DAVID. F. SAYRE. From the Departments of Psychiatry, and. Pharmacology and. Therapeutics,. University of Ftorida,. College of. Medicine,. Gainesville,. Florida.
8883 A
STUDY
OF
THE
FACTORS
AFFECTING
TRIHYDROXYINDOLE
THE
PROCEDURE
ALUMINUM
FOR
THE
OXIDE-
ANALYSIS
OF
CATECHOLAMINES’ AARON the
From
H.
of Psychiatry,
Departments
ANTON
and
AND
Pharmacology
Medicine,
Received The of
plethora
of l)ro(’edures
norepinephrine
and
material emphasizes method still exists von Euler, 1959;
for
and Bone
by
1959). Most of procedures
(1955), involving
catecholamines
onto
aluminum
oxide
terial,
Goodall, procedure
1957), and an organic solvent for tissues also has been
(Shore
and
During utilizing organic laboratory enberg,
Drujan none
factory, the
aluminum
to
the
sulting which
to when
be
the
1951; and
extraction introduced
and
it came
amounts
of catecholamines of the of several
1958) satis-
to estimating in plasma.
factors
oxide-trihydroxyindole
introduction
Olin,
completely
investigation
affecting
A the led re-
procedure
modifications,
in a quantitative and sensitive method is adaptable to urine, plasma and tissue.
Furthermore,
several
1959; Shore
found
particularly
small
detailed
et at.,
our
deficiencies
observations
in current
have
methodology
1 This investigation was supported, P.H.S. research grant H-5251 from Heart Institute, U. S. Public Health
of
some
of the
reported
discrepan-
Each
step
in
this with
experimentation
mainly reagent
The
urine. or
follow
decision a
procedure biological to
particular
use
was maa
par-
procedure
on our objective of obtaining maximum consistent with low blanks and a of quenching. REAGENTS (HIGHEST PURITY). 1. Aluminum oxide (Woelm Neutral Activity Grade 1). The aluminum oxide was prepared in a hood as follows: a. 100 grams of aluminum oxide were added to 500 ml of 2 N HC1 in a 1000-mi beaker, covered with a watch glass and heated at 90#{176} to 100#{176}C for 45 minutes with continuous and rapid stirring (combination magnetic stirrer and hot plate). b. The beaker was removed from the heaterstirrer and the heavier particles of aluminum oxide allowed to settle for 1 minutes. The supernatant fluid (distinctly yellow in color) was discarded along with the finer particles of aluminum oxide. c. The precipitate was washed twice with fresh 250-ml portions of 2 N HC1 at 70#{176}C for 10 minutes, discarding the supernatant with the finer aluminum oxide particles each time. d. In a final acid wash, the aluminum oxide was stirred with 500 ml of 2 N HC1 at 50#{176}C for 10 minutes. e. After decanting the HC1, the precipitate was washed repeatedly (about 20 to 25 times) with fresh 200-mi portions of distilled water until a pH of 3.4 was reached, decanting the finer particles each time. f. Finally, the aluminum oxide was transferred to an evaporating dish, heated at 120#{176}Cfor 1 hour, at 200#{176}C for 2 hours and then stored in an incubator at 37#{176}C to keep the powder dry. Omitting the heat treatment reduced the efficiency of the Al203 by 10%. Before use, each batch of acidwashed aluminum oxide was tested for recovery of
1958).
were
College
was based sensitivity minimum
the l)ast year, several procedures aluminum oxide, ion exchange resins, and solvent extraction were tested in this (Bertler et at., 1958; Cohen and Gold1957; Crout, 1961; de Schaepdryver,
1958a; but
Olin,
for
to
ticular
and of
and Hansson, 1959; Kirschner
of Ftorida,
field.
METHODS.
oxidation of the subsequent eluate to a fluorescent trihydroxyindole derivative, or condensation of the eluate with ethylenediamine. 1\Iore recently ion exchange resins have been used in place of aluminum oxide (Bergstrom Bertler et at., 1958; Du Toit,
University
10, 1962
account
subjected
1950), Euler
and
August
cies in this
chemiintro-
and Weil-Maiherbe the selective adsorption
SAYRE
Therapeutics,
may
biological
Ehrlen (1948), Lund (1949, et at. (1949), Shaw (1938), von
Floding (1952),
the
estimation in
F.
Florida
publication
that the need for a suitable (for reviews on methods see Gaddum, 1959; Strauss and
Wurm, 1960; Weil-1’sialherbe, cal methods are modifications duced Natelson
the
and
Gainesville,
for
epinephrine
DAVID
revealed
which
in part, by the National Service.
standard
high 360
solutions
concentrations)
of
catecholamines
from
urine,
(low
and
was
accepted
and
1962
STUDIES
ON
CATECHOLAMINE
only if the recoveries were at least 95% of those obtained with the previous batch. 2. Perchloric acid (Baker or B & A). a. As a protein precipitant. In an initial purification step all biological material was made 0.4 N using concentrated (11.6 N) perchloric acid. Sulfuric, tnchioroacetic, hydrochloric and nitric acids as well as zinc sulfate were less suitable for this purpose. For example, recovery from urine was about 6% less and blanks were 11% higher with trichloroacetic acid. b. As the eluting agent. A modification of previous methods was the use of 0.05 N perchloric acid as the eluting agent. Various strengths of sulfuric, nitric, oxalic, hydrochloric, tnichloroacetic and acetic acids were less satisfactory. 3. Phosphate buffer 0.5 M pH 7.0. To adjust an aliquot of the perchlonic acid eluate to pH 7.0, several buffers (acetate, citrate, borate and lactate) were tried in various combinations, pH’s and strengths; phosphate buffer (0.5 M, pH 7.0) provided the lowest blanks with the least amount of quenching. 4. Acetic acid solution, 1.6 N. To adjust the perchlonic acid eluate to a low pH (about 2.0) in order to reduce the fluorescence of NE to a minimum without much influence on E fluorescence, a solution of 1.6 N acetic acid (mix 0.5 ml of glacial acetic acid, 17.4 N, with 5.0 ml 1120) was better than the eluate alone or mixed with acetate, glycine-HC1 and KC1-HC1 buffers at various strengths, pH’s and combinations. With this solution the fluorescence of NE was 5 to 6% that obtained with the pH 7.0 buffer. On the other hand, the fluorescence of E was 90 to 95% that at the higher p11. The blanks with the acetic acid solution were about those obtained with the commonly used acetate buffer (pH 3.5, 1.0 M). 5. Potassium ferricyanide (K,Fe (CN)6, 0.25%, Fisher or Baker, freshly prepared each time). Sodium peniodate, sodium nitrite, potassium permanganate, manganese dioxide, sodium hypochlorite, iodine and hydrogen peroxide were unsatisfactory as oxidizing agents for this procedure. 6. Disodium ethylenediaminet etraacet ate (EDTA). Both Baker and Fisher products were satisfactory.
