A method for the detection of superoxide in biological systems

The ability to detect superoxide in biological milieu is filled with a number of difficult problems. For example, the ferricytochrome c assay method cannot be used in the presence of NADPH-cytochrome P-450 reductase since cytochrome c is preferentially reduced by this enzyme. We have found that the superoxide-dependent oxidation of one particular hydroxylamine, 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine, to its corresponding nitroxide, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl, can be used to quantitate superoxide production by hepatic microsomes and purified enzymes. We determined that this assay method is free from most of the problems inherent in other methods for the identification of superoxide.