ilton syringe. For plasma assays, 0.50 ml. of
butyrylcholine iodide solution (0.163M) was
added with a long-tipped glass pipet. These
quantities of substrate were chosen to give a
concentration in the optimum activity range and
still furnish adequate substrate to yield zero-
order kinetics for at least 3 minutes.

Calculations
All activities were expressed as micromoles of
substrate hydrolyzed/minute/ml. of sample.
Calculations were made as follows:
Activity= (S.D./min. ) (0.025) (N) (1,000) (D.F.)
where
S.D.=recorder scale divisions (titrant volume
axis).
min.=minutes corresponding to $.D. on “time”
axis.
0.025= ml. of titrant per S.D.
N=normality of titrant.

D.F.=dilution factor.
D.F, for whole blood is 5.
D.F. for plasma is 10.
D.F. for RBC assay is 5/Het
where

Het is the fractional hematocrit value of the whole
blood.

III. Results and Discussion.

The results of the storage studies are presented
in Figures 1-3. Each point on the graphs rep-
resents the mean of 1 to 10 samples assayed in
duplicate, with the vertical bars representing
-tone standard deviation.

Whole blood :

Whole blood hemolysates maintained 90% of
their original cholinesterase activity for about
6 days when stored at room temperature. When
stored either refrigerated or frozen, they main-
tained at least 90% activity for 50 days.

Those whole blood hemolysates which were
stored frozen occasionally appeared to form a
stringy, gelatinous precipitate on subsequent
thawing. The presence of this precipitate made
it difficult to pipet homogeneous samples.

An interesting and unexplained aspect of the
storage of whole blood was the apparent increase
in activity during the first four days, regardless
of storage temperature, a phenomenon which has
been noted previously’**. Although the aver-

age increase in activity was only 6-8%, with
some individual samples it was as high as 14%.
This increased activity could be the result of any
of several processes.

The erythrocyte enzyme is known to be bound
to the cell membrane. To our knowledge, no
determination has been made of the relative
specific activity of the enzyme in the bound and
solubilized forms, nor of the rate of enzyme
solubilization under different conditions. If the
soluble form of the enzyme has a faster reaction
rate (a larger Vinx) than the bound form, which
would not be illogical, then the observed increase
in activity could be the result of enzyme solu-
bilization.

Other possibilities include an activation of the
enzyme due to conformational changes produced.
by some aspect of the treatment. Also, it has
been established that both the erythrocyte and
plasma cholinesterase molecules are composed of
dissociable sub-units'** which are capable of
reaggregating into multiple molecular forms
(isozymes). The exact sub-unit composition of
these reaggregates has been shown to be influ-
enced by pH, ionic strength, temperature, and
enzyme concentration’, It is not inconceivable
that the treatment and storage conditions in this
study might have favored formation of an iso-
zyme population having an increased reaction
rate.

Plasma:

Cholinesterase activity in the plasma samples
exhibited ‘a remarkable stability when either
refrigerated or frozen; activity remained above
90% for the entire 50-day storage period. At
room temperature, the activity appeared to be
stable for 6 days and then decreased steadily to
about 40% after 3 weeks. There was no differ-
ence in the activity of plasma samples stored
undiluted or diluted 1:10 with Ringers; there-
fore, both values are included in the data.

No difficulty was experienced with the forma-
tion of fibrin strands or any other precipitates
in the stored samples which had been adequately
heparinized.

fed blood cells:

These hemolysates exhibited a very rapid loss
of cholinesterase activity when stored at room
temperature; the activity decreased to about 80%
in the first 24 hours. Refrigeration of the



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REFERENCES

E., Stedman, E, and Easson, L. Ha:
Choline-Esterase: Enzyme Present in Blood-Serum
of Horse. Biochem. J., 26, 2056 (1932).

. Glick, D.: Properties of Choline Esterase in Human

Serum. Biochem. J., 31, 521 (1937).

. Nabb, Dale P. and Whitfield, Florence: Determina-

tion of Cholinesterase by an Automated pH Stat
Method. Arch. Hnviron. Health, 15, 147 (1967).

, Callaway, S. and Davies, D. R.: Blood Cholinesterase

Levels and Range of Personal Variation in a Healthy
Adult Population. Brit. Med. J., 2, 812 (1951).

