JPWO2018216757A1 - Substance measurement method that measures the target substance as a coenzyme - Google Patents

Abstract

To a sample, at least an enzyme having a substance to be measured contained in the sample as a coenzyme and a substrate of the enzyme are added, and a product generated by a chemical reaction catalyzed by the enzyme is measured. The measurement target substance contained in the sample is quantified based on the measurement amount of. When the substance to be measured is thiamine, for example, pyruvate oxidase is used as an enzyme, pyruvic acid is added as a substrate, and a cofactor other than the substance to be measured is added as needed to cause a chemical reaction. Thiamine can be quantified by measuring hydrogen peroxide in the product by a known method.

Description

The present invention relates to a substance measuring method for measuring a physiologically active substance acting as a coenzyme such as thiamine (vitamin B ₁ ) as a substance to be measured.

For example, thiamine (vitamin B ₁ ) is a type of water-soluble vitamin, which greatly contributes to the metabolism of carbohydrates (carbohydrates) and branched-chain amino acids, and regulates the functions of the nervous system, digestive system, heart and blood vessels. Also contributes. It is known that thiamine deficiency causes beriberi or Wernicke encephalopathy. Therefore, the examination of thiamine in a biological sample such as blood has clinical significance as an indicator of vitamin B ₁ deficiency and the like.

  Thiamine is phosphorylated in a living body and exists as a phosphoric acid ester. The phosphoric acid ester includes thiamine monophosphate (TMP), which is a monophosphate, and thiamine diphosphate, which is a diphosphate. (Thiamine pyrophosphate, TPP) and thiamine triphosphate (TTP) which is a triphosphate.

  In the living body, most of thiamine is present as thiamine diphosphate. Since thiamine diphosphate is a coenzyme of α-carboxylase, it is also called cocarboxylase, and is known to function as a coenzyme involved in carbohydrate metabolism or as a coenzyme in various enzymes in the living body. I have.

  Conventionally, high-performance liquid chromatography (HPLC) has been mainly used as a method for measuring thiamine. In a typical post-column (post-label) HPLC method, first, as a pretreatment, a phosphoric acid ester of thiamine contained in a sample is hydrolyzed, and phosphoric acid is eliminated from the phosphoric acid ester. As a result of this pretreatment, all the thiamine in the sample is not phosphorylated. The sample thus pretreated is injected into the HPLC and eluted from the column. At the time of elution, by mixing a labeling reagent with the eluate, the color of the eluted thiamine can be detected by a detector.

Also, recently, in order to cope with a problem assumed in the post-column HPLC method, for example, as disclosed in Non-Patent Document 1, a liquid chromatograph-tandem mass spectrometer (LC / MS / MS) is used. A method for measuring vitamin B ₁ (thiamine) used (for convenience, LC / MS / MS method) has been proposed.

Non-Patent Literature 1 states that the post-column HPLC method has problems that the labeling reagent is a strongly alkaline solution, which strongly damages an analytical instrument, and that there is a concern about an environmental load due to a waste liquid. It has been pointed out that the measurement time per hit takes about 10 minutes and is not efficient. Therefore, Non-Patent Document 1 verifies that the use of the LC / MS / MS method as a method for measuring vitamin B ₁ can provide a measurement result having the same authenticity as the post-column HPLC method. Furthermore, Non-Patent Document 1 describes that the measurement time per sample can be reduced to 4 minutes, and that the use of a strongly alkaline reagent reduces damage to analytical instruments and eliminates the burden on the environment. Have been.

Hidenori Miyagawa, Kazumi Gondo, Hideaki Nakaura, Masako Kato, Naotaka Hashizume, "A Method for Measuring Total Vitamin B1 in Whole Blood by LC / MS / MS," Biological Sample Analysis, Vol. 36, No. 4, September 30, 2013 Date), pp. 327-330

  However, in both the LC / MS / MS method and the post-column HPLC method, hydrolysis for removing phosphoric acid is required as a pretreatment. As described in Non-Patent Document 1, this pretreatment requires a long time of, for example, one hour, which hinders the efficiency of thiamine measurement.

