Identification of radicals formed in the reaction mixture of bovine kidney microsomes with NADPH.
In order to explore the mechanism of myoglobinuric renal toxicity, detection and identification of free radicals was performed for the reaction mixtures of bovine kidney microsomes. EPR measurements showed prominent signals for the control reaction mixture containing 2.0 mg protein/ml bovine kidney microsomes, 5 mM NADPH, 0.1 M 4-POBN and 29 mM phosphate buffer (pH 7.4). Addition of myoglobin (Mb) to the control reaction mixture resulted in increase of EPR peak height. The result indicates that Mb enhances the radical formation. An HPLC-EPR measurement showed three peaks with retention times of 29.4 min (P(1)), 32.4 min (P(2)) and 46.6 min (P(3)). HPLC-EPR-MS analyses of P(1) and P(2) gave ions at m/z 282. The results show that 4-POBN/hydroxypentyl radical adducts form in the reaction mixture. An HPLC-EPR-MS analysis of P(3) gave ions at m/z 266, indicating that 4-POBN/pentyl radical adduct forms in the reaction mixture.
Kumamoto
Kazumasa
K
Department of Pharmacology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan.
Hirai
Tomihiro
T
Kishioka
Shiroh
S
Iwahashi
Hideo
H
D000818:Animals; D002417:Cattle; D005609:Free Radicals / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000166:cytology*; D008861:Microsomes / Q000378:metabolism*; D009211:Myoglobin / Q000378:metabolism; D009249:NADP / Q000378:metabolism*
D005609:Free Radicals; D009211:Myoglobin; D009249:NADP