JP2000253898A - Quantitative determination of substance or enzyme, and quantitative determining reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quantitative determining ammonia, a substance to be directly or indirectly converted into ammonia, or an enzyme, and to obtain a reagent usable for the method. SOLUTION: A glutamine synthase, a purine nucleoside phosphorylase and a xanthine oxidase are reacted with a sample containing ammonia in the presence of glutamic acid, adenosine triphosphate and inosine to convert the ammonia into hydrogen peroxide, and the generated hydrogen peroxide is quantitative determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for quantifying ammonia or a substance or an enzyme which is directly or indirectly converted to ammonia in a sample, and a reagent for quantifying ammonia used in the method.

[0002]

2. Description of the Related Art In the field of clinical examination, the determination of ammonia is an important index for diagnosis of various diseases. For example, blood ammonia level increases in severe liver diseases such as fulminant hepatitis and cirrhosis, congenital urea cycle enzyme deficiency, and intrahepatic / extrahepatic portal vein-cava shunt. In particular, blood ammonia concentration is important for understanding the pathology of hepatic coma.

Conventionally, as a method for quantifying ammonia, a chemical measurement method such as an ion exchange method using indophenol coloring or a direct colorimetric method has been used. However, chemical measurement methods such as an ion exchange method and a direct colorimetric method have problems that the operation is complicated, lacks accuracy and precision, and requires a long time. On the other hand, as a method for quantifying ammonia using an enzymatic method, for example, glutamate is added to ammonia in the presence of α-ketoglutaric acid and reduced nicotine aminoadenine dinucleotide (phosphate) [hereinafter abbreviated as NAD (P) H]. Dehydrogenase is acted on and NAD
There is a method of converting (P) H into oxidized nicotine aminoadenine dinucleotide (phosphate) [hereinafter abbreviated as NAD (P)] and quantifying a decrease in absorbance before and after the reaction.

The method for measuring ammonia using glutamate dehydrogenase measures a decrease in the absorbance of NAD (P) H, and is insensitive and requires a large number of test samples. Abnormally high ammonia samples could not be directly quantified. Therefore, there is a demand for a sensitive method for quantifying ammonia by increasing the absorbance.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a method for quantifying ammonia and a substance or an enzyme which is converted to ammonia by an increasing method in which the amount of a test sample is small and the quantification range is wide, and a reagent used in the method. It is to provide.

[0006]

The present invention provides the following (1) to
(19). (1) Glutamic acid,
A method for quantifying ammonia in a sample, comprising reacting glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase in the presence of adenosine triphosphate and inosine to quantify the generated hydrogen peroxide.

(2) Purine nucleoside phosphorylase and xanthine oxidase are allowed to act on a sample containing ammonia in the presence of inosine to eliminate generated hydrogen peroxide, and then glutamine, adenosine triphosphate, and glutamine are added in the presence of glutamic acid, adenosine triphosphate and inosine. Synthetase,
A method for quantifying ammonia in a sample, comprising reacting purine nucleoside phosphorylase and xanthine oxidase, and quantifying the generated hydrogen peroxide.

(3) Elimination of hydrogen peroxide is carried out by reacting catalase in the presence of oxygen, and glutamic acid,
(2) The method according to (2), wherein the reaction is carried out by inactivating catalase activity with a catalase inhibitor simultaneously with or before the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase in the presence of adenosine triphosphate and inosine. A method for determining ammonia.

(4) Elimination of hydrogen peroxide is carried out by allowing peroxidase to act in the presence of one of the two compounds which becomes a dye when combined with peroxidase in the presence of hydrogen peroxide. And, in the presence of glutamic acid, adenosine triphosphate and inosine, the amount of hydrogen peroxide produced by the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase was determined in the presence of the one compound and the other compound. The method for quantifying ammonia according to (2), wherein the dye produced by the action of peroxidase is quantified.

(5) Quantification of hydrogen peroxide is carried out by quantifying a dye, fluorescence or luminescence intensity produced by the action of peroxidase in the presence of a chromogen, a fluorescent compound or a luminescent compound ( The method according to 1) or (3). (6) A reagent for the determination of ammonia containing glutamic acid, adenosine triphosphate, inosine, glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase.

(7) The reagent for quantifying ammonia according to (6), which comprises a peroxidase and one compound selected from a chromogen, a fluorescent compound and a luminescent compound. (8) a first reagent containing 0, 1 or 2 compounds selected from glutamic acid, adenosine triphosphate, and glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase and catalase, and glutamic acid not contained in the first reagent A reagent kit for determining ammonia, comprising a compound selected from the group consisting of adenosine triphosphate and glutamine synthetase and a second reagent containing a catalase inhibitor.

(9) The quantification kit according to (8), wherein the second reagent contains one compound selected from a chromogen, a fluorescent compound and a luminescent compound and peroxidase. (10) Peroxidase is acted on in the presence of 0, 1, or 2 or less compounds selected from glutamic acid, adenosine triphosphate and glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and hydrogen peroxide A first reagent containing one of the two compounds that becomes a dye by binding to the first reagent and not contained in the first reagent, a compound selected from the group consisting of glutamic acid, adenosine triphosphate and glutamine synthetase and hydrogen peroxide A reagent kit for the determination of ammonia comprising a second reagent containing the other of the two compounds that becomes a dye by the action of peroxidase in the presence of the compound.

