A highly sensitive radioenzymatic assay for simultaneous estimation of norepinephrine, dopamine, and epinephrine

A Highly Sensitive Radioenzymatic Assay for Simultaneous Estimation of Norepinephrine, Dopamine, and Epinephrine

CHENC AND G. FREDERICK WOOTEN

CHUAN-HUAN

We

have developed a radioenzymatic assay for simultaneous estimation of norepinephrine (NE), dopamine (DA), and epinephrine (E) that was the result of the integration of several unique features of previously described assay procedures. Catecholamines in sera or tissue homogenates were enzymatically 0-methylated in the presence of partially purified catechol-0-methyltransferase with S-[methyl3H] adenosyl methionine serving as the methyl donor. The 0-methylated products were then separated by thin-layer chromatography, eluted from the gel, and their tritium content determined. The assay allows measurement of catecholamines with a sensitivity in the range of 15-20 pg. In addition, the assay is highly specific, reproducible, relatively rapid and simple, and inexpensive. Key Words: Radioenzymatic rine; Epinephrine

assay; Catecholamines;

Dopamine;

Norepineph-

INTRODUCTION In recent

years

several

laboratories

matic assays for catecholamines Coyle

and Henry,

and Ziircher,

1973;

1976;

Passon and Peuler,

and Sailer

greater

specificity

(Anton

and Sayre, 1962;

methylate (utilizing mines

using

of 14C-S-adenosyl Subsequently, enosyl

methionine

methionine,

of approaches products

have been

From the Departments

to separate

St. Louis,

Address

reprint

requests

of Medicine,

much

assay procedures was to either

O-

transferase) or N-methylate transferase) catecholagroup

donor.

by the rather low specific (Engleman

and identify

assay procedure Washington

1970).

S-[methyl-3H]-ad-

methylated

and Henry,

(eg, Cuello

The

activity

and Portnoy,

activity

be greatly improved.

(Coyle

procedures

and Pharmacology,

1976; Da Prada

assays possessed

the strategy

high specific

partitioning

a catecholamine

of Neurology

Euclid,

School

solvent

et al., 1973;

as a methyl

limited

of the assays could

and gel chromatographic 1978).

660 South versity

of very

employed

multiple

We wish to describe

In general

methionine were

radioenzy-

(Cuello

fluorometric

used as a cosubstrate

the availability the sensitivity

including

various paper and Zigmond,

S-adenosyl

sensitive

and Kopin,

These

the original

1963).

assay procedures

with

1978).

purified catechol-O-methyl phenylethanolamine-N-methyl

radiolabelled

first radioenzymatic

than

Haggendal,

partially purified

highly

1973; Weise

and Zigmond,

and sensitivity

(utilizing partially

have developed

in body fluids or tissue extracts

A variety reaction

1973) as well as

et al., 1973;

developed University

Saller

by integration School

of Medicine,

Missouri. to Dr.

G. Frederick

Wooten,

Department

of Neurology,

Washington

Uni-

Box

8111, 660 South Euclid, St. Louis, Missouri 63110. Received August 28, 1979; revised and accepted October 23, 1979. 333

Journal of Pharmacological @ 1980 Elsevier North

Methods

Holland,

3, 333-W

(1980)

