chidonylethanolamide (anandamide, AEA 2 ), an endog-A rapid, sensitive, and reliable method for measurenous brain cannabinoid receptor agonist (1-3), into ing anandamide amidase activity in rat brain microarachidonic acid (AA) and ethanolamine (EA) (4). Presomes by reversed-phase high-performance liquid vious studies have demonstrated that the enzyme, chromatography (RP-HPLC) and its applications are which breaks down AEA, is abundantly distributed in described. Enzymatic activity was assayed by the dethe regions where a high density of cannabinoid receptermination of the rates of hydrolysis of anandamide tor exists in the central nervous system (5). Recently, or its analogs at 37ะC. The reaction products were sepathe enzyme was partially purified and characterized as rated using an ODS guard column eluted with aqueous a highly hydrophobic protein with M r ca. 60 kDa (6). phosphoric acid-acetonitrile and quantitated with uv There is much interest at present in the search for detection at 204 nm and an external standard method. AEA analogs which have high binding affinity for the Baseline separation of the acid products from their cannabinoid receptor and are resistant to hydrolysis by substrates was completed in less than 2 min. The detecthe enzyme (7, 8). Moreover, a lot of effort has been tion limits were 1.4 pmol for arachidonic acid and 0.22 made to find selective AEA amidase inhibitors which pmol for anandamide at a signal to noise ratio of 4:1.
do not bind to the receptor (9).
The stability of anandamide in the acidic mobile phase
Previous assays for AEA amidase activity commonly was tested, and no significant decomposition was obrely on the measurement of radioactivity of acid prodserved up to 1 h. The method was successfully applied ucts formed from 3 H-or 14 C-labeled AEA or its analogs.
to the examination of substrate specificity as well as
A normal-phase thin-layer chromatographic (TLC) for testing the ability of amidase inhibitors to block method for the separation of 3 H-labeled arachidonates its hydrolysis. Kinetic constants obtained for (S)-methwas described by Deutsch et al. (4, 9). A reversed-phase anandamide were an apparent K m of 8.6 { 1.3 mM and a V max of 362 { 16 pmol/min/mg of protein. A highly TLC separation coupled with phosphor-imaging quanpotent inhibitor, palmitylsulfonyl fluoride (PSF), was titation was also described by Devane and Axelrod (10). found to have an IC 50 of 50 nM. PSF is 210 times as Non-TLC methods, including minicolumn chromatogpotent as phenylmethylsulfonyl fluoride. The method raphy (5) or a solvent extraction approach using [1,2offers several advantages over existing methodology 14 C]-labeled ethanolamide (11), were recently reported. using radioisotopes or a solvent extraction procedure. Thus far, nonradioactive assays for the amidase activ-