(11) A method for quantifying a substance or an enzyme to be determined in a sample, wherein the substance or the enzyme is involved in ammonia production, and the presence of glutamic acid, adenosine triphosphate and inosine in the sample. Glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase are allowed to act to eliminate the generated hydrogen peroxide, and then a reagent that directly or indirectly converts the substance or enzyme to ammonia, glutamic acid, adenosine triphosphate and inosine. A method for quantifying a substance or an enzyme, which comprises quantifying hydrogen peroxide produced by the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase in the presence of the substance or enzyme.

(12) Elimination of hydrogen peroxide by the action of catalase in the presence of oxygen and the conversion of a substance or enzyme directly or indirectly to ammonia, glutamic acid, adenosine triphosphate and inosine. The method for quantifying ammonia according to (11), wherein the method is performed by inactivating catalase activity with a catalase inhibitor simultaneously with or before the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase in the presence of the enzyme.

(13) Elimination of hydrogen peroxide is carried out by allowing peroxidase to act in the presence of one of the two compounds which becomes a dye when combined with peroxidase in the presence of hydrogen peroxide. And in the presence of reagents that directly or indirectly convert substances or enzymes to ammonia, glutamic acid, adenosine triphosphate and inosine,
Glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase are acted on to determine the amount of hydrogen peroxide produced by the action of peroxidase in the presence of the one compound and the other compound to quantify the produced dye. The method for quantifying ammonia according to (11).

(14) The amount of hydrogen peroxide is determined by using a chromogen,
(11) The method according to (11), wherein a dye produced by the action of peroxidase in the presence of a fluorescent compound or a luminescent compound and the intensity of fluorescence or luminescence are quantified. (15) A reagent for quantifying a substance or an enzyme containing glutamic acid, adenosine triphosphate, inosine, glutamine synthetase, purine nucleoside phosphorylase, xanthine oxidase and a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia .

(16) The ammonia determination reagent according to (15), comprising one compound selected from a chromogen, a fluorescent compound and a luminescent compound and peroxidase. (17) glutamic acid, adenosine triphosphate, glutamine synthetase, A first reagent containing inosine, purine nucleoside phosphorylase, xanthine oxidase and catalase, and a substance or enzyme consisting of a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia and a second reagent containing a catalase inhibitor Reagent kit for quantification.

(18) The quantification kit according to (17), wherein the second reagent contains one compound selected from a chromogen, a fluorescent compound and a luminescent compound, and peroxidase. (19) One of the two compounds that form a dye by binding peroxidase in the presence of glutamic acid, adenosine triphosphate, glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase, and hydrogen peroxide The first reagent containing the compound of formula (1), the reagent that directly or indirectly converts the substance or enzyme to be quantified into ammonia, and the other compound that becomes a dye by binding peroxidase in the presence of hydrogen peroxide A reagent kit for quantifying a substance or an enzyme comprising the second reagent containing the compound of the above.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have found that by reacting ammonia with glutamine synthetase and purine nucleoside phosphorylase in the presence of xanthine oxidase, inosine, adenosine triphosphate and glutamic acid, one molecule of ammonia is converted to 2 molecules of ammonia. The present invention has been completed by finding a simple, accurate, and accurate method for quantifying ammonia, which has a wide quantification range for generating molecular hydrogen peroxide and has a high accuracy.

The reaction of the present invention is carried out according to the following reaction formula (I).

[0021]

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That is, glutamine synthetase acts on ammonia in a sample in the presence of adenosine triphosphate and glutamic acid to generate phosphoric acid, and purine nucleoside phosphorylase acts on the phosphoric acid in the presence of inosine to produce hypoxanthine. Then, xanthine oxidase is allowed to act on the hypoxanthine in the presence of oxygen to generate hydrogen peroxide, and the generated hydrogen peroxide is quantified to determine ammonia.

In the reaction for converting ammonia to hydrogen peroxide by the method of the present invention, for example, a sample containing ammonia containing glutamic acid, adenosine triphosphate, inosine, glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase may be used. The reaction is carried out by adding to an aqueous medium containing an enzyme activity regulator, an enzyme stabilizer, a surfactant, a preservative, etc. The reaction conditions are not particularly limited. The reaction temperature is preferably from 10 to 50 ° C,
-40 is more preferable, and 25-37 degreeC is especially preferable.
The reaction time is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 5 to 15 minutes.

The method for quantifying the generated hydrogen peroxide is not particularly limited, and any method can be used as long as it is a method for quantifying hydrogen peroxide. As a method for quantifying hydrogen peroxide, for example, it can be directly quantified using a hydrogen peroxide electrode, but it is preferable to detect it by a method such as a color development method, a fluorescence method, or a luminescence method using an enzyme reaction.

As a color-forming method, for example, a dye produced by reacting a hydrogen peroxide to be quantified with a peroxidase such as peroxidase in the presence of a chromogen is measured using light of a specific wavelength, and its absorbance is measured. Is a method of obtaining the ammonia concentration from the As the color source, any of those that can be used alone as a dye and those that combine two compounds to form a dye can be used.

Examples of the dye which can be used alone include, for example, JP-A-56-145352 and JP-A-57-2929.
No. 7, JP-A-59-182361, JP-A-59-182361
0-218069 and JP-A-1-128799
And the compounds described in Japanese Patent Application Publication No.

As a specific compound, for example, 10-N-
Carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (hereinafter CCAP)
Abbreviated. ), 10-N-methylcarbamoyl-3,
7-bis (dimethylamino) -10H-phenothiazine (hereinafter abbreviated as MCDP), N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (hereinafter DA-
Abbreviated as 64. ), 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (hereinafter abbreviated as BCMA) and the like. The color reagent used in the present invention is described in, for example, JP-A-57-292.
No. 97 can be synthesized.

