Colorimetric determination of red blood cell acetylcholinesterase activity

Clin. Biochem. 3, 295-306 (1970)

COLORIMETRIC DETERMINATION ACETYLCHOLINESTERASE

C. M. CHOW* Department

of Chemistry,

CELL

M. F. ISLAMt

AND

University (Received

OF RED BLOOD ACTIVITY

of Monclon, May

Moncton,

NB,

Canada

16, 19?‘0)

%iMMARY

1. Effects of various parameters on the production of color from 5:5-dithiobis2-nitrobenzoate as chromogen are described. 2. Slightly higher values were obtained with the hemolysates compared to those with the whole cells. 3. The rates of reaction were linear within the concentrations of the enzyme used in the presence of a number of substrate concentrations. Maximal rate of hydrolysis was obtained at a substrate concentration of about 1.0 X lo+ mol/l. 4. The rate of hydrolysis increased linearly as the temperature of reaction medium rose from 5 to 37. 5. The pH for optimal activity was observed to be within 8 to 9. The rate of non-enzymatic hydrolysis of the substrate increases very rapidly at pH higher than 8.6. 6. The enzyme activity in borate and tris buffers was much lower compared to that in the phosphate or barbital buffers. The inhibitory effect of tris buffer was partially reversed by the presence of phosphate buffer.

A VARIETY OF METHODS ARE AVAILABLE at present for the estimation of acetylcholinesterase (ACHE) activity of different tissues. The electrometric method has been used by many workers (l-4) and has been shown to be reliable and suitable for this purpose in the presence of hemoglobin. This method is used in many laboratories today for the estimation of ACHE activity in clinical samples. This principally involves measurement of the decrease in the pH that occurs when the substrate is incubated with the sample in a standard buffer. Warburg’s Manometric Method has also been conveniently used to follow the hydrolysis of the ACHE substrate (5). It is also suitable in presence of hemo*Present address: Department of Chemistry, University of Saskatchewan, Regina, Saskatchewan, Canada. tcorrespond with: Dr. M. F. Islam, Department of Chemistry, University of Moncton, NB, Canada. Acknowledgments are made to the University of Moncton Research Council for the award of a Research Grant which made this work possible.

296

CHOW&ISLAM

globin. The basis of the method is the manometric estimation of CO2 evolved from a bicarbonate buffer. This method is also used by some laboratories. R,Iethods involving color changes are used to measure the acetic acid formed in presence of an indicator. The acid formed may be titrated with standardised alkali using suitable indicators (G-9), but this is seldom employed at present. However, a number of photometric methods, like the hydroxamate method developed by Hestrin (10) and the dithiobis-nitrobenzoate method of Ellman et al. (II), are also available to measure ACHE, calorimetrically. Recently, Hecht and Stillger reported that the manometric and electrometric methods gave an error of f 3%, while the hydroxamate method had an error of f 5% (11). Ellman et al. (11) have shown that the standard deviation of their method was not more than f 4%. In terms of accuracy, dithiobis-nitrobenzoate method of Ellman appears to be superior to the hydroxamate method, but inferior to the manometric and electrometric methods. However, the manometric method needs quite elaborate arrangements and the electrometric method requires much longer time for the estimations compared to the dithiobis-nitrobenzoate (DTNB) method. We found the latter to be quite sensitive, rapid and quite convenient for such purposes. The experiments reported in this communication were carried out to establish the optimal conditions for the measurement of the red blood cell (RBC) acetylcholinesterase (ACHE) by this method. EXPERIMENT Preparation of red blood cells Human blood, preserved with ACD solution (IS) for 21 days under blood banking conditions, was obtained through the courtesy of the Moncton Regional Laboratory of the New Brunswick Department of Health. The cells were packed by centrifugation at 2,200 rpm for 15 minutes in a Model HN International Centrifuge. The supernatan t plasma and the buff y coats were removed by sue tion with a Pasteur pipette. The remaining cells were then washed four times by resuspension in three volumes of cold sterile physiological saline buffered at pH 7.8 with 0.01 mol/l phosphate buffer, such that the pH of the final suspension became 7.4. At each time the top layer of white cells was removed along with some of the RBC. Practically all of the white cells and the platelets were removed in this way. Suitable suspensions of the cleaned RBC were used to carry out the experiments where RBC were used, and in others hemolysed preparations were employed. A!1 the solutions used were made with reagent grade chemicals. Phosphate buffers were made according to Gomori (14). The carbonate-bicarbonate buffers were made according to Delory and King (15). The tris buffers were made as specified by Gomori (16, 17). Barbital buffers were made according to Holmes cm. Measurement of the acetylcholinesterase (ACHE) activity The ACHE activity was measured essentially according scribed by Ellman et al. (11), with certain modifications.

