VALIDATION OF ASSAY OF CATECHOL-O-METHYLTRANSFERASE ACTIVITY IN HUMANERYTHROCYTES

The multistep assay of specific catechol-O-methyltransferase (COMT) ac tivity in human erythrocytes was validated. Enzyme preparations from l ysed erythrocytes were incubated with a substrate (3,4-dihydroxybenzoi c acid) in the presence of Mg2+ and S-adenosylmethionine. The reaction products (vanillic acid and isovanillic acid) were analyzable by HPLC with electrochemical detection directly in the incubation medium afte r protein precipitation. The precision was calculated in order to defi ne the random variability associated with the method by intra-assay an d inter-assay relative standard deviations (RSDs) for the assays of bo th reaction products and protein. The intra-assay RDSs for the specifi c activities were between 4.8 and 11.9%, (n = 5-6) at two levels of CO MT activity. The inter-assay RSDs were between 6.4 and 14.2%, (n = 5-6 ), respectively. The total variation was mostly caused by the protein assay and the HPLC assay, and contributions From the sample preparatio n and incubation steps were minor.Some results from the clinical appli cation of the erythrocyte COMT assay are also reported. For both norma l volunteers and patients having Parkinson's disease, a single 400 mg dose of entacapone. a peripherally acting COMT inhibitor, decreased th e erythrocyte COMT activity. The application demonstrates that the ass ay was able to detect differences between the subjects and the effect of COMT inhibition in the clinical study.