JP3021611B2 - Composition for potassium ion measurement - Google Patents

Description

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

(Prior Art) Conventionally, potassium ion has been measured by a flame photometer method or an ion electrode method.

However, the operation of the flame photometer method is complicated, and there is a problem in the throughput of the sample.

Further, in the method using the ion electrode method, although the operation is relatively simple, the electrode is deteriorated, and a measurement error at the time of measuring potassium ions has been a problem.

In addition, as a method for measuring potassium ions by the enzymatic method,
A method for measuring the enzyme activation ability of pyruvate kinase by potassium ions using pyruvate kinase and lactate dehydrogenase has been reported (clin, chem. 19).
89; 35: 817-820).

However, this method is a method for measuring a decrease in the absorbance of NADH in the ultraviolet (340 nm), has a low measurement sensitivity, and has a problem of being affected by endogenous lactic acid and the like.

(Problems to be Solved by the Invention) An object of the present invention is to provide a composition for enzymatic measurement of potassium ion which is excellent in operability, quantitativeness, and accuracy. Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and found that pyruvate kinase and pyruvate oxidase or pyruvate dehydrogenase utilize potassium in body fluids. The inventors have found that the ion concentration can be measured easily and with high sensitivity in a short time, and have completed the present invention.

That is, the gist of the present invention is a potassium ion comprising (a) pyruvate kinase, (b) pyruvate oxidase or pyruvate dehydrogenase and (c) adenosine-5'-diphosphate or a salt thereof. In the composition for measurement.

The potassium ion measuring composition of the present invention contains the above-mentioned components (a), (b) and (c) as essential components, but more specifically as a combination with other components. Are preferably compositions such as the following (1) and (2).

(1) (a) pyruvate kinase, (b) pyruvate oxidase, (c) adenosine-5'-diphosphate (AD
P) or a salt thereof, (d) thiamine pyrophosphate or a salt thereof, (e) phosphoric acid or a salt thereof, (f) phosphoenolpyruvate or a salt thereof, (g) reduced chromogen, and (h) peneoxidase A composition for measuring potassium ions, comprising:

(2) (a) pyruvate kinase, (b) pyruvate dehydrogenase, (c) adenosine-5'-diphosphate (ADP) or a salt thereof, (d) thiamine pyrophosphate,
A composition for measuring potassium ion, comprising (e) phosphoric acid or a salt thereof, (f) phosphoenolpyruvate, (g) an oxidized acceptor, and (h) a formazan chromogen.

The composition of the above (1) utilizes the following reaction.

The composition of the above (2) utilizes the following reaction.

Pyruvate kinase which is an enzyme used in the present invention,
The origin of pyruvate oxidase and pyruvate tehydrogenase is not particularly limited. For example, there are pyruvate kinases obtained from rabbits, dogs, pigs, microorganisms and the like, and those derived from rabbit muscle are preferably used. Pyruvate oxidase is obtained from microorganisms and the like, and those derived from the genus Aerococcus or Pediococcus are preferably used. Pyruvate dehydrogenase is obtained from a microorganism or the like, and preferably is derived from Lactobacillus.

The salts of ADP thiamine pyrophosphate, phosphoric acid and phosphoenolpyruvate used in the present invention are not particularly limited as long as they are other than potassium salts, sodium salts, barium salts,
Ammonium salts, trishydroxythimeraminomethane salts, cyclohexylamine salts and the like can be mentioned.

The compounds of the reduced chromogen, oxidized acceptor, and formazan chromogen used in the present invention are not particularly limited. Examples of the reduced chromogen include couplers such as 4-aminoantipyrine and 3-methyl-2-benzothiazoline hydrazone and phenol derivatives such as phenol, 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol or Aniline, N, N-dimethylaniline, N, N-diethylaniline, N, N-diethyl-
m-toluidine, N, N-dithimer-m-anisidine, N
-Ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N-ethyl-N- (β-hydroxyethyl) -m-toluidine, N-ethyl-N- (2-
(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- (2-hydroxy-3-sulfopropyl ) -3,5-Dimethoxyaniline, N-ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N- (3-methylphenyl) -N'-
Succinylethylenediamine, N-ethyl-N- (2-
(Hydroxy-3-sulfopropyl) -m-anisidine;
Combination of aniline derivatives such as N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine 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
-Dihydroxy-1-naphthyl) methane and the like.