This
reagent
was
routinely
added
to all samples with the aluminum oxide. However, the subsequent addition of EDTA to the elu#{224}te (Crout, 1961; Du Toit, 1959) was not necessary in our method and, if anything, slightly depressed the fluorescence. 7. Sodium metabisulfite (Fisher or Baker). This reducing reagent also was routinely added to all samples because of better recoveries, particularly when the catecholamines were adsorbed onto aluminum oxide from acid solutions in the absence
361
METHODOLOGY
of biological metabisulfite or thiosulfate. 8. Alkaline
material. Results were than with ascorbate,
better with glutathione
Mallinckrodt’s sodium blanks than did Fisher and Baker products; the ascorbic acid from Nutnitional Biochemicals provided less interfering fluorescence than did several other brands. The alkaline ascorbate (prepared immediately before use) was made by completely dissolving 10 mg of hydroxide
ascorbic
ascorbate.
gave
acid
lower
in 0.1
ml
of distilled
water
and
then
adding 5.0 ml of 10 N NaOH. 9. Distilled water. Triple glass-distilled water was used throughout this procedure; resin-treated water (Barnstead Demineralizer, Amberlite MB-3) resulted in blanks which were 2 to 3 times those obtained with the triple glass-distilled water. The second distillation was carried out in the presence of EDTA at about 1 g/liter. No rubber was allowed to come in contact with the water since a highly fluorescent contaminant was leached out. Ground glass joints (no lubricant) were u8ed for all connections in the distillation apparatus (Pyrex), and a minimum quantity of water was stored in Pyrex containers. These precautions were particularly necessary to reduce blanks to a minimum when analyzing plasma. PREPARATION OF SAMPLES. 1. Urine. The specimen was collected in the presence of sodium metabisulfite (about 0.5 mg/ml urine) and immediately frozen until used (stable for several weeks under these conditions). The amount of metabisulfite was not critical since 10 times this amount did not alter the results. To reduce quenching to a minimum, 10.0 ml or less were used, e.g., 5.0 ml of rat urine has been sufficient. The urine samples were treated as follows: a. The aliquot of urine was placed in a 50-ml polyethylene taminant
centrifuge was
leached
tube out
of
(a cellulose
yellowish
con-
nitrate
tubes
and they could not be centrifuged at 30,000 X g) and made 0.4 N with concentrated (11.6 N) perchloric acid. b. The tube was capped, shaken vigorously (held in a horizontal position in a test tube rack in a mechanical shaker)2 for 5 minutes and then centrifuged at 30,000 X g for 10 minutes at 10#{176}C. This initial purification step removed such interfering substances as protein, phosphates, and some chromogenic material. c. The clear supernate was removed, adjusted to 25.0 ml with 0.4 N HCIO4 and transferred to a 50-mi beaker containing 400 mg A1203 and 200 mg EDTA. Under constant and rapid stirring (small 2 All manner.
shaking
procedures
were
done
in
this
362
ANTON
AND
stirring motor and glass stirring rod) the mixture was brought to and maintained at pH 8.6 with NaOH, and then the stirring was continued for 5 minutes (although less time, 2 to 3 minutes, gave good results, 5 minutes was chosen as the standard) ; a pH meter had to be used since indicator solutions or papers gave erratic results. A magnetic stirring bar ground the Al20, to a very fine powder which interfered with the subsequent oxidation
step
d. After quickly
the
settled
(also
noted
stirring and
the
by
had
Crout,
1961).
stopped,
supernatant
the was
A12O3
aspirated
off and discarded. e. The precipitated Al203 was washed into a glass centrifuge tube with 10 ml distilled water; the tube was glass stoppered and shaken for 2 minutes, centrifuged for 1 minute in a clinical centrifuge and the supernate aspirated off. This wash was repeated a minimum of four times; the use of buffers (acetate, phosphate) as a wash was unnecessary, and appeared to interfere with the subsequent analysis. f. After the fourth water wash, the catecholamines were eluted by vigorously shaking the Al203 for 15 minutes with 3.0 ml of 0.05 N perchioric acid. At least 85 to 90% of the catecholamines were eluted, and prior treatment of the aluminum oxide with dehydrating agents, e.g., acetone,
ethanol,
did
not
improve
the
recovery.
SAYRE
Vol.
