Brain Research Bulkfin, Vol. 9, pp. xi-xv, 1982. Printed in the U.S.A. Fluorescence of Catechol Amines and Related Compounds Condensed With Formaldehyde”” B. FALCK, N.-A. HILLARP, G. THIEME AND A. TORP Department of Histology, University of Lund, and Department of Pharmacology, University of Giiteborg, Sweden Received for publication August 28, 1961 The noradrenaline cells in the adrenal medulla become a) An amount of the amine hydrochloride corresponding fluorescent when briefly fixed in formalin [6,71, but the na- to 25 mg of base was dissolved in 4.5 ml 0.1 N H,SO, (or ture of the reaction has not been elucidated. Recently HCl). After addition of 0.5 ml 35% formaldehyde the solution Eranko [8] and Falck and Tot-p [lo] found that a very intense was heated at 100°C for 20-30 minutes, then cooled and neu- fluorescence develops in the noradrenaline cells when tralized to about pH 5 with K,C03. freeze-dried sections are exposed to formaldehyde vapour. b) The amine hydrochloride was dissolved in water As a great need exists for highly sensitive histochemical and-where necessary-neutralized to about pH 5 with methods for demonstration of mono-amines--i.a. for lo- K,CO,. Formaldehyde (about pH 6) was added and the solu- calization of the brain catechol amines-it was thought tion heated at 50°C for 20 minutes. worth while to study the reaction between formaldehyde and The reaction mixture was transferred to a cation ex- catechol amines in model systems and to isolate and examine change column (Amberlite XE-64, 22x0.55 cm, equilibrated the reaction products. with 1 M ammonium acetate of pH 6). Elution (5 ml frac- tions) was performed with 0.02 and 0.1 M ammonium acetate (pH 6 and pH 5, respectively) and 0.1 N HCI, and was fol- MATERIAL AND METHODS lowed by determination of the ultraviolet absorption of the Fluorescence microscopy. The amines (usually as hydro- fractions. chlorides) and amine-formaldehyde condensation products The main part of the reaction products from the catechol were dissolved in 1% aqueous solutions of human serum amines (80-90%) was eluted in a single peak with the 0.1 M albumin, gelatin, gliadin (wheat), or sucrose. One microliter buffer. The ultraviolet absorption and the fluorescence spots were placed on object glasses and dried at room tem- spectra of the fractions (at pH 2, 5, and 6.5) were taken, and perature or at 50°C. After microscopy the spots were treated spots containing 0.02-l pg of the compounds in 1% serum with the vapour from a 35% solution of formaldehyde (Merck albumin were examined in the fluorescence microscope as p.a.) for varying periods of time either at room temperature described above. Finally the main fractions were pooled and or at 50°C. In the latter case the glasses were prewarmed at evaporated to a small volume in vacua at 40°C (rotating 50°C to prevent water condensation on the spots. evaporator), dissolved in water, and stored at -30°C. The light from an Osram XBO 2001 high-pressure xenon No unchanged catechol amines were found on paper lamp was passed through a water-cooled 1 mm Schott BG 38 chromatography or on spectrophotofluorimetric examina- filter (for heat absorption) and then through a Zeiss M4 QII tions according to Carlsson and Waldeck [4], or Bertler, monochromator whose exit slit was projected in the dia- Carlsson and Rosengren [2]. phragm of the condenser of a fluorescence microscope. To Preparation of I-methyl-6,7-dihydroxy-I ,2,3,4-tetra- prevent stray light in the visible region from the mono- hydroisoquinoline. Dopamine was condensed with acetal- chromator reaching the microscope a suitable filter (usually a dehyde according to the procedure of Kovacs and Fodor 2 mm Schott BG 12) was placed before the condenser. Ex- [17], which gives a good yield of the tetrahydroisoquinoline. changeable stop filters were placed in the microscope tube The substance was crystallized twice from alcohol-ether. No (usually a Schott GG 4 and above it a Wratten gelatin filter unchanged dopamine was found on paper chromatography. No. 15). Paper chromatography. The amine-formaldehyde prod- Preparation of amine-formaldehyde condensation prod- ucts were examined by ascending paper chromatography ucts. Dopamine, noradrenaline, and adrenaline were treated using n-butanol-glacial acetic acid-water (4:1:5) as the with an excess of formaldehyde either in acid or at pH about 5. solvent system. After drying, the papers were examined in ‘Supported by grants from the Swedish Medical Research Council, the Air Force Ofice of Scientific Research and Development Command, United States Air Force, and by a grant (B-2854) from the National Institutes of Health. *Reprinted with permission from The Journal of Histochemistry and Cytochemistry, Vol. 10, No. 3, May, 1962, Copyright a 1962 by The Williams & Wilkins Co. xi \il FALCK. HILLARP, THIEME AND TORP TABLE 1 TABLE 2 FLUORESCENCE OF CATECHOL AMINES AND RELATED COMPARISON OF FLUORESCENCE INTENSITIES DEVELOPED COMPOUNDS INCLUDED IN A DRIED SERUM ALBUMIN FILM FROM CATECHOL AMINES AND SOME RELATED COMPOUNDS AFTER TREATMENT WITH FORMALDEHYDE VAPOUR AT INCLUDED IN A DRIED SERUM ALBUMIN FILM EXPOSED TO ~50°CFOR I5 MINUTES FORMALDEHYDE VAPOUR AT 50°C FOR 5 OR 15 MINUTES Amount Fluorescence 5 Min IS Min 0.2 0.02 0.01 0.2 0.02 0.01 Phenylalanine @“I: Fc! Tyrosine Meta-tyrosine + 0 0 2t 0 0 Meta-tyrosine Dopa 5+ 3t 2-t 6-c 4+ i- Dopa (3,4-dihydroxy-phenyl- a-methyl-dopa t 0 0 ? 