A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid

Abstract

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their 3H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with 3H-S-adenosylmethionine serving as the 3H-methyl donor. Following the enzymatic reaction, unreacted 3H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the 3H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.