JPH0246300A - Determination of choline esterase activity - Google Patents

Abstract

PURPOSE:To improve the determination accuracy of the activity of choline esterase by reacting a substrate such as phenyllactyl-chloline with choline esterase, reacting the product with an enzyme capable of oxidizing produced phenyllactic acid and determining the increase of absorbance. CONSTITUTION:The activity of the subject enzyme is determined by (1) mixing a substrate with a color-developing reagent composed of a lactic acid oxidase, a chromogen and a peroxidase, heating for a prescribed period, adding a serum and determining the increase of absorbance or (2) mixing a substrate with a lactic acid dehydrogenase and NAD(P)<+>, heating for a prescribed period, adding a serum and determining the increase of absorbance or (3) adding a substrate together with NAD(P)<+> diaphorase and a tetrazolium compound and carrying out the procedure of the process (2). The reaction time and reaction temperature of the above processes are 1-20min and 20-40 deg.C. The present process is a novel method having excellent accuracy, precision and speed and suitable for automatic analyzer.

Description

[Detailed Description of the Invention] [Industrial Application Field] A decrease in serum cholinesterase activity is associated with liver cirrhosis,
It is observed in chronic hepatitis, liver tumors, malignant obstructive jaundo, gastric cancer 2 sepsis, uterine fibroids, liver cancer, and organophosphorus poisoning. - Fishing on a boat is said to be a direct indicator of a decline in liver function.

Increased activity is also observed in nephrotic syndrome. As described above, there is a very close relationship between each disease, especially liver disease, and the serum cholinesterase activity value, and it is one of the important items in clinical tests.

[Prior art]

Traditionally, cholinesterase activity was measured using the pH meter method [Takahashi, Shibata, Medicine and Biology, Vol. 20, p. 96, (195
1)), a method for quantifying the SH group of thiocholine produced using a thiocholine derivative as a substrate [Clinical Chemistry (C1in, Chem, vol. 11, p. 91, (1965)), etc. have been used. In recent years, the development of methods for measuring cholinesterase activity using enzymes has been actively carried out, and a method using choline oxidase (Japanese Patent Application Laid-Open No. 1989-1999)
-136895), method using p-hydroxybenzoic acid hydroxylase [Journal of Biochemistry (J, Biochem), vol. 94, p. 11,
(1983)), a method using choline kinase (Japanese Unexamined Patent Publication No. 52-99888), etc. have also come into use.

However, the pH meter method is complicated to operate and has problems in accuracy, and the method for measuring the SH group of thiocholine derivatives has the disadvantages that the substrate is unstable and the operations are complicated, resulting in problems in measurement accuracy.

Furthermore, the substrate specificity of the enzymes used in the methods using enzymes is not strict, which causes a decrease in measurement accuracy. In addition, methods using choline oxidase are susceptible to interference from choline, which occurs due to the decomposition of phospholipids coexisting in serum. Furthermore, methods using p-hydroxybenzoate hydroxylase or choline kinase are less affected by interfering substances in serum, but have problems with accuracy and precision because they measure the amount of decrease in ultraviolet absorbance.

[Problem that the invention seeks to solve]

The present invention solves a number of problems with the methods for measuring cholinesterase activity that have been reported to date. Furthermore, it solves the following problems in methods using enzymes. In other words, the substrate specificity of the enzyme used is not strict (that is, the enzyme acts on the reaction product produced by the enzyme used), and furthermore, in the case of a method that measures absorbance in the ultraviolet region,
The point is to measure the amount of decrease in absorbance (there are problems with accuracy and precision).

In the present invention, the substrate specificity of the enzyme used is strict (that is, it does not act at all on the reaction products produced by the enzyme reaction), and the increase in absorbance can be measured in ultraviolet absorption measurement. This improves measurement accuracy and also enables application to dye systems.

[Means for solving problems]

According to the present invention, an increase in absorbance is measured by using a reagent composition containing as an essential component an enzyme that oxidizes a choline derivative as a substrate and the other compound, which is not choline, among the decomposition products produced by the action of cholinesterase. A method for measuring cholinesterase activity is provided. More detailed examples include the following methods.

(1) A method in which a substrate, a coloring reagent consisting of lactate oxidase, a chromogen, and peroxidase are added and mixed, and after heating for a certain period of time, serum is added and the increase in absorbance is measured.

(2) A method in which the substrate and lactate dehydrogenase, NAD(P), are added and mixed, heated for a certain period of time, then serum is added, and the increase in absorbance is measured.

