JPH09285297A - Assay of biological specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To assay an ADP-forming enzyme or its substrate in a biological specimen as a test solution based on a formed amount of a reduced NAD(P)s simply and in high accuracy. SOLUTION: In this method for assaying either an ADP-forming enzyme or its substrate in a test solution by using an enzyme reaction, the test solution containing either the ADP-forming enzyme or its substrate is reacted in the presence of the ADP-forming enzyme, its substrate, ATP, glucose, an ADP- dependent hexokinase, an oxidized NAD(P), glucose-6-phosphate dehydrogenase and salts capable of releasing one or more ions of magnesium ion, cobalt ion and manganese ion at 15-45 deg.C. The ADP-forming enzyme or its substrate in the test solution together with AMP formed by the reaction is assayed based on the formed amount of reduced NAD(P).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring one of an ADP-producing enzyme and a substrate thereof in a test solution by using an enzymatic reaction, which comprises a substrate containing one of the ADP-producing enzyme and one of its substrates. The test solution is a reaction reagent of components involved in a reaction based on at least an enzyme that produces ADP and its substrate, ATP, glucose, ADP-dependent hexokinase (ADP-HK), oxidized NAD (P) [oxidized nicotinamide adenine diamine Nucleotide (phosphate)],
Glucose-6-phosphate dehydrogenase (G6PDH) and any one or more ion-releasing salts of magnesium ion, cobalt ion or manganese ion are allowed to react under the temperature condition of 15 to 45 ° C. , Reduced NAD (P) together with AMP produced by reaction of an enzyme or its substrate that produces ADP in a test solution
A method for measuring based on the production amount of [reduced nicotinamide adenine dinucleotide (phosphate)], which is used in the field of clinical tests and the like, and ADP in a test liquid such as serum, plasma, urine, and spinal fluid. The purpose is to measure the produced enzyme or its substrate.

[0002]

2. Description of the Related Art Conventionally, as a method for measuring an enzyme or its substrate for producing ADP in a biological sample, a method in combination with other dehydrogenase or oxidase acting on the reaction product of the substrate by the enzymatic reaction has been used. (For example, in the case of glycerol and glycerol kinase,
AD by activating glycerol kinase in the presence of TP
P and glycerol phosphate are produced, and the glycerol phosphate is measured using glycerol phosphate dehydrogenase or glycerol phosphate oxidase)
There are known methods for measuring ADP produced by enzyme reactions by various methods.

However, the method of measuring in combination with other dehydrogenases or oxidases needs to change the combination of enzymes depending on the enzyme that produces ADP, which lacks versatility, and the measurement is attempted. Depending on the substrate or enzyme used, a combination of enzymes may not exist.
Further, as a method for measuring ADP produced by an enzymatic reaction, a method using liquid chromatography is known, but the liquid chromatography method has a drawback that the operability is complicated.

Furthermore, as an ADP measuring enzyme method having excellent operability, pyruvate kinase [PK (EC 2.7.1.
40)] and lactate dehydrogenase [LDH (EC 1.2.
3.3)] method for reducing reduced NAD (NADH + H ⁺ ) (reaction formula 1), oxidase method using pyruvate kinase and pyruvate oxidase (reaction formula 2), pyruvate kinase and pyruvate decarboxylation Reduced NAD (P) [NAD (P) using enzyme and aldehyde dehydrogenase
A method of increasing H + H ⁺ ] (reaction formula 3) is known (Japanese Patent Laid-Open No. 7-8297).

[0005] These reaction formulas are shown below. In the following formula, PEP is phosphoenolpyruvate, Pi is phosphoric acid, P
DC means pyruvate decarboxylase, AlDH means aldehyde dehydrogenase, and TPP means thiamine pyrophosphate.

[0006]

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[0007]

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[0008]

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However, in the method of reducing reduced NAD, a predetermined amount of reduced NAD is present in the reaction solution in advance, and after the reaction is completed, the reduced NA remaining in the reaction solution.
Since it is a reduction method for measuring the amount of D, (1) the measured value is inaccurate when the component to be measured is small. (2) The upper limit of measurable components is limited by the amount of reduced NAD that is present in the reaction solution before quantification.

(3) It is necessary to change the amount of reduced NAD to be present in the reaction solution before quantification depending on the model of the spectrophotometer used for measuring the amount of reduced NAD. (4) The reduced NAD contained in the analytical reagent is unstable. There are problems such as. Further, a method for quantifying pyruvate, which is measured by using a color development indicator system of hydrogen peroxide generated by using pyruvate kinase and pyruvate oxidase, is widely used. (5) This method is an interfering substance in a biological sample. Since it is affected by (reducing substances) (uric acid, ascorbic acid, etc.) and coloring substances (bilirubin, hemoglobin, etc.), the accuracy of the measured values is not always satisfactory.

Further, reduced NAD using pyruvate kinase, pyruvate decarboxylase and aldehyde dehydrogenase.
The method of increasing (P) requires only a large number of reactions of the enzyme to be used, and is only a complicated method. Also, the ADP used in the present invention
Dependent hexokinases include the extremely thermophilic bacterium Pyrococcus furiosus DSM3638 (Pyrococc
It has been reported that it is present in the bacterial cells of the strain (us, furiosus, DSM3638) (J. Biol. C.
hem. , 269, 17537-17541) has a growth temperature of 90 ° C. to 105 ° C., the optimum temperature of the enzyme derived from the strain is 90 ° C. or higher, and G6PDH used for activity measurement is derived from yeast. In consideration of its thermal stability, the activity is measured at 50 ° C., and ADP-H
The activity was merely measured under the temperature condition different from the optimum temperature of K, and the measurement condition was completely different from the temperature condition of general clinical diagnosis in light of the optimum temperature of ADP-HK.
In addition, regarding the measurement at a reaction temperature of 50 ° C, in the field of clinical diagnosis, the reaction temperature should be 50 ° C (6).
Inaccurate measurement due to inactivation of the combined enzymes.
(7) Thermal denaturation of a biological component as a sample occurs, resulting in cloudiness. Due to such problems, accurate measurement was impossible.

