JPH06311897A - Composition for potassium ion measurement - Google Patents

Abstract

PURPOSE:To obtain a composition for enzymatically measuring potassium ion concentration excellent in operativity, quantitativeness and accuracy without requiring a sodium ion binding agent. CONSTITUTION:The subject composition for measuring potassium ion contains a urea-amide-lyase, urea, adenosine triphosphate or its salt, bicarbonate ion and magnesium ion. The urea-amide-lyase dose not act on sodium ion, thus it is possible to accurately measure potassium ion in a sample without being affected by sodium ion.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION 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 for 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, a flame photometer method, a chemical measuring method, an ion selective electrode method and the like have been used as a method for measuring metal ions in a body fluid including potassium ions. However, the flame photometer method is complicated in operation and has a problem in sample processing capacity. The chemical assay method has a problem that the operation is complicated and the reagent is expensive, and thus it is rarely used in the clinical examination field. 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 enzymatic method has been reported.
(Clin. Chem. 1989; 35: 817-820, JP-A-1-503596, etc.). This method utilizes the fact that pyruvate kinase is activated by potassium ions. However, pyruvate kinase is activated by sodium ions as well as potassium ions. Therefore, there is a problem in that it is necessary to add an expensive sodium binder in order to reduce the influence of sodium ions present in the body fluid in a larger amount than potassium ions.

[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 and does not require a sodium ion binder. Is to provide.

[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. As a result, urea amide lyase was activated by potassium ion. It has been found that it exhibits excellent characteristics that it is activated but is not activated by sodium ions. Therefore, by using urea amide lyase, it was found that the potassium ion concentration in the body fluid can be easily and accurately measured in a short time without using a sodium ion binder,
The present invention has been reached.

That is, the present invention is characterized by containing (a) urea amide lyase, (b) urea, (c) adenosine triphosphate or a salt thereof, (d) bicarbonate ion and (e) magnesium ion. It is a composition for measuring potassium ions.

In the present invention, urea amide lyase (U
RL) catalyzes the following reaction.

[Chemical 1]

To measure potassium ions in a sample using the composition for measuring potassium ions of the present invention, urea and bicarbonate as substrates and ATP are urea in the presence of potassium ions and magnesium ions in the sample. The amide lyase is allowed to act, and the produced ammonia or ADP is measured.

The origin of the urea amide lyase used in the present invention is not particularly limited. For example, those derived from unicellular green algae, yeasts, and other microorganisms are used, and those belonging to the genera Saccharomyces and Candida are preferably used.

As the bicarbonate ion, a bicarbonate salt such as sodium bicarbonate or lithium bicarbonate is used. Potassium salts cannot be used as bicarbonates. As the magnesium ions, magnesium salts such as magnesium sulfate and magnesium chloride are used.

As means for measuring the produced ammonia, for example, ammonia is used as α-ketoglutaric acid and NA.
Glutamate dehydrogenase is allowed to act in the presence of DH or NADPH, and the produced NAD or NADP is measured by a decrease in UV absorption. Glutamine oxidase reacts with glutamine oxidase to measure hydrogen peroxide produced, ammonia reacts with salicylic acid and sodium hypochlorite and sodium nitroprusside, and the produced indophenol is measured by absorption at 560 nm, ammonia electrode There is a method to measure directly.

The enzyme concentration used for the measurement of the urea amide lyase used in the present invention is not particularly limited as long as it is a concentration suitable for the measurement, but usually 0.01 to 1
It is preferably used in the range of 0 U / ml. The use concentration of urea, adenosine triphosphate or its salt, bicarbonate ion and magnesium ion is not particularly limited as long as it is a concentration suitable for measurement, but urea is usually 1 to 500 m
It is preferably used in the range of M, and adenosine triphosphate or a salt thereof is usually preferably used in the range of 0.1 to 10 mM. The bicarbonate ion is preferably used in the range of 5 to 500 mM, and the magnesium ion is preferably used in the range of 1 to 100 mM.

P of the composition for measuring potassium ion of the present invention
H is preferably maintained at pH 6 to 8 by a buffer solution, and any buffer solution may be used as long as it does not contain potassium ions. Examples include triethanolamine buffer, GOOD buffer, Tris buffer and the like.