  According to Non-Patent Document 1, in the measurement of thiamine by the LC / MS / MS method, the measurement time per sample can be shortened as compared with the measurement by the post-column HPLC method. (Batch processing) and not continuous processing. Therefore, there is a limit in shortening the measurement time as compared with the measurement in the continuous processing.

The present invention has been made to solve such a problem, and an object of the present invention is to provide a substance measuring method capable of efficiently measuring a physiologically active substance such as thiamine (vitamin B ₁ ). And

  In order to solve the above-described problems, the substance measurement method according to the present invention includes, for a sample, adding at least an enzyme having a target substance contained in the sample as a coenzyme and a substrate of the enzyme. A product generated by a chemical reaction catalyzed by an enzyme is measured, and a substance to be measured contained in the sample is quantified based on a measured amount of the product.

  According to the configuration, instead of directly measuring the measurement target substance contained in the sample, a chemical reaction is performed using an enzyme that uses the measurement target substance as a coenzyme, and a product generated by the chemical reaction is measured. Indirectly quantifies the substance to be measured, which is a coenzyme. This makes it possible to simplify or omit the pretreatment of the measurement target substance as compared with the method of directly measuring the measurement target substance. Therefore, the measurement target substance can be easily measured.

  Moreover, even if the concentration of the target substance is relatively low, the target substance is indirectly quantified based on the measured amount of the product. Can be measured (quantified) with higher sensitivity. Therefore, by using the substance measurement method according to the present disclosure, it is possible to promote establishment of a system for measuring even a sample having a low concentration of a measurement target substance by an automatic analyzer.

  In the substance measuring method having the above configuration, the measurement target substance may be thiamine.

  Further, in the method for measuring a substance having the above constitution, the enzyme catalyzing the chemical reaction is pyruvate oxidase, the product is hydrogen peroxide, and the measurement of the hydrogen peroxide is performed by measuring the hydrogen peroxide and leuco. A configuration may be employed in which the reaction is performed by reacting a dye with a peroxidase catalysis and measuring the degree of coloration of the leuco dye that has developed color.

  Further, in the substance measuring method having the above configuration, the enzyme that catalyzes the chemical reaction is pyruvate oxidase, the product is hydrogen peroxide, and the measurement of the hydrogen peroxide is performed by measuring the hydrogen peroxide and amino acid. A configuration in which antipyrine and a Trinder reagent are reacted by peroxidase catalysis, and the degree of coloration of the resulting quinone dye is measured may be employed.

  Further, in the substance measuring method having the above configuration, the target substance may be a reduced nicotinamide adenine dinucleotide (NADH).

  Further, in the substance measuring method having the above constitution, the enzyme catalyzing the chemical reaction is lactate dehydrogenase, the product is an oxidized nicotinamide adenine dinucleotide (NAD +), and the oxidized nicotinamide adenine is used. The measurement of the dinucleotide may be performed by a change in absorbance.

  Further, in the substance measuring method having the above configuration, a configuration may be adopted in which a cofactor other than the substance to be measured is added to the sample in addition to the enzyme and the substrate.

According to the present invention, the configuration described above has an effect that a substance measuring method capable of efficiently measuring a physiologically active substance such as thiamine (vitamin B ₁ ) can be provided.

The concentration of nicotinamide adenine dinucleotide (NADH) in a sample and the change in absorbance ΔAbs. 6 is a graph showing a relationship with the graph.

  Hereinafter, typical embodiments of the present disclosure will be specifically described. The substance measurement method according to the present disclosure is such that a sample is obtained by adding at least an enzyme having a substance to be measured contained in the sample as a coenzyme and a substrate of the enzyme to a sample, and performing a chemical reaction catalyzed by the enzyme. Measure the product. Then, the substance to be measured contained in the sample is quantified based on the measured amount of the product.

