A rapid and sensitive potentiometric assay for monoamine oxidase using an ammonia-selective electrode

ANALYTICAL

BIOCHEMISTRY

86,

287-297 (1978)

A Rapid and Sensitive Potentiometric Assay for Monoamine Oxidase Using an AmmoniaSelective Electrode LAURENCE Neurochemistry Houston,

R. MEYERSON,

KENNETH D. MCMURTREY, AND VIRGINIA E. DAVIS

and Addiction Research Laboratory, Veterans Administration Texas 77211, and Departments of Medicine and Biochemistry, Baylor College of Medicine, Houston, Texas

Hospital.

Received August 29. 1977: accepted November 21. 1977 A sensitive and convenient method for the estimation of monoamine oxidase (MAO) activity using an ammonia-selective electrode has been developed. The ammonia in the sample diffuses through thegas-permeable membrane until NH,-N partial pressures are at equilibrium, and the resulting potential (in millivolts) is recorded with a millivolt/pH meter. The amount of ammonia generated enzymatically is obtained by calculating the difference in ammonia concentration in control and test incubation mixtures at pH 12.0. The sensitivity of this method is comparable to radiometric, fluorometric, and polarographic techniques. The millivolt potential corresponding to ammonia formed from substrate by MAO in brain homogenate and mitochondrial preparations is linear to enzyme protein concentration and to time during the 30-min incubation period. The stoichiometric relationship between the amounts of ammonia production and amine depletion is evidenced by the equal amounts of ammonia formed and substrate depleted. The assay technique offers diverse utility. Numerous primary amine substrates can be employed with this method. The procedure is also useful for the study of enzyme-inhibitor interactions. Compared to many other assay procedures, the method outlined offers the advantages of precision. sensitivity. accuracy. and rapidity.

Brain monoamine oxidase (MAO), (monoamine: O2 oxidoreductase (deaminating) EC 1.4.3.4) is an important aminergic enzyme since it participates in the intraneuronal oxidative deamination of free biogenic amines (1). MAO has also been utilized as a marker enzyme for the outer mitochondrial membrane (2). The biochemical and pharmacologic implications of this enzyme have been extensively reviewed (3). Many methods are available for assaying the activity of monoamine oxidase. These techniques are based on the following criteria: (a) disappearance of substrate (4,5). (b) consumption of oxygen (6-8), (c) formation of aldehyde (9-l l), (d) formation of ammonia (12,13), (e) formation of hydrogen peroxide (14,15), and (f) formation of the acid 287

0003-2697/7810861-0287$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved

288

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AND

DAVIS

following oxidation of amine to aldehyde (16). Analytical evaluation of the various reactants or products is performed by calorimetry (43, manometry (6), oxygen electrode (7,8), fluorometry (5,14,15), spectrophotometry (lo), and radiometry (16,17). Several of these techniques possess marked disadvantages. The polarographic oxygen uptake procedure lacks sensitivity and specificity, especially for crude tissue preparations. Fluorometric methods for the detection of hydrogen peroxide are sensitive but involve a fair amount of sample manipulation before fluorometry can be applied. Fluorometric techniques are also subject to error because of potential variation of endogenous levels of fluorescent materials. Spectrophotometric methods give good estimates of initial reaction velocities, but the primary disadvantage is that measurements are carried out in the ultraviolet spectral range where protein and other materials absorb appreciably. Furthermore, the usual substrates are not physiological in nature, i.e., kynuramine, benzylamine, m-iodobenzylamine, and p-dimethylaminobenzylamine. Radiometric procedures generally measure the production of labeled aldehydes or acids. Nonspecific binding of radioactive metabolites to denatured protein as well as differences in extraction coefficients of products prove to be inherent problems in the assay. Unless the conversion of an aldehyde metabolite to its corresponding acid can either be prevented or driven to completion, the varying aldehyde dehydrogenase activity of tissues will lead to variable counts of radiolabel of an acid solvent extract. While aldehydes are not detectable in cellular systems, they are highly reactive and may participate in Schiff base or polymerization reactions (l&19) rather than complete oxidation to acid or reduction to alcohol. Since the substrate specificity of MAO is broad, it is convenient to employ a MAO assay that is applicable to various monoamines, including the neurochemically important biogenic catechol- and indoleamines. This report describes a novel, rapid, and sensitive method for the quantitative estimation of MAO activity. The technique involves the potentiometric measurement with an ammonia-selective electrode of the ammonia formed. MATERIALS

