JPH09271398A - Determination of ammonia and composition therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inexpensive and high-sensitive method of measurement of trace amount of ammonia and/or an ammonium ion with high accuracy. SOLUTION: A sample solution containing ammonia and/or ammonium ion is allowed to react with at least a NAD synthetase, deamide-NAD, ATP, Mg<++> ion and/or Mn<++> ion, in the presence of a dehydrogenase which can form ammonia molecule from an amine substrate and an ammonia substrate. Then, the components which are consumed or generated during the reaction are determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a measuring composition for highly sensitively measuring a trace amount of ammonia and / or ammonium ion using NAD synthase. More specifically, the present invention relates to a test solution containing ammonia and / or ammonium ions, containing at least NAD synthase, deamido-NAD, ATP, Mg ²⁺ ion and / or Mn ²⁺ ion, amine-based substrate and oxidized form. NA
Ammonia and / or ammonium ion in a test solution obtained by reacting D in the presence of a dehydrogenase capable of forming an ammonia molecule and its amine substrate, and then measuring the components consumed or produced in the reaction Method and at least NAD synthase, deamido-NAD, ATP, Mg ²⁺ ion and / or Mn ²⁺ ion, amine-based substrate and oxidized NA
The present invention relates to a composition for measuring ammonia and / or ammonium ion, which comprises a dehydrogenase capable of forming an ammonia molecule from D and an amine substrate thereof.

[0002]

2. Description of the Related Art Ammonia and / or ammonium ions in the living body are produced in the process of protein metabolism and used in the liver for biosynthesis of other nitrogen compounds. Excess ammonia and / or ammonium ions are excreted in the urine after being synthesized into urea, but if the liver is damaged, ammonia metabolism may be abnormal, and the concentration of ammonia and / or ammonium ions in blood may be increased. It is known that measurement of blood ammonia and / or ammonium ions is an indicator of liver damage. However, the methods for measuring ammonia and / or ammonium ions that have been used so far have low sensitivity in the low concentration range and have problems in operability.

Although creatinine is applied mutatis mutandis to the diagnosis of renal function, it has a problem that the existing measurement method has a low sensitivity and cannot make a sufficient diagnosis. Conventionally, NAD synthase (EC6.3.1.5 and EC6.3.5.1)
The method for measuring ammonia and / or ammonium ion using is a method for directly measuring oxidized NAD produced by the enzymatic reaction of NAD synthase as reduced NAD using an oxidoreductase and its substrate, and a trace amount of ammonia. In the case of measuring, the following reactions such as a method of coexisting a diaphorase and a tetrazolium salt in a reaction system to develop and form color as formazan, or a method of combining with an oxidase system to develop and measure hydrogen peroxide produced are known. (JP-A-59-198995)
No. 4,776,712, US Pat. No. 4,767,712, US Pat. No. 5,206,146, JP-A-63-185378.
No. 4,912,786), neither P1 (oxidation product of S1) nor P3 (oxidation product of S3) is accompanied by a reaction product that causes cycling.

[0004]

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E1: A dehydrogenase which catalyzes the reaction of consuming oxidized NAD and S1 as substrates to produce reduced NAD and P1. E2: Oxidized NAD by consuming reduced NAD and S2
And an agent that catalyzes the reaction that produces P2. Reduced substrate of S1: E1. Oxidized substrate of S2: E2. Oxidized substrate of P1: S1. A reduced substrate of P2: S2.

[0006]

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E3: A dehydrogenase which catalyzes a reaction of consuming oxidized NAD as a coenzyme and S3 as a substrate to produce reduced NAD and P3. E4: An agent that catalyzes a reaction that consumes reduced NAD as a coenzyme and S4 as a substrate to produce oxidized NAD and P4. Reduced substrate of S3: E3. S4: E4 oxidized substrate. An oxidized substrate of P3: S3. A reduced substrate of P4: S4.

However, a small amount of ammonia or
For example, in quantitatively measuring creatinine in a living body as a test solution, when directly measuring the reduced NAD in measuring a small amount of ammonia produced by decomposing creatinine with creatinine deiminase, there is a problem that sensitivity is low. However, the method of measuring as formazan has high sensitivity, but there is a problem such as dye adsorption to equipment. Further, the oxidase system is susceptible to a reducing substance such as ascorbic acid, and there is a problem in that the cost is high because many kinds of enzymes are used.

