|
|
|
|
Research Article
|
|
Effects of Antioxidant Vitamin Combination on Pregnancy Induced Hyper-Hepatic State |
|
O.I. Iribhogbe,
J.E. Emordi,
E.O. Nwoke,
B.O. Idonije
and
U. Akpamu
|
|
|
ABSTRACT
|
The purpose of this present study is to determine the influence of varying combinations of antioxidant Vitamins on the hyper-hepatic state of pregnancy. To achieve this, seventy pregnant Wister albino rats weighing between 250-300 g were procured and grouped into 2 control groups treated with distilled water and vehicle- tween-80, respectively and three cohorts (I, II and III) with four sub-groups each (n = 5). Starting from the 7th day, group I received a varying dose combination of Vitamin A+C, group II Vitamin A+E and group III Vitamin C+E respectively for 11 days. Results of liver function assay revealed that supplementation with Vitamin A+C, A+E and C+E caused a significant reduction (p<0.05) in serum protein and a non-significant (p>0.05) alteration in serum albumin. Except for ALT where Vitamin A+E combination produced no significant alteration, serum AST (Aspartate transaminase) and ALP (Alanine transaminase) were significantly reduced (p<0.05) with antioxidant Vitamin combination therapy when compared with control. Antioxidant vitamin combination may be advantageous in pregnancy induced hyper- hepatic state. However, further study is needed in this respect.
|
|
|
|
|
Received: December 08, 2010;
Accepted: March 25, 2011;
Published: May 18, 2011
|
|
INTRODUCTION
It is a known fact that a number of associations between hepatic dysfunction
and pregnancy exist (Rahman and Wendon, 2002; Steingrub,
2004). In recent times, the observation that women with pre-eclampsia have
decreased plasma and placental concentration of antioxidants especially Vitamin
C (Raijmakers et al., 2004; Dehghan
and Dehghanan, 2006) led to the proposal that placental under perfusion
may mediate a state of oxidative stress (Rumbold et al.,
2008). These observations have given rise to increased interest in antioxidants
(Kashinakunti et al., 2010). Despite the proposed
role of oxidant stress in the pathogenesis of Non-Alcoholic Fatty Liver Disease
(NAFLD), antioxidant use have not been investigated sufficiently in NAFLD therapy
(Angula, 2002; Chitturi and Farrell,
2001; Reid, 2001). Our previous study on the effects
antioxidant Vitamin A, C and E mono-therapy on liver function in pregnancy published
in Asian Journal of Medical Sciences (2011), revealed a mixed effect. We then
concluded that the therapeutic benefit of Vitamin A, C and E mono- therapeutic
supplementation in pregnancy is not without impact on the functional and cellular
integrity of the liver (Iribhogbe et al., 2010).
While increased risk of fetal and maternal morbidity and mortality has been
noted in patients with severe hepatic dysfunction in pregnancy (Rahman
and Wendon, 2002), antioxidants have been reported for preventing pre-eclampsia
(Rumbold et al., 2008) and thus, protect the liver
from damage. Also, antioxidant vitamins have been reported to play an important
role in the regulation and eventual outcome of human pregnancy (Dakshinamurti
and Dakshinamurti, 2001). Available data has attached some importance on
the use of several different regimens in combination. This veracity of synergistic
effect lead to the postulation that multi-antioxidant vitamin combination therapy
may be important in managing pregnancy associated abnormal liver state signified
by hyper-hepatic enzymatics. On this premise, this study aim to investigate
the impact of antioxidant Vitamin A, C and E bi-combinations on elevated liver
enzymes in pregnancy.
Table 1: |
Treatment administered to different groups (n = 5 rats per
group) |
 |
MATERIALS AND METHODS This study was conducted between August and December 2009.
Experimental animals: Seventy adult female Wister albino rats weighing
(225 - 300 g) were obtained from the Animal House, College of Medicine, Ambrose
Alli University, Ekpoma between August and October 2009. They were housed in
a stainless steel cage with plastic bottom grid and a wire screen top in physiology
Lab II in the Department of Physiology, Ambrose Alli University, Ekpoma, Edo
State, Nigeria. They were assigned into five groups; a control group (n = 5),
vehicle group (n = 5) and three test groups I, II and III made up of four sub-
groups (n = 5). They were fed ad libitum with tap water and pellated
feeds purchased from Bendel feeds and flour meal Ewu, Nigeria Limited and allowed
to acclimatize for 2 weeks. After which two male Wister albino rats were introduced
into each group to allow for mating. The animals were allowed to mate for 6
days after which the male animals were removed from the cage. Pregnancy was
confirmed using the palpation method (Agematsu et al.,
1983) and vaginal smear microscopy method (Long and Evans,
1922; Daly and Kramer, 1998). From the 7 th day,
administration of the different Vitamin combinations began (Table
1) using orogastric tubes and syringes to minimize the loss of test substance
(Ejebe et al., 2009) and lasted a period of 11
days. The administrations were conducted between the h of 8.00 and 10.00 a.m.
daily.
