JPH0576394A - Composition for potassium ion determination - Google Patents

Abstract

PURPOSE:To provide the title composition excellent in operability, quantifying capability and accuracy, comprising pyruvate kinase, glycerol kinase, glycerol triphosphate oxidase, peroxidase, reduced-type chromogen, adenosine diphosphate (salt), etc. CONSTITUTION:The objective composition comprising (A) pyruvate kinase (pref. derived from rabbit muscle), (B) glycerol kinase (pref. derived from Cellulomonas sp), (C) glycerol triphosphate oxidase (pref. derived from microorganisms), (D) peroxidase (pref. derived from horseradish), (E) reduced-type chromogen (e.g. 4-aminoantipyrine), (F) adenosine diphosphate (salt), and (G) phosphoenolpyruvate (salt). It is preferable that the present composition be maintained at pH 6-9 using a buffer solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition for measuring potassium ion. The measurement of potassium ion in body fluids is of great clinical significance as it provides useful information on renal diseases such as acute renal failure and chronic renal failure, and endocrine diseases such as primary aldosteronism and secondary aldosteronism.

[0002]

2. Description of the Related Art Conventionally, flame photometry, chemical measurement, and ion selective electrode method have been used as methods for measuring metal ions including potassium ions. However,
The flame photometer method is complicated in operation and has a problem in sample throughput. The chemical assay method has a problem that the operation is complicated and the reagent is expensive, and thus it is not practically used at the clinical examination site. The ion selective electrode method is relatively easy to operate, but has a problem that an error occurs during measurement due to deterioration of the electrode. Recently, a method for measuring potassium ion by an enzymatic method has been reported (Clin. Chem. 198).
9; 35: 817-820). However, since the method according to this report is a method of measuring the decrease in the ultraviolet absorbance (around 340 nm) of nicotine adenine dinucleoside (NADH), NADH decreases during storage, and it becomes impossible to measure high-value samples. Since the reaction is a decreasing reaction, the sensitivity cannot be increased above a certain level, or as a means for avoiding the influence of ammonium ion, glutamate dehydrogenase is used to eliminate ammonium ion by the following chemical formula 1. Since the measurement wavelength of the potassium ion concentration and the measurement wavelength of the ammonium ion erasing reaction overlap, there is a problem that it is difficult to set the absorbance or the wavelength when erasing the high concentration ammonium ion.

[Chemical 1]

[0003]

In view of the above situation, an object of the present invention is to provide a composition for enzymatically measuring potassium ion concentration, which is excellent in operability, quantification and accuracy.

[0004]

Means for Solving the Problems The inventors of the present invention have made earnest studies on a method for measuring the concentration of potassium ion in a sample using an enzyme activated by potassium ion. It was found that adenosine triphosphate can be quantified using glycerol kinase, glycerol triphosphate oxidase, peroxidase.

That is, the gist of the first invention of the present invention is: a) pyruvate kinase, b) glycerol kinase,
c) glycerol triphosphate oxidase, d) peroxidase, e) reduced chromogen, f) adenosine diphosphate or a salt thereof, and g) phosphoenolpyruvate or a salt thereof, which is characterized by containing potassium ion It exists in the composition for measurement.

When a sample is measured using this measuring system,
Usually, there is no problem, but it was found that adding ammonium ion in excess has a positive effect. Therefore, as a result of studying a method for avoiding the influence of ammonium ions, it was found that the use of glutamate dehydrogenase can effectively eliminate low-concentration ammonium ions. However, when measuring a sample to which a high concentration of ammonium ions is added, in order to completely eliminate the ammonium ions, it is necessary to use a high concentration of NADH or NAD.
It was found that the addition of PH was necessary. Since NADH or NADPH has a reducing action, NA of high concentration is used.
The presence of DH or NADPH negatively affects the color development reaction system using peroxidase, and sufficient sensitivity cannot be obtained.

