IV. Pfizer ChE-tel Method.

The Pfizer method is a colorimetric determina-
tion based on Garry and Routh’s‘ modification
of the earlier work of Ellman.’ In this reaction,
the artificial substrate, propionylthiocholine, is
hydrolyzed by cholinesterase and the released
thiocholine displaces the yellow anion of 5-thio-
2-nitrobenzoic acid from 5,5’-dithiobis-2-nitro-
senzoate (DTNB). The rate of color production
s measured at 410 nm. Quinidine sulfate, an
nhibitor, is used to stop the reaction at the end
of the incubation period.

Materials and Method. All materials used for
he assay were supplied in the Pfizer #5360
ShE-tel Test Set (Pfizer diagnostics, 300 West
3rd St., New York, N.Y. 10036) and the method
vutline supplied with the kit was followed.
3pectrophotometric measurements were per-
ormed using a Beckman DU monochromator at-
ached to a Gilford Model 222 photometer and
“ower supply.

Individual assays were performed by adding
Onl of plasma to a tube containing 4.0 ml of
uffered DTNB (0.1 mg/ml DTNB in 0.05M Tris
uffer, pH 7.40, with ionic strength increased to
153 with NaCl) previously equilibrated at 37°

With the tube immersed in the incubation
ath, 0.50 ml of substrate (propionylthiocholine
rdide, 0.028M) was added and mixed. After
xactly 3 minutes incubation, 1.0 ml of quinidine
ufate (0.5% w/v) was added to stop the re-
stion and the absorbance was read at 410 nm.

aro absorbance calibration was provided by a
ank prepared exactly as described for the
mple except that the quinidine sulfate was
lded prior to the substrate.

ChE-tel units were calculated by multiplying
e absorbance by 120, the conversion factor for
)-mm cuvettes. Calculated ChE-tel units over
e range of 9.5 to 90 were plotted against pH-
‘at units obtained for the same plasma samples
sayed simultaneously. The linear regression
uations and correlation coefficients were cal-
lated, each point representing the mean of
tplicate assays of the same sample by each
athod. The regression of pH-Stat units on
iE-tel units is shown in Figure 2.

Caraway Method.

The Caraway method is a colorimetric pro-
lure utilizing the acid-base indicator, phenol
i, to measure the reduction in pH which accom-

panies the hydrolysis of acetylcholine.’ The
phenol red absorbance is decreased as the acetic
acid produced lowers the pH of the buffered re-
action mixture. The ratio of the final to initial
absorbance in each sample is compared to a
standard curve, similarly prepared by substitut-
ing known quantities of acetic acid for the
substrate. The method requires equipment ordi-
narily possessed by a clinical laboratory and the
reagents are relatively stable.

Materials and Method. Acetylcholine perchlo-
rate (KX & K Laboratories, Plainview, N.Y.) was
dried overnight in a vacuum desiccator; a 0.663M
solution was prepared by dissolving 4.074 g of the
reagent in distilled water and diluting to 25 ml.
The buffer-indicator mixture was prepared by
grinding 100 mg phenol red with 28 ml of 0.01N
NaOH until dissolved; then 10 ml of the phenol
red solution was added to a solution containing
6.65 g of anhydrous Na,HPO, and 0.43 g of
anhydrous KH.PO,, and diluted to a final volume
of 1 liter with distilled water.

For each assay, duplicate tubes containing 0.5
ml of plasma and 5.0 ml of the buffer-indicator
solution were allowed to come to temperature
equilibrium for 10 minutes in a 25° C. water
bath. The substrate was added (0.5 ml), mixed,

n WR OA N ®@ © O
T

pH - Stat Units (~moles/min/ ml)
T

a ee a a DD
10 20 30 40 50 60 70 80 90 100
Pfizer ChE-Tel Units

°

Ficure 2. Correlation between pH-Stat units and Pfizer
ChE-Tel units. N=25 points; correlation coefficient =
0.980



REFERENCES

. Crane, Charles R., Sanders, Donald C., and Abbott,
John K.: A Comparison of Three Serum Cholin-
esterase Methods, Dept. of Transportation, Office of
Aviation Medicine Report No. AM-70-13, August 1970.

. Michel, Harry 0.; An Electrometric Method for the
Determination of Red Blood Cell and Plasma Cholin-
esterase Activity; J. Lab. Clin. Med. 34:1564, 1949.

