IN VIVO DETECTION OF FREE RADICALS USING NMR

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This project will determine the feasibility of a new NMR methodology for detecting free radical activity in living tissue. The approach will employ the use of a novel free radical spin trap (DEPMPO), recently developed for electron spin resonance (ESR) spectroscopy. Unlike other nitrone spin traps, DEPMPO contains a phosphorus atom, bound closely to its reactive site, which has enabled us to use phosphorus-nuclear magnetic resonance spectroscopy (31P-NMR) for detection. In preliminary experiments we demonstrate that reactions of DBPMPO with specific free radicals result in distinguishable chemical shifts in the phosphorus spectrum. Furthermore, we have found that these shifts represent the "reduced" products of DEPMPO-free radical reactions, which are extremely stable in biological solution. The ability of 31P-NMR to detect the reduced DEPMPO-radical adducts makes it possible to measure the accumulation of free radical activity in the tissue of interest. This greatly enhances the potential sensitivity of the approach for in vivo applications. We hypothesize that spin-trap 31P-NMR technology will allow us to measure free radical activity in living tissue, with sufficient sensitivity to detect and possibly image localized areas of inflammation, as might occur with infection, ischemia-reperfusion, arthritis, inflammatory lung disease and various autoimmune diseases. Therefore, this technology could have a major impact on health care. The specific aims are: 1) To define the characteristic 31P-NMR spectra which occurs from in vitro reactions of DEPMPO with common oxygen- and carbon- based free radicals These experiments involve the generation of free radicals commonly seen in biological systems, analysis of their reactions to form adducts with DEPMPO, the kinetics of adduct formation and decay with both ESR and 31P-NMR, and the determination of their composition by mass spectrometry. 2) To determine the abilIty to detect DEPMPO trapped free radicals for in vitro cell systems using 31P-NMR. We will test the ability of 31P-NMR to detect free radicals generated extracellularly by activation of humin neutrophils. A second series will study isolated hepatocytes undergoing oxidant stress from acetaminophen overdose, resulting in intracellular free radical production. 3) To determine the ability to detect DEPMPO-trapped free radicals in vivo using 31P-NMR In models of localized Inflammation and Ischemia-reperfusion Injury. In these experiments we will test the ability of 31P-NMR to detect localized inflammation in the peritoneum and ischemia-reperfusion in the isolated heart. Although the proposed experiments center around a single existing free radical trap, we envision the development, in future studies, of new NMR-sensitive probes which detect other types of radicals, probes which use other NMR-sensitive isotopes and which compartmentalize to specific organelles or organs.