JPH0664055B2 - Method for quantifying SH compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention mainly relates to a method for quantifying SH compounds, which is intended for use in the field of clinical examination.

[Conventional technology]

Conventionally, there are various methods for quantifying SH groups of SH compounds having SH groups. Among them, the radiochemical method and the method by mercaptide formation reaction are complicated in operation, the former is a radioactive substance, and the latter is mercury. Since a compound is used, there are problems in terms of safety and pollution, and at present, the SH-SS exchange reaction is most often used.

The SH group is quantified by the SH-SS exchange reaction. The SH compound reacts with the SS compound, and the presence of the SH group causes the SS compound to cleave an amount of the SS bond corresponding to the amount of the SH group to generate a thiol. Therefore, the SH group is quantified by spectroscopically measuring the absorption of the thiol amount. Such SS compounds are generally 5,5'-dithiobis (2-nitrobenzoic acid).
(Hereinafter, abbreviated as DTNB), 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, 6,6'-dithiodinicotine, etc. are known.

Since these have a large molecular extinction coefficient and high sensitivity, a small amount of S
Quantitative determination of H group is possible. Among them, DTNB is also called Ehleman's reagent, which itself has an absorption maximum at 325 nm, but the absorption maximum of thiol formed by reaction with SH group is at 412 nm, which does not overlap with it. It is often used because of its advantage.

[Problems to be solved by the invention]

However, when the SH group quantification method by the SH-SS exchange reaction is used for clinical examination, there are the following problems. That is, blood, particularly serum, which is a sample in a clinical test, contains a mixture of compounds having various SH groups including proteins, so-called endogenous SH compounds, which give a non-specific reaction,
This is a cause of lowering the specificity in the quantification. Therefore, first, an endogenous SH compound is reacted with an SS compound to remove the non-specific reaction, and then a specific reaction by the SH compound originally intended for quantification is caused to cause SH.
It is necessary to quantify the groups. However, in general, endogenous S
The H compound often lacks the reactivity with the SS compound, and in reality, it takes a long time for the reaction. Therefore, conventionally, the reaction time of the whole quantification is such an endogenous SH.
It takes about 30 minutes just to avoid the influence of the compound. If the time to remove the non-specific reaction due to this endogenous SH compound is omitted and the desired SH group is immediately measured, such an endogenous SH compound will also be quantified and the specificity will be increased. descend. Therefore, when measuring in a short time, it is impossible to avoid the influence of such an endogenous SH compound, which is a problem.

The purpose of the present invention is to provide SH by conventional SH-SS exchange reaction.
It is to improve the method for quantifying groups so that the measurement can be performed in a short time without being affected by the endogenous SH compound in the sample.

[Means for solving problems]

The present inventors have conducted extensive studies to solve such problems and achieve the above-mentioned object, and as a result, in the determination of SH groups by SH-SS exchange reaction, in the presence of a surfactant,
It was found that the reaction between the endogenous SH compound and the SS compound was remarkably promoted, and the reaction was generally completed in about 1 minute, and as a result of further research, the present invention was completed.

That is, the present invention provides a method for quantifying SH compounds by quantifying SH groups by an SH-SS exchange reaction, in which a substance present in a sample which causes a non-specific reaction is reacted with an SS compound in the presence of a surfactant. The present invention relates to a method for quantifying an SH compound, which comprises a step of removing a nonspecific reaction.

As the surfactant used in the present invention, all those satisfying the object of the present invention can be used, and among them, it can be selected from the group of nonionic surfactants, anionic surfactants and zwitterionic surfactants. . Examples of such surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyoxyethylene alkyl phenyl ethers,
Nonionic surfactants such as polyoxyethylene alkylamine, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, secondary linear alcohol ethoxylate, nonylphenol ethoxylate, polyoxyalkyl sulfate ester salt, alkylbenzene sulfonic acid There are anionic surfactants such as salts, alkyl phosphate ester salts, alkyl naphthalene sulfonate salts, dialkyl sulfosuccinic acid ester salts, alkyl sulfate ester salts, and fatty acid salts. Examples of zwitterionic surfactants include alkyl betaine. However, the present invention is not limited to these examples. The surfactant used in the present invention is usually one type, but two or more types can be used in combination. Further, the concentration of the surfactant used in the present invention is not generally limited depending on the reaction conditions, but is usually 0.
001 to 40 w / v%, preferably 0.002 to 20 w / v% can be used.

In the method of the present invention, in the determination of SH group by SH-SS exchange reaction, in order to prevent a nonspecific reaction by an endogenous SH compound, first, an SS compound and an endogenous SH compound are reacted to remove the nonspecific reaction. At that time, the reaction is carried out in the presence of a surfactant. Thus, although the reaction is slightly different depending on the sample and the added surfactant, the reaction can be terminated almost instantly when the reaction is short.

After removing the non-specific reaction in the above reaction, the original SH group is quantified. For example, an example will be given in which the SH compound produced by the decomposition of the substrate by the in-vivo enzyme is quantified to quantify the in-vivo enzyme. First, when a substrate is put into the sample from which the non-specific reaction has been removed by the above-mentioned reaction, the substrate is decomposed by the enzyme to produce an SH compound.
The H compound causes an SH-SS exchange reaction with the SS compound, and the SS bond of the SS compound is cleaved. By quantifying the thiol compound thus produced, the SH compound produced by the decomposition of the substrate and thus the enzyme in the living body can be quantified. Thus, accurate and rapid quantification of SH groups is possible without non-specific reactions by endogenous SH compounds.

