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REFERENCES

. Merrills, R. J.: An Autoanalytical Method for the

Estimation of Adrenaline and Noradrenaline, NA-
TURE 193 :988, 1962.

. Merrills, R. J.: A Semiautomatic Method for Deter-

mination of Catecholamines,
6 :272-282, 1963.

Robinson, R. L., and D. T. Watts: An Automated Tri-
hydroxyindole Procedure for the Differential Analysis
of Catecholamines, CLIN, CHEM., 11:986-997, 1965.
Sampson, P. A., Jr.: Automated Trihydroxyindole
Procedure for Analysis of Epinephrine and Norepine-
phrine, Aerospace Medical Research Laboratories,
Report No. AMRL~TR-67-96, Wright-Patterson AFB,
Ohio, 1967.

ANAL. BIOCHEM.,

. Hathaway, P. W., L. Jakoi, W. G. Troyer, Jr., and

‘M. D. Bogdonoff: A Method for Semiautomatic Dif-
ferential Analysis of Urinary Catecholamines, ANAL,
BIOCHEM., 20 :466~-476, 1967.

Sampson, P. A. Jr.: Automated Trihydroxyindole
Procedure for Analysis of Epinephrine and Norepine-
phrine, AEROSPACE MED., 39:1128, 1968.

Viktora, J. K., A. Baukal, and F. W. Wolff: New
Automated Fluorometric Methods for Estimati
Small Amounts of Adrenaline and Noradrenaline,
ANAL, BIOCHEM., 23 :513-528, 1968.

Martin, L. E., and C. Harrison: An Automated
Method for Determination of Noradrenaline and
Adrenaline in Tissues and Biological Fluids, ANAL.
BIOCHEM, 23 :529-545, 1968.

Mabry, C. ©. and P. W. Warth: An Automated
Technic for Separate Fluorometric Measurement of
Epinephrine and Norepinephrine in Urine, AM. J.
CLIN, PATH., 52 :57-68, 1969.

Arnold, D. E.: A Sensitive Semiautomated Method
for Determination of Urinary Epinephrine and Nore-
pinephrine, USAF School of Aerospace Medicine
Report No. SAM-TR-70-10, Brooks AFB, Texas,
1970,

Cohen, G., and M. Goldenberg: The Simultaneous
Fluorometric Determination of Adrenaline and Nora-

18

12,

13.

14,

15.

16.

17.

18,

19.

20.

21,

drenaline in Plasma—I, J. NEUROCHEM, 2:58-70,
1957.

Cohen, G., and M. Goldenberg: The Simultaneous
Fluorometric Determination of Adrenaline and Nora-
drenaline in Plasma—II, J. NEUROCHEM., 2 :71-80,
1957.

Fiorica, V.: An Improved Semi-Automated Procedure
for Fluorometric Determination of Plasma Catechol-
amines, CLIN, CHEM. ACTA., 12 :191-197, 1965.
Anton, A. H., and D. F. Sayre: A Study of the
Factors Affecting the Aluminum Oxide-Trihydroxy-
indole Procedure for the Analysis of Catecholamines,
J. PHARMACOL. EXP. THER., 138 :360-375, 1962.
O'Hanlon, J. F., H. C. Campuzano, and S. M. Hor-
yath: A Fluorometric Assay for Subnanogram Con-
centrations of Adrenaline and Noradrenaline in
Plasma, ANAL. BIOCHEM., 34:568-581, 1970.
Crout, J. R.: Standard Methods of Clinical Chemistry
Volume 8, New York, Academic Press, 1961, pp 62-80.
DuToit, C. H.: A Study of Chemical Methods for
Quantitative Measurements of Catechol Amines,
Wright Air Development Center Report No. WADC—
TR-59-175, Wright-Patterson AFB, Ohio, 1959.
Becker, B. J., and F. Kreuzer: Catecholamine Excre!
tion by the Healthy Adult Human, PFLUGERS
ARCH., 316 :95-113, 1970.

Hale, H. B., E. W. Williams, and C. J. Buckley:
Endocrine-Metabolic Effects of Transatlantic Heli-
copter Flight, USAF School of Aerospace Medicine
Report No. SAM-TR-68-68, Brooks AFB, Texas,
1968.

Terry, E. N., R. H. Clauss, and W. Redisch: Deter-
mination of Catecholamines in Arterial Tissue and
Plasma with Simultaneous Measurements of Periph-
eral Blood Flow in the Anesthetized Dog, J. CARI-
VASC. SURGERY (Torino), 9:353-357, 1968,
Manger, W. M., O. S. Steinsland, G. G. Nahas, K. G.
Wakim, and S. Sufton: Comparison of Improved
Fluorometric Methods Used to Quantitate Plasma
Catecholamines, CLIN. CHEM., 15:1101-1123, 1969.



eo j |

e
FILTER SET B js

TOF 4

°

4 FILTER SET A

a

°
T
L

8
el

FLUORESCENCE UNITS
8
T

-5 L 1 L 1 1 1 1
° 25 5.0 75 10.0 12.5 15.0 (7.5

EPINEPHRINE (E) nanograms per sample

Figure 7. Relationship between amount of E in starting sample and fluorescence intensity with filter sets A and
B. The volume of sample was 5.0 ml in all instances and the samples were carried through the entire procedure.
Points represent the means of five measurements ; the flags represent the standard deviation.

