Hepoxilin A3 formation in the rat pineal gland selectively utilizes (12S)-hydroperoxyeicosatetraenoic acid (HPETE), but not (12R)-HPETE.

Hepoxilins A3 and B3 have previously been shown to be formed from 12-hydroperoxyeicosatetraenoic acid (12-HPETE) through a heat-insensitive ferriheme catalysis as indicated by experiments with hemin or hemoglobin, typical of nonenzymatic rearrangement (Pace-Asciak, C. R. (1984) Biochim. Biophys. Acta 793, 485-488; Pace-Asciak, C. R. (1984) J. Biol. Chem. 259, 8332-8337). In this paper, we demonstrate through use of a mixture of (12S)- and (12R)-HPETE that an enzyme system exists in the rat pineal gland that selectively utilizes (12S)-HPETE for the transformation into A3, while the hemin-catalyzed transformation is not selective, with both (12S)- and (12R)-HPETE being utilized. A new procedure was established to rapidly extract (in the absence of acid) and directly derivatize from the incubation mixture the products of incubation using 9-anthryldiazomethane reagent to form the 9-anthryldiazomethane derivatives of the hydroxyeicosatetraenoic acids (HETEs), hepoxilins and trioxilins, which could be detected by high performance liquid chromatography using a flow-through fluorescence detector. The following observations were made: 1) the pineal experiments showed a high preference for the formation of A3 with minor amounts of B3, while experiments with heme catalysis showed formation of both hepoxilins B3 and A3; 2) chiral phase analysis of the HETEs recovered from the incubation mixture showed the predominance of (12R)-HETE from the pineal experiments, indicating that (12S)-HPETE was selectively used up, whereas both (12S)- and (12R)-HPETE were utilized in the hemin experiments; 3) chiral phase analysis of A3 indicated the presence in the pineal experiments of only with the 11S,12S-configuration formed from (12S)-HPETE, while the hemin experiments contained both the native 11S,12S-configuration as well as the unnative or bis-epi-11R,12R-configuration in the epoxide group of A3; 4) reverse phase analysis of the epoxide hydrolase product of A3, i.e. trioxilin A3, showed that the pineal experiments contained only the products of enzymatic hydrolysis derived from the native A3, whose formation was inhibited by the epoxide hydrolase inhibitor, trichloropropene oxide. The pineal system is heat-sensitive, with formation of hepoxilins being abolished by tissue boiling, while the hemin system is insensitive to boiling. These experiments demonstrate that A3 formation in the pineal gland utilizes (12S)-HPETE exclusively, being transformed by a hepoxilin synthase primarily into A3.