JPH06165697A - Composition for potassium ion determination - Google Patents

Abstract

PURPOSE:To provide a reagent capable of accurately, easily determining in a short time the potassium ions in the body fluid. CONSTITUTION:The objective reagent, a composition containing (A) glycerol dehydrogenase, (B) glycerol and (C) NAD or NADP.

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 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, a flame photometer method, a chemical measuring method, and an ion selective electrode method have been used as a measuring method of metal ions. However, the flame photometer method is complicated in operation and has a problem in sample processing ability. The chemical measurement method has a problem that the operation is complicated and the reagent is expensive. Although the ion-selective electrode method is relatively easy to operate, deterioration of the electrode occurs and measurement error during sample measurement has been a problem. Recently, a method for measuring metal ions using an enzyme has been reported (Clin. Chem. 35/5 817-82.
0). However, since this method is a method of measuring the decrease in the absorbance of NADH in the ultraviolet region, there is a problem in that NADH decreases during storage, making it impossible to measure high-value samples.

[0003]

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

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object. As a result, by utilizing glycerol dehydrogenase, the concentration of potassium ion in body fluid can be easily increased in a short time with high sensitivity. The present invention has been completed by finding accurate measurement with. That is, the gist of the present invention is (a) glycerol dehydrogenase,
(B) Glycerol and (c) NAD or NADP
A composition for measuring potassium ion is characterized by containing: Reactions using glycerol dehydrogenase include those exemplified below, but this is an example and does not limit the subject of the present invention.

The above composition utilizes the following reactions.

[Chemical 1]

[0006] The glycerol dehydrogenase used in the present invention is not particularly limited, and even if it uses NAD as a coenzyme (EC 1.1.1.6), NA
Those using DP as a coenzyme (EC 1.1.1.72) may be used.

The origin of the glycerol dehydrogenase used in the present invention is not particularly limited. For example, those derived from rabbit skeletal muscle, Escherichia coli, and other microorganisms are used, and those derived from Aerogenus and Acetobacter are preferably used.

The pH of the reagent of the present invention depends on the pH of the buffer solution.
It is preferable that the buffer solution is maintained at -11, and the buffer solution may be any 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,
You may add a preservative, a stabilizer, an enzyme activator, etc. The surfactant, preservative, stabilizer, enzyme activator and the like are not particularly limited. A nonionic surfactant is preferably used as the surfactant. As a preservative, N
AN3, chelating agents and antibiotics are preferably used. The stabilizer enzyme activator is not particularly limited as long as it shows an effect. Examples thereof include albumin, FAD, magnesium ion and the like.

The enzyme concentration used for the measurement of glycerol dehydrogenase used in the present invention is not particularly limited as long as it is a concentration suitable for the measurement, but it is from 0.01 to
It is preferably used in the range of 10 u / ml. Concentrations used to measure glycerol, NAD, NADP include:
The concentration is not particularly limited as long as it is a concentration suitable for measurement, but 0.001 to 5 M for glycerol and NAD for
The range of 0.1 to 50 mM and NADP of 0.1 to 1 mM are preferably used.

The method for measuring potassium ions using the composition for measuring potassium ions according to the present invention comprises the steps of reacting a sample with the reagent containing glycerol dehydrogenase as described above to generate NADH or NADPH by UV irradiation. It is a method of measuring by the increase of the absorbance. The conditions for measurement using the composition of the present invention are not particularly strictly limited, but the reaction time is preferably 10 to 40 degrees and 37 degrees or 25 degrees. The reaction time is preferably 0 to 10 minutes. It is desirable that the measurement wavelength is around 340 nm.

[0012]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 The potassium ion concentration in a test sample was measured by the following method using the following reagents. Reagent Tris buffer 0.1 M (pH 9.5) Polyethylene glycol monoparaisooctyl phenyl ether 0.1% NAD 1.0 mM Glycerol dehydrogenase 70u / 1 (from Cellulomonas sp.)

Measurement method 10-step dilution of 50 mM potassium chloride aqueous solution and serum 1
Using the 0-step dilution as a sample, 50 μl of each was sampled, 3 ml of the above reagent was added, and the mixture was allowed to react at 37 ° C. for 10 minutes, followed by a time course at 340 nm (enzymatic reaction progress at the measurement wavelength). And the change in absorbance for 1 minute was determined. In the blank, distilled water was used instead of the potassium ion-containing test liquid. 50 mMK in Figure 1
The time course of the Cl aqueous solution and the serum sample is shown in FIG. 2, and the dilution linearity of the serum sample is shown in FIG. 3, and the dilution linearity of the 50 mM KCl aqueous solution is shown in FIG. As is clear from the figure, even if KCl aqueous solution serum is used as a sample, potassium ions can be measured accurately and easily in a short time in the present invention.

[0014]

By using the reagent for measuring potassium ion of the present invention, potassium ion in body fluid can be quantified accurately and easily in a short time.

[Brief description of drawings]

FIG. 1 shows a time course of 50 mM KCl aqueous solution and serum.

FIG. 2 shows the dilution linearity of serum samples.

FIG. 3 shows the dilution linearity of a 50 mM KCl aqueous solution.

Claims (1)

[Claims] 1. (a) Glycerol dehydrogenase,
(B) Glycerol and (c) NAD or NADP
A composition for measuring potassium ion, which comprises: