The Effect of Iron on the Distribution of Superoxide and Hydroxyl Radicals as Seen by Spin Trapping and on the Superoxide Dismutase Assay

Using the spin trap 5,5‐dimethyl‐1‐pyrroline‐1‐oxide we have demonstrated the presence of OH in the xanthine‐xanthine oxidase system when iron and/or iron‐EDTA (ethylenediamine‐tetraacetic acid) is present. With increasing iron (or iron‐EDTA) concentration the intensity of the O 2 ‐ spin adduct decreased while that of OH increased. However, use of diethylenetriaminepentaacetic acid (DETAPAC) as a metal chelator in the reaction mixture suppressed the OH spin adduct signal while maintaining the intensity of the signal from the O 2 ‐ spin adduct. Use of EDTA to eliminate the interfering effects of metal ions in the superoxide dismutase assay employing xanthine oxidase and nitroblue tetrazolium introduces an artifact from the iron present. The interference in the assay from metal ions, including iron, can be eliminated with use of DETAPAC as a metal chelator. Thus, it is possible to make comparisons of measured superoxide dismutase activities even when there are variations in the amount of iron present in the samples.