JP2013208096A - Solution containing uric acid and method of stabilizing uric acid in the solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of stabilizing uric acid in solution by avoiding the deposition or decomposition of uric acid in preservation of solution containing uric acid, uric acid standard solution which contains stabilized uric acid, and a measuring kit which measures uric acid containing this uric acid standard solution.SOLUTION: At least one substance which is selected from bis(2-hydroxyethyl )iminotris(hydroxymethyl) methane (Bis-Tris), N-(2-acetamido)iminodiacetic acid ester (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-tris(hydroxymethyl)-2-aminoethanesulfonic acid (TES) and 3-morpholino propanesulfonic acid (MOPS), is added to solution containing uric acid.

Description

The present invention relates to a uric acid-containing solution, a method for stabilizing uric acid in the solution, a uric acid standard solution, and a uric acid measurement kit including the standard solution.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

Uric acid is an end product of purine metabolism and is produced in the liver, bone marrow, muscle, etc. using endogenous and dietary nuclear proteins and nucleotides and excreted in the urine. When the balance between uric acid production and excretion in the body is lost, various diseases such as hyperuricemia and gout develop. Therefore, reagents and methods for accurately measuring uric acid concentration are required in connection with these diseases.
As a method for measuring uric acid, there are known a reduction method using the reduction property of uric acid, an enzyme method using uricase, a uric acid oxidase, and a separation analysis method using high performance liquid chromatography (HPLC). ing. At present, the enzyme method using uricase is widely used as a general clinical test method, and is applied to various automatic analyzers.

In order to obtain an accurate measurement value of the uric acid concentration, it is necessary to use a standard solution. In measuring uric acid, a uric acid-containing solution having a known concentration is often used as the uric acid standard solution. In this case, the uric acid standard solution is provided in the form of an aqueous solution.
However, since uric acid has a property of being relatively low in water solubility, there is a problem that uric acid is precipitated or decomposed during storage.
Moreover, the water solubility of uric acid also changes depending on the pH, so that the solubility is improved on the alkali side and precipitation tends to occur on the acid side. For this reason, the uric acid standard solution is devised so that the pH does not change by adding a buffer such as a phosphate buffer. Moreover, the device which improves stabilization of the uric acid in a solution is made | formed by adding salts and a chelating agent to a uric acid containing solution (for example, refer patent document 1).

JP 49-30391 A

As described above, since uric acid has poor stability in a solution state, there is a problem that uric acid is precipitated or decomposed when stored in a solution state for a long time as a uric acid-containing solution. Further, when such a uric acid-containing solution is used as a standard solution, there is a problem that an accurate measurement value of uric acid concentration cannot be obtained. Further, in order to suppress the change in water solubility of uric acid, a contrivance has been made so that the pH does not fluctuate by adding a buffering agent such as a phosphate buffer to the uric acid standard solution. Therefore, a method for solving these problems has been desired.
Therefore, an object of the present invention is to avoid precipitation and decomposition of uric acid during storage of a uric acid-containing solution and stabilize uric acid in the solution, a uric acid standard solution containing stabilized uric acid, and the uric acid To provide a measurement kit for measuring uric acid including a standard solution.

As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have found that uric acid-containing solutions contain bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2 -Acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and 3-morpholinopropane It has been found that by adding at least one substance selected from sulfonic acid (MOPS), precipitation and decomposition of uric acid during storage can be suppressed, and uric acid in the solution can be stabilized for a long period of time.
In addition, by using a solution containing uric acid that is stable for a long period of time as a uric acid standard solution in the uric acid measurement method, it is found that accurate uric acid can be measured without error, and the present invention is completed. It came to.

That is, the present invention provides the following inventions.
(1) Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic acid ( A uric acid-containing solution containing at least one substance selected from ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), and 3-morpholinopropanesulfonic acid (MOPS).
(2) To a uric acid-containing solution, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2- Characterized in that it contains at least one substance selected from aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and 3-morpholinopropanesulfonic acid (MOPS). A method for stabilizing uric acid in a solution.
(3) In a standard solution for use in measuring uric acid in a sample, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), At least selected from N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 3-morpholinopropanesulfonic acid (MOPS) A standard solution for measuring uric acid, characterized by containing one substance.
(4) A measurement kit for measuring uric acid in a sample, comprising the standard solution according to (3).

