WO2018056431A1 - Method for measuring l-kynurenine and measurement kit - Google Patents

Abstract

The present invention provides a measurement method and a measurement kit capable of measuring L-kynurenine conveniently, accurately, and with high sensitivity. A method and a measurement kit that measure the L-kynurenine content in a sample, said method and said measurement kit comprising: (A) a step for causing an enzyme reaction between kynurenine 3-monooxygenase and a sample containing L-kynurenine in the presence of an electron donor selected from the group consisting of NADH, NADPH, and derivatives thereof to acquire an enzyme reaction liquid including residual electron donor and produced 3-hydroxy kynurenine; (B) a step for reacting (1) a divalent copper compound and a metal indicator specific to monovalent copper ions or (2) a redox coloring reagent and a charge carrier with the enzyme reaction liquid obtained in step (A); and (C) a step for determining the L-kynurenine content in the sample on the basis of a value obtained by subtracting, from the concentration of the coloring substance obtained in step (B), the concentration of the coloring substance in a sample specimen that does not contain L-kynurenine or a sample specimen to which kynurenine 3-monooxygenase has not been added.

Description

Method and kit for measuring L-kynurenine

The present invention relates to a method and kit for measuring L-kynurenine using an enzyme.

L-kynurenine is known to exist in vivo as a metabolite of L-tryptophan, and various measurement significances have been studied in the medical field. L-kynurenine is generally measured by liquid chromatography, but this measurement is complicated and lacks versatility. As other measurement methods, an antibody method, a colorimetric method using an Ehrlich reagent and an enzyme method are known, but the antibody method is complicated and the colorimetric method has low specificity. As an enzymatic method, a measurement method for detecting fluorescence using kynureninease or kynurenine aminotransferase is known (Non-patent Documents 1 and 2), but biochemical automatic analysis generally used in clinical laboratory settings. There is a problem that the device cannot be used. In addition, the enzymatic method using kynureninase is a technique in which a liquid obtained by extracting an enzyme reaction solution with ethyl acetate is measured by a fluorescence method, and there is a problem that it is very complicated.

On the other hand, Pseudomonas-derived, human-derived, rat-derived, mosquito or yeast-derived enzymes are known as kynurenine 3-monooxygenase using L-kynurenine as a substrate (Non-patent Documents 3 to 7). However, there is no report as to whether kynurenine 3-monooxygenase can be used for the measurement of L-kynurenine.

An object of the present invention is to provide a measurement method and a measurement kit that can measure L-kynurenine simply and accurately by using an enzyme.

Accordingly, the present inventors have examined a method for measuring kynurenine using an enzyme, and as a result, kynurenine 3-mono in the presence of an electron donor selected from the group consisting of L-kynurenine and NADH, NADPH and derivatives thereof. By reacting oxygenase and measuring the product by adding a metal indicator specific for a divalent copper compound and a monovalent copper ion, or a redox coloring reagent and a charge carrier, it is simple, accurate and sensitive. In addition, the inventors have found a method capable of quantifying L-kynurenine and have reached the present invention.

That is, the present invention provides the following [1] to [19].
[1] (A) A sample containing L-kynurenine is subjected to an enzyme reaction with kynurenine 3-monooxygenase in the presence of an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof, and the remaining electrons In the step (B) of obtaining the enzyme reaction solution containing the donor and the produced 3-hydroxykynurenine, the enzyme reaction solution in step (A),
(1) a step of reacting a divalent copper compound and a metal indicator specific for a monovalent copper ion, or (2) a redox coloring reagent and a charge carrier,
(C) Based on the value obtained by subtracting the concentration of the chromogenic substance in the sample not containing L-kynurenine or the sample not containing kynurenine 3-monooxygenase from the concentration of the chromogenic substance obtained in step (B). A method for measuring the amount of L-kynurenine in a sample, comprising the step of determining the amount of kynurenine.
[2] The measuring method according to [1] above, wherein the concentration of the color developing substance by the reaction is measured by absorbance.
[3] The chromogenic material obtained in (1) of the step (B) contains a monovalent copper ion produced by the reaction of 3-hydroxykynurenine and a divalent copper compound, and a metal that specifically acts on the ion. The measurement method according to [1], which is produced by a reaction with an indicator.
[4] The above item [1] or [3], wherein the metal indicator specific for the monovalent copper ion used in (1) of the step (B) is bathocuproin, neocuproin, bicinchoninic acid, a derivative thereof or a salt thereof. ] The measuring method of description.
[5] The measuring method according to [1] above, wherein an enzyme that acts on the phenolic compound is further added before or simultaneously with the reaction of (2) in the step (B).
[6] The measurement method according to [1] or [5], wherein the enzyme cycling method is performed after or simultaneously with the addition of the enzyme that acts on the phenolic compound according to [5].
[7] The item [1] above, wherein the color developing material obtained in (2) of the step (B) is produced by a reaction between an electron donor and a redox coloring reagent via a charge carrier. The measuring method described.
[8] The measuring method according to [1], [5], [6] or [7] above, wherein the redox coloring reagent is a tetrazolium salt.
[9] The measuring method according to [1], [5], [6] or [7] above, wherein the charge carrier is diaphorase.
[10] Any one of [1] to [9] above, comprising a pretreatment step for reducing L-tryptophan and / or a reducing substance in the sample containing L-kynurenine before the step (A). The measuring method as described in.
[11] The measuring method according to [10] above, wherein the reducing substance is bilirubin, ascorbic acid and / or uric acid.
[12] Before the step (A), at least one selected from the group consisting of tryptophanase, tryptophan oxidase, bilirubin oxidase, ascorbate oxidase, uricase, peroxidase, catalase and hydrogen peroxide contains L-kynurenine The measurement method according to [10] or [11] above, which comprises a step of adding the sample to the sample.
[13] The method described in any one of [1] to [12] above, which includes a step of adding a substance having a chelating action and / or a substance that forms a salt with a metal and precipitates before the step (A). Measuring method.
[14] Substances having a chelating action and / or substances that form salts with metals and precipitate are ethylenediaminetetraacetic acid, N, N-bis (2-hydroxyethyl) glycine, diethylenetriaminepentaacetic acid and trans-1,2-diamino The measurement method according to [13], which is at least one selected from the group consisting of cyclohexane-N, N, N, N-tetraacetic acid.
[15] The measurement method according to any one of [1] to [14], including a step of adding a surfactant before the step (B).
[16] L-kynurenine measurement comprising an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof, kynurenine 3-monooxygenase, a divalent copper compound, and a metal indicator specific for monovalent copper ion For kit.
[17] A kit for measuring L-kynurenine, comprising an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof, kynurenine 3-monooxygenase, a redox coloring reagent, and a charge carrier.
[18] A kit for measuring L-kynurenine, wherein the kit according to [17] above further comprises an enzyme that acts on a phenol compound.
[19] A kit for measuring L-kynurenine, wherein the kit according to [18] above further comprises NADH or NADPH regenerating enzyme and a substrate thereof.

According to the present invention, L-kynurenine can be quantified simply, accurately and with high sensitivity. Since a low concentration of L-kynurenine can be measured, a trace amount of L-kynurenine contained in a living body can be measured. This method can be measured by absorbance, and can be used for bioanalytical automatic analyzers that are currently widely used. Therefore, this method is suitable for measuring a large number of samples, and can be put to practical use in the field of clinical tests.

