JPH02100699A - Measurement of organism sample - Google Patents

Abstract

PURPOSE:To measure amounts of dehydrogenated enzyme and matrix of said enzyme in an organism sample in high sensitivity and reproducibility by affecting 1-(3-carboxypropyloxy)-5-ethylphenazines to NADH generated with dehydrogenating reaction and measuring an amount of generated H2O2. CONSTITUTION:In measuring respective contents of dehydrogenated enzyme and matrix of the dehydrogenated enzyme in an organism sample, nicotinamide- adenine dinucleotide of reduction type (NADH) or nicotinamide-adenine dinucleotide phosphate of reduction type (NADPH) generated with dehydrogenating reaction is affected by 1-(3-carboxypropyloxy)-5-ethylphenazines and an amount of generated hydrogen peroxide is measured, thus the organism is measured by detecting respective contents of dehydrogenated enzyme and matrix of the dehydrogenated enzyme in the organism sample.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for measuring biological samples, and more specifically,
It relates to a method for measuring the content of dehydrogenase and dehydrogenase substrates in biological samples.

[Conventional technology]

When measuring the content of dehydrogenase and dehydrogenase substrate in biological samples such as serum and urine in biochemical tests and pathological research, reduction produced by the action of dehydrogenase is used. It has been used to determine the respective contents of dehydrogenase and dehydrogenase substrates in biological samples by measuring the type of nicotine. That is, (1) The ultraviolet absorption (340 nm) of NAD (P)H
) how to measure.

A method in which formazan is produced from a tetrazolium such as nitroblue tetrazolium, and the increase in absorbance near 570 nm, which is the absorption band of formazan, is measured.

However, each of these has major problems.

In the method (1) above, the blind value of the absorbance at 340 nm increases due to other components in the sample that have ultraviolet absorption, increasing the measurement error and making the measurement impossible, and It is greatly affected by the turbidity of the sample itself. Furthermore, in the method (2), the produced formazan has low solubility in water and precipitates are deposited, staining measuring instruments such as cell parts and tubes, and interfering with measurements.

Furthermore, in addition to the methods (1) and (2) above, NAD (
In recent years, a method has been known to measure hydrogen peroxide produced by reacting P)H with an electron carrier, but PMS, which is commonly used as an electron carrier, is extremely unstable in light and alkaline solutions. It is. Generally, in the dehydrogenation reaction of dehydrogenase, the optimum pH is often biased toward the alkaline side, and this limits the measurement operation, and a method for measuring it has not been put to practical use.

[Problems to be Solved by the Invention] The present invention solves the above-mentioned problems of the conventional method.
In addition, the present inventors have been able to determine the respective contents of dehydrogenase and dehydrogenase substrates in biological samples in a short time, not only in alkaline conditions but also in acidic or neutral conditions, without contaminating the measuring instruments. After investigating various methods for measuring the content of dehydrogenase and dehydrogenase substrate with good reproducibility and high sensitivity, we found that hydrogen peroxide can be It has been found that the amount of NAD (P)H produced is proportional to that of NAD (P)H, and that NAD (P)H can be measured even slightly.Based on this knowledge, the present invention was achieved.

That is, the present invention uses reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide produced by a dehydrogenation reaction when measuring the respective contents of dehydrogenase and dehydrogenase substrate in a biological sample. By measuring the amount of hydrogen peroxide produced by the action of 1-(3-carboxypropyloxy)-5-ethyl phenazine on phosphoric acid, we can detect dehydrogenase and dehydrogenase substrates in biological samples. This is a method for measuring biological samples, which is characterized by knowing the content of each component.

Biological samples used in the present invention include samples derived from animals, plants, microorganisms, etc. Typical examples include serum and urine, extracts of various living organisms, and culture fluids of microorganisms and cells. be.

The dehydrogenase used in the present invention may be any enzyme that catalyzes a reaction that produces NAD (P)H.

Representative examples include dehydrogenases and their substrates shown in Table 1.

Table 1 The object of measurement Noboru Goto Substrate alcohol bile acids glycerin Glycerin-3- phosphoric acid glucose glucose-6- phosphoric acid aldehyde formaldehyde Basic alcohol dehydrogenase bile acid dehydrogenase glycerin dehydrogenase Glycerin-3-phosphate deoxidation hydrogen enzyme glucose dehydrogenase Glucose-6-phosphate desorption hydrogen enzyme aldehyde dehydrogenase formaldehyde dehydrogenation Basic lactate dehydrogenase malate dehydrogenase isocitrate dehydrogenase lactic acid malic acid isocitric acid formic acid α-hydroxy butyric acid Tetrahydrofolic acid others Various cholesterol Various alcohol various sugars Various amino acids formate dehydrogenase α-Hydroxybutyric acid dehydrogenation enzyme Tetrahydrofolate dehydrogenase Basic Dehydration of various cholesterols elementary enzyme Dehydration fermentation of various alcohols Basic Dehydrogenase for various sugars Dehydrogenase for various amino acids be able to.