138
with
0.4 N perchlonic acid and transferred to a beaker containing 400 mg Al203, 200 mg EDTA, 10.0 mg sodium metabisulfite, and then the same procedure followed as for urine (c-h). 3. Blood. Heparinized blood was collected in the presence of sodium metabisulfite (about 0.5 mg/mi), immediately centrifuged in the cold and the plasma frozen until used (stable for several weeks under these conditions). It should be noted that we found metabisuifite at a concentration above 0.2 mg/mi interfered with organic extraction procedures for blood serotonin (Davis, 1959; Waalkes, 1959). In agreement with Vaik and Price (1956) the catecholamines were not associated with any of the formed eiements (WBC, RBC, platelets) in blood. The use of sodium fluoride and sodium thiosuifate as preservatives as suggested by Weil-Malherbe and Bone (1952) markedly interfered with our method. This may be due to the effect of these agents on the oxidation of the catecholamines by ferricyanide (used in our method) whereas coupling with ethylenediamine was used by Weil-Maiherbe and Bone. The plasma was then treated as follows: a. Up to 30 ml of plasma were placed in a polyethylene centrifuge tube, made 0.4 N with concentrated HC1O4, stoppered and vigorously shaken for 5 minutes and then spun at 30,000 X g at 10#{176}C for 10 minutes. b. The clear supernate was adjusted to 25 ml with 0.4 N HC1O4 and transferred to a 50-mi beaker containing 400 mg Ai203, 200 mg EDTA, and continued as described for urine (c-h). 50-mi
The use of columns for the adsorption or elution of the catecholamines was inconvenient, resulted in less consistent results, and caused a 2-fold increase in blanks. Also, sensitivity was reFORMATION OF THE FLUORESCENT TRIHYDROXYduced by 50 since 6 ml of acid were necessary for INDOLE DERIVATIVES. Instrument performance eluting the catecholamines from the column. (sensitivity and wavelengths) was initially g. The supernate containing the catecholchecked with a reference solution of quinine sulamines was transferred to a small polyethylene (0.1 mg/mi in 0.1 N H2SO4), and then with centrifuge tube and spun at 30,000 X g at 10#{176}C fate standard solutions of NE and E; this was repeated for 10 minutes. This removed a faint haze which was unnoticed until the samples were examined at intervals during an analysis. These precautions eliminated instrument-caused variations in in a fluorescent light. The maximum force reached results which at times amounted to 15%. with the usual clinical centrifuge did not remove Only one sample at a time was reacted and its this haze which increased the blank. Relative Fluorescence determined. It was more h. After the high speed centrifuging, the clear convenient to do an analysis of all samples at samples were transferred to small glass test tubes pH 7.0 before changing to the lower pH. For conand 0.2-mi aliquots were taken for analysis. sistent results it was essential to maintain the 2. Tissue (immediately frozen until used). a. time relationships between adding the reagents and Up to 2 g of tissue (depending on its anticipated reading the fluorescence as indicated. Particularly catecholamine content) were homogenized in 10 with low concentrations of catecholamines, we ml of 0.4 N perchloric acid in a glass tissue grinder have observed slight shifts in wavelength peaks submerged in an ice bath. (Use 20 ml of acid with during the time that stability of readings has more than 2 g of tissue.) (Catecholamines could been claimed in other methods. An attempt to not be recovered from formaldehyde-treated specimitigate the problem of fluorescence instability mens.) recently has been made through the use of ethylb. The homogenate was transferred to a 50-mi enediamine in the reaction (von Euler and Lispolyethylene centrifuge tube and spun at hajko, 1961); however, this reagent was not neces30,000 X g at 10#{176}C for 10 minutes. sary in our procedure and increased the blanks. c. The clear supernate was adjusted to 25 ml
1962 With least
STUDIES our procedure, readings 10 minutes, blanks were
in fluorescence,
due
ON
CATECHOLAMINE
were stable for lower and variations
to reading
samples
at
NE
WHERE:
was
added,
mixed
E
at different
thoroughly
DIFFERENTIAL AND
CALCULATION
FOR
The
EPINEPHRINE.
NOREPINEPH-
following
calcula-
tions apply:
1.NE-
rFXB1 2.E=[-j-j-_]_1
Amount
100
rDxNExF 100XC
norepinephrine
of the of
ing
B
=
Net
F
=
H
=
Substituting
4
E
NE in which
the pro-
a
net
fluorescence
pH 7.0. The
- (1.05 X B)]
0.43[A
3. NE
of hg/ml)
of 100 blank was 0.2. Percentage of NE oxidized at pH 2.0 as compared to pH 7.0; this was 5%. That concentration of E in the cuvette (0.71 Lg/ml) which produced a net fluorescence of 100 at pH 2.0. The blank was 0.2. Net fluorescence of E (0.71 i.g/mi) at pH 7.0; this was 105. the constants: at
=
at
(0.43
read-
at pH 7.0. pH 2.0.
concentration
duced
D
(fluorescence
fluorescence
cuvette
the
in
eluate.
blank)
minus
C = That
eluate.
epinephrine
acid
the
in
acid
aliquot of the A = Net fluorescence
=
94.75 -
= 0.0045[A P X NE - X(1.050.71 X B)]Mg
ro.71 X B ioo =
JL (0.0071
100XO.43
X B)
- (0.08 X NE)2g
Since a 0.2-mi aliquot of the acid eluate was generally used, the above amounts were multiplied by 15 to give the weight of catecholamines in the original 3-mi aliquot used with the aluminum oxide. Instrumentation. The Aminco-Bowman Spectrophotofluorometer (SPF) was used to measure fluorescence using slit arrangement *5 and the RCA 1P-28 photomultiplier tube.1 RESULTS.
SPF, our Fluorometer
Instrumentation.
In
addition
to
the
method has been used with the Farrand (Model A) employing a modification
of the filter combination and Goldenberg (1957).
recommended Recently,
by the
Cohen contra-
dictory results from two laboratories (Orlans and Brodie, 1960; Sheppard and Zimmerman, 1960a, b) on the effect of small doses of reserpine on brain catecholamine levels apparently was due to the
Procedure.
