ANALYTICAL BIOCHEMISTRY 75, 484-497 (1976)

A Sensitive and Specific Tritium Release Assay for Dopamine-P-Hydroxylase (DpH) in Serum

GILBERT WILCOX AND MICHAEL A. BEAVEN'

Hypertension-Endocrine Branch and Pulmonary Branch, National Heart and Lung Institute, Bethesda, Maryland 20014

Received February 26, 1976; accepted June 4. 1976

The release of tritium from [7-3H,]dopamine was investigated as a possible procedure for the assay for dopamine-phydroxylase (DPH) in rat and human serum. The release was found to have the same characteristics as those described previously for DPH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu 2+ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DPH in serum. Tritium release was blocked by the DPH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of “C-labeled dopamine along with [7-3H,]dopamine revealed that, under the standard assay conditions, the formation of [“Clnorepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DPH activity in serum.

Dopamine-P-hydroxylase [3,4-dihydroxyphenylethylamine, ascor- bate:oxygen oxidoreductase (/3-hydroxylating); EC 1.14.17. l] (DPH) catalyzes the conversion of dopamine to nonrepinephrine. Oxygen, ascorbate, and fumarate are required components of this reaction (1). Enzyme activity is enhanced by the addition of sulfhydryl reagents such as Cu2+ ions (2,3) or N-ethylmaleimide (4) which inactivate unidentified endogenous inhibitors of DPH present in tissues (2,4). The enzyme is localized in chromaffin granules of the adrenal medulla (5) and storage vesicles of the sympathetic nerves (6), and it is released along with the catecholamines during stimulation of these organs (7- 11). Measurable DPH activity is found in serum of man and other species (3,12,13), and the possibility that serum DPH activity may be an index of activity of the sympathetic nervous system (12) in normal and diseased states (3) has interested many research workers and clinicians.

Several procedures are available for the assay of this enzyme. A widely used procedure is a two-step radiochemical assay in which phenyl- ethylamine or tyramine is utilized as a substrate. The hydroxylated product is converted to a labeled N-methyl derivative with purified adrenal

’ Address all correspondence to: Dr. Michael A. Beaven, National Institutes of Health, Building 10. Room 5N107, Bethesda. Md. 20014.

484 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.

DBHASSAY 485

phenylethanolamine -N - methyltransferase (PNMT) and S - adenosyl - L - [methyl-14C]methionine (14,15). Procedures which measure the conver- sion of labeled or unlabeled tyramine to octopamine have been devised. Octopamine is readily converted by reaction with periodate top-hydroxy- benzaldehyde which can be assayed spectrophotometrically (16) or radio- chemically after its extraction into ether (17). A radioimmunoassay has also been described (18,19). The latter assay circumvents the difficulties with endogenous inhibitors but may measure inactive frag- ments of the enzyme as well. The limitations and advantages of these various procedures have been reviewed (20). Another possible approach which has not been fully exploited is the measurement of tritium release from side-chain-labeled [7-3H,]dopamine. Dopamine has an affinity for D/3H similar to tyramine (l), and [7-3H,]dopamine itself loses one of its two tritium atoms during enzymatic hydroxylation (21). A tritium release assay was devised by Goldstein et al. (22) for the measurement of DPH activity in partially purified enzyme preparations from adrenals. At that time, little was known about the need to inactivate endogenous inhibitors of the enzyme, and the procedure was not applied to measurement of DPH activity in serum.

Because of the sensitivity and simplicity of tritium-release assays, we have reinvestigated the possibility of modifying the tritium release assay of Goldstein for the measurement of D@H in serum. The effects of pH, the various cofactors, Cu2+ ions, N-ethylmaleimide, and inhibitors of D/3H and serum monoamine oxidase(s) (MAO) (23), which also release tritium from [7-3H,]dopamine (24), were investigated. The studies will show that tritium release provides a simple, sensitive, and direct measurement of DPH activity in human and rat serum.

MATERIALS AND METHODS

Human Serum

Blood (10ml) was drawn from normal healthy volunteers by ven- ipuncture into a plastic syringe. All samples were obtained between 8 and 9 AM. Posture and activity were not controlled. The blood was allowed to clot at room temperature and centrifuged at 300g for 10 min. The serum was removed and frozen.

Rat plasma

Under quiet conditions, male Sprague-Dawley rats, 200-250 g, were killed by a sharp blow to the head. Blood (2-5 ml) was obtained by cardiac puncture using a syringe and No. 19 needle which contained heparin, 5 units in 0.1 ml of sterile saline. Plasma was obtained by centrifugation of the blood at 3008 for 10 min.

