Simultaneous monitoring of superoxide radical anion and hydrogen peroxide formed in a hypoxanthine plus xanthine oxidase system, using high-throughput HPLC method.
a, concentration of HE, 2-OH-E+, CBA, and COH from incubation of 20 μm HE and 100 μm CBA in the presence and absence of hypoxanthine (HX; 200 μm) and xanthine oxidase (XO; 0.07 milliunits/ml). b, same as a, but HPLC chromatograms of samples analyzed after incubation for 2 h. Line 1, HE alone; line 2, HE + CBA; line 3, HE + CBA + XO; line 4, HE + CBA + XO + HX. c and d, same as a and b, but full system (HX + XO) was used and the effect of SOD (0.1 mg/ml) or CAT (0.1 kilounit/ml) was tested. Line 1, HE + CBA + XO + HX; line 2, with SOD; line 3, with CAT. e and f, same as a and b, but full system (HX + XO) was used, and the oxidation of probes alone was compared with co-incubation with both probes. Line 1, HE + CBA + XO + HX; line 2, HE + XO + HX; line 3, CBA + XO + HX.