CLIN. CHEM.
27/4, 626-628
(1981)
Radioenzymic Measurement of Norepinephrine, Epinephrine, and
Dopamine: Stability, Enzymic Activity, and Sensitivity
Nih Saar, A. W. Bachmann, and R. D. Gordon1
We investigated factors affecting sensitivity and reproducibility of radioenzymic (catechol-O-methyttransferase)
measurement of plasma catecholamines.
There was no
apparent deterioration of catecholamine
during storage
at -20 #{176}C
for 240 days. Accuracy and sensitivity of the
assay depended mostly on the method of transferase
preparation; its preparation by adjusting the pH to 6.8
before fractionation with ammonium sulfate resulted in an
unsuitable enzyme with a relatively high backgroundradioactivity count, particularly for norepinephrine and
dopamine.
Details regarding
stability
of catecholamines
in
samples are unclear. Carruthers
et al. (1) demonstrated
(11 to 106 days) deterioration
of catecholamines
stored
#{176}C.
Sever (2) claimed there is no loss of amines in
during storage for two to three weeks at -20 #{176}C
or six
at -50 #{176}C,
but 50% loss after storage for six months
plasma
rapid
at -20
plasma
months
at -20
OC.
Radioenzymic
assays
plasma norephinephrine,
for simultaneous
epinephrine,
are based on the conversion
determination
and dopamine
of the catecholamines
of
(3-7)
into ra-
diolabeled
O-methylated
derivatives by incubation of plasma
in the presence of catechol-O-methyltransferase
(COMT; EC
2.1.1.6) and radiolabeled methyl donor, [methyl-3H].S-adenosyl-L-methionine.
The methylated
derivatives
are then
extracted
into organic solvents, separated
by thin-layer
chromatography,
and radioactivity
measured in the final
products. Preparation
of COMT according to Axelrod and
Tomchick (8) involves collection of the cytoplasmic
fraction
of rat-liver homogenates by ultracentrifugation,
acid precipitation, fractionation
with various concentrations
of ammonium sulfate, and extensive dialysis.
We used the radioenzymic method of Peuler and Johnson
(7) for simultaneous
measurement
of norepinephrine,
epinephrine,
and dopamine
in our effort (a) to determine the
effect on catecholamines
in plasma of storage at -20 #{176}C
for
as long as 240 days, (b) to compare the activity of different
COMT preparations, and (c) to determine whether K2HPO4,
shown by Anton and Sayre (9) and Hortnagl
eta!. (10) to increase metanephrine
extraction,
would
epinephrine
and dopamine sensitivity.
be useful
to increase
Materials and Methods
We followed the method of Peuler and Johnson (7) unless
stated. Blood was collected in pre-chilled
tubes
containing
1.9 mg of [ethylenebis(oxyethylenenitrilo)]otherwise
tetraacetic acid (EGTA) and 1.2 mg of reduced glutathione
per milliliter of blood. Plasma was separated at 4#{176}C,
divided
into 1-mL aliquots, and stored in polypropylene
containers
at -20 to -25 #{176}C
until 0.1-mL aliquots were assayed. The
COMT
enzyme was prepared according to Axelrod and
Tomchick (8), as modified by Coyle and Henry (11), consisting
of a precipitate from 30 to 55% saturated ammonium sulfate.
Enzyme activity was determined in aliquots of 15 to 75 L,
and the lowest volume of enzyme producing maximal activity
was subsequently
used for catecholamine
determinations.
Other enzyme fractions were prepared according to Axelrod
and Tomchick (8), consisting of the precipitate collected between 30 and 50% saturation with ammonium sulfate fractions, prepared according to the method of Nikodejevic et a!.
(12) in which the pH is adjusted to 6.8 with either 1 mol/L Tris
or phosphate buffers, after precipitation
of proteins by titration to pH 5 with 1 molfL acetic acid.
In routine assays, 45 zg of normetanephrine,
metanephrine,
and methoxytyramine
was added to borate buffer before extraction. The effect of K2HPO4 on extraction was studied by
adding 700 mg to the aqueous phase before addition of toluene-isoamyl
alcohol. In each assay an enzyme blank (water
substituted
for plasma) and an 3H blank (water substituted
for enzyme) were estimated. Significance of differences between means was determined by using Student’s t -test for
unpaired
data.
Results
Stability. No apparent loss in catecholamine
was observed
during storage at -20 #{176}C
for up to 240 days (Table 1).
Enzyme
activity.
We compared activities of four different
COMT preparations
obtained from the same ultracentrifugate
fraction
(Table 2). The enzyme blank of fractions
Table 1. Effect of Storage on Norepinephrine
(NE), Eplnephrine (EPI), and Dopamine (DA) in
Plasma from Two Subjects (A and B)
A
Days after
sampling
1
3
7
10
15
18
22
Address correspondence
to this author at the Department
of
Medicine, Repatriation Hospital, Greenslopes, Brisbane, Queensland
4120, Australia.
