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.