Levels of malondialdehyde (MDA)

The amount of lipid peroxidation was indicated by the content of thiobarbituric acid reactive substances (TBARS) in the brain homogenates as shown by MDA. Tissue MDA was determined by following the production of thiobarbituric acid reactive substances as described previously [39], which was reported by Alirezaei et al. [5]. In short, 40 µl of homogenate was added to 40 µl of 0.9 % NaCl and 40 µl of deionized H₂O, resulting in a total reaction volume of 120 µl. The reaction was incubated at 37 °C for 20 min and stopped by the addition of 600 µl of cold 0.8 M hydrochloride acid, containing 12.5 % trichloroacetic acid. Following the addition of 780 µl of 1 % TBA, the reaction was boiled for 20 min and then cooled at 4 °C for 1 h. In order to measure the amount of TBARS produced by the homogenate, the cooled reaction was spun at 1500×g in a microcentrifuge for 20 min and the absorbance of the supernatant was spectrophotometrically (S2000 UV model; WPA, Cambridge, UK) read at 532 nm, using an extinction coefficient of 1.56 × 10⁵/M cm. The blanks for all of the TBARS assays contained an additional 40 µl of 0.9 % NaCl, instead of homogenate as just described. MDA results were expressed as nanomoles per milligram of tissue protein (nmol/mg protein).