Simultaneous radioenzymatic assay of dopamine and dihydroxyphenylacetic acid: An index of in vivo dopamine release

Simultaneous Radioenzymatic Assay of Dopamine Dihydroxyphenylacetic Acid: An Index of In Vivo Dopamine Release

C. H.

The

CHENC AND G.

relative

brain

metabolite,

tissue

have developed

behavioral,

The concurrent electrical

lease or turnover rectly-acting urement

Dopamine

and its deaminated

estimation

activity in small

of tissue

regions

of brain,

of DA as induced

specific,

DA and DOPAC

drug paradigms.

by D-amphetamine

drugs)

simple,

IO pg and

and relatively concentrations or release

However,

(and perhaps

cannot be meaningfully

we

measurement

the rate of DA turnover

and certain

index

In order to apply this approach

of the assay for DA is approximately

stimulation,

of DA and DOPAC

(DA)

appears to be a reliable

assay for simultaneous

the assay is highly

means of evaluating

dopaminemimetic

Key Words:

neuronal

The sensitivity

to be a reliable

neurons.

radioenzymatic

100 pg. In addition,

inexpensive.

of dopamine

acid (DOPAC),

of dopaminergic a sensitive

of DA and DOPAC.

seems

concentrations

activity of dopaminergic

to the assessment

for DOPAC

F. WOOTEN

dihydroxyphenylacetic

of the functional

and

other

assessed

in

the reindi-

by meas-

alone.

Radioenzymatic

assay;

Dopamine;

Dihydroxyphenylacetic

acid;

release.

INTRODUCTION The activity of central dopaminergic neurons as reflected by dopamine (DA) turnover or release has been estimated by a variety of methods. Some of these methods include determination of the rate of metabolism of radio-labelled precursors (Nyb;ick, 1971), measurement of the rate of change of DA concentration (or some intermediates of DA metabolism) (Walters and Roth, 1974) following inhibition of synthesis (Javoy and Glowinski, 1971) or of inactivation (Javoy et al., 1973), and direct measurement of dopamine release by implantation in vivo of push-pull cannulae or surface collecting cups (Glowinski et al., 1979). Roth et al. (1976) have recently shown evidence suggesting that short-term changes in brain dihydroxyphenylacetic acid (DOPAC) concentration may provide a useful index of the functional activity of dopaminergic neurons. Though not distinguishing among various presumed functional pools of DA, this method allowed a relatively noninvasive means of estimating DA turnover or release in vivo. To utilize this approach for the approxiFrom the Departments of Neurology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri. Address reprint requests to G. F. Wooten, M.D., Department of Neurology, Box 8111, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110. Received October 6, 1980; accepted October 15, 1980. 165 lournalof Pharmacological Methods

5,165-173 (19811

0 1981 ElsevierNorth Holland, Inc..52

Vanderbilt Avenue,

New

York, NY10017

166

C. H. Cheng and C. F. Wooten

mation of DA turnover we have developed a highly sensitive radioenzymatic assay for simultaneous quantification of DA and DOPAC that is applicable to the estimation of DA turnover in very small brain regions. In the course of our studies with several drugs, we encountered an apparently paradoxical effect in which a drug known to release endogenous DA caused a diminution in brain DOPAC levels. MATERIALS AND METHODS Materials Spectroanalyzed grade reagents of toluene, methanol, and chloroform together with ACS reagent grade chemicals of perchloric acid (70%), NaH2P04*2H20, Na,HPO,*7H,O, boric acid, glacial acetic acid, hydrochloric acid (36.5-38.0%), ethyl ether (anhydrous), and ammonium hydroxide (28-30%) were obtained from Fisher Scientific Company, Fairlawn, NJ. S-[methyl-3HladenosyI-L-methionine (1 mCi/0.035 mg/2 ml) and Biofluor were products of New England Nuclear, Boston, MA. Ethanol (pure, USP reagent quality) was obtained from U.S. Industrial Chemical Co., New York, NY. lsoamyl alcohol was from J.T. Baker Chemical Co., Phillipsburg, NJ. Glutathione (reduced form), Trizma base, Trizma HCI, oL-dithiothreitol, EGTA Di-(2ethylhexyl) phosphoric acid, 3-hydroxytyramine HCI (DA), 3,4_dihydroxyphenyl acetic acid (DOPAC), 3-methoxytyramine*HCI, 4 hydroxy-3-methoxyphenyl acetic acid (HVA), S-adenosyl-L-methionine iodide (Grade I), albumine bovine (96-99%), o-amphetamine sulfate, and magnesium chloride were purchased from Sigma Chemical Company, St. Louis, MO. Dowex resin AC 50 W-X 8 (H’, 100-200 mesh) was from Bio-Rad Laboratories, Richmond, CA. Ethylamine (70%) was the product of Eastman Kodak Company, Rochester, NY. Haloperidol was obtained from McNeil Laboratories, Fort Washington, PA, and apomorphine.HCI from Merck & Co. Inc., Rahway, NJ. Thin layer chromatographic plates were silica gel GHL and silica gel G (250 k) uniplates supplied by Analtech Inc., Newark, DE. Catechol-O-methyl

