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 of the decay rate of the superoxide adduct of 5,5-dimethylpyrroline N-oxide (DMPO) revealed that the EPR signal intensity of the system is proportional to the superoxide generation rate. Rapid depletion of oxygen in the sample was utilized to terminate superoxide generation so that the decay rate of the DMPO superoxide adduct (DMPO-OOH) could be determined. For this decay measurement, a controlled atmosphere EPR cavity was developed and was used with an open-air sample cell. Superoxide generation rates determined with this method for stimulated neutrophils 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 supplied from the gas phase is utilized as a source of superoxide.