JPH0236237B2 - PPHIDOROKISHIANSOKUKOSANSOKUTEIYOSHAKUSOSEIBUTSU - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent composition for measuring p-hydroxybenzoic acid.

Recently, in the field of clinical diagnostic reagents, there are expectations for the development of reagents for measuring serum cholinesterase. The conventional method for measuring serum cholinesterase is
Several measurement methods have been discovered in which cholinesterase acts on choline ester to generate organic acid and choline, or to leave choline ester remaining. For example, in the m-nitrophenol method, acetylcholine is hydrolyzed by cholinesterase, and the pH of the system is lowered due to the generated acetic acid. After adding a substrate and heating for a certain period of time, m- There is a method of determining cholinesterase activity by adding a mixture of nitrophenol and measuring the degree of yellow discoloration. However, this method is susceptible to interfering substances in the sample, such as organic acids and buffering substances.
Furthermore, since the reaction is stopped before measurement, it lacks reliability and requires a long reaction time, making it unsuitable for processing multiple samples.

In addition, choline thioesters such as acetylthiocholine, butyrylthiocholine, and propionylthiocholine are used as synthetic substrates, and thiocholine produced by the action of cholinesterase is colored with 5,5'-dithiobisnitrobenzoic acid (DTNB).
There is a DTNB method for colorimetric determination. However, since DTNB reacts with thiol, this method is easily affected when thiol groups coexist in the sample, for example, when glutathione is added to serum.

Furthermore, the substrate benzoylcholine is hydrolyzed by cholinesterase, and the generated choline is decomposed into hydrogen peroxide and betaine by the action of cholinesterase, and the generated hydrogen peroxide is oxidized into phenol and 4-aminoantipyrine by the action of peroxidase. There is an enzyme (choline oxidase) method that condenses with quinone to form a red quinone pigment. However, this method also uses reducing agents such as ascorbic acid,
Sensitive to bilirubin or glutathione. Furthermore, the molecular extinction coefficient of the quinone dye, which is a coloring agent, varies considerably depending on the commercially available measurement reagents from different companies, which poses a problem as a standard method.

In addition to the above method, the substrate benzoylcholine
UV measuring the rate of decrease in absorbance at 240nm
There is a colorimetric method that uses acetylcholine as a substrate and allows Fe ⁸⁺ to interact with hydroxylamine. However, these methods have drawbacks that make it difficult to process a large number of samples.

Compared to the conventional method described above, as a more accurate method for measuring cholinesterase activity, p-hydroxybenzoylcholine is used as a substrate, and the generated p-hydroxybenzoic acid is converted to protocatechuic acid using p-hydroxybenzoic acid hydroxylase and NADPH. A method has been proposed to measure cholinesterase activity by rate-assaying the decrease in NADPH at 340 nm (Journal of the Japanese Society of Clinical Laboratory Automation, Vol. 6 Supplement, p. 166, 1981 Year). This method utilizes the following reaction.

However, in this method, it has been discovered that when the above-mentioned enzyme and reagent are applied to a sample containing a certain amount of cholinesterase, the measured amount of cholinesterase activity increases as the amount of enzyme increases.

The present inventors discovered that in the above method, the protocatechuic acid produced acts as an uncoupler for p-hydroxybenzoic acid hydroxylase, further promoting the oxidation of NADPH, as shown in the reaction formula below. As a result, we found that the measured values were more accurate than they actually were.

Taking these results into consideration, the inventors
In order to find a method for accurately measuring p-hydroxybenzoic acid produced in a method for measuring cholinesterase using p-hydroxybenzoylcholine as a substrate, the present invention was achieved as a result of various intensive studies.

That is, the present invention provides a method for measuring p-hydroxybenzoic acid characterized by containing p-hydroxybenzoic acid hydroxylase, a reduced coenzyme, and protocatechuic acid 3,4-dioxygenase or protocatechuic acid, 4,5-dioxygenase. It is a reagent composition for use.

In the present invention, by using protocatechuate 3,4-dioxygenase or protocatechuate 4,5-dioxygenase, it acts as an uncoupler of p-hydroxybenzoate hydroxylase and prevents the oxidation of NADPH. Furthermore, it becomes possible to remove protocatechuic acid which promotes the process from the system and accurately measure p-hydroxybenzoic acid. Furthermore, when the reagent composition of the present invention is used to measure cholinesterase activity using p-hydroxybenzoylcholine as a substrate, there is no influence of reducing agents (ascorbic acid, bilirubin, glutathione, etc.) compared to conventional measurement methods, and reduced The molecular extinction coefficient of the coenzyme is also clear, making it possible to accurately measure cholinesterase activity, and is expected to become a standard method in the future.

p-Hydroxybenzoic acid hydroxylase used in the present invention belongs to flavin-related external electron donor-requiring atomic oxygenase, and has an enzyme number of
Including those indicated in EC1.14.13.2. Its action is shown by the following formula.

The enzymes may be obtained from bacteria, in particular from bacteria of the genus Pseudomonas, such as Pseudomonas desmolyteica, Pseudomonas fluorescens, Pseudomonas putida, and the like.

