JPH0191798A - Method for analyzing creatinine and pretreating column - Google Patents

Abstract

PURPOSE:To determine the titled compound with high reliability, by pretreating a sample with a column of immobilized enzymes containing immobilized plural species of specific enzymes, removing NH3 and glutamic acid which are interfering substances, enzymically hydrolyzing creatinine and measuring the amount of formed NH3. CONSTITUTION:A sample is introduced into a pretreatment column 21, etc., filled with an immobilized enzyme (A) of immobilized glutamic-pyruvic transaminase (GPT), coexisting with glutamic dehydrogenase (GLDH) and laminated on a immobilized enzyme (B) of immobilized glutamic-oxalacetic transaminase (GOT) coexisting with GLDH in one column container 1 in the column axial direction using a sample introducer 3 to remove ammonia and/or glutamic acid. The resultant treated solution is then treated with a column 8 of immobilized creatinine diiminase to produce ammonia in an amount corresponding to that of creatinine present in the sample. The resultant ammonia is subsequently converted into hydrogen peroxide in a column 5 to emit light with luminol, etc. The emitted light is then detected with a chemiluminescent detector 13 to determine the creatinine.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a creatinine analysis method and a pretreatment column. More specifically, the present invention relates to an analytical method suitable for quantifying creatinine that may be present in a biological sample and a pretreatment column suitably used in this analytical method.

(b) Conventional technology The amount of creatinine in a biological sample can be an indicator of kidney disease, and its quantification has important significance in clinical testing.

Conventionally, the so-called Jaffe method using picric acid and the enzymatic method using various enzymes have been known as analytical methods used for quantifying creatinine, especially using automatic analyzers. A sample of the recommended compound was treated with creatinine deimidase (CNI) to convert creatinine to N-methylhydantoin and ammonia using formula I.
), a method of quantifying the amount of creatinine by measuring the amount of ammonia is known.

Creatinine 10 HtO-”y NH,+N-methylhydantoin (Formula I) Furthermore, as a method for measuring the amount of ammonia, α
- In the presence of ketoglutarate and NADPH (or NADH), treat with glutamate dehydrogenase (GLDIT) to generate glutamate corresponding to NH31i (formula ■), N Hs + α-ketoglutarate - L-glutamate + NADP" + H to
(Formula ■) Furthermore, glutamate oxidase (GLX
D) to generate hydrogen peroxide corresponding to the amount of ammonia (formula ■), and add L-glutamine several tens of * + H * 0i to H2.
O,+α-ketoglutaric acid H s (Formula ■) A method has been proposed in which the amount of hydrogen peroxide is measured by an absorptiometric method or chemiluminescence method, and the measured value is converted (Japanese Patent Application Laid-Open No. 1989-1999).
-41500).

In such enzymatic methods, ammonia and glutamic acid that may coexist in the sample give a large positive error to the quantification of creatinine. Therefore, before the treatment with the above enzyme, ammonia and glutamic acid must be removed in advance (pretreatment).
For example, the sample is prepared in advance by the above (formula ■).
According to glutamate dehydrogenase (GLDH)
The generated glutamic acid and coexisting glutamic acid are then treated with glutamate oxaloacetate transaminase (GOT) or glutamate pyruvate transaminase (GPT) in the presence of oxaloacetate or pyruvate to remove glutamate. (Formula glutamic acid + oxaloacetic acid (or pyruvic acid) GOT + iGPT)
α-ketoglutaric acid + aspartic acid (or alanine) (Formula ■) In order to efficiently carry out such pretreatment, research has been conducted on a method in which the two enzyme reactions for pretreatment described above are carried out using an immobilized enzyme column. It has reached the point where

(c) Problems to be Solved by the Invention However, in the pretreatment, when a GLDH-immobilized enzyme column and a GOT-immobilized enzyme column are connected in series based on the above reaction formula, ammonia and glutamic acid About 20% or more of the initial amount remained, and the removal effect was insufficient.

Although pretreatment was carried out using an immobilized enzyme column, approximately 10% residue was still observed. Therefore, the removal effect of pretreatment using such an enzyme system was not satisfactory and its practicality was limited.

This invention has been made to solve these conventional problems.

(2) Means for solving the problem The inventors of the present invention have conducted intensive research from the above viewpoint, and have found that
We found that the removal rate could be improved by using an immobilized enzyme that co-immobilized not only GLDH and GOT but also GPT.As a result of further research, we found that GLDH and GOT
The present invention has been based on the discovery that the removal rate of ammonia and glutamic acid can be significantly improved by using a multilayer column in which an immobilized enzyme co-immobilized with GLDH and an immobilized enzyme co-immobilized with GLDH and GPT are stacked in one column container. reached.

