Is superoxide demonstration by electron-spin resonance spectroscopy really superoxide?

Abstract

A recent study has indicated that the generation of an oxygen radical in freeze-clamped myocardium on reperfusion can be directly demonstrated using electron-spin resonance spectroscopy (ESR). However, the results need to be analyzed with caution, since artifactual radicals are misleading problems common to this method. To test whether that reported superoxide is truly the biologically existing radical or an artifactual radical, we performed experiments using isolated, perfused rat and rabbit hearts and open-chest canine hearts subjected to ischemia/reperfusion. Radicals were freeze trapped at 77 degrees K, and ESR measurements were made. The ESR spectra exhibited four free radicals. Among these, two radicals which had been previously claimed as superoxide and a nitrogen-centered radical were shown as mechanically yielded artifactual radicals. These were produced by pulverization of the frozen sample. In artifact-free samples, superoxide could not be detected. The radicals native to the myocardium were identified as coenzyme Q10-. and another radical the species of which remains unclear.