Inc., 52 Vanderbilt

Avenue, New York, NY 10017

0160~5402/80/04033312$02.25

334

Chuan-Huan

Cheng and G. Frederick Wooten

of features from a number of other assay systems that allows the simultaneous determination of dopamine, epinephrine and norepinephrine with a sensitivity in the 15-20 pg range and is relatively rapid, simple, and inexpensive. Further, we will demonstrate the utility of the assay in measuring catecholamine concentrations in human serum and in regions of rat brain. MATERIALS AND METHODS Materials The chemical compounds used in the assay procedure were obtained from the following sources: Glutathione (reduced form, Crystal), Trizma base, Trizma HCI, DL-dithiothreitol, ethyleneglycol-bis (p-amino ethyl ether) N, N1-tetraacetic acid (EGTA), DL-metanephrine HCI, DL-normetanephrine HCI, 3-methoxy tyramine HCI, Di-(2-ethylhexyl) Phosphoric acid, 3-hydroxytyramine HCI, L-epinephrine bitartrate, L-arterenol bitartrate, L-p-3,4_dihydroxyphenylalanine methyl ester HCI, DL-alphamethyl-p-tyrosine (crystalline, 98%), magnesium chloride (MgCI,.6H,O), glycerol (99%) were obtained from Sigma Chemical Company, St. Louis, MO. Perchloric acid (70%, reagent ACS), boric acid (crystalline, ACS certified), toluene (spectroanalyzed) and glacial acetic acid (reagent ACS) were provided by Fisher Scientific Company, Fair Lawn, N.J. lsoamyl alcohol, ammonium hydroxide, sodium metaperiodate, methyl amine (40% in H,O), and tertiary-amyl alcohol were from J. T. Baker Chemical Co., Phillipsburg, N.J. Adenosyl-L-methionine S-[methyl-3H] (11.0 Ci/mmol, 1 mCi/2 ml) and Liquifluor were obtained from New England Nuclear, Boston, Mass. Carbidopa was kindly donated by Merk Sharp and Dohme Research Laboratories, West Point, PA; Serpasil (reserpine USP 5 mg/ampule) was purchased from CIBA Pharmaceutical Co., Summit, N.J. Ethanol (pure, ethyl alcohol U.S.P. reagent quality) was purchased from U.S. Industrial Chemical Co., New York, N.Y. Thin-layer chromatographic plates were silica gel G, 250 microns, precoated and prescored uniplates from Analtech Inc., Newark, Del. Plasma Samples Human plasma was obtained from four patient volunteers ages 34 to 58. Whole blood samples were obtained by venipuncture via a 19 gauge indwelling butterfly needle and collected in a 4 ml vacutainer tube (produced by Becton-Dickinson and obtained from Abbott Laboratories). These tubes contained EGTA 7.2 mg and glutathione 4.8 mg. The plasma was quickly separated in a refrigerated centrifuge and stored at -20°C. Rat Brain Samples Male Sprague-Dawley rats (275-325 g) were killed by cervical dislocation and the brains were quickly removed and dissected over an ice-chilled aluminum surface moistened with normal saline. Various brain regions were then frozen on dry ice and stored (for no longer than 10 days) at -20°C until the assay was performed. On the day of assay, frozen brain samples were weighed and homogenized in HCIO, 0.2 N containing reduced glutathione 5 mM to make a 1% homogenate. The ho-

A Radioenzymatic mogenate then

was centrifuged

diluted

for 20 minutes

to appropriate

Catecholamine Fifty ~1 of either

plasma

the reaction

or diluted

and partially

81 pg protein/tube

plus sufficient

quantities

EGTA 2.4 mM,

added.

or norepinephrine

glutathione added

the tubes

and mixed

vigorously.

toluene/isoamyl

alcohol

and added

to another

for 10 seconds

MgCI,

24 mM,

were

incubated

to each

4 mM each of 3-

was added

and discarding

tube,

vigorously

The organic

phase,

to each 2 ml of

mixed

for ten

phase was separated

100 ~1 of acetic acid 0.1 N, mixed

at 800 g for five minutes.

of the organic

rapidly

catecholamines,

The organic

discarded and an additional 1 ml of toluene/isoamyl alcohol (3:2) final wash of the reaction products in the acidic aqueous phase. ration

at 37°C

the tubes were transferred

the 0-methylated

for five minutes.

tube containing

200 mM

epinephrine

tubes.

1 M (pH 11) containing

was added

and centrifuged

protein/hr, in 100 ~1 of

and Tris buffer

100 ~1 final volume

buffer

To

methio-

transferase

100 pg each of dopamine,

and normetanephrine

at 800g

adenosyl

of 46 nmoles/mg

bath. To stop the reaction,

(3:2)

and centrifuged

5 mM,

To extract

was added

and 5 mM HCIO,.