Thus, according to the present invention, an immobilized enzyme formed by co-immobilizing glutamate pyruvate transaminase (GPT) and glutamate dehydrogenase (G-mate oxaloacetate transaminase (GOT) and GLDH) in the column axis direction. There is provided a pretreatment column for removing ammonia and/or glutamic acid, which is packed with an immobilized enzyme formed by stacking or co-immobilizing GPT%GOT and GLDH.Furthermore, according to the present invention, the above-mentioned After introducing the sample into a pretreatment column and performing a treatment to remove ammonia and/or glutamic acid, the treated solution was treated with creatinine diiminase (CNI).
) to produce ammonia corresponding to the amount of creatinine, and a creatinine analysis method is provided in which the amount of creatinine in the sample is determined based on the amount of ammonia.

In the pretreatment column of the present invention, the pretreatment column is formed by stacking and packing the GPT/GLDH co-immobilized enzyme (A) and the GOT/GLDH co-immobilized enzyme (B). After preparing (B), it can be produced by sequentially filling it into a column container.

Here, co-immobilization
) is achieved by using a mixed enzyme as the immobilized enzyme.
This can be carried out according to ordinary enzyme immobilization techniques.

Generally, there are various immobilization methods such as chemical bonding method and entrapment method, but chemical bonding method is suitable for stable immobilization.9 A preferable example is silane cup. Examples include a method in which a ring agent is immobilized on a suitable carrier (eg, glass beads, silica gel, etc.).

On the other hand, GPT/GOT10LDH co-immobilized enzyme CG'
)(1) A packed column can be prepared by filling the immobilized enzyme (C) prepared in the same manner as above into a column container using a conventional method.

Pretreatment of a sample using a pretreatment column is suitably carried out by introducing the sample into the column and passing it through the column while an appropriate carrier liquid is flowing. In order to proceed with the ammonia and glutamic acid removal reaction such as (n) and formula (III), NADPH (
or NADH), α-ketogeltalic acid, oxaloacetic acid, and pyruvic acid. Note that it is suitable to adjust the PL of the carrier liquid to 7 to 8, and it is suitable to use a buffer such as a phosphate buffer.

In addition, in terms of removal efficiency, it is preferable to use a stacked column of immobilized enzymes (A) and (B), and when a stacked column is used, the immobilized enzyme *(A) is first treated with the sample. It is more preferable in terms of removal efficiency to set the

For the measurement of creatinine, the treated solution after the above pretreatment (formula I
), by treating with creatinine iminase (CNI) to generate the corresponding ammonia and measuring the amount.

At this time, it is preferable to treat CNI using a CNI-immobilized enzyme column as it is simple and suitable for automation. In addition, it is suitable for the subsequent measurement of ammonia to be carried out using a hydrogen peroxide generation system and a hydrogen peroxide detection system that use ammonia as a substrate, and GLD is carried out according to the above (formula ①).
A preferred method is to treat with H and further with GLXD according to the above (Formula 2), measure the hydrogen peroxide produced, and calculate from this value. At this time, it is preferable to use an immobilized enzyme column for the treatment with GLDH and GLXD. Two columns in which GLDH and GLXD are immobilized separately may be used, or a column in which they are co-immobilized may be used. .

In addition, suitable methods for measuring hydrogen peroxide concentration include the luminol chemiluminescence method using luminol and red blood salt, the electrochemical detection method using ECD or hydrogen peroxide electrodes, and the colorimetric method using peroxidase. There is.

Note that the pretreatment column in this invention can be used for purposes other than creatinine measurement, and can be used for purposes other than creatinine measurement.
Alternatively, it can be used in various analysis and purification fields that require the removal of glutamic acid.

(e) Effect: In one column, GPTs with different equilibrium constants (K=2
．． It is considered that the presence of 2) and GOT (K = 0.17) promotes the reaction of (Formula 2) above, and further promotes the reaction of GLDH (Formula 2). Furthermore, when a stacked column is used, for example, GPT
After reaching the equilibrium of the reaction by GOT, the overall reaction is promoted by smoothly reaching the equilibrium of the reaction by GOT. It is thought that synergistic reaction promotion is performed and excellent ammonia and/or glutamic acid removal effects are achieved without being restricted by such a low equilibrium constant.

