JPS6060550A - Measuring method of transaminase activity - Google Patents

Abstract

PURPOSE:To measure quickly the respective transaminase activity of glutamic acid oxaloacetic acid and glutamic acid pyruvic acid by using a cell having an enzyme electrode provided with a film fixed with pyruvic acid oxidase and oxaloacetic acid decarboxylase. CONSTITUTION:A buffer soln. 15 for measuring pyruvic acid, a substrate soln. 14 for measuring glutamic acid oxaloacetic acid transaminase (GOT) contg. aspartic acid and alpha-ketoglutaric acid and a sample from a sample injector or a sample 2 for injecting sample are first fed into a cell 3 for reaction and detection provided with an enzyme electrode or hydrogen peroxide electrode 5 formed by fixing pyruvic acid oxidase (POP) and oxaloacetic acid decarboxylase (OAC) on a porous carrier and the GOT activity is measured. A solute soln. 16 for measuring glutamic acid pyruvic acid transaminase (GPT) contg. alanine and alpha-ketoglutaric acid is fed into the cell 3 in succession thereto. The GOT+GPT activity is measured and the GOT activity is subtracted therefore to determine the GPT activity. The measurement is thus performed quickly with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of transaminases (abbreviated as glutamate oxaloacetate transaminase: GOT and glutamate pyruvate transaminase: G
The present invention relates to a method for measuring the activity of OT (abbreviated as OT).

Using the present invention, GO in the same sample in the same reaction cell
Since T and GPT can be measured without replacing the sample, the sample can be measured quickly and accurately.

Transaminase is an enzyme that catalyzes the transamination 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, GOT and GPT measurements 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, cholangitis, and cholelithiasis, myocardial infarction, and progressive disease. They play a useful role in diagnosing muscular dystrophy, infectious diseases, etc., and it is of great clinical significance to easily, accurately, and quickly measure GOT and GPT.

Prior Art Conventionally, GOT was measured by adding a sample to the substrates aspartic acid and α-ketoglutaric acid, and conjugating the generated oxaloacetate in the presence of malate dehydrogenase and coenzyme NADH. The Carmen method, which measures the decrease in the absorbance of 340 jaws of NADH using the initial velocity method, reacts the oxaloacetic acid produced above with 2,4-dinitrophenylhydrazine to produce hydrazone, which is then made alkaline. Reitman creates quinoids and develops color.
There is the Frankl method. However, the Carmen method has problems such as the instability of the reagents used and the need for a special fractional element meter equipped with a constant temperature 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 it is necessary to reduce the amount of the substrate extremely, which may cause the calibration curve to curve and the measurable range to be narrow. There are drawbacks such as.

In addition, a method for measuring GPT involves adding a sample to the substrates alanine and α-ketoglutarate, conjugating the generated pyruvate with lactate dehydrogenase in the presence of the above NA and DH, and measuring the decrease in NADH. There are the Carmen method and the Reitman-Frankl method, in which the pyruvic acid produced as described above is reacted with 2,4-dinitrophenylhydrazine to develop color. However, these methods also have the same drawbacks as the GOT measurement method described above.

In order to solve these drawbacks, we have recently developed GPT and GOT using pyruvate oxidase (abbreviated as pop).
For example, a method for measuring GPT involves a reaction in which a sample is added to the substrates alanine and α-ketoglutarate to generate pyruvate, and the generated pyruvate is reacted 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 conjugated with the pyruvic acid oxidation reaction, and the amount of hydrogen peroxide generated in the pyruvic acid oxidation reaction is determined colorimetrically using a coloring agent.

