Detection by ESR of DMPO hydroxyl adduct formation from islets of langerhans

Abstract

Electron spin resonance (ESR) spectroscopy together with spin trapping techniques and the application of state-of-the-art loop gap resonators was used to provide a direct measure of spontaneous oxygen radical production by homogenates of freshly isolated and cultured rat pancreatic islets. Using the spin trap agent, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), we were able to detect production by islets of an ESR-sensitive 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 consistent with the DMPO-OH adduct. The amplitude of the signal was decreased 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 dimethylthiourea). 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 superoxide dismutase, but was eliminated in a concentration-dependent manner by either potassium cyanide or catalase (but not heat-inactivated catalase). These observations suggest that the origin of the DMPO-OH arose not from free hydroxyl radicals but primarily from endogenous hydrogen peroxide production perhaps of mitochondrial origin. The development of this technology has implications for the potential measure of oxygen radical production in islet homogenates under pathologic conditions as well as to the application of other cell culture systems.