- Email: [email protected]
Plasma levels of free and total catecholamines and two deaminated metabolites in man — rapid deconjugation by heat in acid
199
Clinica Chimica Acta, 137 (1984) 199-209 Elsevier
CCA 02771
Plasma levels of free and total catecholamines and two deaminated metabolites in man - rapid deconjugation by heat in acid Nicolas D. Vlachakis
*, Ella Kogosov, Seiichi Yoneda, and Robert F, Maronde
Section of C&nieal Pharmacology, Department
Natalie Alexander
of Medicine, Universify of Southern Cali@nia, Los Angeles, CA (USA)
(Received
June 24th; revision October
7th. 1983)
Key words: Catecho~~mine; Catecholamine metabolite; Deconjugation
Summary
We have described a procedure for deconjugation of plasma catecholamines, norepinephrine (NE), epineph~ne (E) and dopamine (DA) and two catecholamine metabolites 3,4-dihydroxymandelic acid (DOMA) and 3,4-dihydroxyphenylglycol (DOPEG). Heat at 100°C of the acidified specimen, pH 0.8, produced complete deconjugation of catecholamines in 7 minutes and of metabolites in 5-7 minutes. Subsequently all five products were simultaneously measured with a radioenzymatic assay. However, hydrolysis for 7 minutes produced approximately a loss of 5% in DA and E, 15% in NE and 50% in the metabolites. The percent of free compound in the plasma of 11 normotensive and healthy subjects was 23 + 16 for NE, 20 + 8 E, 0.8 f 1 DA, 20 f 7 DOMA and 42 & 12 for DOPEG. Similar results were obtained in a random specimen of six patients with primary hypertension. In a group of four patients with pheochromocytoma free levels of NE, DOPEG and DOMA were sig~fi~ntly greater than in the other two groups, whereas conjugates were not. The intravenous administration of NE or the activation of sympathetic nervous system by standing combined with exergise for 15 minutes did not produce a change in the levels of plasma conjugates. These findings suggest that short changes in plasma catecholamines are better reflected in the free than the conjugated part.
* Correspondence to Nicolas D. Vlachakis, MD, Department California, 2025 Zonal Avenue, Los Angeles, CA 90033, USA. ~-8981/84/$03.~
0 1984 Eisevier Science Publishers
B.V.
of Medicine,
University
of Southern
200
Introduction The advent of radioenzymatic assays made possible the accurate and specific measurement of catecholamines and catecholamine metabolites in small quantities of specimens [1,2]. The assays are based on the methylation of these compounds by specific enzymes and in the presence of methyl donor S-adenosyl-L-[’ Hlmethionine. However, since the enzymes do not attack the conjugated part of the compound [3], this part (which is the largest) escapes detection [4]. The deconjugation by acidic lyophylization of Buu and Kuchel [4] has been associated with inconsistent results. Nagel and Schiimann [5] have recently achieved consistent deconjugation of plasma catecholamines by acidic hydrolysis at 95°C for 40 min. In a previous study we have successfully deconjugated plasma normetanephrine by acidic hydrolysis at 100°C for 20 min [2]. In the present report we describe a similar procedure for complete and consistent deconjugation of three catecholamines and two major deaminated catecholamine metabolites. Subsequently both the free and conjugated part of these compounds were simultaneously measured with a radioenzymatic assay [6]. Materials and methods All materials used in this assay as well as the isotope S-adenosyl-L-[ were described elsewhere [6].
3Hlmethionine
Plasma specimen collection One ml of blood is drawn in a syringe and transferred into a small glass tube containing reduced glutathione and ethylene-bis(oxyethylene-nitrilo) tetraacetic acid (EGTA) as a preservative [2]. After centrifugation at 600 X g for 10 min in a cold centrifuge, plasma is separated, transferred into a plastic tube and stored at - 70°C until assayed (within 1-2 weeks after collection). Assay procedure On the day of assay the specimens were gradually thawed and placed in ice. Five hundred ~1 of plasma was transferred into a 75 X 13 mm glass disposable tube and its pH was decreased to 0.8 by adding 7.5% of 5 mol/l perchloric acid (v/v) for protein precipitation. Subsequently the specimen was centrifuged and a part of the supernatant (150 ~1) was transferred to a 3-ml Pyrex conical tube for deconjugation by heat, the remainder was used for determination of the free compound. The Pyrex conical tube was tightly covered with a glass stopper and placed into boiling water. Specimens were boiled different times in ord?r to estimate the duration of boiling for optimal deconjugation. Immediately following boiling tubes were placed in ice and the pH was raised to 8 by adding 15% of 5 mol/l Tris-base (v/v). A similar amount of Tris was added to their homologous unboiled supernatant. Subsequently 50 ~1 of the supernatant from each paired tube were transferred into a 13 X 100 mm disposable tube (Kimble, Owen, IL, USA) and to that was added: 5 ~1 of 3H-SAME (2.5 PC;), 10 ~1 of 0.2 mol/l tris buffer containing 0.1 mmol/l benzyloxyamine, pH 8.1, and 10 ~1 of catechol-O-methyl transferase. Internal standards, consisting of 500
201
pg of each compound, were added to another set of tubes together with the specimen and the incubation mixture. Blanks were prepared by replacing the specimen with 50 ~1 of Tris-perchloric acid buffer, pH 8.0. All specimens were run in duplicate. In each assay a known amount of all five compounds was run with and without boiling for estimation of the losses of the free compound by the deconjugation procedure. The incubation tubes were placed in a shaking water bath at 37°C and incubated for 60 min. The subsequent steps of the assay have been described in detail elsewhere [6]. With this assay plasma levels of norepinephrine (NE), epinephrine (E), dopamine (DA), and the metabolites 3,4_dihydroxymandelic acid (DOMA) and 3,4-dihydroxyphenylglycol (DOPEG) were measured simultaneously. The total compound represents the sum of conjugated part (liberated by hydrolysis) and free part in each specimen. Values were adjusted for losses. The conjugated part was calculated by subtraction of the free from the total compound. For statistical analysis the analysis of variance was used and if significant we used the Student’s r test for comparison between groups and the paired t test for the change in the same group. The coefficient of correlation was determined when indicated [7].