In addition, the method for measuring GOT involves a reaction in which a sample is added to the substrates aspartic acid and α-ketoglutaric acid to produce oxaloacetate, and the produced oxaloacetate is reacted with pyruvin in the presence of oxaloacetate decarboxylase (OAC, !: abbreviated). Pyruvic acid is produced by conjugating with a reaction that produces acid. Next, the generated pyruvic acid is subjected to a pyruvic acid oxidation reaction in the presence of POP and a required substrate, and the amount of hydrogen peroxide generated due to the pyruvic acid oxidation reaction is determined colorimetrically using a coloring agent. However, the above measurement method using POP requires disposable, expensive enzymes, long measurement time, and disadvantages of POP.
Since P itself is unstable in a solution, there are problems such as poor reproducibility during measurement.

Therefore, research is being actively conducted to measure GPT and GOT using an enzyme electrode in which POP is immobilized and attached to an electrode for measuring oxidation potential. (Unexamined Japanese Patent Publication No. 55-
No. 111786, No. 56-124396, An
alytica chimica Acta.

118 (1980) 65-71, etc.) However, in these immobilization methods, the activity and stability of immobilized POP cannot be said to be sufficient, and immobilization of POP alone does not improve GO
Since there is a drawback that OAC enzyme must be added during T measurement, GOT using POP and OAC immobilized enzymes with high activity and excellent stability for GOT and GPT measurement is recommended.
There has been a strong desire to provide a method for measuring OAC and GPT. SUMMARY OF THE INVENTION As a result of intensive studies to meet this demand, the present inventors found that POP and OAC have excellent enzymatic activity and stability, and are highly effective in substrate diffusion. By attaching a highly durable and easy-to-clean immobilized enzyme membrane to an electrode for oxidation potential measurement and using it as an enzyme sensor, the drawbacks of conventional measurement methods can be overcome, and trace amounts of transaminases in biological samples can be easily measured. The inventors have discovered that this method can be used to perform measurements quickly, accurately, and over a wide quantitative range, and have completed the present invention.

That is, the present invention provides a cell having an enzyme electrode equipped with an immobilized enzyme membrane in which pyruvate oxidase (POP) and oxaloacetate decarboxylase (OAC) are immobilized on a carrier. A substrate solution for measuring oxaloacetate transaminase (GOT) or a substrate solution for measuring glutamate pyruvate transaminase (GPT) is supplied, GOT activity and GPT activity are measured by the output of the enzyme electrode, and then the cell is not used for the above measurement. The present invention provides a method for measuring transaminase activity in which the total activity of GPT activity and GOT activity is measured by supplying a substrate solution for GPT measurement and a substrate solution for GOT measurement.

3. Detailed Description of the Invention The immobilized enzyme membrane used in the present invention can be manufactured using a porous polymer membrane carrier. As the porous polymer carrier, known vinyl chloride resin carriers, acetyl cellulose, polycarbonate, nylon, polypropylene, polyethylene, Teflon, etc. can be used. Among these, a vinyl chloride resin carrier is particularly preferably used, and the carrier described in JP-A-55-39719 can be used as the carrier.

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

The pre-prepared vinyl chloride resin is dissolved in a solvent (4) such as dimethylbormamide, formed into a desired carrier shape, and then immersed in the solvent (B).

Examples of the vinyl chloride resin (PVR) of the present invention include polyvinyl chloride (PVC), vinyl chloride copolymers, and blends of these and other resins. There are binary or ternary copolymers of vinyl acetate, vinylidene chloride, ethylene, acrylic acid, acrylonitrile, etc.

Solvent (4) is a solvent that can dissolve PVR, and includes dimethylformamide (DMR), dimethylacetamide (DMA), n-methylpyrrolidone (n-MP), hexamethylphos-7-oamide (HIvIPA), and tetrahydrofuran (THF). , a mixed solvent of acetone and benzene, etc. The concentration of PVR solution is 1 to 30% by weight.
are used.

In order for the enzyme-immobilized membrane to exhibit excellent adsorption activity and function, the polymerization degree of PVR is preferably about 1000 and 6 to 12% by weight.

