FATIGUE AND STRESS STUDIES:

An Improved Semi-Automated Procedure for

Fluorometric Determination of Plasma Catecholamines

I. Introduction.

The use of an automated chemical analytical
system (Autoanalyzer, Technicon Instruments
Corporation, Chauncey, New York) as an adju-
vant in routine assay for catecholamines has been
explored by Merrills*:? with promising results.
Based on a fluorometric trihydroxyindole (THI)
method, the procedure uses to best advantage
the constant rates of sample and reagent. intro-
duction and the fixed durations for chemical
reaction provided by the Autoanalyzer. Although
the method described is useful for estimating
catecholamines in tissues or fluids containing
relatively high concentrations of those com-
pounds, it unfortunately lacks sufficient sensitivity
for the determination of catecholamines in human
peripheral plasma.

The present report details an improved semi-
automated procedure based on Merrills’ original
suggestion, Several modifications in both the
isolation and detection steps of the method have
been introduced in order to achieve the sensitivity
and specificity required for the estimation of the
low levels of catecholamines generally found in
resting plasma.

II. Materials and Methods.

A. Reagents ——All chemicals used were reagent-
grade or highest-purity materials. Solutions were
prepared with twice-distilled water, the final
distillation from an all-glass apparatus.

1, Alumina (Woelm), neutral, activity grade
1 for chromatography. Prior to use, the
alumina was purified according to the
procedure described by Anton and Sayre.*

2. Sodium acetate, 0.2 M. This solution was
purified by passage through a 25- by 300-
mm column of Chelex 100 (Bio-Rad Lab-
oratories, Richmond, California), 50-100
mesh, Na+ form. The pH of the purified

buffer was adjusted to 8.40.1 (glass elec-
trode) with 0.5 N sodium carbonate.

8, Ethylenediaminetetracetate disodium, 5%
(w/v).

4, Acetic acid, 0.8 M.

5. Sodium acetate, 1.5 M, containing 0.01%
(w/v) potassium ferricyanide. This solu-
tion was passed through a medium-poro-
sity sintered-glass filter. The pH of this
reagent as prepared is 9.0 +0.1.

6, Ascorbic acid, 0.3% (w/v). The Eastman
product was used without further purifi-
cation,

7. Sodium hydroxide, 2.5 M.

8. Perchloric acid, 4 N.

B. Standards—Stock standard A, 1.0 mg/ml
total catecholamines as the free bases, was pre-
pared from dl-arterenol hydrochloride and 1-
epinephrine bitartrate (Winthrop Laboratories,
New York, New York) in the ratio of 2:1 in
order to simulate that ratio found in human peri-
pheral plasma.‘ Stock standard B, 10 «g/ml, was
prepared from A; stock standard C, 1 pg/ml,
from B. Working standards containing 1 to 5
pg/t total catecholamines were prepared from
stock standard C. All standards were prepared
in a stabilizing solution of 0.01 N HCI containing
0.5 mg/ml sodium metabisulfite. Stock standards
A and B, stored under refrigeration, are stable
for at least 1 month. Stock standard C was pre-
pared weekly; working standards were freshly
prepared before each analysis.

C. Apparatus —

1. Adsorption tubes found most convenient
had the following dimensions: reservoir,
90 by 25 mm (ID); stem, 17 by 4 mm
(ID). A pipette tip (1 mm) was drawn
from the stem to facilitate draining. Reser-
voir capacity was 40 ml. Ten such tubes
can be handled simultaneously by incorp-



plasma sample (and in the catecholamine stand-
ard in the current procedure) insures its availa-
bility during the pH adjustment for optimal
alumina adsorption (8.4). At this pH, without
protection, catecholamines are highly susceptible
to oxidative destruction. The influence of added
antioxidant on retarding the loss of catechola-

mines during assay of biological fluids has been
discussed by Anton and Sayre, Weil-Malherbe,?
and Manger. Whether this point, in itself, is
sufficient to explain the lower values reported for
total catecholamines in normal plasma cannot be
presently decided.