. Larson, E. F.: Microclectromeiric Method for Blood
Cholinesterase Determinations; Div. of Labs., Calif.
State Dept. of Public Health, Oct. 22, 1957.

. Garry, Phil J. and Routh, Joseph I.: A Micro Method
for Serum Cholinesterase, Clin. Chem. 11:91, 1965.

5.

n

Ellman, George L., Courtney, K. Diane, Andress,
Valentino, Jr., and Featherstone, Robert M.: A New
and Rapid Colorimetric Determination of Acetylchol-
inesterase Activity. Biochem. Pharmacol., 7:88, 1961.

;. Caraway, Wendell T.: Photometric Determination of

Serum Cholinesterase Activity. Am. J. Clin Pathol
26 :945, 1956.
Braid, Phillip B, and Nix, Maatje S.: Modification of
a Test Paper Procedure for Determination of Humar
Serum Cholinesterase Activity. Arch. Environ. Healtt
17 :986, 1968.


Aliquots of samples were diluted 1:10 with
Ringer's salts solution. One ml of the diluted
plasma was added to the reaction vessel, the
magnetic stirrer was started and the solution was
allowed to come to temperature equilibrium
(25° C.). The pH was adjusted with the auto-
matic titrator to the pre-set value of 8.1. One-
half ml of butyrylcholine iodide substrate was
added to the reaction vessel using a 1.0 ml
tuberculin syringe and the recorder chart drive
was turned on. The reaction was allowed to con-
until the recorder had plotted a straight
line (titrant volume vs time) for a minimum of
3 minutes, or until a minimum titrant volume
of 0.5 ml had been added.*

Calculations:
All activities were expressed as micromoles of
substrate hydrolyzed/minute/ml of plasma.

Calculations were meade as follows:

Activity = S.D. (0.025) x (N) x (1000) X (D.F.)

t
recorder scale divisions (linear portion)
ime in minutes for linear portion of trace
moles/mmole

i of titrant per S.D.
ormality of titrant
dilution factor (ie. 10)

Il. Michel Method.

The Michel method is an electrometric determi-
nation based on the measurement of the acid
produced by the action of ChE on acetylcholine.?
The acetic acid produced is measured in terms of
the change in pH produced in a buffer-substrate
mixture during a specific incubation period. The
decrease in ChE activity with decreasing pH is
taken into consideration by selecting a buffer
whose capacity closely parallels the activity loss
over the range from pH 8 to pH 6; the buffering
effect of plasma is minimized by sufficient dilu-
tion (1:111). As performed in this laboratory,
the basic method of Michel? as modified by
Larson * was used.

Materials and Method. Acetylcholine perchlo-
rate (IK & K Laboratories, Plainview, N.Y.) was
dried overnight in a vacuum desiccator over
Drierite and stored therein until used. The
substrate was prepared by dissolving 0.405 g of

Where S.D.
t

*Since full scale on the recorder (100 scale divisions)
represents 2.5 ml of titrant, 0.5 ml is represented by 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%.

the salt in a final volume of 10.0 ml of water
This solution is stable for up to four weeks i
refrigerated.

A buffer stock solution was prepared by dis
solving 4,124 ¢ sodium barbital, 0.545 g KH.PO
and 44.730 g KCI in distilled water and dilutin;
to a final volume of 200 ml. Immediately befor:
use, the plasma buffer was prepared by dilutin;
6.4 ml of the stock solution with 75 ml o
distilled water; the pH was adjusted to 8.00 wit)
0.1N HCl and the solution adjusted to a fina
volume of 100 ml with distilled water.

Plasma aliquots of 20 pl were withdrawn usin;
a, Sahli pipet and expelled into 12-ml test tube
containing 1.0 ml of distilled water. One ml o
buffer solution was added and the mixture wa
allowed to equilibrate in a 25° C. water bath fc
10 minutes. After recording the pH to th
nearest 0.01 pH unit, 0.2 ml of substrate wa
added and the tubes were allowed to incubate ¢
25° ©. for one hour. The pH was read again an
the change in pH/hr was calculated. Contrc
tubes containing only distilled water, buffer an
substrate were treated similarly and the chang
in pH in the control tubes was subtracted fro
the values obtained from plasma to correct fc
non-enzymic hydrolysis.

