Metabolism of leukotriene E4 in isolated rat hepatocytes. Identification of beta-oxidation products of sulfidopeptide leukotrienes.

Little is known about the metabolic fate of the sulfidopeptide leukotrienes (LTC4/D4/E4). Earlier studies using radiolabeled leukotrienes have shown that these potent molecules are concentrated and metabolized in the liver when administered to mice and that isolated rat hepatocytes have a high affinity uptake system for LTE4. N-Acetyl-LTE4 has been identified as a metabolite of LTC4 in the bile of rats, but the majority of the metabolites in these studies were not characterized. Based on these earlier reports, incubation of LTE4 with isolated rat hepatocytes was chosen as a model for the study of sulfidopeptide leukotriene metabolism. [3H]LTE4 was incubated with isolated rat hepatocytes and the metabolites formed were purified extensively by ODS flash column chromatography, TLC, and reverse phase-high pressure liquid chromatography. Metabolites were identified by retention of the radiolabel and UV absorbance at 280 nm. Purified metabolites were characterized by UV spectroscopy, fast atom bombardment mass spectrometry, negative ion chemical ionization gas chromatography-mass spectrometry, and electron impact gas chromatography-mass spectrometry. Six LTE4 hepatocyte metabolites were characterized. Metabolite A was determined to be N-acetyl-LTE4. Metabolite B was determined to be the omega-oxidation product 20-carboxy-N-acetyl-LTE4. Metabolite C was characterized as the beta-oxidation product 18-carboxydinor-N-acetyl-LTE4. A further round of beta-oxidation with a concomitant double bond reduction produced Metabolite D, identified as 16-carboxytetranordihydro-N-acetyl-LTE4. The reduction of the 14-15 double bond was most likely the result of the action of 2,4-dienoyl-CoA reductase. The UV spectrum of Metabolite E indicated the presence of a conjugated tetraene, and this metabolite was determined to be 16-carboxytetranor-delta 13-N-acetyl-LTE4. Metabolite F was identified as 14-carboxyhexanor-N-acetyl-LTE4. The observed pathway of beta-oxidation of LTE4 proceeded entirely from the C-20 methyl terminus after omega-oxidation which is in contrast to the known metabolic fate of other eicosanoids. This may be due to the failure to generate the required thioester at C-1 in LTE4 through a strong interaction of the C-5 hydroxy group with the C-1 carboxyl.