Determination of Anandamide Amidase Activity Using Ultraviolet-Active Amine Derivatives and Reverse-Phase High-Performance Liquid Chromatography Ce Qin,* Sonyuan Lin,* Wensheng Lang,* Andreas Goutopoulos,* Spiro Pavlopoulos,* Frank Mauri,† and Alexandros Makriyannis* , ‡ , § ,1 *Department of Pharmaceutical Sciences and ‡Department of Molecular and Cell Biology, §Institute of Materials Science, and †Biotechnology Center, University of Connecticut, Storrs, Connecticut 06269 Received August 6, 1997 Anandamide amidase catalyzes the hydrolysis of anandamide (AEA) to arachidonic acid (AA) and etha- nolamine (EA). Recently, we published a method for determining anandamide amidase activity based on the measurement of arachidonic acid with direct UV detection at 204 nm. However, this method cannot be used to determine the hydrolysis of non-UV-active AEA analogs. It also cannot be used to study AEA ami- dase inhibitors that contain the arachidonic acid tail, and which are also enzyme substrates. Here we report a novel, more general method for measuring amidase activity by o-phthaldialdehyde (OPA) precolumn der- ivatization and reverse-phase high-performance liq- uid chromatography (HPLC). The hydrolysis product, ethanolamine, after separation from protein was deri- vatized with OPA to form a UV-active isoindole deriv- ative which was then detected at 230 nm. The detec- tion limit for derivatized ethanolamine was 1.0 pmol and retention times were typically less than 8 min. Our new method can detect non-UV-active analogs through derivatization of the amine product. It can thus be used after careful selection of the HPLC conditions in competition experiments between AEA and AEA analogs possessing different head groups. The most effective competitive inhibitor tested was (R)-N- (1-methyl-2-hydroxyethyl)arachidonylamide (AM356), which is resistant to enzymatic hydrolysis and yet in- hibits AEA hydrolysis in a competition experiment by 43%. Moreover, this method offers several advantages over existing methodologies using radioisotopes or solvent extraction procedures. Our work to date has shown that small structural changes in the AEA mol- ecule can result in significant variation in both affinity and turnover rate for each analog with respect to AEA amidase. © 1998 Academic Press Arachidonyl ethanolamine or anandamide (AEA) 2 is an endogenous brain cannabinoid receptor (CB1) ligand that has recently been a focus of study (1–3). Anandamide ami- dase, which is also found in mammalian brain, catalyzes the hydrolysis of AEA to arachidonic acid and ethanolamine (4, 5). Currently, there is considerable interest in developing selective AEA amidase inhibitors which do not bind to CB1 (6 –10). Moreover, a serious effort has been made to develop AEA analogs with a high affinity for CB1 and resistance to the enzymatic hydrolysis. The most useful of these analogs is ( R)- N-(1-methyl-2-hydroxyethyl)arachidonylamide which has a high affinity for CB1 and has been shown to be stable toward hydrolysis (8). It should be pointed out that AEA amidase activity is sensitive to variations in the ethanol- amide head group, which can lead to changes in both turn- over rate and affinity for the enzyme’s catalytic site. Thus it is of interest to us not only to determine the ability of these molecules to serve as enzyme substrates, but also to test AEA analogs as AEA amidase inhibitors in competition experiments with AEA. Traditional assays for AEA amidase activity com- monly rely on measuring radioactivity of hydrolysis products (4, 7, 11, 12). We recently described a nonra- dioactive method for AEA amidase assay using direct 1 To whom correspondence should be addressed at School of Phar- macy, U-92, University of Connecticut, Storrs, CT 06269. Fax: 860- 486-3089. E-mail: makriyan@uconnvm.uconn.edu. 2 Abbreviations used: AEA, anandamide; AA, arachidonic acid; EA, ethanolamine; OPA, o-phthaldialdehyde; CB1, brain cannabinoid re- ceptor; 1,2-AP, 1-amino-2-propanol; 2,1-AP, 2-amino-1-propanol. 8 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ANALYTICAL BIOCHEMISTRY 261, 8 –15 (1998) ARTICLE NO. AB982713