IDENTIFICATION OF 3-MI-DERIVED N-CENTERED RADICALS OBTAINED FROM INCUBATION OF 3-MI WITH MICROSOMAL-NADPH SYSTEM BY EPR-HPLC SPIN-TRAPPING

3-Methylindole (3-MI) is a metabolite of tryptophan that causes acute pulmonary edema and emphysema in ruminants when administered orally or intravenously. Electron paramagnetic resonance (EPR) spin-trapping te chniques have been used to investigate the in vitro and in vivo format ion of free radicals during 3-MI metabolism by goat lung. Utilizing C- phenyl-N-tert-butyl nitrone (PBN), a nitrogen-centered free radical ha s been detected from 3-MI in goat lung microsomal incubation. The EPR spectrum of the spin adduct is identical to that observed when 3-MI is irradiated with ultraviolet light. The formation of a nitrogen-center ed 3-MI free radical is followed by the appearance of a carbon-centere d radical in microsomal preparations. The objective of the present stu dy is to prove that the nitrogen-centered radical generated from the 3 -MI incubation system is a 3-MI radical utilizing [C-14]-3 MI and the EPR-HPLC technique. The HPLC chromatogram includes three peaks that gi ve EPR signals. These peaks are assigned to nitrogen-, oxygen- and car bon-centered radical adducts. The polarity of the three peaks follows the order: carbon-centered radical adduct > oxygen-centered radical ad duct > nitrogen-centered radical adduct. The last has a polarity that is weaker than 3-MI. Only the nitrogen-centered peak and the 3-MI peak possessed radioactivity. The retention time of the nitrogen centered radical is the same as the spin adduct generated by 3-MI irradiation w ith ultraviolet light. These results demonstrate that the nitrogen-cen tered radical is a S-MI-PBN spin adduct, and supports the hypothesis t hat 3-MI-induced lung damage results from activation of 3-MI to a free radical. Also, in this study the stability of the radical spin adduct s and the best conditions to produce the radicals in the incubation sy stem was investigated.