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Effect of Simvastin Treatment on MDA Level in Rat Plasma Measured by Sprectrophotometric and HPLC-MS Analysis

Statins, inhibitors of hydroxy-methylglutaryl coenzyme A reductase, are considered first-line therapy for treatment of hypercholesterolemia. However, beneficial effects of statin treatment on cardiovascular morbidity and mortality has been not entirely explained by the reduction in low density lipoproteins (LDL) level but also in antioxidant activity of statins that contribute to their protective cardiovascular effects. There is an extensive evidence that links hypercholesterolemia with increased lipid peroxidation and increased oxidative stress. Several studies correlates levels of LDL oxidation with circulating malondialdehyde (MDA) which is secondary oxidation product . Mainly level of MDA in animal plasma and tissue was determined by the thiobarbituric method which is based on condensation of MDA with two molecules of 2-thiobarbituric acid (TBA) and method was widely used to measure the extent of oxidative deterioration of lipids in various biological systems. The absorbance of the complex is usually measured by spectrophotometry or by spectrofluorometry directly or by specific techniques, also based on TBA adduct, where HPLC separation with spectrophotometric or spectrofluorometric or MS detection was employed. The aim of the present study was to examine effect of simvastatin (SIMV) treatment on LDL oxidation based on the measuring of the rat MDA plasma level as parameter of oxidative stage by two different analytical techniques. Comparison of results between this analytical approaches would contribute in decision which technique is more reliable and accurate for further investigation on statin antioxidant action. Material and Methods. Forty six male Wistar rats (weight 170-200 g), were divided in two control groups (n=7 each) and four experimental groups (n=8 each). Experimental groups were on SIMV treatment which was given orally in the doses 10 and 50 mg/kg/day and for the period of 3 weeks. At the end of drug treatment animals from first two groups were sacrificed and blood samples were taken and other two groups were left additional 9 days and than sacrificed and samples were taken. The level of MDA was measured using the spectrophotometrically or by reverse phase HPLC-MS method. MDA levels are expressed as μ mol concentrations using calibrating curves and expressed as percentage of decrease of MDA level comparing to negative control. Results. SIMV in both doses decreases MDA level in plasma. Spectroscopic analysis of MDA level after SIMV application have shown decrease in plasma concentration by 9, 7% and 16, 1% after administration of 10 mg/kg/day and 50 mg/kg/day dose respectively after 21 day of application. When the same doses and same application period was applied but blood was taken after 30 days, 32, 2% and 64, 5% decrease of MDA level were recorded. However, HPLC-MS analysis has shown 12, 2% and 17, 3% decrease of MDA level after 10 mg/kg/day and 50 mg/kg/day of SIMV dose after examining the same plasma samples. Conclusion. Our results have shown that SIMV shows antioxidant action which was indicated by lowering one of the oxidative markers - MDA. We suggest that both methods, spectroscopic or HPLC-MS gives results that have similar direction, but with difference in magnitude or sensitivity.

Simvastatin; MDA; rat plasma; HPLC-MS analysis

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