The substance to be measured in the substance measuring method according to the present disclosure is not specifically limited, and may be a physiologically active substance that exists in a sample and functions as a coenzyme. Specifically, for example, vitamin B ₁ (thiamine or its phosphate ester), vitamin B ₂ (riboflavin or its nucleotide conjugate), niacin (nicotinic acid or nicotinamide or its adenine nucleotide conjugate, vitamin B ₃₎ ), Vitamin B ₆ (pyridoxine, pyridoxal, and pyridoxamine or its phosphate ester), coenzyme A (pantothenic acid), coenzyme R (biotin), coenzyme F (folate), coenzyme B12 (cobalamin, vitamin B ₁₂ ), vitamin coenzymes such as coenzyme Q (ubiquinone); pyrroloquinoline quinone (PQQ), topaquinone (TPQ), tryptophan-tryptophyll quinone (TTQ), methylamine redox, ricin tyrosylquinone LTQ), cysteinyl-trip Fan quinone (CTQ) quinone coenzyme like; adenosine triphosphate (ATP), uridine diphosphate glucose (UDPG), nucleoside coenzyme such as nicotinamide adenine dinucleotide (NADH / NAD ⁺⁾ (vitamin coenzymes Of these, nucleotide conjugates may be classified as nucleotide coenzymes); α-lipoic acid; and the like.

In this embodiment, as the measurement target substance, vitamin B ₁ That thiamine or a phosphoric acid ester (hereinafter, simply referred to. As thiamine) as an representative example, the substance measurement method according to the present disclosure will be described (to be described later See Example 1).

  The sample to be measured is not particularly limited, and may be any sample that contains the substance to be measured. When the substance to be measured is thiamine, a sample such as whole blood, a food containing (possibly) thiamine, and the like can be mentioned.

The enzyme using thiamine as a coenzyme is not particularly limited, and a known enzyme can be suitably used. Specifically, for example, pyruvate oxidase, pyruvate dehydrogenase, 3-methyl-2-oxobutanoate dehydrogenase, pyruvate synthase, 2-oxoglutarate synthase, oxalate oxidoreductase, 2-oxoacid oxidoreductase, transketolase , Formaldehyde transketolase, acetoin-ribose-5-phosphate transaldolase, 2-hydroxy-3-oxoadipate synthase, acetolactate synthase, 1-deoxy-D-xylulose-5-phosphate synthase, 2-succinyl- 5-enolpyrvinyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase, 3-acetyloctanal synthase, acetoindehydrogenase, sulfoacetaldehyde acetyl Lance Feller Ze, N ₂ - (2-carboxyethyl) arginine synthase, biotin synthase, cyclohexane-1,2-dione hydrolase, pyruvate decarboxylase, benzoylformate decarboxylase, oxalyl -CoA decarboxylase, phenylpyruvic acid decarboxylase, tartronic Acid-semialdehyde synthase, adenosylmethionine decarboxylase, 2-oxoglutarate decarboxylase, branched-chain-2-oxoacid decarboxylase, indolepyruvate decarboxylase, 5-guanidino-2-oxopentanoate decarboxylase, sulfopyruvine Acid decarboxylase, phosphoenolpyruvate decarboxylase, phosphoketolase, fructose-6-phosphate phosphoketo Over Ze, propionic synthase, benzoin aldolase, aconitate hydratase, but like o- succinyl acid synthase include, but are not particularly limited.

  The product to be measured for quantifying thiamine (substance to be measured) is not particularly limited, and may be appropriately selected according to the type of the selected enzyme. Similarly, the method for measuring the product is not particularly limited, and a known measuring method may be employed depending on the type of the product. Further, to the sample, not only the enzyme and its substrate but also other components, for example, a cofactor other than the substance to be measured may be added.

