JPS6181799A - Method of measuring transminase activity - Google Patents

Abstract

PURPOSE:To measure the titled activity rapidly and precisely, by feeding a test specimen solution, a substrate solution for measuring GOT, etc. to a cell provided with an enzyme electrode having an immobilized oxygen membrane of L-glutamate oxidase, and measuring electrode output. CONSTITUTION:The cell 3 is equipped with the enzyme electrode 5 provided with the immobilized enzyme membrane 4 obtained by immobilizing L-glutamate oxidase on a porous high polymer carrier. The cell 3 is charged with the buffer solution 15, the test specimen solution 1, and the substrate solution for measuring glutamic-oxaloacetic transminase (GOT) or glutamic-pyruvic transaminase (GPT) 14 to carry out a reaction. Then, formed L-glutamic acid is reacted with L- glutamate oxidase, evolved hydrogen peroxide or reduced enzyme is detected by the electrode 5, recorded by the recorder 7, so that GOT activity or GPT activity in the test specimen solution 1 is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the use of transaminases (glutamate oxaloacetate transaminase (abbreviated as GOT) and glutamate pyruvate transaminase (abbreviated as GOT)) in biological samples.
The present invention relates to a method for measuring the activity of GPT (abbreviated as GPT).

By using the method of the present invention, G in a sample can be detected using one enzyme electrode.
OT or GPT measurements can be performed quickly and accurately. Furthermore, since GOT and GPT in the same -X material can be measured in the same reaction cell without replacing the sample, the sample can be measured more quickly and with high precision.

Transaminase is an enzyme that catalyzes the transamination of amino acids, and among these, GOT is contained in large amounts in cardiac muscle, liver, skeletal muscle, and kidney, and GPT is contained in large amount in liver and kidney.

Therefore, GOT and GPT measurements can be used to detect liver diseases such as acute viral hepatitis, chronic hepatitis, cirrhosis, liver tumors, acute alcoholic hepatitis, and extrahepatic obstruction such as gallbladder, cholangitis, and cholelithiasis.
It plays a useful role in diagnosing myocardial infarction, progressive muscular dystrophy, infectious diseases, etc., and it is of great clinical significance to easily, accurately, and quickly measure GOTl GPT.

PRIOR ART Conventionally, a method for measuring GOT involves adding a sample to the substrates aspartic acid and α-ketoglutaric acid, and conjugating the oxaloacetic acid of the generated oxaloacetic acid and glutamic acid in the presence of malate dehydrogenase and coenzyme NADH. The Carmen method is used to measure the decrease in the absorbance of NADH in the order of 340 using a modified initial velocity method, and the oxaloacetic acid produced as above is reacted with 2,4-dinitrophenylhydrazine to produce hydrazone. There is the Reitman-Frankel method, which creates quinoids by making them alkaline and develops color. However, the Carmen method has problems such as the instability of the reagents used and the need for a special spectrophotometer equipped with a thermostatic cell. On the other hand, in the Reitman-Frankel method, the substrate amount of α-ketoglutaric acid also develops color and interferes with the color development of the product, so the amount of substrate must be extremely reduced, which may cause the calibration curve to be distorted or the measurable range to be limited. It has drawbacks such as being narrow.

In addition, to measure GPT, a sample is added to the substrates alanine and α-ketoglutaric acid, and of the generated pyruvate and glutamic acid, pyruvate is combined with lactate dehydrogenase and the NAD
The Carmen method, in which the decrease in NADH is measured by conjugation in the presence of H, and the method in which 2,
There is a Reitman-Frankel method in which color is developed by the action of 4-dinitrophenylhydrazine. However, these methods also have the same drawbacks as the GOT measurement method described above.

On the other hand, a method for analyzing GPTXGOT using pyruvate oxidase (abbreviated as POP) has been developed. However, the measurement method using POP has problems such as disposable use of expensive enzymes, long measurement time, and poor reproducibility during measurement because PoP itself is unstable in solution.

Therefore, in order to solve the above drawbacks, POP was fixed,
Many studies are being conducted to measure GPT and GOT using a fermented kidney ball attached to an L:2 ratio stereotaxic electrode. (Unexamined Japanese Patent Publication No. 55-111786, 5
6-124396 publications, Analytica ch
imica Acta, 11th (1980) 65-
However, with these immobilization methods described above, the activity and stability of immobilized POP are not sufficient, and immobilization of POP alone does not allow the enzyme xaloacetate decarboxylase (OAC) to be activated during GOT measurement. There are drawbacks that must be added.