[0012]

First of all, the present inventors
yrococcus, furiosus, DSM363
As a result of culturing 8 strains, purifying ADP-HK, and examining the optimum temperature of the reaction of the enzyme, the optimum temperature was 80 to 100 ° C. Furthermore, the relative activity at 37 ° C is about 10% of that at 100 ° C, and it is generally impossible to carry out an accurate quantitative reaction when the reaction is carried out at 37 ° C using an enzyme having such a property. I thought there was. However, surprisingly, the present inventors carried out a quantitative experiment using the enzyme reaction at 37 ° C., and a reaction temperature condition of 15 to 45 which is a general reaction temperature in general quantification of a biological component containing the enzymatic reaction of 37 ° C. It has been found that the substrate or enzyme activity as a biological component can be measured even at a temperature condition of ° C.

Further, in the method of measuring the enzyme or the substrate thereof for producing ADP in a biological sample by using the following enzymatic reaction (reaction formulas 4 and 5), the present inventors have found that the biological sample produces ADP. In the case of a test liquid for the purpose of measuring the activity of the enzyme, the enzyme that produces ADP in the presence of the substrate of the enzyme and ATP and the reaction reagent of the component involved in the reaction based on the substrate, or the biological sample In the case of a test solution for the purpose of measuring the amount of a substrate, a reaction of an enzyme that acts on the substrate to produce ADP and an enzyme that produces ADP in the presence of ATP and a component involved in the reaction based on the substrate Glucose, ADP-HK and oxidized NAD using reagents
By a reaction based on (P) s, G6PDH and magnesium ion, cobalt ion or manganese ion,
ADP produces AMP and oxidized NAD
A method for quantifying an enzyme or a substrate thereof that performs a reaction of reducing (P) s to reduced NAD (Ps) and produces ADP in the living body based on the amount of reduced NAD (P) s produced is extremely useful. The present invention has been completed by discovering that such a reaction is an enzyme reaction that can be universally used for an enzyme that produces ADP in a biological sample or a substrate thereof.

That is, according to the present invention, an enzyme or its substrate for producing ADP in a biological sample as a test solution is used as well as an enzyme for producing ADP and a reaction reagent involved in a reaction based on the substrate, and ATP or glucose. , ADP-HK
Another object of the present invention is to provide a method capable of reacting in the presence of oxidized NAD (P) s, G6PDH and magnesium ion, cobalt ion or manganese ion, and performing simple and highly accurate measurement.

[0015]

The present invention has been completed on the basis of the above-mentioned findings, and A in a test solution is analyzed by using an enzymatic reaction.
In a method for measuring one of an enzyme that produces DP and its substrate, a test solution containing an enzyme that produces ADP or one of its substrates is at least analyzed for components involved in a reaction based on the enzyme that produces ADP and its substrate. Reaction reagent, A
TP, glucose, ADP-HK, oxidized NAD (P)
, G6PDH and magnesium ion, cobalt ion or manganese ion in the presence of any one or two or more ion-releasing salts at a temperature condition of 15 to 45 ° C. to produce ADP in the test solution The enzyme or its substrate that reacts with AMP produced by the reaction,
This is a method of measurement based on the amount of reduced NAD (P) s produced.

Glucose and ADP used in the present invention
-HK and oxidized NAD (P) s, G6PDH and reduced NADs from oxidized NAD (P) s in the presence of magnesium ion, cobalt ion or manganese ion
The enzymatic reaction for producing the (P) s is shown by the following reaction formulas 4 and 5.

[0017]

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[0018]

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The present invention will be described in more detail below. The ADP-HK represented by the above reaction formula 4 is not limited as long as it is ADP-HK that uses glucose as a substrate and consumes ADP to produce glucose-6-phosphate and AMP. As an ADP-HK producing bacterium, an extremely thermophilic bacterium Pyrococcus furi
The osus DSM3638 strain has been deposited as a reference culture in Deutsche Zammulg von Microorganismen und Cherkuturulen GmbH (DSM) and is described in the DSM catalog (1993), and is available to any person. The highly thermophilic ADP-HK obtained from the strain is preferred.

G6PDH used in the enzyme reaction represented by the above reaction formula 5 is commercially available (Leuconostoc.mesent, Boehringer Mannheim).
derived from oroides, Sigma: baker's yeast, Bacil
lus, stearothermophilus, Le
(from uconostoc.mesentoroides), and is easily available.

If any one or more ion-releasing salts capable of releasing cobalt ion or manganese ion are used in place of the magnesium ion used in the above-mentioned ADP-HK-catalyzed enzymatic reaction (Scheme 4), Often, the salts include chlorides, sulfates, and the like, and preferably include magnesium chloride, cobalt chloride, and manganese chloride, but are not limited thereto.

As shown in the enzyme reaction formula 5, the above G6
Oxidized NAD (P) s as coenzymes used in PDH-catalyzed enzymatic reactions include oxidized NAD and oxidized NAD.
Oxidized thio-NA in addition to oxidized NAD (P) containing P
D, oxidized thio-NAD containing oxidized thio-NADP
(P), oxidized 3-acetyl NAD, oxidized 3-acetyl NAD (P) containing oxidized 3-acetyl NADP, oxidized deamino NAD, oxidized deamino NAD (P) containing oxidized deamino NADP, and the like. However, the present invention is not limited to these.

In the present invention, ADP-HK, G6PDH, glucose, oxidized NAD (P) s and magnesium ions of the enzymatic reactions shown by enzymatic reactions 4 and 5 are used.
The amount of the cobalt ion or the manganese ion may be any amount as long as the enzymatic reaction proceeds smoothly, the type of substance in the test liquid to be measured, the content in the test liquid, the type of enzyme reaction to be conjugated, The concentration of ADP-HK and G6PDH is about 0.1 to 100 U / ml, preferably 1 to 50 U / ml, though it is appropriately adjusted depending on the reaction time and temperature.
It is about ml. The concentration of glucose and oxidized NAD (P) s may be a concentration sufficient to carry out an enzymatic reaction, and glucose is about 0.5 to 100 mM, preferably 1 to 50.
mM, oxidized NAD (P) s are about 0.5 to 50 mM, preferably about 1 to 10 mM, and the concentration of magnesium ion, cobalt ion or manganese ion is about 0.1 to 50 mM, preferably 0. .5-1
It is about 0 mM.

