Determination of superoxide generated by human polymorphonuclear leukocytes by ESR and its clinical application

Abstract

Electron spin resonance (ESR) is regarded as the least ambiguous method for the detection of free radicals. Using spin trapping technique with 5, 5-dimethyl-pyrroline-N-oxide (DMPO) we measured superoxide generated by stimulated polymorphonulcear leukocytes (PMN) . The results were compared to those by chemiluminescence study with Cypridina luciferin analog (CLA) which is highly sensetive to superoxide. ESR spectrum was obtained by JEOL-JES-FE2XG ESR spectrometer. The intensity of the signal of DMPO-OOH adduct was measured as ratio to the intensity of Mn²⁺ signal. Phorbol myristate acetate or opsonized zymosan were used as stimulants of PMN. The ESR signal of DMPO-OOH was completely inhibited by superoxide dismutase, but not affected by catalase or sodium azide. The relative intensity of DMPO-OOH signal and the maximal increase of CLA-dependent chemiluminescence were increased in proportion to the xanthine oxidase unit in hypoxanthine +xanthine oxidase system, and to the cell concentration in PMN system. There were positive correlation between the relative intensity of the ESR signal of DMPO-OOH and CLA-dependent chemiluminescence by stimulatd PMN from patients. By ESR assay, increased generation of superoxide by PMN in patients with fairly controlled diabetes was shown.