title: Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity.
abstract: Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d₄-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d₄-EA) and the internal standard ¹³C₂-EA. Selected reaction monitoring of m/z 66→m/z 48 (d₄-EA) and m/z 64→m/z 46 (¹³C₂-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d₄-AEA hydrolysis obeyed Michaelis-Menten kinetics (K(M)=12.3 μM, V(max)=27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC₅₀=24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Rakers
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Zoerner
authors.forename: Stefan
authors.initials: S
authors.lastname: Engeli
authors.forename: Sandor
authors.initials: S
authors.lastname: Batkai
authors.forename: Jens
authors.initials: J
authors.lastname: Jordan
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004285:Dogs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007554:Isotopes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D007554:Isotopes; D000099252:Fatty Acid Amide Hydrolases