The method of the present invention comprises a suitable buffer solution (eg Tris-hydrochloric acid buffer solution, which does not adversely affect the enzyme reaction system).
Phosphate buffer, mono or diethanolamine buffer,
Good buffer, etc.) (eg pH 6-9, preferably pH
6.5-8). The measuring method is not particularly limited, and an endpoint method, a rate assay method, or the like can be appropriately used.

The test liquid to be measured may be a biological sample containing ADP produced or formed, and examples thereof include serum, plasma, urine and spinal fluid. As such a test liquid, 5 to 200 μl is usually used to carry out the reaction in the above reaction system, and the reaction temperature is, for example, 15 to 45 ° C., preferably 20 ° C. to 40 ° C.
It may be carried out under the reaction temperature condition of ℃, and the reaction time is 1 to 60 minutes, preferably 1 to 10 by the endpoint method.
In the rate assay method, the measurement is carried out within a time period in which the reaction is performed linearly, preferably for 2 to 3 minutes.

The amount of reduced NAD (P) s formed from the used oxidized NAD (Ps) can be measured by various methods, but usually, it can be measured simply and highly accurately. It is performed by an absorbance measurement method. The measurement wavelength is appropriately selected according to the type of reduced NAD (P),
It may be carried out preferably based on the wavelength of the maximum absorption wavelength region of each reduced NAD (P), for example, reduced NAD (P),
Reduced 3-acetyl-NAD (P), reduced deamino-
In the case of NAD (P) etc., 340 nm, reduced thio-
For NAD (P) a wavelength of 405 nm is selected.
As a method for measuring the amount of reduced NAD (P) s produced, phenazine methosulfate (PM) is used as an electron acceptor by using a tetrazonium salt such as indonitrotetrazonium (INT) or nitroblue tetrazonium (NTB).
S) or diaphorase (EC 1.6.4.3) may be used to form a formazan dye, and the coloration of this formazan dye may be measured. In addition, reduced N
The fluorescence of AD (P) s may be measured.

In the measurement of the substrate of an enzyme that produces ADP or one of its enzyme activities in a test solution that is a biological sample in the present invention, the enzyme that produces ADP or the substrate thereof is tested in the presence of ATP. In such a case, the kinase, synthetase, hydrolase or carboxylase and the substance serving as the substrate thereof in various biological samples can be led to ADP by the action of the kinase, synthetase, hydrolase or carboxylase. Therefore, by coupling these enzyme reaction systems with the enzyme reaction systems shown by the enzyme reactions 4 and 5, the amount of the enzyme activity or its substrate induced by ADP in the biological sample is reduced by the reduced NAD (P) s. Can be measured as the production amount of As an example of these, the relationship between the substrate and the enzyme used is shown in Table 1, and either the substrate or the enzyme activity involved in the reaction is measured, and the other component involved in the reaction (provided that the other component involved is involved). ATP may be used as a reaction reagent for the components involved in the reaction.

[0028]

[Table 1]

These reaction formulas are shown below. In the method for measuring either one of urea and urea amide hydrolase as a biological sample and using a reaction reagent as a component involved in the reaction, the reaction formula is shown below, and ADP produced by the enzymatic reaction is shown. Should be measured. The reaction reagents of the components involved in the reaction (however,
As the other component involved, ATP is not included and does not mean: the same shall apply hereinafter) means the components described on the left side and in the lower row of the arrow in the following reaction formula (hereinafter,
The same as the above), and the reaction reagents of the components involved in the reaction when urea is measured as a biological sample include urea amide hydrolase, water molecule, magnesium ion (magnesium chloride), potassium hydrogen carbonate, and biological sample. As the reaction reagents of the components involved in the reaction when measuring the urea amide hydrolase activity, urea, water molecule, magnesium ion (magnesium chloride), potassium hydrogen carbonate can be mentioned, provided that the water molecule is replaced by the molecule of the reaction medium. To be done.

[0030]

[Chemical 6]

In the method for measuring one of creatinine and creatinine amide hydrolase as a biological sample and using a component involved in the reaction as a reaction reagent, a reaction formula is shown below. In addition,
In the case of such a sequential enzymatic reaction, when the left side component and the right side component in the reaction formula are the same, it is the component involved in the reaction because it is a sequential reaction product of the product component in the sequential reaction. Obviously, it is not added separately as a reagent (the same applies hereinafter).

In the following reaction formula, in the method of measuring one of creatinine and creatinine amidohydrolase as a biological sample and measuring one of them, creatine produced by the enzymatic reaction is produced by the reaction of It means that it may be measured as ADP, or that creatine produced in the enzymatic reaction may be measured as ADP produced in the following reaction.

In the reaction system, creatinine amidohydrolase, water molecule, creatine kinase (CK) and magnesium ion are mentioned as the reaction reagents of the components involved in the reaction when creatinine is measured as a biological sample. In the reaction system, creatinine is used. Amidohydrolase,
Examples include water molecules, creatinamide hydrolase, urea, ureidoamide hydrolase, and magnesium ion.As a reaction reagent for components involved in the reaction when measuring creatinine amide hydrolase activity as a biological sample, creatinine, water molecule , Creatine kinase, magnesium ion, and the reaction system includes creatinine, water molecule, creatine amide hydrolase, urea, ureidoamide hydrolase, and magnesium ion.

[0034]

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In the method for measuring one of creatinine and creatinine deiminase as a biological sample and using the components involved in the reaction as reaction reagents, the reaction formulas are shown below and produced by enzymatic reaction. The ADP to be measured may be measured. In this case, creatinine deiminase is used as a reaction reagent for components involved in the reaction when creatinine is measured as a biological sample,
Water molecules and N-methylhydantoinase are listed, and reaction reagents of components involved in the reaction when creatinine deiminase activity is measured as a biological sample include creatinine, water molecules, and N-methylhydantoinase.

