Detection of phagocyte-derived free radicals with spin trapping techniques: Effect of temperature and cellular metabolism

Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20°C with either stimuli, neutrophil superoxide production was manifested predominantely as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37°C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH₃) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.