ANALYTICAL

BIOCHEMISTRY

66,

423-433 (1975)

Reactions

of Biogenic

Aqueous An Application

Potassium to the

Amines Dichromate

Determination

N. ERIC NAFTCHI,

with

of Dopamine

M. ALICE BECKER, AND

ANAND Deparrment

S. AKERKAR

of Biochemicul Phurmacology. Nebc, York University Medical Schwwrz

Mann,

Institute of Rehabilitation Medicine, Cenier. New York. Ne+t’ York 10016

Research and Development, Orangeburg. New York

Mountain 10962

VieKj Avenue.

Received October 31. 1974: accepted January 24. 1975 The reaction of potassium dichromate with a series of phenols, aminophenols, catecholamines, indolealkylamines and metabohtes of the latter two was studied. Reaction required the presence of aromatic who- or pmra-dihydroxy or -diamino groups. Potassium dichromate reacted not only with the vicinal hydroxyl groups of catecholamines but also with the Shydroxy group and the ring nitrogen in the indolealkylamine series. Reaction occurs immediately upon mixing the reagents: the colored products are insoluble in water and most common organic solvents. 30-methylated catecholamines and acids and S-0-methylated indolealkylamines and acids did not react with the dichromate. Physical and chemical data on the products of these reactions suggest lack of reaction with the side chain in the biogenic amines. A method using dichromate oxidation-products to determine dopamine concentrations in urine is presented.

For many years physical and inorganic chemists have shown great interest in the stable metal complexes of aromatic hydroxy and polyhydroxy compounds. Among these, catechol, in which the adjacent hydroxyl groups are readily complexed, has been used in the studies involving nearly one-quarter of the known elements (1). Many reactions occur at basic pH where the hydroxyl groups are ionized and can most easily complex with an electrophilic metal ion. The ease with which the catechol moiety forms metal complexes suggested methods for stabilizing catecholamines, which are known for their sensitivity to light and heat and susceptibility to auto-oxidation. This report presents our initial data on the reactions of biogenic amines and their metabolites with aqueous potassium dichromate and illustrates the usefulness of the dichromate procedure in determining the concentration 423 Copyright 6 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

424

NAFTCHI,

BECKER

AND

AKERKAR

of dopamine in urine. The present method does not require isolation of biogenic amines prior to derivatization and, therefore, the loss of these compounds found in low concentration in biological fluids is minimized. The ability to derivatize these compounds in aqueous solutions has several advantages over other derivatization techniques. For example, the volatile derivatives used for gas-liquid chromatography/mass spectrometry require anhydrous conditions for their preparation (2). Oxidation products of biogenic amines with dichromate can be analyzed by any one of several methods, e.g., radioassay using r51Cr] dichromate, carbon rod atomic absorptiometry (CRA) and radioimmunoassay. METHODS

Chromium nitrate, manganous chloride, zinc sulfate, nickelous ammonium sulfate, as “Baker Analyzed” reagents, were obtained from J. T. Baker Chemical Co. Potassium dichromate (analytical grade) was obtained from Fisher Scientific Co. and K,[51Cr],0, from New England Nuclear. All organic compounds (best grades available) were purchased from Aldrich Chemical Co., Calbiochem, Sigma Chemical Co., K and K Laboratories, Chemical Procurement Laboratories, Inc., and Nutritional Biochemical Co. Sephadex G-25 (60-150 pm) was obtained from Pharmacia Fine Chemicals, and neutral alumina was purchased from M. Woelm, West Germany. Melting points were taken by the capillary method. None of the complexes melted below 300°C. Infrared spectra were taken on a Perkin-Elmer 22 1 Grating infrared spectrophotometer using the potassium bromide pellet technique. Mass spectra were obtained on a DuPont 21-110 spectrophotometer. Atomic absorption and carbon rod measurements were performed on a Varian-Techtron Type AA-5 spectrometer. The biogenic amine-dichromate oxidation. The compound (0.001 mole) was dissolved in water (1 ml). Upon addition of saturated aqueous potassium dichromate (0.05 ml), a black or brown precipitate formed immediately. The product was isolated by suction, washed with water, 0.001 N HCl and water, and then dried in a vacuum desiccator. Fluorescence measurements. An aliquot of a catecholamine-metal salt [Cr (NO,),, Zn(SO,),, Ni(NH,),(SO,),, MnCl,] or catecholaminepotassium dichromate solution (10 nmolelml) was placed in a 7.5 X l.O-cm Pyrex test tube. Phosphate buffer, pH 7.5, was added to obtain a final volume of 1.5 ml. While holding the test tube on a Vortex mixer, 0.5 ml of a solution of fluorescamine (Fluram’) in acetone (16 mg/lOO ml) was added rapidly with vigorous agitation. Blanks contained all reactants except the catecholamine. The fluorescence was read with ’ 4-Phenylspiro[furan-2(3H).