[0028] Examples of the compound that forms a dye by bonding two compounds include a compound that forms a dye by bonding in the coexistence of aqueous hydrogen peroxide and peroxidase.

For example, 4-aminoantipyrine (hereinafter referred to as 4
Abbreviated as -AA. ) Or a coupler such as 3-methyl-2-benzothiazolinone hydrazine and an aniline compound such as N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (hereinafter abbreviated as EMSE),
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5
-Dimethoxyaniline, N-sulfopropyl-3,5-
Dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m -Anisidine, N
-Ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3
-Sulfopropyl) -3-methylaniline sodium salt dihydrate (hereinafter abbreviated as TOOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,
5-dimethoxyaniline, N- (2-hydroxy-3-
Sulfopropyl) -3,5-dimethoxyaniline, N-
Ethyl-N- (2-hydroxy-3-sulfopropyl)
Examples thereof include combinations of -3,5-dimethylaniline, N-sulfopropylaniline, N-ethyl-N-sulfopropylanilinepropyl-m-anidin and the like. In addition, 4-AA and phenol or 3-hydroxy-2,4,
A combination of 6-triiodoacetic acid can be exemplified.

As the fluorescence method, for example, there is a method in which a hydrogen peroxide to be quantified is allowed to act on a peroxidase such as peroxidase in the presence of a fluorescent source to measure the intensity of the generated fluorescence, and the concentration of ammonia is determined from the intensity. can give. Examples of the fluorescent source include 3- (4-hydroxyphenyl) propionic acid. As the luminescence method, there is a method in which a peroxidase such as peroxidase is allowed to act on hydrogen peroxide to be quantified in the presence of a luminescent compound, the light intensity generated is measured, and the ammonia concentration is determined from the intensity. Examples of the light emitting source include luminol, isoluminol, lucigenin, acridinium ester, coumarin derivative, pyrazolopyridopyridazine derivative and the like. As the coumarin derivative, compounds described in WO93-15219 are preferably exemplified. Preferable examples of the pyrazolopyridopyridazine derivative include compounds described in JP-A-9-49831.

The ammonia concentration in the sample is determined by preparing a calibration curve showing the absorbance, the fluorescence intensity or the emission intensity and the ammonia concentration using the absorbance, the fluorescence intensity or the emission intensity and the ammonia at a known concentration for the sample. You can ask. The hydrogen peroxide quantitative reaction may be carried out simultaneously with the hydrogen peroxide producing reaction, or may be carried out after the production reaction is completed. In particular, when the peroxide substance or the like is an enzyme such as peroxidase, it is preferable to carry out the reaction simultaneously with the reaction for producing hydrogen peroxide.

In the present invention, phosphoric acid, hypoxanthine and xanthine contained in a sample such as a serum sample for quantifying ammonia also generate hydrogen peroxide, which affects the quantification of ammonia. For this reason, when it is considered that phosphoric acid, hypoxanthine, xanthine, etc. are present in the sample, it is preferable to quantify the ammonia in the sample after previously eliminating the phosphoric acid, hypoxanthine and xanthine present in the sample. .

The erasing method is not particularly limited, and it can be led to a substance which does not affect the hydrogen peroxide reaction from ammonia by physical removal of a column or the like or an enzymatic reaction. As a method for eliminating phosphate, hypoxanthine and xanthine present in a sample, for example, purine nucleoside phosphorylase and xanthine oxidase are allowed to act on the sample in the presence of inosine to generate hydrogen peroxide,
There is a method of eliminating generated hydrogen peroxide by the following reaction formula (II).

[0034]

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The reaction for converting phosphoric acid, xanthine and hypoxanthine to hydrogen peroxide is, for example, a method in which a sample contains inosine, glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase. It is carried out by adding it to an aqueous medium containing a stabilizer, a surfactant, a preservative and the like. The hydrogen peroxide generation reaction conditions are the same as the above-described conditions for generating hydrogen peroxide from ammonia.

The reaction for eliminating the generated hydrogen peroxide is carried out, for example, by containing peroxidase, which is one of the two compounds that become a dye by binding the sample to peroxidase in the presence of hydrogen peroxide. If necessary, it is added to an aqueous medium containing an enzyme activity regulator, an enzyme stabilizer, a surfactant, a preservative and the like. The conditions for the elimination reaction of hydrogen peroxide are not particularly limited. The reaction temperature is 10 to 50 ° C.
Is preferably 20 to 40 ° C, more preferably 25 to 37.
C is particularly preferred. The reaction time is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 5 to 15 minutes.

Thus, the phosphoric acid, hypoxanthine, and xanthine in the sample are led to hydrogen peroxide, and the generated hydrogen peroxide is converted into water and eliminated. Specific examples of the combination of two compounds that become a dye by binding peroxidase in the presence of hydrogen peroxide to form a dye include 4-aminoantipyrine and 3-methyl-2-benzothiazolinone hydrazine. And a combination of an aniline derivative or a phenol derivative. In addition, aniline derivatives, phenol derivatives and combinations of aniline derivatives and phenol derivatives can be mentioned. Examples of the phenol derivative and the aniline derivative include the above-described derivatives.

Next, a sample in which phosphoric acid, hypoxanthine, and xanthine, which were previously present in the sample, were eliminated by the above-described reaction was added in the presence of the other compound used to eliminate adenosine triphosphate, glutamic acid, and hydrogen peroxide. , Glutamine synthetase, purine nucleoside phosphatase, xanthine oxidase and peroxidase produce a dye.