  According to the present invention, uric acid-containing solution contains bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) Containing at least one substance selected from 2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and 3-morpholinopropanesulfonic acid (MOPS) Thus, even when the uric acid-containing solution is stored for a long period of time, the storage stability of the uric acid-containing solution can be improved by stabilizing the uric acid in the solution by preventing the precipitation and decomposition of uric acid. Then, by using the stabilized uric acid-containing solution as a standard solution, it is possible to accurately measure uric acid without error.

  Hereinafter, the present invention will be described in detail. However, the following embodiments are examples for explaining the present invention, and the present invention is not limited to these embodiments. Moreover, this invention can be implemented with various forms, unless it deviates from the summary.

[1] Outline of uric acid-containing solution (1) In the present invention, uric acid-containing solution contains bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA). N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and 3-morpholinopropanesulfonic acid (MOPS) By containing at least one substance, uric acid in a solution state can be stabilized.
Moreover, these substances used by this invention can be used individually or in combination of 2 or more types.

  The substances used in the present invention are all known as good buffering agents, but not all good buffering agents are effectively used in the present invention. For example, 2-morpholinoethanesulfonic acid ( MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), piperazine-1,4- Bis (2-hydroxy-3-propanesulfonic acid) (POPSO) or the like does not provide a stabilizing effect against uric acid in the target solution of the present invention. In addition, even when a phosphate buffer that is widely used as a buffer for uric acid standard solution is used, the effect of stabilizing the uric acid in the solution intended by the present invention cannot be obtained.

  In the present invention, when the uric acid-containing solution contains at least one substance selected from Bis-Tris, ADA, ACES, TES, and MOPS, the concentration is particularly an amount that can sufficiently stabilize uric acid in the solution. It is not limited, and the optimum concentration varies depending on the concentration of uric acid in the solution, etc., but if it is 5 mM or more, a desired stabilizing effect is obtained, preferably in the range of 5 to 500 mM, More preferably, it is in the range, and particularly preferably in the range of 10 to 100 mM.

(2) Solution pH
The pH of the uric acid-containing solution of the present invention is preferably pH 6 or more, particularly preferably in the range of pH 6-8.

(3) Other components In addition to Bis-Tris, ADA, ACES, TES, and MOPS, the solution of the uric acid-containing solution of the present invention appropriately contains known pH adjusters, preservatives, and the like as necessary. Can be made.
In the present invention, in order to stabilize uric acid in the solution, the uric acid-containing solution only needs to contain at least one substance selected from Bis-Tris, ADA, ACES, TES, and MOPS. The operation of is not particularly necessary.

[2] Standard solution for uric acid measurement The present invention also provides a standard solution for use in measuring uric acid in a sample, wherein bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- ( 2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 3-morpholino The present invention provides a standard solution for measuring uric acid, which contains at least one substance selected from propanesulfonic acid (MOPS).
In the present invention, the concentration in the case of containing at least one substance selected from Bis-Tris, ADA, ACES, TES and MOPS in the standard solution for uric acid measurement can sufficiently stabilize uric acid in the standard solution. The amount is not particularly limited, and the optimum concentration varies depending on the concentration of uric acid in the standard solution, etc., but if it is 5 mM or more, the desired stabilizing effect is obtained, and it may be in the range of 5 to 500 mM. Preferably, it is in the range of 10 to 500 mM, more preferably in the range of 10 to 100 mM.

The concentration of uric acid contained in the uric acid measurement standard solution of the present invention may be appropriately selected from the concentration range normally used as the uric acid measurement standard solution.
The standard solution of the present invention can be prepared by adding one or more of the above substances to a uric acid-containing solution. For example, from the above-mentioned Bis-Tris, ADA, ACES, TES, and MOPS It can be prepared by dissolving at least one selected substance and uric acid in pure water and adjusting the pH with a known pH adjuster or the like.
Moreover, the pH of the standard solution for uric acid measurement of the present invention is preferably pH 6 or more, particularly preferably in the range of pH 6-8.

