Determination of free and conjugated catecholamines and L-3,4-dihydroxyphenylalanine in plasma and urine: evidence for a catechol-O-methyltransferase inhibitor in uraemia.

A sensitive, accurate and reproducible method has been developed for the determination of free and conjugated catecholamines and L-3,4-dihydroxyphenylalanine in plasma and urine. The assay involves the enzymatic conversion of these compounds to their radio-labelled O-methylated derivatives using catechol-O-methyltransferase and S-adenosyl-L-[methyl-(3)H]methionine. Recoveries of 75 + /- 5% for dopamine, 70 + /- 5% for adrenaline and 65 + /- 5% for noradrenaline were obtained. The sensitivities were 0.5 pg for adrenaline and noradrenaline and 5-7 pg for dopamine, and dihydroxyphenylalanine. Measurements of conjugated catecholamines were performed after mild acid hydrolysis for 20 min at 95 degrees C. During this procedure no degradation of the catecholamines was observed. This assay led to the discovery of a dialyzable factor in the plasma of chronic uraemic patients which inhibits catechol-O-methyltransferase activity in vitro. The mean 22% inhibition observed for unhydrolyzed plasma increased to 42% after hydrolysis. The identity of this inhibitor which exists as an inactive conjugated form, probably a sulphate ester, and its implication in physiopathological disorders remain to be established.