drogen bonding chlorohydrocarbon to the acetic acid. There-
fore, the PKB of a nonionic base in acetic acid can be used as
a reliable measure of its apparent base strength in acetic
acid-inert chlorohydrocarbon mixed solvents in which the
aprotic component is dominant.
Evaluation of the slope N in Equation 1 was made from a
Y’ us. log XHoaDgraph, as reported earlier (6). The plot
shown in Figure 2 is based upon the data in Table I1 for both
solvent pairs. The shape of the plot is similar to that for the
acetic acid-chloroform system ; however, the change in slope
occurs at a lower mole fraction ( X = 0.68) in Figure 2. The
final limiting value of N for both of the acetic acid-chloro-
hydrocarbon solvent systems is 0.120 f 0.002 V, indicating
that the solvent composition variable must again be expressed
in terms of the dimeric form of acetic acid (6,11).
The results of the quantitative sub-test on the major emf
variables, using Equation 2, are summarized in Table 111.
The variation in the summation for the six bases (at CE =
0.004M) among the fifteen solvents appears to be a random
one within the precision of the raw data. If this is the case,
then it follows that above a chlorohydrocarbon mole fraction
of about 0.14, the contribution of the sum in Equation 3 to
(E& remains essentially constant even though the propor-
tion of the aprotic component in the mixed solvent becomes
(11) 0.Kolling and W. Cooper, ANAL.CHEM.,42, 758 (1970).
Comparison of Acetylcholinesterase Determinations
by the Michel and Ellman Methods
Kenneth I. Hawkins and C. Edward Knittle
Denver Wildlife Research Center, Bureau of Sport Fisheries and Wildlife, Denver, Colo. 80225
MANYANTICHOLINERGIC AGENTS are being used or are being
studied for use as pesticides. Some means of testing for
effect of the agents is required other than death of the test
animal, such as determination of cholinesterase activity.
The Michel method ( I ) for acetylcholinesterase (AChE)
determination has been used for several years. This method
requires a sensitive pH meter and relatively large samples.
The Ellman method ( 2 ) requires a good laboratory spectro-
photometer/colorimeter but only micro-size samples. We
compared the two methods to estimate the usefulness of the
Ellman method and to relate results from it to work pre-
viously reported for the Michel method.
METHODS
Each AChE method was run on aliquot samples of bird
brain homogenates. To produce a wide response range, the
AChE activity of some of the test birds was lowered by dosing
the animals with different concentrations of fenitrothion
[O,O-dimethyl O-(4-nitro-m-tolyl)phosphorothioate]up to
and including the LDEO dose (3). Forty-seven young (8-10
(1) H.0.Michel, J . Lab. Clin. Med., 34, 1564 (1949).
(2) G. L. Ellman, K. D. Courtney, V. Andres, Jr., and R. M .
Featherstone, Biochem. Pkarmacol., 7,88 (1961).
(3) R. K. Tucker and D. G. Crabtree, “Handbook of Toxicity of
Pesticides to Wildlife,” Bur. Sporr Fish. Wild/. (U.S.), Res. Pub.,
No. 84 (1970).
416 ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
very large.
However, there is no reason to expect that Y, should have
the same numerical value for the hydrogen bonding pair,
HOAc-CHC18,as for the acetic acid-inert chlorohydrocarbon
systems (6).
From potentiometric measurements on a large number of
nitrogen bases, Hall concluded that the apparent base strength
is essentially independent of the solvent for inert aprotic
media, including dichloroethane (12). Although this is con-
sistent with the results above, it should be recognized that
his correlation differs in showing a correspondence between
the PKB of a given base in aqueous solution and the half-
neutralization potential of that base in a mixed solvent con-
taining dioxane as a minor component. Also, by sharp
contrast to the results above, the response of the glass elec-
trode usually gave abnormal slopes in the dioxane-aprotic
solvent pairs, and the effect of the small amounts of water
from the titrant upon these slope variables is unclear.
RECEIVED for review August 4, 1971. Accepted September
15, 1971. Financial support for this study came from the
National Science Foundation Grant No. G P 27634.
(12) H.K.Hall, J . Phys. Chem., 60,63 (1956).
weeks) bob-white quail (Colinus virginianus) were killed in a
COZ chamber. Each skull was quickly opened, the whole
brain excised, weighed, and placed in 50 ml of distilled water
for each gram of brain. The tissue was thoroughly homoge-
nized in a Virtis homogenizer, then divided for analysis by the
two methods.
Procedures for both methods are outlined below, since
each was slightly modified from the original method.
Michel. For each sample, 2 ml of brain homogenate was
pipetted into 2 ml of buffer (0.02M sodium barbital, 0.004M
KH2P04, 0.6M KCl, pH 8.1 at 25 “C). The mixture was
brought to 25 “Cin a water bath, stirred, and pH1 measured.
Then 0.4 ml of acetylcholine bromide (0.09M) was added and
the mixture was stirred again. Forty-five min (k.5 sec) later,
pH2 was measured. The difference in pH values (ApH =
pH1 - pHz) indicates the activity (concentration) of the
AChE. Each ApH was corrected by subtracting the ApH of a
blank test using only buffer and homogenate. Buffer-sub-
strate blanks were run with each batch of tests as a check for
possible substrate breakdown. All solutions were kept at a
final constant volume of 4.4 ml by the addition of either sub-
strate or distilled water. Using untreated samples, the
temperature and time of incubation were selected to ensure a
linear plot of ApH us. time. This procedure eliminates the
need for correction factors as suggested by Michel ( I ) .