[0036]

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In the method for measuring either one of creatine and creatine kinase as a biological sample and using a component involved in the reaction as a reaction reagent, the reaction formula is shown below and the reaction is generated by an enzymatic reaction. ADP may be measured. In this case, the reaction reagents of the components involved in the reaction when measuring creatine as the biological sample include creatine kinase and magnesium ion, and the reaction of the components involved in the reaction when measuring the creatine kinase activity as the biological sample. Examples of the reagent include creatine and magnesium ion.

[0038]

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The creatine of reaction formula 9 may be creatine released based on the above reaction formula 7.
In the method for measuring either one of glycerol and glycerol kinase (GK) as a biological sample and using a component involved in the reaction as a reaction reagent, the reaction formula is shown below, and the reaction is generated by an enzymatic reaction. ADP may be measured. In this case, the reaction reagents of the components involved in the reaction when measuring glycerol as a biological sample include glycerol kinase and magnesium ion.

[0040]

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The glycerol may be glycerol liberated by the action of lipase or pancreatic lipase (however, Co-lipase is appropriately added as an activator) on triglyceride, mono- or diglyceride, or
Glycerol released by allowing phospholipase D to act on phosphatidylglycerol may be used. In particular, it is suitable for the purpose of quantifying triglyceride as a biochemical test item for in vivo components and for measuring pancreatic lipase activity using triglyceride or diglyceride as a synthetic substrate.

For example, lipase, water molecule, glycerol kinase, magnesium ion can be mentioned as the reaction reagents of the components involved in the reaction when measuring for the purpose of quantifying triglyceride as a biological sample. Further, as a reaction reagent of a component involved in the reaction when measuring for the purpose of measuring pancreatic lipase activity, triglyceride or diglyceride synthetic substrate, water molecule, glycerol kinase, magnesium ion, Co-lipase, solubilization of synthetic substrate as appropriate Examples of the agent include a surfactant.

In the method for measuring one of choline and choline kinase containing one of them as a biological sample,
The reaction formula when the components involved in the reaction are used as reaction reagents is shown below, and AD produced by the enzymatic reaction is shown.
It suffices to measure P.

[0044]

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It should be noted that choline is choline or choline ester released by acting phosphatidylcholine (phospholipid component) with phoslipase D, for example, cholinesterase released by acting cholinesterase on a synthetic substrate such as benzoylcholine or orthotoluoylcholine. May be In particular, it is suitable for the purpose of quantifying the phospholipid component as a biochemical test item and the purpose of measuring cholinesterase activity using a synthetic substrate.

The reaction reagents of the components involved in the reaction when measuring for the purpose of quantifying this phospholipid component include phoslipase D, water molecules and magnesium ions, and for the reaction when measuring for the purpose of measuring cholinesterase activity. Examples of the reaction reagents of the components involved include synthetic substrates such as benzoylcholine or orthotoluoylcholine, water molecules, and magnesium ions.

Similarly, in the method for measuring one of a substrate or an enzyme that produces ADP as a biological sample and measuring one of them, a reaction formula when a component involved in the reaction is used as a reaction reagent is as follows. The ADP produced by the enzymatic reaction can be measured as shown in FIG. Further, based on the completion of the measuring method of the present invention, the reaction reagents of the components involved in the reaction in the present invention are obvious from the above and below various reaction formulas, and are appropriately selected and prepared depending on the test liquid to be measured. It is also clear that these are possible, and these reaction reagents are in no way limited to those described.

[0048]

[Chemical 12]

[0049]

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[0050]

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[0051]

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[0052]

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[0053]

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[0054]

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[0055]

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[0056]

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[0057]

[Chemical 21]

[0058]

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[0059]

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[0060]

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[0061]

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[0062]

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[0063]

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[0064]

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[0065]

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The ammonia in the above reaction formula may be ammonia produced by the reaction of urea and urea amide hydrolase exemplified in the above reaction formula 6, and is exemplified in reaction formula 7. Ammonia produced by the reaction between creatinine and creatinine amide hydrolase in the reaction system of, or ammonia produced by the previous reaction for the creatinine and creatinine deiminase of Reaction Formula 8 may be used. The ammonia is not limited at all.

[0067]

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[0068]

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[0069]

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[0070]

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[0071]

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[0072]

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[0073]

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In the above reaction, it is particularly suitable for measuring GOT activity.

[0075]

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In the above reaction, it is particularly suitable for measuring GPT activity. AT added in these reactions
The amount of P is, for example, about 0.1 to 50 mM, preferably 0.1.
It is about 5 to 10 mM. In addition, when quantifying the substrate, the amount of enzyme to be added is about 1 to 100 U / ml.
Furthermore, the substrate to be added when measuring the enzyme activity is 1 to
It is about 100 mM. In the reaction reagents of components other than these substrates and enzymes involved in the reaction, the amount of the components consumed by reacting with the substrate may be the same as or twice the amount of the above substrate used, and the concentration of magnesium ion Is preferably about 0.1 to 50 mM, preferably about 0.5 to 10 mM as magnesium chloride, but it is not excluded to use an excess amount than these unless particularly inhibited in the reaction. .

These reactions may be carried out in the same reaction system as in the above reaction schemes 4 and 5, or may be carried out in different reaction systems, but preferably in the same reaction system.
In particular, in the case of urea, the existing measurement method is

[0078]

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As shown in the above reaction formula 38, the reduced NAD
This is a method of reducing (P), and therefore (8) the measured value is inaccurate when the component to be measured is small. (9) The upper limit of the measurable component is limited by the amount of reduced NAD (P) to be present in the reaction solution before the quantification (the measurement range is narrow). (10) It is necessary to change the amount of reduced NAD (P) to be present in the reaction solution before quantification according to the model of the spectrophotometer used for measuring the amount of reduced NAD (P). (11) Reduced NAD (P) contained in analytical reagent
Is unstable. (12) It is impossible to support a dry reagent. And the like.

