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.