WO2006030866A1

WO2006030866A1 - Method of quantitative determination of uric acid

Info

Publication number: WO2006030866A1
Application number: PCT/JP2005/017055
Authority: WO
Inventors: Yuhko Hirao; Hiroshi Matsui
Original assignee: Denka Seiken Co., Ltd.
Priority date: 2004-09-16
Filing date: 2005-09-15
Publication date: 2006-03-23
Also published as: JPWO2006030866A1

Abstract

A method of quantitative determination of uric acid that is one using peroxidase and excels in the accuracy at low values; and a relevant reagent composition. There is provided a method of quantitative determination of uric acid in an analyte sample, comprising decomposing uric acid in an analyte sample with the use of uricase (enzyme) into hydrogen peroxide and allantoin and measuring the amount of hydrogen peroxide with the use of peroxidase, characterized in that any contact of uric acid with peroxidase before the decomposition of uric acid by uricase (enzyme) is avoided to thereby attain an accurate quantitative determination of uric acid low values.

Description

Specification

Method for quantitative determination of uric acid

Technical field

[0001] The present invention relates to a reagent capable of accurately quantifying uric acid and a method for quantifying uric acid using the reagent. More specifically, the present invention relates to a reagent capable of accurately quantifying a low value in the determination of uric acid and an accurate quantification method of a low value of uric acid using the reagent. Background art

[0002] Uric acid is a final metabolite of purines that are constituents of nucleic acids such as deoxyribonucleic acid and ribonucleic acid, ATP, which is an energy carrier, and other nucleotides. Purines are synthesized mainly from amino acids and related compounds in the human body. Purine metabolism is involved in cell growth and plays a central role in maintaining life.

[0003] Uric acid is produced at a certain rate in the living body, temporarily stored in the body, and then is mainly excreted into the urine of the kidney, and the other part is excreted into the digestive tract and sweat as an extrarenal treatment. Production and excretion are almost the same, so production, storage and excretion are in equilibrium. In normal individuals, the daily production and excretion amount is approximately 700 mg, of which 500 mg is excreted in the kidney urine and approximately 20 Omg is excreted by extrarenal treatment. Extrarenal treatment increases and decreases in parallel with changes in serum uric acid levels.

[0004] Abnormal metabolism or excretion of uric acid in the body results in hyperuricemia or hypouricemia, both of which cause disease.

[0005] Hyperuricemia is a state in which blood is saturated and urate is present, and is mainly caused by abnormal uric acid metabolism and decreased excretion of uric acid. Gout is a typical disease that increases serum uric acid levels, but it also increases in cancer, leukemia, and myeloma.

[0006] Causes of hypouricemia include insufficient intake of purines as raw materials for uric acid, decreased production of uric acid due to debilitating diseases throughout the body, decreased production of uric acid due to metabolic diseases, and increased uric acid excretion. Metabolic diseases include xanthine oxidase deficiency, purine nucleoside phosphorylase deficiency, and 5-phosphoribosyl-1-pyrophosphate (PRPP) synthase deficiency. [0007] Accurate quantification of uric acid in blood and urine is important for the purpose of diagnosing or preventing these diseases.

[0008] In principle, the uric acid measurement method is roughly classified into a reduction method using the reducing property of uric acid under alkaline conditions and an enzymatic measurement method using uric acid-degrading enzyme uricase.

[0009] Reduction methods include a phosphotungstic acid deproteinization method and a phosphotungsten direct method. Enzymatic methods using uricase include uricase 'catalase method, uricase' peroxidase method, and uricase UV method.

[0010] In the past, phosphotungstic acid deproteinization was the main force. Currently, the uricase * peroxidase method is the mainstream. This is because the reduction method causes a large positive error due to the non-uric acid reducing substance in the sample, and pretreatment such as deproteinization is required, so that it is lacking in convenience and speed. The uricase 'peroxidase method is now widely used because of its excellent application to automated analyzers and its simplicity and speed.

[0011] The specific principle of the uricase 'peroxidase method is that uric acid becomes allantoin when it is decomposed by uricase. The peroxyhydrogen (H 0) produced at this time was converted to peroxida.

twenty two

The uric acid content is obtained by colorimetric determination with a chromogenic reaction. This method does not require deproteinization or sample blinding, and is a simple method in which color development is completed in a short time, and can be easily applied to various automatic analyzers.

[0012] In order to diagnose a disease caused by the aforementioned decrease in serum uric acid level, it is required to accurately quantify the low uric acid level.

[0013] As a reagent for measuring uric acid by the uricase 'peroxidase method, a concentrated reagent for dilution immediately before use has been reported (see Patent Document 1).

[0014] In addition, a measurement method for reducing the effect of pyrilvin has been reported for the measurement of low-concentration substances (see Patent Documents 2 to 4).