5500
-
5000
-
4500
-
4000~_ 3300
&
3000
-
2100
-
NE
i ._
H
I
o
::
6
5
9 -w
7 ,”
E
1500
-
so0
-
>
DOPEG
300
-
* 0
I
I
1
I
I
I
1
2.5
5
6
7
a
DURATION
OF
HYDROLYSIS
Fig. 1. Counts per minute produced by the deconjugated testing point represents the average of 7 experiments.
I
1
10
(mid
compound
at different duration of boiling. Each
202
Results Optimal conditions for deconjugation Acidification of plasma did not produce any consistent deconjugation of the compounds. On the other hand, heating of the acidified specimen at 100°C produced complete deconjugation of all compounds. In Fig. 1 the effect of boiling of the acidified specimen at different time intervals is shown, The duration of boiling to get maximal cpm yield was slightly different for the different compounds. Thus, for the amines and DOMA the peak of cpm was at 7 min, whereas for DOPEG, at 5 min. However, the longer the duration of boiling the greater the losses of free compounds. In Fig. 2 the percent of compound recovered at different boiling durations is shown. It is apparent that the amines were more resistant to heat than the metabolites. At 7 min of boiling the losses were approximately 5% for DA and E. 15% for NE and 50% for the metabolites. Therefore, since the duration of boiling determined the amount of compound which was deconjugated as well as destroyed, the optimal time for deconjugation (as determined by the ratio of cpm yield by the specimen to the cpm generated by the added compound) was 5 min for DOPEG, 7 min for NE, E and DA and 8 min for DOMA. Reproducibility of deconjugution Table I shows that the coefficients of variation of the recovered added compounds were low and similar for all compounds tested.
endogenous
and
DOMA -_ 1
o’
1
2.5
DURATION
OF
I
I
5
6
HYDROLYSIS
DOPEG
.
7
0
10
(mid
Fig. 2. Percent of the recovery of the added compound at different duration of boiling. E, epinephrine; NE, norepinephrine; DA, dopamine; DOMA, dihydroxymandelic acid; DOPEG, dihydroxyphenylglycol.
203 TABLE
I
Coefficient
of variation
Compound
assayed
of the recovered Coefficient
Free compound
endogenous of variation
and exogenous
compound
in the recovery
of
NE
E
DA
DOMA
DOPEG
7.5
8.4
14
14
4.4
6.0
4.5
9
16
8
5.0
4.0
5
13
4.8
7.1
6.4
6
14
6.6
Total compound (acidic hydrolysis) Compound
added
after hydrolysis Compound
added
before hydrolysis Each value is the average Standard
deviation
of 5 measurements.
The coefficient
of variation
waS derived
by the formula:
x loo
Mean and is expressed in %. NE, norepinephrine; acid; DOPEG, dihydroxyphenylglycol.
E, epinephrine;
DA, dopamine;
DOMA,
dihydroxymandelic
Plasma levels of freeand total compound in different groups of subjects In Table II are included plasma levels of free and total (free + conjugated) ~atecholamines and catecholamine metabolites in subjects with phe~hromocytoma, with primary hypertension, and in healthy laboratory personnel. Specimens were obtained by venipuncture from the subjects after they had been in the recumbent position for 5 min. The presence of pheochromocytoma was confirmed by surgery in all cases. Patient No. 1 had an inoperable malignant pheochromocytoma. All ph~~hrom~ytoma patients were treated with phenoxybenzamine (an alpha-adrenergic receptor blocker) alone or with alpha-methyltyrosine in~bitor (for patient No. 1). Secondary causes of hypertension were excluded in the patients with primary hypertension by appropriate testing including plasma catecholamine determinations. All patients were off medication for at least 2 weeks at the time of blood collection. The normotensive subjects were healthy laboratory personnel. Normotensive and primary hypertensive groups had similar levels of free and total catecholamines and metabolites. In addition, the percentage of the free to total compound was similar in both groups. In the pheochromocytoma group, free NE ( p -C0.001) and DOPEG ( p -C0.001) were significantly higher than in the other two groups. Two of the pheochromocytoma patients (Nos. 2 and 4) had total NE overlapping the corresponding values of the other two groups. The percentage of the free compound in the phe~hrom~ytoma group was significantly higher than the corresponding value in the other groups for all compounds, but DOPEG. Fig. 3 includes free and total levels of the five compounds in the blood of five normotensive subjects, after (1) 10 min of supine rest, (2) 3 min of isometric handgrip (50% of their maximal capacity) preceded by 12 min of standing, and (3)
II
Results are expressed
% Free
Primary hypertensives % Free
in q/ml,
5
6
3M 2F
4M 2F
F M F M 2M 2F
Sex
1.4 11.2
2.0 51.8
25.8 4.9 19.0 2.8 13.1 k11.1
M, male.
0.23 kO.12 23 + 16
19
0.25 +0.10 20
3.7 1.9 4.2 1.7 2.9 51.2 34 +20
+8
0.04 f 0.02 20
rt8
0.04 io.01 17
13
0.04 0.84 0.09 1.7 0.7 kO.8 33
E free
total
free
metabohtes
NE
and catecholamine
mean rt SD; F, female;
+7
43
+8
50
45 60 24 30 40 f16
1 2 3 4
Pheocbromocytoma
Mean &SD 4; Free
Age (years)
No.
cat~hola~nes
Subjects
Free and total levels of plasma
TABLE
0.33 10.3
0.5 io.2
0.13 2.3 0.26 5.5 2.0 k2.5
total
in humans DA
zk1
0.08 i 0.05 0.8
0.11 kO.14 0.9 kO.5
rtl
0.54 0.1 0.08 0.07 0.2 zt 0.2 2.0
free
8.6 + 10.0
14.0 z?z18.0
19.5 4.9 11.7 2.0 9.5 it: 7.8
total
-t7
2.0 +1.1 20
+S
1.5 + 0.8 13
5.9 4.4 9.8 2.1 5.6 _t 3.2 45 $-13
free
DOMA
9.9 13.4
9.8 k2.9
21 9.9 17.0 4.2 13.0 i 7.5
total
0.81 kO.20 42 +12
0.84 +0.27 40 &13
2.4 4.3 3.6 3.4 kO.8 45 130
3.6
DOPEG ~free
-.
