Bovine erythrocyte sod

SOD activity was determined using an SOD assay kit according to the manufacturer's protocol (Dojindo Laboratories, Kumamoto, Japan). The standard concentration was serially diluted from 100 to 0.01 U/mL of bovine erythrocyte SOD (Sigma Aldrich).
Catalase activity was determined as previously described [20 (link)]. The absorbance of the purple formaldehyde adduct was measured at 550 nm using a spectrophotometer (Molecular Devices Corp.).

We obtained hippocampal homogenates from stored hippocampal tissue (n = 6 to 9 in each group) to verify parameters for oxidative stress and analyze antioxidant components. We used commercial kits for all assays, and all measurements were taken according to the manufacturer’s instructions. The degree of lipid peroxidation was measured by the level of malondialdehyde (MDA) using Lipid Peroxidation Colorimetric/Fluorometric Assay Kit (Catalog ^# K739, BioVision Inc., Milpitas, CA, USA). The assay’s final products were analyzed and quantified using a spectrophotometer (SoftMax, Ver. 5.4, Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 530 nm. Total GSH content was determined using GSH Assay Kit (Catalog ^# CS0260, Sigma-Millipore) and the absorbance was calculated at a wavelength of 405 nm using the spectrophotometer (VersaMax. Ver. 5.1, SoftMax). Superoxide dismutase (SOD) activities were measured using a commercially available kit. As standards, dilutions of bovine erythrocyte Sod (Sigma-Aldrich, St. Louis, MO, USA) in concentrations ranging from 0.01 to 50 U/mL were used. Amplex™ Red Catalase Assay Kit (Catalog ^# A22180, Thermo Fishers, Waltham, MA, USA) was used to measure catalase activities. The absorbance was measured at a wavelength of 450 nm using a plate reader.

Catalase activities in cerebral cortex and hippocampus were assayed as described previously [24] (link). Briefly, 150 µL of phosphatase buffer (250 mM, pH 7.2),150 µL of 12 mM methanol and 30 mL of 44 mM H₂O₂ were mixed with 300 µL of each homogenate sample/standard solutions in a 13×100 mm test tube. The reaction was allowed to proceed for 10 to 20 min and was stopped by the addition of 450 mL of Purpald solution (22.8 mM of Purpald in 2 N potassiumhydroxide). The mixture was kept for 20 min at 25°C, followed by addition of 150 µL of potassium periodate (65.2 mM in 0.5 N potassiumhydrate). The absorbance of the purple formaldehyde adduct formed was measured at 550 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA).
SOD activity in cerebral cortex and hippocampus was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan), according to the protocol from the manufacture. Bovine erythrocyte SOD (Sigma) was used as a standard (0.01 - 20 U/mL).