Next, after forming the obtained PVR solution into a desired carrier shape, it is brought into contact with solvent (3), which is a poor solvent for PVR and a good solvent for solvent (4), to form a carrier layer. form. At this time, the contact with the solvent (B) is preferably carried out before the PVR solution layer becomes white. Before the bleaching,
This is the period until the solution of PVR and solvent (4) becomes opaque with visible cloudiness.

Examples of the solvent (B) include water, alcohol solvents, and ether solvents.

Examples of alcoholic solvents include methyl alcohol, ethyl alcohol, n-propyl alcohol, 1so-7' alcohol, n-7'' alcohol, 5ee-methyl alcohol, and tert-methyl alcohol.
) -hydric alcohols, polyhydric alcohols such as ethylene glycol, diethylene glycol, and glycerin, and glycol monoethers such as ethylene glycol monomethyl ether and ethylene glycol monoethyl ether. The solvent for dipping may be one of these alcoholic solvents alone or a mixed solvent containing 50% by weight or more of an alcoholic solvent 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 may be added to the solution of PVR and solvent 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 done.

The carrier can take various shapes such as a membrane, a tube, a test tube, and a bead. Membrane-like materials are particularly preferred for applications such as enzyme electrodes. The membrane carrier is formed by casting a PVR solution and then dipping it into solvent CB).

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

After forming the carrier, it is preferable to immerse the carrier in water to replace the solvent (g) with water.

The carrier is then brought into contact with a solution containing the enzymes POP and OAC. At this time, preferably, these enzymes are immersed in a solution containing coenzyme flavin adenine dinucleotide (abbreviated as F'AD), coenzyme thiamine pyrophosphate (abbreviated as TPP), and Mg2+ ion and/or Mn2+ ion. An immobilized enzyme used in the present invention is obtained.

In this case, the solution containing the enzyme has a total concentration of POP and OAC of 100 to 1oooη/d11, preferably zo
o-a o omg/as, and the weight composition ratio of pop and OAC is 5015o to 99/1, preferably 70
/30-98/2, particularly 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 Good et al.
Chemistry, 5.467 (1966)) can be used. For example, N-(2-acetamide) is an N-substituted dipolar amino acid.
-2-aminoethanesulfonic acid, N-(2-acetamido)iminodiacetic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfone L N,N-bis(2
-hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid, 2-
(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, piperazine-N, N-bis(2-ethanesulfonic acid), N-4 lis(hydroxymethyl)methyl-3-aminopropanesulfonic acid boric acid, N-
) Lis(hydroxymethyl)methyl-2-aminoethanesulfonic acid, N-tris(hydroxymethyl)methylglycine, and the like. In addition, as the aliphatic amine, 2.2-
Examples include bis(hydroxymethyl)-2,2',2'-nitrilotriethanol, 1,3-bis[tris(hydroxymethyl)methylamine cupropane, glycinamide and the like. Additionally, tris(hydroxymethyl)aminomethane and the like can be used.

Mata, Mg2+ ions and Mn2+ ions are MgC&
and MnCtz. The immobilized enzyme used in the present invention is preferably used in the form of a room-temperature curing, blocked isocyanate compound described in the specification of the patent filed on December 1, 1982. It can also be coated with the compound.

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 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.

, J1, when GOT activity is measured first, a buffer solution for pyruvate measurement (containing FAD, TPP, Mg 2+ and/or Mn 2+ ions, etc.) 15 and at least aspartic acid and α-ketoglutar are added to the measurement cell 3. A substrate solution 14 for GOT measurement containing an acid is pumped into each pump 11.1o.
Inject at Next, a sample is injected into the measurement cell 3 using the syringe 1 or the sampler 2. The oxaloacetate produced in cell 3 by the reaction with aspartic acid, α-ketoglutarate, and GOT in the biological sample is converted into pyruvic acid by the oxaloacetate decarboxylase in the immobilized enzyme Lux4 described above, and then converted into pyruvic acid. It 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 11° C. pole or the oxygen electrode 5, and read by the recorder 7 or the digital multimeter 8.