1.x i_ 0.6
2.0 rt 0.6
20 4.9 15.3 4.3 11.1 * 7.1
total
P
N
205
7000 Total
6000
4000
3000
2000
1000
0
Pra - exercise rest
for
IO
Exarci~e
for
Fig. 3. Effect of activation of the sympathetic compound in the plasma of 5 subjects.
15
min
Post rel)t
min
nervous
-exercise far
30
system of short duration
min
on free and conjugated
30 min of post-exercise supine rest. Basal levels of free and total compounds were: DOMA 1540 + 680 and 7420 f 1490, respectively, DOPEG 980 + 160 and 2120 f 260, NE 265 k 40 and 1485 It 502, E 45 f 22 and 310 f 82 and DA 42 k 28 and 3886 f 650, respectively. During exercise only the change in free NE (+ 355 + 110) and DOPEG ( + 240 + 80) attained significance (p c 0.05, for both). Fig. 4 includes levels of free and total NE in the blood of three normotensive subjects undergoing NE infusion. The protocol of the whole study was approved by the Institutional Research Committee and all participants had signed an informed consent form. Norepinephrine was administered intravenously with an infusion over a period of 15 min. Blood was collected at the pump at 0.05 pg.kgg’-min-’ end of a 30-min period in the recumbent position and several times during and after NE infusion. There was an immediate and significant change in free NE during or after NE administration, as compared to the preceding period, but the conjugated NE did not change. Levels of free and total NE were 260 f 60 and 1140 f 220 before infusion, 1120 + 280 and 2040 f 168 at the end of infusion and 310 f 25 and 1112 i 386 at 30 min after infusion. Table III includes the values of free,and conjugated NE and E under different
206 TABLE
III
Effect of room temperature blood specimens
for 7 days on free and conjugated
noreplnephrine
and epmephrinc
Specimen storage conditions
Norepinephrine free
conjugated
free
conjugated
Kept in freezer at -70°C
304 &48
559 *97
50 +15
196 & 27
Kept in room at +18”C
44 * 14
520 F84
11 +6
176 & 10
Amount
85% +17
lost
Results
Epinephrine
8%
are expressed in pg/ml,
78% +25
*9
i 0.001
p value
m six
ns
11% *12
< 0.01
ns
mean k SEM; ns, not significant.
2000 Total
z Gl a
Free
NE
NE
1400 SE
Y
l-
< z :: ;:
1000
400 200 0
3’ Pre-
6’ NE
9’
12’
15’
’
.d
3’
6’
Q’
15’
30’
Post - infusion
infusion
infusion
TIME
IN
MINUTES
Fig. 4. Plasma levels of free and conjugated norepinephrine, before, during, and after iv. administration of norepinephrine. Norepinephrine was infused in 3 subjects at 0.05 pg.kg-‘.min-’ for a period of 15 min.
201
specimen storage conditions. From six subjects, after sitting for 5 min, blood was withdrawn (as described under ‘Methods’) and each plasma was diquoted into two parts. One part was stored at -7O”C, whereas the other part was kept in room temperature for a period of 7 days, at the end of which free and conjugated amine was simultaneously determined in both parts. There was a substantial loss of the free and not of the conjugated amine in the specimen kept in room temperature. There was no correlation between free and conjugated part for any of the five compounds measured in the primary hypertensives or normotensives, or in both groups together. However, there was a good correlation between these two parts in the pheochromocytoma group. Discussion
Acidic hydrolysis at 100°C is a rapid and effective procedure for deconjugation of plasma catecholamines. It produces complete and reproducible deconjugation of all catecholamines at 7 min. The coefficient of variation of the measurements with the applied radioenzymatic assay was minimal (Table I). The addition of the step of hydrolysis was not associated with a further decrease in the reproducibility. The point of complete deconjugation and the beginning of free amine destruction can be precisely defined. The amount of the free compound lost during hydrolysis was minimal, ranging from 5% for DA and E to 15% for NE (Fig. 2). Both enzymatic [8] and acidic hydrolysis [4,5] have been used for deconjugation of plasma ~at~hola~nes. The simplest procedure is the enzymatic cleavage of sulfate conjugates by the commercially available enzyme arylsulfatase. However, glucuronide conjugates will escape detection, unless both enzymes sulfatase and glucuronase are used. Nevertheless, the addition of sulfatase to the incubation mixture may produce a slight increase in the background [8], whereas results with both enzymes are not available. Since there is a substantial increase in the extraction of all catecholamines (ranging from 41% to 53%) when measurements are made in the supernatant of acidified plasma (used in the present procedure) as against in plain plasma, the sensitivity of our procedure is substantially greater than the enzymatic approach. The acidic hydrolysis by lyophylization described by Buu and Kuchel[4] is a long procedure resulting in a 45% loss of NE and E and inconsistent deconjugation. Our method compares favorably to the recently published acidic hydrolysis of Nagel and Schumann (51 and Demassieux et al 1221.These investigators achieved consistent deconjugation of all catecholamines by heating the acidified sample at 95°C for 20-40 min. The ratio of free to total compound obtained with our procedure in human plasma (Table II) is comparable to that measured with either the enzymatic [S] or the acidic hydrolysis f5]. Conjugates of catechola~nes and their metabolites have been demonstrated in human plasma and urine by several investigators 19-111. We have previously used acidic hydrolysis for deconjugation of plasma normetanephrine, an 0-methylated metabolite of NE [2]. Reports on deconjugation of the deaminated metabolites DOMA and DOPEG are not available in the literature. In the present study acidic
208