Transferase (COMT)

Preparation

This enzyme was partially purified from rat liver according to the method of Axelrod and Tomchik (1958) and was diluted to 10 mg protein per ml with a specific activity of 49 nmoles/mg/hr immediately before assay. Treatment

of Dowex Resin AC !5OW-X8 (H’

form, 100-200 mesh)

Two hundred grams of the resin were soaked in a double volume of NaOH (2N) with stirring at 25°C for one hour. The resin was rinsed with water and dried by suction. The soaking and washing were repeated with HCI (2N). After the rinse became free of acid, the resin was mixed with glass distilled water and stored at 4°C for use. Rat Brain Samples Male Sprague-Dawley rats weighing 275 to 325 g were killed by cervical dislocation and the brains were quickly removed and dissected over an ice-chilled aluminum surface moistened with normal saline. Striatum and olfactory-accumbens were then

Simultaneous Assay of Dopamine and DOPAC frozen on dry ice and stored at -80°C until the assay was performed. On the day of assay frozen brain samples were weighed and homogenized in HC104 0.2 N containing reduced glutathione 5 mM. The 2% homogenate was centrifuged at 27,000 g (4°C) for 20 minutes and the supernatant was then diluted to appropriate concentrations with water. All reagents were made in deionized and glass distilled water. Dopamine

and 3,4-dihydroxyphenylacetic

Acid Assay

Fifty ~1 aliquots of supernatants of rat brain homogenates (diluted to 0.2% with water) were added to glass test tubes containing 10 ~1 of reduced glutathione (GHS) and HCIO, 50 mM each. Two hundred pg of DA and 100 pg of DOPAC were added to appropriate tubes as internal standards. To initiate the reaction 40 ~1 of a solution containing S-[methyl-3H] adenosyl methionine 1 &i (88 pmoles) and partially purified COMT 100 pg protein were added to each tube. This 40~1 solution also contained sufficient amounts of the following to make a final concentration in 100 IJ;I of dithiothreitol 8 mM, EGTA 2.4 mM, pargyline 5 PM, MgCI, 24 mM, bovine serum albumin 0.02%, and tris buffer 200 mM (pH 9.1). After mixing, the tubes were incubated at 37°C for 30 minutes in a shaker water bath. The reaction was stopped by rapidly adding to each tube 50 ~1 of borate buffer 1 M (pH 11) with 4 mM each of 3-methoxytyramine and homovanillic acid. Ten ~1 of S-adenosyl-L-methionine iodide 10 mM was added to each tube to dilute the unreacted tritiated S-adenosyl methionine. Two ml of toluene/isoamyl alcohol (3 : 2) were added to’each tube and vortexed for 30 seconds to extract the 3-0-methylated dopamine (3-methoxytyramine). The mixture was centrifuged at 1200 g for 10 minutes and the top organic layer was transferred into another tube containing 100 ~1 acetic acid 0.1 N for the determination of DA according to the method of Cheng and Wooten (1980). The aqueous layer was used for the determination of DOPAC. One hundred ~1 of HCI 1 N was added to each sample. A 200 ~1 suspension of treated Dowex (about 35 mg) in water was then added to each tube. The resultant solution was mixed for 15 seconds and centrifuged at 1200 g for 10 minutes. The supernatant was transferred to another tube to which was added 2 ml of ethyl ether. After being vortexed and centrifuged, the ether layer containing methylated DOPAC (homovanillic acid) was transferred to another tube containing 100 ~1 NH,OH 4 N. After thorough mixing and centrifugation the ether layer was discarded and the aqueous phase was then mixed with 150 ~1 absolute ethanol and spotted on a silica gel GHL plate. The plate was developed in a solvent system of chloroform : methanol :(70%)ethylamine (10: 3: 3). Development required 45 to 55 minutes and the Rf for HVA was 0.25. A dark brown spot corresponding to the locus of HVA appeared on the plate after exposure to ultraviolet light for 30 minutes or to sunlight for several hours. This spot was scraped and the HVA eluted from the gel with 0.5 ml sodium phosphate buffer 0.1 N (pH 6.0). The eluted HVA was counted in a Searle Delta 300 Scintillation counting system after the addition of 10 ml of di-(2-ethylhexyl) phosphoric acid 2.5% in Biofluor. The results were compared with the corresponding internal standard. The procedure for simultaneous estimation of DA and DOPAC may be further