(F) Examples Example 1 Immobilized enzyme (A) in which glutamate pyruvate transaminase 2Bm9 and glutamate dehydrogenase 28m9 were co-immobilized on a porous glass carrier 17 (
GPT-GLD (immobilized glass) and porous glass carrier 1
Immobilized enzyme (B) in which 28 Mg of glutamate oxaloacetate transaminase and 2829 glutamate dehydrogenase were co-immobilized per g (GOT-GLDll
Immobilized glass) was packed into the column container 21 so as to be stacked in the column axis direction as shown in FIG.
1, Os9 and GLDH65μQ).

The porous glass used was porous glass beads with a mesh size of 20 to 80 and a pore diameter of 500 mm. After propylating, this glass is treated with a 2.5% glutaraldehyde aqueous solution to convert it into glutaraldehyde, and this glass is fixed by mixing a predetermined amount in a mixed aqueous solution (phosphate buffer) of each enzyme intended for immobilization. This was done using a method of The column container 21 used had an inner diameter of 4 cm and was equipped with a liquid inlet and an outlet.
Using an acrylic container with a length of 24 x 11 L, each layer of immobilized enzymes (A) and (B) was filled so that each layer had a length of 12 cm.

This stacked column 1 was attached to the ammonia/glutamic acid removal rate measuring device shown in FIG. 2 to evaluate the removal efficiency. 2 in the figure indicates the carrier solution, 2.5mM oxaloacetic acid, 0.12mM α-ketoglutaric acid, LOmS
I of pyruvic acid, 0.05 m\1 of pyridoxal phosphate,
A 10mM phosphate buffer containing 0.027mM'DNADPI, pH 8.0, was used. In the figure, 3 is the sample introducer, 4 is the liquid pump, and 5 is the GLDII and GLD.
This is a hydrogen peroxide generating column packed with an enzyme co-immobilized with X, and generates hydrogen peroxide corresponding to ammonia and/or glutamic acid according to the above (Formula 1) and (2). Further, 6 is a hydrogen peroxide detection system based on a flow type luminol chemiluminescence method, and 7 is a recorder.

In this apparatus, the liquid feeding rate was set to 1jIQ/min, aqueous solutions of ammonia and glutamic acid (4 μρ) of various concentrations were injected as samples, and the removal rate was calculated based on the amount of hydrogen peroxide generated.

The results are shown in Table 1.

Table 1 As shown above, a high removal effect was obtained, especially at 1mM
It can be seen that ammonia and glutamic acid can be completely removed.

X1 carp 1 Immobilized enzyme CC) (GP
A column (la) as shown in FIG. 1(b) was prepared by filling a column container with an inner diameter of 3RM and a length of 2011M with T-GOT-GLDH (immobilized glass).

On the other hand, GPT-GL was prepared in the same manner as in Example 1 except that the total immobilized amount was the same as that of the immobilized enzyme (C) and the column container had an inner diameter of 3 mm and a length of 20 am.
Laminated filling of DH glass and GOT-GLPH glass (
10 mm:10 mm) columns were prepared and compared.

The removal efficiency of these columns was evaluated in the same manner as in Example 1. The results are shown in Table 2.

Table 2 As shown above, good removal rates were obtained for both, but
It can be seen that the effect is particularly remarkable in stacked columns.

For further comparison, GOT-GLDH glass (A
) and an immobilized enzyme column packed only with GPT-GLDH glass (B), the removal rates were measured at about 80 to 90%, which was not insufficient.

Example 3 GPT-GLDH immobilized glass (A) and GOT-GLDH immobilized in the same manner as in Example 1 except that the carrier used was 80-120 mesh alkoxide glass beads and the amount packed into the column was 20 mm each. A pretreatment column packed with glass (B) was prepared (total quantitative determination was performed using GO
Tl, 0m9. GPT2.0 order 9 and 0LDH130
).

The removal rate of this pretreated column was measured in the same manner as in Example 1. The results are shown in Table 3.

From this result, it was also found that the removal efficiency could be further improved by making the particle size of the carrier finer.

In addition, regarding the above column, GPT-GLDH glass (A
) layer and GOT-GLD) [When the removal rate was evaluated by inverting the glass (B) layer, the removal rates corresponding to Table 3 were 97.3%, 96.8%, and 96.8, respectively. %Met. Therefore, in a stacked column, GPT-GLD
H glass (A) F! It has also been found that it is preferable to set i on the sample introduction port side.

