Arachidonic acid is oxidatively metabolized by rat liver microsomes at a rate of approximately 5 nmol per min per mg of protein at 25C. This reaction is dependent on the presence of NADPH and oxygen. Studies with various inhibitors indicate a role for membrane-bound cytochrome P-450 in the transformation of arachidonic acid to a mixture of hydroxy acid derivatives. The stoichiometry of the reaction conforms to that of a monooxygenase reaction-i.e., one mole of NADPH is oxidized per mole of oxygen utilized-suggesting a reaction mechanism different from that proposed for lipid peroxidation reactions. No evidence for the formation of prostaglandin-like metabolites was obtained. The diene character of some of the metabolites formed suggests another role for cytochrome P-450-i.e., participation in hydrogen abstraction reactions for the activation of various substrates.The oxidative metabolism ofarachidonic acid can lead to a wide variety of metabolites. Interest in the physiological function of prostaglandins, thromboxanes, leukotrienes, and other oxidative metabolites of arachidonic acid, as well as the reactions of lipid peroxidation, have established the need to better understand the role of the various enzymes responsible for oxygen incorporation during the process of arachidonic acid metabolism-e.g., cyclooxygenase, lipoxygenase, etc. (1, 2).The microsomal fraction from rat liver, like that from many other tissues, is rich in phospholipids containing arachidonic acid (3). During the NADPH-and oxygen-dependent function of the liver microsomal cytochrome P-450-containing electron transport system, a significant portion of electrons is diverted to either the formation of hydrogen peroxide or the oxidative transformation of "endogenous substrates" (4, 5). The latter reaction conforms to the stoichiometry of a monooxygenase reaction in that one mole of NADPH is oxidized for each mole of oxygen consumed. The nature of the endogenous substrate(s) of liver microsomes has eluded characterization, although Schenkman et al. (6) have provided evidence that unsaturated fatty acids, in particular oleic acid, might serve in this capacity. In addition, it has been reported that unsaturated fatty acids appear to be released from liver microsomal phospholipids during the course of NADPH oxidation (7,8). In the presence of an iron chelate, such as the ADP-Fe3+ complex, and an electron donor, such as NADPH or ascorbate, malonaldehyde is rapidly formed concomitant with a rapid rate of oxygen utilization (9). It is generally assumed that one source of malonaldehyde is from the oxidation of arachidonic acid (1, 10). Thus, it was of interest to evaluate the oxidative metabolism ofarachidonic acid by the electron transport system associated with rat liver microsomes.It is the purpose of this communication to describe the role of liver microsomal cytochrome P-450 in the oxidation of arachidonic acid. It is suggested that free arachidonic acid, as well as that derived from microsomal phospholipids, may contribute, in part, to the...

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A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.

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