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 or the like is preferably used. As the preservative, NaN ₃ , antibiotics, etc. are preferably used. The stabilizer and the enzyme activator are not particularly limited as long as they exhibit effects, and for example, albumin,
Magnesium ion etc. are mentioned.

The conditions for measuring potassium using the composition of the present invention are not particularly strictly limited,
The reaction temperature is between 20 and 40 ° C, preferably 25 ° C or 30 ° C. The reaction time is preferably between 1 and 10 minutes. The measurement wavelength is preferably around 340 nm, or in the case of using a dye, around λ max of the dye that has developed color.

[0015]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 The potassium ion concentration in a sample was measured by the following measuring method using the following reagents. Reagents Tris buffer (pH 8.0) 0.05M urea amide lyase (from Saccharomyces genus) 0.2U / ml urea 200mM adenosine triphosphate sodium salt 1mM magnesium sulfate 10mM sodium bicarbonate 4mM glutamate dehydrogenase (Proteus genus) 100U / Ml α-ketoglutarate 1 mM NADPH 0.5 mM

Measuring method 10-step dilution of 10 mM potassium chloride solution and serum 1
100 μl of each of the 0-step dilutions was used as a sample, 3 ml of the above-mentioned reagent was added thereto, and the mixture was reacted at 30 ° C. for 5 minutes, and the time course at 340 nm (enzymatic reaction at the measurement wavelength) and 1 The change in absorbance for each minute was obtained. In the blank, distilled water was used instead of the potassium ion-containing test liquid.

FIG. 1 shows the time course of a 10 mM potassium chloride aqueous solution and a serum sample. Figure 2 shows the dilution linearity of serum samples. FIG. 3 shows the dilution linearity of a 10 mM potassium chloride aqueous solution. As is clear from FIGS. 1 to 3, even if an aqueous solution of potassium chloride or serum is used as a sample, the present invention can accurately and easily measure potassium ions in a short time without using a sodium ion binder. it can.

Comparative Example 1 The potassium ion concentration in a sample was measured by the following measuring method using the following reagents. Reagent Tris buffer (pH 7.6) 0.05M Pyruvate kinase (from rabbit muscle) 0.5U / ml Phosphoenolpyruvate 1mM Adenosine diphosphate sodium salt 6mM Magnesium hydrochloride 5mM Lactate dehydrogenase (from microorganism) 10U / ml NADH 0.5 mM measuring method A 10-fold diluted solution of 10 mM potassium chloride was used as a sample, 40 μl of each was sampled, 3.2 ml of the above-mentioned reagent was added thereto, and the mixture was reacted at 37 ° C. for 5 minutes, and the absorbance at 340 nm for 1 minute I asked for change. For the blank, distilled water was used instead of the potassium ion-containing test liquid.

FIG. 4 shows the linearity of dilution of a 10 mM potassium chloride aqueous solution. FIG. 5 shows the change in absorbance when a sodium chloride aqueous solution was used as a sample. Further, FIG. 6 shows a change in absorbance when sodium chloride was used as a sample by the measurement shown in Example 1. As is clear from FIGS. 4 to 6, in the conventional method using pyruvate kinase, the absorbance is changed by the sodium salt, but in the present invention, the potassium ion in the sample can be measured without being affected by the sodium salt. it can.

[0020]

EFFECT OF THE INVENTION By using the composition for measuring potassium ion of the present invention, potassium ion in a sample can be quantified accurately and easily in a short time without using a sodium ion binder.

[Brief description of drawings]

FIG. 1 shows a time course of a 10 mM potassium chloride aqueous solution and a serum sample.

FIG. 2 shows the dilution linearity of serum samples.

FIG. 3 shows the dilution linearity of 10 mM potassium chloride aqueous solution.

FIG. 4 shows the dilution linearity of 10 mM potassium chloride aqueous solution.

FIG. 5 shows the change in absorbance with an aqueous sodium chloride solution.

FIG. 6 shows the change in absorbance with an aqueous sodium chloride solution.

Claims (1)

[Claims] 1. A potassium ion measurement characterized by containing (a) urea amide lyase, (b) urea, (c) adenosine triphosphate or a salt thereof, (d) bicarbonate ion and (e) magnesium ion. Composition.