(3) A method in which a substrate, lactate dehydrogenase, NAD(P)'', diaphorase, and a tetrazolium compound are added and mixed, heated for a fixed period of time, then serum is added, and the increase in absorbance is measured.

In these methods, the reaction time is 1 to 20 minutes, preferably 3 to 10 minutes, and the reaction temperature is 20 to 40''C, preferably 25 to 37°C.The buffer solution may be one that does not inhibit the reaction. For example, phosphate buffer, Tris/hydrochloric acid buffer, etc. can be used.

Although the pH of the reaction can vary depending on the enzyme used, it is preferably carried out at a pH of 7.5 to 8.5.

As described above, the feature of the present invention is that by using an enzyme that acts on substances other than choline among the degradation products of the substrate by cholinesterase, the method is measured by decreasing the absorbance, which is a drawback of the conventional method due to the instability of the substrate. The disadvantages of doing
This method overcomes the drawbacks caused by the lack of strict specificity of the enzyme used and the interference caused by choline, and is a novel method with excellent accuracy, precision, and rapidity, and is suitable for automatic analyzers.

The present invention will be explained in more detail below using experimental examples and examples.

Synthesis of iodine salts of DL and L-phenyllactylcholine 0.34 g of potassium hydroxide was dissolved in 50 ml of methanol, and then 1 g each of OL and L-phenyllactic acid were dissolved. After stirring at room temperature for 5 minutes, the mixture was dried under reduced pressure, and 50 d of isopropyl alcohol and 0.72 g of β-dimethylaminoethyl chloride were added to the residue, followed by refluxing for 2.5 hours. Insoluble matter was removed by filtration, the filtrate was concentrated under reduced pressure, and the remaining oily substance was purified by silica gel chromatography (chloroform:methanol = 200:1 as a developing solvent) to obtain DL and L-phenyllactic acid β-dimethylamino. 0.7 g of ethyl ester was obtained as an oily substance. The results of elemental analysis of this substance were as shown below.

Elemental analysis value = J + JO + CHN Calculated value 65.80% 8.07% 5.90%
Measured value 65.72% 8.17% 5.68%
Next, 0.65 g of the OL obtained above and L-phenyllactic acid β-dimethylaminoethyl ester were added to 10 g of acetone.
1 d, then add 0.35 d of methyl iodide,
Stirred at room temperature for 1 hour. Filter the precipitated crystals and add 20% acetone.
d to obtain 0.67 g of iodine salt of OL and L-3-phenyllactate choline. The results of elemental analysis of this substance were as shown below.

Elemental analysis value = C + Jz□INO.

CHN Calculated value 44.34% 5.85% 3.69%
Measured value 44.20% 5.64% 3.85%
The iodine salts of DL and L-3-phenyllactate choline obtained above can also be used as substrates as they are.

Experimental Example 2 Examination of substrates for cholinesterase 5mM of phenyllactylcholine and 1.0 units of choline oxidase (manufactured by Toyo Jozo Co., Ltd.) were combined with 0.1 mg of 4-aminoantipyrine, 2 μl of phenol, and 10 units of peroxidase (manufactured by Amano Pharmaceutical Co., Ltd.). ) containing 0,1M
) After adding 0.9 d of Lys-HCl buffer (pH 8,0), add 0.05 units, 0.15 units, and 0.25 units of butylcholinesterase (derived from horse serum, manufactured by Sigma), respectively.
unit, 0.40 unit and 0.50 unit added at 37°C
The reaction was carried out for 3 minutes, and the change in absorbance at a wavelength of 505 nm was measured, and the results are shown in FIG.

As can be seen from FIG. 1, a good linear relationship was obtained between cholinesterase activity and the amount of change in absorbance, confirming that phenyllactylcholine serves as a substrate for cholinesterase.

Gol Okuma 6 Reactivity to phenyl lactic acid due to differences in the types of lactate dehydrogenase■ It is a reaction product produced by being degraded by cholinesterase when L-phenyllactylcholine is used as a substrate for measuring cholinesterase activity. 0.1M phosphate buffer (pH 8,0) containing 5mM L-phenyllactic acid and 211IM NAD+ 1. Add 300 units of porcine heart-derived lactate dehydrogenase (Toyobo type) to OmR,
The reaction was carried out at 7°C for 5 minutes. As a result, the wavelength is 340 nm
The absorbance value increased to 0.21, confirming that L-phenyllactic acid is a good substrate for lactate dehydrogenase derived from pig heart.