[3] Measurement kit for measuring uric acid in a sample The present invention further includes a measurement kit for measuring uric acid in a sample containing the above-mentioned standard solution for uric acid measurement of the present invention (hereinafter referred to as “uric acid measurement kit”). It may be said). The uric acid measurement kit of the present invention may follow a conventionally known uric acid measurement kit in a sample, except that the uric acid measurement standard solution described above is included.
For example, the uric acid measurement kit comprises the above-described standard solution for uric acid measurement of the present invention, an oxidizable coloring reagent, a reagent used for normal measurement such as oxidase such as peroxidase, uricase, etc. As other components, components used in the measurement reaction, substrates, dyes, known preservatives, stabilizers and the like can be appropriately used as necessary.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.

[Example 1] (Confirmation of stabilizing effect of uric acid in solution according to the present invention-1)
Prepare an aqueous solution containing a substance having the effect of stabilizing uric acid in the solution of the present invention, potassium phosphate, or other substances together with uric acid, and compare the storage stability for 17 days at 5 ° C. and 37 ° C., respectively. went.

1. Preparation of uric acid standard solution (1) Preparation of standard solution (MES) Dissolve in pure water so that the concentration of 2-morpholinoethanesulfonic acid [MES] is 20 mM and the concentration of uric acid is 10 mg / dL. Then, adjust the pH so that the pH becomes pH 5.5, pH 6.0, pH 6.5, or pH 7.0 at 20 ° C., and prepare a total of four types of “standard solutions (MES)” with different pH. did.

(2) Preparation of standard solution (Bis-Tris) The concentration of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane [Bis-Tris] is 20 mM, and the concentration of uric acid is 10 mg / dL. Each was dissolved in pure water, and adjusted to pH 6.0, pH 6.5 or pH 7.0 at 20 ° C., respectively, so that a total of three types of “standard solutions (Bis-Tris) having different pH were obtained. ) ”Was prepared.

(3) Preparation of standard solution (ADA) Each was dissolved in pure water so that the concentration of N- (2-acetamido) iminodiacetic acid [ADA] was 20 mM and the concentration of uric acid was 10 mg / dL. And pH was adjusted so that pH might become pH 6.0, pH 6.5, or pH 7.0 at 20 degreeC, respectively, and three types of "standard solutions (ADA)" from which pH differs were prepared.

(4) Preparation of Standard Solution (PIPES) Pure piperazine-1,4-bis (2-ethanesulfonic acid) [PIPES] so that the concentration is 20 mM and uric acid is 10 mg / dL. Three kinds of “standard solutions (PIPES)” with different pH were prepared by dissolving in water and adjusting the pH to be pH 6.5, pH 7.0 or pH 7.5 at 20 ° C., respectively. .

(5) Preparation of standard solution (ACES) Pure so that the concentration of N- (2-acetamido) -2-aminoethanesulfonic acid [ACES] is 20 mM and the concentration of uric acid is 10 mg / dL. A total of four types of “standard solutions (ACES) having different pHs, dissolved in water and adjusted to pH 6.0, pH 6.5, pH 7.0, or pH 7.5 at 20 ° C., respectively. Was prepared.

(6) Preparation of standard solution (TES) Each of N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid [TES] was 20 mM and uric acid was 10 mg / dL. Dissolve in pure water and adjust the pH so that the pH becomes pH 7.0, pH 7.5, and pH 8.0 at 20 ° C., and add three types of “standard solutions (TES)” with different pH. Prepared.

(7) Preparation of standard solution (HEPES) The concentration of 2- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid [HEPES] was 20 mM, and the concentration of uric acid was 10 mg / dL. Each was dissolved in pure water, and adjusted to pH 7.0, pH 7.5, and pH 8.0 at 20 ° C., respectively. HEPES) "was prepared.

(8) Preparation of Standard Solution (POPSO) The concentration of piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) [POPSO] is 20 mM, and the concentration of uric acid is 10 mg / dL. In each case, the pH is adjusted to pH 7.5, pH 8.0, and pH 8.5 at 20 ° C., respectively. ) ”Was prepared.

(9) Preparation of Control Standard Solution Each was dissolved in pure water so that the concentration of potassium phosphate was 20 mM and the concentration of uric acid was 10 mg / dL, and at 20 ° C., the pH was pH 6.0, pH 6 PH, pH 7.0, and pH 7.5 were adjusted so that four types of “control standards” having different pH were prepared.

