JPS59109170A - Apparatus for determination of transaminase activity - Google Patents

Abstract

PURPOSE:To enable the rapid and accurate determination of transaminase activity with a simple method, by attaching an easily washable immobilized enzyme membrane having high and stable enzymatic activity and high diffusibility into substrate to an electrode for the determination of oxidation potential, and using the electrode as the enzyme sensor. CONSTITUTION:The determination of GOT activity is carried out as follows. The specimen is injected into the determination cell 3 by the injector 1 or the sampler 2, and the substrate solution 12 containing at least aspartic acid and alpha-ketoglutaric acid and the buffer solution for the determination of pyruvic acid 14 are injected into the cell with the pumps 9 and 10. The oxaloacetic acid produced by the reaction is converted to pyruvic acid by the immobilized enzyme membrane 4, and then reacted with the pyruvic acid oxidase in the membrane. The amount of produced hydrogen peroxide or decreased oxygen is determined by a hydrogen peroxide electrode or an oxygen electrode 5, and recorded by the recorder 7, etc. The activity of GPT can be prepared in the same manner provided that the substrate solution is changed to a solution 13 containing at least alanine and alpha-ketoglutaric acid. A trace of GOT or GPT in a biospecimen can be determined easily, rapidly, in high accuracy by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical device for measuring the activity of glutamate oxaloacetate transaminase (abbreviated as GOT) or glutamate pyruvate transaminase (abbreviated as GPT) in a biological sample.

With the present invention, GOT or QP'l in one cell
”, it is possible to measure samples quickly and accurately.

Transaminase is an enzyme that catalyzes transamidation of amino acids, and among them, 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, measurements of GO'l' and GPT are recommended for acute viral hepatitis, chronic hepatitis, liver cirrhosis, liver tumors, liver diseases such as acute alcoholic hepatitis, extrahepatic obstructive jaundice such as gallbladder disease, cholangitis, and cholelithiasis, and myocardial stiffness. It plays a useful role in the diagnosis of muscular dystrophy, progressive muscular dystrophy, infectious diseases, etc.
It is of great clinical significance to easily, accurately, and quickly measure P′],”.

Conventionally, GOT (7) Measurement Method: A sample was added to aspartic acid and α-ketoglutaric acid, and the resulting oxaloacetate was combined with malate dehydrogenase and coenzyme N.
Conjugated in the presence of ADI-T, at this time NA,
J) The Carmen method, which measures the decrease in the absorbance at 340 m+ of i-(at a wide initial velocity), and the hydrazone obtained by reacting the generated 1-7 oglymeroacetic acid & 2,4-dini)-lophenylhydrazine as described above. There is a Reitman-Frankel method in which the kneading material 5 is made alkaline to produce quinoids and develop color. However, the Carmen method has certain limitations, such as the instability of the reagents used and the need for a special spectrometer 5'e thermometer equipped with a constant temperature cell.

On the other hand, in the Reitman-Frankel method, the substrate amount of α-ketoglutaric acid must be drastically reduced in order to prevent the color development of the product due to the color development 1-, and the 1-' line is inflected. There are drawbacks such as a narrow measurable range.

190 To measure P'I'', a sample is added to the substrates alanine and α-ketoglutaric acid, and the generated pyruvate is conjugated with lactate dehydrogenase in the presence of the above N A ]') H. The Carmen method, which measures the decrease in N A l) I, and the 2.4-
There is a Reitman-Frankel method in which color is developed by the action of dinitrophenylhydrazine. 1. The method of Kashitore β also has the same drawbacks as the 4111 fixed method of G O'['' described above.

Recently, in order to solve these drawbacks, pyruvate oxidase (abbreviated as POP) was used to generate GPT, G
Methods have been developed to analyze OT.

For example, in GPT measurement method 1~, the sample is mixed with the substrates alanine and α-ketoglutaric acid at 1 J [1], and the reaction that generates pyruvate is performed, and the generated pyruvate is oxidized to pyruvic acid in the presence of POP and the required substrate. There is a method in which the amount of hydrogen peroxide generated in the pyruvic acid oxidation reaction is determined colorimetrically using a coloring agent.

