A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol

David J Fulton, J. R. Falck, J. C. McGiff, M. A. Carroll, J. Quilley

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-δ-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-δ-lactone and 5,6-DHT with a t(1/2) ≃ 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (λ = 205) comigrating with 5,6-δ-lactone. Incubation of 5,6-δ-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 ± 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 ± 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 ± 5.5 pg/ml and 21.6 ± 6.3 pg/ml, respectively, by arachidonic acid.

Original languageEnglish (US)
Pages (from-to)1713-1721
Number of pages9
JournalJournal of Lipid Research
Volume39
Issue number8
StatePublished - Aug 1 1998
Externally publishedYes

Fingerprint

Lactones
Acids
High performance liquid chromatography
High Pressure Liquid Chromatography
Kidney
Eicosanoids
5,6-epoxy-8,11,14-eicosatrienoic acid
Reverse-Phase Chromatography
Bioactivity
Dinoprostone
Arachidonic Acid
Gas chromatography
Gas Chromatography-Mass Spectrometry
Purification
Mass spectrometry
Buffers

Keywords

  • 5,6-DHT
  • 5,6-EET
  • 5,6-δ-lactone
  • GC-MS
  • Heart
  • Kidney

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

Cite this

A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol. / Fulton, David J; Falck, J. R.; McGiff, J. C.; Carroll, M. A.; Quilley, J.

In: Journal of Lipid Research, Vol. 39, No. 8, 01.08.1998, p. 1713-1721.

Research output: Contribution to journalArticle

Fulton, David J ; Falck, J. R. ; McGiff, J. C. ; Carroll, M. A. ; Quilley, J. / A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol. In: Journal of Lipid Research. 1998 ; Vol. 39, No. 8. pp. 1713-1721.
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AB - The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-δ-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-δ-lactone and 5,6-DHT with a t(1/2) ≃ 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (λ = 205) comigrating with 5,6-δ-lactone. Incubation of 5,6-δ-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 ± 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 ± 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 ± 5.5 pg/ml and 21.6 ± 6.3 pg/ml, respectively, by arachidonic acid.

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