AND METHODS

Apparatus. An Orion digital millivolt/pH meter (Model 701-A), an ammonia electrode (Orion Model 95lo), a research pH electrode (Orion Model 91-Ol), a single-junction reference electrode (Orion 90-Ol), and a Pharmacia Model 410 servographic recorder were used. Reagents. p-Tyramine hydrochloride, serotonin hydrogen oxalate, dopamine hydrochloride, and kynuramine hydrobromide were purchased from Calbiochem. La Jolla, Calif. o,L-Octopamine hydrochloride, L-norepinephrine bitartrate, histamine dihydrochloride, cadaverine dihy-

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drochloride, spermine tetrahydrochloride, ano tryptamine hydrochloride were obtained from Sigma Chemical Company, St. Louis, MO. Benzylamine and 2-phenethylamine bases, purchased from Aldrich Chemical Company, Milwaukee, Wise., were converted to their respective hydrochloride salts. 5-Methoxytryptamine was purchased from Regis Chemical Company, Chicago, Ill. Clorgyline (M and B 9302), N-methylN-propargyl-3-(2,4-dichlorophenoxy)-propylamine hydrochloride, was supplied by May and Baker Ltd., Dagenham, England. Deprenyl (E-250). 1-phenyl-2-(N-methyl-N-propargyl)-aminopropane hydrochloride, was supplied by Dr. J. Knoll, Semmelweiss University, Budapest, Hungary. Enzyme preparation. Male albino Sprague-Dawley (TEXSDD) rats from Texas Inbred Mice Company, Houston, Tex. were used. Rats weighing between 200 and 250 g were decapitated, and brains were rapidly removed and rinsed in chilled 0.25 M sucrose containing 0.05 M sodium phosphate buffer, pH 7.4. A 10% (w/v) tissue homogenate was prepared in the same buffer in a glass homogenizer equipped with a Teflon pestle. Aliquots of this tissue preparation were utilized for enzyme assays of rat brain homogenate. Rat brain mitochondria were prepared by centrifuging the initial homogenate at 9008 for 10 min in a Sorvall RC-2 refrigerated centrifuge at 4°C. The resultant supernatant was centrifuged at 12,000g for 20 min yielding a crude mitochondrial pellet. The mitochondrial pellet was resuspended and washed in the initial buffer and resedimented at 12,000g for 20 min. The subsequent crude mitochondrial pellet was suspended in the initial buffer and utilized for enzyme assay. Protein determination. Protein concentrations were determined by the method of Hartree (20) with bovine serum albumin as standard. Preparation of ammonia standard curve. Ammonia standard solutions were prepared by serial dilution of 0.1 M ammonium chloride (Orion 9.5-10-06) with triple-distilled deionized water. A 0.2-ml portion of each ammonium chloride solution (lop2 to 10-j M) was added to a mixture of 0.5 ml of 0.2 M sodium pyrophosphate buffer, pH 8.1, and rat brain mitochondrial preparation (1 .O mg of protein) in a final volume of 2.0 ml. Just prior to measurement, 4.0 ml of phosphate-sodium hydroxide buffer, pH 12.0 (Titrisol buffer, EM Laboratories, N. Y.), was added. The ammonia electrode was immersed in successively more concentrated ammonia solutions and stirred with magnetic agitation. The electrode was washed with ammonia-free water between measurements. The equilibrium potential of each solution was recorded after a stable baseline was achieved on the servographic recorder coupled to the millivolt/pH meter. The electrode response time (equilibrium potential) was 7 to 8 min at the lowest ammonia concentration and approximately 1 min for the highest concentration. The millivolt readings are plotted on the ordinate and ammonia concentration on the abscissa in Fig. I.

290

MEYERSON,

5 = = z g I r 3

o+lO+m +3040 +50GO+lO+w+90+lOO+llO+l20+130+140+150+1fi010-e

MCMURTREY,

AND DAVIS

/ I 10-5 IO-4 AYMOIIA COHCEllUllOll

I 10-J [Ml

FIG. 1. Ammonia standard curve. NH,-N concentrations are plotted on the abscissa and potentiometric voltage readings on the ordinate. Each point represents the mean ? SEM of five separate determinations. Details of the preparation and measurement of ammonia standardizing solutions are described in text.

