Current Research Journal of Biological Sciences 3(2): 104-109, 2011
ISSN: 2041-0778
© Maxwell Scientific Organization, 2011
Received: October 11, 2010
Accepted: November 15, 2010
Published: March 05, 2010
Synergistic Effects of Antioxidant Vitamins on Lipid Profile in Pregnancy
1
O.I. Iribhogbe, 1J.E. Emordi, 2B.O. Idonije, 1A. Aigbiremolen, 3E.O. Nwoke and 3U. Akpamu
1
Department of Pharmacology and Therapeutics,
2
Department of Chemical Pathology,
3
Department of Physiology, College of Medicine, Ambrose Alli University,
Ekpoma, Edo State, Nigeria
Abstract: This study was designed to investigate the significance of varying combination of vitamin A, C and
E on lipidemic profile in pregnancy. In a bid to achieve this task, 70 female Wister rats weighing between 250300 g were procured and grouped into 2 control groups treated with distilled H2O and vehicle, tween-80
respectively and three cohorts (I, II and III) with four sub-groups (n = 5). Pregnancy was confirmed on the 6th
day after mating with male Wister rats, after which group I received a combination of vitamin A and C, group
II- vitamin A and E and group III- vitamin C and E respectively for 11 days. Results revealed a significant
(p<0.05) increase in HDL-C in the entire combination group, which was dose dependent. This was more
prominent in group II (vit A + E). Although, there were reductions in serum cholesterol (CHO), serum
triglyceride (TRI) and LDL-C levels, this was not significant (p>0.05). In conclusion, the synergetic potential
of antioxidant vitamin combination appears beneficial in the management of pregnancy related hyperlipedemic
states.
Key words: Antioxidants, hyperlipidemia, pregnancy, synergistic effect, vitamins
show the hypolipidemic effect of antioxidant vitamins A
and E when used in pregnancy as a single agent.
Clinical data has established the use of several
different regimens in combination (selenium, vitamin E,
and beta carotene) to reduce overall cancer mortality
(Fleshner et al., 1999; Ip et al., 2000; Jiang et al., 2001;
Redman et al., 1998; Taylor and Albanes, 1998; Zhong
and Oberley, 2001). Multivitamins and minerals have
been found to be associated with lower body weight, body
mass index, and fat mass (Stephens et al., 1996), other
research shows the administration of the combinations of
vitamins C and E or C and A in trypano-some infections
(Umar et al., 1999, 2007) to boost the reserves of
endogenous antioxidants and reduce the tissue damages
caused by the disease (Umar et al., 2008). Also,
combination of antioxidant vitamin and minerals shows
greater antioxidant ability against oxidative damage
(McDowell, 1989). Thus, we postulate that multiantioxidant vitamin combination therapy may reduce the
severity of hyperlipidemia occurring in pregnancy. This
led to the design of this study which will help to elucidate
the synergetic effect of vitamin A, C and E on lipid
profile in pregnancy.
INTRODUCTION
It is a known fact that higher than normal serum lipid
levels during pregnancy occur. However, epidemiological
studies of the relationship between pregnancy and later
risk for cardiovascular disease have shown mixed results
(Ness et al., 1993). It has been shown that lipid
peroxidation is involved in the oxidative modification of
LDL (Heinecke, 1987; Jargons et al., 1987). The lipid
peroxidation starts only after the depletion of natural
antioxidants such as vitamin- E, vitamin-C, $-carotene,
etc. in the body (Esterbaue et al., 1987). This was
supported by the fact that the low serum levels of
antioxidant vitamins are associated with high risk of
Coronary Artery Disease (CAD) (Ramirez and
Flowers, 1980; Reimersma et al., 1989). Antioxidant
vitamins prevent lipid peroxidation both in vivo and in
vitro (Gey et al., 1993; Reaven et al., 1993). Numerous
epidemiological evidence support the beneficial role of
the dietary antioxidant vitamins (Donaldson, 1982;
Hodis et al., 1995; Stephens et al., 1996). However, some
studies have questioned the beneficial role of antioxidant
vitamins (Gaziano et al., 1995; Zhang et al., 1997).
Vitamins are ideal antioxidants to increase tissue
protection from oxidative stress due to their easy,
effective and safe dietary administration in a large range
of concentrations without harmful side effect (Cadenas
and Packer, 2002). Interestingly, our previous study did
MATERIALS AND METHODS
Animals: Seventy adult female Wister albino rats (250300 g) were obtained from the Animal House of the
Corresponding Author: Dr. O.I. Iribhogbe, Department of Pharmacology and Therapeutics, College of Medicine, Ambrose Alli
University, P.M.B. 14. Ekpoma, Edo State Nigeria. Tel: +2348065794437
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Curr. Res. J. Biol. Sci., 3(2): 104-109, 2011
Table 1: Treatment administered to different groups (n = 5 rats per
group)
Group
Treatment
Control
Negative Control: Normal feed + Distilled water 1 mL
Vehicle: Normal feed + Tween 801 mL
College of Medicine, Ambrose Alli University, Ekpoma,
between August and October 2009 and were housed in
Physiology Lab 1 of the Department of Physiology,
Ambrose Alli University, Ekpoma, Edo State. Nigeria.
