Detection of 5-Hydroxytryptamine by Paper Chromatography

Abstract

IT is well known that enterochromaffin cells acquire a golden-yellow fluorescence after fixation in formaldehyde, due probably to their content of enteramine (5-hydroxytryptamine). We have now carried out chromatographic experiments with synthetic 5-hydroxytryptamine creatinine phosphate to study the sensitivity and specificity of this reaction. We used a butanol – acetic acid – water solvent and a developer composed of nine parts of a solution of potassium dichromate (0.1 per cent) and one part of formaldehyde solution (37–41 par cent). After spraying and heating at 100–110° C. for five minutes, the paper is viewed under ultra-violet light. Under these conditions, 5-hydroxytryptamine (R_F value 0.38) produces a golden-yellow fluorescence immediately after the heating and this persists for days; quantities as small as 0.2 µgm. base can be detected. The behaviour of bufotenine and bufotenidine is quite similar to that of 5-hydroxytryptamine. Tryptamine gives a bright yellow fluorescence if more than 2.5 µgm, are present; but this develops slowly. Tryptophane likewise gives a yellowish fluorescence if 2.5 µgm. are present, while 5-methoxytryptamine gives a yellow-orange fluorescence if similar amounts are present. Bufothionine, histamine, histidine, tyramine, tyrosine and p-norsynephrine produce no fluorescence, while that with adrenaline and nor-adrenaline is very feeble. m-Norsynephrine has already been shown to produce a violet fluorescence under these conditions if 0.5 µgm. is present¹. Such a small amount of 5-hydroxytryptamine (0.2µgm.) can also be detected with alkaline Folin reagent (producing a blue colour), Pauly reagent (red), diazotized p-nitraniline reagent (red-brown), N.N.C.D. reagent of Heinrich and Schuler (red-brown), Gibbs reagent (brownish-violet) and Ehrlich's reagent (blue)2.