Department of Medicine, University of Queensland, Greenslopes,
Brisbane, Queensland 4120, Australia.
Received Sept. 4, 1980; accepted Jan. 9, 1981.
626
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
prepared
150
Mean
SD
CV, %
B
EPI
ng/L
DA
247
52
25
30
53
65
194
234
249
238
46
42
48
48
26
18
27
23
80
113
227
235
45
45
28
231
232
17
7
52
47
3
7
23
24
3
13
NE
EPI
________
Days after
samplIng
ng/L
213
212
217
202
227
204
195
NE
36
45
41
34
39
39
34
34
11
________
208
240
210
38
10
5
4
10
22
Table 2. Noreplnephrlne, Epinephrine, and Dopamine as Determined with Four Different COMT
Preparations
6.8
(NH4)24-satn.
30-50%
fraction
5.0
30-55%
30-50%
30-55%
Norep!nephrine
Blank
Plasma
Plasma + std.
Concn. in plasma, ng/L
Epinephrlne
62 ± 3
± 9
280
1935 ± 74
275
Blank
31 ± 2
Plasma
Plasma
80 ± 1
+ std.
Concn. in plasma, ng/L
Dopamine
Blank
Plasma
Plasma + std.
Concn. in plasma, ng/L
293 ± 33
42 ± 1
627 ± 21
3562 ± 193
238
461 ± 15
4051 ± 245
39 ± 3
489 ± 41
4504 ± 226
244
234
32 ± 3
120 ± 2
3634 ± 261
52
2678 ± 187
39
46 ± 3
52 ± 3
1444 ± 40
-
27 ± 1
24 ± I
122 ± 6
4670 ± 218
108 ± 5
3914 ± 252
45
111±5
31±2
120±2
1988±57
60±3
2260±34
23
-
45
32 ± 1
72 ± 1
2598 ± 85
28
COMT was obtained by eIther 30 to 50% or 30 to 55% ammonlum sulfate fractionation at pH 6.8 or 5.0. Standard (std.) contaIning 209 pg NE, 208 pg EPI, and
175 pg DA was added to plasma. Results expressed In cpm. mean ± SD of triplicates.
at pH 5.0 had lower background
radioactivity
than fractions
prepared
at pH 6.8, and it was similar to 3H-blank, which was
in the range of 30 to 43 cpm for norepinephrine,
24 to 29 cpm
for epinephrine,
and 20 to 27 cpm for dopamine. The high
enzyme blanks for norepinephrine
and dopamine of preparations adjusted to pH 6.8 prevented their accurate determination. Enzyme activity expressed as counts per minute per
picogram (cpm/pg) was derived from the difference between
untreated
plasma and plasma to which known amounts of
standard catecholamines
were added. Enzyme activity was
higher in preparations
obtained on fractionation
with 30 to
55% saturated ammonium sulfate fractionation than with 30
to 50% saturated.
Sensitivity.
(a) Carrier: Increasing the amounts of nonlabeled O-methylated
catecholamine
carriers added to the
borate buffer before the extraction of methylated derivatives
into toluene-isoamyl
alcohol mixture markedly increased the
counts per minute (cpm) for norepinephrine
and epinephrine;
the counts for dopamine were less affected (Table 3). Addition
of non-labeled
O-methylated
catecholamines
to give final
individual concentrations
of 45 zg per incubation volume was
optimal for sensitivity (cpm) and thin-layer separation. Increasing the carriers to 66 tg only slightly increased the cpm,
and resolution on thin-layer chromatographs
was less clear
than with 45-tg carriers.
(b) pH and K2HPO4: The results in Table 4 summarize the
effects of borate-buffer
pH and saturation of the aqueous
phase with K2HPO4. In the absence of K2HPO4, the pH of the
borate buffer was critical; extraction at pH 10 was optimal for
determination
of norepinephrine,
pH 10.5 for epinephrine
determination. Dopamine determinations were not dependent
on the pH of extraction within the range of 9.5 to 11.0. Saturating the aqueous phase with K2HPO4 at each pH used increased the sensitivity of the assay for norepinephrine,
but
decreased the sensitivity of the assay for epinephrine
and
dopamine. In the presence of K2HPO4 the extraction was less
dependent on pH.
Table 3. Dependence of Catecholamine Assay on
Amount of Metanephrine Carriers Added
Carrier
EpIn.phrlne
cpm
Nor.pln.phrlne
Mg
16
33
45
66
Dopamln.
5461 (145)
5871 (246)
5395(121)b
2690(68)
590O(132)
2748(51)
6269(201)
6311 (150)
6403(159)
2776(25)
2751 (67)
6421 (54)
Results are mean (and SD) of ttTee determinations on plasma contaIning 233
and 177pg of dopamlne. Various
amos.jits of normetanepitrine, metanepirine and methoxytyiam1ne carrIers ware
added after the incubatIon step.
p <0.05, #{176}p
<0.005 dIfferent from determinations Inwhith 45 ig of carrier
was used.
pg of norepIneptwlne, 213 pg of epinepitrine,
Discussion
Findings
concerning
the stability
of catecholamines
in
plasma samples are equivocal.
