Arachidonic acid metabolism in isolated pancreatic islets: IV. Negative ion-mass spectrometric quantitation of monooxygenase product synthesis by liver and islets

Abstract

Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-monooxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 μM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 μM) or by nordihydroguaiaretic acid (NDGA, 50 μM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [ 3H]12-HETE and to [ 3H]prostaglandin E 2 (but not to [ 3H]leukotriene C 4, D 4, or E 4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C 4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 μM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.