Using P-KMO, NADH, and copper (II) chloride, the absorbance at 480 nm derived from the chromogenic material produced by reacting with L-Kyn (L-kynurenine) was measured in a sample that did not contain L-Kyn (control). It is the figure which showed the result of having plotted the difference with the light absorbency of 480 nm originating as OD480. After obtaining an enzyme reaction solution by reacting with L-Kyn using P-KMO and NADH, using WST-1 and diaphorase, the absorbance at 440 nm derived from the generated chromogenic material does not contain L-Kyn. It is the figure which showed the result of having plotted the difference with the light absorbency of 440 nm originating in a test substance (control) as OD440. After obtaining an enzyme reaction solution by reacting with L-Kyn using P-KMO and NADPH, using WST-1 and diaphorase, the absorbance at 440 nm derived from the generated chromogenic material does not contain L-Kyn. It is the figure which showed the result of having plotted the difference with the light absorbency of 440 nm originating in a test substance (control) as OD440. Using P-KMO and NADH, react with L-Kyn to obtain an enzyme reaction solution, then act on peroxidase, and then use WST-1 and diaphorase to determine the absorbance at 440 nm derived from the generated color substance. FIG. 6 is a diagram showing the result of plotting the difference from the absorbance at 440 nm derived from a sample not containing L-Kyn (control) as OD440. Using P-KMO and NADH, react with L-Kyn to obtain an enzyme reaction solution, then act on laccase, and then use WST-1 and diaphorase to determine the absorbance at 440 nm derived from the generated color substance. FIG. 6 is a diagram showing the result of plotting the difference from the absorbance at 440 nm derived from a sample not containing L-Kyn (control) as OD440. Using P-KMO and NADH, react with L-Kyn to obtain an enzyme reaction solution, then act on bilirubin oxidase, and then use WST-1 and diaphorase to determine the absorbance at 440 nm derived from the generated color substance. FIG. 5 is a graph showing the result of plotting the difference from the absorbance at 440 nm derived from a specimen (control) not containing L-Kyn as OD440. P-KMO and NADH are used to react with L-Kyn to obtain an enzyme reaction solution, which is then reacted with peroxidase, alcohol dehydrogenase, ethanol, WST-1 and diaphorase to produce 440 nm derived from the generated color substance. It is the figure which showed the result of having calculated the variation | change_quantity per minute about the light-absorbency change, and plotting the value converted into the variation | change_quantity for 4 minutes as OD440. About the absorbance at 480 nm derived from the coloring substance produced by reacting with L-Kyn using P-KMO, NADH and copper (II) chloride after degrading the reducing substance using deproteinized serum, FIG. 7 is a graph showing the result of plotting the difference from the absorbance at 480 nm derived from a specimen not containing −Kyn (control) as OD480. Reacting with L-Kyn using P-KMO and NADH in the presence of serum to obtain an enzyme reaction solution, allowing POD to act, and then using a surfactant, WST-1 and diaphorase 4 is a graph showing the result of plotting the difference between the absorbance at 440 nm derived from the 440 nm derived from the specimen not containing L-Kyn (control) as OD440.

The present invention
(A) A sample containing L-kynurenine is subjected to an enzymatic reaction with kynurenine 3-monooxygenase in the presence of an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof, and the remaining electron donor, And obtaining an enzyme reaction solution containing the produced 3-hydroxykynurenine,
(B) In the enzyme reaction solution of step (A),
(1) a metal indicator specific to a divalent copper compound and a monovalent copper ion, or (2) a step of reacting a redox coloring reagent and a charge carrier, and (C) the color obtained in step (B). Including the step of determining the amount of L-kynurenine in the sample based on a value obtained by subtracting the concentration of the chromogenic substance in the sample not containing L-kynurenine or the sample not containing kynurenine 3-monooxygenase from the concentration of the substance. The present invention relates to a method for measuring the amount of L-kynurenine.

(Process (A))
The present invention includes a step (A) of reacting a sample containing L-kynurenine with an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof and kynurenine 3-monooxygenase.

(Sample containing L-kynurenine)
The sample used in the present invention is not particularly limited as long as it may contain L-kynurenine. For example, biological samples of mammals including humans such as blood (serum, plasma), saliva, urine, cerebrospinal cord Liquid and the like. Among these, blood is preferable, and when blood is used as a sample, it is more preferable to use serum or deproteinized blood.

(Pretreatment process)
The measurement of a biological sample may include a pretreatment step of removing at least one of a reducing substance, a contaminating protein, a metal ion and the like that may interfere with the measurement by a known method. Examples of the reducing substance include bilirubin, ascorbic acid, uric acid and the like. Examples include bilirubin oxidase for reducing bilirubin, ascorbate oxidase for reducing ascorbic acid, and pretreatment using an enzyme such as uricase for reducing uric acid. Furthermore, treatment with hydrogen peroxide or peroxidase can also be performed. Catalase can also be used to reduce the hydrogen peroxide used or produced. In addition, the usage-amount of enzyme, reaction pH, and temperature can be suitably set based on the optimal pH and temperature of each enzyme. The amount of the enzyme used is, for example, 0.1 to 1000 U / mL, preferably 0.5 to 100 U / mL, in the pretreatment solution. The reaction temperature is, for example, 15 to 50 ° C., preferably 20 to 45 ° C., more preferably 25 to 40 ° C. The reaction pH is, for example, 5.0 to 11.0, preferably 5.5 to 10.5, and more preferably 6.0 to 10.0. The reaction time is, for example, 1 to 60 minutes, preferably 1 to 40 minutes.

Contaminating protein can be reduced with a deproteinizing agent such as perchloric acid or trichloroacetic acid. After the treatment, neutralization using potassium carbonate or the like can be used as a sample. It should be noted that reducing substances such as bilirubin and hemoglobin can be reduced by the protein removal treatment.

Metal ions can be reduced, for example, with a metal chelator. The metal chelating agent is not particularly limited, but EDTA (ethylenediaminetetraacetic acid), bicine (N, N-bis (2-hydroxyethyl) glycine), DTPA (diethylenetriaminepentaacetic acid), CyDTA (trans-1,2-diaminocyclohexane- N, N, N, N-tetraacetic acid) and the like are preferably used. In addition, the use density | concentration of a metal chelating agent can be set suitably. For example, in the first aspect of the step (B), it is desirable that the molar concentration is the same or lower than that of the divalent copper compound to be used, more preferably 1/2 or lower. Magnesium ions and the like can be reduced by adding phosphoric acid.
About the liquid which implemented the above-mentioned pre-processing, you may use for a measurement immediately after a pre-processing, and may use it after storage by refrigeration or freezing.

In addition, as a method of reducing the influence of substances that interfere with the measurement, adding a redox agent such as potassium ferricyanide, potassium ferrocyanide, benzoic acid, iodate or nitrite, or adding a surfactant Is mentioned. Examples of usable surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants, such as polyoxyethylene alkyl ethers and polyoxyethylene alkyls. Nonionic surfactants such as phenyl ether, polyoxyethylene sorbitan fatty acid ester, olefin sulfonic acid, polyoxyethylene alkyl phenyl ether sulfate, polyoxyethylene alkyl phenyl ether acetate, sodium lauryl sulfate, sodium cholate, octanoic acid Anionic surfactants such as sodium and sodium dodecylbenzenesulfonate are preferably used. Of these, polyoxyethylene sorbitan monolaurate, polyoxyethylene lauryl ether or polyoxyethylene octyl phenyl ether is more preferred, and the average added mole number of EO (ethylene oxide) is preferably 10 to 30, more preferably 5 to 25. It is. For example, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (23) lauryl ether or polyoxyethylene (10) octylphenyl ether is preferably used. The surfactant is preferably added in an amount of 0.01 to 5.0% (w / v) with respect to the entire measurement system, and an amount of 0.1 to 2.0% (w / v). It is more preferable to add. The addition time is not particularly limited as long as a series of enzyme reactions proceeds, but it is preferably added before the color development reaction.

The liquid subjected to the above pretreatment may be used for measurement immediately after the pretreatment, or may be used after storage in refrigeration or freezing. Moreover, this pre-processing can be implemented at the suitable time before a process (A) and / or a process (B).