2. Preparation of uric acid measurement reagent (1) Preparation of first reagent for uric acid measurement The following measurement reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to 8.0 (20 ° C.). A first reagent for measuring uric acid was prepared.
N- (2-acetamido) iminodiacetic acid [ADA] 10 mM
N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium [HDAOS] 0.6 mM
Peroxidase 1000 units / L

(2) Preparation of second reagent for measuring uric acid The following reagent components were dissolved in pure water so as to have the respective concentrations described above, the pH was adjusted to 6.5 (20 ° C.), and the second reagent for measuring uric acid was measured. Was prepared.
N- (2-acetamido) iminodiacetic acid [ADA] 100 mM
4-Aminoantipyrine 1.97 mM
Uricase 800 units / L

3. Storage of Standard Solution for Stability Study Each standard solution prepared in the above (1) to (9) was sealed and stored at 5 ° C. and 37 ° C. for 17 days.

4). Confirmation of Stability of Stored Standard Solution Using each standard solution stored in 17 above for 17 days as a sample, the first reagent for measuring uric acid prepared in (1) above at the start of storage and 17 days after storage, Using the second reagent for measuring uric acid prepared in (2) above, the uric acid content was measured.

  The measurement of uric acid in each sample (each standard solution) was performed using a Hitachi High-Technologies 7180 type automatic analyzer.

  After adding 160 μL of the first reagent for measuring uric acid as a first reagent to 3.3 μL of the sample and reacting at 37 ° C. for 5 minutes, 40 μL of the second reagent for measuring uric acid is added as a second reagent. A mixed solution (final reaction solution) was obtained.

  Then, the reaction is carried out at 37 ° C. From the increase in absorbance immediately before the addition of the second reagent at the main wavelength of 600 nm and the sub wavelength of 700 nm (16 points) and 5 minutes after the addition of the second reagent (34 points). The uric acid value (uric acid content) in each sample was determined by proportional calculation with the absorbance when a uric acid standard solution whose uric acid concentration was accurately known was measured.

  In addition, the absorbance when measured with pure water as a sample was used as a reagent blind value, and the absorbance obtained by subtracting the reagent blind value at each point from the absorbance measured at each of the 16 points and the 34 points was set to each value. This was used to calculate the uric acid value in the sample.

5. Measurement Results Tables 1 to 4 show the measurement results of the uric acid value (uric acid content) of each standard solution before and after storage in 4 above.

Table 1 shows the measured values of uric acid at the start of storage of each standard solution, and Table 2 shows the measured values of uric acid of each standard solution after storage for 17 days at 5 ° C. and 37 ° C., respectively.
Table 3 shows the value obtained by subtracting the uric acid measurement value at the start of each storage from the uric acid measurement value of each standard solution after storage for 17 days at 5 ° C. and 37 ° C., that is, uric acid stability in the storage for 17 days. It shows the fluctuation of
Table 4 shows the value obtained by subtracting the uric acid measurement value stored at 37 ° C. from the uric acid measurement value stored at 5 ° C. in the uric acid measurement value of each standard solution after storage for 17 days, that is, the storage temperature during the same storage period (17 days). It shows the fluctuation of uric acid stability due to the difference (5 ° C. and 37 ° C.).

6). Summary (1) As is clear from Table 3, the standard solution (Bis-Tris), the standard solution (ADA), the standard solution (PIPES), the standard solution (ACES), after 17 days storage at 5 ° C. In the standard solution (TES), the standard solution (HEPES), the standard solution (POPSO), and the control standard solution, the uric acid value (uric acid content) of each of these standard solutions may or may not change. I understand.
However, it can be seen that the uric acid value (uric acid content) of the standard solution is significantly lowered in the standard solution (MES) after the storage at 5 ° C. for 17 days.

(2) Also, as is clear from Table 3, after storage for 17 days at 37 ° C., in the standard solution (PIPES), standard solution (HEPES), standard solution (POPSO), and control standard solution, It can be seen that the uric acid value (uric acid content) of each of these standard solutions is significantly reduced.
On the other hand, after storage at 37 ° C. for 17 days, the standard solution (Bis-Tris), standard solution (ADA), standard solution (ACES), and standard solution (TES) It can be seen that the uric acid level (uric acid content) has no or almost no change.