They were assigned into three test groups (I, II and III)
made up of four sub- groups (n = 5 rats each). The control
group consist of a negative control and a vehicle/ tween
80 group with 5 rats each. They were allowed to
acclimatize for 2weeks and fed ad libitum with tap water
and pelleted feeds purchased from Bendel feeds and flour
meal Ewu, Nigeria Limited. Two adult male Wister albino
rats were introduced into each sub-group to allow for
mating for 6 days after which the male animals were
removed from the cage. Pregnancy was confirmed
using the palpation method as described by
Agematsu et al. (1983) and vaginal smear microscopy
method typified by the presence of scanty epithelial cells
and leucocytes (Long and Evans, 1922; Daly and
Kramer, 1998). Administration of the different vitamins
combination began on the 7th day using orogastric tubes
and syringes to minimize loss of the test substances
(Ejebe et al., 2009) between the hours of 08.00 am and
10.00 am daily and this lasted for a period of 11 days.
Vitamin A+C 1. Normal feed + Vehicle + Dist H2O + Vit A 0.6 mg/kg
+ Vit C 200 mg/kg
2. Normal feed + Vehicle + Dist H20 + Vit A 0.7 mg/kg
+ Vit C 250 mg/kg
3. Normal feed + Vehicle + Dist H2O + Vit A 0.8 mg/kg
+ Vit C 300 mg/kg
4. Normal feed + Vehicle + Dist H2O + Vit A 1.0
mg/kg + Vit C 400 mg/kg
Vitamin A+E 1. Normal feed + Vehicle + Dist H2O + Vit A 0.6
mg/kg + Vit E 16.4 mg/kg
2. Normal feed + Vehicle + Dist H2O + Vit A0.7 mg/kg
+ Vit E 18.4mg/kg
3. Normal feed + Vehicle + Dist H2O + Vit A 0.8 mg/kg
+ Vit E 19.4 mg/kg
4. Normal feed + Vehicle + Dist H2O + Vit A 1.0
mg/kg + Vit E 22.4 mg/kg
Vitamin E+C 1. Normal feed + Vehicle + Dist H2O + Vit E 16.4 mg
+ Vit C 200 mg/kg
2. Normal feed + Vehicle + Dist H2O + Vit E 18.4 mg
+ Vit C 250 mg/kg
3. Normal feed + Vehicle +Dist H2O + Vit E 19.4
mg/kg + Vit C 300 mg/kg
4. Normal feed + Vehicle + Dist H2O + Vit E 22.4
mg/Kg + Vit C 400 mg/kg
Vitamin preparation: Vitamin A, C and E were
purchased from Clarion Medical Pharmaceuticals Nigeria
Limited and Tween 80 vehicle from Sigma
Pharmaceuticals Limited. 200 mg of the powdered form
of vitamin C was dissolved in 10 mL of distilled water
and the appropriate dose per kg was prepared for
administration. Vitamin A (25,000 IU equivalent to 6 mg
retinal and E, 100 mg) was dissolved in 0.2 mL of tween
80 and water in a ratio of 0.2:0.2:9.6. Group I received
Vitamin A + C combination at 5 different doses. Group II
received vitamin A + E while group III received vitamin
E+ C at 5 different doses, respectively (Table 1).
Table 2: Serum lipid profile (mg/dL) in combine Vitamin A and C
supplementation in pregnancy
TC
TG
HDL-C
LDL-C
Control
4.02±0.43
0.80±0.31
1.08±0.32
2.78±0.49
Tween 80
4.61±0.98
0.66±0.20
1.18±0.21
3.53±0.48*
T1
4.53±0.43
2.07±0.25* 0.98±0.12
4.18±0.37*
T2
4.13±0.32
1.63±0.29* 1.52±0.19* 3.26±0.48
T3
4.04±0.42
1.01±0.17
1.85±0.41* 2.75±0.25
T4
3.78±0.44
0.95±0.24
2.24±0.40* 2.64±0.58
TC: Total cholesterol; TG: Triglyceride; LDL-C: Low Density
Lipoprotein Cholesterol; HDL-C: High Density Lipoprotein Cholesterol;
*: p> 0.05 with control; T: Treatment group
significance level was set at p#0.05. Results were
presented in suitable tables.
