TY - JOUR
T1 - Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs
AU - Nithipatikom, Kasem
AU - DiCamelli, Ralph F.
AU - Kohler, Stephanie
AU - Gumina, Richard J.
AU - Falck, J R
AU - Campbell, William B.
AU - Gross, Garrett J.
N1 - Funding Information:
The authors thank Drs. William S. Edgemond and Phillip F. Pratt, and Anna Hsu and Jeannine Moore for their assistance in synthesis and purification of EETs and DHETs and in performing the dog studies, respectively. These studies were supported by funds from the American Heart Association-Wisconsin (K.N.), NIH Grant HL-51055 (W.B.C.), NIH Grant HL08311 (G.J.G.), and NIH Grant GM31278 (J.R.F.)
PY - 2001/5/1
Y1 - 2001/5/1
N2 - 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.
AB - 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.
KW - 20-hydroxyeicosatetraenoic acid
KW - Cytochrome P450
KW - Dihydroxyeicosatrienoic acids
KW - Endothelium-dependent hyperpolarizing factor
KW - Epoxyeicosatrienoic acids
KW - Ischemia-reperfusion
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U2 - 10.1006/abio.2001.5044
DO - 10.1006/abio.2001.5044
M3 - Article
C2 - 11319825
AN - SCOPUS:0035337058
SN - 0003-2697
VL - 292
SP - 115
EP - 124
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 1
ER -