JPH0653079B2 - Enzymatic determination of urea - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates generally to diagnostic-like assays, and more particularly to methods for quantitatively assaying urea in liquids, particularly blood and urine.

Prior Art Urea is the major end product of protein metabolism in humans and other mammals. This product is formed in the liver, enters the blood, is excreted in the urine through the kidneys, and is one of the major constituents of organic substances present in the urine. An excess of blood urea, called nitremia, almost always impairs renal function. Blood urea measurement is one of the most commonly used clinical chemistry tests. Urea "clearance" or the rate of removal of urea from the blood by the kidneys is measured by the concentration of urea in urine and is used as a measure of glomerular overspeed.

Conventional clinical chemistry methods used to determine the concentration of urea in biological fluids include direct methods by condensation of urea with a suitable reagent capable of forming a measurable chromogen, or urease for urea. Is classified as an indirect method by measuring ammonia as a product produced by the action of

The direct method using diacetyl monooxime has been widely applied to common clinical chemistry by man-made and automated operations. However, it has many drawbacks such as specificity problem due to interfering reaction with citrulline, allantoin and other body fluid components, and rapid disappearance of color due to photosensitivity.

The indirect method is based on the enzymatic conversion of urea to ammonia by urease (urea amide hydrolase, EC 3.5.1.5) according to the formula: The most prevalent method among them is based on the Berthelot reaction in which ammonia ion reacts with phenol and hypochlorous acid in alkaline medium to produce indophenol which is a blue dye.

On the other hand, the method of measuring in the ultraviolet, the glutamate dehumanogenase method is also used. In that way the formula: By measuring the ammonia ion derived from the enzymatic reaction with urease with the enzyme glutamate dehydrogenase (abbreviated as GlDH), the decrease of the absorbance of reduced nicotinamide adenine dinucleotide (abbreviated as NADH) at around 340 nm was measured. It is a method of measuring by wavelength.

Recently, in JP-A-60-54699, urea amide lyase,
A method for measuring urea using pyruvate kinase and pyruvate oxidase has been reported. The method has the formula: Is a method for measuring urea. This method has not been put into practice in the field of clinical diagnostic agents due to the problem of removal of ATP-degrading enzyme (abbreviated as ATPase) in urea amide lyase.

Urea amide lyase is EC 3.5.1.45, and the pre-stage enzyme, urea: carbon dioxide ligase EC
6.3.4.6) and the latter enzyme, allophanate amide hydrolase.

However, urea amide lyase does not
It also has TPase activity, and the above-mentioned composition system does not stabilize the coloration, resulting in spontaneous color increase. That is, ATPase, which is a side reaction of urea amide lyase, decomposes ATP into ADP non-co-synthetically, and further, the produced ADP is converted to pyruvate by pyruvate kinase in the presence of phosphoenolpyruvate. Furthermore, the pyruvic acid thus produced produces hydrogen peroxide by pyruvate oxidase, and the color indicator is colored. The reaction formula is shown below.

In other words, the reaction of (1) is accompanied by the side reaction of (1) ′, so that the phenomenon of gradually increasing color occurs and the reagent blank actually increases in color,
I can't get accurate measurements.

(Means for Solving Problems) The present inventors have reached the present invention as a result of various studies on how to prevent or reduce this side reaction.

That is, according to the present invention, urea in a liquid sample is reacted with urea amide lyase in the presence of adenosine triphosphate, bicarbonate, divalent cation, monovalent cation and alcohols to produce adenosine diphosphate, inorganic A method for enzymatically measuring urea, which is characterized by measuring phosphorus or ammonia.

In the system for measuring adenosine diphosphate produced,
Furthermore, it is a method of causing a color to be developed by allowing a pyruvate kinase and a pyruvate oxidase to act in the presence of phosphoenolpyrupate, thiamine pyrophosphate and a color development indicator, and performing an optical measurement. In addition, the system for measuring adenosine diphosphate produced produces a reduced nicotinamide adenine by allowing pyruvate kinase and lactate dehydrokenase to act in the presence of phosphoenolpyruvate and nicotinamide adenine diphosphate (NAD). There is also a method of optically measuring the production of diphosphate (NADH).