Further, since this measuring method is a method for measuring ammonia in principle, (13) it is necessary to eliminate ammonia in the sample or reagent. On the other hand, the measuring method of the present invention is

[0081]

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[0082]

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[0083]

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As shown in the above reaction formulas 39, 40 and 41,
It was a method of increasing reduced NAD (P), had no drawbacks caused by the method of decreasing NAD (P), and was an excellent measurement method that did not require elimination of ammonia. In the case of creatine kinase, the existing measurement method is

[0085]

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The above reaction formula 42 is shown. In this reaction system, creatine phosphate, which is a substrate, is very expensive and there is a drawback that the cost of the measuring reagent is high. On the other hand, the measuring method of the present invention is

[0087]

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[0088]

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[0089]

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As shown in the above reaction formulas 43, 44 and 45,
Since creatine was used as the substrate, the measurement reagent could be provided at low cost. In the case of creatinine, the existing measurement method is

[0091]

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As shown in the above reaction formula 46, the oxidase method is used, and the measured values are inaccurate due to the influence of the reducing substances (ascorbic acid etc.) in the sample. Furthermore, sarcosine oxidase acts on proline, It has drawbacks such as being affected by the inside of proline. On the other hand, the measuring method of the present invention is

[0093]

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[0094]

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[0095]

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[0096]

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[0097]

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[0098]

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The above reaction schemes 47, 48, 49 and 50,
As shown by 51 and 52, this is a method of increasing reduced NAD (P), and is not affected by reducing substances (ascorbic acid etc.) and proline in the sample at all. In the case of triglyceride, the existing measurement method is

[0100]

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As shown in the above reaction formula 53, the oxidase method is used and the measured value is inaccurate because it is affected by the reducing substance (ascorbic acid etc.) in the sample. On the other hand,
The measuring method of the present invention is

[0102]

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[0103]

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[0104]

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As shown in the above reaction formulas 54, 55 and 56,
It is a method of increasing reduced NAD (P), and is not affected by reducing substances in the sample at all. The same applies to the case of diglyceride or monoglyceride instead of the triglyceride in the above reaction formula 54 (in these cases, a reaction formula in which 2 or 1 molecule of water molecule is consumed to release 2 or 1 molecule of fatty acid). is there.

In the case of phosphatidylcholine, the existing measurement method is

[0107]

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As shown in the above reaction formula 57, the measured value is inaccurate because it is the oxidase method and is affected by the reducing substance (ascorbic acid etc.) in the sample. On the other hand, the measuring method of the present invention is

[0109]

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[0110]

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[0111]

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As shown in the above reaction formulas 58, 59 and 60,
This is a method of increasing reduced NAD (P) and is not affected by reducing substances in the sample. The existing assay method for aspartate aminotransferase (GOT) is

[0113]

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As shown in the above reaction formula 61, the reduced NAD
Therefore, (14) the measured value is inaccurate when the component to be measured is small. (15) The upper limit of measurable components is limited by the amount of reduced NAD to be present in the reaction solution before quantification (measurement range is narrow). (16) It is necessary to change the amount of reduced NAD to be present in the reaction solution before quantification, depending on the model of the spectrophotometer used for measuring the amount of reduced NAD. (17) The reduced NAD contained in the analytical reagent is unstable. (18) It is impossible to deal with dry reagents. And the like. Furthermore, this measuring method was affected by lactate dehydrogenase in the sample and was not able to be accurately measured.

On the other hand, the measuring method of the present invention is

[0116]

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[0117]

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[0118]

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As shown in the above reaction formulas 62, 63 and 64,
It was a method of increasing reduced NAD (P), had no drawbacks caused by the method of decreasing NAD (P), and was an excellent measuring method that did not require elimination of lactate dehydrogenase in a sample. The existing measurement method for alanine aminotransferase (GPT) is

[0120]

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As shown in the above reaction formula 65, the reduced NAD
(19) The measured value is inaccurate when the component to be measured is small. (20) The upper limit value of measurable components is limited by the amount of reduced NAD to be present in the reaction solution before quantification. (Measurement range is narrow) (21) Depending on the model of the spectrophotometer used to measure the amount of reduced NAD, it is necessary to change the amount of reduced NAD to be present in the reaction solution before quantification. (22) The reduced NAD contained in the analytical reagent is unstable. (23) It is impossible to deal with dry reagents. And the like. Furthermore, this measuring method was affected by lactate dehydrogenase in the sample and was not able to be accurately measured. On the other hand, the measurement method of the present invention is

[0122]

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[0123]

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[0124]

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As shown in the above reaction formulas 66, 67 and 68,
It was a method of increasing reduced NAD (P), had no drawbacks caused by the method of decreasing NAD (P), and was an excellent measuring method that did not require elimination of lactate dehydrogenase in a sample. Hereinafter, the method for measuring the enzyme or its substrate for producing ADP in the biological sample of the present invention will be specifically described by way of example, but the method of the present invention is not limited thereto.

[0126]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail based on Examples and Reference Examples, but the present invention is not limited to these Examples and Reference Examples.