simplified

by spotting

both methylated

compounds

simultaneously

on the same

167

168

C. H. Cheng and G. F. Wooten

plate. We combined the 100 ~JJof acetic acid 0.1 N containing -(H-labeled methylated DA (3-methoxytyramine) with the 100 t_dNH,OH 4 N containing 3H-labeled methylated DOPAC (Homovanillic acid) prior to the addition of absolute ethanol (350 f.J). The final solution (250 f-d) was spotted on the hard silica gel GHL plate. The plate was then developed in the same chloroform: methanol: 70% ethylamine (10: 3:3) solvent system. The resulting chromatogram is shown in Figure 1 (R, = 0.74 for 3-methoxytyramine and R, = 0.25 for homovanillic acid). RESULTS Properties of the Assay To determine the limits of sensitivity of the assay for DA and DOPAC, assays were performed in the presence of varying quantities of the two compounds. The results are shown in Figure 2. Blanks for DA ranged from 175-250 cpm whereas DOPAC blanks varied from 550-650 cpm. If the limit of sensitivity of the assay were taken

FIGURE 1. Thin layer chromatogram demonstrating separation of 0-methylated DA and DOPAC. The upper spot is 3-methoxytyramine (R, = 0.74) and the lower spot is homovanillic acid (Rf = 0.25).

Simultaneous Assay of Dopamine

and DOPAC

Dopamme

AMOUNT

OF SUBSTRATE

PER TUBE

(picograms)

FIGURE 2. limits of sensitivity of the assay for DA and DOPAC. The shaded horizontal line parallel to the x-axis denotes the number of CPMs in the blank.

to be twice blank, then the sensitivity of the assay for DA would be approximately 10 pg and for DOPAC 100 pg. The assay for each compound was linear over a wide range of substrate concentrations. Linear conversion of DA to 3-methoxytyramine occurred at DA concentrations of at least 5 ng, while DOPAC 0-methylation was linear at DOPAC concentrations up to 2.0 ng. There was a departure from linearity when more than approximately 11% of the S-adenosylmethionine was demethylated. By utilizing higher concentrations of 3H-labeled or “cold” S-adenosyl methionine, the assay could be made linear for even higher concentrations of DA and DOPAC. However, these changes would either increase the cost per tube or decrease the sensitivity of the assay, respectively. The degree of “cross-reaction” among substrates was quite small. DA “crossover” into DOPAC and vice versa was less than 1%. Likewise, contamination of either assay by dihydroxymandelic acid was less than 0.5%.

169

170

C. H. Cheng and G. F. Wooten

Effects of Drugs on DA and DOPAC

Concentration

in Striatum

DA concentration in control rat striatum was 9.6 ? 0.3 pg/g while the concentration of DOPAC was 1.2 ? 0.1 pg/g (n = 16). The effects of several drug treatments on striatal DA and DOPAC levels are depicted in Figure 3. Administration of apomorphine 0.5 mg/kg SC, a direct-acting DA agonist, produced at 53% reduction in striatal DOPAC concentration at 30 minutes after drug treatment with values returning to control levels by two hours. DA concentration did not differ from the control during the period of observation. In contrast, administration of haloperidol 1 mg/kg SC, a compound that is a potent DA antagonist, caused a greater than three-fold rise in DOPAC concentration two hours after drug treatment. This high level was maintained to the end of experimental period of four hours. No change in striatal DA concentration was noted. Finally, administration of amphetamine 2.5 mg/kg SC, an indirect-acting dopaminemimetic drug, resulted in a 61% reduction in striatal DOPAC concentration 30 minutes after drug administration. One hour after am15or