The reduced coenzyme used in the present invention is
There are NADPH, NADH, etc.

Protocatechuic acid 3,4-dioxygenase used in the present invention is an enzyme that converts protocatechuic acid to 3-carboxy-cis-cis-muconic acid (β-carboxymuconic acid), and is a diatomic enzyme involving non-heme iron. Belongs to oxygenase, enzyme number
It refers to EC1.13.1.3. The action of this enzyme is shown by the following formula.

The enzymes may be obtained from bacteria, particularly from Pseudomonas, Nocardia and Neurospores.

Protocatechuic acid 4,5-dioxygenase used in the present invention is an enzyme that converts protocatechuic acid into 2-hydroxy-4-carboxymuconic acid semialdehyde, and belongs to diatomic oxygenases involving non-heme iron. It is number EC1.13.11.8. The action of this enzyme is shown by the following formula.

The enzyme may be obtained from bacteria, especially from Pseudomonas.

The reagent composition of the present invention contains p-hydroxybenzoate hydroxylase, a reduced coenzyme, and protocatechuate 3,4-dioxygenase or protocatechuate 4,5-dioxygenase. The amounts of enzyme and coenzyme are arbitrarily selected depending on the sample.

The p-hydroxybenzoic acid measured using the reagent composition of the present invention may be derived from any source, and may be p-hydroxybenzoic acid obtained by allowing cholinesterase to act on p-hydroxybenzoylcholine.

When measuring cholinesterase activity,
To the reagent composition of the present invention, p-hydroxybenzoylcholine, a buffer and optionally flavin adenine dinucleotide (FAD) are added. The buffer has a pH of 5.0 to 11.0, preferably 7.5 to 9.0. Further, the buffer may be phosphoric acid, organic acid, or other materials widely used in the field of biochemistry. Concentration is 0.5M
~0.01M is good.

The substrate p-hydroxybenzoylcholine may be a salt thereof, and the concentration is 0.01mM to
100mM, preferably 0.1-10mM is good.

The reduced coenzyme may be its salt, and the concentration is
The amount is about 0.05mM to 1mM, preferably 0.1 to 0.3mM.

FAD can be its salt, and the concentration is 0.001-0.1mM
The amount is preferably 0.01-0.05mM. FMN, riboflavin, riboflavin 4' instead of FAD,
5'-cyclic phosphoric acid, flavin peptide, riboflavin glucoside, and fat-soluble riboflavin derivatives may also be used.

p-hydroxybenzoic acid hydroxylase is 0.01-50
It is preferably about 0.2 to 5 units/ml, especially about 0.2 to 5 units/ml.

The amount of protocatechuate 3,4-dioxygenase or protocatechuate 4,5-dioxygenase is preferably about 0.001 to 10 units/ml, especially 0.02 to 10 units/ml.
1 unit/ml is good.

To measure cholinesterase, a reaction mixture containing the above-mentioned reagents is kept at a constant temperature in advance, an appropriate amount of a sample is added, and the change in reduced coenzyme or the amount of oxygen consumed is measured.

The reaction temperature is preferably about 10 to 45°C, particularly 20 to 40°C. PH is about 5.0 to 11.0, particularly preferably PH7.5 to 9.0.

The sample may be any biochemical substance, and serum, urine, etc. are particularly used.

As a method to measure changes in reduced coenzymes,
Changes in the absorbance of NADPH (around 340 nm), as well as other methods such as fluorescence, luminescence, immunotherapy, and electrode methods, are used.

The amount of oxygen consumed can be measured by polarometry using an oxygen electrode or the like.

In the reaction, the substrate p-hydroxybenzoylcholine is first hydrolyzed by cholinesterase in the sample, and p-hydroxybenzoic acid is liberated (first reaction). Next, p-hydroxybenzoic acid becomes protocatechuic acid by p-hydroxybenzoic acid hydroxylase, which simultaneously consumes an equimolar amount of oxygen molecules and oxidizes an equimolar amount of reduced coenzyme to form an oxidized coenzyme. Becomes an enzyme (second reaction). The produced protocatechuic acid is protocatechuic acid 3,4-
3-carboxy-cis- by dioxygenase
Produces cis-muconic acid. At this time, equimolar amounts of oxygen molecules are consumed at the same time (third reaction).

All of these reactions may proceed simultaneously, or may be conducted separately into the first reaction, the second reaction, and the subsequent reactions.

By using the reagent composition of the present invention, cholinesterase activity can be measured accurately or with good reproducibility. Further, the amount of p-hydroxybenzoic acid hydroxylase added can be small.

The present invention will be explained below with reference to Examples.