■ When 500 units of bacterial-derived lactate dehydrogenase (manufactured by Amano Pharmaceutical) was added instead of pig heart-derived lactate dehydrogenase, no increase in absorbance at a wavelength of 340 nm was observed, and L-phenyl lactic acid was derived from bacteria. It was confirmed that this product is not a substrate for lactate dehydrogenase.

■Instead of L-phenyl lactic acid, the reaction was carried out under the same conditions as in ■ and ■ above. As a result, no matter whether lactate dehydrogenase derived from pig heart or bacteria was added. The absorbance at a wavelength of 340 nm increased. From this, it was estimated that bacterial-derived lactate dehydrogenase can use D-phenyllactic acid as a substrate.

Experimental Example 4 Reactivity of lactate dehydrogenase to phenyllactylcholine ■ 5mM Noshi phenyllactylcholine and 2mM N
Add 500 units of lactate dehydrogenase derived from pig heart (manufactured by Tobo Co., Ltd.) to 0.95 d of 0.1 M Tris-HCl buffer (pH 8,0) containing ``AD'' and incubate at 30°C.
As a result of reacting for 5 minutes, the absorbance at a wavelength of 340 nm hardly increased, confirming that lactate dehydrogenase derived from pig heart does not act on L-phenyllactylcholine.

■ 5mM oral, L-phenyllactylcholine and 211
Add 50 μl of bacterial lactate dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd. > 500 units) to 0.95 mj! of 0.1 M phosphate buffer (pH 8,0) containing IM NAD and incubate at 30″C.
As a result of reacting for 5 minutes, the absorbance at a wavelength of 340 nm hardly increased, confirming that bacterial lactate dehydrogenase did not act on L-phenyllactylcholine.

Based on the predominant acanthus and the acanthus ↓, it was assumed that when phenyllactylcholine was used as a substrate, cholinesterase would act and the absorbance would increase only when phenyllactic acid was produced.

5mM phenyllactylcholine, 2mM NAD
” and 250 units of lactate dehydrogenase from pig heart (
After heating 0.957 0.1 M) Lis-HCl buffer (pH 8,0) containing Toyobo type) at 30°C for 3 minutes,
0.05 units, 0.13 units, and 0.25 μl of butylcholinesterase (derived from horse serum, manufactured by Sigma), respectively.
unit, 0.40 unit and 0.50 unit added, 30'C
.

The reaction was allowed to proceed for 3 minutes, and the amount of increase in absorbance at a wavelength of 340 nm was measured. The results are shown in FIG. As can be seen from FIG. 2, a good linear relationship was obtained between the amount of added cholinesterase activity and the maximum rate of increase in absorbance.

Example 2 5mM D-phenyllactylcholine, 2IIIM NAD'' and 500 units of bacterial lactate dehydrogenase (
After heating 0.95 d of 0.1M phosphate buffer (pH 8,0) containing Amano Pharmaceutical) at 30'C for 3 minutes, 50 μl
Butylcholinesterase (derived from horse serum, manufactured by Sigma) was added at 0.05 units, 0.13 units, 0.25 units, 0.40 units, and 0.50 units, respectively, and incubated at 30°C for 50 minutes.
The reaction was allowed to proceed for minutes, and the amount of increase in absorbance at a wavelength of 340 nm was measured. The results are shown in FIG. As can be seen from Figure 3,
A good linear relationship was obtained between the amount of cholinesterase activity and the maximum rate of increase in absorbance.

Example 3 0.11 μl of 4-aminoantipyrine, 2 μl of phenol, and 10 units of peroxidase (manufactured by Amano Pharmaceutical Co., Ltd.) were added to one bottle of 0.1 M Tris-HCl buffer (pH 8,0
0.05d of a solution containing 5mM phenyllactylcholine and 0.5 units of lactate oxidase (Toyo Jozo type) was added to 0.90d of the coloring solution prepared by dissolving in
After heating for 3 minutes, add 50 μl of butylcholinesterase (derived from horse serum, manufactured by Sigma) to 0.02 units, 0.05 units, 0.10 units, and 0.15 units, respectively.

0.20 units and 0.25 units were added and reacted at 30°C for 5 minutes to measure the amount of increase in absorbance at a wavelength of 505 nm. The results are shown in FIG. As can be seen from FIG. 4, a good linear relationship was obtained between the amount of cholinesterase activity and the maximum rate of increase in absorbance.

