Estimated mean generation of superoxide anion by the hypoxanthine/xanthine oxidase (X/XO) systems used in this study
| System | Hypoxanthine (mM) | Xanthine oxidase (mU) | Estimated generation of superoxide (nmol/min/ml medium) |
|---|---|---|---|
| In order to evaluate estimated mean generation of superoxide anion under controlled conditions (that is, known values of available substrate, enzyme, and enzyme activity, pH, temperature, volume of medium, etc) the methods based on nitroblue of tetrazolium (NBT) reduction21 or dichloro-fluorescein (DCF) fluorescence20 were used. Kinetics of NBT reduction in culture medium at 37°C were found to be essentially linear until substrate consumption in X/XO II and III systems as well as, at least for 45–50 minutes, in the X/XO I system also. The DCF method (performed by taking samples of culture medium at desired time points after starting superoxide generation) was then essentially used to establish the time of disappearance of reactive oxygen species dependent DCF fluorescence, in the presence or absence of 100 U of superoxide dismutase, with the X/XO I system designed to generate low levels of superoxide over a long period of time. | |||
| X/XO I | 0.4 | 2 | 0.8–1.2 |
| X/XO II | 0.2 | 20 | 7–10 |
| X/XO III | 0.05 | 100 | 25–50 |