INTRODUCTION:

Every living cell undergoes chemical reactions involving the oxidation and reduction of molecules. Most of these reactions and pathways lead to the production of free radicals (Bond, 1993). Free radicals are highly reactive molecules generated by biochemical redox reactions (Halliwell, 1993). Free radicals interact with other molecules within the cells and may cause oxidative damage to proteins, membranes and genes (Chung et al., 2001; Abdoljalal, 2006). The end product of lipid peroxidation (MDA) cross links between the amino groups in proteins (Wolff et al., 1986) and may increase viscosity of cell membrane leading to their limited life span. Animals survive the effect of free radicals because of the antioxidant defense system (Lee and Shacter, 1999). Antioxidants protect the cells from being oxidized by free radicals (Wolf, 2005). The discovery of vitamin E as an antioxidant has helped in proper understanding of the biological roles performed by antioxidants.

The continuous fluctuation in power supply in developing countries coupled with the fact that samples collected for blood testing in non-laboratory settings have to be transported over long distances has led to increased risk of interfering factors and consequently errors in laboratory results. The present study therefore evaluated the effect of vitamin-E stored experimental blood samples on Malondialdehyde (MDA) and protein concentrations with a view of suggesting a method of reducing the errors in laboratory results that are due to oxidation.

MATERIALS AND METHODS:

Bovine blood sample procurement and management:

Blood samples were collected immediately as the cows were slaughtered at the Ogige Nsukka Market Abattoir in Enugu State, Nigeria. After centrifugation for 15 minutes, the plasma obtained was divided into two portions, one with vitamin E and the other without vitamin E. Analyses of MDA and protein concentrations were carried out for four days but on day 1, analysis was carried out only on fresh plasma without vitamin E.

MDA determination:

This assay was done by the method of Das et al. (1990) as summarized below. 20% of TCA (1.25ml) was added to 0.25ml of blood plasma and the mixture was allowed to stand for 10 minutes. Then 1.25ml of 0.05N sulphuric acid and 1.5ml of 0.67% TBA were added to the mixture and placed in a water bath for 1 hour. The mixture was then cooled in a water bath and butanol (3ml) was added and centrifuged. The supernatant was collected in butanol layer and the absorbance was read at 532nm. This principle is based upon the reaction of TBA with Malondialdehyde in slightly acidic medium. Values were expressed in % TBARS.

Protein determination:

Protein was determined by the Biuret’s method as described by Amadi et al. (2004). Three test tubes were labeled blank, standard and sample. Distilled water (0.02ml) was added to the blank tube, 0.02ml standard added to the standard tube and serum (0.02ml) was added to the sample tube. To each tube, Biuret’s reagent (1.0ml) was added, mixed and incubated for 30 minutes at 25^oC. The absorbance of the sample and standard were measured against the blank at 540nm.

Statistical Analysis:

Data entry and analysis were done using SPSS version 12.0 and values were represented as Mean ± SD.

RESULTS:

The results obtained are represented in Tables 1 and 2. Table 1 shows the concentration of MDA in experimental blood samples stored with and without vitamin E on different days while Table 2 shows the protein concentration of experimental blood samples stored with and without vitamin E on different days.

Table 1: MDA concentration of experimental blood samples stored with and without vitamin E on different days

Days	MDA concentration (% TBARS)
	Without vitamin E		With vitamin E
Day 1	80		–
Day 2	110		80
Day 3	200		79.0
Day 4	233	79.0

Table 2: Protein concentration of experimental blood samples stored with and without vitamin E on different days

Days	Protein concentration (g/dl)
Days	Without vitamin E	With vitamin E
Day 1	2.5±0.08	–
Day 2	2.5±0.05	2.5±0.03
Day 3	2.0±0.08	2.46±0.09
Day 4	1.7±0.09	2.40±0.01

DISCUSSION:

The results of the MDA concentration (table 1) showed an increase in the samples stored without vitamin E from day 1 (80% TBARS) to day 4 (233% TBARS). Pey et al. (2003) had similar results and accounted the increase to sample deterioration. This probably accounted for the increase observed in this study. In the sample stored with vitamin E, the MDA concentration decreased slightly from day 2 (80% TBARS) to day 4 (79% TBARS). A similar study suggested the decrease to be due to reduction in oxidation caused by the antioxidant activity of vitamin E (Traber and Alkinson, 2007). This explained the important role vitamin E played as antioxidant in this study. The protein concentration decreased significantly (p<0.05) from day 1 (2.5±0.08g/dl) to day 4 (1.7±0.09g/dl) in samples stored without vitamin E. The decrease in protein concentration may be due to protein degradation. This finding agrees with the discovery of Soto, 1999 who reported degradation of protein during storage. The protein concentration in samples stored with vitamin E showed non-significant (p>0.05) decrease from day2 (2.5±0.03g/dl) to day 4 (2.40±0.01g/dl). The only accountable reason is the antioxidant activity of vitamin E in preventing protein degradation.

From the results of this study we therefore strongly suggest that vitamin E should be added to blood after collection to reduce the effect of oxidation especially in situations where there is inconsistent power supply. This will help to reduce the errors in laboratory results.

REFERENCES:

1. Abdoljalal, M.C. (2006). Alterations in plasma lipid peroxidation and antioxidant status during storage of blood. Parkinstan J. Bio.Sci., 9:2500-2523

2. Amadi, B.A., Agomuo, E.N. and Ibegbulem, C.O. (2004). Research Methods in Biochemistry. Supreme Publisher, Owerri, Nigeria.

3. Bond, B.E. (1984). Free radicals. Radio. Biol. Med., 8:545-565

4. Chung, W.Y., Chung, J.K., Szeto, Y.T., Tomlinson, B. and Benzie, I.F. (2001). Plasma ascorbic acid: measurement, stability and clinical utility revisited. Clin. Biochem., 34:623-627.

5. Das, B.S., Thumham, D.I., Patrick, J.K., Das, D.B., Satpathy, R. and Bas, T.K. (1990). Increased plasma lipid peroxidation in riboflavin deficient malaria infected children. Am. J., 5:859-863

6. Halliwell, B. (1993). The Biochemistry of Oxygen free radicals: In free radicals in tropical diseases (Aruroma, O.eds). Hawood. Acada.Pub.London p1

7. Lee, Y.J. and Shacter, E. (1999). Oxidative stress inhibits Apoptosis in human lymphoma cells. J. Biol. Chem., 274: 19792-19798

8. Pey, A., Baborido, A. and Blazquez, I. (2003). Effect of prolonged stanozolol treatment on antioxidant enzyme activities, oxidative stress markers in rat liver. J.Steroid Biochem. and Mol.Biol., 8:269-277

9. Soto, C. (1999). Alzheimer’s and Prion disease as disorders of protein conformation. Implications for the design of noverl therapeutic approaches. J. Mol. Med., 77:412

10. Traber, M.G. and Atkinson, J. (2007). Vitamin E: function and metabolism. FASEB. J., 13:1145-1155.

11. Wolf, G. (2005). The discovery of antioxidant, function of vitamin E. J.Nutr., 135:363-366.

12. Wolff, S.P., Garner, A. and Dean, R.T. (1986). Free radicals, lipids and proteins degradation. ZIBS. 11:27-31.

Received on 07.05.2012 Modified on 02.06.2012

Asian J. Research Chem. 5(6): June, 2012; Page 765-766