Summary
The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas glutationedopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480–485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilibrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.
Similar content being viewed by others
References
Agrup, G., Falck, B., Jacobsson, S., Rorsman, H., Rosengren, A.-M., Rosengren, E.: 5-S-Cysteinyldopa in melanomas of caucasians. Acta Dermatovener (Stockh.) 54, 21–22 (1974)
Björklund, A., Ehinger, B., Falck, B.: A method for differnetiating dopamine from noradrenaline in tissue sections by microspectrofluorometry. J. Histochem. Cytochem. 16, 263–270 (1968)
Björklund, A., Ehinger, B., Falck, B.: Analysis of fluorescence excitation peak ratios for the cellular identification of noradrenaline, dopamine or their mixtures. J. Histochem. Cytochem. 20, 56–64 (1972a)
Björklund, A., Falck, B.: Cytofluorometry of biogenic monoamines in the Falck-Hillarp method. Structural identification by spectral analysis. In: Fluorescence techniques in cell biology (A.A. Thaer and M. Sernetz, eds.), pp. 171–181. Berlin-Heidelberg-New York:Springer 1973
Björklund, A., Falck, B., Jacobsson, S., Rorsman, H., Rosengren, A.-M., Rosengren, E.: Cysteinyl dopa in human malignant melanoma. Acta Dermatovener (Stockh.) 52, 357–360 (1972b)
Björklund, A., Falck, B., Lindvall, O., Svensson, L.-Å.: New aspects on reaction mechanisms in the formaldehyde histofluorescence method for monoamines. J. Histochem. Cytochem. 21, 17–25 (1973)
Björklund, A., Falck, B., Owman, Ch.: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: Methods of investigative and diagnostic endocrinology (S. A. Berson, ed.) Vol. 1: The thyroid and biogenic amines (J. E. Rall and I. J. Kopin, eds.), pp. 318–368 Amsterdam: North-Holland Publ. Comp. 1972c
Björklund, A., Nobin, A., Stenevi, U.: Acid catalysis of the formaldehyde condensation reaction for a sensitive histochemical demonstration of tryptamines and 3-methoxylated phenylethylamines. 2. Characterization of amine fluorophores and application to tissues. J. Histochem. Cytochem. 19, 286–298 (1971)
Björklund, A., Stenevi, U.: Acid catalysis of the formaldehyde condensation reaction for sensitive histochemical demonstration of tryptamines and 3-methoxylated phenylethylamines. J. Histochem. Cytochem. 18, 794–802 (1970)
Corrodi, H., Hillarp, N.-Å.: Fluoreszenzmethoden zur histochemischen Sichtbarmachung von Monoaminen. 2. Identifizierung des fluoreszierenden Produktes aus Dopamin und Formaldehyd. Helv. chim. Acta 47, 911–918 (1964)
Corrodi, H., Jonsson, G.: The formaldehyde fluoresence method for the histochemical demonstration of biogenic monoamines. A review on the methodology. J. Histochem. Cytochem. 15, 65–78 (1967)
Falck, B., Jacobsson, S., Lindvall, O., Nietsche, U.-B.: On the occurrence of cysteinyldopa and dopa in melanocytes and benign nevi cells. Scand. J. plast. Surg., in press (1977)
Falck, B., Jacobsson, S., Olivecrona, H., Olsen, G., Rorsman, H., Rosengren, E.: Determination of catecholamines, 5-hydroxytryptamine and 3,4-dihydroxyphenylalanine (dopa) in human malignant melanomas. Acta Dermatovener (Stockh.) 46, 65–67 (1966a)
Falck, B., Jacobsson, S., Olivecrona, H., Rorsman, H.: Pigmented nevi and malignant melanomas as studied with a specific fluorescence method. Science 149, 439–440 (1965)
Falck, B., Jacobsson, S., Olivecrona, H., Rorsman, H., Rosengren, A.-M., Rosengren, E.: On the occurrence of catechol derivatives in malignant melanomas. Commun. Dept. Anat. Univ. Lund, Sweden, No. 5 (1966b)
Falck, B., Rorsman, H.: Observations on the adrenergic innervation of the skin. Experientia (Basel) 19, 205–208 (1963)
Håkanson, R., Sjöberg, A.-K., Sundler, F.: Formaldehyde-induced fluorescence of peptides with N-terminal 3,4-dihydroxyphenylalanine or 5-hydroxytryptophan. Histochemie 28, 367–371 (1971)
Jonsson, G.: Fluorescence studies on some 6,7-substituted 3,4-dihydroisoquinolines formed from 3-hydroxytyramine (dopamine) and formaldehyde. Acta chem. scand. 20, 2755–2762 (1966)
Lindvall, O., Björklund, A.: The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons. Histochemistry 39, 97–127 (1974)
Paul, E., Hartwig, H.-G., Möller, W., Illig, L.: Fluorescenzhistochemische Untersuchungen und mikrofluorometrische Analysen an pigmentbildenden Tumoren der Haut. Arch. Derm. Forsch. 253, 125–144 (1975)
Paul, E., Illig, L.: Fluorescenzmikroskopische Darstellung pigmentbildender Hauttumoren nach Falck-Hillarp im Vergleich zu ihrem gewöhnlichen lichtmikroskopischen Bild. Arch. Derm. Forsch. 249, 51–64 (1974)
Rorsman, H., Rosengren, A.-M., Rosengren, E.: Fluorometry of a dopa peptide and its thioether. Acta Dermatovener (Stockh.) 51, 179–182 (1971)
Rost, F.W.D., Polak, J.M.: Fluorescence microscopy and microspectrofluorimetry of malignant melanomas, naevi and normal melanocytes. Virchows Arch. Abt. A 347, 321–326 (1969)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Agrup, G., Björklund, A., Falck, B. et al. Fluorescence histochemical demonstration of dopa thioethers by condensation with gaseous formaldehyde. Histochemistry 52, 179–186 (1977). https://doi.org/10.1007/BF00492294
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00492294