CHARACTERIZATION OF THE ENZYMATIC AND NONENZYMATIC REACTION OF 13-OXOOCTADECADIENOIC ACID WITH GLUTATHIONE

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydraxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation o f 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). The se oxidized derivatives of linoleic acid have been shown to be involve d in several cellular regulatory processes. In the present study, we h ave examined the enzymatic and nonenzymatic reaction of 13-OXO with gl utathione;(GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optim um of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HP LC-purified products were examined by FAB MS as well as one- and two-d imensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addit ion of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transf erase 8-8. In the case of the enzymatic reaction there is stereoselect ive product formation. Furthermore, submicromolar concentrations of th e 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provi de insight into the potential metabolic disposition of linoleate oxyge nation products.