Thiobarbituric acid assay kit

The lung tissue GSH-PX and SOD activity was determined on frozen tissue using Xanthine Oxidase assay kits (Jiancheng Bioengineering Institute, Nanjing, China). The MDA content was determined on frozen lung tissue by use of the thiobarbituric acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China).

The frozen hearts were homogenized in 1 mL PBS (pH 6.0) supplemented with 0.5% hexadecyltrimethylammonium hydroxide and centrifuged at 12,000 rpm at 4°C for 20 min. The supernatants were recovered for the determination of MPO activity and MDA level. The protein concentration in the supernatants was determined with a Bio-Rad protein assay kit (Hercules, CA, USA) according to manufacturer's instructions. The MPO activity was detected by using a commercial MPO detection kit (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer's instructions. One unit of MPO activity is defined as degrading 1 μmol of hydrogen peroxide at 37°C. The MPO activity was determined by measuring the absorbance at 460 nm on a PerkinElmer UV/VIS spectrophotometer (Waltham, MA, USA) and expressed as units per gram of total proteins (U/g proteins).
MDA activity was measured by thiobarbituric acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer's instructions. Briefly, the supernatant was incubated with thiobarbituric acid. The absorbance at 532 nm was determined on a PerkinElmer UV/VIS spectrophotometer (Waltham, MA, USA). The MDA level was calculated in the format of nmol/mg proteins.