4 0 0 alanine) 0.2 6+ Meta-tyramine 3-t 0 0 4t i 0 a-Methyl-dopa 1 4+ Dopamine 57 3- ?* 6- 4-r 72 Ij-Phenylethylamine 1 0 Noradrenaline 5+ 3t 21 61 4- 3+ Tyramine (Chydroxy-phenyl- Adrenaline 0 0 0 + 0 0 ethylamine) I 0 Epinine 0 0 0 i 0 0 Meta-tyramine (3-hydroxy- Methoxy- phenylethylamine) 0.2 4+ tyramine 0 0 0 + 0 0 Octopamine (4-hydroxy-phenyl ethanolamine) I 0 Dopamine (3,4-dihydroxy- phenylethylamine) 0.2 6+ Noradrenaline 0.2 6+ Adrenaline I 3+ 15 minutes. An exposure at this temperature for a longer Methoxy-tyramine (3-methoxy- time usually weakened or even destroyed the fluorescence. 4-hydroxy-phenylethylamine) I 31 but at about 20°C an exposure time of up to 24 hours could be Normetanephrine (3-methoxy-4- used. hydroxy-phenylethanolamine) I 0 As seen from Tables I and 2 there are certain structural Phenylephrine (3-hydroxy-N- requirements for the fluorescence reaction. Of the methyl-phenylethanolamine) I + phenylalanine and phenylethylamine derivatives used, a Epinine (3,4-dihydroxy-N- very intense fluorescence is given only by those that are methyl-phenylethylamine) I 3+ primary amines and also have hydroxyl groups at the 3 and 4 Hordenine (4-hydroxy-N,N- positions (dopa, dopamine, noradrenaline). An interesting dimethyl-phenylethylamine) I 0 exception is cw-methyl-dopa (see Discussion). The catechol 3.4-Dihydroxyphenylacetic acid I 0 amines, which are secondary amines (adrenaline and epinine), give a much weaker fluorescence, which further- more develops more slowly in the formaldehyde treatment. Thus it is possible to distinguish between noradrenaline and adrenaline in sections of adrenal medulla by means of the formaldehyde reaction [8,10]. Some data illustrating the ultraviolet light and then sprayed with K,Fe (CN), according sensitivity and the rate of the reaction are found in Table 2. to James [16]. The results obtained favour the view that the 3-OH group Fluorimetry. The fluorescence spectra of the amines and is essential for the fluorescence reaction. Thus the com- their formaldehyde condensation products (l-10 pg/ml), be- pounds with no hydroxyl group or only one in the position fore and after oxidation with HzO, according to Hess and pura to the side chain seem to be non-reactive, whereas the Udenfriend [15], were determined at various pHs with the meta isomers of tyrosine and tyramine show a fairly Aminco-Bowman spectrophotofluorometer. The wave- pronounced fluorescence. Furthermore the substitution of lengths given for activation and fluorescence are the uncor- the 3-OH group by a methoxy group in dopamine and norad- rected instrument values. renaline dramatically inhibits the reaction. The fluorescent products formed from the primary amines in dried spots of serum albumin were quite stable for days at RESULTS room temperature. The fluorescence did not disappear if the Fluorrswnce of amines in model systems: The catechol spots were furth&r dried in vac’uo over P20:, (2 days) or by amines and related compounds examined (see Table 1) heating at 150°C (1 hour). No material decrease of the showed no visible fluorescence in dried spots containing 1 pg fluorescence was found after extraction of the spots with of the substances in serum albumin. After exposure of the water, 0.1 N HCl, ethyl alcohol, xylene, or benzene, or at spots to formaldehyde vapour some of them were trans- acid hydolysis (0.1 N HCl, lOO”C, 10 min). formed into products which fluoresced intensely green to The amines were also examined enclosed in dried spots of yellow. The activation peak lies at 420-440 mp. Serum al- gelatin, gliadin, or sucrose. The fluorescence obtained in bumin itself-after treatment with formaldehyde-showed a gelatin was of somewhat lower intensity, and in gliadin or very faint dirty-green fluorescence. sucrose much weaker than that developed in serum albumin. In most instances a maximum fluorescence was obtained The products formed in gelatin, and especially in gliadin, when the formaldehyde treatment was performed at 50°C for were also to a higher extent extractable with water.