Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells.
We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl-L-methionine and magnesium chloride in sodium-potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl-L-methionine using this method are similar to values reported previously (0.51 and 14 microM, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2-3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.
Allen
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E
Division of Pediatric Pharmacology and Critical Care, Rainbow Babies and Childrens Hospital, Cleveland, OH 44106.
Myers
C
C
Frank
D
D
Pettigrew
A
A
Thompson
S
S
Blumer
J L
JL
D000328:Adult; D002394:Catechol O-Methyltransferase / Q000097:blood*; D004298:Dopamine / Q000737:chemistry; D004563:Electrochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry; D008745:Methylation; D012016:Reference Values; D015203:Reproducibility of Results; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D012997:Solvents
D012997:Solvents; D012435:S-Adenosylhomocysteine; D002394:Catechol O-Methyltransferase; D008274:Magnesium; D004298:Dopamine