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

• Published in: Ensho Vol. 8; no. 5; pp. 443 - 447
• Main Authors: Morita, Yutaka, Itani, Kenji, Tanigawa, Toru, Yoshida, Norimasa, Miyagawa, Haruo, Takemura, Toshiki, Sugino, Shigeru, Ueda, Shigenobu, Yoshikawa, Toshikazu, Tainaka, Kenzo, Kondo, Motoharu
• Format: Journal Article
• Language: Japanese
• Published: The Japanese Society of Inflammation and Regeneration 1988
• Subjects: chemiluminescence; electron spin resonance; polymorphonuclear leukocyte; superoxide
• ISSN: 0389-4290; 1884-4006
• DOI: 10.2492/jsir1981.8.443

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.