Br. J. cin. Pharmac. (1977), 4, 553-558

A SENSITIVE RADIOENZYMATIC ASSAY FOR ADRENALINE AND NORADRENALINE IN PLASMA HEIDE HORTNAGL*, C.R. BENEDICT & D.G. GRAHAME-SMITH MRC Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary, Oxford OX2 6HE

B. McGRATH Department of the Regius Professor of Medicine, Radcliffe Infirmary, Oxford OX2 6HE

I An existing radioenzymatic assay for plasma catecholamines using catechol-o-methyl transferase and 13HI-S-adenosyl-methionine has been modified resulting in a more sensitive assay for the measurement of plasma adrenaline and noradrenaline. 2 The lower limit of sensitivity for this method is 25 pg for adrenaline and 30 pg for noradrenaline/ml of plasma. 3 Resting supine (60 min) plasma adrenaline concentration was (mean ± s.d.) 124 + 76 pg/ml (n= 11) in males and 130 ± 71 pg/ml (n = 7) in females; plasma noradrenaline concentrations were respectively 444 + 129 pg/ml and 550 ± 87 pg/ml. 4 The changes in plasma catecholamine concentrations in response to 40° head-up tilt have been determined in a group of healthy normal subjects and have been shown to be related to changes in blood pressure and heart rate.

Introduction

Recently a radioenzymatic assay for noradrenaline (NA) and adrenaline (A) in human plasma has been described (Passon & Peuler, 1973). That assay is based on the conversion of NA and A to labelled [3Hnormetanephrine and metanephrine using S-adenosyll-(methyl-3H) methionine as a methyl donor. In this report we describe some modifications of this assay, the increased sensitivity thereby achieved, the validation of the modified assay and its use in measuring the concentrations of plasma NA and A in healthy subjects before, during and after a 400 headup tilt. Methods

Chemicals were obtained from the following sources: S-adenosyl-l-(methyl-3H) methionine, specific activity 5-10 Ci/mmol (3H-SAM) (Radiochemical Centre, Amersham); S-adenosyl-methionine iodide salt (SAM) (Calbiochem); (± )-metanephrine {7-3H(N)} (4.65 Ci/mmol), (±)-normetanephrine { 7-3H(N)} (3.2 Ci/mmol) and liquifluour (New England Nuclear *

Permanent address: Pharmakologisches Institut der

Universitit, Innsbruck, Austria.

Corp); L-adrenaline tartrate, L-noradrenaline tartrate, (± )-metanephrine HCI (MN), (± )-normetanephrine HCI (NMN) and reduced glutathione (Sigma); tranylcypromine sulphate (SKF); glass balls (2.5 to 3.5 mm diam) (Hopkins & Williams); thin-layer chromatography plates, silica gel F254 pre-coated plates (layer thickness 0.25 mm) (Merck). All other reagents used were of analytical reagent grade. Catechol-o-methyltransferase (COMT) was prepared according to the method of Axelrod & Tomchick (1958) up to the (NH4)2SO4 precipitation step. The crude COMT preparation was then dialysed for 6 h against two changes of 2 litres of potassium phosphate buffer (1 mmol/l, pH 7.0), the supernatant adjusted to pH 8.1 with 2.0 mol/l tris-HCl pH 8.2 and centrifuged at 3000 g for 10 min at 4°C to remove suspended proteins. The clear supernatant was divided into aliquots and stored at -200C. The protein concentration of the COMT preparation was measured by the method of Lowry, Rosebrough, Farr & Randall (1951). All glassware was silane coated using a 1% solution of dichlorodimethyl silane to prevent absorption of small amounts of catecholamines to the glass. Venous blood samples (2 ml) were collected into lithium-heparin tubes containing sufficient solid

554

HEIDE HORTNAGL, C.R. BENEDICT, D.G. GRAHAME-SMITH & B. McGRATH

reduced glutathione (GSH) to give final concentration of 5 mmol/l. The tubes were immediately placed in ice and centrifuged at 3000 g for 5 min at 4°C. The plasma was stored frozen until assay. The methylation of A and NA was performed in 50 ml glass stoppered tubes containing 300 ,ul of plasma and, in final concentrations, 80 mmol/l trisHCI buffer pH 8.2, 1 gmol/l SAM (including 1 ,jCiP3HJ-SAM), 1 mmol/l GSH, 24 mmol/l MgCI2 and I mmol/l tranylcypromine. The reaction was started by the addition of COMT 3-4 mg. The final volume of the incubation mixture was 1.25 ml. Incubation was carried out at 370C for 45 min and the reaction stopped by the addition of 600-700 d of 1.0 mol/l borate, pH 12, bringing the pH of the mixture to 10.3-10.5. Blanks were determined by using either 300 A of water or 300 ,ul of plasma from which catecholamines had been removed using alumina (Christensen, 1973). Unlabelled MN and NMN (0.5 gmol of each) were added and the mixture saturated with K2HPO4 (2.5 g) (Anton & Sayre, 1966; Siggers, Salter & Toseland, 1970). Five to six glass balls were added and NMN and MN extracted into 12 ml of a toluene: isoamyl alcohol mixture (3:2 v/v) by shaking for 10 min. The subsequent steps involving evaporation of 10 ml of the organic phase, thin layer chromatography (t.l.c.) conversion to vanillin and extraction for counting were carried out as described by Passon & Peuler (1973). The samples were counted in an LKB Wallac 81000 Liquid Scintillation Counter with a mean efficiency of