In the present embodiment, the substance measurement method according to the present disclosure will be described more specifically, taking as an example a case where pyruvate oxidase is selected as the enzyme. Pyruvate oxidase reacts pyruvate (CH ₃ COCOOH), phosphoric acid (H ₃ PO ₄ ) and oxygen (O ₂ ) to produce acetyl phosphate (CH ₃ COH ₂ PO ₄ ), carbon dioxide (CO ₂ ) And reversibly catalyze chemical reactions that produce hydrogen peroxide (H ₂ O ₂ ). Thus, in pyruvate oxidase, pyruvate, phosphate, and oxygen are substrates, and acetyl phosphate, carbon dioxide, and hydrogen peroxide are products. Further, as a cofactor for pyruvate oxidase, flavin adenine dinucleotide (FAD) and magnesium ion (Mg ²⁺ ) are required in addition to thiamine (thiamine diphosphate) as a coenzyme.

Therefore, in the substance measurement method according to the present disclosure, pyruvic acid may be added as a substrate to a sample containing (possibly) thiamine, and FAD and Mg ²⁺ may be added as cofactors. . The method of adding cofactors other than the enzyme, the substrate, and the substance to be measured is not particularly limited. For example, if the sample is a sample of whole blood as in the examples described later, a detection reagent containing an enzyme, a substrate, and a cofactor may be prepared, and the detection reagent may be added to the sample.

  In the chemical reaction catalyzed by pyruvate oxidase, acetyl phosphate, carbon dioxide, and hydrogen peroxide are generated as products as described above, and the product to be measured may be any of these. Good. In the present embodiment, hydrogen peroxide is selected as a measurement target.

  The method for measuring hydrogen peroxide, which is a product, is not particularly limited, but typically includes a measurement method using a leuco dye, a measurement method using a Trinder reagent, and the like.

  In the measurement method using a leuco dye, hydrogen peroxide is measured by reacting hydrogen peroxide with the leuco dye by the catalytic action of peroxidase, and measuring the degree of coloration of the leuco dye formed thereby. A leuco dye is an organic dye that develops or loses color by redox, and reacts with one molecule of hydrogen peroxide.

  The specific type of the leuco dye is not particularly limited, and a known compound can be suitably used. Specifically, for example, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) -10H-phenothiazine salt (DA-67), 10-N-carboxymethylcarbamoyl-3,7-bis ( Phenothiazine derivatives such as dimethylamino) -10H-phenothiazine (CCAP), 10- (N-methylcarbamoyl) -3,7-bis (dimethylamino) -10H-phenothiazine (MCDP); N, N, N ′, N ′ , N ", N" -hexa- (3-sulfopropyl) -4,4 ', 4 "-triaminotriphenylmethane (TPM-PS), 4,4'-tetramethyldiaminotriphenylmethane ( Trife such as leucomalachite green) and tris (4-dimethylaminophenyl) methane (leuco crystal violet) Methane derivative; N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt (DA-64), 4,4′-bis (dimethylamino) diphenylamine, bis [3-bis (4- Diphenylamine derivatives such as chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA); benzidine-based compounds such as diaminobenzidine and tetramethylbenzidine; and other compounds such as o-phenylenediamine and hydroxypropionic acid. it can. Among these, DA-67, DA-64, TPM-PS, MCDP and the like are particularly preferably used.

  The method using a Trinder reagent is a method that utilizes the so-called Trinder reaction, in which hydrogen peroxide, aminoantipyrine, and a Trinder reagent are reacted by the catalytic action of peroxidase, and the degree of coloration of the resulting quinone dye is measured. By doing so, hydrogen peroxide is measured. The Trinder reagent reacts with two molecules of hydrogen peroxide.

  The specific type of the Trinder reagent is not particularly limited, and a known compound can be suitably used. Specifically, for example, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline (TOOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), -Ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxy Aniline (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy Aniline induction such as aniline (HDAOS) and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS) Conductor; phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, 2,4 Phenol derivatives such as 6-tribromophenol, 3,5-dichloro-2-hydroxybenzenesulfonic acid, or 3-hydroxy-2,4,6-triiodobenzoyl acid; toluidine derivatives; and the like. Among these, TOOS is particularly preferably used.

