Clin. Biochem. 3, 327-333 (1970)

A FLUOROMETRIC ASSAY OF THE ACETYLCHOLINESTERASE ACTIVITY IN BLOOD

TETSUO UETE, HIROKO TSUCHIKURA, AND KAZUMI HOSHIDA

Kitano Hospilul Tazuke Kofukai Medical Research Institute, Kita-ku, Osaka City, Japan

(Received March 18, 1970)

SUMMARY

A specific and sensitive fluorometric method for the determination of the activ- ity of acetylcholinesterase in blood was developed, using acetylthiocholine as a substrate. The cholinesterase in blood acts on acetylthiocholine to release thiocholine, which reacts with 0-ph thalaldehyde at pH 8 to produce a fluorophor at an excitation wave length 350 nm and an emission \vave length 420 nm. This fluorophor is stable for at least 2 hr. The present method requires less than 0.05 ml of serum or plasma.

A NUMBER OF METHODS for the determination of the activity of cholinesterase have been reported using acetylcholine as a substrate. These can be classified into three groups: titration, manometric, and calorimetric methods. The acetic acid formed by the hydrolysis of acetylcholine is titrated with standardized alkali using indicators such as phenol red, phenolphthalein, or bromothymol blue (1-S). The enzyme is assayed in a bicarbonate-carbonate dioxide buffered solution. The acetic acid produced by the hydrolysis converts bicarbonate to COZ, which, since the volume of the reaction vessel is maintained constant, is reflected by increase of pressure. The Warburg manometric method has been used (4). A quantitative calorimetric procedure for acetylcholine has been developed, based on the reaction of O-acyl derivatives with alkaline hydroxylamine, and used for the assay of acetylcholinesterase (6).

Recently, acetylthiocholine has also been used as a satisfactory substrate for the measurement of cholinesterase activity (6). Iodometric titration (7,s) and decrease in absorbance of the thioester bond (9) have been used for the assay. The SI-I group of the thiocholine resulting from the enzyme action is determined calorimetrically by the nitroprusside reaction (10) or by the reaction with 5:5-dithiobis-(2-nitrobenzoic acid) (DTNB) (11-14). We determined thiocholine fluorometrically by the reaction with 0-phthalaldehyde (OPT) and developed a fluorometric assay for the activity of acetylcholinesterase. The method is highly sensitive and accurate, and requires less than 0.05 ml of plasma or serum.

325 UETE et al.

MATERIALS AND METHODS

Reagents 1. Tris buffer, 0.2 mol/l, pH 7.4 and 8.0 2. Acetylthiocholine, 0.026 mol/l, dissolved in Hz0 3. HPOa, 25% W/V 4. 0-phthalaldehyde (OPT), O.l%, dissolved in methanol 5. Thiocholine 6. Reduced glutathione (GSH)

PROCEDURE

A serum specimen 0.03 ml is incubated with 0.5 ml of 0.026 mol/l acetylthio- choline and 1 ml of 0.2 mol/l Tris buffer (pH 7.4) at 37 for 3 minutes. The reaction is stopped by the addition of 0.6 ml of 25yo HP03 and the mixture is centrifuged for 10 minutes to precipitate protein. The supernatant 0.1 ml is added to 2.0 ml of Tris buffer (pH 8.0) and 0.1 ml of 0.1% 0-phthalaldehyde, and 15 minutes later fluorescence is determined at an emission wave length 420 nm with an excitation wave length 350 nm at room temperature. As a blank, the reaction mixture was incubated with 0.6 ml of 25% HPOI. The liberation of thiocholine by serum is estimated by the thiocholine standard, treated similarly with 0-phthalaldehyde. One unit of the enzyme activity is defined by the liberation of one micromole of thiocholine in 1 minute at 37. The activity of acetylcholinesterase in blood is expressed as micromoles of thiocholine liberated in 1 minute from 1 ml of serum. Reduced glutathione can be used as the standard for the estimation of sulfhydryl group liberated by serum enzyme. The intensity of thiocholine-OPT fluorescence is approximately 12% of that of GSH-OPT. Therefore, when reduced glutathione is used as the standard, the fluorescence intensity determined in the assay system must be corrected by this factor.

