RADIOENZYMATIC ASSAY OF FEMTOMOLE CONCENTRATIONS OF DOPA IN TISSUES AND BODY-FLUIDS

Abstract— A single isotope radioenzymatic procedure for the measurement of DOPA has been developed. The assay combines O‐methylation of DOPA by purified COMT using [3H]SAM as the methyl donor and subsequent purification as the DNFB derivative of 3‐O‐[methyl‐3H]DOPA. The present method is about 100 times more sensitive than currently available DOPA methods. This is due to decreased blank values and increased enzymatic conversion giving transmethylation values of 50% with tissue extracts and values of almost 100% with pure solutions. Although COMT methylates a wide variety of catechol compounds, specificity of the assay is achieved by selective extraction and purification of the final product by tlc. The method has good inter‐assay reliability, the coefficient of variation being about 3.5%. This ultramicromethod was used to determine the steady‐state concentrations of endogenous DOPA in minute samples of brain areas of the rat. In untreated rats brain DOPA levels varied with the mode of death; the highest levels were found in animals killed by microwave irradiation. Unconjugated DOPA was measured in microlitre aliquots of human body fluids. Copyright © 1979, Wiley Blackwell. All rights reserved