  In the present embodiment, the substance to be measured is thiamine, but since vitamin coenzyme such as thiamine is present in a very small amount in a living body, the concentration in the sample is relatively low. Therefore, by adopting a method of reacting one molecule of the product as a method of measuring the product, measurement with higher sensitivity is possible. In the measurement method using a leuco dye, as described above, the leuco dye reacts with one molecule of hydrogen peroxide. However, in the measurement method using a Trinder reagent, the Trinder reagent reacts with two molecules of hydrogen peroxide as described above. More preferably, a measurement method using a leuco dye is employed.

In the present embodiment, an example in which thiamine (vitamin B ₁ ) in a sample is measured in the substance measurement method according to the present disclosure will be described. Here, as described above, thiamine exists in vivo as phosphate esters of thiamine monophosphate (TMP), 2 thiamine diphosphate (thiamine pyrophosphate, TPP), and thiamine triphosphate (TTP). And most of it is thiamine diphosphate (non-phosphorylated thiamine may also be present). Then, in the method of the above-described embodiment, only the coenzyme thiamine diphosphate is measured.

  Here, the ratio of thiamine diphosphate in the whole thiamine in the living body can be confirmed in advance by a conventional method such as a post-column HPLC method or an LC / MS / MS method. Therefore, from the quantification result of thiamine diphosphate by the substance measurement method according to the present disclosure, it is possible to calibrate the total thiamine amount using the ratio of thiamine diphosphate in all thiamine.

  As described above, in the substance measurement method according to the present disclosure, instead of directly measuring the measurement target substance contained in the sample, a chemical reaction is performed using an enzyme having the measurement target substance as a coenzyme, and the chemical reaction is performed. By measuring the product thus obtained, the substance to be measured, which is a coenzyme, is indirectly determined. This makes it possible to simplify or omit the pretreatment of the measurement target substance as compared with the method of directly measuring the measurement target substance. Therefore, the measurement target substance can be easily measured.

  In addition, vitamin coenzymes such as thiamine and the like have low concentrations in the sample, but when the concentration of the substance to be measured is low, sufficient sensitivity may not be realized even if direct measurement is performed. On the other hand, according to the present disclosure, it is possible to measure (quantify) the measurement target substance with higher sensitivity by indirectly quantifying the measurement target substance based on the measurement amount of the product. .

  In general, if the concentration of the substance to be measured in the sample is too low, there is a possibility that the sensitivity which can be applied to the measurement by the automatic analyzer may not be obtained. However, according to the substance measurement method according to the present disclosure, even with a measurement target substance such as thiamine, measurement with higher sensitivity is possible as described above. Therefore, measurement with sensitivity applicable to an automatic analyzer can be realized.

  In addition, in the case of thiamine measurement, the post-column HPLC method or LC / MS / MS method, which is a conventional measurement method, is a measurement by a batch process, but the substance measurement method according to the present disclosure can be measured by a continuous process. Becomes In addition, in any of the post-column HPLC method and the LC / MS / MS method, in order to measure the total amount of thiamine in a specimen (sample), thiamine phosphate is hydrolyzed to desorb phosphoric acid. Although a pretreatment is required, the substance measurement method according to the present disclosure makes it possible to determine the total amount of thiamine only by quantifying thiamine diphosphate, which accounts for most of the total thiamine. Therefore, as the pre-processing, a simple processing such as the removal of protein is sufficient. Therefore, by using the substance measurement method according to the present disclosure, it becomes possible to easily establish a measurement system for pretreating a sample such as whole blood and measuring the sample with an automatic analyzer.

As another embodiment of the present disclosure, a form in which the substance to be measured is reduced nicotinamide adenine dinucleotide (NADH) can be mentioned. The product measured for quantifying NADH (substance to be measured) is not particularly limited, and may be appropriately selected depending on the type of the selected enzyme. In Example 2 described below, lactate dehydrogenase is used as an enzyme. This lactate dehydrogenase converts pyruvate as a substrate into lactic acid and converts NADH as a coenzyme into oxidized nicotinamide adenine dinucleotide (NAD ⁺ ). The enzyme using NADH as a coenzyme is not limited to lactate dehydrogenase, and may be another known enzyme.