The method for measuring Sunda G O'I' involves a reaction in which a sample is added to the substrates aspartic acid and α-ketoglutarate to produce oxaloacetate, and the produced oxaloacetate is processed using oxaloacetate decarboxylase (abbreviated as OAC). ) in the presence of pyruvic acid to produce pyruvic acid. Next, the produced 1-dipyruvate is subjected to a pyruvate oxidation reaction in the presence of POP and a required substrate, and the amount of hydrogen peroxide generated due to the pyruvate oxidation reaction is determined colorimetrically using a coloring agent. However, the measurement method using POP described above has problems such as the expensive and disposable enzyme, long measurement time, and poor reproducibility during measurement because POP itself is unstable in solution. .

Therefore, POP was immobilized and attached to an electrode for measuring oxidation potential, and GPT was measured using an enzyme electrode.

Research to measure GOT I7 is also actively conducted (Japanese Patent Application Laid-open Nos. 55-111786 and 56-1243).
No. 96 publications, Analytica chirnica
A, cta, +ts (19so) 65-71 etc.)
However, in the above immobilization method, the activity and stability of immobilized POP are insufficient.1. A: First of all, immobilization of POP only has the disadvantage that AC enzyme must be added at the specific time of GOT measurement.
High activity and excellent stability for PT measurement---5-GOT or G using POP and OAC immobilized enzymes
There has been a great need for a PT measuring device.

As a result of intensive studies to meet this demand, the present inventors have developed an immobilized enzyme membrane that has excellent POP and OAC enzyme activity and stability, has good substrate diffusivity, and is easy to wash. By attaching it to an electrode and using it as an enzyme sensor, we can eliminate the shortcomings of conventional measurement methods and measure trace amounts of transaminases in biological samples in a simple manner, quickly, accurately, and over a wide quantitative range. They found this and completed the present invention.

That is, the present invention provides a cell having an enzyme sensor electrode equipped with an immobilized enzyme membrane in which pyruvate oxidase (POP) and oxaloacetate decarboxylase (OAC) are immobilized on a carrier. , the substrate solution [I] supply section containing at least aspartic acid and α-ketoglutaric acid and/or the substrate solution (U) supply section containing at least alanine and α-ketoglutaric acid, and the sample 4-1 solution supply section are connected, respectively. 1-1 Soil in the cell
6- Supply the low-temperature solution, substrate bath solution (1) or (n), and sample solution, and determine the activity of glutamate "oxaloacetate l-transaminase CGOT" or glutamate pyruvate transaminase by the output of the enzyme electrode. The present invention provides a transaminase activity analyzer for measuring (GPT) activity.

The immobilized enzyme membrane used in the present invention can be manufactured using a porous polymer membrane carrier. As the porous polymer carrier, known materials such as vinyl chloride resin 11iT, ff'f3 monolith, acetyl cellulose, polycarbonate, nylon, polypropylene, polyethylene, and Teflon can be used. Among these, a vinyl chloride resin carrier is particularly preferably used, and as the carrier, those described in JP-A-55-39719 can be used.

Particularly preferably, the vinyl chloride resin phase is produced as follows.

First, a vinyl chloride resin is dissolved in a solvent (A) such as dimethylformamide, and this is formed into a desired carrier shape, and then immersed in a solvent (R).

As the vinyl chloride resin (PVR) of the present invention, BoI
J y = Vinyl chloride (PVC), vinyl chloride copolymer, and blends of these and other resins, such as vinyl chloride and vinyl acetate, vinylidene chloride, ethylene, There are binary or monovalent copolymers with acrylic acid, acrylonitrile, etc.