Monoamine oxidase assay procedure. Monoamine oxidase activity in homogenates and mitochondria from rat brain was assayed in a reaction mixture containing the following components: 0.05 M sodium pyrophosphate buffer (pH 8.1), 0.5 to 3.5 mg of protein, 1.0 mM freshly prepared substrate, and triple-distilled deionized water to a final volume of 2.0 ml. After a 30-min incubation period in a shaking metabolic water bath (Research Specialties) at 37.5”C, the reaction was terminated by the addition of 4 ml of Titrisol buffer (pH 12.0). Substrates were added to control tubes after the termination of the incubation. After the reaction solutions were transferred to 20-ml beakers, the ammonia electrode was immersed in the agitated reaction solution and the millivolt equilibrium potential was recorded. Control and test ammonia concentrations were calculated from the standard curve, and the difference between the samples was taken as the amount of ammonia formed enzymatically. As a routine procedure, the ammonia-porous membrane and internal electrode filling solution were changed daily. Determination of Michaelis constants. Studies to determine the K, values for serotonin and tyramine were performed utilizing rat brain crude mitochondrial preparations. Five concentrations, not exceeding 5 x 1O-4 M of amine, each run in duplicate, were utilized for each determination ofK,. Appropriate control tubes were maintained at each of the various amine concentrations. The other details of assay conditions are described above. Kinetic constants were determined from the graphic treatment of data as described by Lineweaver and Burk (21). All kinetic

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data were calculated by the method of least squares and plotted with the aid of a programmed computer. Stoichiometric analysis. The stoichiometry of the MAO reaction was determined with serotonin (0.1 mM) and tyramine (0.1 mM) as substrates using two different mitochondrial protein concentrations as the enzyme source. The molar concentration of ammonia formed was compared to the quantity of amine substrate depleted. Ammonia formation following a 30-min incubation period (T = 30 min) was determined potentiometrically with an ammonia electrode as described above. Similar incubation mixtures were employed to determine depletion of amine substrate. Incubation mixtures evaluating substrate depletion were terminated by the addition of 0.1 ml of 12.0 N HCl, and protein was removed by filtration through a Millipore filtration manifold employing a Type AA O.&pm filter. The resultant amine concentrations of both the test (T = 30 min) and control (T = 0 min) filtrates were then determined by a high-pressure liquid chromatographic analysis (22). Effect of substrate selective inhibitors on MAO activity. Rat brain mitochondrial MAO activity was assayed as described above in the presence of Clorgyline or Deprenyl. The enzyme preparation was preincubated for 15 min with various concentrations of inhibitor before the addition of 1 mrvr serotonin or 1 mM benzylamine to initiate the reaction. MAO activity was expressed as a percentage of the control sample that contained no inhibitor. The percentage inhibition was then calculated and plotted against the negative log of inhibitor concentration from which ISo values were derived. RESULTS

The effects of enzyme concentration using crude homogenate or mitochondrial preparations on the rates of ammonia production measured potentiometrically are shown in Fig. 2. With either MAO source, ammonia production was linear between 0.5 and 3.5 mg of protein with tyramine (1 mM) as substrate. Rates as low as 1 nmol of ammonia produced per minute could be measured. The limit in terms of enzyme protein concentration obviously depends on the activity of the MAO preparation. For this study, samples containing as little as 0.1 mg of protein, in the case of rat brain mitochondrial preparation, or 0.25 mg of protein, for crude homogenate, were employed successfully. The time course (Fig. 3) of the monoamine oxidase reaction showed that ammonia production was linear with respect to time for the first 30 min of the incubation with tyramine (1 mM) as substrate and rat brain mitochondria as enzyme source. A comparison of the specific activities for a number of substrates is shown in Table 1. All substrates were tested at 1.O mM final concentration

292

MEYERSON,

0.0

0.5

MCMURTREY,

I.0

I.5

2.0

AND DAVIS

2.5

3.0

3.5

Y; PROlfll

FIG. 2. Relationship between protein concentration and rate of monoamine oxidase activity. Rat brain homogenate (0) and mitochondria (0) were prepared as outlined in text. Assay of monoamine oxidase utilizing tyramine as substrate was carried out as described in text. Each point represents the mean -+ SEM of four separate determinations.