Vitamin preparation and administration: Vitamin A, C and E were purchased from Clarion Medical Pharmaceuticals Nigeria Limited and Tween 80 vehicle from Sigma Pharmaceuticals Limited. Two hundred milligram 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. Table 1 shows the doses administrated to the test groups. Sample collection: Twenty-four hours after the last administration of vitamins was carried out, the animals were sacrificed after inhalation of chloroform. Cardiac and jugular vein puncture were used to collect blood samples into tubes containing EDTA as anticoagulant and centrifuged plasma preparation assayed for biomarkers of liver function via standard laboratory procedures.
Enzymatic assays: Determination of plasma Albumin (ALB) and Total Protein
(TP) for functionality, Aspartate Amino Transferase (AST) and Alanine Amino
Transferase (ALT) for cellular integrity and Alkaline Phosphatase (ALP) for
conditions linked to the billiary tract were analyzed using standard methods.
Albumin (ALB) and Total Protein (TP) level were determined using a Technicon
RA-XT autoanalyzer (Karakilcik et al., 2005).
Aspartate amino transferase (AST) and Alanine amino transferase (ALT) were determined
using the Randox reagent kit and 2, 4-dinitrophenylhydrazine as substrate (Reitman
and Frankel, 1957). Alkaline Phosphatase (ALP) was determined using the
Randox reagent kit with p-nitrophenylphosphate as substrate (Bassey
et al., 1946).
Data analysis: The Mean±Standard deviation (X±SD) and one-way ANOVA (LSD) statistical test was performed using SPSS version 17 soft ware. The significance level was set at p<0.05. RESULTS
Administration of combinations of Vitamin A+C, A+E and C+E produced a non-steady
reduction in serum protein level in pregnant rats when compared with control
(7.18±0.28 g dL-1).
Table 2: |
Effect of vitamin combinations on enzymes of liver function
in pregnant rats |
 |
Values are Mean±SD, Vit: Vitamin, T: Treatment, *p<0.05
compared with control |
Table 3: |
Effect of vitamin combinations on enzymes of liver integrity
in pregnant rats |
 |
Values are Mean±SD, Vit: Vitamin, T: Treatment, *p<0.05
compared with control |
Table 4: |
Effect of vitamin combinations on enzymes of biliary integrity
in pregnant rats |
 |
Values are Mean±SD, Vit: Vitamin, T: Treatment, *p<0.05
compared with control |
This was significantly (p<0.05) reduced after the 4 th dosing in the entire
vitamin combination groups. Serum albumin levels presented mixed results; dosing
with Vitamin A+C combination showed no significant alteration. However, after
the 1st, 2nd and 3rd dosing there was a significant (p>0.05) increase following
the administration of Vitamin A+ E combination (5.24±0.84, 4.28±0.59
and 3.82±0.41 g dL-1) and after the 3rd dosing (3.74±0.62
g dL-1) with Vitamin C+E combination compared with the control (2.92±0.35
g dL-1) (Table 2).
Enzymes of liver integrity (AST and ALT) were also determined. Vitamin A+C, A+E and C+E combination produced a favourable reduction in serum AST levels. Compared with the control (266.00±3.67 IU L-1), it was significantly higher (p<0.05) after the 1st dosing (276.60±5.13 IU L-1), significantly lower (p<0.05) after the 4th dosing (253.60±9.02 IU L-1) with Vitamin A+C combination, significantly (p<0.05) lower in the entire treatment with Vitamin A+E combination and after the 3rd and 4th dosing with Vitamin C+E combination. Also, Vitamin A+C and C+E combination produced a significantly lower serum ALT levels after the 3rd and 4th dosing while Vitamin A+E presented a significantly higher serum ALT during the entire treatment duration (Table 3). On enzymes of biliary tract integrity with particular reference to serum ALP, Vitamin A+C and C+E combination produced significantly lower changes (p<0.05) after the 3rd and 4th treatment when compared with control (88.00±2.24 IU L-1). Treatment with Vitamin A+E combination showed a higher serum ALP value which becomes non-significantly different with the control (88.00±2.24 IU L-1) after the 3rd and 4th dosing. (Table 4). DISCUSSION
The prsent study has confirmed that during pregnancy, there are changes in
serum protein, albumin, AST, ALP and ALP which are normally associated with
an increased risk of liver disease. Numerous publications have reported on the
specific causes of abnormal Liver Function Test (LFT) in pregnancy (Knox
and Olans, 1996; Castro et al., 1999; Davidson,
1998; Hunt and Sharara, 1999; Sibai
et al., 1993). Abnormal LFT can be mild with no long-term consequences,
or it can be severe, leading to both maternal and fetal mortality (Riely,
1999). Studies show the diagnostic work-up of abnormal Liver Function Test
(LFT) to be challenging, as the conditions peculiar to pregnancy have to be
considered in addition to the causes affecting the non-pregnant population (Riely,
1994; Knox, 1998).