Carruthers
et al. (1), using a
fluorometric
of plasma samples declined by 29 to 71% during storage for 11
to 106 days, but decline and duration of storage were not
clearly correlated. In agreement with our findings, Watson et
al. (13), using a radioenzymic method, reported that norepinephrine decreased by <10% during plasma storage at -20
#{176}C
for four weeks. The decrease in apparent catecholamine
in plasma during storage seen by Carruthers et al. may be related to plasma constituents other than catecholamines.
We
saw no apparent deterioration
of catecholamine
in plasma
samples collected in the presence of antioxidant, as assayed
with the specific enzymic method, after storage for 240 days
at -20 #{176}C.
Although many recent papers deal with modifications
of
methods for the measurement
of plasma catecholamines,
certain important methological aspects have not been detailed. In setting up the radioenzymic assay for simultaneous
measurement of norepinephrine,
epinephrine, and dopamine
in small sample volumes, the procedure for COMT preparation is critical. Several investigators have used the method of
Nikodejevic et al. (12) to prepare COMT, in which COMT is
adjusted to pH 6.8 before the ammonium sulfate fractionation
and subsequently
purified by gel filtration. Because the enzyme is so unstable during the final purification steps (14),
most workers omit them. In the present study, radioactivity
in the enzyme blanks prepared at pH 6.8 did not permit ac-
method,
found that the catecholamine
content
CLINICAL CHEMISTRY,
Vol. 27. No. 4, 1981
627
Table 4. Influence on the Catecholamine
Extraction
pH
Assay of Borate Buffer pH and Saturation of the Aqueous
Phase with K2HP04
Noreplns#{216}vlns
wIthout
KHPO
EpinophirIn.
with
K2HPO4
without
K2HPO4
Dopamine
with
K2HPO4
without
K2HPO4
with
K2HPO4
4993(547)
4712 (120)b
4653 (136)b
2557 (113)’
2749 (49)’
3130(32)’
2094 (187)C
1905 (79)b
1827 (13)b
4037 (175)
2818 (1)
cpm
9.5
10.0
10.5
5750 (318)
5872(245)
5143 (209)C
7025 (677)
6311 (209)
5819 (168)
5358 (243)
11.0
2751(84)d
5175(366)b
4480(134)f
5900 (132)
6429 (196)
Allquots of plasma containIng 233 pg of noreplnephrlne, 213 pg of eplnephrine, and 177 pg of dopamlne were assayed in the same run. Results are cpm mean
(and SD) found In ttwee determInatIons.
‘p <0.05, #{176}p
<0.001, compared with corresponding amine determinatIon at same pH wIthout K2HPO4. C p <0.05, 5p <0.001, compared with norepIneptwlne
determination without K2HPO4 at pH 10.0. #{149}
p <0.05, fp <0.002 compared with correspondIng amIne without K2HPO4 at pH 10.5.
curate
determination
of norepinephrine
and dopamine.
high blank radioactivity
counts could
trace substrate bound to some protein
ration (15). The enzyme prepared at pH
and Henry (11), which consists of the
These
have been caused by
in the enzyme prepa5.0 according to Coyle
30 to 55% saturated
ammonium
sulfate precipitable
fraction, was the most suitable
for assay of catecholamine
in small volumes of plasma, because
of its high enzymic activity and low background
radioactivity,
blank values being equivalent
to 1-2 pg of catecholamine.
The sensitivity
of the assay,
in terms
of counts
per minute
per picogram of catecholamine,
depended not only on the
enzymic activity of COMT, but also on the quantities of
metanephrine
carriers added and the pH used in the extraction subsequent
to incubation.
Most workers extract the
methylated
derivatives at pH 10, but Hortnagl et al. (10)
found that saturating the aqueous phase with K2HPO4 and
increasing
the pH to 10.5 tripled the recovery
of normetanephrine and improved the recovery of metanephrine
by 50%.
In the present
study, the use of K2HPO4
sensitivity
of norepinephrine
determinations,
it for epinephrine
and dopamine.
increased
the
but decreased
The discrepancy
between
our results and those of H#{246}rtnagl
et al. (10) may be related to
the different
assay systems used or, alternatively,
to the different amount of K2HPO4 used, because the amount of solid
K2HPO4 in the saturated
aqueous
phase is critical (9). The
need for optimal amounts
of solid K2HPO4 is crucial in this
type of assay, which relies on consistency rather than absolute
recovery.
Furthermore,
the decrease
nephrine and dopamine determinations
its use in this assay system undesirable.
in sensitivity of epiwith K2HPO4 makes
This investigation was supported by grants from the Department
of Veterans’ Affairs and The National Heart Foundation of Australia.
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