hydrolysis produced complete and consistent deconjugation of plasma DOMA and DOPEG. However, these metabolites are less resistant to heat than parent amines (Fig. 2), resulting in a substantial loss of the free compound at 7 min of hydrolysis. The measurement of plasma NE has been advocated as the best testing for detection of pheochromocytoma in patients with hypertension [12]. In our patients with pheochromocytoma free NE was substantially elevated whereas total NE was less than 2 SD above the mean of the primary hypertensives in two of them (Table II). This finding, which is consistent with previous reports [I3], suggests that free rather than total NE is a better measurement for the detection of pheochromocytoma. By the activity of the conjugating enzyme sulfotransferase and glucuronyltransferase catecholamine sulfate and glu~uronide conjugates are formed from 3’-phosphoadenosine-S’-phosphosulfate [14] and uridine diphosphate glucuronide [IS], respectively. It has been suggested that conjugation to sulfate (predominant in man [16]), or glucuronic acid is one of the pathways of catecholamine metabolism [17], an enzymatic process which can be saturated in the presence of larger amounts of substrate /18]. Hart et al [19] have shown that sulfo-conjugation can be inhibited by large quantities of catecholamines. However, in two of our patients with pheochromocytoma (Nos. 1 and 3) the large quantities of free NE were associated with huge elevations in NE conjugates, suggesting that the saturation of the conjugating process occurs with large amounts of free amine. Our NE infusion studies for 15 min (Fig. 4) did not demonstrate any measurable change in the conjugated NE, suggesting that longer or greater elevations of free NE are needed for a change in the conjugate. Similar results were obtained during exercise and elevations of endogenous catecholamines (Fig. 3). However, the conjugated catecholamines are more stable than the free compounds. Specimens left at room temperature for 1 week suffered a significant loss in the free NE and E, but the conjugates did not change (Table III). Methods for accurate measurement of conjugated catecholamines are available, but Iittle is known about their physiological role. Since both catecholamines and their metabolites exist in both free and conjugated forms, the conjugates have been mainly regarded as inactivated catecholamine metabolites bound for excretion [20]. In our studies there was no correlation between free and conjugated form, a finding that could suggest that the activity of the conjugating enzymes is not determined by the available amount of free amine. Therefore, whereas free ~atecholamines have been shown to change with the activity of the sympathetic system f21], the conjugates may not reflect the amount of catecholamine released from the sympathoadrenal system. References
1 Paler JD, Johnson GA. Simultaneous
single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci 1977; 21: 625-636. 2 Vlachakis ND, Niarchos A. Plasma normetanephrine measurements for detection of pheochromocytoma in patients with hypertension. Clin Chim Acta 1979; 99: 283-288.
209
3 Unger
TH,
Buu NT, Kuchel
0. Renal
handIing
of free and conjugated
surgical stress in the dog. Am J Physiol 1978; 235: FS42-547. 4 Buu NT, Kuchel 0. A new method for the hydrolysis of conjugated 5 6
7 8 9 10 11 12 13
cat~holamines
catecholamines.
folfowing
J Lab Clin Med
1977; 90: 680-685. Nagel M, Schumann HJ. A sensitive method for determination of conjugated catecholamines in blood plasma. J Clin Chem Clin Biochem 1980; 18: 431-432. Vlachakis ND, Alexander N, Velasquez M, Maronde RF. A radioenzymatic microassay for simultaneous m~surement of cateeholamines and their deaminated metabolites. B&hem Med 1979; 22: 323-331. Snedecor GW, Cochran WG. In: Ames G, ed. Statistical methods. Iowa: The Iowa State University Press, 1967. Johnson GA, Baker CA, Smith RT. Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma. Life Sci 1980; 26: 1591-1598. Haggendal J. The presence of conjugated adrenaline and noradrenaline in human blood plasma. Acta Physiol Stand 1963; 59: 255-260. Imai K, Sugiura M, Tamura Z. The presence of conjugated dopamine in normal human plasma. Chem Pharm Bull 1970; 18: 2134. Goodal MC, Alton H. Metabolism of 3,4-dihydrophenylalamine@-dopa) in human subjects. Biochem Pharmacol 1972; 21: 2401-2408. Bravo EL, Tarazi RC, Gifford RW, Stewart BH. Circulating and urinary catecholamines in pheochromocytoma. Diagnostic and pathophysioIo~c implications. N Engl J Med 1979, 310: 682-686. Kuchel 0, Buu NT, Fontaine A et al. Free and conjugated plasma catecholamines in hypertensive patients with and without pheochromocytoma. Hypertension 1980; 2: 177-186.
14 Goldberg III, Delbrueck A. Transfer of sulfate from 3-phosphoadenosine-5’-phosphosulfate to lipids, mucopolysaccharides and aminoalkyl phenols. Fed Proc 1959; 18: 235. 15 Isselbacher K, Axelrod J. Enzymatic formation of corticosteroid glucuronides. J Am Chem Sot 1955; 77: 1070-1071. 16 Jenner WN, Rose FA. Dopamine 3-O-sulfate, an end product of L-dopa metabolism in Parkinson patients. Nature 1974; 252: 237-238. 17 Sharman DF. The catabolism of catecholamines. Br Med Bull 1973; 29: 110-115. 18 Goodal MC, Alton H. Metabolism of 3-hydroxytryptamine (dopamine) in human subjects. Biochem Pharmacol 1968; 17: 90-914. 19 Hart RF, Renskers KJ, Nelson EB, Roth JA. Localization and characterization of phenol sulfotransferase in human platelets. Life Sci 1979; 24: 125-130. 20 Kahane Z, Esser AH, Kline S, Vestegaard P. Estimation of conjugated epinephrine and norepinephrine in urine. J Lab Clin Med 1967; 69: 1042-1050. 21 Reid JL, Kopin IJ. Significance of plasma DbH activity as an index of sympathetic neuronal function. Proc Nat1 Acad Sci USA 1974; 71: 4392-4394. 22 Demassieux S, Corneille L, Lachance S, Carriere S. Determination of free and conjugated catecholamines and L-3,4-dihydroxyphenylalanine in plasma and urine: evidence for a catechol-O-methyltransferase inhibitor in uraemia. Clin Chim Acta 1981; 115: 377-391.