(3) In addition, as is apparent from the values shown in Table 4, ie, the values obtained by subtracting the uric acid measurement value stored at 37 ° C. for 17 days from the uric acid measurement value stored at 5 ° C. for 17 days for each standard solution, the standard In the liquid (MES), the standard liquid (PIPES), the standard liquid (HEPES), the standard liquid (POPSO), and the control standard liquid, it can be seen that the subtracted value is a large negative value. That is, it can be seen that the uric acid value (uric acid content) of each of these standard solutions is significantly reduced in the storage at 37 ° C. compared to the storage at 5 ° C.
On the other hand, in the standard solution (Bis-Tris), the standard solution (ADA), the standard solution (ACES), and the standard solution (TES), this subtracted value may be a slight negative value. I understand. That is, even when stored at 37 ° C., it can be seen that the uric acid value (uric acid content) of each of these standard solutions is not significantly different from the value stored at 5 ° C.

(4) From these, uric acid in the solution is not stabilized by MES, PIPES, HEPES, POPSO, or phosphoric acid or its stabilizing effect is small, but Bis-Tris, ADA, ACES, and TES are not stabilized. It was confirmed that each can effectively stabilize uric acid in the solution even when heated to 37 ° C.
That is, the effect of the present invention could be confirmed.

[Example 2] (Confirmation of stabilizing effect of uric acid in solution according to the present invention-2)
Along with uric acid, a substance having the effect of stabilizing uric acid in the solution of the present invention or an aqueous solution containing potassium phosphate was prepared, and the long-term storage stability at −2 ° C. and 5 ° C. was compared.

1. Preparation of uric acid standard solution (1) Preparation of standard solution (MOPS) [uric acid concentration 10 mg / dL] The concentration of uric acid is 10 mg / dL so that the concentration of 3-morpholinopropanesulfonic acid [MOPS] is 100 mM. And dissolved in pure water such that the concentration of disodium EDTA is 1 mM and the concentration of sodium azide is 0.02%, and the pH is 6.5, 6.8 or 7 at 20 ° C. The pH was adjusted to 0.0, and three types of “standard solution (MOPS) [uric acid concentration 10 mg / dL]” having different pH were prepared.

(2) Preparation of standard solution (MOPS) [uric acid concentration of 15 mg / dL] The concentration of EDTA2 sodium is adjusted so that the concentration of uric acid is 15 mg / dL so that the concentration of 3-morpholinopropanesulfonic acid [MOPS] is 100 mM. Each solubilized in pure water so that the concentration is 1 mM and the concentration of sodium azide is 0.02%, and the pH becomes pH 6.5, pH 6.8 or pH 7.0 at 20 ° C. The pH was adjusted, and three types of “standard solution (MOPS) [uric acid concentration: 15 mg / dL]” having different pH were prepared.

(3) Preparation of standard solution (Bis-Tris) [uric acid concentration 10 mg / dL] The concentration of uric acid was adjusted so that the concentration of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane [Bis-Tris] was 100 mM. Each was dissolved in pure water so that the concentration of disodium EDTA was 1 mM and the concentration of sodium azide was 0.02% so that the concentration was 10 mg / dL, and the pH was 6.5 at 20 ° C. The pH was adjusted to pH 6.8 or pH 7.0, respectively, and a total of three types of “standard solution (Bis-Tris) [uric acid concentration 10 mg / dL]” having different pHs were prepared.

(4) Preparation of standard solution (Bis-Tris) [uric acid concentration 15 mg / dL] The concentration of uric acid was adjusted so that the concentration of bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane [Bis-Tris] was 100 mM. Each was dissolved in pure water so that the concentration of disodium EDTA was 1 mM and the concentration of sodium azide was 0.02% so that the concentration was 15 mg / dL, and the pH was 6.5 at 20 ° C. The pH was adjusted to pH 6.8 or pH 7.0, respectively, and three types of “standard solution (Bis-Tris) [uric acid concentration 15 mg / dL]” having different pH were prepared.