I’-phthalanl-3,3’-dione.

Roche

Diagnostics.

CARBON

the excitation

wavelength

ROD

DOPAMINE

49

ASSAY

at 390 nm and the emission

wavelength

at

480 nm. Analysis

of dopamine in urine. An aliquot (25 ml) of a 24-hr urine specimen, acidified with HCl upon collection, was placed in a 45 ml centrifuge tube, adjusted to pH 8 and adsorbed on 0.5 g of alumina, prepared according to Valori (3). The tubes were shaken for 30 set, centrifuged at 1OOOg for 5 min, and the supernatant fluid decanted. The alumina was washed with cold deionized, double glass-distilled water ( 15 ml), centrifuged for 5 min and the supernatant fluid discarded. This procedure was repeated three times. The catecholamines were eluted by gently shaking the alumina with acetic acid (0.3 N, 3 ml) for 5 min. Standards were prepared by adding 5 pg of dopamine in 0.1 ml water to an aliquot of urine (25 ml) and the samples were treated as above. Two aliquots (0.1 ml and 0.2 ml) of the acetic acid eluate were placed in separate tubes. To each was added 0.25 ml of aqueous potassium dichromate.” Sephadex G-25 columns (5-cm height; h-mm i.d.) were prepared. the packing was checked by applying Blue Dextran 2000, and the flow rate was adjusted to 32 drops/min. In a typical experiment six columns were prepared: two for each urine sample, two for the urine sample containing dopamine as internal standard, and two for the blank containing only the dichromate stock solution (0.25 ml). The catecholaminechromium mixtures were transferred quantitatively to the columns and 5 ml of water was added. Three l-ml fractions were collected, and the chromium content of the eluate was determined either by carbon rod atomic absorption (CRA) or liquid scintillation spectroscopy when K,[“‘Cr],O, was used.

RESULTS

The results for two groups of compounds treated with aqueous potassium dichromate are summarized in Fig. 1 and 2. Potassium dichromate reacts with either ortho- or put-n-dihydroxy, diamino- or amino-hydroxy groups. The products of these reactions are chromium-containing oxidation products of catecholamines. For the sake of brevity we will refer to these as “catecholamine-chromium complexes” or simply as “chromium complexes”. 0-Methylated catechols, indoles. or monohydroxy aromatic compounds do not react with this reagent. Infrared spectra of the chromium-containing oxidation products were broad and undefined. Very strong, broad bands, however, appeared between 3,200 and 3,600 cm-l indicating a large amount of water of crystallization (4). Mass spectral data on the catecholamine-chromium -’ I ml =

I mg Cr:

I nmole

= 2.826

mg K,Cr,O,.

426

NAFTCHI,

3 - Kkj

Q,IA,.P

BECKER

AND

DERIVATIVES

AKERKAR

NJ REAcTrcN

--I6 AMI IERIVATMS

1 - DIVYIRXY (xII)ouHlGAN) ERIVATIMS

M IWTION ND riv+cTIoN

FIG. 1. Reaction of phenols, aminophenols, catecholamines and catecholamine derivatives with aqueous potassium dichromate. An aqueous solution of the organic compound was mixed with saturated potassium dichromate. Reaction occurred immediately with the formation of a brown or black precipitate.

complexes were also not very informative as to their overall structure. Major peaks for the dopamine-chromium complex at m/e 107, 105 and 91 were suggestive of the fragmentation pattern for catechol. A peak at m/e 44 (100%) was indicative of the ethylamine side chain. Fluorescence Properties of Metal-

