Stereochemistry of Leukotriene B₄ Metabolites Formed by the Reductase Pathway in Porcine Polymorphonuclear Leukocytes: Inversion of Stereochemistry of the 12-Hydroxyl Group

Leukotriene B₄ (LTB₄), a potent proinflammatory agent, is a major metabolite of arachidonic acid in polymorphonuclear leukocytes (PMNL). When porcine PMNL were incubated with LTB₄ and the products purified by reversed-phase high-pressure liquid chromatography (HPLC), we previously identified two metabolites: 10,11-dihydro-LTB₄ and 10,11-dihydro-12-oxo-LTB₄ [Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369]. Further analysis of the reaction products by normal-phase HPLC has now revealed the presence of a third major metabolite of LTB₄. This product is not formed in detectable amounts in the first 5 min of the reaction but accounts for about 20-30% of the reaction products after 60 min, when LTB₄ has been completely metabolized. The mass spectrum and gas chromatographic properties of the new metabolite are identical with those of 10,11-dihydro-LTB₄, suggesting that it is a stereoisomer of this compound. This product was identified as 10,11-dihydro-12-epi-LTB₄ [i.e., 5(S),12(R)-di-hydroxy-6,8,14-eicosatrienoic acid] by comparison of its chromatographic properties with those of the authentic chemically synthesized compound. Both 10,11-dihydro-LTB₄ and 10,11-dihydro-12-oxo-LTB₄ were enzymatically converted to 10,11-dihydro-12-epi-LTB₄ by porcine PMNL, the former compound being the better substrate. The reaction was reversible, since both 10,11-dihydro-12-epi-LTB₄ and 10,11-dihydro-12-oxo-LTB₄ could be converted to 10,11-dihydro-LTB₄. When dihydro metabolites of LTB₄ labeled with tritium in the 12-position were incubated with porcine PMNL, only about 15% of the tritium was retained in the 12-position of the product, suggesting that epimerization due to replacement of the 12-hydroxyl group itself was unlikely. Our results would be consistent with an epimerase-catalyzed reacton in which 10,11-dihydro-12-oxo-LTB₄ is an intermediate. This would explain the lack of stereospecificity in the reduction of 10,11-dihydro-12-oxo-LTB₄ as well as the partial retention of tritium in the 12-position after epimerization. Alternatively, it is possible that the reactions could be catalyzed by a combination of stereospecific 12-hydroxy dehydrogenases and 12-keto reductases. These results indicate that the stereochemistry of 12-hydroxy eicosanoids can be reversed by PMNL, which could have important implications for their biological activities.