Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection.
We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day.
Reenilä
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Department of Pharmacology and Toxicology, University of Helsinki, Finland.
Tuomainen
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Männistö
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PT
D000818:Animals; D002394:Catechol O-Methyltransferase / Q000032:analysis* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D003342:Corpus Striatum / Q000201:enzymology; D004355:Drug Stability; D004563:Electrochemistry; D062385:Hydroxybenzoates / Q000378:metabolism; D008832:Microchemistry; D011786:Quality Control; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D014641:Vanillic Acid / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism
D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C009091:protocatechuic acid; C045317:isovanillic acid; D002394:Catechol O-Methyltransferase; D014641:Vanillic Acid