(Reduction of the effects of tryptophan)
In order to perform the L-kynurenine measuring method of the present invention more accurately, it is preferable to substantially eliminate the influence of contaminants in the sample. For example, since the L-tryptophan concentration in a biological sample is generally considered to be about 10 to 100 times higher than the L-kynurenine concentration, it is preferable to suppress the influence of L-tryptophan on the measurement of L-kynurenine. An L-kynurenine measurement method using NADH as an electron donor is preferable, and a measurement method using NADH alone is more preferable.

Alternatively, an L-kynurenine measurement method including a step of reducing L-tryptophan in a sample with an enzyme that acts on L-tryptophan is preferable. This step is preferably performed as a pretreatment prior to the reaction with kynurenine 3-monooxygenase.

For the L-tryptophan reduction step, it is preferable to use an enzyme that acts on L-tryptophan. The enzyme that acts on L-tryptophan is not particularly limited as long as it does not substantially act on L-kynurenine, and examples thereof include tryptophan oxidase and tryptophanase. Tryptophan oxidase is preferably used in combination with catalase.

The amount of enzyme used is not particularly limited as long as L-tryptophan can be reduced. In the case of tryptophan oxidase, the concentration in the pretreatment solution is preferably about 0.1 to 100 U / mL, and about 1 to 10 U / mL. It is more preferable. When combining catalase with tryptophan oxidase, the concentration in the pretreatment solution is preferably about 0.1 to 10,000 U / mL, more preferably about 1 to 1,000 U / mL. In the case of tryptophanase, the concentration in the pretreatment solution is preferably about 0.01 to 10 U / mL, and more preferably about 0.1 to 2 U / mL.

The reaction temperature, reaction pH, and reaction time can be appropriately set based on the optimum pH and temperature of each enzyme. The reaction temperature is, for example, 15 to 50 ° C., preferably 20 to 45 ° C., more preferably 25 to 40 ° C. The reaction pH is, for example, 5.0 to 11.0, more preferably 5.5 to 10.5, and more preferably 6.0 to 10.0. The reaction time is, for example, 1 to 60 minutes, preferably 1 to 40 minutes.

(Kynurenin 3-monooxygenase (KMO))
As kynurenine 3-monooxygenase (KMO), for example, a known enzyme derived from a prokaryotic organism such as Pseudomonas or a eukaryotic organism such as human, rat, mosquito or yeast can be used. The enzyme used may be a recombinant enzyme or a synthesized enzyme. This enzyme is preferably a soluble enzyme, but a surfactant may be combined with the insoluble enzyme, or an enzyme in which the insoluble enzyme is solubilized by fusion with a solubilized protein or deletion of a membrane-bound portion may be used. As this enzyme, an enzyme having a known amino acid sequence can be used. For example, an enzyme having the sequence shown in SEQ ID NO: 1, 2, 3, 4 or 5 can be used. Further, it may be a modified enzyme having kynurenine 3-monooxygenase activity by substituting, adding or deleting one or several amino acids in the sequence shown in SEQ ID NO: 1, 2, 3, 4 or 5. Having a sequence having similarity or identity of SEQ ID NO: 1, 2, 3, 4 or 5, for example 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, A protein having kynurenine 3-monooxygenase activity may be used.

(Electron donor)
Step (A) of the present invention is carried out in the presence of an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof.
NADH or a NADPH derivative can be used to improve the stability of NADH or NADPH, increase the molar extinction coefficient, or the like. In the present invention, the type of NADH or NADPH derivative is not particularly limited as long as the reaction with kynurenine 3-monooxygenase proceeds. For example, a known NADH or NADPH derivative disclosed in JP2012-224638A is known. Things can be used.

(Enzymatic reaction)
The amount of enzyme in the enzyme reaction used in the step (A) is not particularly limited as long as L-kynurenine in the sample can be converted to 3-hydroxykynurenine, but is preferably about 0.0001 to 100 U per sample. About 001 to 10 U is more preferable. The concentration of the electron donor in step (A) is not particularly limited as long as L-kynurenine in the sample can be measured, but is preferably 0.01 to 1 mM, more preferably 0.01 to 0.5 mM. Moreover, the reaction temperature, reaction pH, and reaction time in the step (A) can be appropriately set based on the optimum pH and temperature of the enzyme. The reaction temperature is, for example, 15 to 50 ° C., preferably 20 to 45 ° C., more preferably 25 to 40 ° C. The reaction pH is, for example, 5.0 to 11.0, preferably 5.5 to 10.5, and more preferably 6.0 to 10.0. The reaction time is, for example, 1 to 60 minutes, preferably 1 to 30 minutes, more preferably 1 to 10 minutes.

(Process (B))
The present invention provides the product of step (A)
(1) a step (B) of reacting a divalent copper compound and a metal indicator specific to a monovalent copper ion, or (2) a redox coloring reagent and a charge carrier.

(1) In the first embodiment, a divalent copper compound is used, and a plurality of moles of monovalent copper ions per mole of 3-hydroxykynurenine can be generated by an oxidation-reduction reaction with 3-hydroxykynurenine in the reaction solution. it can. Furthermore, a coloring substance can be produced by reaction with a metal indicator specific for monovalent copper ions.

Although it does not specifically limit as said divalent copper compound, A water-soluble thing is preferable, for example, copper (II) chloride, copper sulfate (II), copper nitrate (II), copper acetate (II), copper citrate (II) ), Copper (II) gluconate and the like are used. Of these, copper (II) chloride or copper (II) sulfate is preferably used.

The metal indicator specific to the monovalent copper ion is not particularly limited, but it does not substantially react with other metal ions and specifically reacts with the monovalent copper ion to form a colored complex. Etc. are used. For example, bathocuproin, neocuproin, bicinchoninic acid, derivatives thereof or salts thereof are preferably used. Of these, bathocuproin or a salt thereof is preferable, and disodium bathocuproin disulfonate is more preferable.

The concentration of each reagent in the reaction is preferably 0.01 to 2.0 mM, more preferably 0.02 to 1.0 mM in the case of a divalent copper compound. The metal indicator specific for monovalent copper ions is preferably from 0.01 to 2.0 mM, more preferably from 0.02 to 1.0 mM. The pH during measurement is preferably from 3.0 to 11.0, more preferably from 4.0 to 10.0. The reaction temperature is preferably 15 to 50 ° C, more preferably 20 to 45 ° C, still more preferably 25 to 40 ° C.

The metal indicator is preferably added to the sample before adding the divalent copper compound so that the production of monovalent copper ions can be discriminated. The produced monovalent copper ions can be accurately quantified by the change in the monovalent copper ion concentration before and after adding the divalent copper compound.

It is known that monovalent copper ions obtained by the action of 3-hydroxykynurenine and divalent copper ions can be detected by bathocuproine or bicinchoninic acid (Biochemistry, 39, 7266-7275, 2000). However, NADH is also known to reduce divalent copper ions (Analyst, 134 (8), 1614-1617, 2009), and NADH or NADPH used in step (A) is 3-hydroxykynurenine. Therefore, it is difficult to easily assume that measurement based on the first aspect can be performed in a solution containing NADH or NADPH.

(Redox coloring reagent)
(2) Among the redox coloring reagents used in the second embodiment, the reducing coloring reagent is an electron selected from the group consisting of NADH, NADPH and derivatives thereof in the reaction solution in the presence of a charge carrier. By reacting with a donor, a reduced coloring material is generated from an oxidized colorless material.

The type of reducing coloring reagent is not particularly limited as long as L-kynurenine in a sample can be measured. For example, 2- (4-indophenol-3- (4-nitriphenyl) -5-phenyl2H-tatrazolium Chloride (INT), 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt (WST-1), 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4, 4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride] (NTB), 2-benzothiazolyl-3- (4-carboxy- Tetrazolium salts such as -methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H-tetrazolium (WST-4), resazurin sodium, etc. can be used, for example, WST-1 In this case, the concentration in the reaction solution is preferably 0.02 mg / mL or more, more preferably 0.1 mg / mL or more, when a coloring reagent having low solubility in water such as MTT is used or when a high concentration is used. When a formazan dye is generated, a surfactant such as sodium dodecyl sulfate (SDS) or dimethyl sulfoxide (DMSO) or an organic solvent may be used for solubilization.