S-[methyL3H]

activity

to appropriate

metanephrine

homogenate

to make a final concentration

standards

to a 4°C bath and 50 ~1 of borate methoxytyramine,

of brain

rat liver catechol-O-methyl

a specific

containing

for one hour in a shaker water

seconds

was

water.

glutathione

containing

purified

with

For internal

were

After vortexing,

tube

glass distilled

supernatant

of the following

reduced

(pH 9.1) were

with

10 ~1 of 50 mM reduced

40 ~1 of a solution

nine 1 FCi (100 pmoles) (see below)

at 6,000 g (4°C) and the supernatant

concentrations

Assay Procedure

to glass tubes containing initiate

Assay for Catecholamines

150 ~1 of absolute

vigorously phase

was

was added for a Following sepa-

ethanol

was added

to the acetic acid extract of the methylated compounds, mixed, and spotted on silica gel G, 250 micron plates (Analtech Inc.). The methylated compounds were separated

in a solvent

(Fig. 1). Substitution gave similar

good

sunlight

several

migration

for

system of tertiary of xylene

separation. hours

of the three

with

1 ml NH,OH

isoamyl The

0.05 M and mixed

was then

counted

overnight

dark

sponding

to metanephrine

with 1 ml NH,OH

adaption

for tertiary-amyl

and

with

were scraped

a counting phosphoric

in a Searle

at a tritium

alcohol

of the plates to to the site of

products.

to 3-methoxytyramine

alcohol/liquifluor/di-(2-ethylhexyl)

mixture

or butyl alcohol

Following development, exposure produced brown spots corresponding

0-methylated

The spots corresponding

(IO : 4 : 5)

amyl alcohol/toluene/methylamine

for toluene

Delta

counting

fluor

300 Scintillation

normetanephrine

were

of 33%.

of toluene/

(700 : 300: 50 : 25).

acid (DEHP)

efficiency

0.05 M, and then 50 ~1 of sodium

into vials and eluted

composed

Counter

after

The spots corre-

scraped

into vials,

eluted

metaperiodate

4% were

added

to each vial and mixed in order to convert these two compounds to vanillin. Exactly five minutes later, 50 ~1 of 10% glycerol was added to stop the reaction followed by the addition of 1 ml acetic acid 0.1 N. After thorough mixing 10 ml of toluene/ liquifluor/DEHP (1,000 : 50 : 25) was added and mixed. The final mixture was counted after overnight

dark adaptation.

335

336

Chuan-Huan

Cheng and C. Frederick Wooten

FIGURE 1. Thin-layer chromatogram demonstrating separation of 0-methylated compounds. The upper spot is 3_methoxytyramine, Rf = 0.60; the middle spot is metanephrine, Rf = 0.47; and the lower spot is normetanephrine, Rf = 0.41.

Preparation

of Partially

Purified

Rat Liver

Catechol-O-Methyl

Transferase

(COMT)

COMT was partially purified from rat liver as described by Axelrod and Tomchick (1958). The entire procedure was carried out at 4°C. Approximately 100 g of rat liver was homogenized

in 400 ml of KCI 0.154 M. The homogenate

was centrifuged

for

30 minutes at 28,000 g (2°C). The supernatant was filtered through cotton gauze, titrated to pH 5 with acetic acid 1 M, and centrifuged at 12,000 g for 20 minutes. The supernatant was decanted and subjected to ammonium sulfate fractionation.

A Radioenzymatic The precipitate

formed

50 ml of sodium two changes

against

dithiothreitol

After

to remove

tris buffer resulting

make

and stored

at -80°C

activity

nephrine

Transferase

as substrate.

methylated

the supernatant

solution

COMT

at 12,000g

was titrated

and

in

with

0.1

activity,

were

mM

diluted

for

to pH 8.1 with added

to the

respectively.

The

appropriately,

ali-

for use in the catecholamine

assay.

(E. C. 2.1.1. I) Assay

by the

Activity

per mg protein

for

was dissolved for 14 hours

1 mM (pH 7.0) containing

and pargyline

of 5 mM

until thawed

was assayed

buffer

of the dialyzed

dithiothreitol

was assayed

Catechol-O-Methyl COMT

phosphate

concentrations

sulfate

(pH 7.0) and dialyzed

centrifugation

Sufficient

final

preparation

quotted

1 mM

the precipitate,

2 M (pH 8.2). to

30% and 50% ammonium

buffer

5 liters of sodium

0.1 mM.

30 minutes solution

between

phosphate

Assay for Catecholamines

method

of Axelrod

was expressed

(1961),

as pmoles

utilizing

norepi-

of norepinephrine

O-

per hour.