[0009]

It has been difficult to measure a very small amount of ammonia and / or ammonium ion with high precision, and there has been a problem of providing an inexpensive and highly sensitive measuring method and a measuring composition. .

[0010]

The present inventors have solved the above-mentioned problems in measuring a trace amount of ammonia and / or ammonium ion using NAD synthase,
As a result of earnest efforts for a highly sensitive measurement method, L-
Ammonia and / or ammonium ion by combining a dehydrogenase capable of forming an ammonia molecule from an amine substrate such as an amino acid dehydrogenase or a D-amino acid dehydrogenase and an oxidized NAD and the amine substrate into a NAD synthesis reaction To ATP, Mg ²⁺ ions and / or M
Deamid-NAD can be reacted with NAD synthase in the presence of n ²⁺ ions to form AMP and oxidized NAD, and this oxidized NAD can form an ammonia molecule from an amine substrate and oxidized NAD. By reacting in the presence of dehydrogenase and its amine-based substrate, the substrate oxide and ammonia and / or ammonium ions forming ammonium ions are cycled, and the accumulated reduced NAD is directly or sequentially reacted. To measure
Further, the inventors have found a method capable of highly sensitively measuring ammonia and / or ammonium ions by sequentially and sequentially measuring a substrate oxide formed from a substrate in association with the production of reduced NAD, and completed the present invention.

The present invention has been completed based on the above findings, and at least NAD synthase, deamid-NAD, ATP, Mg ²⁺ ions and / or a test liquid containing ammonia and / or ammonium ions are added. M
a dehydrogenase capable of forming an ammonia molecule from an n ²⁺ ion, an amine-based substrate and an oxidized NAD and a reaction thereof in the presence of the amine-based substrate, and then measuring the components consumed or produced in the reaction. Method for measuring ammonia and / or ammonium ion in test liquid, and at least NAD synthase, deamido-NAD, ATP, Mg ²⁺ ion and / or Mn ²⁺ ion, amine-based substrate and oxidized NA
A composition for measuring ammonia and / or ammonium ion, comprising a dehydrogenase capable of forming an ammonia molecule from D and an amine substrate thereof.

Hereinafter, the present invention will be described in detail. The reaction system in the present invention is summarized as follows.

[0013]

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E6: A dehydrogenase which catalyzes a reaction to form an ammonia molecule with reduced NAD and P6, using oxidized NAD as a coenzyme and S6 as a substrate. A reduced substrate of S6: E6. P6: S6 oxidation product. As the test liquid of the present invention, any liquid containing ammonia (including ammonium ion) may be used, and further other enzymatic reaction system or acid or alkali hydrolysis reaction capable of liberating and forming ammonia different from the present invention Those containing ammonia liberated and produced by the system, for example, biological components such as blood, serum and urine can be mentioned. The above-mentioned enzyme reaction system is not limited as long as it is an enzyme reaction system that produces ammonia, and examples thereof include the following enzyme reaction in which the enzyme activity or the substrate involved in the reaction is measured. . (1) Creatinine deiminase (EC 3.5.4.
21) Creatinine + H ₂ O → N-methylhydantoin + NH
₄ ⁺ (2) guanine deaminase (EC 3.5.4.3) guanine + H ₂ O → xanthine + NH ₄ ⁺ (3) adenosine deaminase (EC 3.5.4.
4) Adenosine + H ₂ O → Inosine + NH ₄ ⁺ A dehydrogenase capable of forming an ammonia molecule from the amine-based substrate and oxidized NAD used in the present invention and the amine-based substrate thereof are preferably L-amino acid dehydrogenases. And its substrate, for example, an L-amino acid dehydrogenase that forms 2-oxoacid and ammonia from L-amino acid with L-amino acid as a substrate using oxidized NAD formed by NAD synthase as a coenzyme. Without being limited thereto, for example, the following enzymes and L- as a substrate thereof are
Amine substrates such as amino acids, D-amino acids and diamines are exemplified, and they can be used alone, but may be used in a plural number, and dehydrogenases of L-amino acids and their substrates shown in Tables 1 and 2 below are exemplified. To be done.

