Validation of assay of catechol-O-methyltransferase activity in human erythrocytes.

The multistep assay of specific catechol-O-methyltransferase (COMT) activity in human erythrocytes was validated. Enzyme preparations from lysed erythrocytes were incubated with a substrate (3,4-dihydroxybenzoic 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 after protein precipitation. The precision was calculated in order to define the random variability associated with the method by intra-assay and inter-assay relative standard deviations (RSDs) for the assays of both reaction products and protein. The intra-assay RDSs for the specific activities were between 4.8 and 11.9% (n = 5-6) at two levels of COMT 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 preparation and incubation steps were minor. Some results from the clinical application of the erythrocyte COMT assay are also reported. For both normal volunteers and patients having Parkinson's disease, a single 400 mg dose of entacapone, a peripherally acting COMT inhibitor, decreased the erythrocyte COMT activity. The application demonstrates that the assay was able to detect differences between the subjects and the effect of COMT inhibition in the clinical study.