Therefore, in the substance measurement method according to the present disclosure, pyruvate may be added as a substrate to a sample containing (possibly) NADH. In this case, as described above, lactic acid and NAD ⁺ are generated as the products, and the products to be measured may be any of these. In Example 2 to be described later, NAD ⁺ is measured. Since the generation of NAD ⁺ reduces the absorption peak at 340 nm of NADH, NADH in the sample can be quantified by measuring the absorbance with a spectrophotometer.

  The present invention will be described more specifically based on Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

(Example 1)
[Preparation of thiamine diphosphate solution]
10 mg of cocarboxylase (thiamine diphosphate, Wako Pure Chemical Industries, Ltd., product number: 031-03833) is weighed, and 40 mL of 500 mM phosphate buffer (pH) is dispensed, and the volume is adjusted to 100 mL with purified water. Thus, a phosphate buffer solution (pH 6.7) solution of 100 mg / L-thiamine pyrophosphate was prepared.

[Preparation of DA-67 colored thiamine pyrophosphate detection reagent]
10 units of pyruvate oxidase (Asahi Kasei Pharma Inc., product number: T-45), 60 units of peroxidase (Toyobo Co., product number: PEO-301), 189 mg of potassium pyruvate, 40 mg of magnesium chloride, Triton X By weighing 20 mg of each -100 mg and dispensing 4 mL of 500 mM phosphate buffer (pH 6.7) and making up to 10 mL with purified water, the first reagent of the DA-67 thiamine pyrophosphate detection reagent was developed. (R1) was prepared.

  In addition, 311 mg of flavin adenine dinucleotide (FAD, Wako Pure Chemical Industries, Ltd., product number: 062-00164), 0.3 mg of DA-67 (Wako Pure Chemical Industries, Ltd., product number: 046-22341), and Triton X By weighing 10 mg each of -100 mg and dispensing 2 mL of 500 mM phosphate buffer (pH 6.7) and making up to 5 mL with purified water, the second reagent of DA-67 thiamine pyrophosphate detection reagent was developed. (R2) was prepared.

[Blank for standard solution preparation and absorbance measurement]
1 mL of the above-mentioned 100 mg / L-thiamine diphosphate solution and 400 mL of a 500 mM phosphate buffer (pH 6.7) are dispensed, and the volume is adjusted to 1000 mL (1 L) with purified water to prepare a calibration curve. A standard solution (Tstd) was prepared. As a blank (blank test value, Blk) for absorbance measurement, 200 mM phosphate buffer (pH 6.7) was used.

  A predetermined amount of a blank 200 mM phosphate buffer, a standard solution, and a sample (whole blood, Tsam) were dispensed, and each was pretreated for protein removal. In the pretreatment, a predetermined amount of trichloroacetic acid was added to each, and the mixture was acid-treated, and the supernatant was neutralized with a sodium acetate solution.

[Measurement of change in absorbance]
Using a product number: U-3310 manufactured by Hitachi High-Tech Science Co., Ltd. as a spectrophotometer, a blank (Blk), a standard solution (Tstd) or a sample (Tsam), a first reagent (R1), and a second reagent (R2) ) Were mixed at a ratio of 1: 6: 2 (Blk / Tstd / Tsam: R1: R2 = 1: 6: 2), and the change in absorbance was measured under the measurement conditions of a wavelength of 700 nm, a temperature of 37 ° C., and a time scan measurement. did.

  A calibration curve was created from the results of the change in absorbance of the standard solution and the relationship between the concentrations. In addition, the amount of thiamine diphosphate contained in the sample was quantified using this calibration curve. As a result, a calibration curve similar to that of the LC / MS / MS method could be created, and the amount of thiamine diphosphate in the sample could be quantified similarly to the LC / MS / MS method.

(Example 2)
[Preparation of NADH solution]
NADH (manufactured by Nacalai Tesque, Inc., product number: 24335-61) was prepared as a Tris-HCl buffer at a plurality of concentrations (31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM). For a blank for absorbance measurement, only a Tris-HCl buffer solution containing no NADH (NADH was 0 μM) was used.