12


RESERVOIR 95 mm

v

4
STEM 175 mm

WALL THICKNESS
2mm
CAPILLARY TIP v

1D. 2 mm__y 10 mm vy

—,—_

Ficure 1. Diagram of glass adsorption tube used for alumina column chromatography. Capacity of the reservoir is
50 ml. Use of a capillary tip minimizes the-dead volume after the elution step.

3



accepts either urine or plasma samples without
necessitating changes in the AutoAnalyzer sys-
tem. We believe these points represent distinct
advantages over those systems that permit the
analysis of only urine or that require two
analytical runs for differentiation.

In attempting to compare the characteristics
of the present method with those of published
techniques, certain inconsistencies were encoun-
tered which made a direct comparison difficult.
For example, although most authors report data
for recoveries, the techniques used to evaluate
recovery of added NE or E were so varied as to
preclude a comparison of the recovery values
given. In some instances NE and E were added
to the original sample, in other cases the amines
were added to column eluates and in still other
cases no information was presented about how
the recoveries were performed, Further, in several
reports the quantities of NE and E added to
samples were several times the amounts that
might be expected in the sample, biasing the
result in the direction of a higher recovery.

In order to provide reference points for
evaluating the present method in comparison
with other techniques (where possible) the fol-
lowing conditions were observed :

1. The quantities of NE and E used for stand-
ards approximated the quantity of NE and E
likely to be measured in the unknown sample.

2. The quantities of NE and E added to
samples in studies of the recovery of the amines
also approximated the amounts of the amines
present in the sample from which the recovery
was evaluated.

8. All recoveries were based on adding NE, E,
or both to samples of plasma or urine before the
deproteinizing step. In this way the estimate
of recovery considered all of the manipulations
to which the sample was subjected.

We recognize that adherence to the above criteria
has the effect of apparently increasing the vari-
ability of measurement, but we also believe that
such an estimate of the overall variability presents
a more accurate appraisal of the technique than,

‘Taste 6.—Comparison of Catecholamine Values in Urine and Blood Plasma Estimated by Automated (A) or Manual
(M) Trihydroxyindole Techniques.

Human Urine e/24 br
Author and Reference

Human Plasma ng/ml Other Plasma ng/ml

NE E NE E NE E
(A) Sampson (4) - - 129.747.7  7.824.0
(A) Robinson & Watts 224.944.6 6,741.0
(A) Martin & Harrison (8)-. 19.6£8.7 13.743.0  *0,75 0.16 *1.040.33  0.7640.02
(M) Coben & Goldenberg (12) £0,3040.07 0.0640.05
(M) O'Hanlon et al. (15)... 0.17440.052 0,079+0.047
(A) Hathaway et al. (5)--.--. 31.0 8.7
21.0 5.9
(A) Mabry & Warth (9)--.... 10-100 B15
(A) Viktora et al. (7).- -730.742.23  1.740.06
815,841.62 1,440.17

(M). Crout (16)-- 30413 5,643.1
(M)DuToit (17). - 35.8 4.5
(M) Anton & Sayre (14)______* 925.2 8.4 0.97 0.48 191.05 0.41
(M) Becker & Kreuzer (18)... 24.3£6.9 7,142.9 0.12 0.23
(M) Hale et al. (19)_ 1 33.4 7.0
(M) Terry et al. (20) - 101190,2140.14 0,144.0. 08
(M) Manger et al. (21) . 291,641.93  1.0-£1.02
(A) Fiorica & Moses .. 45.7411.5 9141.7 1014 0,5640.19 0.07+0.06

(This study) 624.0461 4,841.2

1All values are Means and SD unless indicated otherwise.  *Means and SE. *Mean of 2 samples. ‘Calf
plasma. Mean and Average Deviation. | ‘Nanograms per mg creatinine. ‘After acid hydrolysis.  *Free
catecholamines. Restated from 0.63 pg NE and 0.21 pg E per 25 mg creatinine; 12-24 hr. urine. Canine plasma.

4128-hour collection interval, “Arterial sample.

Under pentobarbital anesthesia.

4Unanesthetized.

16


1-1.5 ml/min. The maintenance of this rate of
flow is facilitated by the negative pressure mani-
fold. After the buffer solution has passed
through the column, the column is washed with
40 ml glass-distilled water (pH 840.1) at a
flow rate between 1.5 and 2 ml/min. The last ml
of wash is permitted to flow through the column
by gravity. The catecholamines are eluted from
the column with 2.0 ml 0.8 M acetic acid also
allowed to flow by gravity. The eluate is col-
lected in a round bottom polycarbonate tube for
high speed centrifugation (30,000 x g, 10 C).
This is done to precipitate the fine suspension of
alumina occasionally encountered after elution.
The centrifuged eluate is finally decanted into
standard sample cups and processed by the Auto-
Analyzer system described. The fluorescence of
standards (run through columns) and unknowns
from filters sets A and B are recorded on a two-
pen recorder. The fluorescence value of the re-
agent blank is subtracted from the fluorescence
peak values for both standards and unknowns.