RESULTS Properties

of the Assay

To determine performed

the limits of sensitivity

in the presence

norepinephrine. epinephrine

The results

assays varied

in the range

of 125-175

quantities

are shown

assays were

epinephrine,

norepinephrine

of sensitivity

were

and

blanks varied

taken

to be twice

of the assay for each of the three approximately 15 pg, epinephrine

15-20

and

2. Blanks for the dopamine

cpm whereas

If the limit

between

of dopamine,

in Figure

from 200-300 cpm.

three times blank, then the sensitivity amines would be as follows: dopamine pg, and norepinephrine

of the assay for catecholamines,

of varying

to

catecholabout 20

pg.

The assay for each catecholamine was linear over a rather wide range of substrate concentrations as is illustrated in Figure 3. Although the assays were quite linear for

conversion

assays were 25,000

cpm

of up to 50,000 routinely

were

cpm

perfomed

converted

of S-[Methyl-3H]-adenosyl

on tissue

from

concentrations

S-adenosyl

methionine

methionine,

such that

at most

to 0-methylated

the 20cate-

cholamines. Table

1 illustrates

TABLE 1

the degree

Crossover Among Catecholamine

SUBSTRATE

DOPAMINE

ADDED

(500

of “cross-reaction”

PC/TUBE)

Dopamine (DA) Epinephrine (E) Norepinephrine (NE) PERCENT

DA to E 5.6% DA to NE 0

among

various

catecholamine

Substrates EPINEPHRINE

NOREPINEPHRINE

ASSAY

ASSAY

ASSAY

(CPM)

(CPM)

(CPM)

19,746 416 0

1,105 14,880 0

0 267 11,602

CROSSOVER

E to DA EtoNE

AMONG

2.8% 1.8%

SUBSTRATES

NE to DA NE to E

0 0

337

338

Chuan-Huan

Cheng and G. Frederick Wooten

EPINEPHRINE

I

NOREPINEPHRINE

4000-

AMOUNT

OF SUBSTRATE

PER TUBE (picogrcd

FIGURE 2. limits of sensitivity of the catecholamine assay for dopamine, epinephrine, and norepinephrine. The horizontal line parallel to the x-axis denotes the number of cpm in the blank.

substrates.

Presumably

TLC separation, catecholamines epinephrine Human

because metanephrine

migrates

at the intermediate

rate in

the degree of crossover between epinephrine and the other two is greater than the degree of contamination of dopamine by nor-

or vice versa. Plasma

Catecholamine

Concentrations

Venous blood samples from four patient volunteers were obtained after reclining for 30 minutes, and 5 and 10 minutes after standing. Plasma norepinephrine and epinephrine rine control of the upright

values were determined and are shown in Table 2. Whereas epinephlevels of 72 t 38 pg/ml did not change significantly after assumption posture,

norepinephrine

levels

rose dramatically

from

216 2 31 pg!

ml to 627 + 55 pg/ml. Both the control reclining values and the effects of change in body position are similar to results obtained by others (Cryer et al., 1974; Weise and Kopin, 1976; Ziegler, et al., 1977) and serve to illustrate the potential value of the assay in studying

human

plasma catecholamine

concentrations.

A Radioenzymatic TABLE 2 Position

Human Plasma Catecholamine

Norepinephrine Epinephrine n Differs

Studies

control

STANDING

(5 MIN)

(10 MIN)

216 2 31

627 -c 55”

568 2 28”

117 -t 31

99 + 33

by P < 0.01. mean k SEM

of Catecholamine

Rat brains

STANDING

72 k 38

were

339

to Body

(30 MIN) (pg/ml)

represent

Concentrations-Relationship

RECLINING

(pg/ml) from

Numbers

Assay for Catecholamines

for four

volunteer

in Rat Brain

Concentrations

regionally

dissected

patients.

and samples

of ten different

brain

regions

were weighed, homogenized, and their norepinephrine and dopamine contents were determined (Table 3). Norepinephrine levels were highest in the hypothalamus

and lowest

striatum, (neocortex,

in the striatum

whereas

accumbens

and septum,

olfactory

hippocampus,

Effects of Drug Treatments In order treatments

to demonstrate

thalamus,

dopamine

concentration

and extremely

in

regions

and cerebellum).

on Dopamine

Concentrations

in the Striatum

the efficacy of the assay in studying

on brain catecholamine

was highest

low in several

concentrations,

we studied

the effects

of drug

dopamine

concen-

40 0 DOPAMINE qIEPINEPHRINE ANOREPINEPRINE

30 E 8 5.

20

h v

0

0

200 AMOUNT

400

600

OF SUBSTRATE

800

1000

PER TUBE

(plcograms) FIGURE 3. trations.