Clinica Chimica Acta, 137 (1984) 199-209 Elsevier
CCA 02771
Plasma levels of free and total catecholamines and two deaminated metabolites in man - rapid deconjugation by heat in acid Nicolas D. Vlachakis
*, Ella Kogosov, Seiichi Yoneda, and Robert F, Maronde
Section of C&nieal Pharmacology, Department
Natalie Alexander
of Medicine, Universify of Southern Cali@nia, Los Angeles, CA (USA)
(Received
June 24th; revision October
7th. 1983)
Key words: Catecho~~mine; Catecholamine metabolite; Deconjugation
Summary
We have described a procedure for deconjugation of plasma catecholamines, norepinephrine (NE), epineph~ne (E) and dopamine (DA) and two catecholamine metabolites 3,4-dihydroxymandelic acid (DOMA) and 3,4-dihydroxyphenylglycol (DOPEG). Heat at 100°C of the acidified specimen, pH 0.8, produced complete deconjugation of catecholamines in 7 minutes and of metabolites in 5-7 minutes. Subsequently all five products were simultaneously measured with a radioenzymatic assay. However, hydrolysis for 7 minutes produced approximately a loss of 5% in DA and E, 15% in NE and 50% in the metabolites. The percent of free compound in the plasma of 11 normotensive and healthy subjects was 23 + 16 for NE, 20 + 8 E, 0.8 f 1 DA, 20 f 7 DOMA and 42 & 12 for DOPEG. Similar results were obtained in a random specimen of six patients with primary hypertension. In a group of four patients with pheochromocytoma free levels of NE, DOPEG and DOMA were sig~fi~ntly greater than in the other two groups, whereas conjugates were not. The intravenous administration of NE or the activation of sympathetic nervous system by standing combined with exergise for 15 minutes did not produce a change in the levels of plasma conjugates. These findings suggest that short changes in plasma catecholamines are better reflected in the free than the conjugated part.
* Correspondence to Nicolas D. Vlachakis, MD, Department California, 2025 Zonal Avenue, Los Angeles, CA 90033, USA. ~-8981/84/$03.~
0 1984 Eisevier Science Publishers
B.V.
of Medicine,
University
of Southern
200
Introduction The advent of radioenzymatic assays made possible the accurate and specific measurement of catecholamines and catecholamine metabolites in small quantities of specimens [1,2]. The assays are based on the methylation of these compounds by specific enzymes and in the presence of methyl donor S-adenosyl-L-[’ Hlmethionine. However, since the enzymes do not attack the conjugated part of the compound [3], this part (which is the largest) escapes detection [4]. The deconjugation by acidic lyophylization of Buu and Kuchel [4] has been associated with inconsistent results. Nagel and Schiimann [5] have recently achieved consistent deconjugation of plasma catecholamines by acidic hydrolysis at 95°C for 40 min. In a previous study we have successfully deconjugated plasma normetanephrine by acidic hydrolysis at 100°C for 20 min [2]. In the present report we describe a similar procedure for complete and consistent deconjugation of three catecholamines and two major deaminated catecholamine metabolites. Subsequently both the free and conjugated part of these compounds were simultaneously measured with a radioenzymatic assay [6]. Materials and methods All materials used in this assay as well as the isotope S-adenosyl-L-[ were described elsewhere [6].
3Hlmethionine
Plasma specimen collection One ml of blood is drawn in a syringe and transferred into a small glass tube containing reduced glutathione and ethylene-bis(oxyethylene-nitrilo) tetraacetic acid (EGTA) as a preservative [2]. After centrifugation at 600 X g for 10 min in a cold centrifuge, plasma is separated, transferred into a plastic tube and stored at - 70°C until assayed (within 1-2 weeks after collection). Assay procedure On the day of assay the specimens were gradually thawed and placed in ice. Five hundred ~1 of plasma was transferred into a 75 X 13 mm glass disposable tube and its pH was decreased to 0.8 by adding 7.5% of 5 mol/l perchloric acid (v/v) for protein precipitation. Subsequently the specimen was centrifuged and a part of the supernatant (150 ~1) was transferred to a 3-ml Pyrex conical tube for deconjugation by heat, the remainder was used for determination of the free compound. The Pyrex conical tube was tightly covered with a glass stopper and placed into boiling water. Specimens were boiled different times in ord?r to estimate the duration of boiling for optimal deconjugation. Immediately following boiling tubes were placed in ice and the pH was raised to 8 by adding 15% of 5 mol/l Tris-base (v/v). A similar amount of Tris was added to their homologous unboiled supernatant. Subsequently 50 ~1 of the supernatant from each paired tube were transferred into a 13 X 100 mm disposable tube (Kimble, Owen, IL, USA) and to that was added: 5 ~1 of 3H-SAME (2.5 PC;), 10 ~1 of 0.2 mol/l tris buffer containing 0.1 mmol/l benzyloxyamine, pH 8.1, and 10 ~1 of catechol-O-methyl transferase. Internal standards, consisting of 500
201
pg of each compound, were added to another set of tubes together with the specimen and the incubation mixture. Blanks were prepared by replacing the specimen with 50 ~1 of Tris-perchloric acid buffer, pH 8.0. All specimens were run in duplicate. In each assay a known amount of all five compounds was run with and without boiling for estimation of the losses of the free compound by the deconjugation procedure. The incubation tubes were placed in a shaking water bath at 37°C and incubated for 60 min. The subsequent steps of the assay have been described in detail elsewhere [6]. With this assay plasma levels of norepinephrine (NE), epinephrine (E), dopamine (DA), and the metabolites 3,4_dihydroxymandelic acid (DOMA) and 3,4-dihydroxyphenylglycol (DOPEG) were measured simultaneously. The total compound represents the sum of conjugated part (liberated by hydrolysis) and free part in each specimen. Values were adjusted for losses. The conjugated part was calculated by subtraction of the free from the total compound. For statistical analysis the analysis of variance was used and if significant we used the Student’s r test for comparison between groups and the paired t test for the change in the same group. The coefficient of correlation was determined when indicated [7].
