A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol

• Published in: Journal of lipid research Vol. 39; no. 8; pp. 1713 - 1721
• Main Authors: Fulton, D, Falck, J R, McGiff, J C, Carroll, M A, Quilley, J
• Format: Journal Article
• Language: English
• Published: United States Elsevier 01.08.1998
• Subjects: 5,6-DHT; 5,6-EET; 5,6-δ-lactone; 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - analysis; 8,11,14-Eicosatrienoic Acid - chemistry; 8,11,14-Eicosatrienoic Acid - metabolism; Animals; Chromatography, High Pressure Liquid - methods; Chromatography, High Pressure Liquid - standards; Chromatography, High Pressure Liquid - statistics & numerical data; Dinoprostone - metabolism; Gas Chromatography-Mass Spectrometry - methods; Gas Chromatography-Mass Spectrometry - standards; Gas Chromatography-Mass Spectrometry - statistics & numerical data; GC–MS; heart; Hydroxyeicosatetraenoic Acids - metabolism; In Vitro Techniques; kidney; Kidney - metabolism; Male; Myocardium - metabolism; Perfusion; Rats; Rats, Wistar; Reference Standards; Sensitivity and Specificity
• ISSN: 0022-2275
• DOI: 10.1016/s0022-2275(20)32202-1

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.