Samples collection and analysis of lipid profile:
Twenty-four hours after the last administration of the
different vitamin combinations, the animals were
sacrificed after inhalation of chloroform. Cardiac and
jugular vein puncture were used to collect blood samples
into sterilized test tubes containing EDTA as
anticoagulant (Raederstorff et al., 2002).
Serum from blood were obtained by centrifugation at
2500 rpm and serum Total Cholesterol (TC) and
triglyceride (TG) concentrations were determined as
described by (Erickson et al., 1990), while low density
lipoprotein cholesterol (LDL-C) and high density
lipoprotein cholesterol (HDL-C) were determined
according to the method of Nichols et al. (1986).
RESULTS
Vitamin A and C treated group (Group I) showed a
significantly (p<0.05) increased HDL-C from 0.98 mg/dL
after the 1st dosing to 2.21 mg/dL after the 4th dosing.
Vitamin A and C treated group showed a reduced total
CHO, TG and LDL-C level as dose increase; this was not
significant (p>0.05) when compared with control
(Table 2). Vitamin A and E treated group (Group II)
showed a reduced serum CHO, TG, and LDL-C and a
significantly increased (p<0.05) HDL-C levels when
compared with control (Table 3). However, except for a
significant reduction in serum TG levels after the 4th
dosing, none of the other parameters were significantly
reduced when compared with control (p>0.05). As shown
in Table 4, vitamin C and E treated group (group III)
showed a significant (p<0.05) increase in HDL-C,
Data analysis: The mean ± standard deviation was
determined and one-way ANOVA analysis of variance
were performed using SPSS version 17 soft ware. The
105
Curr. Res. J. Biol. Sci., 3(2): 104-109, 2011
Table 3: Serum lipid profile (mg/dL) in combine Vitamin A and E
supplementation in pregnancy
TC
TG
HDL-C
LDL-C
Control 4.02±0.43
0.80±0.31
1.08±0.32
2.78±0.49
Tween 80 4.61±0.98
0.66±0.20
1.18±0.21
3.53±0.48*
T1
5.06±0.29*
2.28±0.33* 1.69±0.30* 3.54±0.37*
T2
4.87±0.41*
1.59±0.38* 2.11±0.29* 3.11±0.27
T3
4.78±0.37*
0.96±0.30
3.02±0.29* 3.07±0.58
T4
4.31±0.41
0.72±0.13
3.81±0.33* 2.90±0.45
TC: Total cholesterol; TG: Triglyceride; LDL-C: Low Density
Lipoprotein Cholesterol; HDL-C: High Density Lipoprotein Cholesterol;
*: p> 0.05 with control; T: Treatment group
boost the reserves of endogenous antioxidants and
reduced the tissue damages caused by the disease
(Umar et al., 1999, 2007). Antioxidant vitamin
supplementations may reduce the severity of trypanosome
infection by offering protection against possible oxidative
injuries associated with the disease (Umar et al., 2008)
and thus, beneficial in pregnancy related infections
(malaria) and complication (hyperlipidemia). Importantly,
Esterbaue et al. (1987) reported reported that lipid
peroxidation starts only after the depletion of natural
antioxidants. In the last 20 years, many basic, clinical and
epidemiological researches has suggested a potential
protective effect of antioxidant nutrients such as beta
carotene, vitamin C, and vitamin E on the risk of cancer
and cardiovascular diseases (Sies et al., 1992;
Diplock, 1991; Weisburger, 1991; Byers and
Perry, 1992). Data provided by cross-sectional, casecontrol and prospective epidemiological studies raise
strong supportive arguments for the relationship between
the intake of antioxidant vitamins and trace elements and
the risk of pathologies (Block et al., 1992; Stampfer and
Rimm, 1993, 1995). Thus it is wise to considered
advantageous a combination of these vitamins since
individually they produce beneficial effects.
Multivitamins and mineral supplementation have been
show to have beneficial effect on lipid profile
(Li et al., 2010). Several studies have shown that the
association of ascorbic acid and their derivatives with
"-tocopherol enhances the antioxidant capability of
both the two vitamins (Madhavi et al., 2009). In studies
with human subjects, vitamin C supplementation
increased plasma lipid standardized "-tocopherol
(Hamilton et al., 2000).
Vitamin C supplementation also led to a higher level
of vitamin E in plasma of the participants who were
administered 800 mg per day of vitamin E than
in participants administered vitamin E alone
(Baker et al., 1996). Vitamin E and vitamin C act as
antioxidants independent of each other and protect cells
when compared to cells lacking both vitamins C and E
(Madhavi et al., 2009). Vitamin C can also regenerate
oxidized Vitamin E by reducing it back to its active form.