REFERENCES

. Merrinys, R. J.: Anal. Biochem., 6:272, 1963.

Menritts, R. J.: Nature, 198 :988, 1962.

. Lup, A.: Acta Pharmacol. Towicol., 6:137, 1950.

. ANTON, A, H., and Sayre, D, F.: J. Pharmacol. Baptt.
Therap., 138 :360, 1962.

. Price, H. L., and Price, M. L.: J. Lab. Clin. Med.,
50:769, 1957.

6. Venpsatu, A.: Acta Physiol. Scand., 49, suppl.: 178,

1960.
7. Weit-Maruerse, H., and Bone, A. D,: Biochem. J.,
51:311, 1952,

Bw

on

8, UNvENrRienp, S.: Fluorescence Assay in Biology and
Medicine, Academic Press, New York, 1962, p. 154.

9, Exatansian, F. in Dorrman, R. I, ed.: Methods in
Hormone Research, Academic Press, New York, vol. 1,
1962, p. 337.

10. Went-Maraterse, H.: Pharmacol. Rev., 11:278, 1959.

11. Conn, G., and Gorpenners, M.: J. Newrochem., 2:71,
1957.

12, Mancer, W. M.: Pharmacol. Rev., 11 :289, 1959.

6763170


worked satisfactorily. At the end of the 4-minute
period, the alumina was transferred as a slurry
to the adsorption tubes. The alumina rapidly
settled as an air-free column. The solutions
above the alumina were then drawn through the
columns at the rate of 1 to 1.5 ml/min. Columns
were next washed with 40-ml glass-distilled water
(pH 8.4 +0.1) at a flow between 1.5 and 2 ml/
min. As the final step, the alumina was eluted
with 2.0 ml of 0.3 M acetic acid, and the eluates
were centrifuged at 30,000g (10°C) to precipi-
tate the fine suspension of alumina occasionally
encountered after elution.

Centrifuged eluates were transferred to stand-
ard sample cups and processed through the Auto-
analyzer at 20 samples/hour. Peaks of unknown
samples were compared with those of standards
run through the entire procedure simultaneously.

All phases of the procedure were conducted
under red illumination (45 foot-candles).

III. Results.
Over the range to 5 y»g/Z original standard,
fluorescence intensity is linearly related to total

TO

60 - }

50b
4ob

3ob
20h L/L
z

i 1 1 L 1 J
° | 2 3 4 5

TOTAL CATECHOLAMINES pgm/L
(NOREPINEPHRINE : EPINEPHRINE = 2:1)

Ficure 2. Standard curve for total catecholamine con-
centration through 5 yg/l. Each point represents
mean fluorescence of 10 determinations. Vertical lines
are the standard deviation at each concentration.

FLUORESCENCE INTENSITY

catecholamine concentration under the conditions
of the procedure described here. Figure 2 shows
the standard curve derived from 10 complete
determinations with a measure of the variability
(SD) at each concentration. A typical record-
ing, from the Autoanalyzer system, of fluores-
cence intensity as a function of concentration is
shown in Figure 3.

Two types of recovery studies were performed
to establish the accuracy of the procedure. Effi-
ciencies of alumina adsorption and elution were
determined by carrying 10.0 ml of standard (1 or
5 ug/l) through the perchloric acidification and
adsorption steps. Columns were eluted with 2.0
ml of 0.3 M acetic acid, and the eluates were
diluted back to 10.0 ml with 0.3 M acetic acid,
pH 3.85 (mean pH of acid eluates).

Fluorescence of diluted samples was compared
to corresponding concentrations of standard in
0.3 M acetic acid, pH 3.85. Ratio of fluorescence,
alumina-treated: direct, is a measure of the effi-
ciency of isolation. Recoveries from plasma were
determined by adding to plasma amounts of total
catecholamines corresponding to quantities likely
to be estimated (0.01 or 0.02 yg/10.0 ml of
plasma). The ratio between the fluorescence incre-
ment of plasma with added catecholamines and a
corresponding standard represents the efficiency
of extraction of the amines from plasma. Recov-
ery data are summarized in the Table.