On the other hand, among the redox coloring reagents, an oxidizing coloring reagent reacts with an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof in the reaction solution in the presence of a charge carrier. A reduced colorless substance is produced from an oxidized color developing substance. The type of this reagent is not particularly limited as long as L-kynurenine in a sample can be measured, and examples thereof include dichloroindophenol (DCIP).

(Charge carrier)
The charge carrier is not particularly limited as long as L-kynurenine in a sample can be measured, but 3-hydroxykynurenine can be reversed without catalyzing the production of a coloring substance by 3-hydroxykynurenine in a reaction solution and a redox coloring reagent. Therefore, it is desirable to inhibit the generation of a color developing substance by the reaction between the electron donor and the redox coloring reagent. Specifically, the use of diaphorase is preferable. When diaphorase is used, the amount used is preferably 0.01 U / mL or more, more preferably 0.1 U / mL or more, in the reaction solution. The type of diaphorase is not limited as long as it reacts with an electron donor. For example, EC number 1.6.5.2, EC number 1.6.99.1, EC number 1.6.99. Those classified into 3 can be used.

The reaction temperature, reaction pH, and reaction time in step (B) can be appropriately set based on the optimum pH and temperature of the enzyme and the like in the first and second aspects. The reaction temperature is, for example, 15 to 50 ° C., preferably 20 to 45 ° C., more preferably 25 to 40 ° C. In addition, you may make it the same temperature in a process (A). The reaction pH is not particularly limited, but for example, pH 3.0 to 12.0 is preferable. In the first embodiment, pH 4.0 to 11.0 is more preferable. In the second embodiment, for example, when diaphorase is used as the charge carrier, pH 6.0 to 10.0 is preferable, and pH 7.0 to 9.0 is more preferable. The reaction pH in step (A) may be the same. The reaction time is, for example, 1 to 60 minutes, preferably 1 to 40 minutes, more preferably 1 to 10 minutes.

In addition, the type of the buffer solution in the first and second aspects is not particularly limited as long as it has a buffering ability at a target pH. For example, bicine, tris, citric acid In the case of a substance that forms a complex with a copper ion, etc., it is necessary to use it with caution because it may inhibit color development in the first embodiment.

(Process (C))
The present invention is based on a value obtained by subtracting the concentration of the coloring substance in the sample not containing L-kynurenine or the sample not added with kynurenine 3-monooxygenase from the concentration of the coloring substance obtained in step (B). A step (C) of determining an amount of L-kynurenine; Samples that do not contain L-kynurenine or samples without kynurenine 3-monooxygenase are called controls, and exclude the influence on assay values when L-kynurenine or kynurenine 3-monooxygenase is not added. is there.

The color-developing substance to be measured in the first aspect is a substance produced by a reaction between a monovalent copper ion and a metal indicator specific to the monovalent copper ion. By measuring the concentration of this coloring substance, the concentration of monovalent copper ions can be measured, and as a result, the concentration of L-kynurenine in the sample can be measured with high sensitivity.

The color-developing substance to be measured in the second aspect is obtained from an oxidation-reduction color-developing reagent, and is an electron selected from the group consisting of NADH, NADPH and their derivatives in the reaction solution in the presence of a charge carrier. A donor is obtained by reacting with a redox coloring reagent.

That is, the L-kynurenine concentration can be measured by the following procedure.
a) Concentration of the coloring substance obtained by adding the electron donor and kynurenine 3-monooxygenase to the reaction solution not containing L-kynurenine, and further donating a charge carrier and a redox coloring reagent. Measure. Alternatively, after adding an electron donor to a reaction solution containing L-kynurenine, no kynurenine 3-monooxygenase is added, and the concentration of the coloring substance obtained by donating the charge carrier and the redox coloring reagent is adjusted. taking measurement.
b) After adding and reacting an electron donor and kynurenine 3-monooxygenase to a reaction solution containing L-kynurenine, the concentration of the chromogenic material obtained by further donating charge carriers and a redox coloring reagent is measured. To do.
c) The L-kynurenine concentration is calculated from the value obtained by subtracting the concentration of b) from the concentration of a).

By the way, when a reducing coloring reagent is used, the concentration of the coloring substance obtained by the reaction of the electron donor with the reducing coloring reagent in the presence of a charge carrier increases depending on the concentration of the electron donor. In addition, when the reaction with L-kynurenine 3-monooxygenase is carried out, the number of electron donors in the reaction solution is the same as the number of moles of L-kynurenine, so in the enzyme reaction solution after the reaction with kynurenine 3-monooxygenase, The chromogenic substance decreases depending on the concentration of L-kynurenine, but the absolute value of the slope is considered to be equal to the absolute value of the slope obtained between the electron donor and the chromogenic substance.

However, as a result of examination by the present inventors this time, it was confirmed that the absolute value of the slope increased at a level exceeding the error range, as shown in the examples. As a result of investigating this cause, 3-hydroxykynurenine produced by the reaction of L-kynurenine with kynurenine 3-monooxygenase causes the reaction between the electron donor and the reducing coloring reagent in the presence of diaphorase, which is a kind of charge carrier. It was found that the above reaction was inhibited depending on the concentration of 3-hydroxykynurenine, and the production of the coloring substance was inhibited.

In other words, the decrease in the concentration of the chromogenic substance accompanying the increase in the L-kynurenine concentration is not only due to the influence of the decrease in the electron donor after the reaction by the kynurenine 3-monooxygenase, but also increases as the L-kynurenine concentration increases. Since the influence of the amount of hydroxykynurenine was also taken into account, it was found that an increase in absolute value of the slope more than expected was obtained. As a result, by measuring the concentration of the coloring material, the concentration of L-kynurenine in the sample can be measured with high sensitivity.

In addition, in the measurement in the second embodiment, before or simultaneously with the provision of the charge carrier and the redox coloring reagent, a substance that acts on 3-hydroxykynurenine is added, whereby the electron donor and the redox coloring by the charge carrier are added. It has been found that the effect of inhibiting the reaction with the reagent is further increased, and as a result, a more sensitive measurement can be performed. The specific substance is not particularly limited as long as it does not inhibit the reaction in a system that does not contain 3-hydroxykynurenine, but is not particularly limited as long as it is a substance that inhibits the reaction in a system that contains 3-hydroxykynurenine. It is an oxidizing agent or a polymerization agent that acts, and more preferably an enzyme that acts on a phenolic compound. Specifically, as the oxidoreductase, peroxidase (EC number 1.11.1.X), laccase (EC number 1.10.3.2), bilirubin oxidase (EC number 1.3.3.5) Tyrosinase (EC number 1.10.3.1 or 1.14.18.1), 3-hydroxyanthranilate oxidase (EC number 1.10.3.5), ferrooxidase (EC number 1.16.3) .1), phenol-2-monooxygenase (EC number 1.14.13.7 or 1.14.14.20), aminophenol oxidase (EC number 1.10.3.4), glyxazone synthase ( EC number 1.10.3.15), 2-aminophenol 1,6-dioxygenase (EC number 1.13.11.74), 2-amino-5-chlorophenol 1 6-dioxygenase (EC number 1.13.1.11.76) and the like are exemplified, and examples of the transferase include N-hydroxyallylamine O-acetyltransferase (EC number 2.3.1.118), glucuronosyltransferase ( EC number 2.4.1.17), phenyl β-glucosyltransferase (EC number 2.4.1.35), allylamine N-acetyltransferase (EC number 2.3.1.5), allyl sulfate sulfotransferase ( EC number 2.8.2.2.2) and the like are exemplified. Among them, peroxidase, laccase or bilirubin oxidase is preferable. The concentration of peroxidase, laccase or bilirubin oxidase hardly inhibits the reaction in a system not containing 3-hydroxykynurenine, but the range is not limited as long as it is a concentration that inhibits the reaction in a system containing 3-hydroxykynurenine. However, it is preferably 0.001 to 1,000 U / mL, more preferably 0.01 to 100 U / mL. The reaction pH, temperature, and time can be appropriately set in accordance with the aforementioned color development reaction and conditions suitable for each enzyme. The timing of addition of the enzyme acting on the phenolic compound may be any time before, during or after the reaction with kynurenine 3-monooxygenase, but preferably after the reaction with kynurenine 3-monooxygenase. The reaction temperature is, for example, 15 to 50 ° C., preferably 20 to 45 ° C., more preferably 25 to 40 ° C. The reaction pH is, for example, 5.0 to 11.0, preferably 5.5 to 10.5, and more preferably 6.0 to 10.0. The reaction time is, for example, 1 to 60 minutes, preferably 1 to 45 minutes, more preferably 1 to 10 minutes.

