JPH06253899A - Reagent composition for measurement of urea nitrogen - Google Patents

Abstract

PURPOSE:To provide a composition for enzymatic measurement of urea nitrogen, convenient for general use, adaptable for an autoanalyzer and excellent in quantitative analysis of high-concentration urea nitrogen. CONSTITUTION:This composition for measurement of urea nitrogen characteristically contains (a) uria amidohase, (b) adenosine triphosphate, (c) phosphoenol pyruvate, (d) pyruvate kinase, (e) pyruvate dehydrogenase and (f) a water-soluble formazan pigment. Even in the case of a solution containing a high-concentration urine nitrogen, urea nitrogen can readily and quantitatively accurately be analyzed in a short time. In addition, this composition is applicable to an automatic analyzer.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a reagent composition for measuring urea nitrogen in liquids, particularly blood and urine.

[0002]

2. Description of the Related Art Conventionally, urease / indophenol method and urease / glutamate dehydrogenase method have been mainly used as a method for quantifying urea nitrogen. These methods are affected by the ammonia produced by urease.Especially when the sample is urine, ammonia is removed by pretreatment due to the high ammonia concentration in urine, or a sample blank is required. It has the problem of lacking in speed and speed. In addition, an enzymatic method for quantifying hydrogen peroxide produced by the action of urea amide lyase, pyruvate kinase, and pyruvate oxidase has been developed (JP-A-62-3800). Although the effect of ammonia can be avoided by this method, the pyruvate oxidase used in this system has a problem that the high-value linearity is poor due to the lack of dissolved oxygen. Further, the problem can be solved by utilizing the pyruvate dehydrogenase reaction (Japanese Patent Laid-Open No. 3-103198). In this system, it is specified that tetrazolium salts or the like are used as the electron accepting indicator substance. However, these tetrazolium salts are sparingly soluble, and precipitate under solution storage conditions, or precipitate due to poor water solubility of formazan formed after color development, and further deposited in a reaction cell in an automatic analyzer, etc. Since line contamination poses a problem, it was difficult to put this system to practical use.

[0003]

SUMMARY OF THE INVENTION In view of the above situation, an object of the present invention is to provide a reagent composition for measuring urea nitrogen, which is convenient for normal use, is suitable for an automatic analyzer, and is excellent in quantifying high-value urea nitrogen. It is to provide things.

[0004]

Means for Solving the Problems The inventors of the present invention have made extensive studies in order to achieve the above-mentioned objects. As a result, a new water-soluble formazan dye is used, whereby the dye does not precipitate in a solution. In terms of compatibility with automatic analyzers,
The present invention has been completed by finding that no complicated operation is required because there is no line contamination such as deposition on the reaction cell, and that urea nitrogen in a liquid sample can be accurately measured to a high value without being affected by dissolved oxygen. .

That is, the present invention comprises (a) urea amide lyase, (b) adenosine triphosphate, (c) phosphoenolpyruvate, (d) pyruvate kinase, (e) pyruvate dehydrogenase and (f) water-soluble. A reagent composition for measuring urea nitrogen, which comprises a formazan dye.

Examples of the reaction in which water-soluble formazan is allowed to coexist include those exemplified below, but these are examples and do not limit the object of the present invention. The assay method and reagent composition of the present invention utilize the following reactions.

[0007]

[Chemical 1]

[0008]

[Chemical 2]

[0009]

[Chemical 3]

[0010]

[Chemical 4]

The urea amide lyase [E used in the present invention
C. The origin of [3.5.1.45] is not particularly limited. For example, those of the genus Candida (Candida sp.), The genus Saccharomyces (Saccharomyces sp.) And the like are used.

The pyruvate kinase used in the present invention [E
C. The origin of [2.7.1.40] is not particularly limited. For example, those obtained from microorganisms, animal muscles, pig hearts, etc. are used.

Pyruvate dehydrogenase [EC. The origin of [1.2.4.1] is not particularly limited. For example, those derived from microorganisms or animal organs are used, and those derived from Lactobacillus delbrueckii are preferably used.

The water-soluble formazan dye used in the present invention has a solubility of 0.1% or more in water, has no precipitation for 24 hours or more at room temperature even after the formation of formazan, and has an automatic analysis. There is no deposition of glass, polymethylmethacrylate, etc. on the reaction cell of the machine. Examples of such dyes include Nitro-TB.QA (nitrotetrazolium blue quarter ammonium salt), IN
T-MIGA (3- (p-indophenyl) -2- (p
-Nitrophenyl) -5-phenyl-2H-tetrazolim chloride-N-methylglucoamide).

