Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs

Kasem Nithipatikom, Ralph F. DiCamelli, Stephanie Kohler, Richard J. Gumina, J R Falck, William B. Campbell, Garrett J. Gross

Research output: Contribution to journalArticlepeer-review

90 Scopus citations

Abstract

Arachidonic acid (AA) can be metabolized by cyto. chrome P450 enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12., and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their role in the ischemic myocardium has not been well investigated. In this study, we used a gas chromatographic-mass spectrometric technique to analyze total EETs, DHETs, and 20-HETE released into coronary venous plasma during coronary artery occlusion and reperfusion in anesthetized dogs. Pentafluorobenzyl esters (PFB-esters) of EETs and PFBesters/trimethylsilyl ethers (TMS-ethers) of DHETs and 20-HETE were detected in the negative ion chemical ionization (NICI) using methane as a reagent gas. Under the conditions used, all four regioisomers of EET eluted from the capillary gas chromatographic column at similar retention times while four regioisomers of DHETs and 20-HETE eluted separately. The detection limits in plasma samples are 5 pg for total EETs, 40 pg for DHET, and 15 pg for 20-HETE. 14,15-DHET is the major regioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the first 5 to 15 min of coronary occlusion, a slight decrease in the concentration of EETs, 14,15DHET, and 20-HETE from the control values was observed in coronary venous plasma. At 60 min of occlusion, their concentrations significantly increased and remained elevated during 5 to 60 min of reperfusion. The concentrations decreased at 120 min of reperfusion. The NICI GC-MS was successfully used as a sensitive technique to determine cP450 metabolites of AA in plasma during prolonged occlusion-reperfusion periods. Furthermore, the results indicate that these metabolites may play a role in mediating ischemic-reperfusion injury.

Original languageEnglish (US)
Pages (from-to)115-124
Number of pages10
JournalAnalytical biochemistry
Volume292
Issue number1
DOIs
StatePublished - May 1 2001

Keywords

  • 20-hydroxyeicosatetraenoic acid
  • Cytochrome P450
  • Dihydroxyeicosatrienoic acids
  • Endothelium-dependent hyperpolarizing factor
  • Epoxyeicosatrienoic acids
  • Ischemia-reperfusion

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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