Linearity with respect to substrate concentration

over a wide range of concen-

340

Chuan-Huan

Cheng and G. Frederick Wooten

TABLE 3 Regional Concentrations in Rat Brain

Olfactory-accumbens Septum Striatum Parietal cortex Hypothalamus Substantia nigra Frontal cortex Hippocampus Thalamus Cerebellum

in the striatum

in striatal

dopamine

phenylalanine hour

the

returned

to control

~%4$9

0.33 0.46 0.11 0.20 1.30 0.59 0.22 0.24 0.41 0.23

2 2 2 k 2 k 2 + 2 +

0.08 0.03 0.01 0.01 0.20 0.08 0.03 0.05 0.11 0.08

6.0 1.2 8.5 0.05 0.05 0.80 0.02 0.03 0.03 0.03

with three following

ester 100 mg/kg concentration

drugs.

i.p. and carbidopa

in the

striatum

dopamine

concentration

resulted

had returned

hydroxylase

in a 40% reduction

a-methyl

in striatal dopamine

i.p.. Within

twice

Reserpine,

control,

2.5 mg/kg

one but i.p.,

concentration within four days the striatal

to about 45% of control.

inhibitor,

the change

of L-dihydroxy-

10 mg/kg

was about

levels by four hours after treatment.

of the tyrosine

shown in

Figure 4 depicts

the co-administration

resulted in a greater than 50% reduction in striatal dopamine four hours and a 90% reduction by 24 hours (Fig. 5). Within tion

k 0.3 -r- 0.3 2 0.5 -c 0.01 2 0.07 k 0.05 + 0.01 f 0.01 k 0.01 k 0.01

mean 2 SEM of the number of determinations

after treatment

methyl

DOPAMINE

~I%&)

(5) (5) (4) (4)

concentration

dopamine

and Dopamine

NOREPINEPHRINE

(6) (6) (6) (6) 6) (6)

Numbers represent parenthesis.

tration

of Norepinephrine

Finally,

para-tyrosine

concentration

administra-

400 mg/kg

i.p.

two hours after drug

treatment. DISCUSSION This assay procedure

is based on the transfer

of a 3H-labelled

methyl

group

from

S-adenosyl methionine to ring meta-hydroxyl groups of catecholamines as catalyzed by rat liver catechol-0-methyltransferase (COMT). The concentraions of S-adenosyl methionine and COMT amine availability was incubation

period,

to completion.

protein in each assay tube were in excess so that catecholrate-limiting in the reaction. Further, with the one hour

the 0-methylation

The pH optimum

of available

for this reaction

catecholamines is about

goes essentially

9.1 (Cuello

et al., 1973)

and the reaction is highly dependent upon magnesium ion (Axelrod and Tomchick, 1958) and inhibited by calcium (Quiram and Weinshilboum, 19761, thus the addition of EGTA 2.4 mM. Reduced glutathione 5 mM is incorporated into the reaction solution When

to maintain COMT in its reduced, active form. 100 pg of dopamine was added as substrate, about

4400 cpm were

pro-

duced. Whereas 100 pg of epinephrine produced 3600 cpm and 100 pg of norepinephrine produced about 2400 cpm. The lower limits of sensitivity based on two to three times blank values were in the IS-20 pg range. Thus the sensitivity of this

A Radioenzymatic

Assay for Catecholamines

2 1

Oi

4

2

8

6

TIME

(hours)

FIGURE 4. Dopamine concentrations in rat corpus striatum at various times after intraperitoneal administration of L-dihydroxyphenylalanine methyl ester 100 mg/kg and carbidopa 10 mg/kg. Points represent mean f SEM (N = 4).

assay exceeds and is similar

that of earlier

Prada and Ztircher, that described amines

with

tographic sitivity

ours,

solvent

studies

and Zigmond,

and Kopin

adsorption

procedures

recent

(1976)

prior

Further,

Our

and Henry,

and Kopin,

method

we employed

of the procedure

a thin-layer

reaction products

we demonstrated

Da than

plasma chroma-

rather than utiliz-

somewhat

to the estimation

1973)

1976;

is simpler

in that we did not concentrate

to assay;

to separate 0-methylated extractions.