33%. For each assay 1.5 and 3 pmol standards of A and NA were freshly prepared in 5 mmol/l reduced

glutathione and assayed in duplicate. Extraction ofMN and NMN

To investigate the effects of K2HPO4 and pH on the extraction of MN and NMN small amounts of 13H]MN and P3H]-NMN (55,000 d/min) were added to the incubation mixture and P3H]-SAM omitted. Unlabelled MN and NMN (0.5 imol) were added as carriers. The pH of this mixture was adjusted to between 9.5 to 1 1.0 with 1 mol/l borate pH 11.0 or 12.0. The extraction was carried out with and without K2HPO4 and the whole procedure of t.l.c. and conversion to vanillin continued. The extraction was also tested with smaller amounts of P3H]-MN and P3HI-NMN corresponding to the counts/min which resulted during plasma catecholamine estimation. Clinical investigations

Resting plasma A and NA concentrations were determined in healthy subjects, eleven males (21-54 years) and seven females (31-70 years), after supine bed rest for 1 h. Venous blood was collected at

between 09.00 h and 10.00 h, approximately 2 h after breakfast. In eight healthy males (23-54 years) and six healthy females (31-56 years) plasma A and NA concentrations were&measured 1 h after supine bed rest, 5 min after a 400 head-up tilt and 15 min after tilting back to the supine position. All estimations were made in duplicate. During this study the blood pressure was measured in the right brachial artery using the Roche arteriosonde 1217 automatic blood pressure monitor and the pulse from the left index finger tip using a Harvard 362 transducer and 2162 amplifier. In each subject blood pressure and pulse rate were averaged from five 1-min recordings immediately before and after the tilt and again between the 10th and 15th min after resumption of the supine posture. The data were analysed using the Students paired ttest (two-tailed). All data are stated as mean + 1 s.d. Results Enzymatic conversion ofA and NA The main modification in the incubation conditions described by Passon & Peuler (1973) was the reduction in the molarity of total SAM from 10 ,umol/l to I 1imol/l and an increase in the specific activity of from 0.25 ,uCi g.molh' 1-1 to P3HI-SAM 0.8 ,uCi iimol-' 1-'. Under the conditions described we did not encounter the high blank values described by Passon & Peuler (1973). The optimum pH of the enzymatic reaction was found to be between 8.2 and 8.5 and a pH of 8.2 was therefore used. Extraction ofMN and NMN In the method of Passon & Peuler (1973) extraction of NMN was 28% and MN 37% at pH 9.5. This extraction has been improved by saturating the aqueous phase with K2HPO4 and by raising the pH of the aqueous phase to 10.5. These modifications improved NMN extraction to 70% and MN to 76% (see Table 1). Linearity of assay A and NA estimation were linear in the 50 pg to 5000 pg range (50-500 pg range illustrated in Figure 1). Passon & Peuler (1973) obtained 67 counts min-' pmolh' NA and 86 counts min-' pmol-' A, while in this method 400 counts min-' pmol-1 NA and A were obtained, an increase in sensitivity of

approximately four-fold. Blanks yielded 20-30 counts/min above ambient background. Influence of the addition ofplasma The amounts of [3H]-MN and [3HJ-NMN formed when NA and A standards were added to

PLASMA CATECHOLAMINE ASSAY

555

Inter-assay variability (six aliquots of the same plasma sample during six consecutive assays) was 9.1% (A) and 5.6% (NA).

Plasma A and NA in healthy subjects

1000

co cD 0 0

600

200

0.6

1.2

,1

,

0.1 0.2

0.1

Figure 1

1.8 ,

0.3

0.2 0.3

3.0 pmol

24 ,

I

,

0.6 ng A

0.4 0.5

0.4

0.5

0.6 ng NA

Linearity of the enzymatic conversion of A

(-) and NA (0) in the range of 50 pg-500 pg of catecholamine concentration.

catecholamine free plasma was 73% (A) and 64% (NA) of those obtained when the standards were added to water in agreement with Passon & Peuler (1973). The addition of tranylcypromine or semicarbazide, a specific plasma amine oxidase inhibitor, did not influence this conversion. The extraction of PHI-MN and [3H]-NMN was not influenced by the presence of plasma. When standards in water were assayed there was approximately 100% enzymatic o-methylation (assuming a final recovery of 70%). Intra- and inter-assay variability

Intra-assay variability (ten aliquots of the same plasma sample) was 7.56% (A) and 3.15% (NA).