. MacDonald, W. E. Jr., Pollard, ©. B. and Gropp,

A. H.: A Rapid Micromethod for Electrometric
Determination of Red Cell Cholinesterase Activity
in Whole Blood. Arch. Ind. Health, 5, 271 (1952).
Strehler, Von EH. and Meyer, H.:
Cholinesterase bei Gesunden und Kranken,
Med. Aeta, 19, 555 (1952).

Die Plasma-
Helv.

. Hobbiger, F.: Chemical Reactivation of Phosphory-

lated Human and Bovine True Cholinesterases.
Brit. J. Pharmacol., 11, 295 (1956).

. Biggs, H. G., Carey, Shirley and Morrison, D. B.:

A Simple Colorimetric Method for Measuring Ac-
tivities of Cellular and Plasma Cholinesterase.
Amer. J. Clin. Pathol., 30, 181 (1958).

9.

10.

ni.

13.

14,

Wendell T.: Photometric Determination
Amer. J. Clin.

Caraway,
of Serum Cholinesterase Activity.
Pathol., 26, 945 (1956).

Kalow, W., Genest, K. and Staron, N.: Kinetic
Studies of Benzoylcholine by Human Serum Cholin-
esterase. Can. J. Biochem. and Physiol., 34, 637
(1956).

Walford, Roy L., Sowa, Masako and Daley, Donald:
Stability of. Protein, Enzyme, and Nonprotein Con-
stituents of Stored Frozen Plasma. Amer. J. Clin.
Pathol., 26, 376 (1956).

. Augustinsson, Klas-Bertil and Heimburger, Gunilla:

‘The Determination of Cholinesterase Activity in
Blood Samples Absorbed on Filter Paper. Acta
Physiol. Seand., 30, 45 (1954).

Main, A. R.: Kinetic Evidence of Multiple Reversible
Cholinesterases Based on Inhibition by Organophos-
phates. J. Biol. Chem., 244, 829 (1969).

LaMotta, Rudolph V., McComb, Robert B. and
Wetstone, Howard J.: Isozymes of Serum Cholin-
esterase: A New Polymerization Sequence. Oan. J.
Physiol. Pharmacol. 43, 813 (1965).

. Grafius, Melba A. and Millar, David B.: Reversible

Aggregation of Acetylcholinesterase. II. Inter-
dependence of pH and Ionic Strength. Biochemistry,
6, 1034 (1967).



hemolysates allowed storage for 6 days before
the 80% level was reached, and freezing main-
tained the activity above 90% for 50 days. As
was observed with whole blood preparations,
freezing of the RBC hemolysates produced a
gelatinous substance which sometimes interfered
with sampling.

Only in the case of frozen RBC hemolysates
was there evidence for an increased activity in
the first few days of storage. This is interesting
when compared with the behavior of whole
blood samples, since most of the measured
activity in whole blood is due to the erythrocyte
enzyme when acetylcholine is the substrate.
Another related anomaly is the observed differ-
ence in stability of the RBC and whole blood
activities when storage is at room temperature.
Once again, since the same enzyme is being
measured in both cases, the obvious difference
in stability is hard to explain. A possible factor
is the presence of stabilizing elements in the
plasma portion of whole blood which decrease
the rate of denaturation of solubilized erythro-
eyte enzyme.

Assay of samples prepared from stored whole
blood:

We have obtained preliminary information on
the storage stability of untreated whole blood.
Two blood specimens were stored without treat-
ment at each of the experimental temperatures.
At intervals during the storage period, RBC and
whole blood hemolysates were prepared from
these stored samples and assayed in duplicate.
After the first few days of storage, it became
difficult to obtain homogeneous whole blood
hemolysates due to red cell agglutination and
formation of a stringy sediment. It was also
difficult to prepare a red cell hemolysate as an
accurate aliquot of the whole blood because of
this agglutination and because of the unknown

relationship between enzyme activity and hema-
tocrit in an appreciably hemolyzed sample.
However, the data indicate that values obtained
during the first week of storage were at least
as high as for samples which were stored as
hemolysates. We, therefore, believe that useful
clinical values can be expected if the assays are
performed within the time limits discussed for
stored hemolysates, namely: for RBC’s, 12 hours
at room temperature or 7 days refrigerated or
frozen; for whole blood, 7 days at any of the
three temperatures.

IV. CONCLUSIONS

From a clinical viewpoint, we consider assay
values to be useful as long as we can expect
them to represent at least 90% of the original
activity. Therefore, we have selected this value
of 90% as a lower limit and store samples under
conditions which will insure at least this level of
activity at the time of assay.

