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We investigated the formation of PGF2α 1-ethanolamide, PGE2 1-ethanolamide, and PGD2 1-ethanolamide (prostamides F2α, E2, and D2, respectively) in liver, lung, kidney, and small intestine after a single intravenous bolus administration of 50 mg/kg of anandamide to normal and fatty acid amide hydrolase knockout (FAAH −/−) male mice. One group of three normal mice was not dosed (naïve) while another group of three normal mice received a bolus intravenous injection of 50 mg/kg of anandamide. Three FAAH −/− mice also received an intravenous injection of 50 mg/kg of anandamide. After 30 min, the lung, liver, kidney, and small intestine were harvested and processed by liquid-liquid extraction. The concentrations of prostamide F2α, prostamide E2, prostamide D2, and anandamide were determined by HPLC-tandem mass spectrometry. Prostamide F2α was detected in tissues in FAAH −/− mice after administration of anandamide. Concentrations of anandamide, prostamide E2, and prostamide D2 in liver, kidney, lung, and small intestine were much higher in the anandamide-treated FAAH −/− mice than those of the anandamide-treated control mice.This report demonstrates that prostamides, including prostamide F2α, were formed in vivo from anandamide, potentially by the cyclooxygenase-2 pathway when the competing FAAH pathway is lacking.