In the present investigation, the combination of antioxidant vitamins reduced serum protein in pregnancy.
Also, albumin was also reduced, though this was not significant. Based on these
results, combination of antioxidant vitamins during pregnancy keep enzymes of
liver function load low as well as ameliorates hyper-proteinemia. Furthermore,
antioxidant vitamin combination ameliorate the reported increased AST, ALP and
ALT in pregnancy, except for Vitamin A+E combination where ALT was not reduced.
In the present investigation, Vitamins A+C, A+E and C+E ameliorated hyper-hepatic
states associated with pregnancy. The present study therefore shows that combined
vitamin supplementation successfully reverted the hyper- hepatic state of pregnancy
in a dose dependent fashion with Vitamin A+C and C+E being more potent. This
finding indicates that there is a synergistic effect of antioxidant vitamins
on pregnancy induced hyper-hepatic state. Corroboratively, Rumbold
et al. (2008) reported no increased risk of abnormal liver function
in randomized trials following supplementation with Vitamin C and E in pregnancy;
this is in consonance with the findings of our study. A study by Ince
(2010) reported Vitamin A+E combination to be a potent liver tonic. This
finding is also in consonance with present study, except that Vitamin A+E combination
did not produce any significant reduction in ALT. In studies with human subjects,
Vitamin C supplementation increased plasma lipid standardized α-tocopherol
(Hamilton et al., 2000). Vitamin E and C act
as antioxidants independent of each other and protect cells when compared to
cells lacking both Vitamin C and E (Madhavi et al.,
2009). Plasma α-tocopherol levels also improved upon supplementation
of Vitamin E and C, this improvement in plasma α-tocopherol levels suggests
synergism of Vitamin C with glutathione peroxidase to revitalize Vitamin E (Madhavi
et al., 2009). The combination of Vitamin A and E and C and E has
been shown to suppress parasitaemia (Umar et al.,
2008). The effect of antioxidant in infections with different species of
trypanosomes was attributed to the protection of membrane and cellular components
against oxidative species by the vitamins (Umar et al.,
2008). Several experimental and clinical studies suggest an interaction
between micro nutrients as suggested by Smith (1980),
Solomons and Russell (1980) and Christian
and West (1998). Their study showed the effect of zinc and vitamin A interaction
in treatment of Vitamin A deficiency. Other studies revealed that Vitamin A
supplementation alone failed to revert Vitamin A deficiency (Rahman
et al., 2002). Another study showed that zinc deficiency reduced
hepatic cellular RBP (cRBP), which is essential for the intracellular transport
of Vitamin A in addition to its well-established role in intercellular transport
(Mobarhan et al., 1992). The association between
zinc deficiency and Vitamin A metabolism is further supported by the simultaneous
reduction in retinol and Retinol Binding Protein (RBP) in zinc deficient rats
(Smith et al., 1974). This suggests that the
low plasma retinol concentrations in zinc deficiency might be caused by an impaired
ability of the deficient animals to mobilize hepatic retinol (Rahman
et al., 2002).
Yakoob et al. (2010) has reported widespread maternal
vitamin and mineral deficiencies and further recommends it logical to consider
supplementation with multiple micronutrient preparations in pregnancy. There
is therefore, a need to consider multivitamins and minerals in pregnant state
based on the findings of this study and the above literature. A nutrient may
affect not only the absorption of other nutrients, but also the transport, tissue
uptake, function and metabolism of other nutrients. Hence, concurrent ingestion
of several nutrients may result in synergistic, antagonistic, or threshold effects
as compared to a single nutrient (Huang et al., 2007).
The clinical benefits of such an approach over single-nutrient supplements are
unclear (Yakoob et al., 2010) as present study
support multiple- vitamin combination therapy in pregnancy.
CONCLUSION Conclusively, lack of antioxidant vitamin combination may be one of the explanations for pregnancy complications associated with elevated hepatic status. Further research to explore the present concepts and limitations of maternal multiple-vitamin supplementation during pregnancy cannot be overemphasized. ACKNOWLEDGMENT The authors sincerely appreciate the technical assistance of Dr. Nwaopara A.O, Head, Department of Anatomy, Ambrose Alli University, Ekpoma, Edo State, Nigeria and Mrs. Oruware, Head of Animal Farm, College of Medicine, Ambrose Alli University Ekpoma, Edo State, Nigeria, for her assistance in rat procurement and technical expertise in the confirmation of pregnancy. We are also grateful to our individual effort toward the success of this research.
|
REFERENCES |
1: Agematsu, Y., H. Ikadai and H. Amao, 1983. Early detection of pregnancy of the rat. Jikken Dobutsu, 32: 209-212. PubMed |
2: Angula, P., 2002. Nonalcoholic fatty liver disease. N. Engl. J. Med., 346: 1221-1223. Direct Link |
3: Bassey, O.A., O.H. Lowery and M.J. Brock, 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimetres of serum. J. Biol. Chem., 164: 321-329.
4: Castro, M.A., M.T. Fassett, T.B. Reynolds, K.J. Shaw and T.M. Goodwin, 1999. Reversible peripartum liver failure. A new persective on the diagnosis, treatment and cause of acute liver of pregnancy, based on 28 consecutive cases. Am. J. Obstet. Gynecol., 181: 389-395. PubMed |
5: Chitturi, S. and G.C. Farrell, 2001. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis., 21: 27-42. CrossRef |
6: Christian, P. and K.P. Jr. West, 1998. Interactions between zinc and vitamin A: An update. Am. J. Clin. Nutr., 8: 435S-441S. PubMed | Direct Link |
7: Dakshinamurti, K. and S. Dakshinamurti, 2001. Blood pressure regulation and micronutrients. Nutr. Res. Rev., 14: 3-43. CrossRef |
8: Daly, T.J.M. and B. Kramer, 1998. Alterations in rat vaginal histology by exogenous gonadotrophins. J. Anat., 193: 469-472. Direct Link |
9: Davidson, K.M., 1998. Intrahepatic cholestasis of pregnancy. Semin. Perinatal., 22: 104-111. Direct Link |
10: Dehghan, H.M. and R. Dehghanan, 2006. Plasma level of vitamin C in women with pre-eclampsia in Ardabil, Iran. Iranian J. Reproductive Med., 4: 35-39. Direct Link |
11: Ejebe, D.E., I.M. Siminialayi, J.O.T. Emudainohwo, S.I. Ovuakporaye and A.E. Ojieh et al., 2009. An improved technique for oral administration of solutions of test substances to experimental rats using Mediflon/Medicut intravenous cannula. African J. Biotechnol., 8: 960-964. Direct Link |
12: Hamilton, I.M.J., W.S. Gilmore, I.F.F. Benzie, C.W. Mulholland and J.J. Strain, 2000. Interactions between vitamins C and E in human subjects. Br. J. Nutr., 84: 261-267. Direct Link |
13: Huang, H., B. Caballero, S. Chang, A.J. Alberg and R.D. Semba et al., 2007. Supplement: Multivitamin/Mineral supplements and chronic disease prevention. Am. J. Clin. Nutr., 85: 265S-268S. Direct Link |
14: Hunt, C.M. and A.I. Sharara, 1999. Liver Disease in Pregnancy. Am. Fam. Physicians, 59: 829-836. Direct Link |
15: Iribhogbe, O.I., J.E. Emordi, A. Aigbiremolen, O.B. Idonije, E.O. Nwoke and U. Akpamu, 2010. Effects of vitamins A, C and E on liver function in pregnancy. Asian J. Med. Sci., 3: 8-13.