Calculations for Differential Fluorometry.
Computation of the quantity of NE and E ina
sample is based on the following consideration :**
let A = total fluorescence of unknown with
filter set A

let B = total fluorescence of unknown with
filter set B

let N, = fluorescence contributed by NE with
filter set A

let E, = fluorescence contributed by E with
filter set A

let Nz = fluorescence contributed by NE with
filter set B

let Ex = fluorescence contributed by E with
filter set B

Then: A = Ny + Ey (1)
and B = Nz + Es (2)
Further:
let X = quantity (ng) of E in the unknown
let Y = quantity (ng) of NE in the unknown
let E, = fluorescence of E with filter set A per
ng E (standard)
let E, = fluorescence of E with filter set B per
ng E (standard)
let N. = fluorescence of NE with filter set A
per ng NE (standard)
let N, = fluorescence of NE with filter set B
per ng NE (standard)

Equation (1) and (2) then become
A= YN, + XE, (3)
B = YN, + XE, (4)

Solving equation (4) for X gives:
B — YN,
= 5

E, @)

x

Substituting (5) into (3) and solving for Y gives:
A= YN. + (B — YN,) E,
E,

YN, + BE, — YNLE.
E,

YN.E, + BE, —YNSE,
B

AE, = YN.E, + BE, — YN,E,
YN.E, — YN,E, = AE, — BE,

__ AE, ~ BE,
NE, —

Y

AE,

—N,

BE,

Normally, 5 ng standards for NE and E are run
through the column procedure in duplicate and
the mean values (corrected for reagent blank)
used to compute N., Ny, E, and Ey.

Linearity. The relationship between quantity
of catecholamine per sample and sample fluo-
rescence for NE, E and a mixture of the two
(2NE : 1E) were examined over the range 0.0 to
20.0 ng for both filter sets A and B. Samples
were taken through the entire procedure of
adsorption, elution and automated analysis to
arrive at the fluorescence per sample.

Recoveries. Studies were made of the recovery
of NE and E from aqueous solution, canine
plasma or human urine. Several levels of added
amine were examined. Additions of NE and E
were made from solutions containing the in-
dividual amines, or from solutions containing
both the amines. In every instance the quantity
of amine to be recovered was added to the ori-
ginal sample (urine or plasma) before the sample


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9.0) and segmented with air. Before its intro-
duction into the liquid stream, air is pulled
through a water scrubber to remove particulate
material (e.g. smoke) which can be a factor in-
fluencing background fluorescence. Oxidation of
the catecholamines by ferricyanide takes place
jn a double mixing coil at pH 6.0+0.1. The
duration of the reaction is determined by the
total flow rate and the length of the coil. In
this system the time permitted for oxidation of
NE and E to noradrenochrome and adreno-
chrome was 100 seconds. After the oxidation
step this stream is joined by the alkaline
ascorbate mixture. High background  fiuo-
rescence, often encountered as the alkaline
ascorbate reagent deteriorates during the course
of a run, is avoided in this system as the mixture
is made just before its addition to the fer-
ricyanide stream. The oxidized catecholamines,
in the form of noradrenochrome and adreno-
chrome, are rearranged in alkaline solution to the
corresponding lutines. The presence of ascorbate
in the alkaline mixture stabilizes the lutines by
retarding their further oxidation. The rear-
rangement reaction also takes place in a double
mixing coil and similarly takes about 100
seconds. Beyond this point air is removed from
the stream and the stabilized lutines are pumped
through the dual fluorometer system.

Because noradrenolutine and adrenolutine have
slightly different fluorescence characteristics, this
variance can be utilized in differentiating the
proportion of the two in a mixture. The use of
two fluorometers in series, each equipped with
filters passing wave lengths near the fluorescence
optima for excitation and emission of one or the
other of the lutines, is the simplest means of
achieving the differential analysis in a continuous
flow system.

The first fluorometer, designated A in Figure
2, is equipped with primary filter Wratten 47B
(Turner 110-813) passing light at wave lengths
of 405 and 436 mu. The secondary combination
consists of a Wratten 2A-12 (Turner 110-818)
and a Corning 3-70. This set provides a sharp
cut filter passing wave lengths longer than 510
mu.

The second fluorometer, B in Figure 2, uses
primary filter RDUV #1, B-3(A), narrow pass
395-410 mu. This filter is available from Baird
Atomic Corp., Cambridge, Mass., and, according
to Hathaway, et al.,* has the following specifica-

tions: “Type B-3(A), Peak center of 395-410
mu, Min. transmission at 455 mu, Block to 690 mu.
Make to provide maximum transmission, size
2x 2 in.” The secondary filter for this fluo-
rometer is a Wratten #8 (Turner 110-817)
passing wave lengths longer than 485 mu.
Several minor changes in the fluorometers were
necessary in order to achieve the sensitivity re-
quired for the assay. As light sources for excita-
tion, continuous emission (400-520 mu) blue
lamps (GE-F4T5-B) were installed in both
fluorometers using the adapters supplied by
Turner (110-856). These lamps replaced the
general purpose lamps normally furnished with
this instrument.

A second change made was the replacement of
the standard cuvet holder in each fluorometer
with a high-sensitivity temperature-stabilized
sample holder. This holder as supplied by the
manufacturer is designed to be used manually
with round glass cuvets. For use with the flow
system, the commercial sample holders were modi-
fied to accept a specially designed flow cuvet
holder (Figure 3). The holder was fabricated
from aluminum tubing stock and sprayed inside
and out with flat black paint. The flow cuvet,
also shown in Figure 3, was constructed from
fused quartz tubing. The volume of the cuvet
chamber, of the dimensions indicated, is 1.00.1
ml. Flow cuvets of this size permit the use of
the maximum slit area of the commercial sample
holder at the smallest practical cuvet chamber
volume.

