We have used spin-trapping techniques to identify radical species formed during the NADPH-stimulated peroxidation of rat hepatic microsomes. Using 5,5-dimethyl-1-pyrroline-1-oxide, we have confirmed the presence of substantial quantities of superoxide but found evidence for the formation of only small quantities of hydroxyl radical. Use of the spin traps Ntert-butyl-a-4-pyridylnitrone-l-oxide and 2-methyl-2-nitroso-lpropanol have allowed us to determine that lipid peroxyl radicals are the predominant "lipid-type" radical found in peroxidizing microsomes under aerobic conditions. These data suggest that microsomal lipid dienyl radicals react with molecular oxygen at near diffusion-controlled rates. can be spin trapped even though other radical species, such as superoxide and lipid hydroperoxyl radicals, have been shown to be generated during lipid peroxidation (1).Here we describe experiments showing that, in the presence ofrat liver microsomes and NADPH, superoxide, hydroxyl, and lipid peroxyl radicals (LOO-) are spin trapped; we could not find any evidence for the existence ofa carbon-centered dienyl lipid radical (L-). General Comments. Hemin, N,amino]ethyl)glycine (DETAPAC), soybean lipoxygenase, and NADPH were purchased from Sigma. Linoleic acid was obtained from Nu Chek Prep (Elysian, MN) and was >99% pure. 2-Methyl-2-nitropropane was purchased from Fluka/ Tridom. Chelex-100 was obtained from Bio-Rad. ESR spectra were recorded by using a Varian Associates model E-9 spectrometer. The spin traps 2-methyl-2-nitroso-1-propanol (MNPOL) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were prepared according to the methods outlined by DeGroot et aL (12) and Bonnett et a! (13). The spin trap N-tert-butyl-a-4-pyridylnitrone-l-oxide (4-POBN) was a gift from William Yamanashi (Department of Ophthalmology, Emory University, Atlanta, GA). Because the rate of spin trapping of some free radicals is slow [e.g., the rate of spin trapping superoxide by DMPO is 10 M-1sec-1 (8)], high concentrations of the probes are essential. For this reason, we chose to use the water-soluble spin traps DMPO, 4-POBN, and MNPOL (Fig. 1). Unless otherwise indicated, all buffers used were passed through a ChelexAbbreviations: EPR, electron paramagnetic resonance; LOO-, lipid peroxyl radical; L, dienyl lipid radical; DMPO, 5,5-dimethyl-1-pyrroline-1-oxide; DMPO-OH, 5-hydroxy-2,2-dimethyl-1-pyrrolidinyloxyl; DMPO-OOH, 5-hydroperoxy-2,2-dimethyl-1-pyrrolidinyloxyl; 4-POBN, N-tert-butyl-a-pyridylnitrone-l-oxide; 4-POBN-OH, tert-butyl(hydroxy)-4-pyridylmethyl nitroxide; 4-POBN-OOH, tertbutyl(hydroperoxy)-4-pyridylmethyl nitroxide; MNPOL, 2-methyl-2-nitroso-l-propanol; MNPOL-H, 2-methylpropyl-1-nitroxide; DETA-PAC, NN-bis(2-[bis(carboxymethyl)amino]ethyl)glycine.
MATERIALS AND METHODS
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