DETECTION BY ESR OF DMPO HYDROXYL ADDUCT FORMATION FROM ISLETS OF LANGERHANS

Electron spin resonance (ESR) spectroscopy together with spin trapping techniques and the application of state-of-the-art loop gap resonator s was used to provide a direct measure of spontaneous oxygen radical p roduction by homogenates of freshly isolated and cultured rat pancreat ic islets. Using the spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect production by islets of an ESR-sensiti ve radical signal consisting of a quartet with intensity ratio of 1:2: 2:1 and hyperfine splitting of a(N) = a(H) = 14.9 Gauss, which is cons istent with the DMPO-OH adduct. The amplitude of the signal was decrea sed by decreasing amount of islets and not detected in the absence of islets. Formation of the DMPO-OH adduct was diminished by the hydroxyl radical scavengers (e.g., ethanol, dimethylsulfoxide, and dimethylthi ourea). Only partial attenuation of signal was produced by incubation with an iron chelator or using chelex-treated buffers. The ESR signal was insensitive to the xanthine oxidase inhibitor, oxypurinol, or to s uperoxide dismutase, but was eliminated in a concentration-dependent m anner by either potassium cyanide or catalase (but not heat-inactivate d catalase). These observations suggest that the origin of the DMPO-OH arose not from free hydroxyl radicals but primarily from endogenous h ydrogen peroxide production perhaps of mitochondrial origin. The devel opment of this technology has implications for the potential measure o f oxygen radical production in islet homogenates under pathologic cond itions as well as to the application of other cell culture systems.