To provide uniform temperature conditions at
the flow cuvets, water was circulated through the
jackets of the sample holders. The temperature
of the sample holders was maintained between
20 and 24 C.

For the conditions described, the fluorometer
sensitivity settings were: A; 10 X excitation
light aperture, span adjustment fully counter-
clockwise, (from the back of the instrument), B;
30 X excitation light aperture, span adjustment
fully clockwise. The limiting factor in establish-
ing fluorometer sensitivities is the response of
epinephrine. With the filter combinations used
and at the fluorometer settings indicated, the
specific fluorescence (fluorescence per ng) of
epinephrine under A conditions is nearly
identical to that under B conditions. The specific
fluorescence of norepinephrine under B conditions
however, is about twice that under A conditions.


for example, do methods that report recoveries
from eluates alone.

Values for NE and E obtained by a number
of different workers, using automated or manual
trihydroxyindole procedures, are shown in Table
6. Although, it is apparent that the reported
values differ widely, it should be realized that
none of the data cited adequately represents a
population sample. Rather than consider the
data shown as “normal” values, it is probably
more reasonable to view the levels given as repre
senting an order of magnitude of levels that have

17

been measured. The reasons for the variations
encountered in reported values are many and
may be the result of small-sample variations, non-
standard conditions surrounding the sampling
interval, the fundamental lability of the sym-
pathetic system and, of course, methodological
differences in measurement techniques. From our
evaluation, data derived by the procedure re-
ported here appear comparable in accuracy, pre-
cision and order of magnitude to results obtained
by other trihydroxyindole techniques, both auto-
mated and manual.



This difference in specific fluorescence forms the
basis for the differential assay.

To maximize sample fluorescence, a sampling
rate of 10 per hour was selected with a wash/
sample ratio of 2:1. This rate and ratio were
achieved by removing one of the lifters from a
standard 20 per hour cam and using this modified
cam to activate the sampler. In this system,
sample pickup occurs over a two-minute period
and wash (0.8 M acetic acid; pH 3.85) pickup
over a four-minute period. Using a 2:1 wash/
sample ratio permits near baseline conditions to
be achieved between samples and minimizes
sample-to-sample interference. At the sample
pumping rate used (0.80 ml/min) two minutes of
pickup required a minimum sample volume of
1.8 ml.

Til. Procedure.

Sample volumes specified in the following
description of procedure were selected by assum-
ing concentrations of NE and E ordinarily en-
countered in plasma and urine. Generally, the
total quantity of catecholamine in the sample
should be between 2.5 and 25 ng.

Plasma. Blood is drawn in_ heparinized
syringes. The sample is transferred to a
centrifuge tube coated with dry sodium metabi-
sulfite (0.5 mg/ml blood), chilled in ice and
centrifuged. Exactly 5.0 ml of plasma are trans-
ferred to a round bottom polycarbonate
centrifuge tube (16 x 100 mm; 12 ml capacity).
To the sample are added 5.0 ml stabilizing solu-
tion and 1.0 ml perchloric acid. The tube is
capped, vigorously agitated, and centrifuged at
30,000 x g (10 C) for ten minutes. The
supernatant liquid is carefully decanted into a
small beaker.

Urine. Urine is acidified as collected in the
approximate proportion of 1 ml 9N H,SO, per
200 ml urine. This schedule of acidification
should give a final pH of 3 or less. Exactly 0.5
ml of the urine sample is transferred to a round
bottom polycarbonate centrifuge tube as above.
To the urine sample are added 9.5 ml stabilizing
solution and 1.0 ml perchloric acid. The tube
is capped, thorougly shaken and centrifuged at
high speed as for plasma. The supernatant liquid
is decanted.

Standards and Reagent Blanks. Standards and
blanks are treated in the same manner as samples.

NE and E standards are prepared in duplicate.
Exactly 5.0 ml of working standard (1 ng/ml)
for NE or E are added to polycarbonate centri-
fuge tubes as above. To the standards are added
5.0 ml stabilizing solution and 1.0 ml perchloric
acid. After the solutions are mixed, the tubes
are centrifuged at high speed and the supernatant
liquid decanted. The reagent blank is prepared
similarly except that 1.0 ml perchloric acid is
added to 10.0 ml stabilizing solution.

Adsorption and Elution. As the method is
presently carried out, each set of unknowns is
accompanied by one reagent blank (0 ng), two
NE standards (5 ng) and two E standards (5
ng). In a full analytical run, nine unknowns can
be processed along with the standards and blank.