486 WILCOX AND BEAVEN

Chemicals and materials

[7-3H,]Dopamine, specific activity 3.4 Ci/mmole, and [7-14C]dopamine, specific activity 50 mCi/mmol, were purchased from New England Nuclear Corporation (Boston, Mass.). Since [7-3H,]dopamine was prepared by catalytic reduction of arterenone with 3H gas, it was assumed to be labeled equally on both positions of the benzylic C-atom. L-Ascorbic acid and semicarbazide hydrochloride were purchased from Fisher Scientific Company (Fair Lawn, N. J.). Catalase, 20 mg/ml, was obtained from Boehringer Chemical Company (Mannheim, West Germany). Fumaric acid, disodium salt (A grade), N-ethylmaleimide (B grade), 3, 4-dihydroxyphenylacetic acid (A grade), 3,Cdihydroxymandelic acid (A grade), (-) arterenol bitartrate (B grade), and 3-hydroxytyramine hydrochloride (A grade) were purchased from Calbiochem (Los Angeles, Calif.). Fusaric acid (5butylpicolinic acid) was obtained from Sigma Chemical Company (St. Louis, MO.). N-methyl-N-benzylpropynylamine hydrochloride (paragyline) and Pphenylisoprophylhydrazine (JB516 or pheniprazine) were supplied by Lakeside Laboratories and Abbott Laboratories, respectively. Six-millimeter Thunberg tubes were obtained from Quickfit, Reeve Angel Inc. (Clifton, N. J.) and Kontes Corporation (Vineland, N. J.).

Assay of DPH Activity

a. Reagents. (i) Sodium acetate buffer, 1.0 M, pH 5.2. (ii) Enzyme source: serum or plasma. (iii) Reagent solution prepared immediately before use. Each 100 ~1 of the reagent solution contains ascorbate, 8 pmol; sodium fumarate dibasic, 11.5 pmol; catalase, 1500 units; N-ethyl- maleimide, 6 pmol; [7-3H,]dopamine, 8 x lo4 dpm (10 pmol); and sodium acetate buffer, pH 5.2, 100 pmol. (iv) Dopamine, 250 pg/ml, in 0.8 N perchloric acid. (v) Solution of monoamine oxidase inhibitors: pargyline, 8.4 mM, and JB516, 2.1 mM.

b. Incubation conditions. Each incubation (total volume 210 ~1) was carried out in the bottom of a Thunberg tube and contained 100 ~1 of undiluted serum or enzyme preparation, 100 ~1 of the freshly prepared reagent, and 10 ~1 of the monoamine oxidase inhibitor solution. In experiments where the various cofactors and inhibitors were studied separately, the composition of the reagent solution was modified accordingly. Each Thunberg tube was flushed with approximately 0.5 liter of oxygen and closed. The tubes were then incubated at 37.5”C for 1 hr with constant shaking. The reaction was stopped by placing the tubes in ice water and adding 200 ~1 of 0.8 N perchloric acid solution of dopamine.

c. Measurement of tritiated water. The acidified incubation mixture was frozen in the bottom of the Thunberg tube by placing the tube on dry ice. The Thunberg tubes were then connected by means of a manifold to a

DPH ASSAY 487

I--/f 3 0 l@ 105 10’ 103 10-Z 10’

Cut+ or N-Ethytmaleimide IM)

FIG. 1. Activation and inhibition of doparnine-@hydroxylase activity in human serum by Cuz+ (0) and Nethylmaleimide (0). Various concentrations of Cuz+ and N-ethylmaleimide were incubated with human serum as described in Materials and Methods. The values obtained for the phosphate buffer blanks were similar for all concentrations of Cu*+ and TV-ethylmaleimide.

vacuum pump and evacuated to a pressure of 10 pm, at which time the tubes were closed. The top head of the Thunberg tube was placed in powdered dry ice; the bottom was left at room temperature. Sublimation of tritiated water into the head of the Thunberg tube was usually complete by 1 hr. The Thunberg tube was then opened and a 100-p aliquot of the sublimate was removed and assayed for radioactivity in a Packard Tri-Carb 3000 Series liquid scintillation spectrometer. Scintillation fluid consisted of 4 g of 2,5-bis [5’-tertiary butylbenzoxazolyl(2’)lthiopente (BBOT obtained from Ciba Pharmaceuticals, Summit, N. J.), 8Og of naphthalene in 1 liter of a mixture of toluene-ethylene glycol monomethyl ether (6:4, v/v) (counting efficiency for 3H, 26%; 14C, 66%).