Taste. Recovery of Total Catecholamines from Aqueous
Solutions and from Plasma.

Quantity of total Number of Recovery of
catecholamines added | determina- | added catechola-
to 500 mg alumina tions mines
ug % +8D
0.01 in 10.0 ml of stabi- 9 92.5 +11.1
lizing solution
0.05 in 10.0 ml of stabi- 9 748 45.1
lizing solution
0.01 in 10,0 ml of human 9 86.4 +8.9
plasma,
0.02 in 10.0 ml of human 9 85.6 45.8
plasma

Considering the nearly fivefold concentration
of catecholamines provided by adsorption from
9.0 ml of original sample and elution with 2.0 ml
of acid, 50 units of deflection (based on a scale of



PROPORTIONING

PUMP
DOUBLE
7 AIR (045)
RECORDER FLUOROMETER MIXING COIL aa
DX 1° 420mp DOUBLE 50S @ ACETATE -FERRICYANIDE (065)
2° S10mp |_MIXING COIL | _@—}— SAMPLE / WASH WATER (.045)
MIXING
mt Q AIR (.040)
50 ® SODIUM HYDROXIDE (.045)
© ASCORBIC ACID (.030)
mn

DEBUBBLER (.081)

q

DISCARD

DISCARD

Fioure 1, Flow diagram for automated fluorometric catecholamine assay. Figures in parentheses are inside dia-
meters of pumping tubes. Model II sampler module was used in the system described. A standard sampler mod-
ule can be incorporated if lines 1 and 8 are attached to a double-crook sampling device to maintain a fixed fluid:
air vatio in the system. Samples are processed at the rate of 20/hour.

orating them in a negative-pressure mani-
fold. Uniformity of flow from tube to
tube was maintained by individual stop-
cock control.

2. Automatic analysis. A standard Auto-
analyzer train (sampler, proportioning
pump, and recorder), coupled with a Tur-
ner fluorometer (Model 111), provided
the automatic system for catecholamine
oxidation, alkaline rearrangement, fluor-
escence detection, and recording. A sche-
matic flow diagram for the system is shown.
in Figure 1. Through the use of a Samp-
Jer II Module, a constant air: fluid ratio
was maintained within the system, mini-
mizing mixing variations and permitting
efficient debubbling at the flow cuvet.
Ferricyanide oxidation and alkaline
rearrangement occurred in separate dou-
ble-mixing coils, permitting reaction times
of 100 seconds for each step.

To increase the sensitivity of the detection
system, a high-sensitivity unit (Turner) was
adapted to permit use of a standard fluorescence
flow cuvet (Technicon). The adapter consisted
of a cylindrical aluminum sleeve, 72 mm long, 12
mm in OD, and 8 mm in ID. Four slits were
milled into the sleeve (25 by 3 mm) at right
angles to each other. This adapter permitted the
use of a 30X light intensity. Excitation light at
420 mp was providing by combining a Wratten
2C gelatin and a 3-mm-thick Schott BG-3, A

secondary filter combination of Wratten 8 and
Wratten 65 provided emission light peaking at
510 mp. A continuous-emission (400 to 520 mp
blue lamp (T-5 envelope) replaced the general-
purpose lamp normally supplied with the Turner
fluorometer.

D. Procedure.—Portions of blood (25 ml), drawn
in syringes lubricated with heparin, were trans-
ferred to 40-ml centrifuge tubes containing 5 mg
of sodium metabisulfite. The tubes were chilled
in shaved ice and centrifuged at 3,000 rpm for
15 minutes. Plasma was separated and stored
at—20°C for analysis.