16: Kashinakunti, S.V., H. Sunitha, K. Gurupadappa, D.S. Shankarprasad, G. Suryaprakash and J.B. Ingin, 2010. Lipid peroxidation and antioxidant status in preeclampsia. Al Ameen J. Med. Sci., 3: 38-41. Direct Link |
17: Karakilcik, A.Z., A. Hayat, N. Aydilek, M. Zerin and M. Cay, 2005. Effects of vitamin C on liver enzymes and biochemical parameters in rats anesthetized with halothane. Gen. Physiol. Biophys., 24: 47-55. PubMed | Direct Link |
18: Knox, T.A., 1998. Evaluation of abnormal liver function in pregnancy. Semin. Perinatol., 22: 98-103. PubMed |
19: Knox, T.A. and LB. Olans, 1996. Liver disease in pregnancy. N. Engl. J. Med., 335: 569-576. CrossRef |
20: Long, J.A. and H. Evans, 1922. The Oestrous Cycle in the Rat and its Associated Phenomena. In: Memoirs of the University of California, Lipman, F. and R. Hedrick (Eds.). Vol. 6, University of California Press, Berkeley, CA., pp: 1-148
21: Madhavi, M., P. Samudram, A. Hemanth and L. Victor, 2009. Effect of antioxidant vitamins C and E supplementation on its plasma levels and on lipid profile in pulmonary tuberculosis patients 26. Am. J. Infect. Dis., 5: 263-272. CrossRef | Direct Link |
22: Mobarhan, S., B. Greenberg, R. Mehta, H. Friedman and D. Barch, 1992. Zinc deficiency reduces hepatic cellular retinol-binding protein in rats. Int. J. Vitam. Nutr. Res., 62: 148-154. PubMed |
23: Rahman, M.M., M.A. Wahed, J.G. Fuchs, A.H. Baqui and J.O. Alvarez, 2002. Synergistic effect of zinc and vitamin A on the biochemical indexes of vitamin A nutrition in children. Am. J. Clin. Nutr., 75: 92-98. Direct Link |
24: Rahman, T.M. and J. Wendon, 2002. Severe hepatic dysfunction in pregnancy. QJM, 95: 343-357. CrossRef |
25: Raijmakers, M.T.M., R. Dechend and L. Poston, 2004. Oxidative stress and preeclampsia; rationale for antioxidant clinical trials. Hypertension, 44: 374-380. CrossRef |
26: Reid, A.E., 2001. Nonalcoholic steatohepatitis. Gastroenterology, 121: 710-723.
27: Reitman, S. and S. Frankel, 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol., 28: 56-63. CrossRef | PubMed | Direct Link |
28: Riely, C.A., 1994. Hepatic disease in pregnancy. Am. J. Med., 96: 18S-22S. Direct Link |
29: Riely, C.A., 1999. Liver disease in the pregnant patient. Am. J. Gastroenterol., 94: 1728-1732. PubMed |
30: Rumbold, A., L. Duley, C.A. Crowther and R.R. Haslam, 2008. Antioxidants for preventing pre-eclampsia. Cochrane Database Syst. Rev. CrossRef | Direct Link |
31: Sibai, B.M., M.K. Ramadan, I. Usta, M. Salamca, B.M. Mercer and S.A. Friedman, 1993. Maternal morbidity and motality in 442 pregnancies with haemolysis, elevated liver enzymes and low platelets (HELLP syndrome). Am. J. Obstet. Gynecol., 169: 1000-1006. PubMed |
32: Ince, S., 2010. What vitamins help the liver?. eHow, conteribution, http://www.ehow.com/about_5092603_vitamins-liver.html.
33: Smith, J.C., 1980. The vitamin A-zinc connection: A review. Ann. N. Y. Acad. Sci., 355: 62-75. PubMed |
34: Smith, J.E., E.D. Brown and J.C. Smith, 1974. The effect of zinc deficiency on the metabolism of retinol-binding protein in the rat. J. Lab. Clin. Med., 84: 692-697. PubMed |
35: Solomons, N.W. and R.M. Russell, 1980. The interaction of vitamin A and zinc: Implications for human nutrition. Am. J. Clin. Nutr., 33: 2031-2040. PubMed |
36: Steingrub, J.S., 2004. Pregnancy-associated severe liver dysfunction. Critical Care Clinics, 20: 763-776. PubMed |
37: Umar, I.A., B.L. Rumah, S.L. Bulus, A.A. Kamla and A. Jobin et al., 2008. Effects of intraperitoneal administration of vitamins C and E or A and E combinations on the severity of Trypanosoma brucei brucei infection in rats. Afr. J. Biochem. Res., 2: 88-91. Direct Link |
38: Yakoob, Y.M., Y.P. Khan, Z.A. Bhutta, E. Manzotti, D. Lie and S. Fleischman, 2010. Maternal mineral and vitamin supplementation in pregnancy CME. Exp. Rev. Obst. Gynecol. Vol. 5. Direct Link |
|
|
|
 |