In the system for measuring the generated inorganic phosphorus, pyruvic acid, thiamine pyrophosphate, and pyruvate oxidase are allowed to act in the presence of a color-developing indicator to cause color to be optically measured.

Urea amide lyase is obtained from various microorganisms. The preparation method is based on the Journal of Biological Chemistry (Journ
al of Biological Chemistry) 294, 4107-4113, (1972) was referred to.

The amount of urea amide lyase used is 0.01 to 10 u / ml, preferably 0.1 to 0.3 u / ml. As the other enzymes, commercially available products can be used.

As the divalent cation, Mg ⁺⁺ or Mn ⁺⁺ can be used,
Mg ⁺⁺ is preferred. The monovalent cation is selected from the group of K ⁺ , NH ₄ ⁺ , Rb ⁺ or Cs ⁺ . K ⁺ is preferred. Any suitable soluble bicarbonate salt can be used as the bicarbonate ion source, such as an alkali metal salt or an alkaline earth metal salt. Sodium or potassium bicarbonate is preferred.
The concentration is 0.1 to 100 mM, preferably 1 to 50 mM.

The color development indicator system refers to a substance that develops color through hydrogen peroxide and peroxidase, for example, 0-dianisidine, leuco dye, 4-aminoantipyrine, 3-methylbenzothiazolinonehydrazone (MBTH) and phenol or a derivative thereof.

The most effective method for adding alcohols is to bring them into contact with urea amide lyase at a high concentration and then immediately use them, but the order of addition is not limited.

The alcohols used in the present invention are aliphatic lower monohydric alcohols, such as methyl alcohol, ethyl alcohol,
It refers to n-propyl alcohol, n-butyl alcohol, allyl alcohol, aluminum alcohol, etc. and their derivatives. The addition concentration is 0.01 to 50% (V / V), preferably 1 to 20% (V / V).

(Effect of the Invention) Surprisingly, the inclusion of alcohols in the measurement system suppressed only the side reaction ATPase, and had almost no effect on the activity of urea amide lyase.

This is a very effective means for practical use. In other words, by suppressing the spontaneous color development of the mixed solution of reagents,
It is possible to perform accurate measurement of urea without having to worry about changes over time in both the application method and the application to an automatic analyzer.

(Examples) Hereinafter, the present invention will be described using examples.

Example 1 A reaction mixture having the following composition was prepared.

Tris-HCl buffer (pH 7.5) 50 mM KHCO ₃ 10 mM MgSO ₄ 10 mM Phosphoenolpyruvate 1.5 mM Thiamine pyrophosphate 0.5 mM 4-Aminoantipyrine 0.01% Phenol 0.02% Phosphate 1 mM FAD 10 μM ATP 2.0 mM Peroxidase 5 u / ml Pyruvate kinase 3 u / ml Pyruvate oxidase 3 u / ml To 2.5 ml of the above reaction mixture was added 0.2 ml of urea solution (final 0.17 mM), and then preheated at 37 ° C for 5 minutes. The blank (blind) was distilled water instead of the urea solution.

Then add 0.3 ml of urea amide lyase solution (with or without addition of 15% ethyl alcohol) (final 0.1 u / ml), and add 3
The reaction was carried out at 7 ° C. for 30 minutes, and the absorbance was recorded with time at a wavelength of 500 nm of a spectrophotometer. The results are shown in FIG. When 15% ethyl alcohol was added to the urea amide lyase solution, the color stability was remarkably excellent as compared with the case where no addition was made.

Example 2 0.3 ml of ethyl alcohol was added to 2.5 ml of the reaction solution of Example 1.
(Final 10%) was added, and then 0.1 ml of urea solution (final)
0.17 mM) was added, followed by pre-heating at 37 ° C. for 5 minutes.
The blank was distilled water instead of the urea solution. Add 0.1 ml of urea amide lyase solution (final 0.1 u / ml),
The reaction was allowed to proceed for 30 minutes, during which time the absorbance was recorded at a wavelength of 500 nm on a spectrophotometer. The results are shown in FIG. The coloration stability was remarkably excellent in the addition of ethyl alcohol as compared with the case of no addition.