As a result of further investigation, NAD oxidized by a reaction using glutamate dehydrogenase.
It was found that H or NADPH can be used for the reaction using glucose dehydrogenase. In other words, by recycling NADH or NADPH, a reaction system for effectively eliminating high-concentration ammonium ions using low-concentration NADH or NADPH has been completed.

Further, by incorporating this ammonium ion elimination reaction system into the above-mentioned potassium ion measuring system, a more accurate potassium ion measuring system has been completed.

That is, the gist of the second invention of the present invention is as follows.
A composition for measuring potassium ion, which comprises the following composition A and composition B. A a) pyruvate kinase, b) glycerol kinase,
A composition containing c) glycerol triphosphate oxidase, d) peroxidase, e) reduced chromogen, f) adenosine diphosphate or a salt thereof, and g) phosphoenolpyruvate or a salt thereof. A composition comprising B a) glutamate dehydrogenase, b) glucose dehydrogenase and c) reduced nicotine adenine dinucleoside (NADH) or reduced nicotine adenine dinucleoside phosphate (NADPH).
The above composition B is a composition for erasing ammonium ions, and is capable of effectively erasing a high concentration of ammonium ions.

As a method for measuring potassium ion using pyruvate kinase, pyruvic acid produced by the reaction using pyruvate kinase is converted into hydrogen peroxide using pyruvate oxidase, and quinoid is produced in the presence of chromogen and peroxidase. Although there is a method of forming a dye and performing colorimetric determination, this method is difficult to be commercially used because it is easily affected by endogenous pyruvate and the cost of pyruvate oxidase is high.

As a method of eliminating ammonium ions using a composition other than the present invention, there is a method of eliminating ammonium ions by combining glutamate dehydrogenase and isocitrate dehydrogenase. In this method, ammonium ion is effectively used. Although it is possible to eliminate ions, isocitrate, which is a substrate of isocitrate dehydrogenase, is more expensive than glucose, and it is necessary to add a metal chelating agent to stop the reaction of isocitrate dehydrogenase. Since the addition has a negative influence on the measurement of potassium ions, it is necessary to limit the addition as much as possible in the measurement of metal ions as well as the measurement of potassium ions.

Therefore, it is difficult to use these methods commercially for measuring potassium ion, and the method for measuring potassium ion using the composition of the present invention is excellent in accuracy, precision, convenience and cost. ing.

As a method for measuring potassium ion in a sample using the above composition, there is a method using a reaction system exemplified below.

[0014]

[Chemical 2]

[0015]

[Chemical 3] PEP Phosphoenolpyruvate GK Glycerol Kinase ADP Adenosine Diphosphate G3POD Glycerotriphosphate Oxidase ATP Adenosine Triphosphate POD Peroxidase 4-AA 4-Aminoantipyrine G1DH Glutamate P Hydrogluconate Dehydrogenase α-KG α-Ketoglutin G Hydroglutanate Acid kinase

The origin of the pyruvate kinase used in the present invention is not particularly limited, and for example, one obtained from rabbit muscle or bovine muscle may be used. The one derived from rabbit muscle is preferably used. The origin of the glycerol kinase used in the present invention is not particularly limited, and may be, for example, Candida Mycoderma.
coderma) and Cellulomonas sp.
lomonas sp.) is used. Suitably, Cell
Those derived from ulomonas sp. are used. The origin of the glycerol triphosphate oxidase used in the present invention is not particularly limited, but a microorganism-derived one is preferably used. The origin of the peroxidase used in the present invention is not particularly limited, but those derived from horseradish are preferably used. The origin of glutamate dehydrogenase used in the present invention is not particularly limited, and bacteria, yeast, and those derived from bovine liver are used,
Those derived from bacteria are preferably used. The origin of the glucose dehydrogenase used in the present invention is not particularly limited, but a microorganism-derived one is preferably used.

