A Sensitive Radioenzymatic Assay for Dopamine,Norepinephrine, and Epinephrine inPlasma and Tissue

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Abstract
A double-isotope, radioenzymatic assay for measuring dopamine, norepinephrine and epinephrine in 1 sample is described. The assay procedure includes incubation, solvent extraction and TLC. Dopamine, norepinephrine and epinephrine were incubated with catechol-O-methyl transferase (COMT) and [3H]S-adenosyl methionine ([3H]SAM) and were converted to the O-methylated tritiated derivatives: [3H]methoxytryramine, [3H]normetanephrine, and [3H]metanephrine, respectively. After several extraction steps, the O-methylated products were purified by means of two-dimensional TLC using silica gel. The TLC system resulted in complete separation of the 3 O-methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30 pg. The addition of large amounts of plasma reduced the activity of COMT, but increasing the Mg concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14C]metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for cross-reactivity. Of the substances tested, only dopa exhibited cross-reactivity. However, the apparent 30% cross-reactivity of dopa with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 .mu.l of trunk plasma from decapitated rats was sufficient for the determination of the 3 catecholamines.