5500
-
5000
-
4500
-
4000~_ 3300
&
3000
-
2100
-
NE
i ._
H
I
o
::
6
5
9 -w
7 ,”
E
1500
-
so0
-
>
DOPEG
300
-
* 0
I
I
1
I
I
I
1
2.5
5
6
7
a
DURATION
OF
HYDROLYSIS
Fig. 1. Counts per minute produced by the deconjugated testing point represents the average of 7 experiments.
I
1
10
(mid
compound
at different duration of boiling. Each
202
Results Optimal conditions for deconjugation Acidification of plasma did not produce any consistent deconjugation of the compounds. On the other hand, heating of the acidified specimen at 100°C produced complete deconjugation of all compounds. In Fig. 1 the effect of boiling of the acidified specimen at different time intervals is shown, The duration of boiling to get maximal cpm yield was slightly different for the different compounds. Thus, for the amines and DOMA the peak of cpm was at 7 min, whereas for DOPEG, at 5 min. However, the longer the duration of boiling the greater the losses of free compounds. In Fig. 2 the percent of compound recovered at different boiling durations is shown. It is apparent that the amines were more resistant to heat than the metabolites. At 7 min of boiling the losses were approximately 5% for DA and E. 15% for NE and 50% for the metabolites. Therefore, since the duration of boiling determined the amount of compound which was deconjugated as well as destroyed, the optimal time for deconjugation (as determined by the ratio of cpm yield by the specimen to the cpm generated by the added compound) was 5 min for DOPEG, 7 min for NE, E and DA and 8 min for DOMA. Reproducibility of deconjugution Table I shows that the coefficients of variation of the recovered added compounds were low and similar for all compounds tested.
endogenous
and
DOMA -_ 1
o’
1
2.5
DURATION
OF
I
I
5
6
HYDROLYSIS
DOPEG
.
7
0
10
(mid
Fig. 2. Percent of the recovery of the added compound at different duration of boiling. E, epinephrine; NE, norepinephrine; DA, dopamine; DOMA, dihydroxymandelic acid; DOPEG, dihydroxyphenylglycol.
203 TABLE
I
Coefficient
of variation
Compound
assayed
of the recovered Coefficient
Free compound
endogenous of variation
and exogenous
compound
in the recovery
of
NE
E
DA
DOMA
DOPEG
7.5
8.4
14
14
4.4
6.0
4.5
9
16
8
5.0
4.0
5
13
4.8
7.1
6.4
6
14
6.6
Total compound (acidic hydrolysis) Compound
added
after hydrolysis Compound
added
before hydrolysis Each value is the average Standard
deviation
of 5 measurements.
The coefficient
of variation
waS derived
by the formula:
x loo
Mean and is expressed in %. NE, norepinephrine; acid; DOPEG, dihydroxyphenylglycol.
E, epinephrine;
DA, dopamine;
DOMA,
dihydroxymandelic
Plasma levels of freeand total compound in different groups of subjects In Table II are included plasma levels of free and total (free + conjugated) ~atecholamines and catecholamine metabolites in subjects with phe~hromocytoma, with primary hypertension, and in healthy laboratory personnel. Specimens were obtained by venipuncture from the subjects after they had been in the recumbent position for 5 min. The presence of pheochromocytoma was confirmed by surgery in all cases. Patient No. 1 had an inoperable malignant pheochromocytoma. All ph~~hrom~ytoma patients were treated with phenoxybenzamine (an alpha-adrenergic receptor blocker) alone or with alpha-methyltyrosine in~bitor (for patient No. 1). Secondary causes of hypertension were excluded in the patients with primary hypertension by appropriate testing including plasma catecholamine determinations. All patients were off medication for at least 2 weeks at the time of blood collection. The normotensive subjects were healthy laboratory personnel. Normotensive and primary hypertensive groups had similar levels of free and total catecholamines and metabolites. In addition, the percentage of the free to total compound was similar in both groups. In the pheochromocytoma group, free NE ( p -C0.001) and DOPEG ( p -C0.001) were significantly higher than in the other two groups. Two of the pheochromocytoma patients (Nos. 2 and 4) had total NE overlapping the corresponding values of the other two groups. The percentage of the free compound in the phe~hrom~ytoma group was significantly higher than the corresponding value in the other groups for all compounds, but DOPEG. Fig. 3 includes free and total levels of the five compounds in the blood of five normotensive subjects, after (1) 10 min of supine rest, (2) 3 min of isometric handgrip (50% of their maximal capacity) preceded by 12 min of standing, and (3)
II
Results are expressed
% Free
Primary hypertensives % Free
in q/ml,
5
6
3M 2F
4M 2F
F M F M 2M 2F
Sex
1.4 11.2
2.0 51.8
25.8 4.9 19.0 2.8 13.1 k11.1
M, male.
0.23 kO.12 23 + 16
19
0.25 +0.10 20
3.7 1.9 4.2 1.7 2.9 51.2 34 +20
+8
0.04 f 0.02 20
rt8
0.04 io.01 17
13
0.04 0.84 0.09 1.7 0.7 kO.8 33
E free
total
free
metabohtes
NE
and catecholamine
mean rt SD; F, female;
+7
43
+8
50
45 60 24 30 40 f16
1 2 3 4
Pheocbromocytoma
Mean &SD 4; Free
Age (years)
No.
cat~hola~nes
Subjects
Free and total levels of plasma
TABLE
0.33 10.3
0.5 io.2
0.13 2.3 0.26 5.5 2.0 k2.5
total
in humans DA
zk1
0.08 i 0.05 0.8
0.11 kO.14 0.9 kO.5
rtl
0.54 0.1 0.08 0.07 0.2 zt 0.2 2.0
free
8.6 + 10.0
14.0 z?z18.0
19.5 4.9 11.7 2.0 9.5 it: 7.8
total
-t7
2.0 +1.1 20
+S
1.5 + 0.8 13
5.9 4.4 9.8 2.1 5.6 _t 3.2 45 $-13
free
DOMA
9.9 13.4
9.8 k2.9
21 9.9 17.0 4.2 13.0 i 7.5
total
0.81 kO.20 42 +12
0.84 +0.27 40 &13
2.4 4.3 3.6 3.4 kO.8 45 130
3.6
DOPEG ~free
-.