Example 3 A reaction mixture having the following composition was prepared.

Tris-HCl buffer pH 7.5 50 mM KHCO ₃ 10 mM MgSO ₄ 10 mM sodium pyruvate 5 mM thiamin pyrophosphate 0.5 mM 4-aminoantipyrine 0.01 phenol 0.02 FAD 10 μM ATP 2.0 mM peroxidase 5 u / ml pyruvate oxidase 3 u / ml The above reaction mixture 2.5 ml After adding 0.2 ml of urea solution (final 0.17 mM) to the above, the mixture was preheated at 37 ° C. for 5 minutes. The blank was distilled water instead of the urea solution. Urea amide lyase solution (with or without addition of 15% ethyl alcohol) 0.3m
l (final 0.1 u / ml) was added and allowed to react at 37 ° C. for 30 minutes, during which time the absorbance was recorded at a wavelength of 500 nm on a spectrophotometer. The results are shown in FIG. The coloration stability was remarkably excellent in the addition of ethyl alcohol as compared with the case of no addition.

Example 4 To 2.5 ml of the reaction mixture shown in Example 1, 0.2 ml of urea solution (final
0.17 mM) was added, followed by pre-heating at 37 ° C. for 5 minutes.
The blank was distilled water instead of the urea solution. Urea amide lyase solution (each alcohol: 15% concentration) 0.3m
l (final 0.1 u / ml) was added and reacted at 37 ° C. for 30 minutes, and the absorbance was read at a wavelength of 500 nm on the spectrophotometer. The results are shown in Table 1. Especially effective was ethyl alcohol and methyl alcohol.

Example 5 2.5 ml of the reaction mixture shown in Example 1 was mixed with 0.2 ml of urea solution (final
0.17 mM) was added, followed by pre-heating at 37 ° C. for 5 minutes.
The blank was distilled water instead of the urea solution. Urea amide lyase solution (each concentration of ethyl alcohol) 0.3 ml
(Final 0.1 u / ml) was added and reacted at 37 ° C. for 30 minutes, and then the absorbance was read at a wavelength of 500 nm on a spectrophotometer,
The results are shown in FIG. Concentrations of 14-18% were most effective.

Example 6 2.6 ml of the reaction mixture shown in Example 1 and 0.1 ml of human serum were added, and then preheated at 37 ° C. for 5 minutes. The blank was distilled water instead of human serum. 0.3 ml of urea amide lyase solution (containing 15% ethanol) (final 0.1 u / ml) was added and reacted at 37 ° C. for 30 minutes.
Absorbance was recorded over time at the wavelength. The result is shown in FIG. The color stability was remarkably superior when ethanol was added, compared to when ethanol was not added.

[Brief description of drawings]

FIGS. 1 to 4 show the coloration stability when the urea amide lyase solution was added to the reaction mixture containing the urea solution. FIG. 5 shows the coloration stability when the urea amide lyase solution was added to the reaction mixture containing human serum.

Claims (3)

[Claims] 1. Urea in a liquid sample is added to adenosine triphosphate,
Urea enzymatically characterized by reacting urea amide lyase in the presence of bicarbonate, divalent cation, monovalent cation and lower alcohol to measure adenosine diphosphate, inorganic phosphorus or ammonia produced. Measurement method. 2. Adenosine diphosphate produced is allowed to develop color by allowing pyruvate kinase and pyruvate oxidase to act in the presence of phosphoenolpyruvate, thiaminepyrophosphate and a color development indicator, and to be optically measured. The method for enzymatically measuring urea according to claim 1, which is characterized in that. 3. The method for producing an inorganic phosphorus by causing pyruvic acid oxidase to act in the presence of pyruvic acid, thiamine pyrophosphate and a color-forming indicator to develop a color, which is optically measured. The method for enzymatically measuring urea according to the above item.