Do human neutrophils make hydroxyl radical? Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry

Using electron paramagnetic resonance spectrometry and the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), neutrophil free radical production in response to phorbol myristate acetate and opsonized zymosan was investigated. Using phorbol myristate acetate and zymosan (3 mg/ml), the superoxide spin-trapped adduct 2-2-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH) and the hydroxyl spin-trapped adduct 2-2-demethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) were detected. Only DMPO-OH was observed with zymosan (0.5 mg/ml). Hydroxy radical production in the presence of dimethylsulfoxide (Me₂SO) and DMPO yields 2,2,5-trimethyl-1-pyrrolidinyloxyl. The only 2,2-trimethyl-1-pyrrolidinyloxyl detected following neutrophil stimulation was that expected from DMPO-OOH degradation. Superoxide dismutase but not catalase inhibited generation of all three spin-trapped adducts. These data indicate that DMPO-OH arose from DMPO-OOH degradation and does not represent hydroxyl radical production. Under certain conditions DMPO-OH is the predominant spin-trapped adduct resulting from neutrophil superoxide production, perhaps due to cellular bioreduction of DMPO-OOH to DMPO-OH. Cytochalasin B, which prevents phagosome closure, inhibited zymosan-stimulated neutrophil oxygen consumption and electron paramagnetic resonance superoxide detection. No hydroxyl radical was detected. Spin trapping with DMPO appears to detect intraphagosomal free-radical formation.