It should be noted that what should be noted here is JP-A-63-185378 and US Pat. No. 492.
NAD synthase shown in 1786 (Baci
llus stearothermophilus H
-804 origin: FERM BP-1408 and FER
MBP-5381) is characterized in that it does not utilize glutamine and asparagine as amino donors, and therefore all of the exemplified dehydrogenases capable of forming an ammonia molecule from an amine substrate and oxidized NAD can be used. Is a NAD synthase that utilizes glutamine and asparagine (Spence)
r, R. L. J. Biol. Chem. 242, 3
85-392, 1967), the latter NA
When using D synthase, a dehydrogenase capable of forming an ammonia molecule from an amine-based substrate and oxidized NAD that does not utilize at least glutamine, asparagine as an amino donor must be used. Examples of enzymatic reactions are shown in Tables 1 and 2 below. However, since some enzymes have low substrate specificity, this reaction formula does not limit the subject of the present invention.

[0016]

[Table 1]

[0017]

[Table 2]

L in the reaction shown in the above (10) is
-Examples of amino acids include L-alanine, L-serine, L-valine, L-leucine, and L-isoleucine, and 2-oxo acid in the reaction is L.
-Means an oxo acid formed from an amino acid, the above (4) to (10), (13) to (15) are positioned as L-amino acids dehydrogenase, and (11) to (1
2) is classified as a diamine dehydrogenase,
(16) to (21) are positioned as D-amino acid dehydrogenases, and these are all L-amino acids, diamines, amine substrates of D-amino acids and oxidized NA.
Included in a dehydrogenase capable of forming an ammonia molecule from D, and included in the present invention are all included in this enzyme.

In carrying out the present invention, each concentration of the composition of the reagent for measuring a target trace amount of ammonia (including ammonium ion) may be at least a concentration at which the reaction rate shows the maximum. The concentration of deamid-NAD used is, for example, 0.01 mM to 100 mM, preferably 0.1 to 5 mM, and ATP is, for example, 0.01 to 200 m.
M, preferably 0.5-20 mM, Mg ²⁺ or Mn ²⁺
Is 0.01 to 200 mM, preferably 0.5 to 20 mM as magnesium chloride or manganese chloride,
A dehydrogenase capable of forming an ammonia molecule from an amine-based substrate and oxidized NAD is, for example, 0.0
5 to 1000 u / mL, preferably 1 to 250 u / m
An amine substrate that is a substrate for a dehydrogenase capable of forming an ammonia molecule from L, an amine substrate, and oxidized NAD, such as an L-amino acid that is a substrate for an L-amino acid dehydrogenase, is, for example, 0.05 to 2
50 mM, preferably 0.5 to 50 mM, NAD synthase is, for example, 0.1 to 100 u / mL, preferably 0.2.
It may be used in a concentration range of up to 20 u / mL.

The reaction temperature is usually 10 ° C. to 50 ° C., preferably 15 ° C. to 40 ° C., and the reaction time is 1 minute to 12 hours, preferably 1 minute to 1 hour, and more preferably 1 minute to 15 minutes. However, these values do not limit the present invention. The present invention has the following reaction formula

[0021]

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E6: A dehydrogenase which catalyzes a reaction to form an ammonia molecule with reduced NAD and P6 using oxidized NAD as a coenzyme and S6 as a substrate. A reduced substrate of S6: E6. P6: S6 oxidation product. As described above, it is possible to easily and easily measure ammonia (including ammonium ion, which may be simply referred to as ammonia hereinafter) by measuring the accumulated reduced NAD, which is a component produced in the reaction. .

In this reaction, the components consumed or produced are measured. For example, as a measurement of the produced components, it is produced by the oxidation reaction of a dehydrogenase capable of forming an ammonia molecule from an amine substrate and oxidized NAD. Of the reduced NAD, and the measurement of the produced components is based on the amine substrate and the oxidized NAD.
It is a measurement of reduced NAD produced by carrying out a cycling reaction of ammonia with ammonia newly produced by the oxidation reaction of dehydrogenase capable of forming an ammonia molecule from.

As a measuring means for the reduced NAD, it is convenient to measure the absorbance at a wavelength around 340 nm which is the maximum absorption wavelength of the reduced NAD. Instead of this absorbance measurement, a dye such as a formazan dye is produced by an enzyme such as diaphorase in the presence of a chromogen and a hydrogen acceptor, and the amount of this formazan dye is absorbed at its maximum absorption wavelength, for example, 550 nm. A method of measuring the amount of increase may be used, and as the hydrogen acceptor used, known acceptors such as methylene blue and tetrazolium salt, for example, nitrotetrazolium blue (NTB) can be used, and they are commercially available. For example, 0.001 to 0.1% of nitrotetrazolium blue is used as a substrate, and diaphorase is adjusted to 0.1%.
01-500u / mL, preferably 0.1-50u / m
It can be measured using a known formazan color development reaction using L.