To 50 ml beakers containing 500 mg acid-
washed alumina and 10 ml 0.2 M sodium acetate,
pH 8.4, are added exactly 9.0-ml aliquots of the
supernatant liquid decanted after high speed
centrifugation. If the unknown samples are
urine, 4 ml 10% EDTA are added to each beaker ;
for plasma samples, 1 ml 5% EDTA is added.
The pH of the alumina—sample mixture is ad-
justed to 8.4:£0.1 (glass electrode) and main-
tained at that pH for 4 minutes by the dropwise
addition of 5 N sodium carbonate. By using a
small turntable (10 rpm) to rotate the beaker
and a motorized glass stirring rod, the mixture
can be agitated continuously through the pH ad-
justment so that the particles of alumina are
evenly distributed throughout the solution.
Using this arrangement for mixing avoids the
problem of pulverizing the alumina, an annoy-
ance generally encountered with the magnetic
mixing bar. After the pH has been maintained
for 4 minutes, the alumina is transferred as a
slurry to the adsorption tubes. The alumina
settles rapidly to form a uniform column in the
stem of the tube. Presently we use a small piece
of glass wool placed just above the capillary tip
to support the column. (We have tried replacing
the glass wool with 100 pore polyurethane filter
material, Belleo Glass Co., Vineland, N.J.
When this material is used to support the column,
a smooth flow of liquid is achieved and the
alumina column formed packs uniformly. Pre-
liminary tests were encouraging and indicated no
difference in the results obtained, whether the
column support was polyurethane or glass wool).
The buffer solution above the column is per-
mitted to flow through the column at the rate of



TECHNICAL REPORT STANDARD TITLE PAGE

T. Report No. 2. Government Accession No. 3. Recipient's Catalog No.
FAA-AM-71-15

4, Title and Subtitle 5. Report Date
AUTOMATED DIFFERENTIAL FLUOROMETRIC ANALYSIS OF April 1971
NOREPINEPHRINE AND EPINEPHRINE IN BLOOD PLASMA 6. Performing Organization Code
AND URINE

7. Kuthor’s) 8. Performing Organization Report No.

Vincent Fiorica, Ph.D.
Russell Moses, B.A.

9. Performing Organization Name and Address 10. Work Unit No.
FAA Civil Aeromedical Institute
P. O. Box 25082 TT. Contract or Grant No.

Oklahoma City, Oklahoma 73125

13. Type of Report and Period Covered

12, Sponsoring Agency Name and Address

Office of Aviation Medicine OAM Report
Federal Aviation Administration
800 Independence Avenue, S.W. 14." Sponsoring Agency Code

Washington, D.C. 20590

15. Supplementary Notes

16. Abstract

An automated fluorometric trihydroxyindole procedure is described for the measure-
ment of norepinephrine (NE) and epinephrine (E) in blood plasma or urine. The
method employs conventional techniques for isolation of the catecholamines by
alumina column chromatography. Column eluates are analyzed in an AutoAnalyzer
system incorporating two fluorometers. Differentiation of the amines is based
on differences in the fluorescence characteristics of the lutines of NE and E.
The accuracy (as estimated by recoveries of added amines) and precision of the
method are comparable to those reported for other trihydroxyindole techniques.
Representative values for urinary excretion and for plasma levels of NE and E
compare favorably with values stated in the literature.

17. Key Words 18. Distribution Statement
Biochemical analysis, Catecholamines, Availability is unlimited. Document may
Norepinephrine, Epinephrine, Urine, be released to the National Technical
Blood Plasma, Fluorometry, Automated Information Service, Springfield,
Analysis Virginia 22151, for sale to the public.
19. Security Classif. (of this report) 20. Security Classif. (of this page) Qi. No. of Pages | 22. Price
Unclassified Unclassified 18 $3.00

Form DOT F 1700.7 (8-69)



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Fieure 8. Diagram of the cuvet holder required for the jacketed high-sensitivity sample holder supplied by Turner
for the Model 111 fluorometer. The dimensions given for the cuvet holder are specific to accommodate the fused
quartz flow cuvet shown. The volume of the cuvet chamber is 1.0 + 0.1 ml.

6


acid/ascorbic acid system, or in differential
fluorescence systems utilizing a single fluorometer.

2. It avoids the need for changing reagents for
the AutoAnalyzer between sets of samples.

8. It is sufficiently sensitive for the analysis
of NE and E in peripheral blood plasma, yet
permits the analysis of NE and E in urine with-
out requiring changes in either the AutoAnalyzer
manifold or adjustments in fluorometer sensi-
tivities.

As in the procedure upon which this method
js based,!* the technique of batch adsorption and
column elution of NE and E has been retained
in order to maximize the recovery of the isolated
catecholamines in a minimum volume of eluate.

II. Materials and Methods.

Reagents. Reagents were prepared from
chemicals of reagent grade or highest purity
available. Solutions were prepared with twice-
distilled water, the second distillation made in an
all-glass apparatus.

1. Alumina (Woelm), neutral, activity grade 1
for chromatography. Alumina was treated by
the procedure described by Anton and Sayre.
The purified alumina was stored in a desiccator.

2. Sodium acetate, 0.2 M, purified by passing
the solution through a 25 x 300 mm column of
Chelex-100 (Bio-Rad Laboratories, Richmond,
California), 50-100 mesh, Na+ form. The pH
of the purified buffer was adjusted to 8.42£0.1
(glass electrode) with 0.5 N sodium carbonate.

8. Ethylenediamine tetraacetate, disodium, 5%
(w/v).

4, Ethylenediamine
10% (w/v).

5. Sodium acetate, 1.5 M containing 0.01%
(w/v) potassium ferricyanide. The pH of this
solution was adjusted to 9.00.1. Traces of
suspended material were removed by passing the
solution through a medium porosity fritted glass
filter.

