A comparison of an improved o-phthalaldehyde fluorometric method and high pressure liquid chromatography in the determination of brain 5-hydroxyindoles of rats treated with L-tryptophan and p-chlorophenyl-alanine

Abstract

1 The determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) by reaction with o-phthalaldehyde (OPT) in the presence of cysteine and subsequent fluorometry was compared with determination by a new high pressure liquid chromatography (h.p.l.c.) electrochemical method. 2 The methods were used to investigate a claim that the OPT method gives falsely high brain 5-HT values for rats given tryptophan (greater than 25 mg/kg i.p.) and as a consequence an inhibition of 5-HT synthesis by tryptophan is obscured. 3 High concentrations of tryptophan caused some increase of fluorescence when added to 5-HT solutions and carried through the OPT method, e.g. tryptophan (80 micrograms/ml) gave fluorescence identical to 5-HT (0.13 micrograms/ml). This interference was decreased by more than half and the sensitivity of the method increased if cysteine was added at more stages and fluorescence developed at 77 degrees C instead of 100 degrees C. 4 The h.p.l.c. and modified OPT methods did not give significantly different mean brain 5-HT or 5-HIAA values in rats given 0,25,100,250 mg/kg L-tryptophan though values were somewhat higher by the OPT method with the highest dose. This method gave significantly higher residual brain 5-HT values (12% of control) than did the h.p.l.c. method (8% of control) after inhibiting 5-HT synthesis by p-chlorophenylalanine (150 mg/kg X 3). 5 There was no indication that tryptophan inhibited 5-HT synthesis even when brain tryptophan was increased about 30 fold (from 3.8 to 124 micrograms/g). 6 Results confirm the general reliability of the OPT method although h.p.l.c. has some advantages i.e. separation of 5-hydroxyindoles from tryptophan, greater sensitivity, ease of automation.