to the method deThis method uses

COLORIMETRIC

DETERMINATION

297

acetylthiocholine iodide (ACTCI) as substrate and 5:5-dithiobis-nitrobenzoate (DTNB) as the chromogen. In all assays, 3 ml of the RBC preparation or dilutions of this with 0.1 mol/l phosphate buffer at pH 8 or hemolysates were taken in cuvettes. To this, 0.025 ml of a lo-mmol/l DTNB solution and 0.020 ml of a 75-mmol/l ACTCI solution were added with micro pipettes and bubbled through with a Pasteur pipette to mix. The progressive formation of the yellow pigment by the reaction of the released thiocholine with DTNB was followed at a wavelength of 412 rnl.cfor at least G minutes in a Beckman Model DB-2 recording spectrophotometer. A Gilford spectrophotometer, Model 2400 fitted with a Lauda K-2/R constant water bath was used to carry out the rates of reaction at different temperatures. The values obtained were corrected for their appropriate blanks. For the other experiments, the values reported were obtained at 25 or were corrected to that of the value at 25, and expressed as units of enzyme (EU). The amount of enzyme present in the solution placed in the cuvette for the assay, corresponding to the production of a change of optical density of 0.001 unit per minute under the assay condition was taken to be one unit of the enzyme. RESULTS The rate of color production as observed with RBC suspension was linear for a considerable length of time, as can be seen from Fig. l(A). Figure l(B) shows the rate of non-enzymatic hydrolysis obtained, about 0.0017 OD units per minute, and is quite close to the value reported by Ellman et al. (21). Some differences were observed in the assay values obtained when using intact RBC or hemolysed preparations as can be seen from Table I. The results with the hemolysed preparations were about 5% higher compared to those with the unhemolysed preparations. It is possible that all of the RBC ACHE are not equally available to the substrate due to the compactness of the constituents in the intact cell membrane. These results support the observations of Firkin et al. (19) that trypsin treatment of RBC could not destroy all of the ACHE activity. Our results are also in close agreement with those of Shinagawa and Ogura (90) in that most of the RBC ACHE is present on the outer side of the RBC membrane. Furthermore, Vincent e.! al. (61) observed hemolysates to have about 3% more activity compared to the whole cell suspension, which is in line with our observations. The results obtained with various substrate concentrations are presented in Fig. 2. This figure depicts that with a suitably fixed enzyme concentration an increase of substrate concentration leads, at first, to a rapid rise in the velocity of reaction. As the substrate concentration continues to rise, however, the rate increase slows down until with a larger substrate concentration there is appreciable inhibition of the reaction rate. The maximal velocity is reached at a substrate concentration of approximately 1 X 10d3 mol/l. This is more appropriate with the higher concentration of the enzyme (Fig. 2(A)). V ariations in the rates of reaction with a number of substrate concentrations are shown in Fig. 3. The rates of

CHOW & ISLAM

298

1.2

O.D. c

0,8

0.4

B 0

0

A 5

TIME

n

#a I

10

15

IN MIN.

FIG. 1. Changes in OD at 412 ma with the time of incubation. (B) non-enzymatic hydrolysis.

(A) Enzymatic

4

20 hydrolysis, and

COLORIMETRIC

DETERMINATION TABLE

VALUES

OF ACHE

MEASUREMENTS

RBC suspension (hematocrit 10%) cc

Volume of 0.004 mol/l phosphate buffer atpH8 added cc

2 2 2 2 2 2

98 98 98 -

WITH

299

I THE INTACT

RBC AND HEMOLYSED

Volume of Volume of 0.01 mol/l 0.02 mol/l phosphate phosphate Volume of buffer buffer distilled Value atpH8 atpH8 water Total of ACHE added added added volume obtained cc cc cc CC EU/3CC 398 398 398