One milliliter of 4 N perchloric acid was added
to each 10.0 ml aliquot of plasma. The mixture
was agitated thoroughly and centrifuged at
30,000g for 10 minutes at 10°C to remove all
protein, Aliquots (9 ml) of the protein-free
supernatant solution were added to 50-ml beakers
containing 10 ml of 0.2 M sodium acetate, 1.0 ml
of 5% EDTA, and 500 mg of acid-washed
alumina. Stabilizing solution, treated as sample
and carried through the entire procedure, served
as the column (reagent) blank. The pH of the
mixture was adjusted to 8.4 +0.1 (glass electrode)
and maintained at that pH for 4 minutes (timed)
by the dropwise addition of 5.0 N sodium car-
bonate. The mixture must be agitated continu-
ally during this adjustment in order to distribute
the particles of alumina evenly throughout the
solution. A motorized glass stirring rod and a
small turntable (10 rpm) to rotate the beaker


ment if fluorescent contaminants do not appear in
the final sample. By obviating the use of an
internal pH indicator, the present procedure
excludes the need for dialysis and the attendant
loss of about 75% of the catecholamine activity
initially present in the eluate.

Isolation of catecholamines by adsorption from
plasma onto alumina is a common step in both
the THI and ethylenediamine conjugation proce-
dures. Experience in this laboratory has shown
that performing the adsorption from plasma as
generally described *7* yields an eluate that is
not devoid of protein (as indicated by the for-
mation of a fine precipitate after addition of
4 N perchloric acid). Fluorescence intensities
obtained after direct adsorption from plasma are
approximately 2.5 times higher than those secured
by making the adsorption from a protein-free
supernate of plasma. These results indicate that
traces of plasma protein must also be adsorbed
onto alumina in quantities sufficient to interfere
with fluorescence measurement of the catechol-
amines. Prior removal of protein with per-
chloric acid avoids this difficulty.

Although column chromatography is considered
a more efficient process than “batch” adsorp-
tion,’ ?° the present modifications include a com-
bination of the two. Thorough mixing of alu-
mina, sample, and buffer during pH adjustment,
followed by a column step, resulted in column
recoveries of greater than 90% when physiological
quantities of both amines were present. At
higher concentrations (5 »g/2), column recoveries
averaged around 75%, indicating some reduction
in the efficiency of either adsorption or elution at
this concentration. Prior experiments with solu-
tions containing only epinephrine indicated that
column adsorption alone was sufficient to extract
the amine efficiently. If both amines were present
in the ratio of 2NE:1E, however, a reduction in
senistivity of approximately 30% resulted if only
column adsorption was used (Figure 4).
Increased sensitivity was achieved with the com-
bined standard by using the combination proce-
dure finally adopted. Such data suggest that the
rates of adsorption of the two amines may not be
similar under the conditions described although
the adsorption of both is optimized in the present
procedure.

Recoveries of catecholamines added to human
plasma before protein precipitation in quantities
equivalent to 1 or 2 pg/Z (0.01 pg/10 ml or 0.02

80 -

° /
©
/ eo

Yo

(ne

| 2 3 4 5
CONCENTRATION pgm/L

60 F

50

INTENSITY

40

FLUORESCENCE
QS

30 F

Fieure 4, Sensitivity of the assay (slope) as a func-
tion of the amines present in the original standard
and the mode of adsorption. Epinephrine, column ad-
sorption only, 0-0; norepinephrine-epinephrine mixture
(2:1), column adsorption only, @@; norepinephrine-
epinephrine mixture (2:1), bateh plus column adsorp-
tion, 0-0.

vg/10 ml) averaged 86.4% and 85.6%, respec-
tively.