The solvent (A) is a solvent that can be understood as PVR,
Dimethylformamide (DIVFR), dimethylacetamide (DMA), n-methylpyrrolidone (1
1-MP), hexamethylphosphoramide (I-IMP)
A), THF, a mixed solvent of acetone and benzene, etc. The concentration of the PVR solution used is 1 to 30%. In order for the enzyme-immobilized membrane to exhibit excellent adsorption activity and function, it is preferable that the PVR concentration is approximately 1,000 and the weight ratio is 6 to 12%.

Next, the thus obtained P VR bath solution is formed into a desired carrier shape, and then brought into contact with solvent 03) which is a poor solvent for P VR and a good solvent for solvent traps to form the entire carrier layer.

At this time, the contact with the solvent (I3) is preferably carried out before the I) VR solution layer becomes white. Here, the term "before whitening" refers to the period in the trench where the solution of PVR and solvent (4) becomes opaque with visible cloudiness.

Examples of solvents include water, alcohol solvents, and ether solvents.

Examples of alcoholic solvents include monohydric alcohols such as methyl alcohol, ethyl alcohol, n-propyl alcohol, 1so-7′ lobil alcohol, n-butyl alcohol, 5ec-butyl alcohol, and tert-butyl alcohol, ethylene glycol, and diethylene glycol. , polyhydric alcohols such as glycerin, and glycol monoethers such as ethylene glycol monomethyl ether and ethylene glycol monoethyl ether. The solvent to be immersed may be one of these alcoholic solvents alone or a mixed solvent containing 50% 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.

The solution of PVR and solvent (A) contains a compound that is a non-solvent for PVR, such as polyethylene glycol, 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 be thrust (1).

The carrier can take various shapes such as a membrane, a tube, a test tube, and a bead. Membrane-like carriers are particularly preferred for applications such as enzyme electrodes17,3 and are formed by casting a PVIR solution and then immersing the carrier in bath m (B).

At this time, a reinforcing material such as a 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 -(solvent G) with water.

The carrier is then brought into contact with a solution containing the enzymes POP and OAC. At this time, along with these enzymes, coenzyme flavin adenine dinucleotide (abbreviated as FAD), coenzyme thiamine pyropho-1 (abbreviated as TPP), and Mg2+ ion and/or Mnz+ ion are added. The immobilized enzyme used in the present invention is obtained by immersing it in a solution containing the following.

In this case, the solution containing the enzyme has a total concentration of POP and OAC of 100 to 1000/d7! , preferably 2
00~800■/d7! and the weight composition ratio of pop and OAC is 50150 to 99/1, preferably 70/1.
30-98/2, especially preferably 85/15-97/
3, and the pH of the bath solution is in the range of 6 to 8.5, preferably 6.5 to 7.0.

The above buffer solution used in the present invention is described by Qood et al.
iochemistry, 5.467 (1966)
) can be used. For example, N-substituted dipolar amino acids include N-(2-acetamido)-2-aminoethanesulfone m, N-(2-acetamido9iminodiacetic acid, N,N-bis(2-hydroxyethyl)-2-amino Ethanesulfonic acid, N, N-bis(2-hydroxyethyl) f Lysine, N-(2-
hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid,
3-(N-morpholino)propanesulfonic acid, hyperazine-N, N'-bis(2-ethanesulfonic acid), N-)
Examples include IJs(hydroxymethyl)methyl-3-aminopropanesulfonic acid, N-)lis(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and N-tris(hydroxymethyl)methylglycine. In addition, as aliphatic amines, 2,2-bis(hydroxymethyl)-2,
2',2''-nitrilotriethanol, 1,3-bis[
Tris(hydroxymethyl)methylamine]propane,
Examples include glycinamide. Additionally, tris(hydroxymethyl)aminomethane and the like can be used.

Furthermore, Mg 2+ ions and Mn2+ ions are Mg
Preference is given to using it in the form of Cl2 and MnC6z.

The immobilized enzyme used in the present invention was released on December 1, 1981.
It can also be coated with an aqueous solution of a room-temperature-curable blocked isocyanate compound described in the patent specification of the Japanese patent application.