Catechol Complexes

Aqueous solutions of chromium, zinc, nickel and manganous salts and dopamine or norepinephrine and the norepinephrine-dichromate reac-

CARBON

ROD

DOPAMINE

ASSAY

MO aiJ IR

kWTIW

HO CIJ IR

NO REACTION

427

H

N H

R=o!-$ty+;

cm; ani

u!$m

FIG. 2. Reaction of indolealkylamines and indolealkylacids with aqueous potassium dichromate. An aqueous solution of the organic compound was mixed with saturated aqueous potassium dichromate. Reaction occurred immediately with the formation of a brown or black precipitate.

tion product were treated with fluorescamine reagent (5-7). In one experiment fluorescamine was added to the mixture immediately after the catecholamine and metal solutions were mixed; in another, fluroescamine was added after the catecholamine and metal solutions remained for 24 hr in the cold. In a third set of experiments, fluorescamine was added after the catecholamine-metal salt solution remained for 24 hr at room temperature. In all cases the fluorescence levels were the same, i.e., two to four times that of the blank. The fluorescence is stable for up to 8 hr with the first four mixtures and about 24 hr for the dichromate oxidation-product. The detection limit is about 1 nmole. Fluorescence values for free norepinephrineand dopamine-fluorescamine mixtures are higher than those of the complexed amine. Detection limits are about 0.1 nmole. Determination of Dopamine in Urine

The dopamine-dichromate oxidation-product is used to measure dopamine levels in urine. The catecholamines from urine were adsorbed onto alumina, eluted with acetic acid, reacted with dichromate and separated on a Sephadex G-25 column. The dopamine-chromium product is retained on the column while the effluent contains other catecholchromium oxidation-products and excess dichromate. Employing K,[51Cr],0, the amount of dopamine can easily be determined from the

428

NAFTCHI,

BECKER

AND

AKERKAR

2.0I .8s

a

1.6-

2

1.4-

5

1.2-

6

l.O-

Fk

0.8-

i3

0.6-

$

0.40.2 o.o-

0

5

IO

I5

20

25

I 30

1 35

v 40

1 45

I 50

I 55

1 60

CHROMIUM CONCENTRATION (pg /ml 1 FIG. 3. Measurement of dopamine by atomic absorption spectroscopy. The chromiumcatechol amine mixture is chromatographed on Sephadex G-25. The dopamine-chromium oxidation-product remains on the column and the chromium content of the effluent containing the other catecholamine-chromium oxidation products plus unreacted dichromate is measured. The dopamine concentration is calculated using Eq. [l-3].

amount of chromium not adsorbed by Sephadex G-25. The short halflife of 51Cr (27 days) may limit the application of this method if not applied routinely. Dopamine levels in urine can also be determined by analyzing the dopamine-chromium complex using either atomic or carbon rod absorptiometry. The carbon rod method is extremely sensitive and can detect less than 5 ng of chromium; O.OOl-ml samples are sufficient for analysis. Dopamine levels determined by atomic absorption are linear over a wide range as shown in Fig. 3. A standard curve is prepared, and unknown samples can be read directly from the curve. A trace of carbon rod responses for the measurement of dopamine in urine is shown in Fig. 4. The amount of dopamine is calculated by measuring peak height and using the formula below: Chromium-dopamine

product = K2Cr20,

standard - (effluent);

[I]

CARBON

ROD DOPAMINE

429

ASSAY 0

= nq

dopaminelml

urine

123.81

STANDARD

I

SAMPLE I DUPLICATES

STANDARD

I

SAMPLE

2

STANDARD

2

SAMPLE 3 DUPLICATES

FIG. 4. Measurement of dopamine in urine by carbon rod absorption spectroscopy. The chromium-catecholamine mixture is chromatographed on Sephadex G-25. The dopaminechromium oxidation product remains on the column and the chromium content of the effluent containing the other catecholamine-chromium oxidation-products plus unreacted dichromate is measured.

Effluent = (unreacted ucts). Since

K,Cr,O,

M, dopamineiM,

+ other catecholamine-chromium chromium

= 153152 = 2.976,

prod[21

therefore [Dopamine]

= 2.976 [(CRA

response for K,Cr,O, standard) - (the response for the effluent)],

[3]

where CRA = carbon rod absorptiometry. standard = amount of potassium dichromate used for reaction of the catecholamines. The mean normal urinary excretion of dopamine (pg/mg of creatinine) was found to be 0.18 -+ 0.04. The method is reproducible within one or among several samples.