[0127]

[Reference example]

 ADP-HK enzyme activity measurement method Measurement reagent 50 mM Tris-hydrochloric acid buffer solution (pH 7.5) 20 mM Glucose 2 mM ADP 2 mM MgCl_Two  5 U / ml G6PDH 0.025% NBT (nitrotetrazonium blue) 1 mM Oxidized NADP 1% Triton X-100 5 U / ml Diaphorase (NADH) 1 ml of reagent was preheated at 37 ° C. for 1 minute, and then 0.
Add 02 ml of enzyme solution and react for 10 minutes. reaction
Thereafter, 2 ml of 0.1N hydrochloric acid was added to stop the reaction, and 5
Using a cell with a layer length of 1.0 cm within a
The absorbance at m is measured (As). Also blind
Same operation using 0.02 ml of distilled water instead of enzyme solution
Absorbance is measured (Ab).
Absorbance difference (A) between absorbance (As) and absorbance (Bb) of blind test
The enzyme activity is determined from s-Ab). One unit of enzyme activity is 3
Generate 1 μmol reduced NADP per minute at 7 ° C.
The amount of enzyme is used, and the calculation formula is as follows. Enzyme activity (U / ml) = (As-Ab) × 0.795 ×
Enzyme dilution ratio Acquisition of ADP-HK Pyrococcus furiosus DSM36
Culture medium composition of 0.1% yeast extract 0.5% tryptone 0.72% maltose 2.39% NaCl 0.4% Na_TwoSO_Four 0.07% KCl 0.02% NaHCO_Three 0.01% KBr 0.03% H_ThreeBO_Four 1.08% MgCl_Two 0.15% CaCl_Two 0.0025% SrCl_Two 0.025% NH_FourCl 0.014% K_TwoHPO_Four 0.1% CH_ThreeCOONa 0.0015% N (COOH)_Three 0.0005% MnSO_Four 0.0014% FeSO_Four 0.0002% NiCl_Two 0.0001% CoSO_Four 0.0001% ZnSO_Four 0.00001% CuSO_Four 0.000001% Na_TwoWO_Four 0.000001% Na_TwoMoO_Four 0.1% cysteine hydrochloride 500 ml of a liquid medium (pH 7) containing the above medium components
Dispense into 10 00 ml Erlenmeyer flasks at 120 ° C, 2
After heat sterilization for 0 minutes, Pyrococcus
・ Furiosus ・ DSM3638 strain 1
Transfer 0 ml and incubate at 95 ° C for 20 hours while stirring.
Nourishing was used as a seed culture. Liquid medium 2 containing the above medium components
After sterilizing the 00l / 300l tank, transplant the seed culture
Culturing at 95 ° C. for 15 hours while stirring, and 5 mU
200 l / ml of culture were obtained.

Purification of ADP-HK 200 l of the obtained culture solution was centrifuged, and the obtained cells were washed once with 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 0.9% NaCl. did. 20m for washed cells
M Tris-HCl buffer (pH 7.5)
And the ultrasonic crusher manufactured by Kubota (INSONAT)
OR 201M) at 180 W for 30 minutes to obtain a disrupted cell suspension.

The disrupted solution was centrifuged at 8000 rpm for 30 minutes to obtain 1.8 l (enzyme activity 980 U) of supernatant. The supernatant was dialyzed overnight at 5 ° C. against 8 liters of 10 mM Tris-HCl buffer (pH 7.5) using a dialysis tube, and then 10 mM Tris-HCl buffer (pH 7.0).
DEAE-Sepharose FF buffered in 5)
(Pharmacia) 200ml (2.6 × 38cm)
And elution was performed with a linear gradient of 0 to 1 mol of NaCl. As a result, an active fraction (950 U) was eluted at a NaCl concentration of 0.08 to 0.1 mol. The active fraction thus obtained was adjusted to 4M with NaCl.
Phenyl buffered with 4M NaCl
-Sepharose FF (made by Pharmacia) 20
Pass through a 0 ml (2.6 x 38 cm) column, 4-0M
Elution was performed with a linear gradient of NaCl.

As a result, 0.02 to 0.07 mol of N
An active fraction (900 U) was obtained at an aCl concentration. The obtained active fraction was treated with 10 mM Tris-hydrochloric acid (pH 7.5).
After dialysis against 8 liters at 5 ° C. overnight, 100 ml (2.6 × 19 cm) of hydroxyapatite (manufactured by Pentax) buffered with 10 mM Tris-hydrochloric acid buffer (pH 7.5).
Through a column of 0-0.5M phosphate buffer (pH
Elution was performed with a linear gradient of 7.5). As a result, an active fraction (850 U) was eluted at a phosphate buffer concentration of 0.02 to 0.03 M. This enzyme solution was freeze-dried to obtain 5 mg of enzyme powder (170 U / mg).

The physicochemical properties of ADP-HK were as follows. Physicochemical properties of ADP-HK (1) Enzyme action The enzyme action using glucose as a substrate is shown below.

[0132]

Embedded image

(2) Molecular Weight Tosoh TSK-G3000SW _XL (0.75 × 3)
ADP-H measured by a gel filtration method using
The molecular weight of K was 100,000 ± 5000. (3) Optimum pH is pH 6.0-7.0 (phosphate buffer)
Met.

(4) Since the optimum temperature was 80 to 100 ° C., it was confirmed to be highly thermophilic ADP-HK.

[0135]

Example 1 Quantitative determination of urea using magnesium ion at 37 ° C. Reagent 50 mM Tris-hydrochloric acid buffer (pH 7.5) 30 U / ml urea amide lyase 2 mM ATP 10 mM KCl 8 mM KHCO ₃ 20 mM glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM MgCl ₂ 5 U / ml ADP-HK measurement method Urea 2.6 mM, 5.2 mM, 7.8 mM, 10.4
A urea sample was prepared by adjusting to an aqueous solution of mM and 13 mM. Add 20 μl of urea sample to 1 ml of measuring reagent
The absorbance at 340 nm after heating at 7 ° C. for 5 minutes was measured using a reagent blank as a control. As shown in FIG. 1, urea could be quantitatively measured.

[0136]

Example 2 Quantification of creatinine using magnesium ion at 37 ° C. (1) Measuring reagent 50 mM Tris-HCl buffer (pH 7.5) 100 U / ml creatinine amide hydrolase 5 U / ml creatine kinase 2 mM ATP 20 mM glucose 5U / Ml G6PDH 1 mM Oxidized NADP 2 mM MgCl ₂ 5 U / ml ADP-HK Method of measurement Creatinine 2.6 mM, 5.2 mM, 7.8 mM,
A creatinine sample was prepared by adjusting to an aqueous solution of 10.4 mM and 13 mM. 20 μl of creatinine sample was added to 1 ml of the measurement reagent, and the mixture was heated at 37 ° C. for 5 minutes and then 34
Absorbance at 0 nm was measured using a reagent blank as a control. As shown in FIG. 2, creatinine could be quantitatively measured.

