JPS58135000A - Method and apparatus for determination of glutamic acid oxaloacetic acid transaminase and glutamic acid pyruvic acid transaminase - Google Patents

Abstract

PURPOSE:To determine the content of the titled enzyme necessary for the diagnosis of diseases, in high accuracy, by adding alpha-ketoglutaric acid to the specimen solution, adding L-aspartic acid or L-alanine to the mixture to cause the enzymatic reaction, and determining the produced carbonate. CONSTITUTION:A specimen solution containing glutamic acid oxaloacetic acid transaminase (abbreviated to GOT) and glutamic acid pyruvic acid transaminase (abbreviated to GPT) is added with alpha-ketoglutaric acid. L-aspartic acid or L- alanine is added to the mixture to effect the enzymatic reaction of formulaI or formula II. The produced L-glutamic acid is made to react with glutamic acid decarboxylase to effect the enzymatic reaction of formula III, and the produced carbon dioxide gas is absorbed through a membrane to an absorbent. GOT and GPT and can be determined from the variation of the electrical conductivity of the absorbent liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to glutamate oxaloacetate transadenase (hereinafter abbreviated as "GOTJ"), which contains a 1fK to be measured in blood, etc. used for diagnosis of myocardial infarction, liver failure, etc. The present invention relates to a method and apparatus for quantifying glutamate pyruvate totunsaminase (hereinafter abbreviated as "OFTJ").

Conventionally, ultraviolet spectroscopy and colorimetric methods have been known as such quantitative methods, but these methods require pretreatment operations to convert the blood into serum suitable for measurement, and they require color development. Recently, the so-called coenzyme electrode method, which combines a cofactor and a redox substance, has been used. However, there is a drawback that it is often difficult to accurately quantify GOT4-GPT due to the interference of coexisting substances contained in the mold to be measured 11. The present invention has been developed in view of these drawbacks. The purpose of this method is to provide a method and apparatus for quickly and accurately quantifying GO'r+GP'r contained in the 1 [K] to be measured, with all of the above-mentioned drawbacks eliminated. .

A feature of the present invention is that in the method and apparatus for quantifying GOT + GPT in blood, the analyte 1 [Kg-ketoglutaric acid is added, and then each L
- Adding aspatugic acid or L-alani/ to cause the respective enzyme reactions, converting the oxalate produced by the enzyme reaction into carbon dioxide and absorbing it into the absorption liquid through the diaphragm, changing the electrical conductivity of the axillary 1 , to quantify GO or GPT from the amount of quantification. Next, the attached drawing will be explained in detail using the drawings. In the drawing, 11 to 1 g are containers, and 21 is, for example, 10 mol Oa-ketogel tar acid or 6 g/e The carrier liquid 2b is a mixture of sodium chloride, etc., for example, 20x10-
3 mol OL-aspartic acid iI solution, 2oif7A, for example, 20xlOJEL/L-Ara=/IIIm, 2d, for example, acidic pllllmll consisting of 0.1 mol xo3,
2. is, for example, an absorption liquid that is not from country h, 2f is a waste liquid such as a carrier liquid, 2g is a recovered liquid such as the above absorption liquid, and 3
1 to 3 are inlets, 3f and 3g are outlet ports, 4 is a series of peristaltic pumps, 5 is a temple injector that injects a predetermined amount of the stirrup to be measured into the 111th road 11aO first AC point 12 & described later, 6 companies A mixer for mixing liquids, 7 is a mixing 6
9-nitriding enzyme, 6 is arranged downstream of flow 7, 9
is an electrode that detects the amount of change in conductivity of the absorption liquid, and 10 is an electrode!
The signal detected by J611 is used to calculate the temperature of 011 degrees and display it once. No. 101, which leads to the flow cell via the housing 6 and the immobilized enzyme 7! 11b is introduced from the inlet 3b, and the aspa2ginic acid**zb is introduced into the peristaltic pump 4.
205th! via the first ON111i valve 131!
The 20th injected from the flow point 12b into the first flow path 11 &
The flow path 11C is introduced from the inlet 3C and the flow path 11C is connected to the peristaltic pump 4 and the second on-off valve 13b.
The S-th flow path 11d is injected into the first flow path 111L from the S-th AC point 12c via the inlet 3d. A 40th flow path is injected from the AC point 12d to the first flow path 111; 11 is a first sO flow path through which the absorption I12 introduced from the inlet 3 is guided to the flow cell S via the peristaltic pump 4; 11f is a 60th flow path through which the carrier liquid etc. are guided from the flow cell 6 to the discharge port 3f;
11g is the 70111th path through which the fIk collected liquid is guided from the cell 6 to the discharge port 3g via the electrode 9.

Further, the flow cell $ is divided into a first part 82 and a second part 83 by a gas-permeable diaphragm 81 made of, for example, a polycarbonate membrane, and an inlet 84 of the first part 82 is divided into two parts.
1 and the outlet port 85& are respectively connected to the fifth flow path 11 and the seventh flow path 11.
The flow path figK is connected and the inlet 84b of the second wealth 83
The outlet port 85b is connected to the first flow path 11m and the sixth flow path 11f, respectively. Further, the first and second on-off valves 13m and 13b, the mixing 6, the immobilized enzyme 7, the flow cell S, and the electrodes are often housed in a constant temperature bath 14 maintained at, for example, 37°C. The immobilized enzyme 7 is often produced in the following manner, for example, by performing a KO-alkyl reaction on the inner wall of a nylon chip and then cross-linking the carboxylase. Fixed.

