Determination of catechol-O-methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection.
A rapid assay employing HPLC with electrochemical detection for catechol-O-methyltransferase (COMT) activity in red blood cells is described. Enzyme activity is determined from erythrocyte lysates using S-adenosyl-L-methionine as methyl donor and 3,4-dihydroxybenzoic acid as substrate. The 3-O- and 4-O-methylated reaction products are measured by high-performance liquid chromatography with electrochemical detection. Human erythrocyte soluble form of COMT had Km values of 6.1 microM and 26.0 microM for S-adenosyl-L-methionine and dihydroxybenzoic acid, respectively. The mean O-methylation ratio for the soluble form of COMT was 5.3. An O-methylation ratio of 15.5 was estimated in the membrane fraction of an erythrocyte pool from three samples. The activities of soluble COMT in erythrocytes of some animal species are also reported. The procedure is easily automated, and a large number of samples can be processed during one working day.
Schultz
E
E
Orion Pharmaceutica, Research Center, Espoo, Finland.
Nissinen
E
E
Kaakkola
S
S
D000818:Animals; D002394:Catechol O-Methyltransferase / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004563:Electrochemistry; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D051381:Rats; D013552:Swine
D002394:Catechol O-Methyltransferase