Determination of anandamide amidase activity using ultraviolet-active amine derivatives and reverse-phase high-performance liquid chromatography

Abstract

Anandamide amidase catalyzes the hydrolysis of anandamide (AEA) to arachidonic acid (AA) and ethanolamine (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 amidase 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 derivatization and reverse-phase high-performance liquid chromatography (HPLC). The hydrolysis product, ethanolamine, after separation from protein was derivatized with OPA to form a UV-active isoindole derivative which was then detected at 230 nm. The detection 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 inhibits 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 molecule can result in significant variation in both affinity and turnover rate for each analog with respect to AEA amidase.