-
of
Amount
use in one of the ment of questionable
DXH 100-
=
and
allowed to stand for exactly 1 minute. 3. 0.2 ml of the alkaline ascorbate solution was added, and immediately and thoroughly mixed. 4. Within 15 to 30 seconds 0.5 ml distilled water was added, mixed thoroughly and the sample transferred to the cuvette. It must be emphasized that the 10 N alkali is somewhat viscous and must be completely mixed with the ascorbate solution before use. After adding the alkaline ascorbate, immediate and through mixing is absolutely necessary in order to obtain satisfactory results. 5. In not less than 1 minute nor more than 5 minutes after the addition of the alkaline ascorbate the fluorescence of the sample was read in the SPF. The activation and fluorescence wavelengths (uncorrected) for NE (409-519) were used at pH 7.0 whereas E peaks (422-529) were used at the lower pH. This procedure made the readings for NE more dissimilar and those for E more similar at the two pH’s. The use of zinc sulfate to catalyze the reaction at the lower pH (von Euler and Floding, 1955) was not necessary and, if anything, caused a slight quenching of the fluorescence. BLANKS. The same procedure was foilowed as for the sample except the fernicyanide was omitted. To ensure lowest blanks, glassware must be scrupulously clean. INTERNAL STANDARDS. During the developmental stages of this procedure, internal standards were included to check recoveries and quenching. However, with the present method, this procedure has been unnecessary except in pathologicai urine specimens which are very dark in color or produce a large amount of precipitated material when acidified. RINE
=
aiiquot
intervals, were avoided. 1 . A 0.2-mi aiiquot of the perchlonic acid eluate was placed in a small test tube and 0.1 ml of buffer (phosphate 0.5 M, pH 7.0 or the acetic acid solution) was added and the contents thoroughly mixed. 2. 0.02 ml of a 0.25% solution of potassium ferricyanide
363
METHODOLOGY
laboratories specificity.
Aluminum
Relative Fluorescence is equal to the per cent by the Meter Multiplier multiplier.
oxide,
of a filter which
instruhas
been
(RF), as used herein transmission multiplied setting of the Photo-
364
ANTON
AND
TABLE The
effect
of various
treatments
of Al203
Vol.
SAYRE
1 on recoveries
of norep’inephrine
(NE)*
Brand
Treatment
w
of AlsO,
Alcoa (A-303)
#{176}#{176}
Per cent
1. 2. 3. 4.
Standard Without Standard Standard
Preparations (NE, 0.5 Lg)I Standard Preparation (NE, 0.5 g)J Preparations (NE, 200 g)J Preparations (NE, 200 ug; eluted with
Extracted
*
from
t Woelm
as described
acetic
lOOt 64 96 94
acidll)
under
taken as 100% (actual uncorrected as described under METHODS.
sample prepared
A12O3
0.4 N HC1O4
25 ml
138
and
Baker (0537)
relative
Cat.
No.
Fisher (A-950)
recovery
96 89
Merck (71707)
of NEt
87
92
71
72 62
86 79 86
40 64 85
METHODS.
recovery
was
83%).
§ Al,O3
used as is without acid washing. #{182} NE, 0.5 Lg or 200 ig present in 25 ml of 0.4 N HCIO4. II These samples eluted with 3 ml 0.2 N acetic acid; 3 ml 0.05 more have
critically the ion
evaluated exchange
for resins,
this procedure was selected
than as the
adsorbent. In our hands, the use of ion exchange resins (Bertler et at., 1958) resulted in recoveries which oxide Also, from
were about 10% less than with aluminum as well as causing slightly higher blanks. the necessity for neutralizing the acid eluate the resin was a decided disadvantage be-
cause
of
problem the salt
the
time
of higher concentration
trihydroxyindole
and caused sample.
derivatives
enediamine catecholamines;
condensation several
the validity of the and Goidenberg, 1954; Nagatsu and Yagi tion
required quenching of the
additional
by increasing Oxidation to
rather
than
ethyl-
was used to detect the reports have impugned
condensation method (Cohen 1957; Holzbauer and Vogt, Yagi, 1962; Price et at., 1960;
and Nagatsu, 1959; Zilei etat., 1958). Oxidaat two different pH’s rather than the use of
different
filter
analysis (E); ratio
sets
was
used
of norepinephrine
for
and
the
ticularly afforded
at low concentrations, more reliable results
influenced
by
a
conclusion
similar
(1961). Effect
errors
other
in
methods.
procedure
was
or
minum hands, ployed
PH method it was less fluorescence;
suggested
by
Crout
Alcoa)
Woeim
pharm
Chemicals,
La.) provided
and
brands Neutral P.O.
preparation (Baker, Activity Box
best and most
of atuminum Fisher, Merck, Grade
755,
New
1 (AluOrleans,
consistent recoveries
others.
blanks. Only with Woelm was recovery of high as well as of catecholamines obtained NE with
the
was extracted various alu-
Goldfien et at., 1961 ; Jacobs et at., 1961 ; Price et al. , 1960), resulted in poor and erratic recoveries of catecholamines also show that, the
acid
used
(table depending for
1). The on the could
elution
the recoveries obtained. there was little difference perchioric acid (treatments Merck Al203, acetic acid of NE chioric
about acid.
brands. was an almost
markedly
NE
from than
impossible
with
obtained (62%) the
of Alcoa fine powder to
charcoal, Reagent
adsorbents
It must A12O3 used
a
obtain
withof the
Baker
Celite
product by finding had been figure other
not
although
cellulose, be used
that were
as
activated
very efficient adsorbent, could not be recovered from
be emphasized in this method
it
supernatant.
methyl could
procedure;
perhigh
A12O3 (activated) which made
a clear
kaolin, and
in this
charcoal was catecholamines
influence
the solution, whereas this 98% in the case of the
The sample extremely
Activated
in table 1 of Al203,
In the case of Woelm, between acetic acid and 3 and 4), whereas with increased the recovery
30% over that The poor recovery of
data brand
3) was explained, in part, 89% of the catecholamine
adsorbed was more
Lloyd’s of brand Of several
the
for
oxides as described in METHODS. In our nonacid washed aluminum oxide, as emby some investigators (Drujan et at., 1959;
(treatment that only
par-
used
1). In this experiment 25 ml 0.4 N HC1O4
concentration
Therefore, the since
(table from
differential
the pH method provided a differentiating of E to NE which was more than twice that with
as well as lowest relatively complete low concentrations
epinephrine
the
(NE)
obtained
oxide.