These conditions allow storage of red cell
hemolysates for less than 12 hours at room tem-
perature, up to three days at 4° C., and up to
6 weeks at —20° C. Whole blood hemolysates
and plasma may be stored for 6 days at room
temperature and in excess of 6 weeks if refrig-
erated or frozen.

We recommend that samples for shipment be
drawn into heparinized tubes, adequately mixed,
and refrigerated until shipment. Shipment
should be made in an insulated container con-
taining sufficient ice to last during transit. If
the samples are to be shipped frozen, one-half
of each whole blood sample should first be sepa-
rated into plasma and red cells, and these three
preparations placed in separate containers; other-
wise the hemolysis resulting from freezing whole
blood prevents a separate assay on plasma and
renders the accurate measurement and assay of
red cells more difficult.


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AM 70-4

STUDIES ON THE STORAGE STABILITY OF HUMAN
BLOOD CHOLINESTERASES: I

Charles R. Crane, Ph.D.
Donald C. Sanders, M.S.
John K. Abbott, B.A.

Approved by Released by
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Curr, Civ AEROMEDICAL Feprran Ai Surcrow
InstrruTE

January 1970

Department of Transportation

FEDERAL AVIATION ADMINISTRATION
Office of Aviation Medicine





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constant-pH titrations, and consisted of the
following units:

PHM26—expanded scale pH meter.
TTT 11b—electronic titration control unit.
ABUtb—semi-micro automatic buret for
titrant delivery, 2.5 ml. capacity.
TTA3—glass-jacketed reaction vessel (3
ml.) with plastic-coated magnetic
stirring disc.
VTS18c—constant-temperature water bath
and circulator, set to maintain
25° C. in reaction vessel.
SBR2c—recorder for indicating volume of
titrant delivered per unit time.

Blood collection:

Blood for studies lasting more than 7 days was
secured by advance arrangement with a local
blood bank. Processing began within 2 hours
after collection in heparinized, 500 ml. vacuum
bottles. Blood for short-term studies was drawn
from laboratory personnel into heparinized
Vacutainers (Becton, Dickinson & Co., Ruther-
ford, N.J.) and processed immediately.

Preparation of storage samples :

Fresh, whole blood, with sufficient solid hep-
arin added to prevent clotting over the planned
storage period, was added to a volumetric flask
five times the size of the blood aliquot. To each
flask was added 20% Triton X-100 in the ratio
of 0.05 ml. per ml. whole blood. The contents
were mixed thoroughly, allowed to stand for 8
to 5 minutes and diluted to volume with Ringers
salts solution. Duplicate 1-ml. samples of this
whole blood hemolysate were assayed immediately
to determine the “zero-day” activity. The re-
mainder of the hemolysate was divided into three
parts and each stored at a different temperature,
namely, room temperature (23°-94° C.), re-
frigerator temperature (4° C.) and freezer tem-
perature (—20° C.).

Plasma samples were collected from fresh,
heparinized, whole blood after centrifugation at.
800 X g for 10 minutes and were diluted 1:10
with Ringers salts solution. This diluted plasma
was divided for immediate assay and for storage
at the three temperatures designated for whole
blood.

Red blood cells were collected from fresh,
heparinized whole blood by centrifuging at 800 X

g for 10 minutes, carefully removing the plasma
with a Pasteur pipet, resuspending the cells in
Ringers salts solution and recentrifuging. This
washing was repeated three times. Triton X-100,
20%, (0.05 ml. per ml. whole blood) was added
to the washed cells and mixed by gently touching
to a rotary mixer (SUPER-MIXER, Matheson
Sci.). The resulting hemolysate was quantita-
tively transferred to a volumetric flask five times
the volume of the whole blood sample and di-
luted to volume with Ringers salts solution.
Aliquots of the diluted hemolysate were prepared
for immediate assay and for storage at the three
temperatures designated for whole blood.*

Assay Procedure:

One ml. of the enzyme solution (whole blood
hemolysate, diluted plasma or RBC hemolysate)
was added to the reaction vessel, the magnetic
stirrer was turned on and the solution was al-
lowed to come to temperature equilibrium
(25° C.). The pH was adjusted to 8.10 by ad-
dition of dilute aqueous NaOH from a Pasteur
pipet for whole blood or RBC, for plasma the
adjustment was made with the automatic titrator.
The appropriate type and volume of substrate
(see below) was added and the titrator and re-
corder chart drive turned on.

