Development of procedures for the determination of catecholamines, indoleamines, related metabolites, and monoamine oxidase activity.

Abstract

A procedure was also employed for the determination of monoamine oxidase activity in tissue samples. Focusing on the analysis of the acidic metabolites of dopamine and/or 5-hydroxytryptamine, preincubation and incubation parameters were optimized to provide maximal in vitro activity. The assay requires the addition of exogenous aldehyde dehydrogenase to transform the intermediate aldehyde into the acid. Quantitation of the products by liquid chromatography with electrochemical detection may employ the conditions mentioned above. Alternatively, the mobile phase may be altered to yield maximum throughput if one is only concerned with monoamine oxidase activity.

A procedure was developed for the routine determination of eleven different compounds involved in the tyrosine and tryptophan pathways of nervous tissues. Using a high resolution, microparticulate reverse phase liquid chromatographic column with an electrochemical detector, the capacity factor was investigated as a function of mobile phase pH, sodium octyl sulfate (ion-pairing agent) concentration, and methanol concentration for many different metabolites. The final procedure is applicable to milligram samples of tissue and employs only a single column with simple isocratic conditions. Direct injection of a homogenized supernatant fraction provides quantitation of 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, homovanillic acid, 5-hydroxytryptophol, 5-hydroxytryptamine, and 3-methoxytyramine. Following the application of an alumina isolation step to the remaining supernate, injection into the same column provides quantitation of 3,4-dihyroxyphenylalanine, norepinephrine, epinephrine, 3,4-dihydroxyphenylacetic acid, and dopamine.