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 techniques have been used to investigate the in vitro and in vivo formation of free radicals during 3-MI metabolism by goat lung. Utilizing C-phenyl-N-tert-butyl nitrone (PBN), a nitrogen-centered free radical has 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-centered 3-MI free radical is followed by the appearance of a carbon-centered radical in microsomal preparations. The objective of the present study is to prove that the nitrogen-centered radical generated from the 3-MI incubation system is a 3-MI radical utilizing [14C]-3 MI and the EPR-HPLC technique. The HPLC chromatogram includes three peaks that give EPR signals. These peaks are assigned to nitrogen-, oxygen- and carbon-centered radical adducts. The polarity of the three peaks follows the order: carbon-centered radical adduct > oxygen-centered radical adduct > 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 with ultraviolet light. These results demonstrate that the nitrogen-centered radical is a 3-MI-PBN spin adduct, and supports the hypothesis that 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 adducts and the best conditions to produce the radicals in the incubation system was investigated.
Department of Nutritional Sciences, University of Guelph, Ontario, Canada.
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Chen
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Janzen
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Bray
D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D012756:Sheep; D012862:Skatole / Q000378:metabolism*
D005609:Free Radicals; D009249:NADP; D012862:Skatole