200 200 200 -

100 100 100 -

400 400 400 400 400 400

89 84 85 84

RBC

Remarks Hemolysed Hemolysed Hemol&ed Not lvsed Not &ed Not lysed

were all found to be linear. Variations in the rates of reaction with different concentrations of RBC ACHE are shown in Fig. 4; within the limits used, the rates of reaction were found to be linear. As usual, the temperature of incubation of the reaction medium was found to be very important and the results of our experiments are shown in Fig. 5. The rate of reaction was found to vary with a linear relation over a wide range of temperature (5-37). This is particularly interesting as it gives an easy way to convert the observed values at any suitable temperature to a standard temperature with the help of the difference in temperature and one common factor. The rate of increase per degree rise of the temperature of reaction, as can be observed from Fig. 5, is very close to 2% X lOmaOD units. Changes in the rates of reaction are shown in Figure 6. It can be seen that the pH for optimal reaction lies between 8 and 9. It can also be seen that the rate of non-enzymatic hydrolysis sharply increases above pH 8.6. The influence of buffer composition on the value of ACHE assay is shown in Table II. The phosphate and barbital buffers were found to be quite favourable for the ACHE activity. Tris buffer and boric acid-borax buffer had marked inhibitory effects. With 0.01 mol/l tris only 66% of the activity was obtained, with 0.05 mol/l tris buffer 64% and with 0.05 mol boric acid-borax buffer it was 75% of the activity obtained with the phosphate buffer. Interestingly enough the inhibitory effect of tris buffer can be partially reversed by the addition of phosphate buffer.

reaction

DISCUSSION

It is evident from the results presented that a variety of conditions have an important bearing on the results of estimation of acetylcholinesterase. Buffers made of tris and boric acid are inhibitory (Table II). Michaeli et al. (%?, SS) used tris buffer to prepare ACHE fractions from RBC. From our results it appears that precautions should be taken in the assay of the enzyme such that the concentration of buffers like tris and borax, in the final assay system, is regulated so that the inhibitory effect is negligible.

CHOW&ISLAM

300

h

8 ‘1

A ’

.

I 5

(

X

1O-4

CONCENTRATION

FIG, 2. Variations in the rate of concentrations in presence of different arbitrary enzyme solution). (A) Four

10 ) OF

, !

M ACETYLTHIOCHOLINE

,

I

10

6 (

X

loo3

)

M

IODIDE

acetylcholine iodide hydrolysis with different substrate enzyme concentrations (shown in relative volumes of an volumes, (B) two volumes, (C) one volume and (D) blank without the enzyme.

CHANGES IN O.D./MIN. P 02

( EIO-l) h)

CHOW & ISLAM

302

O.D.

TIME

IN MIN.

FIG. 4. Rates of ACHE reaction with acetylthiocholine iodide, 0.5 mM, as substrate and different concentrations of the enzyme. (A) 96 units; (B) 64 units; (C) 48 units and (D) 24 units.

COLORIMETRJC

0

DETERMINATION

10 TEMPERATUREOF FIG. 5. Effects of incubation

20 30 MEDIUM(°C)

temperature

on the rate of reaction.

303

40

304

CHOW & ISLAM

13f

@

PkDSPHhTEBUFFER

8

CARBONATE-BICARBONATE

BUFFER

lO( A a '2 x V

960 4 0 E 3 P 0 20

6.6

8.0

7.0

9.0

10.0

PH FIG. 6.

Rates of ACHE activity at different pH. (A) Enzymatic hydrolysis the blank and (B) non-enzymatic hydrolysis.

after correction

for

COLORIMETRIC

DETERMINATION TABLE

305

II

EFFECTS OF A NUMBER OF COMMON BUFFERS ON THE RBC ACHE ACTIVITY. AN ARBITRARY CONCENTRATION OF ACHE WAS USED IN THIS EXPERIMENT. THE ACTIVITY IN PHOSPHATE BIJFFER AT pH 8 WAS TAKEN AS 100%

pH of the final assay system 8 8 8 8

Buffer used in the assay system (mol/l) 0.05 0.01 0.05 0.01 0.05 0.05 0.05 0.05 0.05 0.05

EU observedineach (corrected for blank)

PC of activity (in phosphate, 100%)

231 152 148 198

100 66

182

79

229 252 174

99 109 75

phosphate tris tris tris and phosphate tris and phosohate barbital barbital boric acid-borax