The key step is the reaction between the tocopheroxyl
radical and vitamin C (Böhm et al., 1997). Vitamin C
regenerates active vitamin E and increases cholesterol
excretion (Bisby and Parker, 1995). Plasma "-tocopherol
levels also improved upon supplementation of vitamins E
and C, this improvement in plasma "-tocopherol levels
suggests synergism of vitamin C with glutathione
peroxidase to revitalize vitamin E (Madhavi et al., 2009).
Conclusively, concerning the effect of antioxidant vitamin
combination, we did observe a significant increase in
HDL-C with the entire vitamins combination groups. This
improvement was more potent with vitamin A and E
combination. The synergetic potential of antioxidant
Table 4: Serum lipid profile (mg/dL) in combine Vitamin C and E
supplementation in pregnancy
TC
TG
HDL-C
LDL-C
Control
4.02±0.43
0.80±0.31
1.08±0.32
2.78±0.49
Tween 80
4.61±0.78
0.66±0.20
1.18±0.21
3.53±0.48*
T1
6.28±0.53*
2.02±0.33* 1.07±0.14
4.94±0.50*
T2
6.01±0.32*
1.64±0.32* 1.48±0.38* 4.89±0.30*
T3
5.92±0.30*
1.05±0.16
1.94±0.28* 4.64±0.28*
T4
5.02±0.28*
0.52±0.62
2.32±0.35* 4.08±0.31*
TC: Total cholesterol; TG: Triglyceride; LDL-C: Low Density
Lipoprotein Cholesterol; HDL-C: High Density Lipoprotein Cholesterol;
*: p> 0.05 with control; T: Treatment group
which was accompanied with a significant increase in
total CHO and LDL-C, except for TG levels which was
only significantly elevated after the 1st and 2nd dosing.
DISCUSSION
In this in vivo study with pregnant rats, combination
of antioxidant vitamins A, C and E reduced the in vivo
lipid profile as measured by serum assay. To our
knowledge this is the first animal study evaluating the
effect of multiple vitamins supplementation on
hyperlipidemia occurring in pregnancy. Eleven days of
daily supplementation significantly improved HDL-C in
the entire treatment group with a non significant
difference in CHO following vitamin A and C
administration. The associated decrease LDL-C
concentrations in a dose dependent fashion, was not
significantly different in the vitamin A + C and A + E
combination group. This however, was not the case in the
vitamin C + E combination group which was significantly
higher when compared with control even though it was
decreasing.
Findings in earlier studies have reported that
supplementation with vitamins C and E is beneficial
in
women
at
high
risk
of preeclampsia
(Chappell et al., 1999). However, Rumbold et al. (2006)
report in a randomized, placebo-controlled trial revealed
that giving healthy nulliparous women supplements of
1000 mg of vitamin C and 400 IU of vitamin E daily
during pregnancy did not reduce their risk of
preeclampsia, the risk of death or serious outcomes in
their infants, or the risk of intrauterine growth restriction.
Also of benefit is that, combinations of vitamins C
and E or C and A in trypano-some infections are shown to
106
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vitamin combination appears beneficial in the
management of pregnancy related hyperlipidemic states
as demonstrated by this study. The efficacy of antioxidant
vitamin combination in pregnancy influenced HDL-C and
LDL-C levels. However, there is need for concern of
toxicity when combining antioxidant vitamins since its
benefit increases with increasing dose..
ACKNOWLEDGEMENT
The authors sincerely appreciate the technical
assistance of Dr. Nwaopara, A.O., Head of Department of
Anatomy, Ambrose Alli University, Ekpoma, Edo State,
Nigeria. and Mrs. Oruware the Head of Animal Farm,
Department of Physiology, Ambrose Alli University,
Ekpoma, Edo State, Nigeria., for her assistance in rat
procurement and detection of pregnancy. We are also
grateful to our individual effort toward the success of this
research.
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AUTHOR’S CONTRIBUTION
sample collection. Akpamu, U. performed the analysis
and interpretation of data. Iribhogbe, O.I. and Akpamu,
U., were responsible for review the existing literature and
for writing the first draft of the paper. All authors
performed a critical revision of the manuscript for
important intellectual content. All authors read and
approved the final manuscript.
Iribhogbe, O.I, Aigbiremolen, A. and Akpamu, U;
contributed to the study conception and design. Emordi,
J.E. Idonije B.O. Aigbiremolen, A. and Nwoke E.O. were
responsible for the daily feeding and monitoring of
animals. Emordi, J.E. Idonije B.O., Aigbiremolen, A.
Nwoke E.O. and Akpamu, U. were responsible for blood
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