The reaction conditions are not particularly limited. The reaction temperature is preferably from 10 to 50C, more preferably from 20 to 40C, particularly preferably from 25 to 37C. The reaction time is 1 to
60 minutes is preferable, 2 to 30 minutes is more preferable, and 5 minutes
Especially preferred is １５15 minutes. Further, as a method for eliminating phosphate, hypoxanthine, and xanthine present in a sample,
There is a method in which purine nucleoside phosphorylase, xanthine oxidase, and inosine are allowed to act to generate hydrogen peroxide, and the generated hydrogen peroxide is eliminated by the following reaction formula (III).

[0040]

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The reaction for converting phosphoric acid, xanthine and hypoxanthine to hydrogen peroxide can be carried out, for example, in the same manner as described above. Phosphoric acid, hypoxanthine, a reaction for eliminating hydrogen peroxide generated from xanthine, for example, an aqueous solution containing catalase and optionally containing an enzyme activity regulator, an enzyme stabilizer, a surfactant, and a preservative Performed by adding to the medium.

Thus, the hydrogen peroxide is converted into water by catalase and eliminated. Then, a catalase inhibitor is added to completely inhibit catalase activity. The catalase inhibitor is added to the reaction solution before or simultaneously with the action of glutamine synthase. Examples of the catalase inhibitor include NaN ₃ , H ₂ S, HCN, NH ₂ OH, 3-amino-1,2,4-triazole and the like.

Next, glutamine synthetase, purine nucleoside phosphatase and xanthine oxidase are allowed to act in the presence of adenosine triphosphate and glutamic acid, and the amount of hydrogen peroxide produced is quantified. The method for quantifying the generated hydrogen peroxide is not particularly limited, and any method may be used as long as it is a method for quantifying hydrogen peroxide. Specific examples include the above-mentioned method for determining hydrogen peroxide.

Further, according to the present invention, not only can ammonia be quantified according to the above reaction formula, but also substances involved in the reaction system for producing ammonia and enzyme activities can be quantified. Examples of substances and enzyme activities involved in such a reaction system for producing ammonia or an enzyme system for producing ammonia include the following.

When the substance to be quantified is urea, urease, when the substance to be quantified is nicotinamide, nicotinamide, when the substance to be quantified is glutaminyl peptide, glutaminyl peptide-glutaminase, and the substance to be quantified is guanine. When the substance to be quantified is adenosine, adenosine deaminase, when the substance to be quantified is threonine, threonine dehydrolase, when the substance to be quantified is aspartic acid, the aspartate ammonia lyase Examples include methionine lyase when methionine is used, guanine deaminase when the substance to be quantified is guanine, and creatinine deimidase when the substance to be quantified is creatinine. When the substance to be quantified is creatine, creatinase and urease are included, and when the substance to be quantified is creatinine, creatininase, creatinase and urease are exemplified.

In the present invention, the reagent containing these substances or enzymes is referred to as a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia. When quantifying a substance or an enzyme involved in the reaction system that produces ammonia, the presence of ammonia or inorganic phosphorus in the sample in advance gives a non-specific quantification result for the substance or enzyme to be quantified. It is desirable to eliminate ammonia, inorganic phosphoric acid, xanthine, and hypoxanthine present therein.

The elimination involves adding adenosine triphosphate, adenosine triphosphate to the sample before converting the substance or enzyme to be quantified into ammonia.
Glutamine synthetase, purine nucleoside phosphorylase, and xanthine oxidase are allowed to act in the presence of glutamic acid and inosine, and the produced hydrogen peroxide is eliminated by the method described above.

When the elimination of hydrogen peroxide is carried out by the reaction formula (II), the substance to be quantified is a reagent for converting the substance to be measured into ammonia after completion of the elimination reaction and the compound used for elimination of hydrogen peroxide. Can be carried out by adding the other compound that forms a pair with and quantifying the resulting dye. The reaction conditions are the same as described above.

When the elimination of hydrogen peroxide is carried out by the reaction formula (III), ammonia, phosphoric acid, hypoxanthine and xanthine are led to hydrogen peroxide, and then catalase is added to eliminate the produced hydrogen peroxide. After the hydrogen peroxide is eliminated by catalase, a catalase inhibitor is added to inhibit catalase activity. The catalase inhibitor is added to the reaction solution before or simultaneously with the addition of a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia.

Glutamine synthetase in the present invention is an enzyme number [EC6.3.1.2. Glutamine synthetase collected from a microorganism or an enzyme produced by modifying them by genetic engineering. For example, commercially available products such as Kikkoman and Unitika are available. Some glutamine synthetases which are commercially available or prepared are mixed with adenosine triphosphatase, but in the present invention, mixed with adenosine triphosphatase is not preferred. The mixing of adenosine triphosphatase is preferably 20 U or less with respect to 1000 U of glutamine synthetase, and
U or less is more preferable, and 5U or less is particularly preferable.

Further, even when adenosine triphosphatase is present, an enzyme prepared to have the above-mentioned enzyme activity by adding an adenosine triphosphatase inhibitor can also be used. The method for measuring glutamine synthetase activity is described in Enzymes, page 45. The method for measuring unitica and adenosine triphosphatase activity is described in Biochemistry Laboratory, 15 , 341-361 (19).
75), the amount of ADP change on page 343, "Method for combining pyruvate kinase and lactate dehydrogenase".

[0052] The origin of the purine nucleoside phosphorylase used in the present invention is not particularly limited, and examples thereof include enzymes derived from bacteria, yeast, erythrocytes, and animal tissues, and enzymes derived from bacteria are preferred. The origin of the xanthine oxidase used in the present invention is not particularly limited, and examples thereof include enzymes derived from bacteria, animal tissues, and milk.

