Spin Trapping has been the most successful method for the detection of highly reactive free radical molecules in vivo. This technique involves the direct detection of primary free radicals that cannot be directly observed by conventional ESR due to low steady-state concentrations or to very short relaxation times, which lead to very broad lines. All the reported in vivo spin-trapping investigations have used the nitrone spin traps (PBN, POBN, DMPO). Although the major difficulty of the spin-trapping technique in vivo is the mere detection of a radical adduct, other factors such as absorption, distribution, metabolism and excretion must be considered when spin traps are administered in vivo. In addition, artifacts and ambiguities in the assignment of radical adduct structure must be taken into serious consideration. The group has pioneered the application of the ESR spin-trapping technique to biochemical, pharmacological and toxicological problems by using two major approaches for in vivo spin-trapping and ex vivo ESR detection of free radicals: lipid extraction of radical adducts generated in tissues and detection of radical adducts in biological fluids. ? i. Metal-mediated Free Radical Production: ESR spin-trapping investigations of acute cadmium- and/or diesel exhaust particle poisoning have shown the in vivo formation of either the hydroxyl radical in the liver or lipid-derived radicals in the lungs. ? ii. Characterization of Endogenous Radical Adducts by HPLC/ESR/MS: The combination of an on-line high performance liquid chromatography (HPLC)/electron spin resonance (ESR) system with mass spectrometric analysis (MS) was used to identify a variety of lipid-derived radicals formed from lipid peroxidation both in vitro and in vivo. For the first time, POBN adducts of linoleic acid carbon-centered pentadienyl radicals were detected and identified. ? iii. Novel Methanol and Ethylene Glycol Free Radical Metabolites: The investigations of ethanols free radical metabolism have been extended using knockout mice, chemical analogues such as ethylene glycol and methanol, and their metabolites such as formic acid. The evidence for formate-derived radical metabolites in vivo, during acute sodium formate poisoning is provided by ESR spin trapping of the radicals from the metabolism of formate, which are detected in bile and urine as radical adducts.
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