Pull! Subsequently, a GPT measurement substrate solution 16 containing at least alanine and α-ketoglutaric acid is injected into the measurement cell 3 using the pump 9. At this time, the amount of hydrogen peroxide generated or the amount of oxygen decreased is the amount of pyruvic acid generated from the GOT and oxaloacetate decarboxylase reaction, and newly added 7-lanine and α
- Pyruvate generated by the GPT reaction caused by injecting ketoglutarate simultaneously reacts with pyruvate oxidase in the immobilized enzyme membrane 4, and as above, the amount of hydrogen peroxide generated or decreases. The amount of oxygen is detected by a hydrogen peroxide electrode or an oxygen electrode 5 and read by a recorder 7 or a digital multimeter 8. Therefore, first G
The output due to the GPT reaction can be calculated by measuring only the output due to the OT reaction, then measuring the output from the two-component reaction of the GOT reaction and the GPT reaction, and first correcting the output due to the measured GOT reaction. After measurement, pump 1
3, the liquid in the cell is discarded.

When measured in this way using a sample having known GOT activity values and G, PT activity values, a reaction curve of the enzyme electrode output shown in FIG. 2 is obtained, and the GOT activity value and G
A good linear relationship as shown in FIG. 3 was observed between the PT activity value and the rate of change in current value.

In addition, in measuring GOT activity, pyruvate buffer solution 15
is supplied to the measurement cell 3, the sample is injected, and then the GOT
The measurement can be performed in the same manner even if the measurement substrate solution 14 is injected into the measurement cell 3.

On the other hand, when GPT activity is first measured and then two-component activity is measured, GPT measurement containing at least alanine and α-ketoglutaric acid is performed using a GOT measurement substrate solution containing at least aspartic acid and α-ketoglutaric acid. The measurement can be carried out in the same manner as described above by changing the GPT measurement substrate solution 16 to a GOT measurement substrate solution containing at least aspartic acid and α-ketoglutaric acid.

As detailed above, by using the method for measuring transaminase activity of the present invention, trace amounts of GOT and GPT in biological samples can be measured quickly and accurately using a simple method.
The quantitative range can also be measured over a wide range.

[Brief explanation of drawings]

FIG. 1 is a system diagram of a transaminase activity analyzer using the measuring method of the present invention. FIG. 2 is an output characteristic diagram of an enzyme electrode obtained using the measurement method of the present invention. FIG. 3 shows the GO obtained by measuring using the measuring method of the present invention.
FIG. 3 is a correlation diagram between T activity value, GPT activity value, and current value change rate. 1... Sample injector, 2... Sampler for sample injection,
3... Reaction and detection cell, 4... Immobilized enzyme cell, 5
. . . Oxygen electrode or hydrogen peroxide electrode, 6. Amplifier, 7. Recorder, 8. Testital multimeter, 9. ]0.11.12.13. Pump, 14.
16... Substrate solution tank, 15... Buffer solution tank for pyruvate measurement, 17... Waste liquid tank. Patent Applicant Mitsubishi Yuka Co., Ltd. Agent Patent Attorney Hidetoshi Furukawa Agent Patent Attorney Masahisa Hase Figure 1 Figure 2

Claims (1)

[Claims] (1) Pyruvate measurement buffer solution, sample solution, and glutamic acid oxaloacetic acid were placed in a cell equipped with an enzyme electrode equipped with an immobilized enzyme membrane in which pyruvate oxidase (POP> and oxaloacetate decarboxylase (OAC) were immobilized on a carrier). Substrate solution for measuring transaminase (GOT) A substrate solution for measuring glutamate pyruvate transaminase (GPT) is supplied, GOT activity or GPT activity is measured by the output of the enzyme electrode, and then GPT, which was not used in the above measurement, is added to this cell. Transaminase activity measurement method 0 that measures the total activity of GPT activity and GOT activity by supplying a substrate solution for measurement or a substrate solution for GOT measurement