Biochemical Analysis
Protein determination in the tissue was performed by Biuret protein analysis using bovine serum albumin as a standard [14].
MDA, an indicator of lipid peroxidation, was assessed according to the method described by Uchiyama and Mihara [15]. The rat kidney sample was homogenized in a 1.15% KCl solution on ice for 1 minute at 15000 rpm to obtain a 10% homogenate. This homogenate was used directly in the MDA analysis, and the results were expressed as nmol/gram tissue.
GSH was determined according to Ellman’s method [16]. The rat kidney sample was homogenized on ice to form a 10% homogenate at 15000 rpm for 1-2 minutes. The homogenate was then centrifuged at 3000 rpm, +4 degrees, for 15 minutes. The resulting supernatant TCA solution was added, mixed, and centrifuged again, making the sample ready for GSH analysis, and the results were expressed as nmol/g tissue.
Tissue SOD activity was measured according to the method described by Sun et al. [17]. The rat kidney sample was homogenized on ice for 1 minute at 15000 rpm to obtain a 10% homogenate. The homogenate was then centrifuged at 10000 rpm for 20 minutes. A 3:5 ratio of chloroform/ethanol (3 parts chloroform to 5 units of ethanol) was added to the supernatant, and the samples were centrifuged again at 5000 rpm at +4 degrees for 20 minutes. The top clear white chloroform phase was carefully taken with a pipette and used in Cu/Zn-SOD analysis. The enzyme activity was expressed as U/g protein.
Tissue CAT activity was measured according to Luck’s method [18]. The rat kidney sample was homogenized on ice for 1 minute at 15000 rpm to obtain a 10% homogenate. The homogenate was then centrifuged at 10000 rpm for 20 minutes and used for the supernatant CAT analysis. The results were presented as K/g protein.