1.x i_ 0.6
2.0 rt 0.6
20 4.9 15.3 4.3 11.1 * 7.1
total
P
N
205
7000 Total
6000
4000
3000
2000
1000
0
Pra - exercise rest
for
IO
Exarci~e
for
Fig. 3. Effect of activation of the sympathetic compound in the plasma of 5 subjects.
15
min
Post rel)t
min
nervous
-exercise far
30
system of short duration
min
on free and conjugated
30 min of post-exercise supine rest. Basal levels of free and total compounds were: DOMA 1540 + 680 and 7420 f 1490, respectively, DOPEG 980 + 160 and 2120 f 260, NE 265 k 40 and 1485 It 502, E 45 f 22 and 310 f 82 and DA 42 k 28 and 3886 f 650, respectively. During exercise only the change in free NE (+ 355 + 110) and DOPEG ( + 240 + 80) attained significance (p c 0.05, for both). Fig. 4 includes levels of free and total NE in the blood of three normotensive subjects undergoing NE infusion. The protocol of the whole study was approved by the Institutional Research Committee and all participants had signed an informed consent form. Norepinephrine was administered intravenously with an infusion over a period of 15 min. Blood was collected at the pump at 0.05 pg.kgg’-min-’ end of a 30-min period in the recumbent position and several times during and after NE infusion. There was an immediate and significant change in free NE during or after NE administration, as compared to the preceding period, but the conjugated NE did not change. Levels of free and total NE were 260 f 60 and 1140 f 220 before infusion, 1120 + 280 and 2040 f 168 at the end of infusion and 310 f 25 and 1112 i 386 at 30 min after infusion. Table III includes the values of free,and conjugated NE and E under different
206 TABLE
III
Effect of room temperature blood specimens
for 7 days on free and conjugated
noreplnephrine
and epmephrinc
Specimen storage conditions
Norepinephrine free
conjugated
free
conjugated
Kept in freezer at -70°C
304 &48
559 *97
50 +15
196 & 27
Kept in room at +18”C
44 * 14
520 F84
11 +6
176 & 10
Amount
85% +17
lost
Results
Epinephrine
8%
are expressed in pg/ml,
78% +25
*9
i 0.001
p value
m six
ns
11% *12
< 0.01
ns
mean k SEM; ns, not significant.
2000 Total
z Gl a
Free
NE
NE
1400 SE
Y
l-
< z :: ;:
1000
400 200 0
3’ Pre-
6’ NE
9’
12’
15’
’
.d
3’
6’
Q’
15’
30’
Post - infusion
infusion
infusion
TIME
IN
MINUTES
Fig. 4. Plasma levels of free and conjugated norepinephrine, before, during, and after iv. administration of norepinephrine. Norepinephrine was infused in 3 subjects at 0.05 pg.kg-‘.min-’ for a period of 15 min.
201
specimen storage conditions. From six subjects, after sitting for 5 min, blood was withdrawn (as described under ‘Methods’) and each plasma was diquoted into two parts. One part was stored at -7O”C, whereas the other part was kept in room temperature for a period of 7 days, at the end of which free and conjugated amine was simultaneously determined in both parts. There was a substantial loss of the free and not of the conjugated amine in the specimen kept in room temperature. There was no correlation between free and conjugated part for any of the five compounds measured in the primary hypertensives or normotensives, or in both groups together. However, there was a good correlation between these two parts in the pheochromocytoma group. Discussion
Acidic hydrolysis at 100°C is a rapid and effective procedure for deconjugation of plasma catecholamines. It produces complete and reproducible deconjugation of all catecholamines at 7 min. The coefficient of variation of the measurements with the applied radioenzymatic assay was minimal (Table I). The addition of the step of hydrolysis was not associated with a further decrease in the reproducibility. The point of complete deconjugation and the beginning of free amine destruction can be precisely defined. The amount of the free compound lost during hydrolysis was minimal, ranging from 5% for DA and E to 15% for NE (Fig. 2). Both enzymatic [8] and acidic hydrolysis [4,5] have been used for deconjugation of plasma ~at~hola~nes. The simplest procedure is the enzymatic cleavage of sulfate conjugates by the commercially available enzyme arylsulfatase. However, glucuronide conjugates will escape detection, unless both enzymes sulfatase and glucuronase are used. Nevertheless, the addition of sulfatase to the incubation mixture may produce a slight increase in the background [8], whereas results with both enzymes are not available. Since there is a substantial increase in the extraction of all catecholamines (ranging from 41% to 53%) when measurements are made in the supernatant of acidified plasma (used in the present procedure) as against in plain plasma, the sensitivity of our procedure is substantially greater than the enzymatic approach. The acidic hydrolysis by lyophylization described by Buu and Kuchel[4] is a long procedure resulting in a 45% loss of NE and E and inconsistent deconjugation. Our method compares favorably to the recently published acidic hydrolysis of Nagel and Schumann (51 and Demassieux et al 1221.These investigators achieved consistent deconjugation of all catecholamines by heating the acidified sample at 95°C for 20-40 min. The ratio of free to total compound obtained with our procedure in human plasma (Table II) is comparable to that measured with either the enzymatic [S] or the acidic hydrolysis f5]. Conjugates of catechola~nes and their metabolites have been demonstrated in human plasma and urine by several investigators 19-111. We have previously used acidic hydrolysis for deconjugation of plasma normetanephrine, an 0-methylated metabolite of NE [2]. Reports on deconjugation of the deaminated metabolites DOMA and DOPEG are not available in the literature. In the present study acidic
208
