Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells.
A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.
Nithipatikom
Kasem
K
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee 53226, USA. kasemn@mcw.edu
Laabs
Nathan D
ND
Isbell
Marilyn A
MA
Campbell
William B
WB
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008297:Male; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011453:Prostaglandins / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014407:Tumor Cells, Cultured
D011453:Prostaglandins; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases