2.8. Analysis of erythrocyte antioxidant status

Hemolysate of erythrocyte was prepared by adapting the method of Beutler et al., as mentioned earlier [41]. Erythrocyte suspension (0.2 ml) in saline was added to 1.8 ml of β-mercaptoethanol-EDTA stabilizing solution (0.05 ml of β-mercaptoethanol and 10 ml of neutralized 10% EDTA to a volume of 1 L with water) to obtain 1:20 hemolysate. The tubes containing hemolysate were stored at 4 °C for assay of enzymes within 1–2 h of preparation unless otherwise stated. Hemoglobin in hemolysate was estimated and activity of the enzyme was expressed as international units/gram haemoglobin (IU/gHb) unless otherwise stated. Activity of catalase enzyme was determined considering extinction coefficient of H₂O₂ as 0.071 cm⁻¹ mol⁻¹ and expressed as IU x 104/g Hb at 37 °C. Total reduced glutathione content and glutathione peroxidase enzyme activity was measured, wherein the cell lysate was prepared by adding 2.0 ml of water to 0.2 ml of packed erythrocytes. Superoxide dismutase enzyme activity was assayed after removal of hemoglobin from the hemolysate.