The enzyme 7 may be completely removed, and the predetermined enzyme to be immobilized on the immobilized enzyme may be mixed in the carrier 112a in a liquid state. An example operation will be explained. In the figure, when the peristaltic pump 4 is driven, carrier liquids 2*thL-aspartic acid solution @2b, L-acetic acid solution @2a, pH adjustment 2d, and absorption I12. *1st to 1st SO channels 11&~
You will be led to 11. Front, first and second on-off valves 1
Both 3m and 13b are in vso state, for example 10~5oy
The liquid to be measured consists of the blood of e! When the liquid to be measured is injected into the first flow path 111 by the sample injector 5, the liquid to be measured is drawn into the middle tube 112m and transferred to the mixer 4, where the liquid to be measured and the carrier liquid are sufficiently mixed. After that, the container to be measured is placed in the container again and placed on the immobilized enzyme 7, and the glutacic acid present in the container to be measured undergoes an enzymatic reaction as shown in the following formula (1). .

Glutamic acid - some bacteria →,,, r-aminobutyric acid + ■
2(1) The O product of the above formula (1) and the liquid to be measured are then transported again to the IEK and passed to the fourth exchange point 12d.
, and the detected 41 odd is detected by the compensation 4 #l & the master section 10, and becomes, for example, the first detected compensation odd -OO.
For example, L-Atsuen II Ryo 2C is injected into the 10th channel 111 from the 12° StO AC point at a flow rate of 10 to
50 doses of blood are injected into the 111th channel 11a using the nozzle injector 6. The measured object i
I and the L-alanine solution were transported to the middle box and warmed in the mixed house 111, and the a-ketodaltaric acid in the carrier, the L-alanine, and 7 in the liquid to be measured were heated. It reacts as shown in equation (4) below.

a-ketotalic acid + L-azene-pyruvic acid + L
- Dacitaric acid (4) was added to the product of formula (4) again as a carrier l[K was transported and stabilized enzyme 7KIIl, OL-glutaζ) acid in the product was stabilized enzyme 7 was stabilized 1 The above formula (1) is used between glutamic acid decal xylase and
) 0-like enzyme reaction. Therefore, through the above (4) formula shi and 佽) formula, the result is 90, a, the above side) formula shi and [
) through the formula and change the conductivity of Absorption II by 1 by using the above-mentioned ooo2o field and Oka 11K.The amount of change in the axillary electrical conductivity is the electrode! , and the detected signal is detected by 10 and the first detection signal is 1, for example, j12 detection 14IK. A calculation is performed to subtract the reference signal 14#I.Thus, the concentration of IGO in the liquid to be measured is determined, and the reference signal is subtracted from the first detection correction Cc. According to this embodiment, in addition to the shortcomings of the conventional example, it has the advantage of being able to move quickly and precisely at a fixed speed.

[Brief explanation of the drawing]

Attachment - One example from the company's headquarters
...Container, 2a... Life Yaria box, 2b... L-aspartic acid $11, 2c... Shealanine 111L
2 (1-...pii style @IE, 2e""aJK house, 2f
-...Abandoned house, 2g-...■Cool cooling, 31-3...Inlet, 3f, 3g...Outlet, 4-...Peristal pump, G...Nample injector, 6... Onkai work, 7.
...-electroenzyme, ...) 1-cell,! ···electrode,
10... Confucian 4J6 Riku, iim-11g" IIIE Road, 12a-1ad... Exchange point, 13a, i3b ---
-99% valve, 14-... constant temperature bath.

Claims (1)

[Scope of Claims] (1) After adding 11EKg of ketodaltaric acid to be measured containing gourmet 2-phosphate transaminase and glutamic acid pyruvate transaminase, L-aspartic acid or L-alanine is added, respectively. The enzymatic reaction of glutamate oxaloacetate Flanmeaminase and glutamate oxaloacetate Flanmeaminase and glutamic acid Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase OVa determination method 0 characterized by quantifying pyruvate transaminase, respectively. a sample injector for injecting a predetermined amount of the liquid to be measured into the channel; a 20th flow channel for injecting L-aspartic acid into the first flow channel via the tenth on-off valve; a fifth flow path for injecting L-alanine into the flow path; a mixer provided in the first flow path for mixing the casing flowing in the flow path; In the first waterfall passage, nine immobilized enzymes are disposed under the mixer O 11, and the $111 downstream of the immobilized enzymes! a 40th flow path through which a predetermined 01E tone** is injected into the flow path; a 50th flow path through which a predetermined absorption liquid flows;
#I below the immobilized enzyme O! It forms part of the flow path through the gas-permeable 01li membrane. jlI wealth and two rattans forming a part of the first flow path are provided so as to be adjacent to each other; It is equipped with an electrode for detecting the amount of conductivity concentration of the collected IIO, and a series of peristaltic pumps for delivering a predetermined amount of OII to the first to third SO@ channels, respectively, and the OII is detected by the electrode. Glue and mic acid transaminase are used to indirectly quantify glutamic acid transaminase and glutamic acid pyruvate transaminase and glutamate pyruvic acid transaminase contained in the liquid to be measured from the amount of change in conductivity. Fixed device for acetate transaminer-V and glutamate pyruvate transaminase ζ (3) The immobilized enzyme is removed and the predetermined enzyme is dissolved in the carrier Il.
A measuring device containing K as described in Patent ■Kyo Range Paragraph (2). (4) The first and second on-off valves, the mixer, the immobilized enzyme, the flow cell, and the electrodes are housed in a constant temperature bath kept at a predetermined temperature. The measuring device described in WAjI (2) or Hiro (3).