the
N HC1O4
the it.
the amounts of optimum in rela-
1962
STUDIES
tion to the adjusted eluting
fluid.
were
less
ing
and
were
varied
If
if less
too the
volume
of
volume A12O3
more was
was
of
of sample,
CATECHOLAMINE
supernate used,
blanks
used,
high. Although amount
ON
and
recoveries
and
quench-
other investigators
A12O3
according
we found
this
to
procedure
constant
volume
more
(adjusted)
consistent
Effect
of
agreement von
pH,
that
EDTA
and
of NE
aluminum lines).
In the
oxide
berg
(1957),
which
no mention
was
plasma and suspension
aluminum of the to the
made
1949)
at pH
of Cohen
were
we
Lund,
unable
of adjusting
8.6 and
we
absence
1,
Golden1)11 of the
oxide mixture. The pH of a aluminum oxide, prepared
directions
of Cohen
and
of
also
ment
better
Golden-
of recovery an
in
the
was present prevented in
or
metabisulfite,
the
distillation.
recovery
from
distilled
or
In
of NE
at
the
this
experi-
pH
7.0
than
was due to the fact that the at the higher pH was a more
factor
than
the
poorer
adsorption
onto A12O3 at the lower pH. Effect of alkaline ascorbate made with \TQJ The use of 10 N NaOH to make kaline
ascorbate
5 N was
cedure
used it was
by all others. In our oxidation observed that with increasing
was
umes
of
standard
a
by
Resin-
protected
destruction.
8.6 probably destruction
distilladata in
HC1O4)
partially
from
the
pH greater
lack
N
metabisulfite effect was
tissue
acetate
(second The
water
during
water
an almost tamed with
a critical
10 N the al-
innovation
since provoi-
catecholamine
theoretical alkaline
linear ascorbate
solution
response made
was obwith 10 N
but not with 5 N NaOH (fig. 2). This observation suggests that a considerable error may be introduced with 5 N alkali if the aliquot is var-
100’
90-
ied 80-
as
advised
with
the
but
all
7o-
alkali.
‘4-
60-
in
5 N
Also,
less
40
pink
sodium
for
ascorbate
30’ for
20
using
the of o
o
o
0
I0
pH
intensity
1. Effect of pH, metabisulfite the recovery of norepinephrine, HC1O4, from aluminum oxide. 0, acid made with EDTA-distilled metabisulfite; #{149}, acid made with no
EDTA-distilled
metabisulfite;
water,
and 0.5
EDTA g/25
on ml
water, with EDTA-distilled A, acid made with nonno metabisulfite.
almost
This
difference
half
from
the
hand,
This
with
due
oxidation
dopamine
fluoresced
to
cysteine,
not
substitute
agent.
the
One
eluting
reason
agent
presence
the
trihydroxyindole
that was
acetic acid had other
whereas began
was
fluores-
derivative
(3 ,4-dihydroxyphenylethylamine)
was
the
as
in its
of the
day
NaOH
agent.)
as the eliding acid
10 N
10 N alkaline
the
could
that
dopamine
during FIG.
acid
and
the
(Glutathione,
reducing
perchloric
observation
cence
0
1 hour.
the
with
5 N
same
stable,
of the
with
blanks
the
more
during
made
as
were
a problem
metabisulfite
Perchioric #{163}
much
of
The
NaOH
observed
after
and
methods.
N
no discoloration was
the ascorbate turn
10 were
was
ascorbate
50-
several
and
readings
quenching
water,
(no
. This
EDTA
sodium
8.05.
the 0.4
with
of EDTA
significant
to reproduce, the
made
experiment)
treated
show
in 25 ml
presence
the of
be distilled of EDTA.
line)
jg
solution
at
(fig.
a pH
catecholamines
of catecholamines
occurred
method
In
although
had
had to presence
(0.5
acid
the
1958a;
of 6.8
step
1 (dotted
in this
metabis-utfite.
a P11 one
Our water tion) in the figure
provided
Schaepdryver,
adsorption
solid
according
and (de
Orw#{233}n, 1955;
onto
at
using
others
optimum
had
used
ap-
results.
with
Euler
found
of sample
berg,
the
l)licabie only over a limited range. In our hands the use of constant amounts of reagents with a
365
METHODOLOGY
equally
concentration
acetic acid (table 2).
to the step,
and
presence not
of acetate because
the
eluted twice as much of the A12O3 as had the HCIO4. the well of
derivatives
of
in
acids
both
perchloric
acid
NE
the On
and (l)H (0.05
E 7.0). N)
366
ANTON
AND
Vol.
SAYRE
138
5, ‘I, 5,
0 ‘4-
5’
5,
ml of NE Standard
2. Comparison
FIG. (NE).
See
of the effect
Each point represents text for details.
the
results
of iON from
with
SN
at least
Contoining
alkali two
of relative
fluorescence
of trihydroxyindole (E) and dopamine
on the Relative
experiments
TABLE Comparison
0.I67g/mI
which
Fluorescence
of norepinephrine
did not
by more
NE 0.034 ,ig/ml
(1:5)
Present
(1: 10) von Euler (i955)1 (i:50)* de Schaepdryver (l:25)* Crout (l96l)T
and
0.034
derivatives of norepinephrine by several method8*
E g/m1
than
5%.
