2.8. Assay of malondialdehyde

Concentration of malondialdehyde (MDA) in hemolysate, an index of lipid peroxidation, was determined spectrophotometrically (Ohkawa et al., 1979). A mixture of 200 μL of 8% sodium dodecyl sulfate, 200 μL of 0.9% thiobarbituric acid, and 1.5 mL of 20% acetic acid were added to a 200 μL supernatant of hemolysate and 1.9 mL of distilled water was used to make the volume up to 4 mL. After boiling for 1 h, the mixture was cooled and 5 mL of an n-butanol and pyridine (15:1) solution was added. The mixture was centrifuged at 5000 rpm for 15 min and the absorbance was measured at 532 nm. MDA levels were calculated using tetraethoxypropane as standard.