HISTOCHEMISTRY OF HISTAMINE: MICROSPECTROFLUOROMETRIC CHARACTERIZATION OF THE FLUOROPHORES INDUCED BY o-PHTHALDIALDEHYDE

Abstract

o-Phthaldialdehyde (OPT) has been used as a reagent for the histochemical demonstration of histamine by fluorescence microscopy. OPT induces a blue fluorescence in gastric epithelial cells of the rat and, under the same conditions, a yellow fluorescence in mast cells. Microspectrofluorometric analysis of the histamine-OPT fluorophore in model systems (silica gel thin layer or dried albumin droplets) revealed a concentration-dependent shift of the emission maximum from blue (at low histamine concentrations) to yellow (at high concentrations). The characteristics of the fluorophores, which were very ultraviolet-labile, were not overtly affected by pH, but the red shift was more pronounced upon hydration. The blue fluorescence was maximally excited by the 365 mµ line of the mercury lamp; the excitation maximum for the yellow fluorescence was approximately 405 mµ. This strongly suggests that at least two different fluorophores are formed as a consequence of the condensation between histamine and OPT. This was confirmed by thin layer chromatography of the OPT-histamine fluorophore and by the finding that ultraviolet exposure converts the yellow fluorophore into a blue one. It is suggested that the blue and yellow OPT-induced fluorescence in cells reflect markedly different concentrations of histamine. The present observations may offer a new possibility for the histochemical quantitation of cellular histamine based on the concentration-dependent shift of the emission and/or excitation maxima.