The buffer used for measuring ammonia was pH 6
Any of those having a buffering capacity between -8 and 8, such as Tris buffer, phosphate buffer, citrate buffer, imidazole buffer, sodium maleate buffer, 3-morpholinopropanesulfonic acid (MOPS), etc. (GOOD) buffer and the like. The concentration of the buffer solution when measuring ammonia is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 10 to 1000 mM, and more preferably 20 to 100 mM.
Is more preferable, and 30 to 50 mM is particularly preferable.

In the present invention, an enzyme activity inhibitor, a surfactant, a preservative, a stabilizer, an enzyme activator, and the like are added to the reaction solution, if necessary. Examples of adenosine triphosphatase inhibitors include P1, P5-bis (adenosine-5 ')-pentaphosphate, β-eudesmol, ouabain, quinidine, ethacrynic acid, 2,3-butanedione,
Quinidine sulfate, benzamil hydrochloride, 4-acetamide-4 '
-Isothiocyanatostilbenzene-2,2'-disulfonate disodium (SITS), sodium potassium adenosine triphosphatase inhibitor-1
(SPAI-1), fisetin, myricetin, etc., organic acids such as glycylglycine, succinic acid, and maleic acid, salts such as sodium acetate and ammonium sulfate, chelating agents such as sodium fluoride and EDTA, and SH such as N-ethylmaleimide. Reagents, peptide condensing reagents such as dicyclohexylcarbodiimide, denaturing agents such as guanidine, antibiotics such as streptomycin and neomycin sulfate, and adenosine triphosphatase inhibitors such as nucleic acids such as cytidine triphosphate.

For the purpose of avoiding turbidity of the specimen, a surfactant can be added. Nonionic HS21 is used as the surfactant.
A nonionic surfactant such as 0 (Nippon Yushi Co., Ltd.) is preferably used. As a preservative, NaN ₃ (when catalase is used, as long as the catalase activity is not inhibited) and antibiotics are suitably used.

The stabilizer and the enzyme activator are not particularly limited as long as they exhibit an effect. For example, magnesium ion, manganese ion and the like can be mentioned. As uricase activity inhibition, for example, oxonate can be added, and as phosphatase activity inhibition, for example, sodium fluoride or the like can be added. As a stabilizer for xanthine oxidase, for example, boric acids, EMSE, 4-AA, (added Mg
Ethylenediaminetetraacetic acid (hereinafter E)
Abbreviated as DTA. ), Bovine serum albumin, sodium glutamate, N- (3,5-dimethoxyphenyl) -N '
-Succinylethylenediamine sodium salt (DOS
E), a surfactant and an aldehyde supplement can be added. Examples of aldehyde scavengers include 4-phenyl semicarbazide, phosphoric acid iproniazide, and ethyl carbazate.

For the purpose of avoiding the effect of bilirubin, for example, potassium ferrocyanide can be added. For the purpose of avoiding the effects of ascorbic acid, for example, ascorbic acid oxidase can be added. Glutamine synthetase, purine nucleoside phosphorylase,
Xanthine oxidase, catalase, and peroxidase are not particularly limited as long as they are concentrations suitable for measurement.

Preferably, glutamine synthetase is 1 to 1
It is suitably used at a concentration of 00 U / mL, especially 1 to 10 U / mL. Purine nucleoside phosphorylase is 1-1
It is preferably used at a concentration of 00 U / ml, especially 2 to 10 U / mL. Xanthine oxidase is 2-100 U / m
L, especially at a concentration of 2 to 10 U / mL.
Catalase is 200-1000 U / mL, especially 250-
It is preferably used at a concentration of 500 U / mL. Peroxidase is suitably used at a concentration of 4 to 100 U / mL, particularly 5 to 10 U / mL.

Glutamic acid is not particularly limited as long as it is a concentration suitable for measurement, but preferably 1 to 100 mmol / g.
L, particularly preferably at a concentration of 2 to 10 mmol / L. Adenosine triphosphate is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 0.2 to 100 mmol.
/ L, particularly 0.5 to 5 mmol / L. Inosine is not particularly limited as long as it is a concentration suitable for measurement, but 3 to 100 mmol / L can be used, and particularly, 4 to 10 mmol / L is suitably used.

The catalase inhibitor is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 1 to 10 mmol.
It is preferably used at a concentration of 1 / L, particularly 1.5 to 3 mmol / L. The ammonia determination reagent of the present invention comprises a reagent containing glutamic acid, adenosine triphosphate, inosine, glutamine synthetase, purine nucleoside phosphorylase, and xanthine oxidase. The reagent preferably contains a compound selected from peroxidase and a chromogen, a fluorescent compound and a luminescent compound.

The ammonia determination reagent kit of the present invention comprises 0, 1, or 2 compounds selected from glutamic acid, adenosine triphosphate and glutamine synthetase, and inosine, purine nucleoside phosphorylase,
A kit comprising a first reagent containing xanthine oxidase and catalase and a second reagent containing a catalase inhibitor and a compound selected from the group consisting of glutamic acid, adenosine triphosphate and glutamine synthetase which are not contained in the first reagent. .

The ammonia determination reagent kit of the present invention comprises 0, 1, or 2 or less compounds selected from glutamic acid, adenosine triphosphate and glutamine synthetase, inosine, purine nucleoside phosphorylase,
A first reagent containing one of the two compounds that becomes a dye by binding peroxidase in the presence of xanthine oxidase, peroxidase and hydrogen peroxide, and a glutamic acid, adenosine trioxide not contained in the first reagent. A kit consisting of a compound selected from the group consisting of phosphate and glutamine synthetase and a second reagent containing the other compound of the compound that becomes a dye by binding peroxidase in the presence of hydrogen peroxide .

