Journal of Chromatography A, 1074 (2005) 47–51 Improved assay for catechol-O-methyltransferase activity utilizing norepinephrine as an enzymatic substrate and reversed-phase high-performance liquid chromatography with fluorescence detection Nozomi Aoyama a , Makoto Tsunoda a,∗ , Kazuhiro Imai b a Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan Received 9 December 2004; received in revised form 17 February 2005; accepted 10 March 2005 Available online 2 April 2005 Abstract We have previously established a rapid catechol-O-methyltransferase (COMT) assay using norepinephrine (NE) as a natural substrate and flow-injection analysis. In this study, the method is improved for screening of COMT inhibitors or activators using reversed-phase high- performance liquid chromatographic separation with fluorescence detection. The excess substrate, NE, was removed by the addition of borate in the eluent for HPLC to make an ionic complex with NE, which was eluted faster than the enzymatic product, normetanephrine. The method had good precision and accuracy, and was able to assay one sample in 5min, showing the usability for screening of COMT inhibitors or activators. © 2005 Elsevier B.V. All rights reserved. Keywords: Catechol-O-methyltransferase; Norepinephrine as substrate; Borate complex; HPLC-fluorescence detection; Hypertension; Parkinson’s disease 1. Introduction Catechol-O-methyltransferase (COMT) catalyzes the methylation of o-hydroxyl group of compounds with cate- chol structure, using S-adenosyl-l-methionine (SAMe) as a methyl donor [1]. It plays an important role in the metabolism of catecholamines (CAs) and the deactivation of exogenous catechol compounds. We have studied the blood pressure regulation in spon- taneously hypertensive rats (SHR) in relation to CAs metabolism by COMT. As a result, it was found that com- pared to Wistar-Kyoto (WKY) rats, SHR had less ability for methylation of CAs, especially norepinephrine (NE) [2]. Also the activity and amount of membrane-bound COMT in liver, which should be an important isozyme in the metabolism of CAs, was lower in SHR [3]. When SAMe was given to SHR, blood pressure decreased because of the accelerated ∗ Corresponding author. Tel.: +81 3 5841 4761. E-mail address: makotot@mol.f.u-tokyo.ac.jp (M. Tsunoda). 3-O-methylation of CAs catalyzed by COMT to afford 3- O-methyl metabolites (especially normetanephrine (NMN)). Moreover, it was reported that COMT inhibition elicits a moderate, dose-dependent pressure response in the setting of severely impaired baroreflex buffering [4]. These results suggest that activation of COMT should cause blood pressure reduction. Thus, compounds that have the ability to activate COMT may be the candidates to control blood pressure under a new mechanism. On the other hand, COMT inhibitors also attract attention because of their medical use for Parkinson’s disease [5–7]. The brain of patients with Parkinson’s disease is deficient of dopamine due to a defect in the dopamine nerves. l-dopa is, as its precursor, now administered to supply dopamine. To prevent the peripheral metabolism of l-dopa by COMT, the COMT inhibitor together with aromatic l-amino acid decar- boxylase inhibitor is administered. According to these observations, it was suggested that the search for the COMT activators and inhibitors is an impor- tant issue for the treatment of hypertension or Parkinson’s 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.03.037