O. Lindvall, A. Björklund, L. Svensson
May 3, 1974
Citations
1
Influential Citations
25
Citations
Quality indicators
Journal
Histochemistry
Abstract
SummaryCondensation with glyoxylic acid is a highly sensitive method for fluorescence histochemical demonstration of biogenic monoamines and related compounds. In the present study the fluorophore formation from phenylethylamines in the histochemical glyoxylic acid reaction has been investigated. From an analysis of 32 different phenylethylamine derivatives it was found that strong fluorescence is obtained from 3-hydroxylated and certain 3-methoxylated, primary phenylethylamines, whereas the fluorescence yield from secondary, tertiary, and N-acetylated phenylethylamines is very low.The fluorophore formation, which was analysed in detail for the primary catecholamine, dopamine, proceeds in two steps: first a Pictet-Spengler cyclization to the weakly fluorescent 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid derivative that, in the second step, reacts with another glyoxylic acid molecule yielding the strongly fluorescent 2-carboxymethyl-3,4-dihydroisoquinolinium derivative. The yield of the fluorophore from dopamine in the glyoxylic acid vapour reaction was found to be about 10%, the tetrahydroisoquinoline derivative constituting the main reaction product, about 80%. When the glyoxylic acid reaction was performed under conditions simulating the perfusion-immersion procedure used on tissue, the yield of the fluorophore from dopamine is strongly enhanced, and under these conditions it is almost quantitative.The catecholamine fluorophores demonstrated a pH-dependent tautomerism between the non-quinoidal and quinoidal states, the two forms of the fluorophore having different spectral properties. It was also shown that the 4-hydroxy group of the noradrenaline fluorophore can be split off by hydrochloric acid vapour yielding the fully aromatic isoquinoline. The fluorophores formed from DOPA and dopamine in a two-step reaction with formaldehyde and glyoxylic acid showed markedly different excitation and emission spectra. The DOPA fluorophore formed in this combined reaction is proposed to be a fully aromatic isoquinoline, the 2,4-dicarboxymethylisoquinolinium compound. This provides a possibility for the direct histochemical differentiation between DOPA and dopamine by microspectrofluorometry.