[Detection of free radicals generated in NADPH-dependent lipid peroxidation in ischemic brain homogenates--use of the spin trapping technic].
Spin trapping technique has been applied to the detection of free radicals generated in NADPH stimulated lipid peroxidation process in ischemic brain homogenate. Using male Wistar rats, complete cerebral ischemia for 30 min, 60 min or 120 min was produced by decapitation followed by preservation of the heads at 37 degrees C. Global cerebral ischemia of 30 min or 60 min duration was induced by occlusions of three vessels (bilateral common carotid and basilar artery) in the ventilated rats. In some animals, bilateral carotid occlusions were released for 30 min following 30 min of ischemia to study postischemic event. Two reaction mixtures containing of brain homogenate, NADPH, Fe-EDTA and spin trapping reagent, phenyl-t-buthylnitrone (PBN), were prepared from each brain sample--one to be incubated in air and the other to be incubated in nitrogen gas. After the incubation for 20 min at 37 degrees C, free radical adducts of PBN were measured by electron spin resonance (ESR). In preliminary experiments, no ESR signals were obtained from the reaction mixtures without the addition of NADPH and Fe-EDTA. And the dependence of ESR signal intensity upon the NADPH concentration was observed. The six-line signals (triplet of doublets), which hyperfine splitting constants were AN = 16.2-16.5 G and A beta H = 3.6-3.8 G, were obtained from both ischemic models. These signals were dependent upon the presence of oxygen in the reaction systems, as evidenced by the fact that the signal intensity obtained from aerobic incubation was consistently stronger than that obtained from anaerobic incubation in each brain sample.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism*; D004492:Edetic Acid; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005290:Ferric Compounds; D005609:Free Radicals; D008054:Lipid Peroxides / Q000378:metabolism*; D008297:Male; D009249:NADP; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D005290:Ferric Compounds; D005609:Free Radicals; D008054:Lipid Peroxides; D009249:NADP; D004492:Edetic Acid; C019179:Fe(III)-EDTA; D010100:Oxygen