In Table 2 are listed the normal plasma A and NA concentrations determined in this study and in eleven other studies using radioenzymatic techniques. In fourteen subjects (eight male, six female) resting supine plasma noradrenaline concentrations (490 + 135 pg/ml) increased markedly after 5 min of 40° head-up tilt (2210 + 1571 pg/ml, P< 0.001) and returned towards resting concentrations 15 min after resumption of the supine position (570 + 153 pg/ml) (Figure 2) but were still greater than the initial resting value (P < 0.05). In parallel with the changes in plasma NA with tilt, heart rate increased (from 65.4 + 9.0 beats/min to 72.0 + 8.6 beats/min, P < 0.02) as did diastolic blood pressure (from 76.5 + 8.2 mmHg to 83.1 + 9.0 mmHg, P < 0.001). These also returned to control values 10-15 min after resumption of the supine posture (heart rate to 62.7 + 2.0 and diastolic blood pressure to 77.8 + 2.7 mmHg). Plasma A did not change with the tilt (120 + 71 pg/ml to 106 + 64 pg/ml) (Figure 2).

Discussion By increasing the specific activity of the 3H-methyl groups donated by SAM in the o-methylation of A and NA and by improving the extractions of the [3H]MN and [3H1-NMN, the sensitivity of Passon & Peuler's (1973) method has been improved approximately four-fold. In that method 1 ng A yields 470 counts/min and 1 ng NA 400 counts/min with blanks of 10 counts/min. With the modifications described 1 ng of A or NA yields 2220-2300 counts/min with blanks of 20-30 counts/min and the lower limits of measurement are 25 pg A and 30 pg NA/ml of plasma.

Table 1 Influence of pH and K2HP04 on the extraction of [3HI-MN and [3H1-NMN. The mean +s.d. values given for the extraction percentage of [3H]-MN and [3H1-NMN were obtained after thin layer chromatography and conversion to vanillin pH of aqueous

PHI-MN

9.5 10.0 10.5 11.0 10.5

61 + 1.5 65+ 3.0 70± 1.5 71.5+ 1.1 23.4+ 0.2

61.5 ±4 64.5±4

9.5

28

phase With K2HP04

Without K2HP04 Passon & Peuler (1 973)

% extraction

PHI-NMN

77+3 76 ± 3

44.5+0.8 37

n=4 n=4 n=6 n=4 n=3

HEIDE HORTNAGL, C.R. BENEDICT, D.G. GRAHAME-SMITH & B. McGRATH

566

Table 2 Catecholamine contents (mean ± s.d.) of venous plasma samples from normal human subjects Number of subjects

Sex

Age (years) range

Plasma content

(mean + s.d.)

(pg/mi) A

11 t

Males

21-54

NA

Resting

124 + 76

444+ 129 Present

Resting

130± 71

550 ± 87

Resting

170 ± 63 130±24

Resting and fasting Resting and fasting

40±28

290 ± 158 Callingham & 410 ± 122 Barrand (1976) 220± 57 Cryeretal.

50 ± 40

220 ± 80

90±100

420±10

(38.1 + 12.3) 7t

Females

31-70

Reference

study

(47.7 +14.5) 1ot 6t 8t 16

Males Females Males (4) and females (4) Males (8) and females (8)

18-78 21-77

(44)

4

Males Females

20-42

Resting and fasting

16*

Males

10-65

Resting

22*

Females

12 10

(1974)

60± 139 40±95

7*

Christensen

(1973) Siggers et al. (1970) 200±277 Engelman & 210± 285 Portnoy (1970) 250± 530 Henry et a.

(1975) 270± 164 Ziegler et a/. (1976) 307 + 145

(30±16) 11-61

Resting

(30.6+17.4) 10-70 (32.7 + 16.3)

74*

25

Males

26

Males

20-65

(40.3) 6

Females

23-65

(39.8) 8t

Resting Resting and fasting Resting and fasting Resting and fasting Resting

58 ± 30 45

(00-160) 58 (20-190) 160± 57

292 ± 138 Lake et al. (1976) 133± 70 Miura & De Quattro (1975) Pedersen & 258 (80-750) Christensen 238 (1975)

(12-430) 160± 141

Moerman et at.

(1976)

tAssay performed directly in plasma. Phenyl ethanolamine-N-methyl transferase used ftor assay. All other values obtained by using COMT for

I

enzymatic methylation. We have not yet determined why lower values for A and NA are obtained when standards are made up in plasma rather than in water. Powis (1974, 1975) and Branco, Torrinha & Osswald (1974) reported binding of catecholamines to plasma proteins protecting them from metabolic degradation (Whitby, Axelrod & WeilMalherbe, 1961). Christensen (1973) using isolation in alumina found no difference in the recovery of catecholamines added to plasma or buffer. Engelman & Portnoy (1970) used pH 6.0-6.2 for column chromatography and low pH has been shown to decrease plasma protein binding. Carruthers, Conway, Taggart, Bates & Somerville (1970) found that

prolonged storage of plasma samples at -200C influenced plasma catecholamine concentrations. We found no decrease in catecholamine concentrations after 3 weeks storage at -200C in agreement with Christensen (1973). Normal values

The range of normal values found in this study may be compared with those of other studies (Table 2). Only two other groups (Siggers et al., 1970; Callingham & Barrand, 1976) have assayed plasma without first employing separation techniques and though the

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