The reducing chromogen used in the present invention is not particularly limited. For example, a coupler such as 4-aminoantipyrine, 3-methyl-2-benzothiazoline hydrazone and a phenol derivative such as phenol, 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol or aniline, N, N-dimethyl- m-anisidine, N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N-
Ethyl-N- (β-hydroxyethyl) -m-toluidine, N-ethyl-N- (hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl -N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N- (2
-Hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-m-
Anisidine, N-ethyl-N- (3-methylphenyl)
-N'-succinylethylenediamine, N-ethyl-N
A combination of aniline derivatives such as-(2-hydroxy-3-sulfopropyl) -m-anisidine, or 10-N-
Methylcarbamoyl-3,7-dimethylamino-10H
-Phenothiazine, bis [3-bis (4-chlorophenyl) methyl-4-dimethyl-aminophenyl] amine,
4,4-bis (dimethylamino) diphenyl- (2,7
Leuco dyes such as -dihydroxy-1-naphthyl) methane.

The adenosine diphosphate and phosoenolpyruvate used in the present invention are not particularly limited as long as they do not contain potassium ions, and sodium salts, trishydroxymethylaminomethane salts, cyclohexylamine and the like salts are usable. Can be mentioned. The reduced nicotine adenine dinucleoside (NADH) and the reduced nicotine adenine dinucleoside phosphate (NADPH) are not particularly limited, and any one containing no potassium ion can be used.

The pH of the reagent of the present invention depends on the pH of the buffer solution.
It is preferably maintained at 6 to 9, and any buffer may be used as long as it does not contain potassium ions. Examples include triethanolamine buffer, GOOD buffer, and Tris buffer.

If necessary, the reagent of the present invention contains a surfactant,
Preservatives, stabilizers, enzyme activators and the like may be added. As the surfactant, a nonionic surfactant is preferably used. As the preservative, NaN3 or an antibiotic is preferably used. The stabilizer and the enzyme activator are not particularly limited as long as they are effective. Examples thereof include albumin, FAD, magnesium ion and the like.

The enzyme concentration used in the measurement of pyruvate kinase used in the present invention is not particularly limited as long as it is a concentration suitable for the measurement, but it is 0.01-10 u / ml.
It is preferably used in the range of. Pyruvate oxidase is preferably used in the range of 1 to 30 u / ml. Glycerol kinase is preferably used in the range of 0.05 to 50 u / ml. Glycerol triphosphate oxidase is preferably used in the range of 0.1 to 50 u / ml.
Peroxidase is preferably used in the range of 1 to 100 u / ml. Phosphoenolpynvic acid or a salt thereof, adenosine diphosphate or a salt thereof, and a reduced chromogen are not particularly limited as long as they are concentrations suitable for measurement, but phosphoenolpyruvate or a salt thereof is not limited. It is preferably used in the range of 0.5 to 5 mM, and adenosine diphosphate or a salt thereof is preferably used in the range of 1 to 20 mM. The reduced chromogen is preferably used in the range of 0.01 to 10 mM. The reduced nicotine adenine dinucleoside (NADH) and the reduced nicotine adenine dinucleoside phosphate (NADPH) are not particularly limited as long as they have a concentration suitable for measurement, but are preferably used in the range of 0.05 to 0.5 mM. Be done.

The method for measuring potassium ion using the composition for measuring potassium ion of the present invention is as follows, wherein the sample is reacted with a reagent containing pyruvate kinase, glycerol kinase and glycerol triphosphate oxidase. There is a method of producing H ₂ O ₂ in the presence of a chromogen and peroxidase to form a quinoid pigment and performing colorimetric determination. The composition A and the composition B of the second invention may be mixed from the beginning and used as a one-pack type reagent, or may be separately used as a two-pack type reagent. The conditions for measurement using the composition of the present invention are not particularly limited, but the reaction temperature is preferably 10 to 40 ° C, preferably 25 ° C or 37 ° C. The reaction time is preferably 0 to 10 minutes. It is desirable that the measurement wavelength is around λ max of the colored dye.