430

NAFTCHI,

BECKER

AND

AKERKAR

DISCUSSION

Since catecholamines are extremely labile and the analysis of these compounds is often hampered by their rapid degradation, we began a search for methods of stabilizing the biogenic amines prior to their determination. Jameson and co-workers (8-10) have shown that catechol, epinephrine and norepinephrine form stable complexes with transition metals by coordination through the phenolic groups rather than the side chain. Copper (II), manganese (II), cobalt (II), and zinc (II) react to form 1: 2 complexes (8,9). Nickel (II) appears to chelate intermolecularly through both the phenolic group and the side chain (9). Potassium dichromate has been used as a staining agent for the preservation of the dense core vesicles of adrenergic nerves (I 1). The staining ability is most likely due to rapid oxidation of the biogenic amines contained in the vesicles and formation of colored products. It appeared, therefore, that complexation and/or reaction of the catecho1 moiety with a metal ion would provide a simple, rapid method for stabilizing the catecholamines. Initially we decided to investigate the reaction of potassium dichromate with biogenic amines and then proceeded to other metal-complexation reactions using methods previously established (1). A series of phenols, aminophenols, catecholamines, indolealkylamines, and metabolites of the latter two groups of compounds were treated with aqueous potassium dichromate (Fig. 1 and 2). We examined aromatic dihydroxy, diamino- and amino-hydroxy compounds with and without a side chain to determine the structural requirements for the reaction. Phenylalkylamines or acids and indolealkylamines or acids do not react with dichromate. SHydroxyindole derivatives, however, yield oxidation products. Aqueous potassium dichromate will react with two OH groups, two NH, groups, or an OH and an NH2 group provided they are situated in an ortho or para relationship. Quinone, which is oxidized hydroquinone, also will react with the reagent. If one hydroxyl group is blocked by a methyl group, if one hydroxyl group is removed, or if the hydroxyl groups are meta to each other no reaction occurs. These data all suggest that dichromate oxidizes the catechol moiety to a quinoid structure either prior to or concomitant with complexation with chromium. All the compounds in Fig. 1 and 2 contain ortho or para reactive groups and can easily form stable quinoid-type structures as shown below:

CARBON

ROD

DOPAMINE

ASSAY

43 1

Resorcinol, which cannot form a stable quinoid species, does not react with aqueous dichromate. Elemental analysis of the dopamine-complex shows the presence of chromium, and the amount present is approximately one chromium atom for every molecule of catechol compound. The exact structure of the dichromate oxidation-product has not yet been elucidated, but presumably the electrophilic metal coordinates with the electron-rich oxygens and the benzene ring. In order to gather further evidence that the side chain does not enter into reaction and to investigate further the stability of biogenic amines when the catechol portion is unavailable for reaction, we measured the fluorescence of several catecholamines after they had been mixed with solutions of transition metal salts or potassium dichromate. We used fluorescamine reagent’ I (5-7) that reacts with primary amines I1 to form fluorescent derivatives III. If the side chain containing the primary

I

II

111

amine were to enter into oxidation or complex formation no reaction between fluorescamine and the complexed or oxidized catecholamine could occur. The use of solutions of transition metal salts was suggested by earlier work (1,8-IO). We reasoned that the metal will chelate to the catechol