Calculated Michel units (ApH/hr) wei
plotted against pH-Stat units obtained for tk
same plasma samples assayed simultaneousl:
The linear regression equations and correlatic
coefficients were calculated, each point represen
ing the mean of duplicate assays of the san
sample by each method. The statistical regre
sion of the pH-Stat units on Michel units
graphically represented in Figure 1.

10
9

pH - Stat Units (zmoles/min/ ml)
yw boon @

° L 1 1 L 1
0.10 0.20 030 040 050 060 0.

Michel Units (ApH/hr.)
Ficure 1. Correlation between pH-Stat units and Mick
units. N=20 points; correlation coefficient =0.986


and the absorbance was determined at 540 nm on
an instrument zeroed with a distilled water blank.
After 30 minutes of incubation, the absorbance
was determined a second time and the ratio of the
final absorbance (A.) to the initial absorbance
(A;) was determined (ie. A,/A,). Two control
tubes, with 0.5 ml of distilled water substituted
for the plasma, were prepared and incubated,
then the A,/A, ratio was obtained as before. The
average value of A,/A, for the control tubes was
subtracted from the ratio for each test replicate
to correct for non-enzymic hydrolysis.

A standard curve was prepared by reading the
absorbances of a series of eight tubes, each con-
taining 5.0 ml of buffer-indicator solution, 0.5
ml of pooled plasma, and 0.5 ml of 0.01N-0.15N
acetic acid (corresponding to 10-150 Caraway
activity units/ml) and plotting the ratios of each
of these absorbances to the absorbance of a tube
similarly prepared but containing no acetic acid.
These ratios were plotted on the log axis of semi-
log paper against the corresponding Caraway
units on the linear axis and the sample activity
was read directly from the curve.

Calculated Caraway units were plotted against
the pH-Stat units obtained for the same plasma
samples assayed simultaneously. Regression
equations and correlation coefficients were cal-
culated, with each point representing the mean
of duplicate assays by each method. The regres-
sion of pH-Stat units on Caraway units is
depicted in Figure 3.

yok aon oo d

pH - Stat Units (zmoles/min / ml)

° L L 1 4 1 1
10 20 30 40 50 60 70

Caraway Units

Ficure 8. Correlation between pH-Stat units and
Caraway units. N=20 points; correlation coefficient =
0.987

VI. Acholest Method.

The Acholest method is a simple “test-paper”
procedure of the type used for field screening
where multiple cases of suspected pesticide ex-
posure have occurred. The method is based on
the time required for the enzyme activity of a
plasma sample to change the color of a piece of
filter paper impregnated with buffer, substrate.
and pH indicator to match a filter paper
similarly treated but containing no substrate
Under stable temperature conditions, we have
found the results from this method to correlate
well with those obtained by more sophisticatec
techniques. The principal advantages are inex
pensive materials with long storage life and very
simple equipment requirements. These factor:
might induce some clinical laboratories having
few requests for ChE assays to maintain thi:
capability.

Materials and Method. All materials used fo:
the assay were supplied in the Acholest Tes
Paper Kit (donated by E. Fougera & Co., Inc.
Hicksville, New York, Distributor) and th
modified procedure described by Braid and Nix
was followed. These modifications consisted o
cutting both test and control strips into 14-ine!
dises with a paper punch prior to use and utiliz
ing an incandescent light source for viewing th
color change against a dull-black background.

Individual plasma samples were assayed b
placing six 7-~] drops on a clean microscor
slide; three test discs and three control dises wer
lowered onto the drops with tweezers and th
timer was activated. A second slide was lowere
onto the first and pressed gently to assure saturt
tion of the discs with the plasma. The time r
quired for the color of the test discs to matc
the color of the control discs was recorded. A
tests performed during the unit conversion stud
were at 25° C.

Reciprocal Acholest units (i.e., 1/minutes 1
color match) were plotted against pH-Stat uni
obtained by simultaneous assays of the san
samples. Regression equations and correlatic
coefficients were calculated, each point represen
ing the mean of duplicate assays of the san
sample by each method. The regression of pI
Stat units on Acholest reciprocal units is show
in Figure 4.