it

Barbital buffer is as good as phosphate buffer at pH 8, and can be safely used for this purpose. Besides, the rate of ACHE activity at pH 8 is quite high, about 98% compared to that at the maxima (Fig. 6). The rate of non-enzymatic hydrolysis of the substrate rises rapidly as the pH increases above 8. The non-enzymatic hydrolysis product is appreciable only at higher concentrations, where there is also a lot of substrate inhibition of the enzyme activity (Fig. 2). It appears from our results that even with a very high concentration of the enzyme (96 enzyme units were used in our experiments, Fig. 5) the rate of reaction stays linear for a fairly long period (Fig. 4). When varying the substrate concentration we observed that the highest color production was obtained at about 1 X lF3 mol/l substrate concentration (Fig. 2). We therefore recommend the use of a substrate concentration of 1 X low3 mol/l instead of 5 X 10m4mol/l as used by Ellman et al. (11). According to our results this higher concentration of the substrate is quite safe and assures the highest possible rate available, with a greater degree of flexibility in the use of the enzyme concentrations. It was suggested by Ellman et al. that hemolysis of RBC in the blood samples is not necessary (11). We feel however from our results (Table I) that a hemolysed system is better for this purpose as it gives a more dispersed suspension with a much lower tendency towards sedimentation compared to the intact cells and also the values obtained are somewhat higher. REFERENCES 1. MICHEL, H. 0. An electrometric method for the determination of red cell and plasma cholinesterase. J. Lab. Clin. Med. 34, 1564, 1949. 6. ANGUSTINSSON, K. B. Methods of biochemical analysis, Vol. 5. Glick, D., ed., Interscience Publishers, Inc., 1957, pp. l-63. S. BURMAN, D. Modifications of Michel’s electrometric method for the estimation of red cell cholinesterase. Am. J, Clin. Pathol. 37, 134, 1962. 4. LESLIE, W. LEE. Rate recording modification of Michel’s cholinesterase. Am. J. Med. Technol. 32, 255, 1966.

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CHOW

& ISLAM

R. The enzymatic hydrolysis of acetylcholine. Pflugers Arch. ges. Physiol. 233, 6. AMMON, 486,1933. E., el al. Cholinesterase an enzyme present in the blood serum of the horse. 6. STEDMAN, Biochem. J. 26, 2056, 1932. B. The esterase activity of human plasma. Arch. Physiol. 72, 133, 1935. Y. VAHLQUIST, D. Enzymatic histochemistry XXV. A micromethod for the determination of 8. GLICK, cholinesterase and the activity pH relationship. Gen. Physiol. 21, 289, 1938. C. H. Cholinesterase and the behavior in ambystoma. Distribution in nerve and 9. SAWYER, muscle throughout development. J. Exp. Zool. 94, 1, 1943. S. Reaction of acetylcholine and other carboxylic acid derivatives with hy10. HESTRIN, droxylamine and its analytical application. J. Biol. Chem. 180, 249, 1949. G. L., et al. A new and rapid calorimetric determination of acetylcholinesterase 11. ELLMAN, activity. Biochem. Pharmacol. 7, 88, 1961. G. & STILLGER, E. Normal values and individual variations of acetylcholin12. HECHT, esterase in blood. 2. Klin. Chem. Klin. Biochem. 5, 156, 1967. methods and procedures of the American association of blood banks. Rev., 1962. IS. Technical AABB, Chicago, 1962. G., after Sorensen, S. P. L. Preparation of buffers for use in enzyme studies. 14. GOMORI, Methods in Enzymol. 1, 143, 1955. G. E. & KING, E. J. A so d ium carbonate-bicarbonate buffer for alkaline 16. DELORY, phosphatase. Biochem. J. 39, 245, 1945. G. Buffers in the range of pH 6.5 to 9.6. Proc. Sot. Exp. Biol. Med. 62, 33, 1946. 16. GOMORI, G. Histochemical determination of site of cholinesterase activity. Proc. Sot. Exp. 1Y. GOMORI, Biol. Med. 68, 354, 1948. G., after Holmes, W. Preparation of buffers for use in enzyme studies. hIethods 18. GOMORI, in Enzymol. 1, 145, 1955. B. G., et al. The effects of tripsin and chymotrypsin on the acetylcholinesterase 19. FIRKIN, content of human erythrocytes. Aust. Ann. Med. 12, 26, 1963. Y. & OGURA, M. Cholinesterase in erythrocyte membrane. Kagaku 31, 20. SHINAGAWA, 554,196l. 21. VINCENT, D., el al. The cholinesterase of human blood. Compt. Rend. Sot. Biol. 155, 662, 1961. 22. MICHAELI, D., et al. Restoration of enzyme activity of heat-denatured acetylcholinesterase by antibodies to the native enzyme. Nature (London) 213, 77, 1967. 23. MICHAELI, D., et al. Immunology of acetylcholinesterase. Il. Effect of antibody on the heat-denatured enzyme. Immunochemistry 6, (3) 351, 1969.