2475 multi fluorescence detector

The formation of MDA was used as an indicator of lipid peroxidation. MDA concentrations were measured according to the method described by Mateos et al. [28] . For each sample 500 mL of the brain homogenate was mixed with 100 mL of 0.01% solution of butyl-4-hydroxytoluene in acetone and 500 mL of 5% trichloroacetic acid (TCA).The samples were incubated for 10 min at room temperature and centrifuged for 10 min at 4000 g. Then 500 mL of supernatant was mixed with 4.5 mL of 15% (w/v) TCA solution in 25 mM HCl and 0.375% (w/v) thiobarbituric acid (TBA) in 25 mM HCl (hydrochloric acid) and samples were kept for 20 min in a boiling water bath. After cooling, the samples were centrifuged at 12 000g for 10 min. From each sample 200 mL volume of supernatant was transferred to a vial, then a 50 mL aliquot was injected into the high-performance liquid chromatography (HPLC) system. Excitation and emission wavelengths were of 532 and 553 nm, respectively. The mobile phase was composed of methanol in 50 mM ammonium formate buffer of pH 6.5 (40: 60, v/v), the flow rate was 1 ml/min. MDA concentration was measured by HPLC connected to a fluorescence detector (binary HPLC pump 1525 and 2475 multi fluorescence detector, Waters, Milan, Italy), equipped with a Polaris C18 column, 150 Â 4.6 mm ID, particle size 5 mm (Varian, USA). The concentration of MDA was expressed in nmol/mg protein.