Identification of 5,6-trans-epoxyeicosatrienoic acid in the phospholipids of red blood cells

J Biol Chem. 2004 Aug 27;279(35):36412-8. doi: 10.1074/jbc.M403962200. Epub 2004 Jun 22.

Abstract

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard. Hydrogenation of the extracted 5,6-erythro-DHET with platinum(IV) oxide/hydrogen resulted in an increase of the molecular mass by 6 daltons and the same retention time shift as an authentic standard in gas chromatography, suggesting the existence of three olefins as well as the 5,6-erythro-dihydroxyl structure in the metabolite. Match of retention times by chromatography indicated identity of the stereochemistry of the red blood cell 5,6-erythro-DHET vis à vis the synthetic standard. High pressure liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the phospholipase A(2)-hydrolyzed lipid extracts from red blood cells revealed match of the mass spectrum and retention time of the compound with the authentic 5,6-trans-EET standard, providing direct evidence of the existence of 5,6-trans-EET in red blood cells. The presence of other trans-EETs was also demonstrated. The ability of both 5,6-trans-EET and its product 5,6-erythro-DHET to relax preconstricted renal interlobar arteries was significantly greater than that of 5,6-cis-EET. In contrast, 5,6-cis-EET and 5,6-trans-EET were equipotent in their capacity to inhibit collagen-induced rat platelet aggregation, whereas 5,6-erythro-DHET was without effect. We propose that the red blood cells serve as a reservoir for epoxides which on release may act in a vasoregulatory capacity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 8,11,14-Eicosatrienoic Acid / analogs & derivatives*
  • 8,11,14-Eicosatrienoic Acid / chemistry*
  • 8,11,14-Eicosatrienoic Acid / metabolism*
  • Animals
  • Arteries / pathology
  • Blood Platelets / metabolism
  • Chromatography, Gas
  • Chromatography, High Pressure Liquid
  • Collagen / metabolism
  • Erythrocytes / metabolism*
  • Kidney / blood supply
  • Lipids / chemistry
  • Mass Spectrometry
  • Models, Chemical
  • Phospholipases A / metabolism
  • Phospholipids / metabolism*
  • Platelet Aggregation
  • Rats
  • Rats, Sprague-Dawley
  • Spectrometry, Mass, Electrospray Ionization
  • Time Factors

Substances

  • Lipids
  • Phospholipids
  • 5,6-epoxy-8,11,14-eicosatrienoic acid
  • Collagen
  • Phospholipases A
  • 8,11,14-Eicosatrienoic Acid