[Preparation of pyruvic acid solution]
By mixing with 10 mM Tris-HCl buffer (pH 9.5) so that the concentration of pyruvic acid becomes 2 mM, a pyruvic acid solution as the first reagent was prepared.

[Preparation of lactate dehydrogenase solution]
The second reagent is mixed with 20 mM Tris-HCl buffer (pH 7.0) so that the concentration of lactate dehydrogenase (manufactured by Toyobo Co., Ltd., product name: LCD-209) becomes 30 units / mL. A lactate dehydrogenase solution was prepared.

[Measurement of change in absorbance]
A solution A for absorbance measurement was prepared by mixing 25 μL of the NADH solution as a sample to be measured (sample) and 450 μL of the first reagent, and after heating at 37 ° C. for 5 minutes, the absorbance of the solution A was measured. Thereafter, 150 μL of the second reagent was further mixed with the A solution to prepare a B solution for absorbance measurement. After heating at 37 ° C. for 5 minutes, the absorbance of the B solution was measured. Abs. A, and the absorbance of the solution B is Abs. B, absorbance change ΔAbs. Is Abs. A and Abs. B (ΔAbs. = Abs.A−Abs.B).

  For the NADH solutions having different concentrations, the absorbance change ΔAbs. Was measured and compared with the concentration of the NADH solution. As shown in FIG. 1, the absorbance change ΔAbs. Is substantially linear, indicating that NADH in the sample can be quantified.

  It should be noted that the present invention is not limited to the description of the above embodiments, and various changes can be made within the scope of the claims, and the present invention is disclosed in different embodiments and a plurality of modified examples. Embodiments obtained by appropriately combining the technical means described above are also included in the technical scope of the present invention.

  From the above description, many modifications and other embodiments of the present invention are obvious to one skilled in the art. Accordingly, the above description is to be construed as illustrative only and is provided for the purpose of teaching those skilled in the art the best mode of carrying out the invention. Details of its structure and / or function may be substantially changed without departing from the spirit of the invention.

  INDUSTRIAL APPLICABILITY The present invention can be widely and suitably used in the field of measuring a substance that functions as a coenzyme contained in a sample.

Claims (7)

For a sample, at least an enzyme having a substance to be measured contained in the sample as a coenzyme and a substrate of the enzyme are added, and a product produced by a chemical reaction catalyzed by the enzyme is measured.
Based on the measured amount of the product, characterized by quantifying the measurement target substance contained in the sample,
Substance measurement method. The substance to be measured is thiamine,
The substance measuring method according to claim 1. The enzyme that catalyzes the chemical reaction is pyruvate oxidase, and the product is hydrogen peroxide,
The measurement of the hydrogen peroxide is performed by reacting the hydrogen peroxide with the leuco dye by the catalytic action of peroxidase, and measuring the degree of coloration of the leuco dye thus developed,
The method for measuring a substance according to claim 2. The enzyme that catalyzes the chemical reaction is pyruvate oxidase, and the product is hydrogen peroxide,
The measurement of the hydrogen peroxide is characterized in that the hydrogen peroxide, aminoantipyrine and a Trinder reagent are reacted by the catalytic action of peroxidase, and are measured by measuring the degree of coloration by a quinone-based dye to be produced.
The method for measuring a substance according to claim 2. The measurement target substance is reduced nicotinamide adenine dinucleotide (NADH),
The substance measuring method according to claim 1. An enzyme that catalyzes the chemical reaction is lactate dehydrogenase, and the product is oxidized nicotinamide adenine dinucleotide (NAD +);
The measurement of the oxidized nicotinamide adenine dinucleotide is characterized by being performed by a change in absorbance,
The method for measuring a substance according to claim 5. The sample, in addition to the enzyme and the substrate, characterized by adding a cofactor other than the substance to be measured,
The method for measuring a substance according to claim 1.

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