v i ty
processed for testing the lipid peroxidation level, determination
superoxide dismutase, and glutathione peroxidase and Glutathione peroxidase activity was determined
catalase activities. according to the method described by Sedlak and
26
Lindsay . To the homogenate 10% TCA was added,
Testosterone assay followed by centrifugation. 1.0ml of the supernatant
Testosterone was quantified using the enzyme-linked was treated with 0.5ml of Ellman's reagent (19.8mg of
immunosorbent assay from Monobind Inc (Lake DTNB in 100ml of 0.1% sodium nitrate) and 3.0ml of
Forest, CA 93630, USA, cat no: 3725-300) according to phosphate buffer (0.2M, pH 8.0). The absorbance was
the manufacturer's procedures. The assay utilized read at 412nm. The GSH concentration was calculated
antibody, enzyme-antigen conjugate and native 4
using a molar extinction coefficient of ∑ = 1.34 x 10
antigen. Mean absorbance values from duplicating 1 1
M- cm-
experiments were used to determine the corresponding
concentration of T in ng/ml from a standard curve.
Statistics
Statistical analysis was performed using the statistical
Journal of Anatomical Sciences 2018: Vol. 9 No. 1
29
Vitamin K: Its Antioxidant Effect on Testicular Oxidative Stress and Male
package for social sciences (SPSS) version 22 (IBM, other groups. This difference was significant to the
Chicago IL, USA). Data obtained were expressed as the VK1low and MK-4low group at four and eight weeks
mean ± standard error of the mean (SEM). The level of respectively. Both the VK1high and the VK1low
statistically significant differences between the groups groups showed an increase in serum testosterone level
was determined using Bonferroni's correction for when compared with the MK-4low group at four-
multiple testing for each of the phases. The least weeks. The WVDF group had the lowest level of
significant difference (LSD) was set at p < 0.01 testosterone in both feeding regimens and the
difference was statistically significant only when
RESULTS compared with the MK-4high and VKI high groups at
Serum levels of testosterone four-weeks (see figure 1).
The serum testosterone level in the group that was fed
with the high MK-4 diet was higher than that of the
Figure 1: Serum testosterone level at four and eight
weeks. Values are expressed as means of five animals,
error bars represent standard error of the mean and the
statistically significant difference (* and â) is placed at
P> 0.01. , Asterisk represents significant when
compared with MK-4high, alpha (á) when compared to
VK1high and (â) when compared to WVDF.
Levels of lipid peroxidation
At four-weeks feeding regimen, the WVDF group
showed an elevated MDA level, which is not significant
when compared to the other groups. However, for
eight-weeks feeding regimen, there was a significant
difference in the MDA level between the MK-4high
and WVDF group. The MK-4low group also shows a
non-significant decrease in the MDA level when
compared with the vitamin K1 supplemented groups as
shown in Figure 2.
Figure 2: Malondialdehyde level at four and eight
weeks feeding regimen. Values are expressed as means
of five animals, error bars represent standard error of the
mean and the statistically significant difference (*) is
placed at P>0.01. Asterisk represents significant when
compared with MK-4high
Testicular glutathione levels
The testicular glutathione levels in all groups were
shown in figure 3. At four weeks, the VK1low group had
the lowest glutathione levels followed by the WVDF
groups. Nearly the same level was observed between the
two high vitamin K supplemented diets, however
MK4high has the highest level. At eight weeks of
feeding on the respective diet, there was a collective
increase in the glutathione levels in all the groups. The
MK4high group maintains the highest level followed by
the VK1high group while the WVDF had the lowest
level.
Journal of Anatomical Sciences 2018: Vol. 9 No. 1
30
Arinola OS, Oremosu AA Osinubi AA and Bakare AA
,
Figure 3: glutathione level at four and eight weeks
feeding regimen. Values are expressed as means of five
animals, error bars represent standard error of the mean
and the statistically significant difference (* and á) is
placed at P>0.01. Asterisk represents significant when
compared with MK-4high and alpha (á) when
compared to Vk1high.
Levels of SOD activities
Figure 4 shows the SOD level in the various groups. At
four weeks the two (high and low) vitamin K1
supplemented diet has the least SOD activity levels
while the WVDF had the highest level when compared
with all other groups. At eight weeks the SOD level
decreases in the WVDF group while the MK4high
group had the highest SOD activity level that is
statistically significant to others.
Figure 4: Superoxide dismutase level at four and eight
weeks feeding regimen. Values are expressed as means
of five animals, error bars represent standard error of
the mean and the statistically significant difference (*,
á andâ) is placed at P>0.01. Asterisk represents
significant when compared with MK-4high, alpha (á)
when compared to VK1high and (â) when compared to
WVDF.
Levels of catalase activities
The level of catalase activities in the various groups
was shown in Figure 5 shows that at four weeks the
WVDF group has the highest CAT level followed by
the MK-4low and VK1high group respectively. Then at
eight-weeks, there is a decrease in the CAT activity
across the groups. The difference that was observed
among the various groups for the four and eight-weeks
feeding regimen was not statistically significant.
Figure 5: Level of catalase at four and eight- weeks
feeding regimen. Values are expressed as mean, standard
error of the mean and the statistically significant
difference (*) is placed at P>0.01.
DISCUSSION
The activities in the testes will continually lead to the
generation of reactive species, hence the need for the
testes to develop a high degree of complex antioxidant
system which includes both enzymatic (Superoxide
dismutase (SOD), glutathione peroxidase (GPX) and
catalase) and non-enzymatic (vitamin C, vitamin E, plant
polyphenol) in nature. This study shows evidence that
vitamin K insufficiency result in an increase in the
testicular oxidative stress that may also impair the
steroidogenic process in the male Sprague-Dawley rats.
The antioxidant potentials of vitamin K1 and MK-4 in
the testes are dependent on their dietary concentration.
The high dietary concentration of MK-4 was observed to
be more potent than K1 in attacking testicular oxidative
stress in this study. This may explain some of the reasons
why it is the most abundant form of vitamin K in the
testes. Generally, dietary nutrients, protein, and amino
acids provides the necessary materials needed for the
synthesis of antioxidant enzymes that plays an important
role in the body defensive mechanism. The decrease
observed in the MDA level in the groups that receive the
highest dietary concentration of vitamin K is a possible
evidence of its involvement as a free radical scavenger of
ROS in the testes. In order to regulate the ROS activities
in an organ there must be an increase in the antioxidant
enzyme synthesis and activities. However, the
continuous increase in ROS generation and presence in a
tissue can leads to a state of oxidative stress. The initially
increase that was observed in the SOD level in the
WVDF group over the four-week study period may be
regarded as an attempt to counter the presence of ROS in
the testes. Although, SOD plays an important role in the
defense system of the testes, however, the GSH and the
catalase are also important in testes protection against
oxidative stress. GSH provides electrons that reduce
H O to water and are concentrated in the mitochondria,
2 2
nucleus and acrosomal domain of differentiating
27, 28
spermatozoa .
The increase observed in the level of GSH in the testes is
marked by the high dietary intake of both forms of
vitamin K (KI and MK-4). The GSH level was not totally
low in the Vitamin K insufficient group and this may be a
Journal of Anatomical Sciences 2018: Vol. 9 No. 1
31
Vitamin K: Its Antioxidant Effect on Testicular Oxidative Stress and Male
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Vitamin K: Its Antioxidant Effect on Testicular Oxidative Stress and Male