Example 4 5mM phenyllactylcholine, 2mM NAD
” and 250 units of lactate dehydrogenase derived from pig heart (Toyobo type) and 0.3 units of diaphorase (manufactured by Amano Pharmaceutical)
and 0.15■ containing nitroblue tetrazolium
．． 95d 0.1M) Lis-HCl buffer (pH 8.0)
and heated at 30"C for 3 minutes. Butylcholinesterase (derived from horse serum) was added to this solution.

(manufactured by Sigma) 0.02 units, 0.05 units, respectively.
0.10 unit, 0.15 unit, 0.20 unit and 0
．． Add 25 units and react at 30°C for 3 minutes at wavelength 57
The amount of increase in absorbance of 0nn+ was measured. The result is the fifth
As shown in the figure. As can be seen from FIG. 5, a good linear relationship was obtained between the amount of cholinesterase activity and the maximum rate of increase in absorbance.

Example 5 5mM phenyllactylcholine, 2mM NAD
After heating 24.5d 0.E) Lis-HCl buffer (pH 8,0) containing 600 units of pig heart-derived lactate dehydrogenase (Toyobo type) at 37°C for 3 minutes, human serum was added. Add 50 μ2 of
The increase in absorbance in nm was measured. As a blank, the serum was replaced with distilled water and the same operation was performed. The amount of increase in absorbance per minute due to cholinesterase activity in serum was calculated by subtracting the amount of increase in absorbance per minute of the blank from the amount of increase in absorbance per minute measured using serum.

At the same time, for comparison with the currently used cholinesterase measurement method, a commercially available kit for cholinesterase measurement using p-hydroxybenzoylcholine as a substrate (C
The cholinesterase value of the same serum was determined using h-ε Neo UV "Ginotest" (manufactured by Ginotest). The results are shown in FIG.

As can be seen from FIG. 6, a good correlation was obtained between the constant values on both sides.

Example 6 Add 5mM to the 2,45d coloring solution prepared in the same manner as Example 3.
of phenyllactylcholine and 1.5 units of lactate oxidase were added. After heating this solution at 37°C for 3 minutes, add 50μ of human serum! The amount of increase in absorbance at a wavelength of 505 nm was measured after reaction at 37°C for 3 minutes.

As a blank, the serum was replaced with distilled water and the same operation was performed. The amount of increase in absorbance per minute due to cholinesterase activity in serum was calculated by subtracting the amount of increase in absorbance per minute of the blank from the amount of increase in absorbance per minute measured using serum.

At the same time, for comparison with the currently used cholinesterase measurement method, a commercially available kit for cholinesterase measurement (C
The cholinesterase value of the same serum was determined using h-E Neo UV" (manufactured by Cynotes Vicino Test Co., Ltd.). The results are shown in FIG.

As can be seen from FIG. 7, a good correlation was obtained between the constant values on both sides.

〔Effect of the invention〕

The present invention can solve many of the problems of conventional methods for measuring cholinesterase activity using enzymes, and provide a highly accurate and precise measuring method.

[Brief explanation of the drawing]

Figure 1 shows the relationship between cholinesterase activity and absorbance in Experimental Example 2, Figure 2 shows the relationship between cholinesterase activity and absorbance in Example 1, and Figure 3 shows the relationship between cholinesterase activity and absorbance in Example 2. Figure 4 shows the relationship between the absorbance and the absorbance of Example 3.
Figure 5 shows the relationship between cholinesterase activity and absorbance in Example 4, and Figure 6 shows the relationship between the method of the present invention and the commercially available cholinesterase measurement method in Example 5. This shows the correlation between the two measurement methods when using the kit (Ch-E Neo UV "Ginotest" manufactured by Ginotest), and Figure 7 shows the correlation between the method of the present invention in Example 6 and the commercially available method for measuring cholinesterase. This shows the correlation between the two measurement methods when using a kit (Ch-E Neo UV "Ginotest" manufactured by Ginotest).

Claims (1)

[Claims] (1) General formula [▲There are mathematical formulas, chemical formulas, tables, etc.▼] (In the formula, X represents a halogen atom, OH or OSO_3R, and R is H or CH_3.) Cholinesterase is produced using a compound represented by the substrate as a substrate. 1. A method for measuring the activity of cholinesterase, which is characterized by allowing an enzyme to act on the generated phenyl lactic acid and measuring the increase in absorbance.