CPEP utilized in the present invention refers to tilphenazine or 1-(3-carboxypropyloxy)-5-ethylzenadinium salt.

1-(3-carboxypropyloxy)-5-ethylzenadinium salt is, for example, 1-(3-carboxypropyloxy)-5-ethylphenadinium alkyl sulfate or 1-(3-carboxypropyloxy)-5-ethylzenazinium salt. )
-5-ethylzenadinium chloride and the like.

CPEP is 1-(3-
It can be obtained by synthesizing ethoxycarbonylpropyloxy) phenazine, ethylating the 5-position with diethyl sulfate, and then acid hydrolyzing it. −”
''' -CPEP is a reddish-purple powder, and an aqueous solution at a normal concentration of use is pale pink to pale reddish-purple in the vicinity of neutrality. In an aqueous solution with a pH of 7.
There are maximum absorption values at 8nm, 386nm, and 510nm, and the molecular extinction coefficient at 273nm is 47mM-1cm.
-'.

CPEP is also an electron carrier that is stable in light and alkaline solutions. CPEP has relatively high stability; for example, even if it is irradiated with scattered light in 0.2 M sodium acetate buffer (pH 4.0) at room temperature for one month, the electron transport activity and spectrum do not change at all. Furthermore, even if CPEP was stored at a concentration of 22 μm at 30°C for 3 days in a buffer solution ranging from pH 4 to 9, it did not lose its activity at all, and even at pH 10 it had 92% residual activity;
It can be used in the range of 4 to 10.

The method for quantifying NAD (P)H of the present invention usually involves a step of reacting NAD (P)H produced by a dehydrogenase with CPEP to produce hydrogen peroxide, and a step of reacting the produced hydrogen peroxide with a known method. The measurement is carried out in two stages:

The reaction between CPEP and NAD (P)H is a coupling reaction. CPEP and NAD+ or NADP" can be added in small amounts.
The reaction with AD (P)H is usually performed at an alkaline pH, which is the optimum pH for the dehydrogenation reaction, when measuring the respective contents of phosphate buffer, Tris-HCl slow dehydrogenase, and dehydrogenase substrate. It is also possible to generate (P)H with high efficiency. The concentration range of CPEP in the reaction solution is not particularly limited, and is appropriately set depending on the type of sample, reaction conditions, and measurement time. Usually 0.01~10m
A concentration range of M is preferred. NAD (P)H, NAD”
The concentration range of "or NADP" is also not particularly limited, and is usually used in the concentration range of 0.01 to 10 mM.The reaction temperature is also not particularly limited, but it is usually carried out in the range of 20 to 40 °C; There is no need for heating or cooling as long as the temperature is within the range, but this does not preclude heating or cooling.

Since CPEP has a carboxyl group in the carboxyprobyloxy group at the 1st position, it can be linked to enzymes, coenzymes, resin carriers, etc. using crosslinking reagents such as carbodiimides.
It is possible to bind directly or via a spacer such as a polyethylene glycol (hereinafter abbreviated as PEG) neck. Combining CPEP with other materials such as those described above provides various advantages. For example, C.P.E.
When P is combined with a resin carrier and packed in a column or the like, it becomes a reactor that converts it into hydrogen peroxide, NAD (P)H. In addition, when combined with an enzyme or coenzyme, the reaction takes place within the molecule, which not only increases the concentration of the reaction partner and allows the reaction to proceed rapidly, but also allows the enzyme, coenzyme, or CPEP to be present in large excess. It is no longer necessary to use it for In addition, CPEP and NAD+ or NA are added to the dehydrogenase.
When CPEP is combined, oxygen is consumed within the molecule and hydrogen peroxide is produced, so dehydrogenase can be artificially converted into oxidase.Also, CPEP can be combined with other substances to form a polymer. This makes it possible to create a reactor that is confined within the reaction system, making recovery and reuse possible.

There are no particular limitations on the method for quantifying hydrogen peroxide produced in the reaction between CPEP and NAD (P)H.

Practically speaking, representative methods include an electrode method, a coloring method, and a luminescent method. As the electrode method, there is a method using a hydrogen peroxide electrode. As a coloring method, peroxidase is used to measure the color development caused by phenols such as phenol or aromatic amines such as O-phenylenediamine, or peroxidase is used to produce 4-aminoantipyrine (hereinafter referred to as 4-AA). A method that measures the color development due to the oxidative condensation of aniline and aniline, or a method that uses catalase is often used. As a luminescence method, there is a method of detecting the chemiluminescence generated by allowing luminol to interact with a complex, which enables highly sensitive measurement.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example I NAD (P)H was quantified by measuring hydrogen peroxide produced by the reaction between NAD (P)H and CPEP.

First solution 0.5mM CP E P 0
．． 13mQ 20mM Glycine-NaOH Slow Massage (pH 9,0) 0.87ynQ Distilled Pure Water
``Soken [2,00 shrimp 2nd night 10mM 4-AAO,
2, 10mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-)luidine (hereinafter referred to as T).