240 210 -

z Ia0 t L r” 150 p. E 120 5 X ; go-

FIG. 3. Time course of monoamine oxidase assay. Rat brain mitochondrial preparation (20 mg of protein) and 1.0 mM tyramine were incubated in 50 mM sodium pyrophosphate buffer, pH 8. I, at 37S”C in a total volume of 20 ml. Two-milliliter aliquots were pipetted out from the reaction mixture at the times indicated. The concentration of ammonia formed at each time interval was determined as described in text. Each point represents the mean ?SEM of four separate determinations.

MAO POTENTIOMETRIC TABLE SUBSTRATE

SPECIFICITY

Substrate Serotonin Tryptamine Tyramine 5-Methoxytryptamine Dopamine Kynuramine Benzylamine Octopamine Phenethylamine Norepinephrine Histamine Cadaverine Spermine

OF RAT

BRAIN

ASSAY

293

1

MITOCHONDRIAL

MONOAMINE

Specific activity (nmol of ammonia formediminlmg 6.31 6.07 5.63 4.44 4.03 3.46 2.68 2.07 1.15 0.63 0.00 0.00 0.00

k 2 t 5 i k k 2 2 ” 2 2 +

OXIDASE” of protein)

0.66 0.61 0.47 0.15 0.51 0.33 0.32 0.10 0.25 0.18 0.00 0.00 0.00

a Specific activity of mitochondrial monoamine oxidase toward various substrates was determined by measuring ammonia production. The reaction mixtures contained rat brain mitochondrial enzyme preparation (1.0-2.0 mg of protein), 50 mM sodium pyrophosphate buffer (pH S.l), and 1 mM selected substrate in a total volume of 2.0 ml. Incubations were initiated by addition of substrate and conducted at 37.5”C for 30 min. Ammonia production was determined as described in text. Specific activities are expressed as means + SEM of at least four separate determinations.

using rat brain mitochondrial enzyme preparation. The highest specific activities were obtained with serotonin, tryptamine, tyramine, 5methoxytryptamine, and dopamine, while kynuramine, benzylamine, and octopamine possessed intermediate activity values, and 2-phenethylamine and norepinephrine exhibited the lowest specific activities. As anticipated, secondary and diamines such as histamine, cadaverine, and spermine showed no evidence of metabolism in this assay system. Michaelis constants for tyramine and serotonin using rat brain mitochondria as the source of MAO activity are graphically depicted in Fig. 4. The apparent K, (air environment) and V,,, values for tyramine were 1.0 -c 0.3 x lo-* M and 5.6 k 0.5 nmol of ammonia formed/min/mg of protein and for serotonin were 8.0 * 2.0 x 10e5 M and 6.3 ? 0.7 nmol of ammonia formed/min/mg of protein. The stoichiometric relationship between the amount of ammonia formed and substrate (tyramine or serotonin) depleted using varying concentrations of rat brain mitochondrial enzyme preparation is given in Table 2. Since the amount of ammonia produced from either tyramine or serotonin

294

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MCMURTREY,

AND DAVIS

1.2 I

L

l/TX

-30 -20

-10

0

IO 20 30 I IURAMINt mM )

40

l/[StROlONlN ml ) FIG. 4. Lineweaver-Burk plots representing oxidative deamination of tyramine and serotonin. Assay conditions are described in text. l/V is expressed as nanomoles of ammonia formed per minute per milligram of rat brain mitochondrial protein. Each point represents the mean -r-SEM of three separate experiments. The cross-hatching on the ordinate and abscissa on either side of each intercept indicates the upper and lower statistical limits of the derived Michaelis constants.