When an enzyme that acts on a phenolic compound is used, an enzyme that acts on a phenolic compound can be combined with an enzyme cycling method after or simultaneously with the addition of an enzyme that acts on the phenolic compound, thereby enabling further highly sensitive measurement. This enzyme cycling method specifically refers to NADH or NADPH by donating an appropriate NADH or NADPH regenerating enzyme and its substrate to NAD ⁺ or NADP ⁺ obtained by a reaction using a charge carrier. In this method, the reaction using charge carriers is performed again.

Examples of NADH or NADPH regenerating enzyme include alcohol dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and phosphoglucocone. Examples include acid dehydrogenase and glycerol dehydrogenase.

As a combination of NADH or NADPH regenerating enzyme and substrate used in the above reaction, the type of enzyme or its substrate is not limited as long as the purpose is satisfied. For example, alcohol dehydrogenase and ethanol, glucose dehydrogenase and glucose, and glutamate dehydrogenation. Enzyme and glutamate, formate dehydrogenase and formate, malate dehydrogenase and malate, glucose 6-phosphate dehydrogenase and glucose 6-phosphate, phosphogluconate dehydrogenase and phosphogluconate, glycerol dehydrogenase and glycerol Etc. are known.

When alcohol dehydrogenase is used as NADH regenerating enzyme, the amount of enzyme is not particularly limited as long as L-kynurenine can be measured in the sample, but the concentration of alcohol dehydrogenase in the reaction solution is about 0.5 to 30 U / mL. It is preferably about 3 to 15 U / mL. Moreover, reaction temperature, reaction pH, and reaction time can be suitably set based on the optimum pH and temperature of each enzyme.

(Measuring means)
The concentration measuring means for the color developing substance is not particularly limited, and for example, measurement of absorbance change by a spectrophotometer can be exemplified. In the measurement with a spectrophotometer, it is preferable to select appropriately from the rate method or the endpoint method. Examples of spectrophotometers used for measurement include commercial products sold by various companies such as JASCO, Hitachi High-Technologies, Shimadzu Corporation.

In the first embodiment, the measurement wavelength in the absorbance measurement varies depending on each metal indicator. For example, when bathocuproine is used as a metal indicator specific for monovalent copper ions, absorbance at a wavelength of around 480 nm, neocuproine at around 450 nm, and bicinchoninic acid at around 562 nm can be used. It may be changed.

In the second embodiment, the measurement wavelength for quantifying the generated color substance is not particularly limited as long as each color substance has absorption, but a wavelength indicating the absorption maximum of each color substance is desirable. For example, when WST-1 is used, it is preferably 400 to 480 nm, and more preferably measured at a measurement wavelength around 440 nm. However, when measuring at a wavelength that exhibits an absorption maximum with a high NADH or NADPH concentration, the absorbance may be too high and the accuracy may be lost. It is also possible to measure by shifting to either.

(Optional component)
In the L-kynurenine measurement method of the present invention, other optional components known to those skilled in the art may be appropriately contained to enhance the stability of the reagent components such as the enzyme. The optional component is not particularly limited as long as it does not affect the measurement. For example, flavin adenine dinucleotide (FAD), bovine serum albumin (BSA), ovalbumin, saccharide, sugar alcohols, carboxyl group-containing compound, antioxidant Examples include agents, surfactants, amino acids having no activity with enzymes, and the like. In particular, kynurenine 3-monooxygenase derived from Pseudomonas tends to increase in stability and activity when the salt concentration is high. For example, the final concentration of the buffer solution or sodium chloride of the reagent composition is 10 mM or more. Preferably, it is more preferably 100 mM or more.

(kit)
The measurement of the present invention can be performed using a kit for measuring L-kynurenine containing kynurenine 3-monooxygenase and the electron donor. The kynurenine 3-monooxygenase included in the kit may be an enzyme that converts L-kynurenine into 3-hydroxykynurenine. Specifically, any enzyme that catalyzes the reaction of producing 3-hydroxykynurenine, the electron acceptor and water in the presence of the electron donor, H ⁺ and oxygen using L-kynurenine as a substrate may be used.

The kit of the present invention contains at least one selected from the group consisting of tryptophanase, tryptophan oxidase, bilirubin oxidase, ascorbate oxidase, uricase, peroxidase, catalase and hydrogen peroxide for pretreatment. May be.

In the first embodiment, a divalent copper compound is used, and a metal indicator that specifically reacts with monovalent copper ions is included. In the second embodiment, the redox coloring reagent and the charge carrier are used. That is, in the first aspect, an electron donor selected from the group consisting of NADPH, NADP and derivatives thereof, kynurenine 3-monooxygenase, a divalent copper compound, and a metal indicator specific for a monovalent copper ion are included. L-kynurenine measurement kit is used. In the second embodiment, an L-kynurenine measurement kit including the electron donor, kynurenine 3-monooxygenase, a redox coloring reagent and a charge carrier is used. The kit in the second aspect may further contain an enzyme that acts on a phenolic compound, and may further contain NADH or NADPH regenerating enzyme and its substrate.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

[Example 1]
(Measurement with copper (II) chloride / bathocuproine system)
L-kynurenine at each concentration was quantified by measuring the resulting monovalent copper ion using kynurenine 3-monooxygenase (P-KMO) and Pseudomonas (Pseudomonas) -derived copper chloride (II).
2.64 mL of the reagent mixture was placed in a quartz cell having an optical path length of 1 cm and incubated at 25 ° C. for 5 minutes. Then, 0.06 mL of P-KMO was added and mixed, and the enzyme reaction was started at 25 ° C. The composition of the enzyme reaction solution was 100 mM HEPES buffer (pH 7.5), 0.5, 1 or 2 μM L-kynurenine, 0.05 mM NADH, and 0.01 U / mL P-KMO (Note: reaction) The concentration of the liquid is indicated by the concentration after addition of copper (II) chloride). As a control, ultrapure water was used instead of the substrate.

P-KMO is a protein having the amino acid sequence described in SEQ ID NO: 1, and is published by UniProt as Q84HF5 (http://www.uniprot.org/uniprot/Q84HF5). An enzyme purified by culturing a recombinant microorganism into which a gene encoding the amino acid sequence shown in SEQ ID NO: 1 has been inserted and recovering P-KMO from the culture.

30 minutes after the start of the enzymatic reaction, 0.15 mL of 1 mM of bathocproin disulphonate, a metal indicator, was added to each L-kynurenine concentration and control cell, mixed, and the spectrophotometer (V-660, Japan) Absorbance at 480 nm was measured with a spectrophotometer. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 0.15 mL of 1 mM copper (II) chloride solution was added to each L-kynurenine concentration and control cell, mixed, and the absorbance at 480 nm was measured over time at 25 ° C. As described above, when a copper (II) chloride solution is added, hydroxyquinurenine converted from L-kynurenine by P-KMO reduces plural moles of divalent copper ions. Ions and bathocuproine produce a complex with an absorption maximum at 480 nm.