The second reagent selected from peroxidase and a chromogen, a fluorescent compound and a luminescent compound
Preferably it contains two compounds. Further, the reagent for quantifying the substance or enzyme of the present invention is glutamic acid, adenosine triphosphate, inosine, glutamine synthetase, purine nucleoside phosphorylase, xanthine oxidase and a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia. Consisting of a reagent containing

The reagent preferably contains one compound selected from peroxidase and a chromogen, a fluorescent compound and a luminescent compound. Further, the reagent kit for quantifying the substance or enzyme of the present invention includes glutamic acid, adenosine triphosphate, glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase,
It comprises a first reagent containing catalase, a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia, and a reagent kit for quantifying ammonia comprising a second reagent containing a catalase inhibitor.

The second reagent of the reagent is selected from peroxidase and a chromogen, a fluorescent compound and a luminescent compound.
Preferably it contains two compounds. Further, the reagent kit of the present invention for quantifying a substance or enzyme binds to glutamic acid, adenosine triphosphate, glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase, and peroxidase in the presence of hydrogen peroxide. Reacting peroxidase in the presence of a first reagent containing one of the two compounds that becomes a dye, a reagent that directly or indirectly converts the substance or enzyme to be quantified into ammonia, and hydrogen peroxide The kit comprises a second reagent containing the other of the two compounds that combine to form a dye.

These reagents and reagent kits may be freeze-dried products, or may be dissolved in an aqueous medium such as a buffer in advance. The concentration of the reagent is not particularly limited, but is preferably adjusted so as to have the aforementioned concentration in the reaction solution. Examples of the present invention will be described below, but the present invention is not limited to these examples. The reagents and enzymes used were from the following manufacturers.

MOPS buffer (manufactured by Dojindo), Nonion HS210 (manufactured by NOF Corporation), TOOS (manufactured by Dojindo), sodium glutamate (manufactured by Wako Pure Chemical Industries),
Adenosine triphosphate (Kanto Chemical Co., Ltd.), inosine (Hansui Chemical Co., Ltd.), purine nucleoside phosphorylase (microbial origin, Toyobo Co., Ltd.), xanthine oxidase (microbial origin, Toyobo Co., Ltd.), peroxidase (Western horseradish origin, Toyobo Co., Ltd.) Sulfate) (manufactured by Kanto Kagaku), 4-AA (manufactured by Kanto Kagaku), glutamine synthetase (manufactured by Seishin), magnesium sulfate (Kanto Chemical), N
aN ₃ (manufactured by Dojin Kagaku), phosphate buffer (manufactured by Kanto Kagaku), disodium α-ketoglutarate (manufactured by Kyowa Hakko Kogyo), tetrasodium EDTA (manufactured by Dojindo), N
ADPH (manufactured by Oriental Yeast) and catalase (manufactured by Sigma).

[0068]

Example 1 A kit for the determination of ammonia in a sample was prepared as follows. First reagent MOPS buffer (pH 7.25) 30 mmol / L Nonionic HS210 0.3% TOOS 1.4 mmol / L Sodium glutamate 6 mmol / L Adenosine triphosphate 1 mmol / L Inosine 4 mmol / L Purine nucleoside phosphorylase 2 U / mL xanthine Oxidase 5 U / mL Peroxidase 5 U / mL Second reagent MOPS buffer (pH 7.25) 30 mmol / L Nonionic HS210 0.1% Magnesium sulfate 16 mmol / L 4-AA 2.5 mmol / L Glutamine synthetase 2 U / mL Oxidase 30U / mL

Example 2 Ammonia in a sample was quantified by the following method. Sample liquid Dilute ammonium sulfate with distilled water, 7, 14, 21,
28, 35, 42, 49, 56, 63, 70 μmol /
L and 35, 70, 105, 140, 175, 21
Standard solutions for ammonium sulfate calibration curves of 0, 245, 280, 315, and 350 μmol / L were prepared.

Procedure The sample solution was added to 2.25 mL of the first reagent prepared in Example 1.
After adding 05 mL and incubating at 37 ° C. for 5 minutes, 0.75 mL of the second reagent prepared in Example 1 was added and reacted at a wavelength of 555 nm at 37 ° C. for 5 minutes.
The absorbance after one minute was measured. Blind operation was performed in the same manner using purified water instead of the sample solution. The calibration curves are shown in FIGS.

When the ammonia concentration in the sample is determined using the calibration curve, the ammonia concentration in the sample (μmol /
L) is quantified as a double concentration of the ammonium sulfate concentration corresponding to the obtained absorbance.

Example 3 A kit for the determination of ammonia in a specimen was prepared as follows. First reagent MOPS buffer (pH 7.25) 30 mmol / L Nonionic HS210 0.3% TOOS 1.4 mmol / L Sodium glutamate 6 mmol / L Adenosine triphosphate 1 mmol / L Inosine 4 mmol / L Purine nucleoside phosphorylase 2 U / mL xanthine oxidase 5 U / mL catalase 300 U / mL Second reagent MOPS buffer (pH 7.25) 30 mmol / L Nonionic HS210 0.1% magnesium sulfate 16 mmol / L 4-AA 2.5 mmol / L NaN ₃ 1.5 mmol / L Glutamine synthetase 2U / mL peroxidase 30U / mL

Example 4 Ammonia in a sample was quantified by the following method. Sample liquid Dilute ammonium sulfate with distilled water, 7, 14, 21,
28, 35, 42, 49, 56, 63, 70 μmol /
L and 35, 70, 105, 140, 175, 21
Standard solutions for ammonium sulfate calibration curves of 0, 245, 280, 315, and 350 μmol / L were prepared.