Furthermore, without exchanging samples within the same cell, the G in the same sample
It is extremely difficult to measure OT and GPT activity simultaneously.

Therefore, G. improves the shortcomings of these conventional methods.

A simple method for measuring T activity and GPT activity, and
It has been desired to provide a method for measuring GOT activity and GPT activity in a sample in a short time.

Summary of the Invention As a result of intensive studies to meet this demand, the present inventors discovered the recently developed L-glutamate oxidase (
The present invention has been completed based on the discovery that the above-mentioned drawbacks can be solved by utilizing an immobilized enzyme membrane using the enzyme membrane (see Japanese Patent Application Laid-Open No. 57-43685).

That is, the present invention provides a cell having an enzyme electrode equipped with an immobilized enzyme membrane in which L-glutamate oxidase is immobilized on a porous polymer carrier, a buffer solution, a sample solution, and glutamate oxaloacetate transaminase (GOT) measurement. Provided is a method for measuring transaminase activity in which a substrate solution for measuring glutamate pyruvate transavanase (GPT) is supplied, and GOT activity is measured by the output of the enzyme electrode. According to the method of the present invention, GPT activity in biological samples, GO
T activity can be easily measured in a short time, and GPT activity and GOT activity in the same sample can be measured easily and continuously in a short period of time.

In addition, when using a known immobilized F of POP, enzymes such as oxaloacetate decarboxylase (OAC), flavin adenine dinucleotide (FAD), and thiamine pyrophosphate-1-(TPP), ¥! l:
Although it is necessary to use Sung in combination, according to the final method, there is no need to use other enzymes or coenzymes in addition to the L-glutamate oxidase immobilized enzyme.

Furthermore, the powder method using an immobilized enzyme of L-glutamate oxidase has good stability during use and maintains G for a long period of time.
PTX GOT activity can be stably measured.

DETAILED DESCRIPTION OF THE INVENTION The immobilized enzyme membrane used in the present invention can be constructed using a porous polymer carrier. As the porous polymer carrier, known vinyl chloride resin carriers, acetyl cellulose, polycarbonate, nylon, polyethylene, polyethylene, Teflon, etc. can be used. Among these, a standardized vinyl resin carrier is preferably used for the load, and the carrier is
The material described in JP-A-55-39719 can be used.

Particularly preferably, one vinyl coated fat carrier is
Manufactured by

First, vinyl chloride resin is dissolved in a solvent such as dimethylformamide, this is formed into a desired carrier shape, and then the solvent (
Perform the immersion treatment in B).

The vinyl chloride resin (PVR) of the present invention includes polyvinyl chloride (PVC), 'a, vinyl copolymer, and blends of these and other resins. For example, there are binary, ternary or more copolymers of imized vinyl and vinyl acetate, vinylidene chloride, ethylene, acrylic acid, acrylonitrile, and the like.

Solvent 4 (A) is a solvent that can dissolve PvR, and contains dimethylformamide (DMR), dimethylacetamide (
DMA), n-methylpyrrolidone (n-MP), hexamethylphosamide (HMPA), tetrahydrofuran (THF), and a combination of acetone and benzene. The PVR solution used has a concentration of 1 to 30%.

In order for an enzyme-immobilized membrane to exhibit excellent adsorption activity and function, the degree of polymerization of PVR must be 6 to 6 at about 1000.
1% by weight is preferred.

Next, the PVR solution obtained in this way is formed into a desired carrier shape, and then brought into contact with a solvent (B) that is a poor solvent for PVR and a good solvent for solvent (N) to form a carrier layer. It is preferable to contact the solvent (contact) before the PVR solution layer whitens.
R and volume I This is the period until a certain solution (5) becomes cloudy and becomes opaque.

@1% (EJ includes water, alcohol solvents, ether solvents, etc.