Examples of the oxidized acceptor include 5-methylphenazinium thymer sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, and the like.

As formazan chromogen, for example, 3- (p-iodophenyl) -2- (p-nitrophenyl) -5-phenyl-2H tetrazolium salt (INT), 3- (4,5-dimethyl-2-thiazolyl) ) -2,5-Diphenyl-2H tetrazolium salt (MTT), 3,3 '-(1,1'-biphenyl-4,4'
-Diyl) -bis (2,5-diphenyl-2H tetrazolium salt) (Neo-TB), 3,3 '-[3,3'-dimethoxy-
(1,1'-biphenyl) 4,4'-diyl] -bis [2-
(P-nitrophenyl) -5-phenyl-2H tetrazolium salt] (Nitro-TB), 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4,4'-diyl] -Screw [2,
5-bis (p-nitrophenyl) -2H tetrazolium salt] (TNTB), 3,3 '-[3,3'-dimethoxy- (1,1'
-Biphenyl) -4,4'silyl] -bis (2,5-diphenyl-2H tetrazolium salt) (TB).

The pH of the reagent of the present invention is preferably maintained at pH 5 to 11 by a buffer, and the buffer may be any buffer as long as it does not contain potassium ions. For example, triethanolamine buffer, MES buffer, Tris buffer and the like can be mentioned.

If necessary for the reagent of the present invention, a surfactant, a preservative,
Further, a stabilizer, an enzyme activator and the like may be added. Surfactants, preservatives, stabilizers, and enzyme activators are not particularly limited. As the surfactant, a nonionic surfactant is suitably used. As the preservative, NaN ₃ , a chelating agent and an antibiotic are suitably used. The stabilizer and the enzyme activator are not particularly limited as long as they exhibit an effect. For example, albumin, FAD, magnesium ion and the like can be mentioned.

The pyruvate kinase used in the present invention, pyruvate oxidase, the enzyme concentration used for the measurement of pyruvate dehydrogenase is not particularly limited as long as it is a concentration suitable for the measurement, but pyruvate kinase is 0.001.
~ 0.1 U / ml, pyruvate oxidase is preferably used in a range of 0.1-30 U / ml, and pyruvate dehydrogenase is preferably used in a range of 0.01-10 U / ml.

Phosphoenolpyruvate or a salt thereof, ADP, thiamine pyrophosphate or a salt thereof, phosphoric acid or a salt thereof,
The concentrations used for measurement of reduced chromogen, peroxidase, oxidized acceptor, and formazan chromogen,
The concentration is not particularly limited as long as it is suitable for measurement, but phosphoenol pyruvic acid or a salt thereof is 0.01 to
10 mM, thiamine pyrophosphate or a salt thereof is 0.01 to 10 mM,
Phosphoric acid or its salt 0.001 to 100 mM, reduced chromogen is 0.00
1-10 mM, peroxidase 0.1-100 U / ml, acceptor (oxidized form) 0.01-10 mM, formazan chromogen 0.01
It is preferably used in the range of 1010 mM.

As a method for measuring potassium ion using the composition for measuring potassium ion of the present invention, H ₂ O ₂ formed by reacting a sample with the reagent containing pyruvate kinase and pyruvate oxidase as described above is used. In the presence of a chromogen and peroxidase, a quinoid dye is formed and colorimetrically determined, and as described above, pyruvate dehydrogenase is followed and colorimetrically determined using a formazan dye.

The conditions for measurement using the composition of the present invention are not particularly strictly regulated, but the reaction temperature is preferably from 10 to 40 ° C, and preferably 37 ° C or 25 ° C. Reaction time is 0
A period of up to 10 minutes is preferably used. The measurement wavelength is preferably measured around λmax of the colored dye.