RESULTS

Development of fluorescence of thiocholine and redmed glutathione with O-phthalalde- Hyde. Thiocholine and reduced glutathione react with 0-phthalaldehyde at pH 8 and develop fluorescence at an excitation wave length 350 nm and an emission wave length 420 nm. The fluorescence spectra of thiocholine-OPT were quite similar to that of GSH-OPT fluorophor. However, the intensity of the fluorescence of thiocholine-OPT was approximately 12% of that of GSH-OPT, as is shown in Fig. 1.

The intensity of fluorescence of thiocholine-OPT was reduced in the presence of acetylthiocholine and serum protein. Therefore, proteins and acetylthiocholine in the incubation system is better to be eliminated for the determination of the fluorescence intensity. Efect of the incubation time on the liberation of thiocholine from the acetylthiocholine by serum. Previous workers (12, IS) have studied the conditions of the choline-

ASSAY OF ACETYLCHOLINESTERASE ACTIVITY 329

10

t gcitation spectra gpisslml spectra

60 -

50 -

40 -

30 -

20 l

10 *

0 -

i%iocholins

250 300 350 400 m)l 350 400 455 500 mp

FIG. 1. The fluorescence spectra of thiocholine and reduced glutathione developed with 0-phthalaldehyde.

sterase assay using the acetylthiocholine. In this study the basic conditions used were similar to those of Garry and Rout11 (IS). A serum specimen, 0.03 ml, was incubated 2,4, 6, and 8 minutes. The liberation of thiocholine and the incubation time were linearly correlated, as is shown in Fig. 2. The fluorescence measured was maximal at an emission wave length 420 nm with an excitation wave length 350 nm, indicating the fluorescence spectra of thiocholine-OPT. This fluorescence was stable for at least 2 hours at room temperature. The amount of serum and the units of the activity of cholinesterase. These amounts were linearly correlated under the conditions of this assay, as is shown in Fig. 3. ,?Zfect of various anticoagulants on the determination. The results are shown in Table I. EDTA markedly decreased the activity of cholinesterase, possibly chelating serum Mg.++ Sodium fluoride also decreased the cholinesterase activity. However, citrate, oxalate, and heparin did not affect the activity of enzyme. Efect of bilirubin and hemolysis on the determination. No effect of bilirubin was observed on the determination of the cholinesterase activity in blood. As is shown in Table II, when the activity of cholinesterase in red blood cells and in plasma of 1 ml of blood was determined separately, the activity of enzyme in red blood cells was 2 to 5 times greater than that of plasma. This indicates that hemolysis seriously influences the determination of the activity of cholinesterase in serum or plasma. Comparison of the cholinesterase activity in serum determined by the calorimetric method and present jluorometric method. The results are shown in Fig. 4. The cholinesterase activity in serum of healthy subjects and patients with hepatic disease was determined by the method of Garry and Rout11 (IS) and the present method. A good correlation was observed between the activities measured by the

UETE et al.

DC1 tat1on sp2ctrs

350 400 4>3 5J’J ‘91

FIG. 2. The effect of the incubation time on the liberation of thiocholine from the acetylthio- choline by serum.

0.3

0.2

0.1

0

0 0.02 0.04 0.06 0.08 0.10 ml

FIG. 3. The amount of serum and the units of the activity of cholinesterase.