The immobilized enzyme membrane obtained as described above is laminated 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 sensor 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 sensor electrode and showing an example of the present invention.

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

When measuring GOT activity in FIG. 1, a sample is injected into a measurement cell 3 using a syringe 1 or a sampler 2. On the other hand, a substrate solution 12 containing at least aspartic acid and α-ketoglutaric acid and a buffer solution for measuring PIA-vic acid (FA
D, TPP, Mg'' and/or Mn2+ ions, etc.) are injected into the measuring cell 3 using pumps 9 and 10. In the cell 313-, aspartic acid, α-ketoglutaric acid and GOT in the biological sample Oxaloacetic acid produced by the reaction with
It becomes pyruvate by oxaloacetate decarboxylase in the immobilized enzyme 11Q4 mentioned above, and then reacts with pyruvate oxidase in the immobilized enzyme membrane 4. At this time, the amount of hydrogen peroxide generated or the amount of oxygen that decreases is detected by the hydrogen peroxide electrode or oxygen electrode 5 and read by the recorder 7 or digital multimeter 8. After measurement, pump 11
The liquid inside the cell is discarded. In this way, the known G
When measuring using a sample having an OT activity value, a good linear relationship as shown in FIG. 2 was observed between the GOT activity value and the rate of change in current value.

- When measuring 10,000 GPT activity, the substrate solution is at least 1 ml of a solution containing aspartic acid and α-ketoglutaric acid.
2 to solution 13 containing at least alanine and α-ketoglutaric acid, and the other measurements can be carried out in the same manner as in the case of GOT. In the measurement of GPT activity, there was a good linear relationship between the GPT activity value and the current value change rate of 14 degrees, as shown in FIG. 3, similar to that in the GOT activity measurement.

Furthermore, by using the apparatus of the present invention, it is also possible to measure, for example, lactic acid, pyruvic acid, etc. in biological samples other than GOT and GPT by changing the substrate solution.

As described in detail above, by using the transaminase activity analyzer of the present invention, microscopic GO in biological samples can be detected.
T or GPT can be measured quickly, accurately, and over a wide quantitative range using a simple method.

[Brief explanation of drawings]

FIG. 1 is a system diagram of a transaminase activity analyzer showing an example of the present invention. Figure 2 is a correlation diagram between the GOT activity value and the rate of change in current value, each measured using the device of the present invention, and Figure 3 is a diagram of the correlation between
It is a correlation diagram between a PT activity value and a current value change rate. 1... Sample injector, 2... Sampler for sample injection,
3... Reaction and detection cell, 4... Immobilized enzyme membrane, 5
. . . Oxygen electrode or hydrogen peroxide electrode, 6. Amplifier, 7. Recorder, 8. Digital multimeter, 9. 10, 11-.- Pump, 12- Substrate bath for GOT measurement, 13-...Substrate solution tank for GPT measurement, 14-
...Buffer tank for pyruvate measurement. Applicant for prefectural approval Mitsubishi Yuka Co., Ltd. Agent: Patent attorney Hidetoshi Furukawa Agent: Hisashi Hase (Figure 1, Figure 2) GOT activity 1 raw value IU/1)

Claims (1)

[Claims] rl) Pyruvate oxidase (POP) and oxaloacetate decarboxylase (OAC) are immobilized on a carrier.1- An immobilized enzyme membrane is attached to a cell having an enzyme sensor electrode. Measurement buffer supply section,
A substrate solution containing at least aspartic acid and α-ketoglutaric acid [13 supply unit and/or a substrate solution containing at least alanine and α-ketoglutaric acid [11] A supply unit and a sample liquid supply unit are connected to each other 1-1 The cell Supply the above buffer solution, substrate solution [1] or [■], and sample solution to 1~
, a transaminase activity analyzer for measuring glutamate oxaloacetate transaminase (GOT) activity or glutamate pyruvate transaminase (GPT) activity by the output of the enzyme sensor's awakening electrode.