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

• Published in: The Journal of biological chemistry 1994-09, Vol.269 (39), p.23976-23980
• Main Authors: Reynaud, D, Demin, P, Pace-Asciak, C R
• Format: Article
• Language: English
• Subjects: 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - isolation & purification; 8,11,14-Eicosatrienoic Acid - metabolism; Animals; Chromatography, High Pressure Liquid; Leukotrienes - metabolism; Male; Pineal Gland - metabolism; Rats; Rats, Wistar; Spectrometry, Fluorescence; Stereoisomerism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)51034-1

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 hepoxilin 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 hepoxilin A3 with minor amounts of hepoxilin 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 hepoxilin A3 indicated the presence in the pineal experiments of only hepoxilin 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 hepoxilin A3; 4) reverse phase analysis of the epoxide hydrolase product of hepoxilin A3, i.e. trioxilin A3, showed that the pineal experiments contained only the products of enzymatic hydrolysis derived from the native hepoxilin 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 hepoxilin A3 formation in the pinea