Results were calculated either in terms of percentage of tritium released or as picomoles of v-3H,]dopamine hydroxylated per milliliter of serum per hour. The assumption was made that 50% of the tritium was released during hydroxylation. The minimum activity that could be measured by the assay was in the range 0.2-0.3 pmol/g/hr.

d. Blanks. For routine assays the reagent blank consisted of 100~1 of sodium acetate buffer, pH 5.2, in place of serum.

Identification of the Reaction Products by Chromatography

[7-14C]dopamine (0.01 PCi) and [73H,]dopamine (0.04 PCi) were incubated with cofactors and MAO inhibitors under the standard conditions described above, except that the 200 ~1 of perchloric acid solution added at the end of the reaction contained unlabeled nonrepineph- rine (50 pg) in addition to unlabeled dopamine (50 pg). The reaction

488 WILCOX AND BEAVEN

FIG. 2. Effect of serum volume on dopamine+hydroxylase activity in the presence of 10 (0) and 80 PM (A) Cu*+ (A) and N-ethylmaleimide, 30 mM (0) (B). Incubations contained varying amounts of serum, Cuz+ , or N-ethylmaleimide. The shift in the curve with the higher copper concentration was attributed to (i) inhibition of D/3H activity by Cu2+ ions and (ii) reversal of inhibition by the binding of Cuz+ ions to serum proteins in the presence of larger amounts of serum.

mixture was then freeze-dried. The amines were extracted from the residue into 500 ~1 of methanol (dehydrated with anhydrous MgSO,). The methanolic extract was centrifuged, insoluble material was discarded, and the supernatant extract was evaporated to dryness under a stream of an-. The procedure was repeated two more times by dissolving the resultant residue in 100 ,~l of dehydrated methanol and finally in 40 ~1 of methanol. These procedures removed most of the constituents of the reaction mixture which would otherwise interfere in the chromatographic separation of the amines. The final residue was dissolved in 40 ~1 of 0.01 NHCI, and 10 ~1 of this solution applied in a 2-mm-wide band on thin-layer cellulose powder

1553-

30 60 so 120

Time Uninf

FIG. 3. Time course of 3H release from [7-3H,]dopamine in the presence of human serum. The incubations contained 100 ~1 of serum and the various reagents as described in text. A 60-min time point was adopted for the routine assay of DPH.

DPH ASSAY 489

TABLE 1

EFFECT OF ADDED DOPAMINE ON DPH ACTIVITY IN HUMAN SERUM AS MEASURED

BY TRITIUM RELEASE FROM [73H2] DOPAMINE

Final dopamine concentration

(/W Percentage of Percentage of 3H released” control

0.05 (control)* 6.15’ 100 0.25 4.83 87 0.5 6.10 99 1.0 5.80 95

10.0 5.50 89 50.0 4.10 77

a Values corrected for the tritium released in the absence of serum (i.e., blank). * [7JH]Dopamine only; the concentration was that used in the standard assay procedure. c DPH activity in the serum sample was equivalent to 12.9 pmol/mVhr.

plates. The chromatograms were placed in a solvent system consisting of butanol saturated with 1 .O N HCl for 120 min. The sheets were air-dried and the amines visualized by exposure to iodine vapor. The distribution of radioactivity was determined by scraping 2.5mm-wide sections of the chromatograms into vials, and 14C and 3H were assayed by liquid scintillation counting.

The presence or absence of acidic metabolites was checked by mixing 200 ~1 of the incubation mixture with suspension of 50 mg of Dowex 50 (H+ form) in 200 ~1 water. After shaking the mixture on a Vortex mixer and depositing the resin by centrifugation, lOO-~1 aliquots of the supematant fluid were assayed for 14C and 3H.

Assay of Monoamine Oxidase Activity

Monoamine oxidase activity of plasma samples was assayed by a modification of the method of Robinson et al. (25). The concentration of substrate, [oG4C] benzylamine, was lops M (100 @/ml). Activity was expressed as nanomoles of [&4C]benzylamine deaminated per hour of incubation per milliliter of plasma.