432

NAFTCHI,

BECKER

AND

AKERKAR

group, stabilize the catecholamine and perhaps increase or extend the life of the fluorescent product. The observation of fluorescence that is stable for several hours suggests that the side chain remains free and reaction or complexation occurs only at the adjacent hydroxyl groups. Since the Auorescence of the complexed catecholamine-fluorescamine product is lower than that of the free catecholamine-fluorescamine product and since the detection limits are tenfold less for the former than the latter, the fluorescamine technique may not represent a sensitive analytical procedure for the determination of total catecholamines in blood. It should also be noted that the use of fluorescamine in structural studies as well as in analytical methods further increases the usefulness of the reagent. The determination of dopamine levels in urine illustrates the utility of the dichromate procedure. The method is simple, sensitive, and adaptable to several instruments. Moreover, the immediate stabilization and preservation of the amines is an important aspect of this procedure. The mean urinary excretion of dopamine for eight normal subjects was found to be 0.18 4 0.04 pglmg of creatinine. This compares very well with the value of 0.15 -t- 0.05 pgg/mg of creatinine previously found using gas-liquid chromatography ( 12) and with the value of 0.19 -+ 0.03 Fg/mg of creatinine determined by a fluorescence method (13). In conclusion, we have found that dichromate oxidation products of catecholamines are easily prepared, stable and can be used in simple, sensitive analytical techniques. We are presently exploring the use of these derivatives in the analysis of total catecholamines in blood and urine and of several enzymes involved in the synthesis and degradation of biogenic amines. ACKNOWLEDGMENTS Fruitful discussions with Dr. Margaret acknowledged.

Demeny and Dr. Gisela Witz are gratefully

REFERENCES 1. A partial list of relevant studies: (a) Weinland, R., and Denzel, W. (1914) Berichte 47, 2753-2759, 2990-2994; (b) Weinland, R., and Sperl, H. (1925) Z. Anorg. ANg. Chem. 150, 69-83; (c) Weinland, R., and Walter, E. (1923) Z. Anorg. A//g. Chem. 126, 141-166; (d) Brown, H. P., and Austin, J. A. (1941) J. Amer. Chem. SOL.. 63, 2054-2055; (e) Thomas, L. H. (1946) J. Chem. Sor. 820-22; (f) Rosenheim, A., and Barattschesky, M. (1925) Berirhte 58B, 891-893; (g) Rosenheim, A., Raibmann, B., and Schendel, G. (1931) Z. Anorg. Allg. Chem. 196, 160-176; (h) Schalder, R., and Wolf, M. (1933) Z. Anorg. Al/g. Chem. 210, 184-l 94; (i) Garreau, Y. (1940) Bull, Sot. Chim. Fr. 7, 920-927; (j) Weinland, R. and Dottinger, A. (1918) Z. Anorg. A&. Chem. 102, 223-240: (k) Shafer, H. (1948) Angew. Chemie A60, 73-76; (1) Prasad, S. and Kacker, K. P. (1958) J. Indian Chem. Sot. 35, 890-892; (m) Dubey, S. N., and Mehrota, R. C. (1964) J. Inorg. Nucl. Chem. 26, 1543-50.

CARBON

ROD DOPAMINE

433

ASSAY

2. Handbook of Silylation (1972) 1st ed., pp. 10-21, Pierce Chemical Co., Rockford, IL. 3. Valori, C.. Renzini, V., Brunori, C. A., Porcellati. C., and Corea. L. (1969) Ital. J. Biochem. 18, 394-405. 4. Conley. R. T. (1972) Infrared Spectroscopy. 2nd ed., p. 206. Allyn and Bacon, Boston. 5. Weigele, M., De Bernardo, S., Tengi, J., and Leimgruber. W. (1972) J. Amer. Chrm. Sot.

94. 5927-5928.

6. Ildenfriend. S.. Stein, S., Bohlen, P., and Dairman, W. (June, 1972) Third American Peptide Symposium, Boston, MA. 7. Udenfriend, S., Stein, S., Bohlen, P., Dairman. W.. Leimgruber. W.. and Weigele. M. ( 1972) Science 178, 87 I-872. 8. Jameson. R. F. and Neillie. W. F. S. (1965) J. Inoye. NM-/. Chcjm. 27, 2b23-2b34. 9. Jameson, R. F. and Neillie, W. F. S. (1966) J. Inorg. NK/. Chem. 28, 2667-2675. 10. Gorton, J. E. and Jameson, R. F. (1968)J. Chem. Sot. A 2615-2618. I 1. Tranzer, J. P. (1972) in Histochemistry, Theoretical and Applied (Everson Pearse, A. G., ed.) 3rd ed., Vol. 2, pp. 1452-53, Churchill Livingstone, London. 12. Clarke, D. D., Wilk, S., Gitlow, S. E., and Franklin, M. J. (1967). J. Gas Chromafog. 7, 307-3

10.

13. Anton. A. H., and Sayre, D. F. ( 1964) J. Pharmacol.

E.xp.

Thu.

145,

326-336,