was equal to the quantity of amine depleted, the enzymatic formation of detectable ammonia appears to be stoichiometric with the oxidative deamination of substrate. The application of the assay technique for enzyme-inhibitor interactions was demonstrated by testing the substrate selective inhibitors Clorgyline (MAO Type A) and Deprenyl (MAO Type B). Plots of percentage inhibition of MAO activity with serotonin (MAO Type A substrate) and benzylamine (MAO Type B substrate) versus concentration of inhibitors are shown in Fig. 5. The data presented show that Clorgyline inhibited serotonin oxidation to a much greater degree than benzylamine oxidation. Deprenyl, on the other hand, strongly inhibited the oxidation of benzylamine at concentrations which had little effect on the oxidation of serotonin. The ISo values of Clorgyline for the oxidations of serotonin and benzylamine were 4.5 x IO+ and 1.8 x 10e5 M, respectively. When serotonin and benzylamine oxidation was examined in the presence of Deprenyl, the corresponding ZSOvalues were 1.3 x 10e5 and 1.0 x 10m7 M, respectively. These results closely agree with other published values utilizing different assay techniques (23-25).

MAO POTENTIOMETRIC TABLE STOICHIOMETRIC

ANALYSIS MONOAMINE

Mitochondrial protein (mg)

Substrate

OF THE OXIDASE

Amine concentration, T = 0 min (nmol)

295

ASSAY

2 RAT BRAIN MITOCHONDRIAL REACTION”

Ammonia formed, T = 30 min (nmol)

Amine depleted, T = 30 min (nmol)

Amine remaining, T = 30 min (nmol)

Tyramine (1.0 X 1o-4

M)

0.5 1.0

200 200

42 85

40 82

160 118

Serotonin (1.0 X 1o-4

M)

0.5 1.0

200 200

59 120

63 125

137 75

n Rat brain mitochondria was used as the source of MAO, was used as substrate. Details of incubation constituents are formation was measured by the present method. T = 0 initial = 30 remaining concentration were determined by high-pressure

and tyramine or serotonin described in text. Ammonia amine concentration and T liquid chromatography (22).

-SfROlOl ILiz BWVI

10 8 8 7 6 5 4 3

IO B I 7 6 5 4 3 WRfIVL (-106 COMAAl] FIG. 5. Percentage inhibition of rat brain mitochondrial monoamine oxidase versus the negative log of Clorgyline or Deprenyl concentration with serotonin (1 mM) or benzylamine (1 mM) as substrates. Incubation conditions are described in the text. Point values are the means of at least three separate determinations performed in duplicate.

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DISCUSSION

The electrode employed in this study is a gas-detecting electrode, sensing the level of dissolved ammonia in aqueous solutions. When ammonia is dissolved in water it will, to some extent, react with hydrogen ions to form ammonium ions. NH,

+ H+ = NH4+

pK, = 9.3.

Therefore, the relative amount of ammonia and ammonium ion is determined by the solution pH. Virtually all ammonium ion is converted to detectable ammonia at a pH of 12.0. Enzymatically formed ammonia concentrations are readily obtained from calibration curves prepared from direct millivolt potentiometric readings. Sample color and turbidity do not affect measurements, nor do anions, cations, or dissolved species interfere with potentiometric readings. The ammonia electrode responds logarithmically to the level of ammonia in solution. Each tenfold increase in ammonia concentration gives rise to about a 55mV change in electrode potential. Volatile amines are reported to interfere with the potentiometric readings of the ammonia electrode (26). The volatile amines employed in this study were benzylamine and phenethylamine. Due to their volatility, when control and test incubations were run with these amines, slightly lower potentiometric readings were obtained. Therefore, the corresponding calculated specific activity values tend to reflect marginally higher values. Normalization of potentiometric readings rendered from these volatile substrates eliminates this aberration. The specific activity of rat brain MAO obtained with the present methodology is slightly higher toward various substrates than is commonly reported in the literature (27). This is due to the difference in pH of the incubation mixture and temperature. Most other studies conduct MAO assay incubations at pH 7.0 to 7.5 and a temperature of 25 to 37°C. In the present study, the pH optimum (8.1) of the brain enzyme activity was utilized (28,29). There are several advantages of the present method for measuring MAO activity. The assay technique can be used to determine MAO activity with many primary monoamines as substrates. Of course, secondary monoamines such as epinephrine and N-methyl tryptamine cannot be used since methylamine rather than ammonia is rendered during oxidative deamination. This highly sensitive potentiometric assay technique offers particular advantage in studying the effects of inhibitors on this enzyme system. Unlike several other assay procedures, enzyme activity can be monitored in crude homogenates with minimal interference from endogenous ammonia. Compared to many other laborious MAO assay procedures, the method outlined offers the advantages of rapidity, accuracy, and simplicity, since isolation of reaction products is not required.