The method for measuring the activity of each enzyme is as follows.

p-Hydroxybenzoic acid hydroxylase: Add 50 μl of enzyme dilution to 3.0 ml of reaction solution (37°C) of Tris-malate buffer mM (PH8.2) p-hydroxybenzoic acid 0.5mM NADPH 0.3mM FAD 0.02mM , measure the decrease in absorbance at 340 nm. 1
The amount of enzyme that changes 1 μmole of NADPH per minute is defined as 1 unit. (Molecular extinction coefficient of NADPH 6.22
cm ³ /micromole) Protocatechuic acid 3,4-dioxygenase: Add 50 µl of the enzyme dilution solution to 3.0 ml of a reaction solution containing 0.4 mM protocatechuic acid and 50 mM Tris-acetate buffer (PH 7.5) (at 24°C). Measure the decrease in absorbance.
The amount of enzyme that changes 1 μmole of protocatechuic acid per minute is defined as 1 unit (molecular extinction coefficient of protocatechuic acid: 3.8 cm ² /micromole).

Protocatechuic acid 4,5-dioxygenase: 0.1ml of enzyme dilution solution (50mM phosphate buffer, containing 10% methanol) in 3.0ml (24℃) of reaction solution of 0.33mM protocatechuic acid and 50mM phosphate buffer (PH7.0) and measure the decrease in absorbance at 250 nm. 1
The amount of enzyme that changes 1 μmole of protocatechuic acid per minute is defined as 1 unit (molecular absorption coefficient of protocatechuic acid: 8.0 cm ² /micromole).

Example 1 A reaction mixture as shown below was prepared and p-hydroxybenzoic acid was measured.

50mM Tris-malate buffer PH8.2 0.3mM NADPH 0.02mM FAD p-hydroxybenzoate hydroxylase 1U/ml protocatechuate 4,5-dioxygenase
0.1U 4.0ml of the above reaction mixture was prewarmed at 37°C for 10 minutes. When the absorbance at 340 nm became constant, 50 μl of p-hydroxybenzoic acid aqueous solution was added. The absorbance at 340 nm decreased and the change in absorbance was recorded after 15 minutes.

p-hydroxybenzoic acid aqueous solution concentration is 0,
The concentrations were 0.25, 0.50, 0.75, and 1.0mM.

As shown in Figure 1, the additive-free product does not show accurate values and produces a regular difference, but protocatechuic acid 4,5
-The one with dihydrooxygenase added matched the theoretical value.

Example 2 The following reaction mixture was prepared and the titer of serum cholinesterase was measured.

50mM Tris-malate buffer PH8.2 0.75mM Hydroxybenzylcholine 0.3mM NADPH 0.02mM FAD p-hydroxybenzoic acid hydroxylase
0 to 2 U/ml (protocatechuic acid 3,4-dioxygenase 0.1 U/ml) 4.0 ml of the above reaction mixture was preheated at 37° C. for 10 minutes. When the absorbance at 340 nm becomes constant, control serum (Monitorol, DADE)
50 μl was added to start the reaction. 5-10 minutes
The change in absorbance at 340 nm was recorded. The amount of enzyme that changes 1 μmole of NADPH per minute is 1
The titer of cholinesterase is expressed as a unit (molecular extinction coefficient of NADPH 6.22 cm ² /micro).
omole).

Using this measurement method, it was found that when the amount of p-hydroxybenzoate hydroxylase was changed, the serum cholinesterase value changed depending on the amount of p-hydroxybenzoate hydroxylase, as shown in FIG. There, protocatechuic acid 3,4-dioxygenase is added.
When 0.1 U/ml was added, the serum cholinesterase level became constant. In addition, the reaction proceeded quickly, and the amount of p-hydroxybenzoic acid hydroxylase added could be reduced.

Reference example 1 The choline oxidase method was performed as follows.

Reaction mixture 50mM Tris-malate buffer PH8.2 Phenol 100mg/dl 4-aminoantipyrine 20mg/dl Peroxidase
1000 U/dl (purpurogalin unit) Choline oxidase 100 U/dl Benzoylcholine chloride 50 mg/dl 2.0 ml of the reaction mixture was prewarmed at 37°C for 10 minutes, and then 20 μl of the sample was added. After heating at 37°C for exactly 5 minutes, 2.0 ml of a stop solution (neostigmine sulfate 100 mg/dl) was added, and after heating at 37°C for 10 minutes, measurement was performed at 500 nm. In the blind test, the same operation was performed without adding the sample.

This method X (Example 2) and choline oxidase method Y
The correlation was good as Y=1.82×36 γ=0.995 (n=48).

[Brief explanation of drawings]

Figure 1 shows the absorbance at 340 nm when protocatechuic acid 4,5-dioxygenase was added (●――――●) and when it was not added (○――――○) when measuring p-hydroxybenzoic acid. Show change. Second
The figure shows the relationship between cholinesterase activity and p-hydroxybenzoate hydroxylase when protocatechuate 3,4-dioxygenase (0.1 unit/ml) is added (●――――●) and when it is not added (○―― ―
○).

Claims (1)

[Claims] 1. A reagent composition for measuring p-hydroxybenzoic acid characterized by containing p-hydroxybenzoic acid hydroxylase, a reduced coenzyme, and protocatechuic acid 3,4-dioxygenase or protocatechuic acid 4,5-dioxygenase .