The blank value was subtracted from each L-kynurenine concentration and the measured value 4 minutes after the control (subtraction value A and subtraction value B). Subsequently, a value obtained by subtracting the control subtraction value B from the subtraction value A of each L-kynurenine concentration was plotted in the figure as OD480. This measurement was performed three times on different days. The measured values and average values are shown in FIG.

FIG. 1 shows that the value of OD480 increases with increasing L-kynurenine concentration, indicating that L-kynurenine can be quantified by P-KMO. In all measurements, reproducible data was obtained even when the L-kynurenine concentration was as low as 2 μM or less, and it was shown that L-kynurenine can be quantified with high sensitivity by P-KMO. It was done.

From the above, it was found that L-kynurenine can be quantified with high sensitivity by using an electron donor such as kynurenine 3-monooxygenase, NADH, disodium bathocuproin disulfonate and copper (II) chloride.

[Example 2]
(Measurement with diaphorase / WST-1 system (using NADH))
The L-kynurenine solution at each concentration was used as a sample, and the amount of NADH decreased by the P-KMO reaction was measured using diaphorase as a charge carrier and WST-1 as a reducing coloring reagent. 1.8 mL of the enzyme reaction solution was added to and mixed with a quartz cell having an optical path length of 1 cm, and the enzyme reaction was carried out at 25 ° C. for 15 minutes.

The composition of the enzyme reaction solution was 50 mM potassium phosphate buffer (pH 7.5), 1, 2, 3 or 4 μM L-kynurenine, 0.03 mM NADH, and 0.2 U / mL P-KMO. As a control, ultrapure water was used instead of the substrate (Note: The concentration of the reaction solution is shown by the concentration after addition of diaphorase).

After completion of the reaction, 10 mg / mL WST-1 (2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt ( 100 μL of Dojindo Laboratories) was added to each cell and mixed, and the absorbance at 440 nm was measured with a spectrophotometer (V-660, manufactured by JASCO Corporation). This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 100 U / mL of diaphorase (manufactured by Oriental Yeast) was added to and mixed with 100 μL of each cell, the enzyme reaction was carried out at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer.

The blank value was subtracted from each L-kynurenine concentration and the measured value 10 minutes after the control (subtraction value C and subtraction value D). FIG. 2 shows the result of plotting the value obtained by subtracting the subtraction value C of each L-kynurenine concentration from the subtraction value D of the control as OD440. FIG. 2 shows that the OD440 value increases with increasing L-kynurenine concentration, indicating that L-kynurenine can be quantified.

Separately from this test, 100 U was added to 1.8 mL of a solution in which NADH concentration was 0.027, 0.028, 0.029 or 0.030 mM in 50 mM potassium phosphate buffer (pH 7.5). / ML diaphorase (manufactured by Oriental Yeast), 10 mg / mL WST-1 was added in an amount of 100 μL, mixed, and subjected to an enzyme reaction at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer. As a result, the obtained slope was an increase of 0.029 [abs] per 1 μM NADH. Since the slope obtained in the previous test was an increase of 0.045 [abs] per 1 μM of L-kynurenine, it was confirmed that the sensitivity was increased by a factor other than a simple change in the amount of NADH.

In addition, 10 mg / mL for 180 μL of each reagent added in 50 mM potassium phosphate buffer (pH 7.5) so that the final NADH concentration is 0.03 mM and 3-hydroxykynurenine concentration is 0 to 5 μM. WST-1, 100 U / mL diaphorase (made by Oriental Yeast) 10 μL each, mixed, and subjected to an enzyme reaction at 25 ° C. for 10 minutes, and absorbance at 440 nm was measured by a plate reader (SpectraMax Plus 384, manufactured by Molecular Devices). As a result of measurement, it was confirmed that the value at 440 nm decreased as the 3-hydroxykynurenine concentration increased.

From the above, it was found that L-kynurenine can be quantified with high sensitivity by using a reducing coloring reagent such as kynurenine 3-monooxygenase, NADH, diaphorase and WST-1.

[Example 3]
(Measurement with diaphorase / WST-1 system (using NADPH))
The L-kynurenine solution at each concentration was used as a sample, and the amount of NADPH decreased by the P-KMO reaction was measured using diaphorase as a charge carrier and WST-1 as a reducing coloring reagent. 1.8 mL of the enzyme reaction solution was added to and mixed with a quartz cell having an optical path length of 1 cm, and the enzyme reaction was carried out at 25 ° C. for 15 minutes.

The composition of the enzyme reaction solution is 50 mM potassium phosphate buffer (pH 7.5), 0.5, 1 or 2 μM L-kynurenine, 0.04 mM EDTA, 0.03 mM NADPH, and 0.2 U / mL P-KMO. (Note: The concentration of the reaction solution is indicated by the concentration after addition of diaphorase). As a control, ultrapure water was used instead of the substrate.

After completion of the reaction, 10 mg / mL WST-1 was added to and mixed with 100 μL of each cell, and the absorbance at 440 nm was measured with a spectrophotometer. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 3 U / mL of diaphorase (manufactured by Asahi Kasei Pharma) was added to and mixed with 100 μL of each cell, the enzyme reaction was carried out at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer.

The blank value was subtracted from each L-kynurenine concentration and the measured value 10 minutes after the control (subtraction value C and subtraction value D). Next, FIG. 3 shows the result of plotting the value obtained by subtracting the subtracted value C of each L-kynurenine concentration from the subtracted value D of the control as OD440. FIG. 3 shows that the value of OD440 increases with increasing L-kynurenine concentration, indicating that L-kynurenine can be quantified. The slope was an increase of 0.058 [abs] per μM of L-kynurenine, and even when NADPH was used, the L-kynurenine concentration could be quantified with high sensitivity.

[Example 4]
(Measurement with diaphorase / WST-1 / POD system)
Using L-kynurenine solution of each concentration as a sample, after reaction with P-KMO, peroxidase (POD) was mixed, and the reduced NADH amount was reduced using diaphorase as a charge carrier and WST-1 as a reducing coloring reagent. It was measured. 1.85 mL of the enzyme reaction solution was added to and mixed with a quartz cell having an optical path length of 1 cm, and the enzyme reaction was carried out at 25 ° C. for 30 minutes.

The composition of the enzyme reaction solution was 100 mM Tris-HCl buffer (pH 8.5), 0.25, 0.5, 1 or 2 μM L-kynurenine, 0.1 mM EDTA, 0.03 mM NADH, and 0.2 U / mL P. -KMO (Note: The concentration of the reaction solution is indicated by the concentration after addition of diaphorase). As a control, ultrapure water was used instead of the substrate.

After completion of the reaction, 40 U / mL POD (manufactured by Wako Pure Chemical Industries) was added to a 50 μL cell, and an enzyme reaction was carried out at 25 ° C. for 3 minutes. After completion of the reaction, 50 mg of 20 mg / mL WST-1 was added to each cell and mixed, and the absorbance at 440 nm was measured with a spectrophotometer. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 20 U / mL of diaphorase (manufactured by Nipro) was added to 50 μL of each cell, mixed, subjected to an enzyme reaction at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer.

The blank value was subtracted from each L-kynurenine concentration and the measured value 10 minutes after the control (subtraction value C and subtraction value D). FIG. 4 shows the result of plotting the value obtained by subtracting the subtracted value C of each L-kynurenine concentration from the subtracted value D of the control as OD440. As the concentration of L-kynurenine increased, the value of OD440 increased, indicating that L-kynurenine can be quantified by adding POD. The slope was an increase of 0.2 [abs] per 1 μM of L-kynurenine, and measurement with higher sensitivity than in Example 2 was possible.

