title: Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs.
abstract: Arachidonic acid (AA) can be metabolized by cytochrome 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 PFB-esters/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, 14,15-DHET, 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.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
authors.forename: K
authors.initials: K
authors.lastname: Nithipatikom
authors.forename: R F
authors.initials: RF
authors.lastname: DiCamelli
authors.forename: S
authors.initials: S
authors.lastname: Kohler
authors.forename: R J
authors.initials: RJ
authors.lastname: Gumina
authors.forename: J R
authors.initials: JR
authors.lastname: Falck
authors.forename: W B
authors.initials: WB
authors.lastname: Campbell
authors.forename: G J
authors.initials: GJ
authors.lastname: Gross
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000097:blood*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001685:Biological Factors / Q000097:blood; D003331:Coronary Vessels / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004285:Dogs; D015777:Eicosanoids / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood*; D007511:Ischemia / Q000097:blood*; D012015:Reference Standards; D015424:Reperfusion
chemical_list: D001685:Biological Factors; D015777:Eicosanoids; D006893:Hydroxyeicosatetraenoic Acids; C067410:endothelium-dependent hyperpolarization factor; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D015126:8,11,14-Eicosatrienoic Acid