RESULTS AND COMMENTS

The Effect of CLP and N-ethylmaleimide on Tritium Release

The tritium-releasing activity of human serum increased with increasing concentrations of Cu2+ up to 0.01 mM and then markedly decreased with higher concentrations of Cu2+ (Fig. 1). With N-ethylmaleimide an increase in activity was observed over a much broader range of concentrations than

490 WILCOX AND BEAVEN

I 4 5 6 7 6

PH

FIG. 4. Relationship of pH and tritium release. Incubations contained 100 ~1 of serum and the various reagents including pargyline, 4 x lo-’ M, and JB516, 10-s M, in sodium acetate buffer, 1 M (0, A), or potassium phosphate buffer, 1 M (0, A). The blanks (A, A) contained buffer but not serum. The pH indicated is that of the final incubation mixture.

with Cu2+; optimum release was observed at 10 mM (Fig. 1). This pattern of increase was similar to that observed by Nagatsu and Udenfriend (16).

Effect of Serum Volume, Substrate Concentration, and pH on Tritium Release

With 0.01 mM Cu2+, tritium release was proportional to serum volume in the range O-80 4, but, in the presence of a higher concentration (0.08 mM)

TABLE 2

EFFECTOFOXYGENANDVARIOUSCOMPONENTSON SERUM D/3H ACTIVITY

Omission

None Oxygen* Ascorbated Catalased Fumarate

Activity (% of control)

loo 45,37c

25 17 40

Q Values are averages of duplicate experiments. Incubations were performed using the standard assay condition except that various components were omitted as indicated.

* Incubations carried out in air. c Values from two studies. d Omission of these agents increased spontaneous 3H release, i.e., gave high values for

enzyme blanks.

DPH ASSAY 491

TABLE 3

COMPAIUSON oP -ME EFFECT OF MAO INHIBITORS ON PLASMA DPH AND MAO ACTIVITIES’

Inhibitor

Concen- Plasma DPH activity Plasma MAO activity nation @M) (pmoliml/hr) (% of control) (nmol/ml/hr) (% of control)

Control (no MAO inhibitors)

Pargyline

JB516

Pargyline + JB.516

Semicarbazide

- 12. I

0.4 12.0 0.4 12.8

1.0 1.6 0.1 11.6 0.02 12.5

0.4 5.9 1.0 0.1 12.2

100

99 106

62 96

103

48 0 0

101 0.3 8

3.9 100

3.9 100 - -

0 0 0 0 - -

cL Plasma DPH activity was assayed by tritium release from [7-3H,]dopamine and MAO activity by measurement of the deamination of [a-raC]benzylamine as described in text.

of Cu2+, the release was completely inhibited in the presence of small volumes of serum (O-40 ~1) and partially inhibited with larger volumes (Fig. 2A).

Because of the complex relationship between Cu2+ concentration and serum volume, N-ethylmaleimide was adopted for the assay procedure. With this compound the relationship between serum volume and tritium release was linear over the entire range of volumes studied (Fig. 2B). The rate of tritium release was constant with time up to 60 min (Fig. 3) and with

TABLE 4

EFFECT OF FUSARIC ACID ON PLASMA DPH AND MAO ACTIVITY”

Concentration of fusaric

acid W-M

Inhibition (% of control)

DPH MAO

0.01 64 100 0.1 17 100 1.0 0.1 88

a DPH activity was measured by the tritium-release assay and MAO by measurement of deamination of [cr-r4C]benzylamine as described in Materials and Methods. The values for DPH are the averages of three experiments. The effect on MAO was studied separately with pooled serum from four individuals.

492 WILCOXANDBEAVEN

FIG. 5. Identification of reaction products by thin-layer chromatography. Incubations with acetate buffer (blank) (A) and serum (B) were carried out with a mixture of [7-3H,]dopamine and [7J4C]dopamine, cofactors, and MAO inhibitors. The chromatograms were prepared with methanolic extracts of the incubation mixtures on thin-layer cellulose-powder plates and chromatographed in a butanol:HCl system as described in the text. Chromatograms prepared from standard compounds are depicted in the lower part of the figure: DHMA = dihy- droxymandelic acid; NE = norepinephrine; DA = dopamine; DHPAA = dihydroxy- phenylacetic acid. The upper figure shows the ratio of ‘YYH for the different parts of the chromatogram. The increase in ratio from 0.46 in the DA peak to 0.9 in the NE peak indicates the loss of one-half of the tritium label upon hydroxylation.

different concentrations of substrate up to 10 PM (Table 1). An optimum rate of release was observed at pH 5.2 (Fig. 4).