6. Ascorbic acid, 0.8% (w/v). The Eastman
product was used without additional purification.

7, Sodium hydroxide, 2.5 M.

8. Perchloric acid, 4.4 N.

9. Acetic acid, 0.3 M.

10. Acetic acid, 0.3 M, adjusted to pH 3.85.

11. Stabilizing solution, 0.01 N HCl contain-
ing 0.5 mg/ml sodium metabisulfite.

tetraacetate, disodium,

Standards. Stock standards for NE and E
were prepared from dl-arterenol hydrochloride
and l-epinephrine bitartrate (Winthrop Labora-
tories, New York) respectively. The primary
standards for each were prepared in stabilizing
solution at a concentration of 50 pg/ml (as the
free base). All subsequent dilutions were also
made with stabilizing solution. Another set of
catecholamine stock standards containing both
NE and E in the ratio 2:1 were prepared to
contain a total of 50 pg/ml of both amines as
free bases (33.3 »g NE and 16.7 pg E per ml).
Working standards for NE, E and the combina-
tion of NE and E were prepared from dilutions
of the respective stock standards. Appropriate
dilutions were made to give working standards
of 1 ng/ml of NE or §, or in the case of the
combined standard, 1 ng total catecholamines per
ml. In routine use, only the individual stand-
ards for NE and E are used. Under usual con-
ditions 5.0 ml of each working standard were
taken as the sample; the total quantity of each
amine used as a standard was 5 ng.

Stock standards for either NE or E, prepared
in stabilizing solution, are stable under refrigera-
tion for at least 6 months. All working stand-
ards were prepared freshy before each run.

Apparatus.

1. Glass adsorption tubes for column chroma-
tography were slightly modified from those de-
scribed previously.* The tubes presently used
(Figure 1) have the following dimensions;
reservoir, 95 x 28 (I.D.) mm, stem 175 x 4
(LD.) mm. A capillary tip, 10 x 2 (LD.) mm
is sealed to the stem. The wall thickness of both
the reservoir and stem is 2 mm. Capacity of the
reservoir is 50 ml.

The adsorption tubes are used in a negative
pressure manifold which facilitates washing of
the column. The flow through each tube is con-
trolled independently so that uniformity of flow
from tube to tube can be maintained. One
analyst can conveniently handle 14 tubes in such
a manifold system.

2, Automated analysis was achieved with a
standard AutoAnalyzer system consisting of a
sampler (II), pump, two fluorometers (Turner,
Model 111) and a two-pen recorder. A schematic
flow diagram of the system is shown in Figure 2.
In this scheme, sample -(column eluate, pH 3.85)
is mixed with ferricyanide in acetate buffer (pH


80

70

60

& 8

FLUORESCENCE UNITS
$
T

Lo
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Jo

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20

5 1 1 1

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FILTER SET B

s

a
—

1

So
a
ar

FILTER SET A

vy

25 5.0 75

10.0
TOTAL CATECHOLAMINES

12.5 15.0 17.5 20.0
nanograms per sample

Ficure 8. Relationship between amounts of total catecholamine (NE + E) in starting sample and fluorescence in-
tensity with filter sets A and B. The volume of sample was 5.0 ml in all instances and the samples were carried
through the entire procedure. The ratio of catecholamines in each sample was 2 NE:1E. Points represent the
means of five measurements; the flags represent the standard deviation.

standard the sensitivities are intermediate to the
sensitivities obtained under both A and B con
ditions with standards containing only NE or E.

Recovery data are given in Tables 1-3. Several
aspects of the recovery of NE and BE, added in
physiological amounts to samples of plasma or
urine, were examined in order to evaluate the
accuracy of the procedure. To provide a point
of reference, we first examined the simplest case,

the recovery of added NE and E from an aqueous
solution containing both amines in amounts likely
to be found in peripheral plasma (Table 1).
Under this set of conditions added NE and E
were recovered to about the same extent (NE,
91.2% ; E, 98.1%). In a second series of recovery
experiments, we examined the effect of adding
two levels of each amine and the effect of adding
both amines together on recovery from canine



mens ranged from 2.7 to 4.1 (mean 3.2). Values
for the 24-hour excretion of the catecholamines
in this sample of men are given in Table 4. So
that comparisons may be made with the existing
literature, the data for urinary excretion have
been expressed in the three most common forms,
ng/24 hours, ng/mg creatinine and ng/minute.
Results of multiple analyses for NE and E in
canine arterial plasma are summarized in Table 5.

Although such data are not intended as an esti-
mate of the population means for NE and E
levels in canine plasma, they do indicate the
plasma concentration of the catecholamines that
can. be anticipated and give an approximation of
the variability encountered. For this study
dogs were prepared with chronically indwelling
arterial catheters. Blood samples were taken
from the catheter in nonanesthetized animals,

Taste 4.—24 Hour Urinary Excretion of NE and E of Twelve Male Laboratory Workers (Mean and Standard Deviation)

Quantity Per Unit Creatinine Per Unit Time
pg/24 hours ng/mg creatinine ng/min
45.7411.5 24,046,1 31.748.0

9.14 1.7 4.8412 6341.2
54.84£12.7 28.847.0 38.04£8,8

avoiding both the effects of anesthetics and the
trauma of venipuncture on the catecholamine
levels. The animals were permitted an adequate
period of recovery (several days) between sam-
plings. The red cell volume was measured daily
(hematocrit) to monitor the effects of repeated
blood sampling; no changes were detected.