The value for resting concentration of plasma
total catecholamines determined by this procedure
is lower than that reported by Anton and Sayre
(E + NE)‘ although both are somewhat higher
than others obtained with the THI procedure.**
The reason for these differences is not immedi-
ately apparent, although the higher value of
Anton and Sayre may in part reflect the sam-
pling situation. It is interesting to note, however,
that in none of the other THI procedures cited
is an antioxidant added to plasma prior to
adsorption. In both this method and that of
Anton and Sayre, sodium metabisulfite is rou-
tinely added to blood immediately after sampling.
The presence of this reducing material in the



AM 66-6

FATIGUE AND STRESS STUDIES:

An Improved Semi-Automated Procedure for

Fluorometric Determination of Plasma Catecholamines

Vincent Fiorica

Approved by Released by
5 o
hae. (USdge€ wo
J. Rosert Drie, M.D. P. V. Srecen, M.D.
Cuter, Civ AEROMEDICAL Feperan Air SuRGEON
Instrrure
April 1966

FEDERAL AVIATION AGENCY
Office of Aviation Medicine







CM
TITMIM

Fiaure 8. Recorder tracing of five standards carried through the entire procedure. Column blank represents 0 pg/l.
RBL is the reagent baseline level. Concentrations are are in terms of total catecholamines (free bases), norepi-

nephrine: epinephrine =2:1.

100) were obtained with original solutions con-
taining approximately 4 »g/Z. Given this sensi-
tivity, total catecholamines can be readily esti-
mated from 1.5 to 2.0 ml of acid eluate containing
as little as 0.0020 pg/ml of the amines.

Resting levels of total catecholamines in plasma
were determined in 20 male subjects, 20 to 29
years old. Subjects were postabsorptive and
remained at bedrest for 30 minutes prior to
sampling. The mean resting concentration of
total catecholamines, determined from analysis of
10.0-ml aliquots of plasma, was 0.89 £0.15 (SD)

ng/l.

IV. Discussion.

Application of the principles of automated
chemical analysis in the estimation of plasma
catecholamines provides the immediate advantage
of reproducibility in two critical steps of the THI
assay: (a) the time during which the catecho-
lamines are oxidized by ferricyanide and (b) the

duration of alkaline rearrangement of adreno-
chrome and noradrenochrome to the correspond-
ing fluorescent lutines. The importance of rigid
standardization of these reaction times has been
emphasized by several authors.+* In the system
described here, reaction times are determined
solely by the lengths of the mixing coils in which
those reactions occur, since flow in those channels
is maintained constant. Once appropriate coil
lengths have been determined, these steps remain
unvarying from sample to sample and require no
further attention. Introduction of the ferri-
cyanide reagent together with buffer also allows
automatic pH adjustment of the acid eluate.
In this system, the 0.3 M acetic acid eluate has
a final pH of 3.85 0.05. Mixing eluate (sample)
and buffered ferricyanide in the molar strength
and ratio designated yields an oxidation mixture
of pH 5.8. This pH is favorable for the oxidation
of both epinephrine and norepinephrine.
Incorporation of the dialyzer module as
described by Merrills * is an unnecessary refine-




FOREWORD

Stress may be regarded as an operational condition that challenges the self-
regulating systems of man and demands a corrective response. In general,
most of the stresses encountered during the course of ordinary life experiences
are adequately compensated for and are usually of little concern. Those
stresses that overwhelm man’s compensating mechanisms are, however, often
debilitating and result in generalized performance decrements.

The common denominator to many stresses is the response of the sympatho-
adrenal system. Regardless of the origin of the stress, the application of the
stressor is followed by a liberation of catecholamines (norepinephrine and
epinephrine) from the sympathetic nerve endings and from the adrenal med-
ulla. The present report describes a new application of the chemical technique
for the estimation of catecholamines in blood. Such measurements may permit
a quantitative evaluation of the magnitude of stresses encountered in specific
work situations and may provide an index to man’s tolerance for various
specific stress exposures.



ACKNOWLEDGMENTS

The author expresses his gratitude to Dr. J. R. Dille and Mr. William F.
Scott for their services throughout the study and to Mr. Don R. Mayer for
valuable technical assistance.

Qualified requestors may obtain Aviation Medical Reports from Defense Documentation
Center. ‘The general public may purchase from Clearinghouse for Federal Scientific and
Technical Information, U.S. Dept. of Commerce, Springfield, Va. 22151