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

John Turk, Bryan A. Wolf, Patricia G. Comens, Jerry Colca, Barbara Jakschik, Michael L. McDaniel

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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 E2 (but not to [3H]leukotriene C4, D4, or E4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C4 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.

Original languageEnglish
Pages (from-to)1-17
Number of pages17
JournalBiochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Volume835
Issue number1
DOIs
StatePublished - Jun 14 1985

Keywords

  • (Rat liver and pancreas)
  • Arachidonate metabolite
  • Eicosanoid
  • Insulin release
  • Mass spectrometry
  • Monooxygenase
  • Prostaglandin synthesis

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