HEPOXILIN A(3) FORMATION IN THE RAT PINEAL-GLAND SELECTIVELY UTILIZES(12S)-HYDROPEROXYEICOSATETRAENOIC ACID (HPETE), BUT NOT (12R)-HPETE

Hepoxilins A(3) and B-3 have previously been shown to be formed from 1 2-hydroperoxyeicosatetraenoic acid (12-HPETE) through a heat-insensiti ve ferriheme catalysis as indicated by experiments with hemin or hemog lobin, typical of nonenzymatic rearrangement (Pace-Asciak, C. B (1984) Biochim. Biophys. Acta 793, 485-488; Pace-Asciak, C. R. (1984) J. Bio l. 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 t he rat pineal gland that selectively utilizes (12S)-HPETE for the tran sformation into hepoxilin A(3), while the hemin catalyzed transformati on 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-anthr yldiazomethane derivatives of the hydroxyeicosatetraenoic acids (HETEs ), hepoxilins and trioxilins, which could be detected by high performa nce 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 hepoxilin A(3) with minor amou nts of hepoxilin B-3, while experiments with heme catalysis showed for mation of both hepoxilins B-3 and A(3); 2) chiral phase analysis of th e 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 uti lized in the hemin experiments; 3) chiral phase analysis of hepoxilin A(3) indicated the presence in the pineal experiments of only hepoxili n with the 11S,12S-configuration formed from (12S)-HPETE, while the he min experiments contained both the native 11S,12S-configuration as wel l as the unnative or ''bis-epi''-11R,12R-configuration in the epoxide group of hepoxilin A(3); 4) reverse phase analysis of the epoxide hydr olase product of hepoxilin A(3), i.e. trioxilin A(3), showed that the pineal experiments contained only the products of enzymatic hydrolysis derived from the native hepoxilin A(3), whose formation was inhibited by the epoxide hydrolase inhibitor, trichloropropene oxide. The pinea l system is heat-sensitive, with formation of hepoxilins being abolish ed by tissue boiling, while the hemin system is insensitive to boiling . These experiments demonstrate that hepoxilin A(3) formation in the p ineal gland utilizes (12S)-HPETE exclusively, being transformed by a ' 'hepoxilin synthase'' primarily into hepoxilin A(3).