In the measurement, the substrate oxide of the amine substrate produced by the oxidation reaction of dehydrogenase capable of forming an ammonia molecule from the amine substrate and oxidized NAD may be measured. In measuring the amount of hydrogen peroxide produced or consumed by using an oxidase that catalyzes a reaction of consuming at least oxygen to produce hydrogen peroxide using the substrate oxide as a substrate, To be

The relationships between the dehydrogenase capable of forming an ammonia molecule from the amine-based substrate and the oxidized NAD, and the relationship between the amine-based substrate and the substrate oxide are listed in Tables 1 and 2 above. In measuring a substrate oxide, such as pyruvic acid, which is included in the reaction shown in (4) of Table 1, a reaction in which at least oxygen is consumed and hydrogen peroxide is produced using pyruvic acid as the substrate oxide as a substrate Using pyruvate oxidase, which is an oxidase that catalyzes

[0027]

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(However, oxidase means pyruvate oxidase). For example, by using pyruvate oxidase and peroxidase, pyruvic acid produced by using alanine dehydrogenase

[0029]

[Chemical 6]

For example, simply by measuring the amount of oxygen, which is a consumed component, or the amount of hydrogen peroxide, which is a produced component, the reaction represented by The ammonia in the test solution in this reaction may be ammonia that is present in the test solution in advance, and, for example, in the reactions (1) to (3) that can release and produce ammonia different from the present invention. Release as a measure of the indicated substrate or enzyme activity,
Produced ammonia may be used, preferably for the test solution containing creatinine, it may be prepared as a composition containing creatinine deiminase, which is a component that releases ammonia from creatinine as a composition for measurement, and may be prepared. Ammonia released and produced from creatinine by the action of minase may be measured.

In the above reaction, L-alanine dehydrogenase (EC 1.4.1.1.) And L-alanine as its substrate were used, and pyruvic acid was used as the substrate oxide. As shown in Tables 1 and 2, preferably L-alanine dehydrogenase (E
C 1.4.1.1. ) And its substrate instead of L
-Glutamate dehydrogenase (EC 1.4.1.
2. ) And L-glutamic acid as substrate oxides, 2-oxoglutarate, L-serine dehydrogenase (EC 1.4.1.7) and L-serine as substrate oxides, hydroxypyruvate, L-valine dehydrogenase. (EC 1.4.1.8) and 2-oxoisovaleric acid, L as substrate oxide using L-valine
-Leucine dehydrogenase (EC 1.4.1.9)
2-oxoisocaproic acid, glycine dehydrogenase (EC
1.4.1.10) and L-glycine as substrate oxide glyoxylic acid, L-amino acid dehydrogenase (EC 1.4.1.5) and L-amino acid as substrate oxide 2-oxo. Acid, L-lysine dehydrogenase (EC 1.4.1.15) and 1,2-didehydropiperidino-2-carboxylic acid as substrate oxide, L-tryptophan dehydrogenase (EC 1.4.1.15) using L-lysine. 1.1.19) and L-tryptophan as substrate oxide (indole-3-
) Pyruvate, L-phenylalanine dehydrogenase (EC 1.4.1.20) and L-phenylalanine are used as the substrate oxides for phenylpyruvate, and at least oxygen is consumed together with these substrate oxides. For example, simply by measuring the amount of oxygen, which is a component consumed, or the amount of hydrogen peroxide, which is a component produced, by an enzymatic action that catalyzes a reaction that produces hydrogen oxide.

The oxygen amount can be conveniently measured using an oxygen electrode. The amount of hydrogen peroxide may be measured using a hydrogen peroxide electrode or an indicator composition that reacts with hydrogen peroxide and changes into a detectable product. Further, as the indicator composition, a coloring composition that is normally visible in a change in color tone, a fluorescent composition that emits fluorescence by ultraviolet irradiation, a light emitting composition, and the like are spectroscopically optically at a wavelength in its maximum absorption wavelength range. A composition is used that can quantify the amount of change. For example, as the coloration composition, a substance containing a substance having a peroxidase action and a color forming reagent is used. As a substance having a peroxidase action, horseradish-derived peroxidase is usually often used and is usually 0.05 to 100 u / m.
L, preferably used at 0.1-20 u / mL.

