A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol.
The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.
Fulton
D
D
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
Falck
J R
JR
McGiff
J C
JC
Carroll
M A
MA
Quilley
J
J
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D015232:Dinoprostone / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000378:metabolism; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D012680:Sensitivity and Specificity
D006893:Hydroxyeicosatetraenoic Acids; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; D015126:8,11,14-Eicosatrienoic Acid; D015232:Dinoprostone