[Example 5]
(Measurement with diaphorase / WST-1 / laccase system)
L-kynurenine solutions at various concentrations were used as samples. After reaction with P-KMO, laccase was mixed, and the decreased amount of NADH was measured using diaphorase as a charge carrier and WST-1 as a reducing coloring reagent. 1.84 mL of the enzyme reaction solution was added to and mixed with a quartz cell having an optical path length of 1 cm, and the enzyme reaction was carried out at 25 ° C. for 30 minutes.

The composition of the enzyme reaction solution is 100 mM potassium phosphate buffer (pH 7.0), 0.25, 0.5, or 1 μM L-kynurenine, 0.05 mM EDTA, 0.04 mM NADH, and 0.2 U / mL P. -KMO (Note: The concentration of the reaction solution is indicated by the concentration after addition of diaphorase). As a control, ultrapure water was used instead of the substrate.

After completion of the reaction, 100 U / mL laccase (from Kawaratake) (Sigma Aldrich) was added to a 10 μL cell, and an enzyme reaction was carried out at 25 ° C. for 3 minutes. After completion of the reaction, 10 mg / mL WST-1 was added to each 100 μL cell and mixed, and the absorbance at 440 nm was measured with a spectrophotometer. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 20 U / mL of diaphorase (manufactured by Nipro) was added to 50 μL of each cell, mixed, subjected to an enzyme reaction at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer.

The blank value was subtracted from each L-kynurenine concentration and the measured value 10 minutes after the control (subtraction value C and subtraction value D). FIG. 5 shows the result of plotting the value obtained by subtracting the subtracted value C of each L-kynurenine concentration from the subtracted value D of the control as OD440. Up to 0.5 μM L-kynurenine, the value of OD440 increased with increasing concentration, indicating that L-kynurenine can be quantified by adding laccase. In the range up to 0.5 μM L-kynurenine, the slope was an increase of 0.12 [abs] per 1 μM L-kynurenine, and measurement with higher sensitivity than in Example 2 was possible.

[Example 6]
(Measurement with diaphorase / WST-1 / bilirubin oxidase system)
L-kynurenine solution of each concentration was used as a sample. After reaction with P-KMO, bilirubin oxidase was mixed, and the decreased amount of NADH was measured using diaphorase as a charge carrier and WST-1 as a reducing coloring reagent. . 1.84 mL of the enzyme reaction solution was added to and mixed with a quartz cell having an optical path length of 1 cm, and the enzyme reaction was carried out at 25 ° C. for 30 minutes.

The composition of the enzyme reaction solution was 100 mM Tris-HCl buffer (pH 8.5), 0.25, 0.5, 1, or 2 μM L-kynurenine, 0.05 mM EDTA, 0.03 mM NADH, and 0.2 U / mL. P-KMO was used. As a control, ultrapure water was used instead of the substrate (Note: The concentration of the reaction solution is shown by the concentration after addition of diaphorase).

After completion of the reaction, 2 U / mL bilirubin oxidase (manufactured by Sigma Aldrich) was added to a 10 μL cell, and an enzyme reaction was performed at 25 ° C. for 3 minutes. After completion of the reaction, 10 mg / mL WST-1 was added to each 100 μL cell and mixed, and the absorbance at 440 nm was measured with a spectrophotometer. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 20 U / mL of diaphorase (manufactured by Nipro) was added to and mixed with 50 μL of each cell, an enzyme reaction was performed at 25 ° C. for 15 minutes, and the absorbance at 440 nm was measured with a spectrophotometer.

The blank value was subtracted from each L-kynurenine concentration and the measured value 15 minutes after the control (subtraction value C and subtraction value D). FIG. 6 shows the result of plotting the value obtained by subtracting the subtracted value C of each L-kynurenine concentration from the subtracted value D of the control as OD440. Up to 1.0 μM L-kynurenine, the OD440 value increased with increasing concentration, indicating that L-kynurenine can be quantified by adding bilirubin oxidase. In the range up to 1.0 μM L-kynurenine, the slope increased by 0.1 [abs] per 1 μM L-kynurenine, and higher sensitivity measurement than in Example 2 was possible.

[Example 7]
(Measurement by enzyme cycling method in diaphorase / WST-1 / POD system)
The L-kynurenine solution at each concentration was used as a sample, and the reaction with P-KMO was performed with 0.4 mL of the reaction solution A. The composition of the reaction solution A is 100 mM potassium phosphate buffer (pH 7.5), 0.25, 0.5, 0.75, 1 μM L-kynurenine, 2 mM EDTA, 0.04 mM NADH, and 0.4 U / mL. P-KMO was used. As a control, ultrapure water was used instead of the substrate. The reaction was carried out at 25 ° C. for 15 minutes.

After completion of the reaction, it was mixed with 1.5 mL of reaction solution B. By mixing reaction solution B, 200 mM potassium phosphate buffer (pH 7.5), 1 mM EDTA, 4% (v / v) ethanol, 5 U / mL alcohol dehydrogenase (made by Oriental Yeast), 1 U / mL POD, and 0.2 mg / mL WST-1 (Note: The concentration of this reaction solution is shown by the concentration after addition of diaphorase).

Next, 100 μL of 10 U / mL diaphorase (manufactured by Nipro) was added to each cell, mixed, enzyme reaction was performed by enzyme cycling at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer by the rate method. In the case of this enzyme cycling method, the absorbance change due to the measurement time lasts continually in the rate method. Therefore, the endpoint method as in Examples 1 to 6 has a large measurement error. by.

The final concentration of L-kynurenine at this time is 0.05, 0.1, 0.15, and 0.2 μM by adding the reaction solution B and the diaphorase solution.

For each L-kynurenine concentration and control, the amount of change at 440 nm per minute was calculated from the data from 0 to 4 minutes after the start of enzyme cycling. Subsequently, in order to calculate the amount of change of 440 nm in 4 minutes of enzyme reaction, the result of plotting the value obtained by multiplying the amount of change of 440 nm per minute by 4 as OD440 is shown in FIG. As a result, the value of OD440 increased as the concentration of L-kynurenine increased, and it was shown that L-kynurenine at a low concentration can be quantified by combining the enzyme cycling method. An increase of 0.046 [abs] per 0.1 μM of L-kynurenine, that is, an increase of 0.46 [abs] when converted to 1 μM of L-kynurenine, which is a higher sensitivity measurement than in Examples 2 and 4. It was possible.

[Example 8]
(Measurement in the copper (II) chloride / vasocproin system in the presence of serum)
When it was verified whether the measurement in the presence of serum was possible by the method shown in Example 1, in the measurement using the serum as it is, the measurement was difficult because the effect of the reducing substance was large. I understood. Therefore, the pretreatment was performed in the following manner and the measurement was performed.

After adding 0.5N perchloric acid solution to human normal serum and mixing the supernatant by centrifugation, neutralize it by adding 180 μL of 2N potassium carbonate solution to 1000 μL of the resulting solution, After refrigerated storage, deproteinized serum was obtained by removing excess potassium perchlorate as a precipitate. 2.445 mL of an enzyme reaction solution containing 1.0 mL of deproteinized serum was added to and mixed with a quartz cell having an optical path length of 1 cm, and a pretreatment was performed to reduce reducing substances at 37 ° C. for 20 minutes.

The composition of the enzyme reaction solution was 200 mM HEPES buffer (pH 7.5), 40 μM EDTA, 1 U / mL ascorbate oxidase (manufactured by Wako Pure Chemical Industries), 0.5 U / mL uricase (manufactured by Wako Pure Chemical Industries), 10 U / It was made into mL catalase (made by Wako Pure Chemical Industries).