The pH of the reagent composition of the present invention is preferably maintained at pH 5 to 11 with a buffer solution. Any buffer may be used, and examples thereof include Tris buffer, phosphate buffer, and Good's buffer.

The reagent composition of the present invention may optionally contain a surfactant, a preservative, a stabilizer and the like. The surfactant, preservative, stabilizer and the like are not particularly limited. As the surfactant, a nonionic surfactant is preferably used. As the preservative, NaN ₃ , a chelating agent, or an antibiotic is preferably used. The stabilizer is not particularly limited as long as it shows an effect.

The enzyme concentration of the urea amide lyase used in the present invention is not particularly limited as long as it is a concentration suitable for measurement, but it is preferably used in the range of 0.01 to 10 U / ml. The enzyme concentration of pyruvate kinase is not particularly limited as long as it is a concentration suitable for measurement, but 0.0
It is preferably used in the range of 01 to 10 U / ml. The enzyme concentration of pyruvate dehydrogenase is not particularly limited as long as it is a concentration suitable for measurement, but 0.01 to 20
It is preferably used in the range of U / ml. The concentrations of adenosine triphosphate, phosphoenolpyruvate, and water-soluble formazan dye are not particularly limited as long as they are concentrations suitable for measurement, but adenosine triphosphate is 0.1 to 2
0 mM, phosphoenolpyruvate 0.1-20 m
The M and water-soluble formazan dyes are preferably used in the range of 0.01 to 10 mM.

The method for measuring urea nitrogen using the reagent composition for measuring urea nitrogen of the present invention comprises the steps of preparing a sample containing urea amide lyase, pyruvate kinase, pyruvate dehydrogenase and a water-soluble formazan dye as described above. It is a method of reacting with a reagent to measure the amount of generated formazan color. The conditions for measurement using the reagent composition of the present invention are not particularly strictly limited, but the reaction temperature is preferably 10 to 40 ° C., and 37 ° C. or 30 ° C. is preferably used. The measurement wavelength is not particularly limited as long as it is suitable for measurement within the range in which the generated formazan has absorption. The shape of the reagent composition of the present invention may be either a freeze-dried product or a non-lyophilized product and is not particularly limited.

[0019]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 The concentration of urea nitrogen in a test sample was measured by the following method using Reagent 1 and Reagent 2 having the following compositions. Reagent 1 Phosphate buffer 50 mM (pH 6.5) KHCO ₃ 10 mM MgCl ₂ · 6H ₂ O 14 mM KCl 70 mM FAD 42 μM TPP 0.84 mM ATP 7 mM 1-m-PMS 7 μM pyruvate dehydrogenase 7 U / ml (Lactobacillus delbruii)

Reagent 2 Phosphate buffer 150 mM (pH 7.5) Phosphoenolpyruvate 12 mM Urea amide lyase (from Candida sp.) 8 U / ml Pyruvate kinase (from Rabbit muscle) 8 U / ml Nitro-TB-QA 2. The 86 mM abbreviation means the following. FAD: flavin adenine dinucleotide TPP: thiamine pyrophosphate Nitro-TB / QA: nitrotetrazolium blue quarter ammonium sulfate 1-m-PMS: 1-methoxy-5-methylphenazine methyl sulfate

Measurement method Using 10-step diluted solution of 300 mg / dl urea nitrogen aqueous solution as a sample, 30 μl of each was sampled, 3 ml of reagent 1 was added thereto, and the mixture was heated at 37 ° C. for 5 minutes, and then 1 ml of reagent 2 was added. Then, the absorbance after completion of the reaction was measured at 600 nm. In the blank, distilled water was used instead of the urea nitrogen-containing test liquid. Figure 1 shows the dilution linearity.

[0022]

According to the present invention, the amount of urea nitrogen can be accurately and easily quantified in a short time even in a solution containing a high concentration of urea nitrogen, and it can be adapted to an automatic analyzer. .

[Brief description of drawings]

FIG. 1 is a diagram showing the dilution linearity of a urea nitrogen diluted solution.

Claims (1)

[Claims] 1. (a) urea amide lyase, (b) adenosine triphosphate, (c) phosphoenolpyruvate,
A reagent composition for measuring urea nitrogen, which comprises (d) pyruvate kinase, (e) pyruvate dehydrogenase, and (f) water-soluble formazan dye.