ANALYSIS OF FLUORESCENCE EXCITATION PEAK RATIOS FOR THE CELLULAR IDENTIFICATION OF NORADRENALINE, DOPAMINE OR THEIR MIXTURES

Abstract

Treatment with HCl induces changes in the excitation spectra of the formaldehyde condensation products of dopamine and noradrenaline that permit the distinction of these two catecholamines on the cellular level. Using histochemical models and tissue sections, these spectral shifts and changes in the ratio of the two main excitation peaks at about 320 and 370 nm have been measured. Acidification of the dopamine fluorophore rapidly leads to the conversion of the quinoidal dihydroisoquinoline fluorophore into its tautomeric nonquinoidal form. The process probably goes to completion within a few seconds following treatment with HCl. The fluorophore remains stable during prolonged acid treatment. The conversion of the noradrenaline fluorophore into its nonquinoidal state is equally rapid; however, the subsequent dehydration to the fully aromatic form is a slower process, and it is apparently not complete even after several minutes, when the rising over-all tissue fluorescence makes measurement increasingly difficult. The results, however, indicate that this characteristic fluorophore is formed from the noradrenaline fluorophore in a high degree also in tissue sections. In the present report, the various states of the catecholamine fluorophores are characterized in more detail, and the evaluation of excitation spectra for the cellular identification of dopamine and noradrenaline, alone or in mixtures, is discussed. A detailed description of the practical performance of the microspectrofluorometric differentiation procedure is given.