2
High pHt
Method
differ
(NE),
Low Dopamine 0.340 pg/mI
NE 0.034 pg/mi
Direct
Blank
Direct
Blank
Direct
Blank
7.80
0.20
5.10
0.20
1.90 (3.lO)t
0.20 (0.20)t
3.80 6.00 4.00
0.37 0.31 0.41
4.80 5.00
0.37 0.31 0.41
Direct
Blank
epinephr-ine
p1St
E
Dopansine 0.340 pg/mI
0.034 pg/mI Direct
Blank
0.80
0.20
4.70
0.20
0.41 0.35 1.10
0.30 0.26 0.52
4.50
0.30 0.26 0.52
Blank
Direct
0.19 (0.39)t
0.18 (O.20)
0.87
0.62
Floding (1958a)
2.50
3.30
0.52
2.50
5.50
catecholamines were made up in the acid used for elution in the respective method. An amount of NE and E was used so final concentration in the reaction mixture (and in the cuvette) was the same (0.034 pg/mI) for all methods. The volumes of used and the procedures followed were exactly as described in the respective method. Note that the concentration of dopamine that of NE and E. Crout’s method uses iodine as the oxidant whereas K,Fe(CN)* is used in the others. high and low pH refer to the different buffers used in the particular method. These samples differed from the others in that 0.2 N acetic acid rather than 0.05 N HCIO4 was used as the solvent. § This is the ratio between the aliquot of the catecholamine solution used and the final volume of the reaction mixture as described in the respective method. #{182} Reference to the method used. * The that the reagents is 10 times The
was
readily
oxidation
buffered step
without
to the prior
pH
required
neutralization
for
the
which
may be necessary when more concentrated acids are used for elution. Also, in the case of urine extracts, quenching was about 10% less with perchloric acid than with acetic acid eluates.
If the ness for was used
acid
eluate
was
parently were destroyed under these conditions. aluminum
to be evaporated
paper chromatography, for elution since the
oxide
with
0.033 catecholamines
by the Although the
perchloric recoveries
hydrochloric
to dryN
HC1 apacid from and
1962
STUDIES
Perchloric less with
in the
strated
the
freezer
fluorescence of NE, E commonly
for
stability
perchloric and Comparison
several
months
the
catecholamines
of
hydrochloric of several
of and
acids. methods.
used
methods
is
The
final
concentration
had
been
added)
cuvette
was
with
The
(loser
ratio
fluorescence
tions
is
is to unity
high
blanks
Also
note
dopamine
the
of the if
with
the
that in
approached the
our
of sample mixture.
relative
Reducby using resulted
(K3Fe(CN)6
A. ROOM
a in
readings.
obtained was
(1961) of
BLANK,
obtained
(1958)
reported
products obtained
appear
that
to
by
be
our
with
the
iodine
iodine.
function
of
a
than
This
time
Attempts
were
to reduce
with
and quenching the salt saturated
various
ethylene
organic chloride,
with
chloride,
butanol,
the
heptane)
prior
B.
to
intensity
is particularly
nonspecific
(ethyl
acetate,
carbon
tetra-
alcohol,
xylene,
onto
aluminum
(5#{176}C)
ACID
ACID
______ BLANK,
DIRECT,
META META
0
DIRECT,
ACID
BLANK DIRECt
META META
DIRECT, DIRECT,
4
ACID BLANK,
BLANK,
that
3 to required
reassayed. catecholarnines.
blanks,
isoamyl
REFRIGERATOR
than less
of urine samples by acidified samples
solvents
adsorption
no was
within were
chloroform, ether,
parphe-
ferricyanide.
factor
need to be and urinary
made
the
the
with
if samples conditions
l)e ob-
general
of ferricyanide
important Storage fluorescence extracting
may
with ferricyanide quenching was
advantage
fern-
these
procedure,
a problem
the
However,
than
was greater (table 2) and
(4
DIRECT,
that
quenching
a
of fluorescence with iodine An important
iodine
produce half the with iodine. Also,
iodine.
rather
Thus,
when
method).
with
method
nomenon.
that
reported
a problem
ticular
(25#{176}C)
(U
Olin
cyanide oxidation fluorescence of that
for is
concentra-
methods.
and
than (Crout’s
the reaction could be completed minutes whereas 30 to 60 minutes
sensitive
the direct
fluorescence method
were obwe had
same
similar
in both
in
specimen
in the other procedures or less diluent (water) which
the
less
oxidant
servations
highest
more
original
only
obtained
of catecholamines
ing the ratios larger aliquot
volume reaction
was
the
Crout
in
all
lowest blanks even though
lowest ratio between the the final volume of the this
ours
(after
the and
the method since less required; this is true
to
in all cases. The
same
was
less
of catecholamines
(direct) and our procedure
fluorescence
tained
the
relative
derivatives with three
compared
2.
oxidant) Shore
in
367
METHODOLOGY
demon-
The
the trihydroxyindole dopamine obtained
reagents the
CATECHOLAMINE
acids were similar, blanks were slightly the perchioric acid. Analysis of eluates
stored
table
ON
0
0
02468101214
02468101214 DAYS
3. Comparison of acid and refers to calculations based mnetabisulfite added to urine; A, urine FIG.
Direct
metabisulfite as preservatives for on the net Relative Fluorescence. layered with toluene.
urinary
#{149}, acid
catecholamines. added to urine;
o,
368
ANTON
oxide.