As the color-developing reagent, a combination of an electron acceptor and a phenol derivative or an aniline derivative is usually often used. Further, as an electron acceptor, for example, 4-
Aminoantipyrine, 4-amino-3-hydrazino-5
-Mercapto-1,2,4-triazole, 2-hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydratin, 2-aminobenzothiazole and the like are used. For example, in the case of 4-aminoantipyrine, it is usually It may be used at 0.01 to 15 mM, preferably 0.1 to 5 mM.

Examples of the phenol derivative include phenol, sodium p-hydroxybenzoate, p-chlorophenol, 2,4-dichlorophenol, 4,6-dichloro-o-cresol, 2,4-dibromophenol,
3,5-dichloro-2-hydroxybenzenesulfonic acid Na, 3,5-xylenol and the like can be mentioned. For example, in the case of phenol, it is usually 0.001 to 0.2%, preferably 0.005 to 0.1%. You can use it with.

Further, as the aniline derivative, for example, 3
-Methyl-N-ethyl-N- (β-hydroxyethyl)
Aniline, N, N-dimethylamylline, N, N-diethylamylline, N, N-diethyl-m-toluidine, N,
N-diethanol-m-toluidine, N, N-dimethyl-m-methoxyaniline, N-ethyl-N-sulfopropyl-m-toluidine Na, N-ethyl-N- (3-methylphenyl) -N'-acetyl Ethylenediamine, 3
-Acetamino-N, N-diethylaniline, N-ethyl-N-sulfopropyl-m-anisidine, 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- (2-hydroxy-3-sulfopropyl)
-M-anisidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, 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) 3,5
-Dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-sulfopropylaniline and the like can be mentioned, for example, N-ethyl-
N- (2-hydroxy-3-sulfopropyl) -3,5
In the case of dimethoxyaniline usually 0.05-10 m
M, preferably 0.1 to 2 mM may be used.

As the luminescent substrate in the fluorescent composition and the luminescent composition, various known ones can be mentioned, for example, 2,4,6-trichlorophenol, oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenyl. Acetic acid, vanillylamine, 3-methoxytyramine, phloretinic acid, hordenine, luminol monoanion, acridinium ester, lumigenin, loffin and the like can be mentioned. To obtain light emission by chemiluminescence reaction, as a catalyst for promoting decomposition of hydrogen peroxide K ₂ S ₂ O ₂ , N
aClO, Fe (II) salt, Mn (II) salt, NH ₃ −
Cu ²⁺ , K ₃ Fe (CN) ₆ , Co (II) amine complex salt, hemoglobin, hemin, peroxidase, etc. may be used. Further, during the luminescence reaction, the coexistence of a fluorescent substance can enhance the detection sensitivity, and at the same time, change the emission wavelength to the fluorescence wavelength of the fluorescent agent. Preferred fluorophores are 2-methylumbelliferone, fluorescein, 9,
Examples thereof include 10-diphenylanthracene.

In measuring the above components, any of a late assay, an end point assay or a combination thereof may be selected, and the reaction may be a one-step reaction or a two-step or more reaction The reagent can be appropriately selected even if the reagent is a one-reagent system or a reagent system in which two or more reagent systems are divided into a plurality of compositions.

[0038]

BEST MODE FOR CARRYING OUT THE INVENTION Next, examples of the present invention will be described in detail, but the present invention is not limited thereto.

[0039]

Example 1 40 mM potassium phosphate buffer (pH 7.0) as a reaction solution, 1 mM deamid-NAD ⁺ , 5
mM ATP, 5 mM magnesium chloride, 50 mM L-alanine, 50 u / mL L-alanine dehydrogenase (manufactured by Asahi Kasei Corp.), 1 u / mL NAD synthase (Bacillus stearothermoph).
Derived from ilus H-804 producing bacterium: FERM BP-5
381, manufactured by Asahi Kasei Kogyo Co., Ltd .: hereinafter means the same producing bacteria), and at 37 ° C., using 1 mL of this reaction solution, ammonium chloride concentration 0, 2
0, 40, 60, 80, 100 μg / dl sample solution 3
The reaction was started by adding 0 μL.

The absorbance at a wavelength of 340 nm was measured using a spectrophotometer for 2 to 5 minutes after the start, and the result obtained by subtracting the ammonium chloride concentration of 0 as a blank is as shown in FIG. Can be quantified up to 100μg / dl, 5μg / dl
However, it became clear that it had a sufficiently measurable sensitivity.

From these results, 10 cycling reactions were performed per minute, and the number of cycles can be increased by further increasing the amount of NAD synthase.