Example 4: Creatinine analysis) The creatinine analyzer shown in FIG. 3 was constructed using the above-mentioned pretreatment column. In the figure, ■ is a pretreatment column, 3 is a valve switching type sample introducer, and 5 is a GLDH-GLXD
Hydrogen peroxide generation column filled with co-immobilized enzyme, 8 is C old immobilized enzyme column, 9 is blank column, 10a,
lOb is a flow path switching valve, 11.12 is a branch flow path, 13
14 indicates a chemiluminescence detector, and 14 indicates a recorder. Also, I
5 is a distilled water supply line (supply amount 0.511ff/min), 16
has the following composition: Sodium phosphate dibasic 5725z9/2 1st
Potassium phosphate 543m9/(1α-ketoglutarate 70m9/(IEDTA disodium (2H,O) 7752m9/I2oxaloacetate 10mM Sodium pyruvate 40mM pyridoxal phosphate
Supply path for buffer solution containing 0.2mM and adjusted to pH 7.25 (supply ff10.25x+77 minutes)
, 17 is NADPH20029/(2 aqueous solution supply path, 1
8 is red blood salt 6, 69/σ aqueous solution supply path, 19 is Luminol LOzg/Q aqueous solution (potassium hydroxide 11.29/σ
f2. (containing 12.49 # of boric acid).

This device is configured to be able to quantify creatinine in a sample based on the detector output when the branch channel 11 is selected. That is, the treated liquid from which ammonia and glutamic acid have been substantially completely removed in pretreatment column 1 as described above is introduced into column 8 to convert creatinine to ammonia, which is converted to hydrogen peroxide in column 5. Here, the amount of hydrogen peroxide is detected by the detector 13 using the luminol emission method. Since this value corresponds to the amount of creatinine in the sample, it is possible to convert and quantify creatinine based on this value. Note that the branch flow path 11 is for background measurement, and by using the output at the time of selecting this flow path as a base, more accurate measurement can be performed.

Incidentally, creatinine analysis in serum (8 .mu.e) was carried out using such an apparatus, and the correlation with the conventional Jaffe method was investigated. The results are shown in FIG.

In this way, the correlation was excellent.

(g) Effects of the Invention According to the pretreatment column of the present invention, ammonia and/or glutamic acid in a sample can be removed with extremely high efficiency. Therefore, by using a sample pretreated using such a pretreatment column, highly reliable creatinine analysis can be performed.

[Brief explanation of the drawing]

FIGS. 1(a) and (b) are configuration explanatory diagrams showing one embodiment of the pretreatment column of the present invention, and FIG. 2 is an apparatus used to evaluate the removal efficiency of the pretreatment column of the present invention. Fig. 3 is an explanatory diagram of the configuration of an embodiment of the apparatus for carrying out the creatinine analysis method of the present invention, and Fig. 4 shows the correlation between the measured values of the creatinine analysis method of the present invention and the conventional method. FIG. (A)...GPT-GLDH immobilized glass, (
B)...GOT-GLDH immobilized glass, (
C)...GPT-GOT-GLDEI immobilized glass, l...Laminated column, la...
・Column, 2...Carrier solution, 3...Sample introducer, 5...Hydrogen peroxide generation column, 6...
Hydrogen peroxide detection system, 8...CNI immobilized enzyme column, 13...
...Chemiluminescence detector, 15... Distilled water supply line, 16... Buffer solution supply line, 17... NADPH aqueous solution supply line, 18...
... Red blood salt aqueous solution supply path, 19 ... Luminol aqueous solution supply path, 2!・・・・・・Column container.

Claims (2)

[Claims] (1) An immobilized enzyme obtained by co-immobilizing glutamate pyruvate transaminase (GPT) and glutamate dehydrogenase (GLDH) and glutamate oxaloacetate transaminase (GLD) in one column container.
OT) and an immobilized enzyme formed by co-immobilizing GLDH are stacked in the column axis direction, or GPT, GOT
A pretreatment column is prepared by filling an immobilized enzyme obtained by co-immobilizing GLDH and GLDH, and a sample is introduced into this pretreatment column to remove ammonia and/or glutamic acid, and then the treated solution is treated with creatinine diiminase. 1. A creatinine analysis method, which comprises treating the sample with ammonia to produce ammonia corresponding to the amount of creatinine that may be present in the sample, and quantifying creatinine in the sample based on the amount of ammonia. (2) The analytical method according to claim 1, wherein quantification of creatinine based on the amount of ammonia is performed using a hydrogen peroxide generating enzyme system using ammonia as a substrate and a hydrogen peroxide detection system. (3) An immobilized enzyme formed by co-immobilizing glutamic acid pyruvate transaminase (GPT) and glutamate dehydrogenase (GLDH) together with an immobilized enzyme formed by co-immobilizing glutamic acid oxaloacetic transaminase (GOT) and GLDH in one column container. Enzymes and enzymes are stacked in the axial direction of the column, or GP
A pretreatment column for removing ammonia and/or glutamic acid packed with an immobilized enzyme obtained by co-immobilizing T, GOT, and GLDH.