However,
and
in
none
some
added
of
instances
catecholamines.
from
normal, might
cannot
be said.
against
the
acid
as
was
observed
be of value The
in
NE
been
had
with
from
metabisulfite placed
the
were
or
or (3)
refrigerator
10
layered
mg
it
per-
out
in the
(the
pH effect
samples
the
acidified
tor
temperature;
samples
tremendous samples
were
prepared
increase
in
at also
room
1.6
m,g
as
there
OF
FIG. 4. Standard Each point represents the See text for details.
was
2.0
24
EACH
curves results
(0 the
well
as
a
2.8
refrigera-
concomitant
of
con-
samples
NE
trend
catecholamine Lishajko
(1961)
content at pH’s
of urine below 1.0
was
Although
marked
0.5). in
was
was
keep
the
In most
other
optimum for many other
and
instances
less because
necessary
blanks
several
other
same
in all.
reagents highest
variations
The in
in
the
quenching
0.4-mi
case to
aliquots
(with
were used for plasma very low concentration
water)
of their
catecholamines.
of the
of and
series
the
there (room
metabisulfite as the of any preservative loss
as
mixture
minimum.
time).
blanks
catecholamine
Euler
not
138
of the method. A 1:5 ratio between of the aliquot and the final volume of to
of
the
Von
acidified
was
reaction
the
the
of our
urine
correspondingly
time
significant
of urine,
tory; samples were then analyzed at the intervals indicated and related to the results obtained at Note
a
Sensitivity volume
the
labora-
free
containing the absence
In
24 hours.
this
of
were
apparent
also reported increases in stored at room temperature
the
toluene.
series
was
within
sodium
with
each
or left
there
under
and to which (1) made 0.4 N
(2)
of
advise
became
either
HC1O4
in the
in both samples. At the same time only a slight change in those samples
and refrigerator) preservative.
in the freezer, 25-ml aliquots
source
tent was
unines
Since
urine
samples
in the
literature.
same
added)
added
3 may
of urine
when stored was done:
the
concentrated
Representative
figure
acidified
that
ceptibly darker even the following experiment
were such pro-
abnormal
or storage
recommended
(all
in
of
specimens whether
with
data
recovery
Vol.
SAYRE
increase
helped
the
humans;
collection
of urine
treatments
These
healthy
cedures
the
reduced
AND
volume and the reaction
sensitivity and
concentration mixture were
with lowest combinations
blanks; were
3.2
CATECHOLAMINE/mI
of norepinephrine (NE) and from at least two experiments
epinephnine which did
not
(E) at pH 7.0. differ by more
than
5%.
a
1962
STUDIES
ON
CATECHOLAMINE
369
METHODOLOGY
2.40 2.20-
aoo1.801.60I40I’
(U L ,‘ -1
1.20-
1.00.80.6040.20-
1.2
.8
4
2.4 2.8 32 35 4.0 4.4 4.8 52 5.6 6.0
1.6 2. m,.g
FIG. 5. Standard Each point represents the See text for details.
tried,
and
found
to
be
of Each
curves results
less
Catecholamlne/mI
of norepinephrine from at least two
In
satisfactory.
the
course of several hundred analyses, plasma blanks were the same as, or 5 to 10 higher than those obtained from an liver blanks kidney blanks.
when the catecholamines acid solution. Tissue
and
kidney,
also
from urine ranged from The reaction
were
until
of acetic this
in this
range.
Most
specimens and liver and 10 to 30% higher than plasma at the lower pH is particu-
larly sensitive to slight fluorescence of the standard 90 to 95% that obtained volume
were extracted blanks, except for
acid
changes in pH. The E solution should be at pH 7.0. If not, the
solution
relationship
should
and
using
a O.2-ml
aliquot
oxidation step); less could be detected rather than a 0.2-mi aliquot of the was
used
in the
his
apparent magnitude
About
5 mg
(Kuntzman (1958)
mg be
of E meas-
Strauss
reaction.
results were extrapolated from standards which were several more concentrated than his samples
review
published
the
his
5.
method the
the
fluores-
of the blank (Udenfniend,
limit of
et al.,
is not 1959).
of sensitivity
in
1961)
for
oxidized
4 and
E
reaction
These
curves highest curves
fig. 4) with
of the
aliquots
concentration known
indicate
reproduced aniounts
in up
the In
this
solutions
together) those the to 430
tested.
A (dotted
of NE
were The
were
(also linear
over
method.
Mvnons.
curves
mixture
of no is (locu-
of standard
under
remained
knew point
and
in the
and
linearity this
(separately
was
they
5 which
linearity
Shore
methodology,
that range;
figures
shown
final
the
demonstrated
as described
centrations
of
could
procedure.
stated
that
O.2-ml and
that
on eatecholamine
and
experiment
tissue
extraction
concentration
sensitivity
these
than
Although
between
amount
Wurm
data
a 10-fold
the
was
organic
and
NE
00
be weighed); this was about 8 times more than a recently reported! micro-modifica-
accurately sensitive
of
of
range.
difference
any
tion
in the
linearity
sample and that to be reliable
(using
mented
9.6
9.2
(E) at pH 2.0. differ by more
the
the
of the enough
tissue
not
concentration
of epmephrme, cence great
8.8
sensitivity of our method is of a similar we feel that at such low concentrations
Olin
Recently, Vendsalu (1960) indicated that he was able to measure less than 0.1 g epinephrine per liter of plasma using about 10 ml of plasma. However, catecholamine hundred-fold
large
8.0 84
7.6
did
indicating this
varied
if a O.4-ml acid eluate
72
Mixture
which
In their
of NE and 0.75 mixture could
6.8
epinephrine
he
ured. In terms of plasma catecholamine concentration, a minimum of 0.5 g NE and 0.75 zg E per liter could be detected (based on a 20-ml sample
without over
6.4
Reaction
(NE) and experiments
is obtained.
As little as 0.5 mjg per ml of final reaction
plasma
of Final
and
conin the
cuvette). mug/mI, portion lines E added
of in
370
ANTON
TABLE Recovery
%
Recov
from
75, 85,
0.20 0.50
83, 85 83 ± 1.2
ered
fromt
1.00
82,
Tissue
Urine
The
added
(12)
75,
77 ± 2.8 74, 77
amounts
(0.4 N HCIOs), human urine.
76
74, 77
84
of NE
20 ml human
(18)
were
75
80, 80,
75 ± 1.9 74, 76
81 81
from
1 gram
(10)
acid
and
10 ml
rat liver
t Results are shown for at least two experiments at each concentration. Where experiments were repeated many times (at the 0.50 level) the mean, the standard deviation and the number (in I)arefltheses) been made precipitation
to
of determinations for losses except in plasma and
plasma
tion
and
the
procedure
entire
followed
Recoveries
and
extraction
(based
90%
of the
counted
for.