Apomorphme

0.5 mg/kg SC

o Dopamlne . DOPAC

t-ialoperldol

I 15Or

1 mg/kg SC

I

I

1

Amphetamine

2.5mg/kg

S.C.

TIME (hours) FIGURE 3. Effects of administration of apomorphine, haloperidol, or amphetamine on the concentration of DA and DOPAC in striatum. * differs from control p < 0.05; ** differs from control p < 0.01.

Simultaneous TABLE 1

Assay of Dopamine

and DOPAC

Effects of Drugs on Ratio of DA to DOPAC TIME AFTERINIECTION (HOURS) CONTROL

0.5

1

2

8.5 ZL 2.0 5.1 * 1.7 8.1 + 1.9

21.2 2 3.2” 22.3 2 3.5”

16.0 2 2.8= 1.9 2 0.6” 19.2 ” 3.1”

8.5 2 1.9 1.5 * 0.5a 9.6?1.8a

DRUG (DOSE)

Apomorphine (0.5 mg/kg) Haloperidol (1 mg/kg) Amphetamine (2.5 mg/kg)

4

1.7 t -

0.5”

Each number represents mean ? SEM (n = 6) ratio of DA to DOPAC. a Differs from control (i.e., at time = 0) p < 0.01.

phetamine

treatment

elevation.

The

striatal

DA concentration

concentrations

showed

at the end of two hours. The ratio of striatal DA/DOPAC

content

with

apomorphine,

or amphetamine

with

either

DOPAC

haloperidol,

apomorphine

ratio while

a small

of both DA and DOPAC as a function

or amphetamine

haloperidol

resulted

to control

levels

of time after drug treatment

is shown

produced

treatment

(20%) but significant

returned

in Table

1. Treatment

a large increase

in the DA/

in a large reduction.

DISCUSSION DOPAC oxidase

formation

(Tipton,

to be formed

from DA is catalyzed by the intracellular

1979).

primarily

rapidly.

Electrical

DOPAC

concentration,

sions

acutely

As Roth

in dopaminergic

stimulation this

concentration.

(or gamma butyrolactone),

impulse

led to a dramatic

drug(s)

reduction or no change in the concentration led Roth and coworkers (1976) to conclude of DOPAC, tration,

particularly

provided

dopaminergic morphine, However, activity rometric

neurons.”

which

technical

with

dopaminergic

limitations

for estimating

reduce nigrostriatal

DA concentration

estimation

in the functional

haloperidol, impulse

were imposed DA and DOPAC

which

flow,

on the application

neurons

le-

of gamma hyand a small

of striatal DOPAC. These observations that short-term changes in brain levels

of alterations

Our findings

decreases

of dopaminergic method

index

striatal

by structural

administration

when coupled with simultaneous

a “useful

appears

over relatively

increased

flow

that markedly

in striatal

monoamine

DOPAC

and turns

impulse

Further,

increase

striatal

pathway acutely

of nigrostriatal

droxybutyrate flow,

nerve terminals

of the nigrostriatal

and blockade

decreased

enzyme(s)

et al. (1976) have shown,

increases,

support

of their

of DA concen-

activity this

method

of central and apo-

conclusion.

for estimating

by the low sensitivity

of the fluo-

available at that time.