The reaction was allowed to continue until the
recorder had plotted a straight line (titrant
volume vs time) for a minimum of 3 minutes or
until a titrant volume of 0.5 ml. had been
added.**

Substrates:

For whole blood and RBC assays, 100 micro-
liters of acetylcholine perchlorate solution
(0.102M) was added with a 250 microliter Ham-

*The activity of 1 ml. of this hemolysate multiplied
by a dilution factor of five gives the activity of the
volume of red blood cells contained in 2 ml. of whole
blood. Multiplication of this value by 1/hematocrit
yields the activity of exactly 1 ml. of packed red blood
cells. This technique was used in order to avoid the
direct measurement and quantitative transfer of packed
red blood cells.

** Since full scale on the recorder (100 scale divisions)
represents 2.5 ml. of titrant, 0.5 ml. represents 20 scale
divisions. The recorder trace can be read to +0.2 scale
division; therefore, the precision in reading titrant
volume is +1%. With a chart speed of 2 cm. per min.,
the precision in reading elapsed time of 3-min. trace is
also +1%.



STUDIES ON THE STORAGE STABILITY OF HUMAN BLOOD
CHOLINESTERASES: I.

I. Introduction.

The widespread use of organophosphate pesti-
cides by aerial and surface applicators has
necessitated the development of methods for
assessing the degree of exposure to these toxic
chemicals. The principal biochemical action of
this class of compounds is the inhibition of
hydrolytic enzymes, such as the cholinesterases;
therefore, the measurement of blood cholinesterase
activity has become the standard clinical method
for evaluating exposure. Although many tech-
niques are used for assaying blood cholinesterase,
the method of titration at constant pH (pH-Stat)
is one of the most preciset ? *.

It has been recommended that aerial appli-
cators have periodic cholinesterase determinations
during the spraying season, and particularly if
a known spill occurs, but the scarcity of clinical
laboratories equipped to perform this test often
dictates shipping blood samples elsewhere for
this analysis. Changes in cholinesterase activity
which occur during such shipments, or during
storage, are of some concern but have received
little consideration. The interpretation of cho-
linesterase values in blood stored or shipped
under unknown conditions is questionable be-
cause subnormal values may be due to either
pesticide poisoning or enzyme denaturation.

The present study resulted from two problems
which had confronted our laboratory period-
ically: (1) Receipt of blood samples shipped by
a physician who was unable to obtain cholin-
esterase assays in his locale, and (2) the receipt
of more samples (freshly-drawn or not) than
could be assayed in one day, requiring their
storage overnight, over a weekend, or occasionally
for longer periods. Most storage studies have
dealt with changes in activity over periods of a
few hours to a few days‘**7*, and many of the
earlier studies used less precise methodology

than is currently available’ *°*. We have, there-
fore, examined the changes in cholinesterase
activity over a longer period of time and under
various storage conditions.

IL. Materials and Methods.

Reagents:

1. Acetylcholine perchlorate (K and K
Laboratories, Plainview, N.Y.) was dried over-
night at 110° C. and stored in a desiccator over
Drierite. Aqueous stock solution was 0.102M
(25 mg./ml.) prepared fresh weekly and stored
at 4° C.

2. Butyrylcholine iodide (Mann Research
Laboratories, New York, N.Y.) was dried over-
night at 110° C. and stored in a desiccator over
Drierite. Aqueous stock solution was 0.163M
(49 mg./ml.), prepared fresh weekly and stored
at 4° C.

3. Triton X-100 (Rohm and Haas, Phila-
delphia, Penna.) ; a 20% (v/v) aqueous solution
was prepared and stored at room temperature.

4. Sodium hydroxide, standard solution,
0.005N. Aqueous stock solution of approximate
normality was prepared using CO,-free distilled
water. Solution was stored in titrant reservoir
of pH-Stat and protected from CO, absorption
by a drying tube filled with Mallcosorb, 30-50
mesh, indicating, CO, absorbent (Mallinckrodt).
This solution was standardized daily by titration
with potassium hydrogen phthalate (primary
standard grade).

5. Ringers salts solution: 0.9 g. NaCl, 0.03
g. KCl, 0.025 g. CaCl, 0.02 g. NaHCO, per
100 mi. distilled water. :

Titration equipment:
Radiometer pH-Stat (Radiometer A/S;
Copenhagen, N.V., Denmark) was used for all



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