[0023]

【Example】

Example 1 The potassium ion concentration in a sample was measured by the following measuring method. As a control, the same sample was measured by the electrode method. Reagent A 0.1 M triethanolamine-HCl buffer pH 7.6 0.1% Triton X-100 5 mM MgCl ₂ 0.2 M glycerin 4 mM ADP 0.7 mM PEP tris salt 0.5 mM 4-aminoantipyrine 2 mM phenol 0. 4 u / ml pyruvate kinase 3.0 u / ml glycerol kinase 10 u / ml glycerotriphosphate oxidase 30 u / ml peroxidase

Reagent B 0.1 M triethanolamine-HCl buffer pH 7.6 0.1% Triton X-100 5 mM MgCl ₂ 0.2 M glycerin 4 mM ADP 0.7 mM PEP tris salt 0.5 mM 4-aminoantipyrine 2 mM Phenol 0.4u / ml Pyruvate kinase 3.0u / ml Glycerol kinase 10u / ml Glycerotriphosphate oxidase 30u / ml Peroxidase 50mM Glucose 5mM α-Ketoglutanate 0.2mM NADPH 50u / ml Glutamate dehydrogenase 10u / ml Glucose dehydrogenase

Measurement method Using a 10-step diluted solution of 100 mM potassium chloride aqueous solution and a 10-step diluted solution of serum as samples, 40 μl of each was sampled, 4 ml of the above reagent was added thereto, and the mixture was reacted at 37 ° C. for 10 minutes to give 400 nm. The time course (behavior of the enzymatic reaction proceeding at the measurement wavelength) and the change in absorbance for 1 minute were determined. The blank used distilled water instead of the potassium ion-containing test liquid. 100 mM in Figure 1
Figure 2 shows the time course of the potassium chloride aqueous solution and the serum sample.
3 shows the linearity of dilution between the 100 mM potassium chloride aqueous solution and the serum sample. As is clear from the figure, even if potassium chloride aqueous solution serum is used as a sample, the present invention can accurately and easily measure potassium ions in a short time. Furthermore, ammonium chloride aqueous solution 100 mM 10
The same measurement was performed using the serial dilution as a sample. 4 and 5 show the time courses of the 100 mM potassium chloride aqueous solution and the 100 mM ammonium chloride aqueous solution.

Tables 1 and 2 show the results of measuring each sample by the reagents A and B and the electrode method.

[Table 1]

[0027]

[Table 2]

Table 3 shows the results of measuring the samples obtained by adding 100 mM potassium chloride aqueous solution and 100 mM ammonium chloride aqueous solution to live serum by the reagents A and B and the electrode method.

[Table 3]

[0029]

Industrial Applicability According to the present invention, there is provided a composition for enzymatically measuring potassium ion concentration, which is excellent in operability, quantification and accuracy.

[Brief description of drawings]

FIG. 1 shows a time course of a 100 mM potassium chloride aqueous solution and a serum sample.

FIG. 2 shows the dilution linearity of a 100 mM potassium chloride aqueous solution.

FIG. 3 shows the dilution linearity of serum samples.

FIG. 4 shows time courses of a 100 mM potassium chloride aqueous solution and a 100 mM ammonium chloride aqueous solution when Reagent A is used.

FIG. 5 shows time courses of 100 mM potassium chloride aqueous solution and 100 mM ammonium chloride aqueous solution when reagent B is used.

Claims (2)

[Claims] 1. A) pyruvate kinase, b) glycerol kinase, c) glycerol triphosphate oxidase, d) peroxidase, e) reduced chromogen, f) adenosine diphosphate or a salt thereof, and g) phospho. A composition for measuring potassium ion, which comprises enolpyruvic acid or a salt thereof. 2. A composition for measuring potassium ion, comprising the following composition A and composition B. A a) pyruvate kinase, b) glycerol kinase,
A composition containing c) glycerol triphosphate oxidase, d) peroxidase, e) reduced chromogen, f) adenosine diphosphate or a salt thereof, and g) phosphoenolpyruvate or a salt thereof. A composition comprising B a) glutamate dehydrogenase, b) glucose dehydrogenase and c) reduced nicotine adenine dinucleoside (NADH) or reduced nicotine adenine dinucleoside phosphate (NADPH).