[0042]

Example 2 The same reaction solution as in Example 1 was prepared, and the temperature was 37 ° C.
In 1 mL of this reaction solution, the ammonium chloride concentration was 0, 250, 500, 750, 1000, 12
The reaction was initiated by the addition of 5 μL of 50, 1500, 1750, 2000 μg / dl sample solution. The absorbance at a wavelength of 340 nm was measured using a spectrophotometer for 2 to 1/3 minutes after the start, and the result obtained by subtracting ammonium chloride concentration 0 as a blank is as shown in FIG. Quantitation was possible up to 2000 μg / dl.

[0043]

Example 3 As a reaction solution, 40 mM potassium phosphate buffer (pH 7.0), 1 mM deamide-NAD ⁺ , 5
mM ATP, 5 mM magnesium chloride, 10 mM L-glutamic acid, 20 u / mL L-glutamate dehydrogenase (Sigma), 1 u / mL NA
Prepare a reaction solution containing D synthase (Asahi Kasei Co., Ltd.),
At 37 ° C., using 1 mL of this reaction solution, ammonium chloride concentration 0, 20, 40, 60, 80, 100 μ
The reaction was initiated by the addition of 30 μL of g / dl sample solution.

The absorbance at a wavelength of 340 nm was measured using a spectrophotometer for 2 to 5 minutes after the start, and the result obtained by subtracting the ammonium chloride concentration of 0 as a blank is as shown in FIG. Can be quantified up to 100 μg / dl, 5 μg / d
It was revealed that even 1 had a sufficiently measurable sensitivity.

[0045]

Example 4 As a reaction solution, 40 mM potassium phosphate buffer (pH 8.0), 1 mM deamido-NAD ⁺ , 5
A reaction solution containing mM ATP, 5 mM magnesium chloride, 10 mM L-leucine, 5 u / mL L-leucine dehydrogenase (manufactured by Sigma) and 1 u / mL NAD synthase (manufactured by Asahi Kasei Co., Ltd.) was prepared, At 37 ° C., using 1 mL of this reaction solution, ammonium chloride concentration 0, 250, 500, 750, 1000, 125
The reaction was started by adding 50 μL of a sample solution of 0, 1500, 1750, 2000 μg / dl.

The absorbance at a wavelength of 340 nm was measured using a spectrophotometer for 2 to 5 minutes from the start, and the result of subtracting the ammonium chloride concentration of 0 as a blank is as shown in FIG. Was quantified up to a concentration of 2000 μg / dl.

[0047]

Example 5 As a reaction solution, 40 mM potassium phosphate buffer solution (pH 7.0), 1 mM deamido-NAD ⁺ , 5
mM ATP, 5 mM magnesium chloride, 50 mM L-alanine, 50 u / mL L-alanine dehydrogenase (Asahi Kasei), 1 u / mL NAD synthase (Asahi Kasei), 1 u / mL lactate dehydrogenase (Manufactured by Sigma), 1 u / mL lactate oxidase (manufactured by Asahi Kasei Corporation), 3 u / mL peroxidase, 0.03% 4-aminoantipyrine, 0.03%
A reaction solution containing TOOS was prepared, and 1 mL of this reaction solution was used at 37 ° C.
The reaction was started by adding 30 μL of a sample solution of 20, 40, 60, 80, 100 μg / dl. The absorbance at a wavelength of 555 nm was measured using a spectrophotometer for 2 to 5 minutes from the start, and the result obtained by subtracting the ammonium chloride concentration of 0 as a blank is as shown in FIG. It became clear that quantification was possible up to 100 μg / dl, and that even 5 μg / dl had sufficient measurable sensitivity.

[0048]

Example 6 40 mM potassium phosphate buffer (pH 7.0) as a reaction solution, 1 mM deamide-NAD ⁺ , 5
mM ATP, 5 mM magnesium chloride, 50 mM L-alanine, 50 u / mL L-alanine dehydrogenase (manufactured by Asahi Kasei), 1 u / mL NAD synthase (manufactured by Asahi Kasei), 15 u / mL creatinine day Prepare a reaction solution containing minase (manufactured by Sigma),
At 37 ° C., 1 mL of this reaction solution was used to add 50 μL of a sample solution having a creatinine concentration of 0, 1, 2, 3, 4, 5 mg / dl to start the reaction.