Procedure The sample solution was added to 2.25 mL of the first reagent prepared in Example 3.
After adding 05 mL and incubating at 37 ° C. for 5 minutes, 0.75 mL of the second reagent prepared in Example 3 was added, and reacted at 555 nm at 37 ° C. for 5 minutes.
The absorbance after one minute was measured. Blind operation was performed in the same manner using purified water instead of the sample solution. The calibration curves are shown in FIGS.

When the ammonia concentration in the sample is determined using the calibration curve, the ammonia concentration in the sample (μmol /
L) is quantified as a double concentration of the ammonium sulfate concentration corresponding to the obtained absorbance.

Example 5 The same operation as in Example 2 was performed using human serum as the sample liquid and the first and second reagents of Example 1,
When the concentration was determined from the calibration curve obtained from Example 2,
A value of 23.5 μmol / L was obtained. When this sample was separately measured using a commercially available kit, Determiner NH ₃ (manufactured by Kyowa Medex), 22.8 μmol / L
And agreed well.

Comparative Example 1 The following ammonium sulfate quantitative reagent was prepared. This reaction is performed according to the following reaction formula (IV).

Embedded image

First reagent phosphate buffer (pH 8.25) 0.1 mol / L disodium α-ketoglutarate 5 mmol / L NADPH 100 μmol / L Second reagent phosphate buffer (pH 8.25) 0.1 mol / L Glutamate dehydrogenase 40 U / mL EDTA-tetrasodium 25 mmol / L Sample solution Dilute ammonium sulfate with distilled water, and add 7,14,21,
28, 35, 42, 49, 56, 63, 70 μmol /
L and 35, 70, 105, 140, 175, 21
Standard solutions for ammonium sulfate calibration curves of 0, 245, 280, 315, and 350 μmol / L were prepared.

Operating method 0.05 mL of the sample solution was added to 2.25 mL of the first reagent,
After incubating at 37 ° C. for 5 minutes, the second reagent was added to 0.
75 mL was added and reacted at a wavelength of 340 nm at 37 ° C. for 5 minutes, and the absorbance 5 minutes after the start of the reaction was measured. Absorbance is 34
Performed at 0 nm. Blind operation was performed in the same manner using purified water instead of the sample solution. The calibration curves are shown in FIGS.

When the ammonia concentration in the sample is determined using the calibration curve, the ammonia concentration in the sample (μmol /
L) is quantified as a double concentration of the ammonium sulfate concentration corresponding to the obtained absorbance. Sample volume is 0.05
When a sample as small as mL, that is, a sample having a low ammonia concentration in the sample was quantified by this method, variation was observed in the measured value on the low concentration side.

Comparative Example 2 Ammonium sulfate was quantified by the following method.

Operating Procedure 0.32 mL of the sample solution prepared in Comparative Example 1 was added to 2.25 mL of the first reagent prepared in Comparative Example 1, and the mixture was incubated at 37 ° C. for 5 minutes. 0.75 mL of the reagent was added and reacted at a wavelength of 340 nm at 37 ° C. for 5 minutes, and the absorbance 5 minutes after the start of the reaction was measured. Blind operation was performed in the same manner using purified water instead of the sample solution. The calibration curves are shown in FIGS.

When the ammonia concentration in the sample is determined using the calibration curve, the ammonia concentration in the sample (μmol /
L) is quantified as a double concentration of the ammonium sulfate concentration corresponding to the obtained absorbance. Sample volume is 0.36
When a sample as large as mL, that is, a sample having a high ammonia concentration in the sample was quantified by this method, the quantification was reduced on the high concentration side.

[0084]

According to the present invention, there is provided a method which requires a small amount of a test sample, has a wide quantification range, can accurately measure ammonia by an increasing method of colorimetry, and a reagent used therefor. .

[Brief description of the drawings]

FIG. 1 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 70 μmol / L) and absorbance when quantified by the method of the present invention.

FIG. 2 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 350 μmol / L) and absorbance when quantified by the method of the present invention.

FIG. 3 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 70 μmol / L) and absorbance when quantified by the method of the present invention.

FIG. 4 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 350 μmol / L) and absorbance when quantified by the method of the present invention.

FIG. 5 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 70 μmol / L) and absorbance when quantified by a conventional method. The sample volume is 0.05 mL.

FIG. 6 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 350 μmol / L) and absorbance when quantified by a conventional method. The sample volume is 0.05 mL.

FIG. 7 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 70 μmol / L) and absorbance when quantified by a conventional method. The sample volume is 0.36 mL.

FIG. 8 is a calibration curve showing the relationship between ammonium sulfate concentration (0 to 350 μmol / L) and absorbance when quantified by a conventional method. The sample volume is 0.36 mL.

