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A fluorometric assay of the acetylcholinesterase activity in blood
Clin. Biochem. 3, 327-333 (1970)
A FLUOROMETRIC
TETSUO Kitano
Hospilul
UETE, Tazuke
ASSAY OF THE ACETYLCHOLINESTERASE ACTIVITY IN BLOOD
HIROKO Kofukai
TSUCHIKURA, Medical
(Received
AND
Research Institute, March
KAZUMI Kita-ku,
HOSHIDA Osaka
City,
Japan
18, 1970)
SUMMARY
A specific and sensitive fluorometric method for the determination of the activity 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 acetylthiocholine 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-phthalaldeHyde. 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 10
OF ACETYLCHOLINESTERASE
gcitation
ACTIVITY gpisslml
spectra
329 spectra
t 60
-
50
-
40
-
30
-
20
l
10
*
0
i%iocholins
-
250
FIG.
1.
The
300
350
fluorescence
spectra
400
m)l
350
of thiocholine and reduced 0-phthalaldehyde.
400
glutathione
455
developed
500
mp
with
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.
DC1tat1onsp2ctrs
350
FIG. 2.
The effect of the incubation
time on the liberation choline by serum.
400
of thiocholine
4>3
5J’J ‘91
from the acetylthio-
0.3
0.2
0.1
0 0 FIG. 3.
0.02
0.04
0.06
0.08
0.10
The amount of serum and the units of the activity of cholinesterase.
ml
ASSAY
OF ACETYLCHOLINESTERASE TABLE
ACTIVITY
331
I
THE EFFECT OF VARIOUS ANTICOAGULANTS ON THE DETEKhIINATION OFTHEACTIVITYOFCHOLINESTERASEINSERUM Thiocholine ~moles/min/ml Addition
Expt.
None
2.42 1.40 2.49 2.57 2.25 2.25 1.79
EDTA 1 mg/ml serum Sodium citrate 1 mg/ml serum .%~~monium oxalate 1 mg/ml serum Potassium oxalate 1 nig/nil serum Heparin 0.1 mg/ml serum Sodium fluoride 0.25 mg/ml serum TABLE THE ACTIVITIESOF
CHOLINESTERASE
1
liberated serum Expt.
2
1.34 0.70 2.02 1.87 2.02 1 .9.5 1.09
II IN PLASMA
AND RED BLOOD
CELLS
Activity of cholinesterase in 1 ml of blood thiocholine, liberated ~moles/min Case
Sex
-4s h-1
Hct (%)
Plasma
1 2 3 4
F F F F
23 22 21 26
40 42 40 37
2.44 2.03 2.48 2.40
3.23 6.48 14.40 2.12C.
5
NI
26
47
1.54
7.21
calorimetric method and the present the liberation of thiocholine from subjects measured by the calorimetric respectively 2.81 f 0.42 and 2.72 f liver cirrhosis, showed a decrease in is seen in Fig. 4.
Red blood
cells
fluorometric method. The average values of acetylthiocholine by serum of 16 healthy method and the fluorometric method were 0.50 ccmoles per minute per ml. Patients with the level of this enzyme activity in serum, as
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 determination 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
UETE
332
el al.
l
Healthy subjects
0 0
2.0 1.o 3.0 ~iocholin13, liberated, measured
FIG. 4.
Comparison
4.0 pmolea/mln./ml by fluorometric
of the cholinesterase activity in serum measured and the present fluorometric method.
5.0 serum, method.
by the calorimetric
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 acetylthiocholine. 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
333
ACTIVITY
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 hydrolyzed 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 Research Foundation.