ASSAY OF ACETYLCHOLINESTERASE ACTIVITY 331

TABLE I

THE EFFECT OF VARIOUS ANTICOAGULANTS ON THE DETEKhIINATION OFTHEACTIVITYOFCHOLINESTERASEINSERUM

Thiocholine liberated ~moles/min/ml serum

Addition Expt. 1 Expt. 2

None 2.42 1.34 EDTA 1 mg/ml serum 1.40 0.70 Sodium citrate 1 mg/ml serum 2.49 2.02 .%~~monium oxalate 1 mg/ml serum 2.57 1.87 Potassium oxalate 1 nig/nil serum 2.25 2.02 Heparin 0.1 mg/ml serum 2.25 1 .9.5 Sodium fluoride 0.25 mg/ml serum 1.79 1.09

TABLE II

THE ACTIVITIESOF CHOLINESTERASE IN PLASMA AND RED BLOOD CELLS -

Case Sex

1 F 2 F 3 F 4 F

5 NI

-4s Hct h-1 (%)

23 40 22 42 21 40 26 37

26 47

Activity of cholinesterase in 1 ml of blood thiocholine, liberated

~moles/min

Plasma Red blood cells

2.44 3.23 2.03 6.48 2.48 14.40 2.40 2.12 C.

1.54 7.21

calorimetric method and the present fluorometric method. The average values of the liberation of thiocholine from acetylthiocholine by serum of 16 healthy subjects measured by the calorimetric method and the fluorometric method were respectively 2.81 f 0.42 and 2.72 f 0.50 ccmoles per minute per ml. Patients with liver cirrhosis, showed a decrease in the level of this enzyme activity in serum, as is seen in Fig. 4.

DISCUSSION

The reaction of 0-phthalaldehyde with various compounds to yield a highly fluorescent reaction product, is used for the assay of histamine (15), histidine (15,16), carnosine (16), arginine (l7), agmatine (17), and glutathione (18). In this investigation, it was found that thiocholine also reacts with 0-phthalaldehyde at pH 8 to yield a highly fluorescent product, indicating the most sensitive chemical method for the assay of thiocholine. With this fluorometric determina- tion of thiocholine, it is feasible to determine the activity of cholinesterase in blood, using acetylthiocholine as a substrate. The method appears to be highly specific and sensitive.

The nature of the thiocholine-OPT reaction or its product is not clear. Cohn and Lyle (18) have suggested that under alkaline conditions the sulfhydryl group

332 UETE el al.

0

l Healthy subjects

0 1 .o 2.0 3.0 4.0 5.0 ~iocholin13, liberated, pmolea/mln./ml serum,

measured by fluorometric method.

FIG. 4. Comparison of the cholinesterase activity in serum measured by the calorimetric method and the present fluorometric method.

of reduced glutathione might form a hemimercaptal with an aldehyde. The nature of the thiocholine-OPT reaction may be similar to that of GSH-OPT. At pH 8, reduced glutathione, histamine, and histidine in blood develop fluorescence, but by using a serum blank these interfering materials in blood are effectively eliminated from the assay of thiocholine. The intensity of fluorescence of the thiocholine-OPT was reduced in the presence of serum protein and acetylthio- choline. Therefore, in this study serum protein was precipitated by HP03 and eliminated, and the dilution of the concentration of acetylthiocholine was made, when the fluorescence intensity of the thiocholine-OPT was measured.

Recently, a calorimetric method for the assay of thiocholine using 5:5-dithiobis- (2-nitrobenzoic acid) has been developed (11-14). In these methods, the reading of colour intensity should be made within a shorter period (11-14). However, the fluorophor of thiocholine developed with 0-phthalaldehyde is stable for at least 2 hours. Therefore, this method is well suited for use in the routine clinical investigation as well as experimental works. However, this fluorometric method is little more complicated compared with that of the calorimetric method (13).

ASSAY OF ACETYLCHOLINESTERASE ACTIVITY 333

Therefore, at the present time a study is in progress to develop more simple assay system.

Guilbault and Kramer (19) have reported a highly sensitive fluorometric method for the determination of the cholinesterase activity using resorfin butyrate and indoxyl acetate as fluorogenic substrates. These substrates are also hydro- lyzed by acylase, acid phosphatase, and chymotrypsin. The specificity of the determination of the cholinesterase activity in serum using these substrates is not known.

ACKNOWLEDGMENT

This work was supported by a grant from the Tazuke Kofukai Medical Research Foundation.

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