TECHNICAL REPORT STANDARD TITLE PAGE

T. Report No. 2. Government Accession No. Recipient's Catalog No.
FAA@AM-72=12
@ Title ond Subtitle 5. Report Date

March 1972

6. Performing Organi zetion Code

A COMPARISON OF SERUM CHOLINESTERASE METHODS: II
7 Authors) 8. Performing Organization Report No.

Crane, Charles R., Ph.D., Sanders, Donald C., M.S.,
Abbott, John Ks, BsAs

9. Performing Organization Name and Address 10. Work Unit No.
FAA Civil Aeromedical Institute
P. 0, 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

Federal Aviation Administration

800 Independence Avenue, S.W.
ton, D.C, 20591

5. Supplementary Notes

OAM Report
14." Sponsoring Agency Code

This work was performed under Task No, AM-B-72-PHB-19,

6. Abstract

Among aerial applicator personnel, the primary value of periodic blood
cholinesterase (ChE) assays is the detection of pesticide poisoning indicated by a
decrease in enzyme activity since the previous (or pre-season) assay, Comparison
of these values is difficult if they are based on different methodologies and
expressed in different units, which is frequently the case, This report provides
an evaluation of four serum or plasma ChE methods which are currently in use and
establishes the relationships for interconversion among their respective units,
This was accomplished by performing simultaneous assays by each method on a series
of samples whose activities covered the range from normal levels down to the very
low level found in organophosphate-poisoned individuals, The resulting conversion
data (regression equations) are also compatible with those described in Report

No, AM-70~13, thereby providing direct interconversion capability among the units
of seven commonly-used serum ChE methods,

17 Key Words 18. Distibution Statement

\erial applicators Availability is unlimited. Document may be
Irganophosphate pesticides released to the National Technical
fethodology Information Service, Springfield,
tonversion Factors Virginia 22151, for sale to the public,

7 Security Classif. (of this report) 0. Security Clossif. (of this page) 21. No. of Pages [ 22, Price
Inclassified Unclassified 6 $3.00

am DOT F 1700.7 (8-69)



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“2 4 6 8 10 12 14 16 18 20
Acholest Units '/T (min) x 10?

‘tovre 4. Correlation between pH-Stat units and
Acholest units, N=13 points; correlation coefficient =
0.991

‘II. Conclusions.

The relative precision of the methods in this
tboratory as represented by the coefficient of
ariation (standard deviation/mean) for repli-
ute samples was 1.70%, 1.45%, 2.0%, 2.79% for
1e pH-Stat, Michel, Caraway, and Pfizer ChE-
‘1 methods respectively. A precision study was
ot performed for the Acholest method, however
raid advises that the procedure produces a co-
ficient of variation of approximately 8%, Any
* the methods discussed would be satisfactory
w confirming cases of acute organophosphate
sisoning. The more precise methods would ob-
ously be more satisfactory for the routine
onitoring of low-level chronic exposure.

Regression equations were calculated from the
:perimental data for the following conversions:

a. To convert Michel units to pH-Stat units:
pH-Stat units = (8.32) (Michel units) + 0.41

b. To convert. pH-Stat units to Michel units:
Tichel units = (0.117) (pH-Stat units) — 0.039

ce. To convert Pfizer (ChE-tel) units to pH-
Stat units:

pH-Stat units = (0.08) (ChE-tel units) + 0.80

d. To convert pH-Stat units to Pfizer (ChE-
tel) units:

ChE-tel units = (12.07) (pH-Stat units) — 1.88

e. To convert Caraway units to pH-Stat units:
pH-Stat units = (0.092) (Caraway units) + 0.366

f. To convert pH-Stat units to Caraway units:
Caraway units = (10.67) (pH-Stat units) — 8.15

g. To convert Acholest units (minutes to color
match at 25° C.) to pH-Stat units:
pH-Stat units = (44.9/T) + 0.827 where T=

minutes to color match by the Acholest method

h. To convert pH-Stat units to Acholest units:

Acholest units = (81.4/pH-Stat Units) — 5.19
Conversion equations for the relationships be-
tween the units of the Michel, Pfizer, Caraway,
and Acholest methods may be readily derived
from the preceding equations. Also, since the
same pH-Stat primary method was used in an
earlier paper to interconvert the units of the
Sigma and Boehringer methods,’ these two
papers provide the clinical worker with the neces-
sary data to effect a conversion of units between
any of seven commonly used serum (or plasma)
ChE methods.