Examples of alcoholic solvents include methyl alcohol, ethyl alcohol, n-propyl alcohol, 1qo-7'' lobil alcohol, n-butyl alcohol, 5eC-butyl alcohol, and tert-butyl alcohol.
i) Alcohol, polyhydric alcohols such as ethylene glycol, diethylene glycol, and glycerin, and glycol monoethers such as ethylene glycol monomethyl ether and ethylene glycol monoethyl ether are used. The solvent for dipping may be one of these alcohol-based solvents alone or a mixed solvent containing 50% by weight or more of alcoholic solvents in which the vinyl chloride resin is insoluble.

In particular, when methyl alcohol or ethyl alcohol is used, an immobilized product with high immobilized enzyme activity can be obtained, which is preferable. - In addition, a non-solvent compound for PVR such as polyethylene glycol is added to the PVR and solvent solution in order to adjust the degree of swelling of the carrier, that is, to adjust the water content of the carrier, adjust the pore size, etc. can do.

The carrier can take various shapes such as a membrane, a tube, a test tube, and a beam. Membrane-like materials are particularly preferred for applications such as enzyme electrodes. The membrane carrier is made by casting a PVR solution,
Thereafter, it is formed by immersing it in solvent a3).

At this time, a reinforcing material such as nonwoven fabric may be used to improve the strength of the carrier.

Note that after forming the carrier, it is preferable to immerse the carrier in water to replace the solvent () with water.

Next, the above carrier is brought into contact with an f8 solution containing the enzyme L-glutamate oxidase to obtain the immobilized enzyme used in the present invention.

In this case, the solution containing the enzyme has a m% K of L-glutamine oxidase of 50 to xooorM! /a1 is preferably #1100 to 50011 FJ/dt, and 7)H of the solution is in the range of 4 to 7, preferably 5 to 6. The buffer solutions used at this time are phosphoric acid-based, citric acid-based, acetic acid-based, and Qood et al.
The buffering agent known by Emistry, 5.467 (1966) can be used.

The immobilized enzyme used in the present invention is
It is also possible to use a water-soluble 11K curable block German cyanate compound described in Japanese Patent Application No. 393.

The immobilized enzyme membrane obtained as described above is bonded with a known porous polymer membrane, such as an acetyl cellulose membrane, a polycarbonate membrane, a nylon membrane, a polypropylene membrane, a polyethylene membrane, a Teflon membrane, etc., to form an immobilized enzyme membrane. An enzyme electrode is manufactured and used by attaching it to a known, for example, hydrogen peroxide electrode so that it is in contact with the sample solution.

FIG. 1 is a system diagram of a transaminase activity analyzer using this enzyme electrode and showing an example of the present invention.

Hereinafter, a method for measuring GOT and GPT using the method for measuring transaminase activity of the present invention will be explained using FIG.

(Measurement of GOT Activity) In FIG. 1, a buffer solution 15 and a substrate solution for GOT measurement (containing at least aspartic acid and α-ketoglutaric acid) 14 are respectively injected into the measurement cell 3 using a pump 1019. Next, a sample is injected into the measurement cell 3 using the syringe 1 or the sampler 2. Monoglutamic acid produced in the cell 3 by the reaction with aspartic acid, α-ketoglutaric acid, and GOT in the biological sample reacts with the monoglutamic acid oxidase in the immobilized enzyme 4 described above. At this time, the amount of hydrogen peroxide generated or the amount of oxygen that decreases is detected by the hydrogen peroxide electrode or the FiWl element electrode 5 and read by the recorder 7 or the digital multimeter 8. FIG. 2 shows the results measured using samples having known GOT activity values. As is clear from FIG. 2, a good linear relationship was observed between the GOT activity value and the rate of change in current value.

(Measurement of GPT activity) In Fig. 1, GOT measurement substrate solution (containing at least aspartic acid and α-ketoglutaric acid) 14 was
By changing to the substrate solution for T measurement (containing at least alanine and α-ketoglutaric acid), measurement can be performed in the same manner as described above. The measurement results are shown in Figure 2. As is clear from FIG. 2, a good linear relationship was observed between the GPT activity value and the rate of change in current value.

