Lipid peroxidation assay

The end product of lipid peroxidation was estimated by measuring the level of MDA according to the method of Satoh K.³² 0.5 ml of tissue homogenate was added to 1.5 ml of 10% TCA, vortexed and incubated for 10 min at room temperature. 1.5 ml of supernatant and 2 ml of thiobarbituric acid (0.67%) were added and placed in a boiling water bath in sealed tubes for 30 min. The samples were allowed to cool at room temperature. 1.25 ml of n-butanol was added, vortexed and centrifuged at 2000 g for 5 min. The resulting supernatant was removed and measured at 532 nm on a spectrophotometer. MDA concentrations were determined by using 1,1,3,3-tetraethoxypropane as standard. MDA concentration was expressed as µmol/mg protein.