hydrolysis produced complete and consistent deconjugation of plasma DOMA and DOPEG. However, these metabolites are less resistant to heat than parent amines (Fig. 2), resulting in a substantial loss of the free compound at 7 min of hydrolysis. The measurement of plasma NE has been advocated as the best testing for detection of pheochromocytoma in patients with hypertension [12]. In our patients with pheochromocytoma free NE was substantially elevated whereas total NE was less than 2 SD above the mean of the primary hypertensives in two of them (Table II). This finding, which is consistent with previous reports [I3], suggests that free rather than total NE is a better measurement for the detection of pheochromocytoma. By the activity of the conjugating enzyme sulfotransferase and glucuronyltransferase catecholamine sulfate and glu~uronide conjugates are formed from 3’-phosphoadenosine-S’-phosphosulfate [14] and uridine diphosphate glucuronide [IS], respectively. It has been suggested that conjugation to sulfate (predominant in man [16]), or glucuronic acid is one of the pathways of catecholamine metabolism [17], an enzymatic process which can be saturated in the presence of larger amounts of substrate /18]. Hart et al [19] have shown that sulfo-conjugation can be inhibited by large quantities of catecholamines. However, in two of our patients with pheochromocytoma (Nos. 1 and 3) the large quantities of free NE were associated with huge elevations in NE conjugates, suggesting that the saturation of the conjugating process occurs with large amounts of free amine. Our NE infusion studies for 15 min (Fig. 4) did not demonstrate any measurable change in the conjugated NE, suggesting that longer or greater elevations of free NE are needed for a change in the conjugate. Similar results were obtained during exercise and elevations of endogenous catecholamines (Fig. 3). However, the conjugated catecholamines are more stable than the free compounds. Specimens left at room temperature for 1 week suffered a significant loss in the free NE and E, but the conjugates did not change (Table III). Methods for accurate measurement of conjugated catecholamines are available, but Iittle is known about their physiological role. Since both catecholamines and their metabolites exist in both free and conjugated forms, the conjugates have been mainly regarded as inactivated catecholamine metabolites bound for excretion [20]. In our studies there was no correlation between free and conjugated form, a finding that could suggest that the activity of the conjugating enzymes is not determined by the available amount of free amine. Therefore, whereas free ~atecholamines have been shown to change with the activity of the sympathetic system f21], the conjugates may not reflect the amount of catecholamine released from the sympathoadrenal system. References
1 Paler JD, Johnson GA. Simultaneous
single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci 1977; 21: 625-636. 2 Vlachakis ND, Niarchos A. Plasma normetanephrine measurements for detection of pheochromocytoma in patients with hypertension. Clin Chim Acta 1979; 99: 283-288.
209
3 Unger
TH,
Buu NT, Kuchel
0. Renal
handIing
of free and conjugated
surgical stress in the dog. Am J Physiol 1978; 235: FS42-547. 4 Buu NT, Kuchel 0. A new method for the hydrolysis of conjugated 5 6
7 8 9 10 11 12 13
cat~holamines
catecholamines.
folfowing
J Lab Clin Med
1977; 90: 680-685. Nagel M, Schumann HJ. A sensitive method for determination of conjugated catecholamines in blood plasma. J Clin Chem Clin Biochem 1980; 18: 431-432. Vlachakis ND, Alexander N, Velasquez M, Maronde RF. A radioenzymatic microassay for simultaneous m~surement of cateeholamines and their deaminated metabolites. B&hem Med 1979; 22: 323-331. Snedecor GW, Cochran WG. In: Ames G, ed. Statistical methods. Iowa: The Iowa State University Press, 1967. Johnson GA, Baker CA, Smith RT. Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma. Life Sci 1980; 26: 1591-1598. Haggendal J. The presence of conjugated adrenaline and noradrenaline in human blood plasma. Acta Physiol Stand 1963; 59: 255-260. Imai K, Sugiura M, Tamura Z. The presence of conjugated dopamine in normal human plasma. Chem Pharm Bull 1970; 18: 2134. Goodal MC, Alton H. Metabolism of 3,4-dihydrophenylalamine@-dopa) in human subjects. Biochem Pharmacol 1972; 21: 2401-2408. Bravo EL, Tarazi RC, Gifford RW, Stewart BH. Circulating and urinary catecholamines in pheochromocytoma. Diagnostic and pathophysioIo~c implications. N Engl J Med 1979, 310: 682-686. Kuchel 0, Buu NT, Fontaine A et al. Free and conjugated plasma catecholamines in hypertensive patients with and without pheochromocytoma. Hypertension 1980; 2: 177-186.
14 Goldberg III, Delbrueck A. Transfer of sulfate from 3-phosphoadenosine-5’-phosphosulfate to lipids, mucopolysaccharides and aminoalkyl phenols. Fed Proc 1959; 18: 235. 15 Isselbacher K, Axelrod J. Enzymatic formation of corticosteroid glucuronides. J Am Chem Sot 1955; 77: 1070-1071. 16 Jenner WN, Rose FA. Dopamine 3-O-sulfate, an end product of L-dopa metabolism in Parkinson patients. Nature 1974; 252: 237-238. 17 Sharman DF. The catabolism of catecholamines. Br Med Bull 1973; 29: 110-115. 18 Goodal MC, Alton H. Metabolism of 3-hydroxytryptamine (dopamine) in human subjects. Biochem Pharmacol 1968; 17: 90-914. 19 Hart RF, Renskers KJ, Nelson EB, Roth JA. Localization and characterization of phenol sulfotransferase in human platelets. Life Sci 1979; 24: 125-130. 20 Kahane Z, Esser AH, Kline S, Vestegaard P. Estimation of conjugated epinephrine and norepinephrine in urine. J Lab Clin Med 1967; 69: 1042-1050. 21 Reid JL, Kopin IJ. Significance of plasma DbH activity as an index of sympathetic neuronal function. Proc Nat1 Acad Sci USA 1974; 71: 4392-4394. 22 Demassieux S, Corneille L, Lachance S, Carriere S. Determination of free and conjugated catecholamines and L-3,4-dihydroxyphenylalanine in plasma and urine: evidence for a catechol-O-methyltransferase inhibitor in uraemia. Clin Chim Acta 1981; 115: 377-391.