Kofukai
Medical
REFERENCES STEDYIAN, E., STEDMAN, E. & EASSON, L. H. Choline-esterase. An enzyme present in the blood-serum of the horse. Biochem. J. 26, 2056-2066 (1932). 2. VAHLQUIST, B. On esterase activity of human blood plasma. SKand. Arch. f. Physiol. 72, 133-160 (1935). D. Studies on enzymatic histochemistry. XXV. A micromethod for the determinas. GLICK, tion of cholinesterase and the activity-pH relationship of this enzyme. J. Gen. Physiol. 21, 289-295 (1938). AMMON, R. Die fermentativeSpaltungdesAcetylcholins. Pflugers Arch. ges. Physiol. 233, 486-491 (1933). HERSTIN, S. The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J. Biol. Chem. 180, 249-261 (1949). KOELLE, G. B. The histochemical differentiation of types of cholinesterase and their localizations in tissue of the cat. J. Pharmacol. Exp. Therap. 100, 15&179 (1950). of plasma and red AUGUSTINNSON, K. B. A titrimetric method for the determination cell cholinesterase activity using the thiocholine esters as substrates. Stand. J. Clin. Lab. Invest. 7, 284-290 (1955). W. The determination of choiinesterase activity in human 8. MEYER, A. & WILBRANDT, blood. Helv. Physiol. Pharmacol. Acta 12, 206-216 (1954). method for determination of cholinesterase 9. GAL, E. M. & ROTH, E. Spectrophotometric activity. Clin. Chim. Acta 2, 316-326 (1957). 10. McOSKER, D. E. & DANIEL, L. J. A calorimetric micromethod for the determination of cholinesterase. Arch. Biochem. Biophys. 79, l-7 (1959). G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77 (1959). Ii. ELLMAN, G. L., COUTNEY, D. K., ANDRE& V., JR. & FEATHERSTONE, R. M. A 1.2. ELLMAN, new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95 (1961). IS. GARRY, P. J. & ROUTH, J. I. A micro-method for serum cholinesterase. Clin. Chem. 11, 91-96 (1965). J. G. & STILES, D. E. Serum cholinesterase activity. A 14. SIDER, D. B., BATSAKIS, calorimetric microassay and some clinical correlation. Amer. J. Clin. Path. 50,344-350 (1968). A. & COHN, V. H. A method for the Auorometric assay 16. SHORE, P. A., BURKHALTER, of histamine in tissues. J. Pharmacol. Exptl. Therap. 127, 182-186 (1959). 16. PISANO, J. J., WILSON, J. D., COHEN, L., ABRAHAM, D. & UDENFRIEND, S. Isolation of r-aminobutylhistidine (homocarnosine) from brain. J. Biol. Chem. 236, 499-502 (1961). method for the determination of agma17. COHN, V. H. &SHORE, P. A. A microfluorometric tine. Anal. Biochem. 2 237-241 (1961). 18. COHN V. H. & LYLE, J. A fluorometric assay for glutathione. Anal. Biochem. 14, 434-440(1966). 19. GUILBAULT, G. G. & KRAMER, D. N. Resorufin butyrate and indoxyl acetate as fluorogenic substrates for cholinesterase. Anal. Chem. 37, 120-123 (1965). 1.
A FLUOROMETRIC
TETSUO Kitano
Hospilul
UETE, Tazuke
ASSAY OF THE ACETYLCHOLINESTERASE ACTIVITY IN BLOOD
HIROKO Kofukai
TSUCHIKURA, Medical
(Received
AND
Research Institute, March
KAZUMI Kita-ku,
HOSHIDA Osaka
City,
Japan
18, 1970)
SUMMARY
A specific and sensitive fluorometric method for the determination of the activity 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 acetylthiocholine 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-phthalaldeHyde. 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 10
OF ACETYLCHOLINESTERASE
gcitation
ACTIVITY gpisslml
spectra
329 spectra
t 60
-
50
-
40
-
30
-
20
l
10
*
0
i%iocholins
-
250
FIG.
1.
The
300
350
fluorescence
spectra
400
m)l
350
of thiocholine and reduced 0-phthalaldehyde.
400
glutathione
455
developed
500
mp
with
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.
DC1tat1onsp2ctrs
350
FIG. 2.
The effect of the incubation
time on the liberation choline by serum.
400
of thiocholine
4>3
5J’J ‘91
from the acetylthio-
0.3
0.2
0.1
0 0 FIG. 3.
0.02
0.04
0.06
0.08
0.10
The amount of serum and the units of the activity of cholinesterase.
ml
ASSAY
OF ACETYLCHOLINESTERASE TABLE
ACTIVITY
331
I
THE EFFECT OF VARIOUS ANTICOAGULANTS ON THE DETEKhIINATION OFTHEACTIVITYOFCHOLINESTERASEINSERUM Thiocholine ~moles/min/ml Addition
Expt.
None
2.42 1.40 2.49 2.57 2.25 2.25 1.79
EDTA 1 mg/ml serum Sodium citrate 1 mg/ml serum .%~~monium oxalate 1 mg/ml serum Potassium oxalate 1 nig/nil serum Heparin 0.1 mg/ml serum Sodium fluoride 0.25 mg/ml serum TABLE THE ACTIVITIESOF
CHOLINESTERASE
1
liberated serum Expt.
