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, John R. Falck, William B. Campbell, Garrett J. Gross

Research output: Contribution to journalArticle

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

Fingerprint Dive into the research topics of 'Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs'. Together they form a unique fingerprint.

  • Cite this

    Nithipatikom, K., DiCamelli, R. F., Kohler, S., Gumina, R. J., Falck, J. R., Campbell, W. B., & Gross, G. J. (2001). Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs. Analytical Biochemistry, 292(1), 115-124. https://doi.org/10.1006/abio.2001.5044