[0137]

Example 3 Quantification of creatinine using magnesium ion at 37 ° C. (2) Measuring reagent 50 mM Tris-hydrochloric acid buffer (pH 7.5) 10 U / ml creatinine deiminase 10 U / ml N-methylhydantoinase 2 mM ATP 20 mM Glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM MgCl ₂ 5 U / ml ADP-HK Method of measurement Creatinine 2.7 mM, 5.3 mM, 7.9 mM,
A creatinine sample was prepared by adjusting to an aqueous solution of 10.5 mM and 13 mM. 20 μl of creatinine sample was added to 1 ml of the measurement reagent, and the mixture was heated at 37 ° C. for 5 minutes and then 34
Absorbance at 0 nm was measured using a reagent blank as a control. Creatinine could be quantitatively measured as shown in FIG.

[0138]

Example 4 Measurement of creatine using magnesium ion at 37 ° C. Measurement reagent 50 mM Tris-HCl buffer (pH 7.5) 5 U / ml creatine kinase 2 mM ATP 20 mM glucose 5 U / ml G6PDH 1 mM oxidized NADP 2 mM MgCl ₂ 5U / ml ADP-HK measuring method creatine 2.6mM, 5.2mM, 7.8mM, 1
A creatine sample was prepared by adjusting to 0.4 mM and 13 mM aqueous solutions. 20 μl of creatine sample was added to 1 ml of measurement reagent, and heated at 37 ° C. for 5 minutes to 340 nm.
Absorbance was measured using the reagent blank as a control. As shown in FIG. 4, creatine could be quantitatively measured.

[0139]

Example 5 Measurement of Glycerol When Using Magnesium Ion at 37 ° C. Measurement Reagent 50 mM Tris-HCl Buffer (pH 7.5) 5 U / ml Glycerol Kinase 2 mM ATP 20 mM Glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM MgCl ₂ 5U / ml ADP-HK measuring method 2.6mM glycerol, 5.2 mM, 7.8 mM,
A glycerol sample was prepared by adjusting to an aqueous solution of 10.4 mM and 13 mM. 20 μl of glycerol sample was added to 1 ml of measurement reagent, and the mixture was heated at 37 ° C. for 5 minutes and
Absorbance at 0 nm was measured using a reagent blank as a control. Glycerol could be quantitatively measured as shown in FIG. Further, glycerol released by allowing lipase to act on triglyceride and glycerol released by allowing phospholipase D to act on phosphatidylglycerol could also be measured.

[0140]

Example 6 Measurement of choline using magnesium ion at 37 ° C. Measurement reagent 50 mM Tris-hydrochloric acid buffer solution (pH 7.5) 5 U / ml choline kinase 2 mM ATP 20 mM glucose 5 U / ml G6PDH 1 mM oxidized NADP 2 mM MgCl ₂ 5U / ml ADP-HK measuring method choline chloride 2.6mM, 5.2mM, 7.8mM, 1
A choline chloride sample was prepared by adjusting to 0.4 mM and 13 mM aqueous solutions. 20 μl of choline chloride sample was added to 1 ml of the measurement reagent, and the mixture was heated at 37 ° C. for 5 minutes and then 340 nm.
Absorbance was measured using the reagent blank as a control. As shown in FIG. 6, choline could be quantitatively measured. In addition, phospholipase D was allowed to act on phospholipids, and the released choline could also be measured.

[0141]

Example 7 Measurement of L-Glutamic Acid When Using Magnesium Ion at 37 ° C. Measurement Reagent 50 mM Tris-HCl buffer (PH 7.5) 10 U / ml Glutamine synthetase 10 mM NH ₄ Cl 2 mM ATP 20 mM Glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM MgCl ₂ 5 U / ml ADP-HK measurement method L-glutamic acid was 2.6 mM, 5.2 mM, 7.8 m
An L-glutamic acid sample was prepared by preparing an M, 10.4 mM, 13 mM aqueous solution. L- for measuring reagent 1 ml
20 μl of a glutamic acid sample was added, and the absorbance at 340 nm after heating at 37 ° C. for 5 minutes was measured using the reagent blank as a control. As shown in FIG. 7, L-glutamic acid could be quantitatively measured. In addition, aspartate transferase (GOT) and alanine transferase (GP)
L-glutamic acid released by the action of T) could also be measured.

[0142]

Example 8 Quantification of urea when using cobalt ion at 37 ° C. Reagent 50 mM Tris-hydrochloric acid buffer (pH 7.5) 30 U / ml urea amide lyase 2 mM ATP 10 mM KCl 8 mM KHCO ₃ 20 mM glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM CoCl ₂ 10 U / ml ADP-HK measuring method Urea 2.3 mM, 4.6 mM, 6.9 mM, 9.2 m
A urea sample was prepared by adjusting to an aqueous solution of 11.5 mM of M. Add 20 μl of urea sample to 1 ml of measuring reagent,
The absorbance at 340 nm after heating at 37 ° C for 5 minutes was measured using a reagent blank as a control. As shown in FIG. 8, urea could be quantitatively measured.

[0143]

Example 9 Quantitative Assay for Creatinine Using Manganese Ion at 37 ° C. Reagent 50 mM Tris-HCl buffer (pH 7.5) 100 U / ml creatinine amide hydrolase 5 U / ml creatine kinase 2 mM ATP 20 mM glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM MnCl ₂ 10 U / ml ADP-HK Method of measurement Creatinine 2.5 mM, 5.0 mM, 7.5 mM,
A creatinine sample was prepared by adjusting to an aqueous solution of 10.0 mM and 12.5 mM. 20 μl of creatinine sample was added to 1 ml of the measurement reagent, and the absorbance at 340 nm after heating at 37 ° C. for 5 minutes was measured using the reagent blank as a control. As shown in FIG. 9, creatinine could be quantitatively measured.

