Malondialdehyde Quantification in Splenic Tissue

The thiobarbituric acid reactive substances were used for measuring the malondialdehyde (MDA) levels [30 –32 (link)]. Briefly, the splenic tissue was homogenized in phosphate-buffered saline using a tissue homogenizer (IKA, RW 20.n, Germany), and the homogenate obtained was centrifuged. A volume of 0.5 mL supernatant of the homogenate was mixed with a 2 mL reagent (0.37% thiobarbituric acid, 0.24 N HCl, and 15% TCA). The mixture was boiled at 100°C for 15 minutes, cooled, and centrifuged at 3,000 rpm for 10 minutes. The absorbance of the supernatant was measured at 532 nm, and the unknown concentration of the TBA-MDA adducts was calculated from the standard curve generated using different concentrations of 1, 1, 3, 3-tetra methoxy propane and expressed as nmol/mL.