DETERMINATION OF THE RATE OF SUPEROXIDE GENERATION FROM BIOLOGICAL-SYSTEMS BY SPIN-TRAPPING - USE OF RAPID OXYGEN DEPLETION TO MEASURE THE DECAY-RATE OF SPIN ADDUCTS

A method was developed to measure the superoxide generation rate from biological systems using the spin trapping method. Kinetic treatment o f the decay rate of the superoxide adduct of 5,5-dimethylpyrroline N-o xide (DMPO) revealed that the EPR signal intensity of the system is pr oportional to the superoxide generation rate. Rapid depletion of oxyge n in the sample was utilized to terminate superoxide generation so tha t the decay rate of the DMPO superoxide adduct (DMPO-OOH) could be det ermined. For this decay measurement, a controlled atmosphere EPR cavit y was developed and was used with an open-air sample cell. Superoxide generation rates determined with this method for stimulated neutrophil s and for the hypoxanthine-xanthine oxidase system were comparable to those obtained with the cytochrome c reduction method. This method is specifically applicable to the system in which dissolved oxygen suppli ed from the gas phase is utilized as a source of superoxide.