2
1.34 0.70 2.02 1.87 2.02 1 .9.5 1.09
II IN PLASMA
AND RED BLOOD
CELLS
Activity of cholinesterase in 1 ml of blood thiocholine, liberated ~moles/min Case
Sex
-4s h-1
Hct (%)
Plasma
1 2 3 4
F F F F
23 22 21 26
40 42 40 37
2.44 2.03 2.48 2.40
3.23 6.48 14.40 2.12C.
5
NI
26
47
1.54
7.21
calorimetric method and the present the liberation of thiocholine from subjects measured by the calorimetric respectively 2.81 f 0.42 and 2.72 f liver cirrhosis, showed a decrease in is seen in Fig. 4.
Red blood
cells
fluorometric method. The average values of acetylthiocholine by serum of 16 healthy method and the fluorometric method were 0.50 ccmoles per minute per ml. Patients with the level of this enzyme activity in serum, as
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 determination 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
UETE
332
el al.
l
Healthy subjects
0 0
2.0 1.o 3.0 ~iocholin13, liberated, measured
FIG. 4.
Comparison
4.0 pmolea/mln./ml by fluorometric
of the cholinesterase activity in serum measured and the present fluorometric method.
5.0 serum, method.
by the calorimetric
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 acetylthiocholine. 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
333
ACTIVITY
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 hydrolyzed 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 Research Foundation.
Kofukai
Medical
REFERENCES STEDYIAN, E., STEDMAN, E. & EASSON, L. H. Choline-esterase. An enzyme present in the blood-serum of the horse. Biochem. J. 26, 2056-2066 (1932). 2. VAHLQUIST, B. On esterase activity of human blood plasma. SKand. Arch. f. Physiol. 72, 133-160 (1935). D. Studies on enzymatic histochemistry. XXV. A micromethod for the determinas. GLICK, tion of cholinesterase and the activity-pH relationship of this enzyme. J. Gen. Physiol. 21, 289-295 (1938). AMMON, R. Die fermentativeSpaltungdesAcetylcholins. Pflugers Arch. ges. Physiol. 233, 486-491 (1933). HERSTIN, S. The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J. Biol. Chem. 180, 249-261 (1949). KOELLE, G. B. The histochemical differentiation of types of cholinesterase and their localizations in tissue of the cat. J. Pharmacol. Exp. Therap. 100, 15&179 (1950). of plasma and red AUGUSTINNSON, K. B. A titrimetric method for the determination cell cholinesterase activity using the thiocholine esters as substrates. Stand. J. Clin. Lab. Invest. 7, 284-290 (1955). W. The determination of choiinesterase activity in human 8. MEYER, A. & WILBRANDT, blood. Helv. Physiol. Pharmacol. Acta 12, 206-216 (1954). method for determination of cholinesterase 9. GAL, E. M. & ROTH, E. Spectrophotometric activity. Clin. Chim. Acta 2, 316-326 (1957). 10. McOSKER, D. E. & DANIEL, L. J. A calorimetric micromethod for the determination of cholinesterase. Arch. Biochem. Biophys. 79, l-7 (1959). G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77 (1959). Ii. ELLMAN, G. L., COUTNEY, D. K., ANDRE& V., JR. & FEATHERSTONE, R. M. A 1.2. ELLMAN, new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95 (1961). IS. GARRY, P. J. & ROUTH, J. I. A micro-method for serum cholinesterase. Clin. Chem. 11, 91-96 (1965). J. G. & STILES, D. E. Serum cholinesterase activity. A 14. SIDER, D. B., BATSAKIS, calorimetric microassay and some clinical correlation. Amer. J. Clin. Path. 50,344-350 (1968). A. & COHN, V. H. A method for the Auorometric assay 16. SHORE, P. A., BURKHALTER, of histamine in tissues. J. Pharmacol. Exptl. Therap. 127, 182-186 (1959). 16. PISANO, J. J., WILSON, J. D., COHEN, L., ABRAHAM, D. & UDENFRIEND, S. Isolation of r-aminobutylhistidine (homocarnosine) from brain. J. Biol. Chem. 236, 499-502 (1961). method for the determination of agma17. COHN, V. H. &SHORE, P. A. A microfluorometric tine. Anal. Biochem. 2 237-241 (1961). 18. COHN V. H. & LYLE, J. A fluorometric assay for glutathione. Anal. Biochem. 14, 434-440(1966). 19. GUILBAULT, G. G. & KRAMER, D. N. Resorufin butyrate and indoxyl acetate as fluorogenic substrates for cholinesterase. Anal. Chem. 37, 120-123 (1965). 1.