Continued on the front page F-term (reference) 4B063 QA01 QA19 QQ03 QQ21 QQ89 QQ90 QR02 QR03 QR06 QR20 QR42 QR49 QR66 QR67 QS20 QX01 QX02

Claims (19)

[Claims] 1. A method comprising the steps of: adding a sample containing ammonia in the presence of glutamic acid, adenosine triphosphate and inosine;
A method for quantifying ammonia in a sample, which comprises reacting glutamine synthetase, purine nucleoside phosphorylase, and xanthine oxidase, and quantifying the generated hydrogen peroxide. 2. A sample containing ammonia is reacted with purine nucleoside phosphorylase and xanthine oxidase in the presence of inosine to eliminate generated hydrogen peroxide, and then, in the presence of glutamic acid, adenosine triphosphate and inosine, glutamine synthase. A method for quantifying ammonia in a sample, characterized by quantifying the generated hydrogen peroxide by reacting a protease, purine nucleoside phosphorylase and xanthine oxidase. 3. Elimination of hydrogen peroxide by the action of catalase in the presence of oxygen and the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase in the presence of glutamic acid, adenosine triphosphate and inosine. The method for quantifying ammonia according to claim 2, wherein the catalase activity is deactivated with a catalase inhibitor at the same time as or before. 4. Elimination of hydrogen peroxide is carried out by allowing peroxidase to act in the presence of one of the two compounds that becomes a dye when combined with peroxidase in the presence of hydrogen peroxide. In addition, in the presence of glutamic acid, adenosine triphosphate and inosine, quantification of hydrogen peroxide produced by the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase was performed in the presence of the one compound and the other compound. The method for quantifying ammonia according to claim 2, wherein the method is performed by quantifying a dye produced by the action of oxidase. 5. The method for quantitatively determining hydrogen peroxide in the presence of a chromogen, a fluorescent compound, or a luminescent compound by quantifying a dye, fluorescence, or luminescence intensity produced by the action of peroxidase. 4. The method according to 1 or 3. 6. A reagent for the determination of ammonia containing glutamic acid, adenosine triphosphate, inosine, glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase. 7. The reagent for determining ammonia according to claim 6, comprising one compound selected from a chromogen, a fluorescent compound and a luminescent compound, and peroxidase. 8. 0, 1 or 2 selected from glutamic acid, adenosine triphosphate and glutamine synthetase.
Compounds, inosine, purine nucleoside phosphorylase, a first reagent containing xanthine oxidase and catalase and not contained in the first reagent, a compound and a catalase inhibitor selected from the group consisting of glutamic acid, adenosine triphosphate and glutamine synthetase. A reagent kit for quantifying ammonia comprising a second reagent to be contained. 9. The quantification kit according to claim 8, wherein the second reagent contains one compound selected from a chromogen, a fluorescent compound and a luminescent compound, and peroxidase. 10. A method for producing peroxidase in the presence of 0, 1 or 2 or less compounds selected from glutamic acid, adenosine triphosphate and glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and hydrogen peroxide. A first reagent containing one of the two compounds that becomes a dye by being acted upon, and not contained in the first reagent,
A second compound containing a compound selected from the group consisting of glutamic acid, adenosine triphosphate and glutamine synthetase, and the other of the two compounds that become dyes when peroxidase is acted on in the presence of hydrogen peroxide. A reagent kit for the determination of ammonia consisting of reagents. 11. A method for quantifying a substance or an enzyme to be quantified in a sample, wherein the substance or the enzyme is involved in ammonia production, and wherein the sample or glutamic acid, adenosine triphosphate and inosine are present in the sample. , Glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase to act to eliminate the hydrogen peroxide produced and then directly or indirectly convert the substance or enzyme to ammonia, glutamic acid, adenosine triphosphate and inosine A method for quantifying a substance or an enzyme, characterized by quantifying hydrogen peroxide produced by the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase. 12. Elimination of hydrogen peroxide in the presence of oxygen
Simultaneous with the action of glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase in the presence of glutamic acid, adenosine triphosphate and inosine, a reagent that performs the reaction with catalase and converts the substance or enzyme directly or indirectly into ammonia. The method for quantifying ammonia according to claim 11, wherein the method is carried out by inactivating catalase activity with a catalase inhibitor before that. 13. Elimination of hydrogen peroxide is carried out by allowing peroxidase to act in the presence of one of the two compounds that becomes a dye when combined with peroxidase in the presence of hydrogen peroxide. And in the presence of glutamic acid, adenosine triphosphate and inosine, a reagent for directly or indirectly converting a substance or an enzyme into ammonia, glutamine synthetase, purine nucleoside phosphorylase and xanthine oxidase are used to determine the amount of hydrogen peroxide produced. The method for quantifying ammonia according to claim 11, wherein the method is performed by quantifying a dye produced by the action of peroxidase in the presence of the one compound and the other compound. 14. The method according to claim 11, wherein the determination of hydrogen peroxide is carried out by quantifying a dye, a fluorescence or a luminescence intensity produced by the action of peroxidase in the presence of a chromogen, a fluorescent compound or a luminescent compound. The described method. 15. Glutamate, adenosine triphosphate,
A reagent for quantifying a substance or an enzyme comprising inosine, glutamine synthetase, purine nucleoside phosphorylase, xanthine oxidase and a reagent for directly or indirectly converting the substance or enzyme to be quantified into ammonia. 16. The reagent for quantitatively determining ammonia according to claim 15, comprising one compound selected from a chromogen, a fluorescent compound and a luminescent compound, and peroxidase. 17. Glutamate, adenosine triphosphate,
Consists of a first reagent containing glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase and catalase, a reagent for directly or indirectly converting a substance or enzyme to be quantified into ammonia, and a second reagent containing catalase inhibitor Reagent kit for the quantification of substances or enzymes. 18. The kit according to claim 17, wherein the second reagent contains one compound selected from a chromogen, a fluorescent compound and a luminescent compound and peroxidase. 19. Glutamate, adenosine triphosphate,
Glutamine synthetase, inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a first reagent containing one of the two compounds that become a dye when combined with peroxidase in the presence of hydrogen peroxide, and quantification It consists of a reagent that directly or indirectly converts the substance or enzyme to be converted to ammonia and a second reagent containing the other compound of the compound that becomes a dye by binding peroxidase in the presence of hydrogen peroxide Reagent kit for the quantification of substances or enzymes.