In the present invention, the SH compound having an SH group to be quantified is not particularly limited, but preferably, in a clinical test, an SH compound liberated from a substrate by the action of an enzyme or the like in a living body is exemplified. Specifically, acetylthiocholine halide, butyrylthiocholine halide,
Examples include 2,3-dimethoxybenzoylthiocholine halide, isobutyrylthiocholine halide, cyclohexanecarbonylthiocholine halide, propionylthiocholine halide, succinylthiocholine halide, 2,3-dimercaptopropan-1-ol, and tributyroate. To be done. Examples of the enzyme in the living body include cholinesterase, lipase, esterase and the like. Therefore, as the sample in the present invention,
Body fluids, especially blood, especially serum and plasma, and urine are exemplified as typical ones.

Examples of the SS compound used in the present invention include those which cause an SH-SS exchange reaction with the above-mentioned SH compound, such as DTNB, 2,2′-dithiodipyridine,
Examples include 4,4'-dithiodipyridine and 6,6'-dithiodinicotine.

[Operation and effect of invention]

According to the present invention, the reaction between the endogenous SH compound and the SS compound can be completed in a very short time, so that the reaction with the SH compound for the purpose of quantification can be started in a short time. For example, when the SH compound released from the substrate by the action of the enzyme cholinesterase in the sample is quantified and the activity of the enzyme is measured, conventionally, as described above, the reaction with the endogenous SH compound in the sample takes a long time. Therefore, there is a problem that it cannot be applied to an automatic analyzer, or if it is forcibly applied, the specificity becomes low. Since the reaction with the endogenous SH compound can be terminated in a very short time in the present invention, it became possible to measure cholinesterase with an automatic analyzer without lowering its specificity.

In particular, in recent years, high-speed automatic analyzers have become widespread in clinical tests, and more rapid methods are required. Therefore, the present invention is useful as a method widely applicable to clinical tests.

〔Example〕

Examples of the present invention are shown below, but the present invention is not limited to these examples.

Example 1 100 mM Tris buffer containing 15 mg / d of DTNB (pH
8.0) each of which is a polyoxyethylene alkylphenol ether-based product as a surfactant: HS-240
[Nippon Yushi Co., Ltd. product], polyoxyethylene alkylphenyl ether type trade name: Triton X-100 [Katayama Chemical Co., Ltd. product], secondary linear alcohol ethoxylate type trade name: ADECATOL (registered trademark) ) SO-160 [Asahi Denka Kogyo Co., Ltd. product], alkyl sulphate salt type sodium lauryl sulphate, alkyl betaine type trade name:
Amphitol (registered trademark) 24B [Kao Atlas Co., Ltd.]
Product] is added at 1 g / d. 2.5 ml of this was taken, respectively, and 3 cases of serum were prepared as samples, and 0.1 ml of each was added and reacted. The concentration of each surfactant at this time is 0.96 (w / v)%. Reaction temperature 37
The absorbance at a wavelength of 412 nm was checked at ℃, and the reaction time from the start to the end of the reaction was examined. The results are shown in Table 1, and it was found that the reaction was completed within 1 minute in each case. When the surfactant was not added, the reaction continued for about 20 minutes.

Example 2 With respect to the surfactant HS-240 used in Example 1, the addition concentration was changed and the same operation as in Example 1 was carried out to examine the reaction time. The results are shown in Table 2, and it was found that the reaction time was shortened from the addition concentration of 0.05 w / v%, and particularly when the concentration exceeded 1.0 w / v%, the reaction was completed in a very short time.

Example 3 100 mM Tris buffer containing 12 mg / d of DTNB (pH
8.0 g of surfactant HS-240 is added at 1 g / d. (As a control, prepare one without the addition of a surfactant and perform the same procedure below.) Take 2.4 ml of this and add 0.1 ml of each of 10 serum samples as a sample to it.
Heat for 5 minutes. At this time, the concentration of the surfactant HS-240 is 0.8 (w / v)%. Further, 0.5 ml of an aqueous solution containing 0.25 g / d of 2,3-dimethoxybenzoylthiocholine iodide was added to measure an increase in absorbance at a wavelength of 412 nm, and then converted into a cholinesterase activity value from a calibration curve previously obtained for each. .
The results are shown in Table 3, showing that the values were all higher when the surfactant was not added, suggesting that the sample was affected by the endogenous SH compound in the sample.

The numerical value shows the cholinesterase activity value (IU /).

Example 4 Of the sera used in Example 3, the amount of the endogenous SH compound in each of the three samples (Nos. 1 to 3) was measured and converted into a cholinesterase activity value (sample blank). That is, 100 containing 12 mg / d of DTNB
Take 2.4 ml of mM Tris buffer (pH 8.0), add 0.1 ml of serum (No. 1-3) as a sample, and heat at 37 ° C. for 30 minutes. Further, 0.5 ml of purified water was added and the increase in absorbance at a wavelength of 412 nm was measured, and then converted into a cholinesterase activity value from a calibration curve previously obtained for each. The results are shown in Table 4, and the results obtained in Example 3
When this sample blank value was subtracted from the value when no surfactant was added, it was found that it was almost the same as the value when the surfactant was added. From this, it was confirmed that the method of the present invention is not affected by the endogenous SH compound in the sample when measuring the activity value of cholinesterase.

The numerical value shows the amount equivalent to the cholinesterase activity value (IU /).

Claims (1)

[Claims] 1. A method for quantifying an SH compound for quantifying an SH group by an SH-SS exchange reaction, which comprises a substance which causes a non-specific reaction present in a sample in the presence of a surfactant and SS.
A method for quantifying an SH compound, comprising the step of reacting with a compound to remove a non-specific reaction.