In recent years several radioenzymatic assays based on the transfer of 3H-labeled methyl groups from S-adenosyl methionine to meta-hydroxyl groups of DOPAC (as catalyzed

by rat liver

catechol-0-methyl-transferase)

have been reported

(Ke-

babian, et al., 1977; Argiolas and Fadda, 1978; Saller and Zigmond, 1978; Meller, et al., 1980). We have integrated features from several of these assay systems to allow for the simultaneous estimation of DA and DOPAC that is relatively rapid, simple, and inexpensive. Our assay is more sensitive than the original radioenzymatic procedures

(Kebabian,

et al., 1977;

Argiolas

and Fadda, 1978) and is of com-

171

172

C. H. Cheng and C. F. Wooten parable

sensitivity

to those

reported

more

recently

(Sailer

and Zigmond,

1978;

Meller, et al., 1980). Sailer and Zigmond (1978) discussed modifications that allowed greater assay sensitivity but which sharply increased the cost per tube and required two dimensional

chromatography;

one single

spot required

The sensitivity

of the assay for DA and DOPAC

concentration

of these

techniques regions

(Palkovits,

then allows

ronal projections Our

finding

DOPAC

compounds

to a variety that

of brain obtained

assay of DA and DOPAC

of functional

activity

of the

by “punch” in small

brain

of dopaminergic

neu-

of brain regions.

amphetamine

(Roth et al., 1976; Braestrup,

a 20 x 20 cm LQF plate.

for accurate estimation

regions

1973). Simultaneous access to an index

and a large increase

evidence

in small

allows

administration

causes

in the DA: DOPAC 1977) and requires

that amphetamine

is an indirect

a reduction

ratio confirms

further

comment.

dopaminemimetic

in

findings There

drug.

striatal

of others is abundant

Administration

of amphetamine results in the release of endogenous brain dopamine (Besson et al., 1969). If the mechanism of amphetamine-induced DA release were entirely analogous to striatal

DA release induced

then one would

expect amphetamine

tal DOPAC There

concentration.

are several DOPAC

concentration.

affinity

DA re-uptake

that most DOPAC to the apparent

dogenous minergic

neurons

diated via DA autoreceptors loop from

striatum

of amphetamine other

between

neuronal

Therefore, However,

neurons,

the

amphetamine

DA

appears to be

of amphetamine

with

indirect firing

of en-

neurons

and/or via a short

feedback

nigra. The net effect of each of these mechanisms dopaminemimetic

rate and effective

reflect

release

reduction in the firing rate of dopameet al., 1973); this effect is presumably

(Bunney,

action

the attendant

in a marked

on dopaminergic

to substantia

levels

of dopaminergic

Second,

dopaminergic

the use of DA to DOPAC

ratios

the rate of release of DA is not valid by amphetamine, but for a variety of theless the DA: DOPAC ratio remains behavioral and electrical stimulation

levels. drugs,

Thus,

amphetamine,

produces

and

a dissociation

release of dopamine.

even in the case of amphetamine,

that DOPAC

of high

may reduce the access of released

action is to reduce brain DOPAC

perhaps

blocker

Roth et al (1976) have shown

of monoamine oxidase and therefore would compete diDA for the catalytic site to produce DOPAC (Miller, et al.,

of DA results

nigrostriatal

is a potent

in terminals

site of deamination.

the administration stores

nigra, in stria-

action that may serve to reduce

1969). Since

block of DA re-uptake

primary

in an increase

occurs.

o-amphetamine

appears to be formed

a competitive inhibitor rectly with endogenous 1980). Third,

First,

of the substantia

to result

of amphetamine

(Coyle and Snyder,

amphetamine-induced

stimulation

administration

Yet, the opposite

mechanisms

striatal

by electrical

the suggestion

neuronal

firing

to estimate

of Roth et al. (1976)

rate is apparently

functional

valid.

activity defined

as

in the case of amphetamine. DA is released reasons DOPAC levels are reduced. Neververy useful as an index of DA release rate in paradigms. It would seem that in order to

assess DA release rates induced by drugs with multiple mechanisms of action like amphetamine, all metabolites of DA must be quantified simultaneously. Recent advances in simultaneous

estimation

of DA and its metabolites

tography with electrochemical detection (Felice, et al., 1978; Hefti, 1979).

may represent

a useful

by liquid technical

chromaapproach

Simultaneous This

work

Institute

was supported

for Medical

is a George

by research

Research

C. Cotzias

grants

from

of the City of St. Louis,

Research

Fellow

Assay of Dopamine

the American

Parkinson’s

and NIH-NINCDS

of the American

Disease

Association,

Grant POI-NS-14834-01.

Parkinson’s

Disease

173

and DOPAC the

C.F.W.

Association.

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