A simple determination of serotonin, 5-hydroxyindoleacetic acid and 5-hydroxytryptophan decarboxylase activity in rat brain areas and parallel correlation among the levels

Abstract

We devised a simple method for the separation of serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) using small P-cellulose and DEAE-Sephadex columns, respectively. Both substances were estimated fluorometrically by the reaction with o-phthalaldehyde (OPA). In this method, some known interfering substances in addition to many primary amines and amino acids were separated from 5HT and 5HIAA. The recoveries of 5HT and 5HIAA were 80 and 71%, respectively. The fluorescence intensities of 30 pmol of 5HT and 15 pmol of 5HIAA were about twice over the reagent blank. The complete separation of 5-hydroxytryptophan (5HTP) from 5HT made it possible to determine 5HTP decarboxylase activity in rat brain areas without using an isotope-labeled substrate. The method allows for quantitation of the enzyme activity in mg amounts of the brain tissues. The activities in discrete areas were in good parallel with the 5HT contents, except for the striatum. The levels of 5HIAA were also much in parallel with those of 5HT.