The absorbance at a wavelength of 340 nm was measured using a spectrophotometer from 2 minutes to 5 minutes after the start, and the result of subtracting the creatinine concentration of 0 as a blank is shown in FIG.
And the creatinine concentration was 5 mg.
It was clarified that it is possible to quantify up to / dl, and sufficient measurement is possible even at a concentration of 1 mg / dl or less.

[0050]

Example 7 As a reaction solution, 40 mM potassium phosphate buffer (pH 7.0), 1 mM deamide-NAD ⁺ , 5
mM ATP, 5 mM magnesium chloride, 50 mM L-alanine, 50 u / mL L-alanine dehydrogenase (manufactured by Asahi Kasei), 1 u / mL NAD synthase (manufactured by Asahi Kasei), 5 u / mL diaphorase ( Asahi Kasei Kogyo Co., Ltd.), a reaction solution containing 0.025% of nitrotetrazolium blue (Wako Pure Chemical Industries, Ltd.) was prepared and 37
At 0 ° C., using 1 mL of this reaction solution, ammonium chloride concentration 0, 5, 10, 15, 20, 25 μg / dl
The reaction was started by adding 30 μL of the sample solution of.

The absorbance at a wavelength of 550 nm was measured using a spectrophotometer for 2 to 7 minutes from the start, and the result of subtracting the ammonium chloride concentration of 0 as a blank is as shown in FIG. It was clarified that the concentration could be quantified up to 25 μg / dl, and the sensitivity was sufficient even at 5 μg / dl or less.

[0052]

Example 8 As a reaction solution, 40 mM potassium phosphate buffer solution (pH 7.0), 1 mM deamid-NAD ⁺ , 5
mM ATP, 5 mM magnesium chloride, 50 mM L-alanine, 50 u / mL L-alanine dehydrogenase (Asahi Kasei), 1 u / mL NAD synthase (Asahi Kasei), 15 u / mL pyruvate Oxidase (manufactured by Asahi Kasei Corp.), 3 u / mL peroxidase, 0.03% 4-aminoantipyrine, 0.0
A reaction solution containing 3% TOOS was prepared, and 30 mL of a sample solution having an ammonium chloride concentration of 0, 20, 40, 60, 80, 100 µg / dl was added to 1 mL of this reaction solution at 37 ° C to conduct the reaction. Started.

The absorbance at a wavelength of 555 nm was measured using a spectrophotometer for 2 to 5 minutes from the start, and the result of subtracting the ammonium chloride concentration of 0 as a blank is as shown in FIG. Can be quantified up to 100 μg / dl, 5 μg / d
It was revealed that even 1 had a sufficiently measurable sensitivity.

[0054]

Example 9 Instead of the L-alanine dehydrogenase and the substrate L-alanine of Example 1, L-serine dehydrogenase and the substrate L-serine, L-valine dehydrogenase and the substrate L-valine and glycine, respectively, were used. A reaction solution was prepared in the same manner as in Example 1 using dehydrogenase and L-glycine as a substrate, L-amino acid dehydrogenase and L-alanine, L-serine, L-valine, L-leucine or L-isoleucine as a substrate. However, the same procedure as described below can be performed on the sample solution of ammonium chloride.

[0055]

According to the present invention, a highly sensitive reagent for measuring ammonia and / or ammonium ions can be provided.

[Brief description of drawings]

FIG. 1 shows a calibration curve of ammonia using a measurement reagent containing L-alanine dehydrogenase.

FIG. 2 shows a calibration curve of ammonia using a measurement reagent containing L-alanine dehydrogenase.

FIG. 3 shows a calibration curve of ammonia using a measurement reagent containing L-glutamate dehydrogenase.

FIG. 4 shows a calibration curve of ammonia using a measurement reagent containing L-leucine dehydrogenase.

FIG. 5 shows a calibration curve of ammonia using a measurement reagent containing L-alanine dehydrogenase, lactate dehydrogenase and lactate oxidase.

FIG. 6 shows a calibration curve of creatinine using a measurement reagent containing L-alanine dehydrogenase and creatinine deiminase.

FIG. 7 shows a calibration curve of ammonia using a measurement reagent containing L-alanine dehydrogenase.

FIG. 8 shows a calibration curve of ammonia using a measurement reagent containing L-alanine dehydrogenase and pyruvate oxidase.