1978).

(Coyle

(Weise

higher

sen-

of catecholamines

homogenates.

method

of Da Prada and Ztircher

employed

reaction

a more

products

composed highly system

by Weise

alumina

in more

Sailer

as well as application

in tissue The

1976;

procedure

ing multiple

radioenzymatic

to that attained

of tertiary

prior

(1976),

time-consuming to thin-layer

while

procedure chromatography;

amyl alcohol/toluene/methylamine

of comparable to isolate also,

sensitivity

to

the 0-methylated our

solvent

system

(IO : 4: 5) gave discrete

and

reproducible separation of the reaction products. The well-characterized of Saller and Zigmond (1978) utilized the addition of phosphotungstic acid

to terminate acid-treated

the COMT reaction followed supernatant on TLC plates.

by direct spotting of the phosphotungstic In our hands this procedure resulted in

341

342

Chuan-Huan

Cheng and G. Frederick Wooten

04

0

24

TIME

I

I

48

72

(hours)

I

96

FIGURE 5. Dopamine concentration in rat striatum at various times after intraperitoneal administration of reserpine 2.5 mg/kg. Points represent mean f SEM (N = 4).

marked phrine,

“tailing”

of the spots

and metanephrine

gel plates.

Thus

we were

phosphotungstic

corresponding

in a variety unable

acid treatment

to 3_methoxytyramine,

of solvent

to resolve

prior

the

to spotting

systems

normetane-

and on a variety

reaction

products

of silica

utilizing

only

of the plates.

The primary sources of the high sensitivity are the high specific activity of tritiated S-adenosyl methionine, the organic extraction with toluene/isoamyl alcohol, the periodate droxylated scintillation

cleavage reaction

media (Temple highly

specific.

epinephrine achieving

reduces

the counts

and Gillespie,

1966).

greater

expensive

As demonstrated

The small cross-contamination

can be easily eliminated

plate). When less

(which

attributable

to non-p-hy-

catechols) and the addition of di-(2-ethylhexyl) phosphoric acid to the fluor, which facilitates the extraction of amines from aqueous to organic

separation

epinephrine

by using

(eg, the values

levels are relatively

plates can be used.

Thus

in Table

1 the assay

is also

of norepinephrine

and dopamine

a larger thin-layer

plate and thereby

in Table

by

1 are based on a 10 x 20 cm

negligible

as in brain, the smaller

the assay provides

a highly

sensitive

and and

specific means of simultaneously determining the concentration of dopamine, epinephrine, and norepinephrine. Our estimation of both supine and upright plasma norepinephrine and epinephrine levels are quite similar to recent observations made by others (Ziegler et al., the sensitivity of our assay for dopamine 1977; Cryer et al., 1974). Unfortunately coupled with the low levels of dopamine in human serum preclude an accurate determination of human plasma dopamine levels as our results were usually at or

A Radioenzymatic Assay for Catecholamines slightly

less than twice blank. The application

levels

of norepinephrine

and dopamine

exploit its sensitivity. Because “punch” technique of Palkovits to study

the effects

regions

of various

of this assay to measurement

using

regional

dissections

of brain

does not fully

of this high sensitivity, regional studies using the (1973) would be facilitated. Finally it is quite feasible drugs

on catecholamine

concentrations

in very small

of brain.

In addition procedure, required

to the

sensitivity,

it is both simple

specificity,

and excellent

and inexpensive.

to assay 40 samples

is about

The

12. The

reproducibility

average number

COMT

of this

of man-hours

can be purified

in

large

batches, aliquotted, and stored for further use. The reaction mixture minus the S[methyl-3H] adenosyl methionine and enzyme can also be produced in large quantities,

aliquotted

and stored.