(abbreviated as O8) 0.2% 100tJ/Peroxidase 0.2% I
M Tris-)1cI buffer M (pt17.5)
-MeAi-1,0 reaction was carried out in a thermostat at 37°C. The first liquid was preheated at 37°C. NAD, (P)H (0 to 2
0.05 mQ of mM) was added and reacted for 5 minutes. The second liquid was added to this, and the absorbance at 550 nm was measured 1 minute after the addition.

The results are shown in FIG.

FIG. 1 shows that the relationship between the concentration of added NAD (P)H and the absorbance is a linear relationship, and that NAD (P)H can be quantified with high accuracy. This method allows NAD (
The concentration of P)H can be determined up to 10mM.

Example 2 The hydrogen peroxide production reaction in the coenzyme-enzyme-CPEP tripartite conjugate was investigated.

PEG-CPE that combines aminated PEG and CPEP
PEG combined with P and aminated PEG and NAD+
-NAD"" was prepared.

The terminal amino groups of PEG-CPEP and PEG-NAD were activated with 3,3'-(1,6-thioxo-1,6-hexanediyl)-bis-2-thiazolidinethione, and lactate dehydrogenase (hereinafter abbreviated as LDH) was activated. ) to obtain a NAD"-LDH-CPEP ternary conjugate.

This three-way conjugate and the coloring solution shown below were mixed and reacted.

Coloring solution 1mM 3-methyl-2-penzothiazolinone hydragosin hydrochloride (hereinafter abbreviated as MBTH) 0.3mg 300mM L-lactic acid 0.5m
Q10mM Ethylenediaminetetraacetic acid 0.3 IU/peroxidase 0.4 250
The color development of the oxidized condensate of MBTH and primaquine due to the generated hydrogen peroxide was measured by absorbance at 510 nm.

Table 2 shows the oxygen concentration and dye production rate.

Table 2 From this result, lactate dehydrogenase is NAD”LDH-C
It can be seen that by creating a PEP three-way conjugate, it was artificially converted into lactate oxidase.

Example 3 One'S N A D produced by the dehydrogenation reaction of LDH
Serum LDH was quantified by reacting H with CPEP and measuring the generated hydrogen peroxide.

1st liquid 70% D sodium monolactate 13.3mg4.4
mM NAD” 0.4mQ2mM
CPEP 0.2 to 0.1M potassium phosphate buffer (pH 7,0) 1.4 to 2.0 second solution 10mM 4-AA 0.1
10mM TOO50, 1mQ 100U/peroxidase 0.1 0.1M
Potassium phosphate buffer (pH 7,0) 0.7 comparison and 1.0 comparison The reaction was carried out in a thermostat at 37°C. The first liquid was allowed to react. The second solution was added to this, and the absorbance at 550 nm was measured immediately after the addition.

The results are shown in FIG.

Figure 2 shows that the concentration of added serum LDH can be determined by absorbance at 550 nm.

Example 4 The stereotypicality of lactic acid, which is a substrate for the dehydrogenation reaction of LDH, was investigated.

1st night 501J/+y+Q LDH0,1ynQ2mM
NAD” 0.
2mQ0.5mM CP E P
O, ITILQ distilled pure water
0.6m! Q20mM glycine/Na0fl
Loose? I? Night (pH 9,0) 1,0rrl-Q-2, OaQ 2nd solution 10mM 4-AA 0.2Shark 10mM TOOS 0.
2mQ1001J/Peroxidase 0.2 comparison IM Tris・ItCI F5j? ``3 liquid (pH 7,
5) The 1,0 reaction was carried out in a thermostat at 37°C. The liquid No. 1 was preheated at 37°C. 0 and monolactate water soluble? a (0~
50mM) was added in an amount of 0.05mQ and reacted for 5 minutes. The second liquid was added to this, and the absorbance at 550 nm was measured 1 minute after the addition.

The results are shown in FIG.

Figure 3 shows the concentration of lithium monolactate added to 550n.
This shows that it can be quantified by absorbance at m.

By this method, the concentration of lithium monolactate can be determined up to 50 mM.

〔Effect of the invention〕

As described above, according to the method of the present invention, it is possible to measure the dehydrogenase content or the dehydrogenase substrate content in a biological sample with higher sensitivity and better reproducibility than before. It can be used for various clinical tests.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the concentration of added NAD(P)H and the absorbance. FIG. 2 shows the relationship between the activity of added LDH and the absorbance. FIG. 3 shows the relationship between the concentration of added lithium monolactate and the absorbance.

Claims (1)

[Claims] When measuring the content of dehydrogenase and dehydrogenase substrate in a biological sample, 1- By measuring the amount of hydrogen peroxide produced by the action of (3-carboxypropyloxy)-5-ethylphenazine, we can determine the content of dehydrogenase and dehydrogenase substrate in a biological sample. A method for measuring biological samples characterized by the following.