(5) Preparation of standard solution (TES) [uric acid concentration 10 mg / dL] The concentration of uric acid was 10 mg / day so that the concentration of N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid [TES] was 100 mM. Each solution was dissolved in pure water so that the concentration of disodium EDTA was 1 mM and the concentration of sodium azide was 0.02% so as to be dL, and at 20 ° C., the pH was pH 6.5, pH 6 The pH was adjusted to 8 or pH 7.0, respectively, to prepare a total of three “standard solutions (TES) [uric acid concentration 10 mg / dL]” having different pHs.

(6) Preparation of standard solution (TES) [uric acid concentration 15 mg / dL] The concentration of uric acid was 15 mg / day so that the concentration of N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid [TES] was 100 mM. Each solution was dissolved in pure water so that the concentration of disodium EDTA was 1 mM and the concentration of sodium azide was 0.02% so as to be dL, and at 20 ° C., the pH was pH 6.5, pH 6 The pH was adjusted to 8 or pH 7.0, and three types of “standard solution (TES) [uric acid concentration of 15 mg / dL]” having different pH were prepared.

(7) Preparation of standard solution (ACES) [uric acid concentration 10 mg / dL] The concentration of uric acid was 10 mg / dL so that the concentration of N- (2-acetamido) -2-aminoethanesulfonic acid [ACES] was 100 mM. So that the concentration of disodium EDTA is 1 mM and the concentration of sodium azide is 0.02%, and each is dissolved in pure water, and at 20 ° C., the pH is pH 6.5, pH 6. The pH was adjusted to 8 or 7.0 to prepare three types of “standard solution (ACES) [uric acid concentration of 10 mg / dL]” having different pH.

(8) Preparation of standard solution (ACES) [uric acid concentration 15 mg / dL] The concentration of uric acid was 15 mg / dL so that the concentration of N- (2-acetamido) -2-aminoethanesulfonic acid [ACES] was 100 mM. So that the concentration of disodium EDTA is 1 mM and the concentration of sodium azide is 0.02%, and each is dissolved in pure water, and at 20 ° C., the pH is pH 6.5, pH 6. The pH was adjusted to 8 or 7.0 to prepare 3 types of “standard solution (ACES) [uric acid concentration 15 mg / dL]” having different pH.

(9) Preparation of control standard solution [uric acid concentration 10 mg / dL] The concentration of disodium EDTA is 1 mM so that the concentration of uric acid is 10 mg / dL so that the concentration of potassium phosphate is 100 mM. And dissolved in pure water so that the concentration of sodium azide is 0.02%, and adjusted to pH 6.5, pH 6.8 or pH 7.0 at 20 ° C., respectively, Three types of “reference standard solutions [uric acid concentration: 10 mg / dL]” with different pH were prepared.

(10) Preparation of control standard solution [uric acid concentration 15 mg / dL] The concentration of disodium EDTA is 1 mM so that the concentration of uric acid is 15 mg / dL so that the concentration of potassium phosphate is 100 mM. And dissolved in pure water so that the concentration of sodium azide is 0.02%, and adjusted to pH 6.5, pH 6.8 or pH 7.0 at 20 ° C., respectively, A total of three “control standards [uric acid concentration: 15 mg / dL]” having different pHs were prepared.

2. Preparation of uric acid measurement reagent (1) Preparation of first reagent for uric acid measurement The following measurement reagent components were dissolved in pure water so as to have the respective concentrations, and the pH was adjusted to 8.0 (20 ° C.). A first reagent for measuring uric acid was prepared.
N- (2-acetamido) iminodiacetic acid [ADA] 10 mM
N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium [HDAOS] 0.6 mM
Peroxidase 1000 units / L

(2) Preparation of second reagent for measuring uric acid The following reagent components were dissolved in pure water so as to have the respective concentrations described above, the pH was adjusted to 6.5 (20 ° C.), and the second reagent for measuring uric acid was measured. Was prepared.
N- (2-acetamido) iminodiacetic acid [ADA] 100 mM
4-Aminoantipyrine 1.97 mM
Uricase 800 units / L

3. Storage of Standard Solution for Stability Study Each standard solution prepared in (1) to (10) above was sealed and stored at a temperature of −2 ° C. for 6 months.
Each of the standard solutions prepared in (1) to (10) above was sealed and stored at a temperature of 5 ° C. for 12 months.

