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

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-epox yeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic a cids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their ro le in the ischemic myocardium has not been well investigated, In this study , we used a gas chromatographic-mass spectrometric technique to analyze tot al EETs, DHETs, and SO-METE released into coronary venous plasma during cor onary artery occlusion and reperfusion in anesthetized dogs. Pentafluoroben zyl esters (PFB-esters) of EETs and PFB-esters/trimethylsilyl ethers (TMS e thers) of DHETs and SO-METE were detected in the negative ion chemical ioni zation (NICI) using methane as a reagent gas. Under the conditions used, al l four regioisomers of EET eluted from the capillary gas chromatographic co lumn at similar retention times while four regioisomers of DMETs and 80-HET E eluted separately. The detection limits in plasma samples are 5 pg for to tal EETs, 40 pg for DHET, and 15 pg for SO-METE. 14,15-DHET is the major re gioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the f irst 5 to 15 min of coronary occlusion, a slight decrease in the concentrat ion of EETs, 14,15-DHET, and 20-HETE from the control values was observed i n coronary venous plasma. At 60 min of occlusion, their concentrations sign ificantly increased and remained elevated during 5 to 60 min of reperfusion . The concentrations decreased at 120 min of reperfusion, The NICI G:C-MS w as successfully used as a sensitive technique to determine cP450 metabolite s of AA in plasma during prolonged occlusion-reperfusion periods. Furthermo re, the results indicate that these metabolites may play a role in mediatin g ischemic-reperfusion injury. (C) 2001 Academic Press.