[0144]

Example 10 Measurement of creatine using magnesium ion at 20 ° C. Measurement reagent 50 mM Tris-HCl buffer (pH 7.5) 5 U / ml creatine kinase 2 mM ATP 20 mM glucose 5 U / ml G6PDH 1 mM oxidized NADP 2 mM MgCl ₂ 20 U / ml ADP-HK measurement method Creatine 2.8 mM, 5.6 mM, 8.4 mM, 1
A creatine sample was prepared by adjusting to 1.2 mM and 14 mM aqueous solutions. 20 μl of creatine sample was added to 1 ml of measurement reagent, and heated at 20 ° C. for 5 minutes to 340 nm.
Absorbance was measured using the reagent blank as a control. As shown in FIG. 10, creatine could be quantitatively measured.

[0145]

Example 11 Measurement of Glycerol When Using Magnesium Ion at 40 ° C. Measurement Reagent 50 mM Tris-HCl Buffer (pH 7.5) 5 U / ml Glycerol Kinase 2 mM ATP 20 mM Glucose 5 U / ml G6PDH 1 mM Oxidized NADP 2 mM MgCl ₂ 5U / ml ADP-HK measuring method 2.7mM glycerol, 5.4 mM, 8.1 mM,
A glycerol sample was prepared by adjusting to an aqueous solution of 10.8 mM and 13.5 mM. 20 μl of a glycerol sample was added to 1 ml of the measurement reagent, and the absorbance at 340 nm after heating at 40 ° C. for 5 minutes was measured using the reagent blank as a control. As shown in FIG. 11, glycerol could be quantitatively measured. Further, glycerol released by allowing lipase to act on triglyceride and glycerol released by allowing phospholipase D to act on phosphatidylglycerol could also be measured.

[0146]

According to the measuring method of the present invention, the reduced NAD is used.
Since biological substances are measured based on the amount of (P) produced, the measurement limit is high, and since reduced NAD (P) whose molecular extinction coefficient is clear is measured, the reliability of the measured values is high. high. Furthermore, the measuring method of the present invention has an advantage that it is not affected by a reducing substance in a sample. Therefore, according to the present invention, the enzyme that produces ADP and its substrate in a biological sample can be simply and highly accurately measured.

[Brief description of drawings]

FIG. 1 shows a calibration curve for urea measurement when magnesium ions were used at 37 ° C.

FIG. 2 shows a calibration curve for creatinine measurement (1) when magnesium ions were used at 37 ° C.

FIG. 3 shows a calibration curve for creatinine measurement (2) when magnesium ions were used at 37 ° C.

FIG. 4 shows a calibration curve for creatine measurement when magnesium ions were used at 37 ° C.

FIG. 5 shows a calibration curve for glycerol measurement using magnesium ions at 37 ° C.

FIG. 6 shows a calibration curve for choline measurement when magnesium ions were used at 37 ° C.

FIG. 7 shows a calibration curve for glutamic acid measurement using magnesium ions at 37 ° C.

FIG. 8 shows a calibration curve for urea measurement when cobalt ions were used at 37 ° C.

FIG. 9 shows a calibration curve for creatinine measurement when manganese ions were used at 37 ° C.

FIG. 10 shows a calibration curve for creatine measurement when magnesium ions were used at 20 ° C.

FIG. 11 shows a calibration curve for glycerol measurement using magnesium ions at 40 ° C.

Claims (15)

[Claims] 1. A method for measuring one of an enzyme that produces ADP and its substrate in a test solution by using an enzymatic reaction, wherein at least the test solution containing one of the enzyme that produces ADP and its substrate is ADP. Reagents for components involved in reactions based on the enzyme that produces glucose, its substrate, ATP, glucose, ADP-dependent hexokinase, oxidized NAD
(P), glucose-6-phosphate dehydrogenase, and magnesium ion, cobalt ion, or manganese ion in the presence of any one or more ion-releasing salts at a temperature condition of 15 to 45 ° C. A method of reacting an enzyme that produces ADP or a substrate thereof in a test solution together with AMP produced by the reaction, and measuring the amount of reduced NAD (P) s. 2. The measuring method according to claim 1, wherein the enzyme that produces ADP is urea amide hydrolase, and the substrate thereof is urea. 3. The method according to claim 1, wherein the enzyme that produces ADP is creatine kinase, and the substrate thereof is creatine. 4. The method for measuring according to claim 3, wherein the creatine is creatine released by the action of creatinine on creatinine. 5. The method according to claim 1, wherein the enzyme that produces ADP is N-methylhydantoinase, and the substrate thereof is N-methylhydantoin. 6. The method according to claim 5, wherein the N-methylhydantoin is N-methylhydantoin released by the action of creatinine deiminase on creatinine. 7. The enzyme producing ADP is glycerol kinase, and its substrate is glycerol.
The described measuring method. 8. The method according to claim 7, wherein the glycerol is glycerol released from triglyceride, diglyceride or monoglyceride by the action of lipase or glycerol released from phosphatidylglycerol by the action of phospholipase D. 9. The method according to claim 1, wherein the enzyme that produces ADP is choline kinase, and its substrate is choline. 10. The method according to claim 9, wherein choline is phosphatidylcholine by the action of phospholipase D or choline released from choline ester by the action of cholinesterase. 11. The method according to claim 1, wherein the enzyme that produces ADP is glutamine synthetase or glutamate kinase, and its substrate is L-glutamic acid. 12. The method according to claim 11, wherein L-glutamic acid is L-glutamic acid formed from L-aspartic acid and α-ketoglutarate by the action of aspartate aminotransferase. 13. The method according to claim 11, wherein L-glutamic acid is L-glutamic acid formed from L-alanine and α-ketoglutarate by the action of alanine aminotransferase. 14. The method for measuring according to claim 1, wherein the ADP-dependent hexokinase is a highly thermophilic ADP-dependent hexokinase. 15. The measuring method according to claim 1, wherein the temperature condition is 20 to 40 ° C.