Ellman. For each sample, 50 bl of homogenate was mixed
with 10 ml of phosphate buffer (pH 8.0, O.lM), and 3-ml
 The “normal” AChE level in the bird brains for each
Table I. Acetylcholinesterase Levels in Brains from Untreated method was determined by running samples of untreated
Bob-White Quail as Determined by the birds. Ten samples were run by the Michel method, and 20
Michel and Ellman Methods samples were run by the Ellman method. Table I lists the
Test statistic Michel method Ellman method findings.
Mean 1 ,548 ApH/45 min 16.5bmoles/min/g Since the Ellman method as presented here is being com-
Standard deviation 0.119 1.7 pared to the more accepted Michel method, a standard
Coefficient of variation 7.7 % 10.3% cholinesterase preparation was used to check the accuracy of
n 10 20
the colorimetric procedure. Bovine erythrocyte cholinesterase
of known activity was purchased (Sigma Chemical Company)
Table 11. Differences in Michel and and dissolved in saline to give a calculated activity of 2020
Ellman Methods Used in This Report pmoles/l./min at 30 “C. Thirteen analyses of this standard
Michel Ellman solution, over several weeks of time, indicated a mean activity
Sample size 2 ml 50 pl of 2010 pmoles/l./min with a standard deviation of 158 and
Incubation time 45 min 10 min correlation coefficient of 1.9Z.
Special equipment Beckman Research Spectronic 70
pH meter spectrophotometer DISCUSSION
Reporting units ApH/45 min pmoles/min/g
Chemicals required 2 ml buffer 10 ml buffer The high degree of correlation between the two methods
(concentrations (0.01M barbital, and the good precision experienced in each method indicates
are for final test 0.002M phosphate) (0.1M phosphate) little or no difference in the methods as far as numerical
solution) Acetylcholine bromide Acetylthiocholine
(8 X lW3M) iodide (4.5 X values are concerned. The use of quinidine in the Ellman
10-4 M ) method eliminates possible interference from plasma (pseudo)
D T N B ( ~ Sx 10-5,141 AChE ( 4 ) . However, additional tests without quinidine
Esterine sulfate showed no increase of activity that could be due to plasma
(3 x 10-5~)
AChE.
Ideally, in a comparison of this kind, the regression line
should pass through the origin. It is highly improbable that
aliquots were pipetted for “test” and “blank” determinations. this occurred in our study, although neither test method in-
The “test” sample was brought to 30 OC in a water bath, then dicated a zero activity for any sample. Similarly, when
50 ~1 of color-substrate reagent [2.7 X 10-2M acetylthio- Pearson and Walker (5) compared the Michel and pH-Stat
choline iodide, 4.5 x lO-3M 5,5’-dithiobis-(Z-nitrobenzoic methods on plasma and erythrocytes, neither of their re-
acid) (DTNB) and 2.4 X 10-4Mquinidine sulfate] was added. gression lines passed through the origin. However, the ideal
The reaction was stopped after 10 min (+5 sec) with 0.1 ml situation is not necessary for a valid comparison. Many
of eserine sulfate (0.001M in phosphate buffer). The acetyl- factors can contribute to the offset of the intersect, and they
thiocholine iodide (43.4 mg) and DTNB (10 mg) were dis- can be accepted as long as they are accounted for in the
solved in 4.5 ml of pH 8 phosphate buffer; then 1 ml of regression equation.
quinidine sulfate (0.1 g/100 ml phosphate buffer) was added. Perhaps the most significant differences in the two methods
The solution was mixed, aliquoted into 1-ml samples, and are the sample size and the incubation time (Table 11).
frozen. The reagent was discarded when a definite yellow The close correlation shown in this test indicates that the
coloration was observed. The quinidine sulfate and eserine Ellman method can be used and the results compared to
sulfate were also stored frozen (stable for at least a month). previous work performed by the Michel method. The
The “blank” tube was prepared by adding the eserine sulfate, regression formula may apply only to the tissue used in this
mixing, and then adding the color-substrate solution. study. No attempt was made to compare results of other
Absorbance ( A ) of both samples was determined at 412 nm, tissue analyses. The choice of method can be determined by
and the difference ( A A = “test” A - “blank” A ) was used in the investigator on the basis of available equipment, sample
calculations. size, and time factors. The use of pipetting devices, such as
the Oxford “Sampler” or Eppendorf micropipet, and of a
RESULTS flow cell in the calorimeter greatly reduces the work load in
the Ellman procedure. Likewise, semiautomatic pipetting
The regression formula, Q, and the standard error of esti-
devices can reduce the work involved in the Michel method.
mate, S, .z,for the data obtained are:
0= 1.58 + 9.88~ RECEIVEDfor review July 16, 1971. Accepted September 15,
1971. Reference to trade names does not imply endorsement
Sff,z= 0.70
of commercial products by the Federal Government.
where y represents the Ellman values and x represents the
Michel values. The correlation coefficient ( 7 ) for the data is
(4) J. C. Sabine, Blood, 10, 1132(1955).
0.99, indicating very close agreement of the two methods for (5) J. R. Pearson and G. F. Walker, Arch. Ellairon. Health, 16,
the analysis of AChE in a sample. 809 (1968).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972 417