(Examples) Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 The potassium ion concentration in a test solution was measured by the following method using the following reagent.

1. Reagent A Triethanolamine buffer 0.1 M (pH 7.6) MgCl ₂ 2.5 mM ADP 4.5 mM Phosphoenolpyruvate 0.5 mM Thiamine pyrophosphate 0.5 mM Pyruvate kinase 0.05 unit / ml Pyruvate oxidase 3.0 unit / ml FAD 10 μM 4-aminoantipyrine 0.1 mM N-ethyl-N- (3-sulfopropyl) -m-anisidine peroxidase 5.0 unit / ml H ₃ PO ₄ 1 mM 2. Measurement method a. A serum was used as a sample, and 50 μl of each sample was collected.3 ml of the above-mentioned reagent A was added thereto, and reacted at 37 ° C. for 10 minutes.
The time course at 0 nm (the behavior of the enzymatic reaction at the measurement wavelength) and the change in absorbance for one minute were determined.
As a blank, water was used instead of the test solution containing potassium ions.

FIG. 1 shows the time course of the 50 mM KCl aqueous solution and the serum sample, and FIG. 2 shows the dilution linearity of the 50 mM KCl aqueous solution and the serum sample. As is clear from the figure, even if a KCl aqueous solution or serum is used as a sample, the reagent of the present invention can accurately and easily measure potassium ions in a short time.

Example 2 The potassium ion concentration in the test solution was measured by the following method using the following reagent.

1. Reagent B MES buffer (pH 6.3) 0.1 M MgCl ₂ 2.5 mM ADP 4.5 mM Phosphoenolpyruvate 0.5 mM Thiamine pyrophosphate 0.5 mM Pyruvate kinase 0.05 unit / ml Pyruvate dehydrogenase 3.0 unit / ml FAD 10 μM 1- methoxy-5-methylphenazinium methylsulfate 0.2mM nitrotetrazolium blue _{1 mM H 3 PO 4 1 mM} 2. measurement methods a. 10 serial dilutions of an aqueous potassium chloride solution 50mM serum 10 serial dilutions of each as a sample, each 50 μl was collected, 3 ml of the above reagent B was added thereto, and reacted at 37 ° C. for 10 minutes to obtain 57 μl.
The time course at 0 nm and the change in absorbance for 1 minute were determined. As a blank, water was used instead of the test solution containing potassium ions.

FIG. 3 shows the time course of the 50 mM KCl aqueous solution and the serum sample, and FIG. 4 shows the dilution linearity of the 50 mM KCl aqueous solution and the serum sample. As is clear from the figure, even when a KCl aqueous solution or serum is used as a sample, the sample of the present invention can accurately and easily measure potassium ions in a short time.

(Effect of the Invention) By using the reagent for measuring potassium ions of the present invention, potassium ions in body fluids can be accurately and easily colorimetrically determined in a short time.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 3 show a 50 mM KCl aqueous solution and a tycose of a serum sample. FIG. 2 and FIG. 4 show the dilution linearity of a 50 mM KCl aqueous solution and a serum sample.

Claims (3)

(57) [Claims] 1. A composition for measuring potassium ion, comprising (a) pyruvate kinase, (b) pyruvate oxidase or pyruvate dehydrogenase and (c) adenosine-5'-diphosphate or a salt thereof. object. (2) pyruvate kinase, (b) pyruvate oxidase, (c) adenosine-5'-diphosphate or a salt thereof, (d) thiamine pyrophosphate or a salt thereof, (e) phosphate or A composition for measuring potassium ions, comprising: a salt thereof; (f) phosphoenolpyruvate or a salt thereof; (g) a reduced chromogen; and (h) peroxidase. (3) pyruvate kinase, (b) pyruvate dehydrogenase, (c) adenosine-5'-diphosphate or a salt thereof, (d) thiamine pyrophosphate or a salt thereof, (e) phosphate or A composition for measuring potassium ions, comprising a salt thereof, (f) phosphoenolpyruvate or a salt thereof, (g) an oxidized acceptor, and (h) a formazan chromogen.