Superoxide dismutase sod activity

Rat pheochromocytoma (PC12) cell lines were a kind gift from the Institute of Neuroscience, Soochow University. All cell lines were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Langley, OK, USA). Cells were seeded in culture flasks, 24- or 96-well plates, to a confluence of 60–70%. Cells were treated for 24 hrs with MPP+ (Sigma-Aldrich, St. Louis, MO, USA) (0.5 mMol/L) in RPMI-1640 medium, Paeoniflorin (50 uM) (Sigma-Aldrich, St. Louis, MO, USA), and Rapamycin (0.2 μg/mL) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteasome activity (Promega, Madison, WI, USA), catalase (CAT) activity, and superoxide dismutase (SOD) activity (Cayman Chemical, Ann Arbor, Michigan, USA) were measured with the appropriate assay kits according to the manufacturer's protocols.

Plasma thiobarbituric acid reactive substances (TBARS; Cayman Chemical, Ann Arbor, MI, USA), Catalase activity (Cayman Chemical, Ann Arbor, MI, USA), Superoxide Dismutase (SOD) activity (Cayman Chemical, Ann Arbor, MI, USA), and Hydrogen Peroxide (Amplex UltraRed assay; Molecular Probes, Eugene, Oregon, USA) were analyzed by a spectrophotometer (xMark microplate spectrophotometer, Bio-Rad Laboratories, Mississauga, ON, Canada) in duplicate as per the manufacturer's instructions. One unit of Catalase activity is defined as the amount of enzyme required to cause the formation of 1.0 nmol of formaldehyde per minute at 25°C. One unit of SOD activity is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. TBARS is expressed in nmol/ml of malondialdehyde equivalents. Plasma hydrogen peroxide is expressed in nmol/ml. Intra-assay coefficients of variation were determined from each duplicate for all participants and resulted in a coefficient of variation of 1.05, 1.99, 4.91, and 1.88% for TBARS, SOD, catalase and hydrogen peroxide, respectively. Inter-assay coefficients of variation for assay standards were 0.65, 3.65, 2.30, 1.20, for TBARS, SOD, catalase, and hydrogen peroxide, respectively.