Claims (14)

[Claims] 1. A test solution containing ammonia and / or ammonium ions, which contains at least NAD synthase, deamido-NAD, ATP, Mg ²⁺ ions and / or
Or Mn ²⁺ ion, amine-based substrate and oxidized NAD
Reacting in the presence of a dehydrogenase capable of forming an ammonia molecule and its amine substrate,
Next, a method for measuring ammonia and / or ammonium ions in a test solution, which comprises measuring the components consumed or produced in the reaction. 2. A dehydrogenase capable of forming an ammonia molecule from an amine-based substrate and oxidized NAD is a dehydrogenase of L-amino acids, a dehydrogenase of D-amino acids, or L-erythro-3,5-diaminohexanoate. Dehydrogenase (EC 1.4.1.
11) and 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12). 3. A dehydrogenase of L-amino acids,
L-alanine dehydrogenase (EC 1.4.1.
1), L-glutamate dehydrogenase (EC 1.
4.1.2, and EC 1.4.1.3), L-amino acid dehydrogenase (EC 1.4.1.5), L-
Serine dehydrogenase (EC 1.4.1.7), L
-Valine dehydrogenase (EC 1.4.1.8),
L-leucine dehydrogenase (EC 1.4.1.
9), glycine dehydrogenase (EC 1.4.1.
10), L-lysine dehydrogenase (EC 1.4.
1.15), L-tryptophan dehydrogenase (E
C 1.4.1.19) or L-phenylalanine dehydrogenase (EC 1.4.1.20). 4. The measurement of the produced component is carried out by carrying out a cycling reaction of ammonia with ammonia newly produced by the oxidation reaction of dehydrogenase capable of forming an ammonia molecule from an amine-based substrate and oxidized NAD. The method according to claim 1, wherein the reduced NAD is measured. 5. The measurement of the produced component forms an ammonia molecule from the amine substrate and the oxidized NAD produced by the oxidation reaction of dehydrogenase capable of forming the ammonia molecule from the amine substrate and the oxidized NAD. The method according to claim 1, which is a measurement of a substrate oxide of dehydrogenase that can be used. 6. A substrate oxide is measured by using an oxidase which catalyzes a reaction of consuming at least oxygen and producing hydrogen peroxide using the substrate oxide as a substrate. Oxygen consumed or hydrogen peroxide produced. The measuring method according to claim 5, which comprises measuring 7. The measuring method according to claim 1, wherein the test liquid containing ammonia and / or ammonium ions is ammonia and / or ammonium ions released or produced from creatinine. 8. At least NAD synthase, deamide-
Dehydrogenase capable of forming an ammonia molecule from NAD, ATP, Mg ²⁺ ion and / or Mn ²⁺ ion, amine substrate and oxidized NAD, and ammonia and / or ammonium ion containing the amine substrate Measuring composition. 9. A dehydrogenase capable of forming an ammonia molecule from an amine-based substrate and oxidized NAD is a dehydrogenase of L-amino acids, a dehydrogenase of D-amino acids, or L-erythro-3,5-diaminohexanoate. Dehydrogenase (EC 1.4.1.
11) and 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), The measuring composition according to claim 8. 10. The L-amino acid dehydrogenase is L-alanine dehydrogenase (EC 1.4.
1.1), L-glutamate dehydrogenase (EC
1.4.1.2, and EC 1.4.1.3), L-
Amino acid dehydrogenase (EC 1.4.1.5),
L-serine dehydrogenase (EC 1.4.1.
7), L-valine dehydrogenase (EC 1.4.
1.8), L-leucine dehydrogenase (EC 1.
4.1.9), glycine dehydrogenase (EC 1.
4.1.10), L-lysine dehydrogenase (EC
1.4.1.15), L-tryptophan dehydrogenase (EC 1.4.1.19) or L-phenylalanine dehydrogenase (EC 1.4.1.10), The measuring composition according to claim 9. 11. The measuring composition according to claim 8, which contains a reducing NAD measuring composition.
The composition for measurement described. 12. The composition for measurement according to claim 8, which comprises a composition for measurement of a substrate oxide of dehydrogenase capable of forming an ammonia molecule from the amine-based substrate and oxidized NAD produced. The composition for measurement according to claim 8. 13. A substrate oxide measuring composition for measuring hydrogen peroxide produced using an oxidase that catalyzes a reaction of consuming at least oxygen and producing hydrogen peroxide using the substrate oxide as a substrate. The composition for measurement according to claim 12, which is a composition. 14. The measuring composition according to claim 8, which is a measuring composition containing a component that liberates or produces ammonia and / or ammonium ions from creatinine.