The major expense

for expendables

in this assay is for

S-[methyl-3H] adenosyl methionine and by utilizing a minimal quantity (ie, I PCi per tube in our case), the cost per tube is relatively low. When less sensitivity is needed,

even less

labelled

belled S-adenosyl

methionine

in the 0-ethylation

reaction.

Numerous viously.

and minimize

This

work

Dr.

of other

be utilized

with

this substrate

for catecholamines

here reported procedures

reflects

the addition

from becoming

have been described

an attempt

to maximize

of unla-

rate-limiting

to combine

sensitivity,

specificity

pre-

many of

and utility,

cost.

was supported

Parkinson’s

Wooten

assays

assay predure

the best features

might

to prevent

radioenzymatic

The

American

substrate

by research

Disease

is a George

grants

Association

C. Cotzias

from

the Pharmaceutical

and the Institute

Research

Fellow

Manufacturers

for Medical

Research

of the American

Association,

the

of the City of St. Louis.

Parkinson’s

Disease

Association.

REFERENCES Anton

AH,

Sayre

affecting

the

DF (1962) A study aluminum

procedure

for the analysis

Pharmacol

Exp Ther 138:360-365.

Axelrod

J, Tomchik

and other

demic

Colowick Press,

JT,

Henry

O-methyl-

catechols.

/ Biol

of human

rivative

method: Hiley

M.

Zurcher

/ Neurochem

sue

adrenaline,

within

the

G (1976) Simultaneous

determination

of plasma

noadrenaline

femtomole

York:

Aca-

Engleman isotope

K, Portnoy derivative

and

range.

ra-

and tis-

dopamine

Life

Sciences

epinephrine: ] Neurochem

in fetal

B (1970) A sensitive

Normal

resting

JV, Shah S (1974) Measurements

rometric

determinations

in small vol-

renaline

and nrarenaline

isotope

Acta Physiol

and epinephrine by a single

response R, lversen

techol-0-methyltransferase

to the

Metab

upright

depos-

LL (1973) for

the

Use

Palkovits

J (1963) An improved

of ca-

enzyme

ra-

Stand

M (1973)

amic or other

39:1025-1029.

human

and

plasma lev-

els. Circ Res 26:53-57. Haggendal

plasma

double-

assay for norepinephrine

21:61-67.

ture. / C/in Endocrinol AC,

New

D (1973) Catecholamines

of norepinephrine umes

Vol.

in Enzymology,

and Kaplan.

rat brain.

Cryer PE, Santiago

Da Prada

dioenzymatic

pp. 748755.

and newborn

assay of dopamine.

19:1161-1174.

J (1961) In Methods

5. Eds.,

Cuello

/

233:702-705.

Axelrod

Coyle

diochemical 21:1337-1340.

of catecholamines.

R (1958) Enzymatic

ation of epinephrine Chem

of the factors

oxide-trihydroxyindole

method

of small

for fluo-

amouts

in plasma

of ad-

and tissue.

59:242-254. Isolated

brain

nuclei

removal

of

hypothal-

of the rat. Brain Res

59:449-450. Passon

PC,

Peuler

JD

(1973) A simplified

radio-

343

344

Chuan-Huan metric

assay for plasma norepinephrine

inephrine. Quiram

Cheng and G. Frederick Wooten

Analyt

Biochem

DR, Weinshilboum

methyltransferase other

tissues:

erties

after removal

rochem Sailer

CF,

and three

of biochemical

of inhibitory

prop-

calcium. / Neu-

DM,

Gillespie

amines. Weise

Nature

V, Kopin

in human

(London)

(1966)

Liquid

ion-ex-

studies

procedure.

active

209:714-715.

I] (1976) Assay

plasma:

radioenzymatic

R

of some physiologically

of catecholamines of a single

isotope

Life Sciences

19:167-

IT (1977) The

sympa-

1686.

27:1197-1203. Zigmond

Temple

change extraction

RM (1976) Catechol-O-

in rat erythrocyte

comparison

and ep-

51:618-631.

MJ (1978)

A radioenzymatic

Ziegler

MC,

assay for catecholamines

and dihydroxyphenyl-

thetic

acetic acid. Life Sciences

23:1117-1130.

potension.

Lake CR, Kopin

nervous

system

in primary

orthostatic

N Engl I Med 296:293-297.

hy-