Assessment of catechol-O-methyltransferase activity and its inhibition in erythrocytes of animals and humans

✍ Scribed by Gerhard Zürcher; Mosé Da Prada; Jasper Dingemanse

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 475 KB
• Volume: 10
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/(sici)1099-0801(199601)10:1<32::aid-bmc550>3.0.co;2-n

✦ Synopsis

A non-radiometric method to measure the catechol-0-methyltransferase (COMT) activity in erythrocytes was modified to increase its sensitivity four-fold as well as its reproducibility and applicability. The method is based on the COhlT-mediated O-methyiation of 44naphtho [1,2-d] thiazol-2-yl) pyrocatechol, the product of which was determined fluorometrically. COMT activities down to less than 1% of those present at baseline could be measured precisely and accurately. The intra-and inter-assay coefficients were below 3 and 5.3'/0, respectively. Basal COMT activity and the distribution between soluble and membrane-bound COMT was shown to be variable among different species (ten species tested). The applicability of the method was demonstrated by the characterization of COMT activity-time courses in human erythrocytes after oral administration of the COMT inhibitor tolcapone. The assay developed will be useful in the rapid screening and clinical development of new COMT inhibitors.

EXPERIMENTAL

Reagents. 2,ZDithio-bis (1-aminonaphthalene) (DTAN) and SAM were purchased from Sigma (St. Louis, MO, USA): dithiothreitol (DIT, ultrol grade) from Calbiochem (La Jolla, CA, USA); magnesium chloride (p.a.), sodium dihydrogenphosphate (p.a.), sodium sulphite (p.a.), 3.4dihydroxy-benzaldehyde (pract.) and silica gel 60. particle size 40-63 pm from Merck (Darmstadt. Germany); Nucleosil