Abstract: The optimal conditions for a HPLC-EC method for measuring human mononu-clear cell catechol-O-methyltransferase (COMT) activity with dobutamine as substrate are reported. The major metabolite of dobutamine by COMT is 3-O-methyldobutamine (3-O-MD). COMT reaction should be conducted in pH 7.4 phosphate buffer. In order to saturate COMT, the concentrations of S-adenosyl-L-methionine (SAM; a methyldonor) and dobutamine should be more than 200 μmol·L^-1 and 0.5 mmol·L^-1 respectively. The apparent Michaelis constants for S-adenosyl-L-methionine and dobutamine are 0.05 μmol·L^-1 and 50 μmol·L^-1 respectively. The COMT activity increases linearly with increase of incubation time up to 1 h and with increase of protein concentrations ranged from 1.7 to 6 mg·ml^-1 respectively. The optimal concentration of magnesium chloride is ten times higher than that previously reported. The betweenday relative standard deviation for 3-O-methyl dobutamine is 9.7% and that of within-day is 8.9%. This method is ideally adapted to clinical studies.

Key words: Dobutamine, COMT, HPLC