Effect of Oxygen, Ascorbate, Fumarate and Catalase on Tritium Release

Since oxygen is known to participate in the reaction, the effect of oxygen on DPH activity was studied. Tritium release was diminished by more than half when incubations were performed under air (Table 2). Omission of ascorbate, fumarate, or catalase also reduced tritium release (Table 2).

Injluence of MAO on Tritium Release. Studies with MAO and DPH Inhibitors

At pH 5.2, tritium release was unaffected by the addition of JB516 or semicarbazide in a concentration (O.lmM) that inhibited serum MAO activity completely (Table 3). Higher concentrations of JB516 partially inhibited tritium release. Pargyline, an inhibitor of mitochondrial MAO, neither inhibited tritium release nor serum MAO activity (Table 3).

DPH ASSAY 493

TABLE 5

SERUM DPH ACTIVITY ININDIVIDUALHUMAN SUBJECTS

Subject Sex -4s Serum D/3H activity (yr) (pmol/hr/ml)

1 2 3 4 5 6 7 8 9

10 11

Mean f SEM

12 13 14 15 16 17 18 19 20

Mean + SEM

M 17 M 23 M 25 M 28 M 30 M 30 M 34 M 36 M 38 M 44 M 68

20 23 24 28 35 50 52 68 84

11.2 16.0 4.7 6.0 8.4 5.8 6.7 1.7 1.7 2.7 6.8

6.3 2 4.3

15.9 13.2 3.7 4.9 2.7 8.6 5.9 6.8 0.8

6.9 + 4.9

In contrast to the MAO inhibitors, tritium release was inhibited by the DPH inhibitor fusaric acid. Serum MAO activity was only minimally affected by this drug (Table 4).

Stoichiometry of Tritium Release and IdentiJcation ofReaction Products. Studies with Y- and 3H-Labeled Dopamine

Chromatography revealed only two labeled compounds in the incubation mixture, dopamine and a second peak which migrated in a fashion identical to norepinephrine and dihydroxymandelic acid (Fig. 5). Since all (< 97%) of the nonvolatile labeled material in the incubation mixture was taken up by Dowex 50, the second peak was identified as norepinephrine. The ratio of 14C to 3H in the “norepinephrine” peak was twice that in the dopamine peak (Fig. 5). This suggested that only half of the tritium label was displaced during the conversion of dopamine to norepinephrine.

A number of other published chromatographic systems was tested. These did not give adequate separation of dopamine from norepinephrine, but they gave no indication that other labeled products were formed.

494 WILCOX AND BEAVEN

DPH Activity in Human Serum and Rat Plasma

D/3H activity in serum of 20 normal subjects ranged from 0.8 to 16.0 pmol/hr/ml (Table 5). No significant difference was observed between values for male (6.3 ? 4.3) and female (6.9 + 4.9 pmoYml/hr) subjects. Values for rat serum were lower than for human subjects and ranged from 0.3 to 1.1 pmol/hr/ml (mean 0.9 + 0.2, n = 5). Even with low values, the assay was highly reproducible. In two typical experiments, a serum sample with an activity of 3.9 pmol/g/hr gave duplicate values of 129, 131 cpm and 134, 144 cpm of 3Hz0 above the values for blanks of 72,71 cpm and 77,81 cpm of 3H, respectively. Serum samples could be stored frozen at -20°C for 2 months without significant loss of activity.

DISCUSSION

The present study shows that, in the presence of rat or human serum and appropriate cofactors and inhibitors, one molecule of [3Hlwater is released for each molecule of [7-3H,]dopamine converted to [7-3H]norepinephrine. The tritium release has characteristics similar to those described by other authors for serum DPH activity: an optimum rate of reaction at pH 5.2 (16); a requirement for ascorbate, catalase, and fumarate (1,14-16), an enhancement of activity upon the addition of reagents such as Cu2+ and N-ethylmaleimide (14- 16), and an inhibition by the D/3H inhibitor fusaric acid (26) at concentrations that do not inhibit serum monoamine oxidase(s). The curves obtained for Cu2+ andiV-ethylmaleimide (Fig. 1) are identical to those obtained with the spectrophotometric assay by other workers (16). The study shows in addition that, in an atmosphere of oxygen, activity is enhanced several-fold from that in air and the values are made more reproducible. Enhancement (see citations in Ref. (1)) or loss of activity (3) has been noted previously when incubations were carried out under oxygen or nitrogen, respectively.