After completion of the above pretreatment, the mixture was added to 100 μM NADPH, 0.5, 1, 2 μM L-kynurenine, 0.08 U / mL P-KMO, and reacted at 30 ° C. for 30 minutes. The enzyme reaction solution at this time was adjusted to 2.55 mL (Note: the concentration of the reaction solution is indicated by the concentration after addition of copper (II) chloride). As a control, ultrapure water was used instead of the substrate.

After the enzyme reaction, 0.15 mL of 2 mM bathocuproin disulphonate, a metal indicator, was added to each L-kynurenine concentration and control cell, mixed, and a spectrophotometer (V-660, manufactured by JASCO Corporation) ), The absorbance at 480 nm was measured. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 0.3 mL of 4 mM copper (II) chloride solution was added to each L-kynurenine concentration and control cell, mixed, and the absorbance at 480 nm was measured over time at 37 ° C.

The blank value was subtracted from each L-kynurenine concentration and the measured value 10 minutes after the control (subtraction value A and subtraction value B). Subsequently, a value obtained by subtracting the control subtraction value B from the subtraction value A of each L-kynurenine concentration was plotted in the figure as OD480.

As shown in FIG. 8, the OD480 value increased as the L-kynurenine concentration increased, indicating that L-kynurenine can be quantified by this method.

As described above, even in a system containing serum, L-kynurenine is highly sensitive by pretreatment, using kynurenine 3-monooxygenase, NADH, disodium bathocuproine disulfonate and copper (II) chloride. It was found that it can be quantified.

[Example 9]
(Measurement with diaphorase / WST-1 / POD system in the presence of serum)
When it was verified whether measurement in the presence of serum was possible by the method shown in Example 4, in the measurement using serum as it was, the influence of the reducing substance was large, and the color reaction of WST-1 was inhibited. Therefore, the measurement which added polyoxyethylene (20) sorbitan monolaurate (brand name: Tween20) which is surfactant was performed. An enzyme reaction solution (1.5 mL) containing 0.2 mL of human normal serum was added to and mixed with a quartz cell having an optical path length of 1 cm, and an enzyme reaction was performed at 25 ° C. for 10 minutes.

The composition of the enzyme reaction solution was 200 mM potassium phosphate buffer (pH 7.5), 0.1, 0.2, 0.3 or 0.5 μM L-kynurenine, 1 mM EDTA, 0.04 mM NADH, 0.2 U / mL P-KMO, 0.5 U / mL POD. As a control, ultrapure water was used instead of the substrate (Note: The concentration of the reaction solution is shown by the concentration after addition of diaphorase). As a comparative example, evaluation in a system without addition of serum was also performed.

After completion of the reaction, 200 μL of 3% (w / v) Tween 20 was added in the serum-added group (200 μL of ultrapure water was added in the serum-free group). Thereafter, 10 mg / mL WST-1 was added to and mixed with 100 μL of each cell, and the absorbance at 440 nm was measured with a spectrophotometer. This measured value was used as a blank value for each L-kynurenine concentration and control.

Next, 200 μL of 5 U / mL diaphorase (manufactured by Nipro) was added to and mixed with each cell, an enzyme reaction was performed at 25 ° C. for 10 minutes, and the absorbance at 440 nm was measured with a spectrophotometer.

The blank value was subtracted from each L-kynurenine concentration and the measured value 10 minutes after the control (subtraction value C and subtraction value D). Subsequently, FIG. 9 shows the result of plotting the value obtained by subtracting the subtraction value C of each L-kynurenine concentration from the subtraction value D of the control as OD440. Even in the presence of serum, the OD440 value increased as the concentration of L-kynurenine increased, indicating that L-kynurenine can be quantified. The slope increased by about 0.1 [abs] per 0.5 μM L-kynurenine regardless of the presence of serum, that is, increased by about 0.2 [abs] when converted to 1 μM L-kynurenine. The result was equivalent to Example 4.

Claims (19)

1. (A) A sample containing L-kynurenine is subjected to an enzymatic reaction with kynurenine 3-monooxygenase in the presence of an electron donor selected from the group consisting of NADH, NADPH and derivatives thereof, and the remaining electron donor, And obtaining an enzyme reaction solution containing the produced 3-hydroxykynurenine,
   (B) In the enzyme reaction solution of step (A),
   (1) a metal indicator specific for a divalent copper compound and a monovalent copper ion, or (2) a step of reacting a redox coloring reagent and a charge carrier, and (C) the color obtained in step (B). Including the step of determining the amount of L-kynurenine in the sample based on a value obtained by subtracting the concentration of the chromogenic substance in the sample not containing L-kynurenine or the sample not containing kynurenine 3-monooxygenase from the concentration of the substance. Of measuring the amount of L-kynurenine.
2. The measuring method according to claim 1, wherein the concentration of the color developing substance due to the reaction is measured by absorbance.
3. The chromogenic material obtained in (1) of the step (B) comprises a monovalent copper ion produced by the reaction of 3-hydroxykynurenine and a divalent copper compound, and a metal indicator that specifically acts on the ion. The measuring method according to claim 1, wherein the measuring method is produced by a reaction.
4. The measurement method according to claim 1 or 3, wherein the metal indicator specific for the monovalent copper ion used in (1) of the step (B) is bathocuproin, neocuproin, bicinchoninic acid, a derivative thereof or a salt thereof.
5. 2. The measuring method according to claim 1, wherein an enzyme acting on the phenolic compound is further added before or simultaneously with the reaction of (2) in the step (B).
6. 6. The method according to claim 1 or 5, wherein an enzyme cycling method is performed after or simultaneously with the addition of an enzyme that acts on the phenolic compound of claim 5.
7. 2. The measuring method according to claim 1, wherein the coloring substance obtained in (2) of the step (B) is produced by a reaction between an electron donor and a redox coloring reagent via a charge carrier. .
8. The measurement method according to claim 1, 5, 6, or 7, wherein the redox-based color-developing substance is a tetrazolium salt.
9. The measurement method according to claim 1, 5, 6 or 7, wherein the charge carrier is diaphorase.
10. The measurement method according to any one of claims 1 to 9, further comprising a pretreatment step of reducing L-tryptophan and / or a reducing substance in the sample containing L-kynurenine before the step (A).
11. The measurement method according to claim 10, wherein the reducing substance is bilirubin, ascorbic acid and / or uric acid.
12. Before the step (A), at least one selected from the group consisting of tryptophanase, tryptophan oxidase, bilirubin oxidase, ascorbate oxidase, uricase, peroxidase, catalase and hydrogen peroxide is contained in the sample containing L-kynurenine. The measuring method of Claim 10 or 11 including the process added to.
13. The measurement method according to any one of claims 1 to 12, comprising a step of adding a substance having a chelating action and / or a substance that forms a salt with a metal and precipitates before the step (A).
14. Substances having a chelating action and / or substances which form salts with metals and precipitate are ethylenediaminetetraacetic acid, N, N-bis (2-hydroxyethyl) glycine, diethylenetriaminepentaacetic acid and trans-1,2-diaminocyclohexane-N The measurement method according to claim 13, which is at least one selected from the group consisting of N, N, N, N-tetraacetic acid.
15. The measurement method according to any one of claims 1 to 14, further comprising a step of adding a surfactant before the step (B).
16. A kit for measuring L-kynurenine comprising an electron donor selected from the group consisting of NADP, NADPH and derivatives thereof, kynurenine 3-monooxygenase, a divalent copper compound, and a metal indicator specific for monovalent copper ions.
17. A kit for measuring L-kynurenine comprising an electron donor selected from the group consisting of NADP, NADPH and derivatives thereof, kynurenine 3-monooxygenase, a redox coloring reagent, and a charge carrier.
18. A kit for measuring L-kynurenine, wherein the kit according to claim 17 further comprises an enzyme that acts on a phenolic compound.
19. A kit for measuring L-kynurenine, which further comprises NADH or NADPH regenerating enzyme and a substrate thereof in the kit of claim 18.
    

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