2.7 Oxidative Stress Parameters Analysis
2.7.1 Superoxide Dismutase (SOD)
Superoxide dismutase activity was measured by Marklund and Marklund [58]. After
the homogenization of tissues in 100 mM Tris-HCl buffer (pH 7.4), the contents
were centrifuged at 12,000 rpm for 15 min at 4
C. The obtained supernatant of
50 mL was added to the reaction solution (50 mM Tris-HCl buffer, pH 8.4 with
1 mM EDTA and 2.64 mM pyrogallol), and the absorbance was read at 420 nm in
UV spectrophotometer.
2.7.2 Lipid Peroxidation (LPO)
LPO was measured following thiobarbituric acid reactive substances (TBARS)
assay [59]. Pooled tissue samples were homogenized in 100 mL ice-cold potassium
phosphate buffer (pH 7.4), and 100 mL of 5% trichloroacetic acid (TCA) was added.
Shortly the contents were kept undisturbed on ice for 10 min, and 100 mL of 0.67%
thiobarbituric acid was added. After centrifugation (2,200 g, 10 min at 4
C), 250 mL
of supernatant was incubated in boiling water bath for 10 min preceded by cooling,
and the absorbance was determined at 535 nm in UV spectrophotometer.
2.7.3 Catalase (CAT)
The catalase activity was estimated by adapting the method of Sinha [60]. Briefly,
the sampled tissues were homogenized manually in Tris-HCl buffer (100 mM,
pH 7.4) and cold centrifuged at 12,000 rpm for 15 min. After centrifugation,
100 mL of supernatant with 3 mL of reaction mixture (containing 5% potassium
dichromate and acetic acid (1,3) and phosphate buffer (10 mM, pH 7.0)) was
incubated in water bath for 20 min, and the absorbance was read at 570 nm in UV
spectrophotometer.
2.7.4 Glutathione S-Transferase (GST)
GST activity was measured by the method of Habig and Jakoby [61]. From each
treatment, the sampled tissues were homogenized in potassium phosphate buffer
(0.1 M, pH 6.5). Subsequently the homogenate is centrifuged at 9,000 g for 30 min at
4
C, and 50 mL of the obtained supernatant is added to 100 mL of the reaction
solution (10 mM GSH and 60 mM 1-chloro2, 4-dinitrobenzene). The absorbance of
the samples was read at 340 nm in UV spectrophotometer for 5 min.
198
S. Siddeswaran et al.
2.7.1 Superoxide Dismutase (SOD)
Superoxide dismutase activity was measured by Marklund and Marklund [58]. After
the homogenization of tissues in 100 mM Tris-HCl buffer (pH 7.4), the contents
were centrifuged at 12,000 rpm for 15 min at 4
C. The obtained supernatant of
50 mL was added to the reaction solution (50 mM Tris-HCl buffer, pH 8.4 with
1 mM EDTA and 2.64 mM pyrogallol), and the absorbance was read at 420 nm in
UV spectrophotometer.
2.7.2 Lipid Peroxidation (LPO)
LPO was measured following thiobarbituric acid reactive substances (TBARS)
assay [59]. Pooled tissue samples were homogenized in 100 mL ice-cold potassium
phosphate buffer (pH 7.4), and 100 mL of 5% trichloroacetic acid (TCA) was added.
Shortly the contents were kept undisturbed on ice for 10 min, and 100 mL of 0.67%
thiobarbituric acid was added. After centrifugation (2,200 g, 10 min at 4
C), 250 mL
of supernatant was incubated in boiling water bath for 10 min preceded by cooling,
and the absorbance was determined at 535 nm in UV spectrophotometer.
2.7.3 Catalase (CAT)
The catalase activity was estimated by adapting the method of Sinha [60]. Briefly,
the sampled tissues were homogenized manually in Tris-HCl buffer (100 mM,
pH 7.4) and cold centrifuged at 12,000 rpm for 15 min. After centrifugation,
100 mL of supernatant with 3 mL of reaction mixture (containing 5% potassium
dichromate and acetic acid (1,3) and phosphate buffer (10 mM, pH 7.0)) was
incubated in water bath for 20 min, and the absorbance was read at 570 nm in UV
spectrophotometer.
2.7.4 Glutathione S-Transferase (GST)
GST activity was measured by the method of Habig and Jakoby [61]. From each
treatment, the sampled tissues were homogenized in potassium phosphate buffer
(0.1 M, pH 6.5). Subsequently the homogenate is centrifuged at 9,000 g for 30 min at
4
C, and 50 mL of the obtained supernatant is added to 100 mL of the reaction
solution (10 mM GSH and 60 mM 1-chloro2, 4-dinitrobenzene). The absorbance of
the samples was read at 340 nm in UV spectrophotometer for 5 min.
198
S. Siddeswaran et al.