Regardless
and
oxida-
on 75 % recovery).
reproducibility.
at least
catecholamines was retained
are shown. No correction has that incurred as a result of protein tissue.
By
this could
be ac-
of the
concentration
of 15% the
had
been
adsorbed
In
and
normal
in
volume
incurred
tion. Table bility range
of
(de Schaepdryver,
reproduci-
occurring
substances
have
reported
been
to interfere abnormally in
tumors
could
the
same
the
Lishajko,
1959;
Vendsalu,
that fluorescence droxyl substituent hydrogen on the attached
to
the
catechol 1951;
relatively
the
speci-
1960).
Studies
on
reaction
have
high l)eaks)
nucleus Lund,
been
Dopa oxidized
case
number
fluorescence of methyldopa,
in
and
However, only
a few
of compounds.
(even
more which
at does
naturally
by
a
dagger)
concentration However, the and dopamine with
patients problem
neural
[dopa.
normal
up
to
urine
can be distinguished products have dopamine.
are
However,
(Carisson 1959) can
presence substance (Lockett,
from different
they
whereas
of
be
the other
and used
Waldeck, to detect
of NE. Traces of an have been detected 1954, 1957) but the
3% NE equivalents) After treatment with fluorescence of 3 ,4-diacid would be negligible amount found in the urine of
large
stance) established
of
a small
amount
togral)hic a molar differences
aluminum
the
with neuroblastoma [90 mg/24 hr vs. 0.4 mg/24 hr (von Studnitz, 1960)]. apparent purity (single fluorescent subthe interfering catecholamines was by paper chromatography. However,
substance could missed with the
suggest
(Bu’lock 1949).
quantified!
instances and with a small its fluorescent
Euler
requires the presence of a hyon the beta carbon and a alpha carbon of the alkyl amine
Harley-Mason, studies
von
in the
patients normal, The
were
; dopamine hr vs. about 50 g/24 hr in Studnitz, 1960; Voorhess and in
oxidative procedures 1958; Drujan et at.,
even
from of
enough
of a
characteristics
in
is compared compounds
method. of dopa
urine
15 mg/24 vs. none up to 100 mg/24 normal urine (von
fluorescent
g/ml
(indicated
present
1962)]. their
1960) prompted of this problem. NE peaks) of a
none
with this large amounts
excreted
of the (Axeirod,
in parentheses
in high
hydroxyphenylacetic
concentration results were
1958a;
Specificity of the method. ficity of the tnihydroxyindole
The
precipita-
good
a large Similar
and loss
with E, and we have not noted any differences in the recovery of NE and biological material as reported by some
investigators
such
protein
the
of the method over with diverse material.
obtained significant E from and
because
3 also emphasizes
plasma for the
catabolites
eluted
urine
small amounts (about would be inconsequential. aluminum oxide, the
loss, the corrected
identi-
interfering
were tissue
this were
of the
recent
at 0.43
dopamine in the isoproterenol-like in animal tissue
for
carbon
figures
first elution. Since this amount was ignored, the recoveries of NE and E from biological material were at least 75% (table 3). Although recoveries not corrected concentrations
alpha
urinary
possibly
138
as the
catecholamines
4 ; the
after
NE since
catecholamines
present, a constant 10 to the aluminum oxide after
in
table
in
Gardner,
method
new
of compounds
NE
oxide.
25 ml
as well
occurring
obtained
74, 75
on the 4),
1960; Goodall, 1959; Kirschner, a more thorough investigation The Relative Fluorescence (at to
81, 81
extracted
plasma,
(table of many
number
77
74,
a hydrogen
chain
naturally
77. 77 85
have
side fication
Plasma
0.02 0.05 0.10
Vol.
SAYRE
not
(NE)
-
Acid
*
3
of norepinephrine various media*
NE Add&
AND
of a contaminating
fluorescent
be detected in the SPF but far less sensitive paper chroma-
procedures.
Expressing
the
basis would not significantly except that the fluorescence
norepinephrine rine would
results
on
alter these of ethyl-
and isopropylethylnorepinephbe more alike.
Comparative distribution of catecholamines in several species. Table 5 shows the comparative distribution of norepinephnine and epinephrine in plasma,
urine
and
tissues
of
man
and
several
1962
ON
STUDIES
CATECHOLAMINE
TABLE Relative
fluorescence
(RF)
371
METHODOLOGY
4
of analogs
of norepinephrine5 a
H-C Compound
.fii........_
Norepinephninet (NE) Epinephninet (E) N-Ethylnorepinephnine Isoproterenolt (Isuprel) Norhomoepinephnine (Cobefrin) Ethylnorepinephnine (Butanefnin) Isopropylethylnorepinephnine (Dilabron) Dopaminet Methyldopaminet (Epinine)
Relative Fluorescence (RF)
NH
I \ .1.
OH OH OH OH OH OH OH
OH OH OH OH OH OH OH
OH OH OH OH OH OH OH
H H H H CH3 C2H5 C2H5
H H H H H H H
H CH3 C2H5 C3H7 H H C3H7
100.0 (80.0) 70.0 (56.0) 65.8 (51.6) 41 .2 (33.0) 9.5 (6.1) 5.9 (3.5) 4.9 (3.1)
Methyldopa (Aldomet) Normetanephninet Dichloroisopnoterenol Synephnint
OH OH OH OH OH Cl OH H
H H H H OH OH OH OH H H OH H H H
H H H CH3 H H H H CH3 CH3 CH3 CH, H H
H H COOH COOH H H H H H H H CH, H H
H CH3 H H H C3H7 CH3 CH3 H H CH3 CH3 H H
2.2 1 .0 12.1 17.2 0.06