Thus, it is possible to directly compare pre-
exposure ChE units with post-exposure units,
even when these values were determined by dif-
ferent techniques. Such a comparison can fre-
quently identify a case of low-level chronic ChE
inhibition which would’ be undetectable by com-
paring a single ChE value with the broad normal
range of values for a specific method. With
direct interconvertibility of units, any one of
these methods best suited to the resources and
facilities of the laboratory can be used for the
medical monitoring of aerial applicator per-
sonnel or for confirmatory diagnosis in acute
organophosphate poisoning. Regardless of the
method selected, however, each aerial applicator
should have a pre-exposure test from which any
subsequent depression of ChE activity can be
measured.





A COMPARISON OF SERUM CHOLINESTERASE METHODS: II

- Introduction.

The utility of measurement of the activity of
ne acetylcholine-hydrolyzing enzyme present in
lood plasma as a diagnostic tool in organophos-
hate poisonings and for monitoring the health-
ifety aspects of aerial applicator operations has
een previously discussed.

The activity of this enzyme in unexposed in-
ividuals varies widely, with the normal range
xtending above and below the population mean
y as much as 30%. Therefore, any such value
otained after potential contact with an anti-
1olinesterase should be compared to the in-
\vidual’s normal, pre-exposure value to deter-
ine whether inhibition is present, Such a
mparison can be made only if both values are
spressed in the same units. Unfortunately for
© purpose of comparison, the various methods
x cholinesterase (ChE) assay produce dis-
milar units, and no single “standard” method
tists; consequently, a means of converting
tivity units obtained by one analytical method
those of another is required.

This paper represents the second in a series
aich will provide conversion data to transform
1E activity units obtained by one method into
e corresponding units of other methods. Most.
the methods developed primarily to measure
e activity of the pseudocholinesterase present in
e fluid portion of blood, including those com-
red in this study, customarily utilize serum for
e determination. However, plasma can be used
r the measurement, with identical results, pro-
led that the presence of fibrinogen does not
croduce interference. There is a distinct ad-
ntage in performing the assay on plasma if
2 method permits, since it allows the measure-
mt of erythrocyte enzyme activity on the same
sod specimen. Therefore, plasma was utilized
this study.

The procedure used to develop these compari-
1 data was to perform simultaneous assays by
th method on aliquots of the same sample of

plasma, using the pH-Stat method as a primary
reference. Heparinized, whole blood was pur-
chased from a local blood bank. Part of the
separated plasma was heated in a water bath at
60° C. until pH-Stat assay indicated a loss of
about 95% activity (10-20 minutes). The in-
activated plasma was then mixed in varying
proportions with fresh plasma to obtain a
gradient of enzyme activity.

II. pH-Stat Reference Method.

Materials and Method. Butyrylcholine iodide
(Mann Research Laboratories, New York, N.Y.)
was dried overnight in a vacuum desiccator and
stored therein until used. An aqueous solution,
0.163M (49 mg/ml), was prepared weekly and
stored at 4° C. The sodium hydroxide titrant
was prepared at a concentration of approximately
0.005N using CO,-free distilled water. The solu-
tion was stored in the titrant reservoir of the
pH-Stat and protected from CO, absorption by
a drying tube filled with Mallcosorb, 30-50 mesh,
indicating type CO. absorbent (Mallinckrodt).
This solution was standardized daily by titration
of a potassium hydrogen phthalate standard.
Ringer’s salts solution for diluting plasma
samples was prepared as needed using 9.00 g
NaCl, 0.20 g KCl, 0.26 g CaCh, and 0.20 g
NaHCO; brought to a 1 liter final volume with
distilled water.

A Radiometer pH-Stat (Radiometer A/S;
Copenhagen, N.V., Denmark) was used for all
constant-pH titrations, and consisted of the fol-
lowing units:

PHM26—-expanded scale pH meter

TTT11b—electronic titration control unit

ABUlb—semi-micro automatic buret for
titrant delivery, 2.5 ml capacity

TTA3—glass-jacketed reaction vessel, 3 ml,
with plastic-coated magnetic strring disc

VTS13c—constant-temperature water bath
and circulator, set to maintain 25° C. in
reaction vessel

SBR2c—tecorder for indicating volume of
titrant delivered per unit time