4). Confirmation of Stability of Stored Standard Solution Using each standard solution stored at −2 ° C. for 6 months in 3 above as a sample, at the start of storage, after 1 month of storage and after 6 months of storage, The uric acid content was measured using the first reagent for measuring uric acid prepared in 1) and the second reagent for measuring uric acid prepared in (2) above.
In addition, each standard solution stored at 5 ° C. for 12 months at the above 3 is used as a sample, at the start of storage, after 1 month of storage, after 6 months of storage, and after 12 months of storage, the above (1) The uric acid content was measured using the first reagent for measuring uric acid prepared in 1 and the second reagent for measuring uric acid prepared in (2) above.

  The measurement of uric acid in each sample (each standard solution) was performed using a Hitachi High-Technologies 7180 type automatic analyzer.

  After adding 160 μL of the first reagent for measuring uric acid as a first reagent to 3.3 μL of the sample and reacting at 37 ° C. for 5 minutes, 40 μL of the second reagent for measuring uric acid is added as a second reagent. A mixed solution (final reaction solution) was obtained.

  Then, the reaction is carried out at 37 ° C. From the increase in absorbance immediately before the addition of the second reagent at the main wavelength of 600 nm and the sub wavelength of 700 nm (16 points) and 5 minutes after the addition of the second reagent (34 points). The uric acid value (uric acid content) in each sample was determined by proportional calculation with the absorbance when a uric acid standard solution whose uric acid concentration was accurately known was measured.

  In addition, the absorbance when measured with pure water as a sample was used as a reagent blind value, and the absorbance obtained by subtracting the reagent blind value at each point from the absorbance measured at each of the 16 points and the 34 points was set to each value. This was used to calculate the uric acid value in the sample.

5. Measurement Results Table 5 and Table 6 show the measurement results of the uric acid value (uric acid content) of each standard solution before and after storage in 4 above.

Table 5 shows the uric acid measurement value of each standard solution when stored at -2 ° C for 6 months.
Table 6 shows the measured uric acid values of each standard solution when stored at 5 ° C. for 12 months.

6). Summary (1) As is clear from Table 5, the standard solution (MOPS), standard solution (Bis-Tris), standard solution (TES), and standard solution (standard solution (TES)) after storage at -2 ° C for 6 months In ACES), the uric acid level (uric acid content) of each of these standard solutions has no or little change regardless of whether the concentration of uric acid in each of these standard solutions is 10 mg / dL or 15 mg / dL. I understand that.
However, after this 6-month storage at -2 ° C, in the control standard solution, the uric acid level of this standard solution (uric acid level) was measured regardless of whether the concentration of uric acid in this standard solution was 10 mg / dL or 15 mg / dL. It can be seen that the (content) is greatly reduced.

(2) As is clear from Table 6, the standard solution (MOPS), the standard solution (Bis-Tris), the standard solution (TES), and the standard solution (after the storage at 5 ° C. for 12 months) In ACES), the uric acid level (uric acid content) of each of these standard solutions has no or little change regardless of whether the concentration of uric acid in each of these standard solutions is 10 mg / dL or 15 mg / dL. I understand that.
However, after 12 months of storage at 5 ° C., in the control standard solution, the uric acid value (uric acid content) of the standard solution was measured regardless of whether the concentration of uric acid in the standard solution was 10 mg / dL or 15 mg / dL. ) Is significantly reduced.

(4) From these results, it was confirmed that MOPS, Bis-Tris, TES, and ACES can each effectively stabilize uric acid in the solution for a long period of time.
That is, the effect of the present invention could be confirmed.

Claims (4)

  Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), A uric acid-containing solution containing at least one substance selected from N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and 3-morpholinopropanesulfonic acid (MOPS).   To the uric acid-containing solution, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfone A solution characterized by adding at least one substance selected from acids (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and 3-morpholinopropanesulfonic acid (MOPS) Of stabilization of uric acid in water.   In a standard solution for use in measurement of uric acid in a sample, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- ( At least one substance selected from 2-acetamido) -2-aminoethanesulfonic acid (ACES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), and 3-morpholinopropanesulfonic acid (MOPS) A standard solution for measuring uric acid, which contains   A measurement kit for measuring uric acid in a sample, comprising the standard solution according to claim 3.