2.2.3. MDA content (lipid peroxidation LPO)
This assay is used to determine MDA levels in liver tissue as described by Okhawa et al[12]. The tissue was homogenized (5%W/V) in 50 mM phosphate buffer (pH 7.0) containing 0.1 mM ethylene diamine tetraacetic acid (EDTA).The homogenates were centrifuged at 10 000 rpm for 10 min at 0 oC in cold centrifuge. The separated supernatant part was used the estimation 200 毺L of the tissue extract was added to 50 毺L of 8.1% sodium dodecyl sulphate (SDS), vortexed and incubated for 10 min at room temperature. 375 毺L of 20% acetic acid (pH 3.5) and 375 毺L of thiobarbituric acid (0.6%) were added and placed in boiling water bath for 60 min. the sample were allowed cool at room temperature. A mixture of 1.25 ml of butanol: phyridine (15: 1) was vortexed and centrifuged at 1000 rpm for 5 min. the colored layer (500 毺L) was measured at 532 nm using 1,1,3,3,- tetraethoxypropane as a standard. The values were expressed in 毺 moles of
malondialdehyde formed/ gram wet weight of the tissue.