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Although the method was developed in the present study for assaying brain monoamine oxidase activity, the same procedure could be fruitfully applied to other tissues and to evaluation of other enzymatic systems that either consume or produce ammonia in the course of the reaction. ACKNOWLEDGMENTS The authors wish to express their appreciation to Ben Pashkoff and Alice Tucker for their excellent technical assistance. This work was supported by USPHS Grant S-ROI-AA-00226 and the Veterans Administration.

REFERENCES 1. Blaschko, H. (1952) Pharmacol. Rev. 14, 415-458. 2. Schnaitman, C.. Erwin, V. G.. and Greenwalt, J. W. (1967) .I. Cell Viol. 32, 719-735. 3. Costa, E., and Sandler. M. (1972) in Advances in Biochemical Psychopharmacology (Costa, E.. and Greengard, P.. eds.), Vol. 5. pp. l-454, Raven Press, New York. 4. Udenfriend, S., Weissbach, H., and Clark, C. T. (1954) J. Bio/. Chem. 215, 337-344. 5. Weissbach, H., Smith. T. E., Daly, J. W., Witkop, 9.. and Udenfriend, S. (1960)5. Biol. Chem. 235, l160- 1163. 6. Creasey, N. H. (1956) Biochem. J. 64, 178- 183. 7. Tipton, K. F. (1971)in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17, pp. 717-722, Academic Press, New York. 8. Weetman, D. F., and Sweetman. A. J. (1971) Anal. Biochem. 41, 517-521. 9. Tabor. C. W., Tabor, H.. and Rosenthal, S. M. (1954) J. Biol. Chem. 208, 645-661. 10. Deitrich. R. A., and Erwin, V. G. (1969) Anal. Biochem. 30, 395-402. 1I. Otsuka, S., and Kobayashi, Y. (1964) Biochem. Pharmacol. 13, 995-1006. 12. Nagatsu, T.. and Yagi, K. (1966) J. Biochem. 60, 219-221. 13. Harada, M., and Nagatsu, T. (1973) Anal. Eiochem. 56, 283-288. 14. Snyder, S. H., and Hendley, E. D. (1968)J. Pharmacot. Exp. Ther. 163, 386-392. 15. Guilbault. G. G., Brignac, P. J.. and Juneau, M. (1968) Anal. Chem. 40, 1256-1263. 16. Southgate, J., and Collins, G. G. S. (1969) Biochem. Pharmacol. 18, 2285-2287. 17. Jain, M., Sands. F., and Von Korff, R. W. (1973) Anal. Biochem. 52, 542-554. 18. Alivisatos, S. G. A., and Ungar, F. (1968) Biochemistry 7, 285-292. 19. Kveder, A., and Iskric, S. (1967) Croatia Chem. Acfa 39, 185-188. 20. Hartree. E. F. (1972) Anal. Biochem. 48, 422-427. 21. Lineweaver. H.. and Burk, D. (1934) J. Amer. Chem. Sot. 56, 658-666. 22. McMurtrey, K. D.. Meyerson. L. R.. Cashaw. J. L., and Davis, V. E. (1976) Anal. Biochem.

72, 566-572.

23. Hall, D. W. R., Logan, B. W., and Parsons, G. H. (1969) Biochem. Pharmacol. 18, 1447-1454. 24. Christmas, A. J., Coulson, C. J., Maxwell. D. R.. and Riddell, D. (1972) Brir. J. Pharmucol.

45, 490-503.

25. Meyerson, L. R.. McMurtrey, K. D., and Davis, V. E. (1976) Biochem. Pharmacol. 25, 1013-1020. 26. Instruction Manual (1974) Ammonia Electrode Model 95-10. p. 9, Orion Research, Inc.. Cambridge, Mass. 27. Nagatsu, T. (1973) Biochemistry of Catecholamines, pp. 206-207. University Park Press, Baltimore, Md. 28. Harada, M.. Mizutani, and Nagatsu, T. (1971) J. Neurochem. 18, 559-569. 29. Tabakoff, 9.. Meyerson. L. R., and Alivisatos, S. G. A. (1974)Brain Res. 66,491-508.