Xanthine solutions

SOD levels were examined as described previously [6 (link)]. A SOD enzyme solution (100 units/mL, Sigma-Aldrich, St. Louis, MO, USA) was used as a standard solution. The supernatant was mixed with a reaction cocktail (pH 7.8), which contained a 216 mM potassium phosphate buffer, pH 7.8 (K₂HPO₄, Ajax Finechem, Auckland, New Zealand), 10.7 mM ethylenediaminetatraacetic acid or EDTA (Sigma-Aldrich, St. Louis, MO, USA), 1.1 mM cytochrome C (Sigma-Aldrich, St. Louis, MO, USA) and 0.108 mM xanthine solutions (Sigma-Aldrich, St. Louis, MO, USA). A xanthine oxidase enzyme (XOD) solution (0.1 units/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to the mixture and then vortexed. The OD was read at 540 nm and the absorbance was read at t = 0 min and t = 5 min.

A standard curve was prepared using 100 units/mL of SOD enzyme solution (Sigma-Aldrich, MO, USA) as a standard compound. SOD levels were determined by adding the supernatant into the reaction cocktail (pH 7.8) containing 0.108 mM xanthine solutions (Sigma-Aldrich, MO, USA), 216 mM potassium phosphate buffer (pH 7.8, Ajax Finechem, Auckland, New Zealand), 1.1 mM cytochrome C (Sigma-Aldrich, MO, USA), and 10.7 mM ethylenediaminetatraacetic acid (Sigma-Aldrich, MO, USA). After that, the reaction mixture was reacted with 0.1 units/mL of xanthine oxidase enzyme solution (Sigma-Aldrich, MO, USA) before analyzing the absorbance at 540 nm at 0 and 5 min in triplicate.