Measurement of antioxidant enzyme activities

The activities of antioxidant enzymes, including GPx, CAT and SOD in the heart tissues of mice were assayed^67,68.

GPx activity was examined by measuring the regeneration of GSH from GSSG based on the action of glutathione reductase with NADPH. The heart homogenates were added into 100 mM phosphate buffer containing 0.5 mM EDTA, 1.0 mM NaN₃, 0.25 mM NADPH, 2.25 mM GSH and 1.0 U ml⁻¹ glutathione reductase. The change of NADPH optical density was assayed through recording the absorbance at 340 nm for 2 min. GPx activity was calculated with a molar extinction coefficient for NADPH of 6.22 × 10³ M⁻¹ cm⁻¹.

The activity of CAT was measured through adding the tissue homogenates into 100 mM phosphate buffer containing 10 mM H₂O₂. Absorbance values were recorded at 240 nm for 30 s, and the CAT activity was expressed according to a H₂O₂ extinction coefficient of 43.6 × 10³ M⁻¹ cm⁻¹.

Total SOD activity was measured based on the reaction mixture including 0.1 mM EDTA, 10 mM acetylated cytochrome c, 50 mM hypoxanthine, and 8 mU xanthine oxidase in 50 mM potassium phosphate. Absorbance was read at 418 nm for 2 min. SOD activity was expressed as U per min per mg protein.