(Continuous measurement of GOT and GPT activities) When first measuring GOT activity and then measuring the total activity of GOT and GPT in Figure 1, add buffer solution 15 and GO to measurement cell 3.
A substrate solution for T measurement (containing at least aspartic acid and α-ketoglutaric acid) 14 is injected using each pump 10.9. Next, a sample is injected into the measurement cell 3 using the syringe 1 or the sampler 2. Aspartic acid in cell 3,
L-glutamic acid produced by the reaction with α-ketoglutaric acid and GOT in a biological sample is the immobilized enzyme 4 described above.
reacts with L-glutamate oxidase in At this time, the amount of hydrogen peroxide generated or the amount of oxygen that decreases is detected by an electrode on peroxide water or an oxygen electrode 5, and read by a recorder 7 or a digital multimeter 8.

Subsequently, 1 G of a substrate solution for GPT measurement (containing at least alanine and α-ketoglutaric acid) is injected into the measurement cell 3 using the pump 11. The amount of hydrogen peroxide generated or the amount of oxygen decreased at this time is the amount of L generated from the reaction of the GOT.
- Glutamic acid and L-glutamic acid produced by the GPT reaction caused by newly injecting alanine and α-ketoglutaric acid are simultaneously immobilized by fermentation [・"This is produced by reacting with L-glutamate oxidase in N4, Similarly to the above, the amount of hydrogen peroxide generated or the amount of oxygen decreased is detected with the electrode or oxygen electrode 5 and read with the recorder 7 or digital multimeter 8. Therefore, the output from the GOT anti-core is first only, then GO
By measuring the output of the two components of T reaction and GPT reaction and first correcting the output from the measured GOT reaction, GP
The output due to the T reaction can be determined. After the measurement is completed,
The pump 13 discards the yffl in the cell.

In this way, when measuring using a sample with known GOT activity value and GPT activity value, a reaction curve of the direct output of the enzyme shown in Figure 43 is obtained, and GOT activity value and GPT activity value
A good linear relationship was observed between the activity value and the current value change rate.

On the other hand, when GPT activity is measured first and then two-component activity is measured, GOT measurement substrate solution (containing at least aspartic acid and α-ketoglutaric acid) 14 is
The determination can be made in the same manner as described above by changing the substrate solution for T measurement (containing at least alanine and α-ketoglutaric acid) and further using JS' Isol y, 16 for GPT measurement as the substrate solution for GOT measurement.

As detailed above, by using the method for measuring transaminase activity of the present invention, it is possible to measure small quantities of GOT and GPT in biological samples in a simple manner, quickly, with high precision, and over a wide constant Q range. I can do it

[Brief explanation of drawings]

FIG. 1 is an example of a system diagram of a transaminase activity analyzer using the measuring method of the present invention. Figure 2 shows the results obtained by measuring using the measuring method of the present invention.
FIG. 3 is a correlation diagram between T activity value, GPT activity jet, and current value change rate. FIG. 3 is an output characteristic diagram of an enzyme electrode obtained using the measurement method of the present invention. 1... Sample injector, 2... Templar for sample injection 1
3... Reaction and detection cell, 4... Immobilized enzyme membrane, 5
...Oxygen electrode or superhydrogen electrode, 6.. Amplifier, 7.. Recorder, 8.. Digital multimeter, 9.10.11, 12.13.. Pump, 14.1
6...Substrate lysis tank, 15...Buffer solutions, 17.
...Waste liquid tank. Patent applicant Mitsubishi Yuka Co., Ltd. Agent Patent attorney Hidetoshi Furukawa Agent Patent attorney Masahisa Hase Figure 1 Figure 2 Transaminase activity/pressure (KU/mi) Figure 3 Time wJ1 (min)

Claims (2)

[Claims] (1) A buffer solution, a sample solution, and a substrate solution for measuring glutamate oxaloacetate transaminase (GOT) are placed in a cell equipped with an enzyme electrode equipped with an immobilized enzyme membrane in which L-glutamate oxidase is immobilized on a porous polymer carrier. or glutamate pyruvate transaminase (GP
T) A method for measuring transaminase activity in which a substrate solution for measurement is supplied and GOT activity or GPT activity is measured by the output of the enzyme electrode. (2) Supply a buffer solution, a sample solution, and a substrate solution for GOT measurement or a substrate solution for GPT measurement to the cell, measure GOT activity or GPT activity by the output of the enzyme electrode, and then apply the above measurement to the cell. The method for measuring transaminase activity according to claim 1, characterized in that the total activity of GPT activity and GOT activity is measured by supplying an unused substrate solution for GPT measurement or substrate solution for GOT measurement.