The precision of the method was evaluated
from multiple analyses on a single sample of
urine or plasma. With urine, 18 measurements

of a single sample yielded the following informa-
tion: NE; 14.9 + 1.8 (mean and 8.D.) ng/ml,
C.V. (Coefficient of variation), 8.9%, E; 5.6 +
0.5 ng/ml, C.V., 9.0%. Eighteen measurements
on a single sample of plasma gave: NE, 0.80 +
0.09 ng/ml, C.V., 11.8%, E; 0.35-£0.06 ng/ml,
C.V., 16.5%. As expected, measurements of
sample containing smaller amounts of the amines
showed a slightly greater variability.

TaBLE 5.—Multiple Measurements of NE and E in Canine Arterial Plasma from Four Dogs (Mean and Standard

Deviation)
Dog No. No. of Observations NE E NE+E
N ng/ml ng/ml ng/ml
8 0.5340.13 0.1540.08 0.6840. 09
23 0.67+0.17 0.0540. 04 0.7240.17
43 0. 5140. 22 0.08+0.05 0.5940. 20
23 0.5640, 11 0.03 0.03 0.59+0.11
97 0. 5640.19 0.07+0.06 0. 6340.18
V. Discussion. can analyze nine unknown samples (and five

None of the automated methods that have been
developed thus far for the differential estima”
tion of NE and E in plasma or urine are as rapid
or direct as one might wish for studies involv-
ing large numbers of samples or for the clinical
laboratory. The technique outlined in this paper
is no exception. One operator working alone

15

standards) per day. Two operators with equiv-
alent training and working together can process
six standards and 22 unknown samples per day.
The merit of the scheme proposed resides in the
reduction of the number of manipulations re-
quired to conduct the automated portion of the
assay, and in the ease with which the procedure



AUTOMATED DIFFERENTIAL FLUOROMETRIC ANALYSIS OF
NOREPINEPHRINE AND EPINEPHRINE IN BLOOD PLASMA AND URINE

I. Introduction.

During the past several years an increasing
number of reports has appeared in the literature
describing the application of the AutoAnalyzer
(Technicon Instruments Corp., Tarrytown, N.Y.)
system to the analysis of the catecholamines,
norepinephrine (NE) and epinephrine (E).
This trend seems to indicate a growing interest
in modifying or adapting existing manual
techniques for NE and E to the more routinely
manageable AutoAnalyzer procedures. The de-
velopment of such automated analytical systems
has given this difficult analysis much broader
appeal for general clinical usage or for studies
involving large numbers of samples.

Aviation studies dealing withthe problems of
stress and fatigue frequently require measure-
ments of adrenal catechol hormones in order to
evaluate the magnitude of response to the stressor
under study. Because this analysis is an intricate,
demanding and time-consuming procedure, the
additional requirement for large numbers of
samples (as in work shift rotation studies,
circadian rhythm studies and time-zone crossing
studies) intensifies the problem of keeping
chemical analysis time commensurate with the
time required for the analysis of other data
acquired. A practical solution seems to be the
automation and simplification, to the extent
possible, of this complex analysis to reduce exces-
sive manipulation and to increase the speed and
accuracy of the test. The method described in
this paper was devised to meet those needs.

Of the automated techniques presently avail-
able (1-10) all are based, completely or in part,
m the methodology proposed by Merrills in
1962.12, The key feature of Merrills’ differential
scheme is the use of a solution (1% v/v) of
thioglycolic (mercaptoacetic) acid as a stabilizing
reagent. After oxidation of both NE and E ina

sample eluate by ferricyanide at pH 6.0, only the
fluorescent noradrenolutine is stabilized in the
presence of thioglycolic acid. The NE in the
sample is thereby measured directly. If
thioglycolic acid is replaced by ascorbic acid
(0.3% v/v) and a second aliquot of the sample
eluate is analyzed, both the noradrenolutine and
the adrenolutine are stabilized and the total
NE + E in the sample is measured. The E con-
tent of the sample is then obtained by difference.
Although several modifications of Merrills’ pro-
cedure have been proposed, these have been con-
cerned chiefly with improving the sensitivity of
the assay.

The present method is based on the differential
system of Cohen and Goldenberg  ”? a procedure
used successfully in its original form, but which,
until now, has not been adapted for automated
analysis. Differential analysis of the catecho-
lamines is achieved in the Cohen-Goldenberg
scheme by utilizing differences in the fluorescence
characteristics of noradrenolutine and adrenolu-
tine. By expanding our existing system for
automated analysis of total catecholamines,*
differential fluorometry could be achieved with-
out further modification. This was accomplished
by the simple expedient of adding, in series with
the existing fluorometer (Turner, Model 111), a
second fluorometer, differing from the first only
in the wave lengths selected by the primary and
secondary filter combinations.

