Sample preparations Rats were sacrificed by decapitation and the hippocampi were removed quickly and immediately stored at ?20 °C. The tissue samples were thawed and homogenized in 1 ml of distilled water. The aliquots of the homogenates were separated and used to determine protein concentration, whereas the remaining homogenates were centrifuged at 3000 s?1 for 10 min. The supernatants were used for enzyme assays. Proteins were determined by the Bio Rad protein assay kit using bovine serum albumin as a standard according to Bradford
SOD assay In order to measure SOD activity, the method employing xanthine and xanthine oxidase to generate superoxide radicals was used. These radicals react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye (Erakovi? et al., 2000). The degree of the inhibition of this reaction was measured to asses SOD activity. The standard assay substrate mixture contained 0.05 mmol/l xanthine 0.05, 0.025 mmol/l INT and 0.94 mmol/l EDTA, in 40 mmol/l CAPS (pH 10.2). The sample aliquot (50 μl) was added to 1.7 ml of the substrate mixture. After 30 s, initial absorbance was recorded and the timer was started. Final absorbance was recorded after 3 min. The reaction was followed at 406 nm. Purified bovine erythrocyte SOD (Randox Laboratories, UK) was used under identical conditions in order to obtain a calibration curve showing the correlation of the inhibition percentage of formazan dye formation and SOD activity. The activity of SOD in the samples was determined from this curve. All samples were diluted with 0.1 mmol/l phosphate buffer pH 7.0 to cause an inhibition between 30 and 60% of the diluent rate (i.e., the uninhibited reaction). One unit of enzyme activity was defined as the quantity of SOD required to cause a 50% inhibition of the absorbance change per minute of the blank reaction (diluent rate).