Tritium-release assays (27) have been utilized in this laboratory for the assay of histaminase (28) in a variety of basic and clinical studies (see citations in Ref. (29)). Such assays have a number of advantages. They do not require isolation of product from excess substrate. Water, being the vehicle, is not subject to decomposition, adsorption, or metabolic reactions. The release of one molecule of water from each molecule of substrate hydroxylated provides a simple direct measurement of enzyme reaction. The factor limiting sensitivity is the extent of spontaneous release in the absence of enzyme, about 0.5% in this assay. The reproducibility is such that the release of an additional 0.2% tritium (equivalent to the hydroxylation of 2 x lo-l4 mol of substrate) can be measured reliably. Interference from endogenous substrate does not appear to be a complication. Dilution with endogenous substrate will alter the rate of tritium release once the concentration of substrate is sufficient to remove

DBH ASSAY 495

the reaction from first-order kinetics.’ Concentrations of 50 j.,&M (7.5 pg/ml) dopamine were needed before a perceptible decrease (23%) in tritium release was noted. While other assays of DPH may share some of these attributes, the simplicity and sensitivity of the tritium-release assay are probably its most useful assets.

There are precautions that one should consider with tritium-release assays. These are the possible lack of specificity and isotope effects. Our earlier studies have shown that tritium is released from [7-3H,ldopamine upon deamination with soluble beef serum MAO but not with mitochon- drial MAO (24). For this reason, MAO inhibitors are included in the reaction mixture. The effects of MAO inhibitors at different pH, the inhibition of tritium release with fusaric acid, and the demonstration that 1 mol of water is released for each molecule of norepinephrine formed indicate, however, that MAO activity contributes little or no tritium-releasing activity of human or rat serum at pH 5.2. The MAO inhibitors serve also to prevent destruction of substrate by monoamine oxidase and are utilized for such a purpose by other authors (14- 17). Pargyline, which is usually employed as the MAO inhibitor (14- 17), does not inhibit soluble MAO, which is present in human plasma (23). A mixture of pargyline and JB516 would be preferable, although the concentration of JB5 16 should not exceed 0.1 mM because of inhibition of DPH (Table 3).

Because of the stoichiometric relationship between tritium release and norepinephrine formation, there does not appear to be an isotope effect. The loss of one of the two tritium atoms on the benzylic carbon is consistent with the earlier findings of Senoh and co-workers (21) and the more recent studies of Taylor (30) which show that the hydroxylation involves the stereospecific removal of one proton from the benzylic carbon and retention of configuration.

The problem of endogenous inhibitors is common to all of the enzymatic and chemical assays of DPH (14- 18,20). Because of the inhibition of activity with high concentrations of Cu2+ (Fig. 1) and the variable effects produced with different amounts of serum (Fig. 2), our preference is to use N-ethylmaleimide to block the endogenous inhibitors.

In principle, the tritium-release assay could be applied to measurement of the enzyme in tissue extracts. In view of the long availability of

’ As with all assays in which a labeled substrate is used, the tritium-release assay has different requirements than assays in which nonlabeled substrate is used. In the latter, it is generally advantageous to have the substrate at concentrations that saturate the enzyme to maximize the amount of product formed. In the tritium-release assay, it is desirable to have the maximum amount of label converted to labeled product. This occurs when substrate concentrations are within the linear portion of the Henri plot. The proportion of substrate converted to product decreases once the concentration of substrate approaches saturation. The reproducibility of the assay is not impaired by having low concentrations of substrate. As the data in Table I indicate, the [7-3H]dopamine can be diluted 1000 times before significant changes in tritum release are observed.

496 WILCOX AND BEAVEN

[7-3H,]dopamine on the market, it is surprising that the method of Goldstein et al. (15) has not been more widely utilized. In some respects, the procedure was introduced too early in that details about D/IH inhibitors were not fully understood. Further modifications of the procedure were necessary to make it practical. Since that time, the PNMT assay, a sensitive but complex procedure, and the spectrophotometric assay, a relatively simple procedure, were introduced and widely adopted. In addition, Nagatsu and co-workers (3 1, and cited by Evans (27)) have raised the question of possible nonspecific release of tritium from [7-3H,]dopamine with tissue extracts. They reported that tritium was released from [7-3H,]dopamine when incubated with extracts of both normal and sympathetically denervated kidneys without detectable formation of norepinephrine (from ring-labeled [3H]dopamine. However, no ex- perimental data were presented to support this statement, and extra- neuronal sources of DPH have now been shown to exist at least in salivary gland (32). The ability to suppress tritium release with the DPH inhibitor fusaric acid could be used to test specificity of the tritium- release assay in individual tissues.

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DPH ASSAY 497

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