A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine inplasma and tissue

A double-isotope, radioenzymatic assayfor measuring dopamine, norepinephrine, and epi-nephrine in one sample is described. The assayprocedure includes incubation, solvent extraction, and thin-layer chromatography. Dopamine, norepi-nephrine, and epinephrine were incubated withcatechol-O-methyl transferase (COMT) and [³H]S-adenosyl methionine ([³H]SAM) and were convertedto the O-methylated tritiated derivatives: [³H]meth-oxytyramine, [³H]normetanephrine, and [³H]meta-nephrine, respectively. After several extractionsteps, the O-methylated products were purified bymeans of two-dimensional, thin-layer chromatogra-phy using silica gel. The thin-layer chromatographicsystem resulted in complete separation of the threeO-methylated compounds with an overlap of only1-2%. The assay was linear from 0 to 5 ng for eachcatecholamine and had a sensitivity of 10-30 pg. Theaddition of large amounts of plasma reduced theactivity of COMT, but increasing the magnesiumconcentration in the incubation mixture and the addition of EGTA to plasma samples improved the re-coveries. Each sample was corrected for lossesincurred during extraction and chromatography byusing [¹⁴C]methoxytyramine, [¹⁴C]normetanephrine, and [¹⁴C]metanephrine that were added at the endof incubation. Several catechol compounds known tobe O-methylated by COMT were examined for cross-reactivity. Of the substances tested, only dihydroxy-phenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPAwith dopamine was due to the presence of decar-boxylase activity in the COMT preparation. As littleas 50 μl of trunk plasma from decapitated rats wassufficient for the determination of the three cate-cholamines.