The system described in this paper offers
several advantages over existing automated
procedures :

1. It permits the use of a smaller initial sample
because it eliminates the need for dividing the
eluate (from the adsorption step) for the two
runs required on the AutoAnalyzer in the dif-
ferential oxidation/pH system, the thioglycolic



ACKNOWLEDGMENTS

The authors thank Floyd Passmore for technical support and Miss
Patsy Fowler for assistance with some of the analyses. The contributions of
Miss Betty Gatliff and William V. Flores for the research data drawings and
of Mrs. La Donna Cook, Mrs. Dickie Price, and Mrs. Freida Ditto for their
work on the manuscript are gratefully appreciated.


was run through the full procedure. The recovery
was stated as:
Quantity measured Quantity measured

in sample with — in sample without
added amine added amine

% Recovery = Quantity of amine added

xX 100

IV. Results.

Typical recordings from the AutoAnalyzer
system for graded sample quantities of NE and
E are shown in Figures 4 and 5. The first peak
(reading from right to left) of each pair repre-
sents sample fluorescence from the A filter set;
the second peak represents sample fluorescence
from the B filter set. In practice the two chan-
nels of recording are distinguished from each
other by use of differently colored inks.

Standard curves for NE, E and a combined
standard containing both catecholamines in the
ratio of 2NE : 1E are shown in Figures 6, 7,
and 8. In all of these curves each point repre-

sents the mean of five separate analyses of stand-
ard carried through the complete procedure. Over
the range 0.0 to 20.0 ng/sample, fluorescence is
linearly related to the amount of NE in the
sample for both the A and B filter sets. With
filter set B (and B fluorometer sensitivity) NE
fluorescence is about twice that obtained with
filter set A (and A fluorometer sensitivity) per
ng NE. With standards containing E alone,
fluorescence with A and B filter sets is also
linearly related to the quantity per sample (Fig.
7). For E, however, the sensitivity (slope)
under A conditions is approximately the same
as under B conditions over the range 0.0 to 17.5
ng per sample.

With standards containing both catecholamines
(2NE : 1E) fluorescence under A and B condi-
tions is also a linear function of the total quantity
of amine in the sample over the range 0.0 to
20.0 ng per sample. In the case of the combined

50 r 7 T T T 1 T T

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NOREPINEPHRINE (NE) nanograms per sample

Ficure 6. Relationship between amount of NE in starting sample and fluorescence intensity with filter sets A and
B. The volume of sample was 5.0 ml in all instances and the samples were carried through the entire procedure.
Points represent the means of five measurements; the flags represent the standard deviation.

11



Tasix 1.—Recovery of a Mixture of NE and E from an Aqueous Sample (5.0 ml) Containing 3.33 ng NE and 1.67 ng E

Number of Recoveries Quantity Added (ng) *+Quantity Recovered (ng) *Recovery (%)
NE E NE E NE E NE E
15 15 3.33 1.67 8.1240.64 1.4740.27  91.2423.6  93.1419.2

*Mean and standard deviation

plasma (Table 2). Adding 5.0 or 10.0 ng of each
amine to samples of canine plasma yielded re-
coveries of approximately 81% for NE and 95%
for E. When the NE and E were added together,
but in smaller quantities, the recovery of E re-
mained at about 95% but the NE recovery de-
creased to about 70%. In a third series, we
evaluated the recovery of the amines from urine
(Table 3). NE or E added to urine at a single
level (5.0 ng) was recovered to the extent of
about 80% for NE and about 94% for E. When
the amines were added to the urine sample to-

gether, the recoveries for NE and E respectively
were 101% and 76% at the low level (5.0 ng
total) and 85% and 98% at the high level (10.0
ng total). It appears that the greatest vari-
ability in recovery is encountered when attempt-
ing to recover small quantities from samples
already containing relatively larger amounts of
the amines. This is especially true for recoveries
from urine. The overall means for recoveries
of added NE and E from 130 separate analyses
were 82.9 + 22.1 (S.D.) % for NE and 93.1 +
13.4 (S.D.) % for E.

Tasim 2.—Recovery of NE and E Added Individually or Together to Canine Plasma (5.0 ml)

Number of Recoveries Quantity Added (ng)

*Quantity Recovered (ng)

*Recovery (%)

NE E NE E NE E NE E

32 32 5.0 5.0 4,0341.11 4.7740. 60 80.74+22.2 94.8411.1
16 16 10.0 10.0 8.06+0.99 9.6340.32 80.629.9 96.343.2
20 20 ¥3,.33, **1,67 2,230.77 1.6140.20 67.14 23.1 96.4411.8

*Mean and standard deviation
** Added together

To compare values for NE and E obtained by
the present method with data reported in the
literature, measurements were made of the 24-
hour urinary excretion of NE and E in man and
of the arterial plasma levels in the dog. Values re-
ported were not corrected for recovery. Twenty’

four hour urine specimens were collected from
12 men of similiar work routines (laboratory
personnel). The urine specimens were kept well-
acidified through the collection period by the
prior addition of 6 ml 9N H,SO, to the collect-
ing vessel. The final pH of the 24-hour speci-

‘Tanz 3.—Recovery of NE and E Added Individually or Together to Human Urine (1.0 ml)

Number of Recoveries Quantity Added (ng)

¥Quantity Recovered (ng)

*Recovery (%)

NE E NE E NE E NE E

16 16 5.0 5.0 4,0140.78 4.6940. 41 80.2415.5 93.848.4
15 15 3.33, **1,67 3.3740.79 1.2640.27 101.14.23.8 75,5415.9
16 16 #6, 67 #4333 5,824 1.23 3.2840. 28 87.2418.5 98.448.4

*Mean and standard deviation
** Added together

14