Patent Document 1: Japanese Patent No. 2862817

Patent Document 2: Japanese Patent No. 3283348

Patent Document 3: Japanese Patent No. 3520874

Patent Document 4: Japanese Patent No. 3357667 Disclosure of the invention

Problems to be solved by the invention

[0015] An object of the present invention is to provide a quantification method and reagent composition excellent in accuracy at a low value over a uric acid quantification method using peroxidase, and in a wide measurement range. It is to provide a method for quantifying uric acid and a reagent composition with excellent accuracy.

Means for solving the problem

[0016] The inventors of the present invention conducted extensive research on the problem of low accuracy in the uric acid quantification using the uricase 'peroxidase method. As a result, the peroxidase in the reagent reacted with uric acid to react with uric acid. It was found that uric acid in the low concentration range could not be accurately quantified.

[0017] Peroxidase is a reagent composition involved in the production of a quinone dye, and is usually used after a uricase enzyme reaction. Since the uricase enzyme is usually added to the second reagent, it is often added to the first reagent due to the stability and component balance of the whole reagent. Until the inventors of the present application found the above problems, the problems of the conventional reagent composition were overlooked.

[0018] In view of the above problems, the inventors of the present application transferred the first reagent force to the second reagent except for peroxidase so that uric acid and peroxidase do not contact before uric acid and uricase react. The inventors have found that uric acid in a low concentration range can be accurately quantified, and have completed the present invention.

That is, the present invention is as follows.

[0020] [1] Uric acid in the test sample is decomposed into peroxyhydrogen and allantoin by uricase enzyme.

Is a method for quantifying uric acid in a test sample by quantifying its peroxyhydrogen using peroxidase, and lowering uric acid by not contacting uric acid with peroxidase prior to degradation of uric acid by uricase enzyme. A method for quantifying uric acid, characterized in that

[2] The method for quantifying uric acid according to [1], wherein a first reagent containing a hydrogen donor compound is added to a test sample, and then a second reagent containing uricase, 4-aminoantipyrine, and peroxidase is added. , [3] The method for quantifying uric acid according to [1] or [2], wherein the second reagent is added 1 to 10 minutes after the addition of the first reagent,

[4] Quantification of uric acid according to [1], characterized in that the first reagent containing 4-aminoantipyrine is added to the test sample, and then the second reagent containing hydrogen donor, uricase, and peroxidase is added. Method,

[5] A first reagent containing a hydrogen-donating compound is added to a test sample, then a second reagent containing 4-amaminoantipyrine is added, and then a third reagent containing uricase and peroxidase is added. [1] uric acid quantification method,

[6] Add the first reagent containing 4-aminoantipyrine to the test sample, then add the second reagent containing the hydrogen-donating compound, and then add the third reagent containing uricase and peroxidase. [1] The method for quantifying uric acid according to [1],

[7] The method for quantifying uric acid according to any one of [2] to [6], wherein the first reagent further contains sodium azido

[8] A uric acid determination reagent comprising a first reagent containing a hydrogen donor and a second reagent containing peroxidase and uricase, wherein the first reagent contains peroxidase, and

[9] The uric acid assay reagent according to [8], wherein the first reagent further contains sodium azido.

The invention's effect

[0021] By the method of the present invention, it is possible to quantify uric acid with excellent accuracy at a low value. In addition, uric acid can be quantified accurately over a wide measurement range.

[0022] This specification includes the contents described in the specification of Japanese Patent Application No. 2004-270366, which is the basis of the priority of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

[0023] The principle of the present invention is represented by the following formula.

[0024] Λ Licase

First reaction: Uric acid + 0 ₂ + 2 0 → Allantoin + C0 ₂ + H ₂ 0 ₂

POD

Second reaction: 4AA + hydrogen donor + 2H ₂ 0 ₂ → quinone dye + 4H ₂ 0 As shown in the equation, the method of the present invention has a two-step reaction force. In the first reaction, uric acid in serum and urine is broken down into allantoin, carbon dioxide and hydrogen peroxide by uricase. The peroxyhydrogen generated here produces a colored quinone (quinone dye) in the presence of peroxidase, 4-aminoantipyrine (4AA) and phenolic or alpha phosphorus hydrogen donor compounds in a second reaction. To do.

[0025] The method of the present invention can also be applied to a reaction system in which hydrogen peroxide generated in the first reaction oxidizes a leuco dye in the presence of a peroxidase and a leuco dye to produce a blue dye. In general, an automatic analyzer in a clinical test generally puts a predetermined amount of one or more reagents into a test sample in order, and measures the absorbance, scattered light, etc. at a certain wavelength after a certain amount of time for reagent loading, and within a predetermined time. By determining the amount of change and the rate of change, the amount of the test substance in the test sample can be quantified.

[0026] When adjustment of reaction sequence and reaction rate is required for each reagent, the reagent composition necessary for quantitative reaction is arbitrarily allocated to multiple reagents, but each reagent is kept for a long period of several months to several years. Therefore, the chemical reaction proceeds before being used for clinical testing, and the reagent composition necessary for the quantitative reaction is degraded, or a composition that inhibits the quantitative reaction is generated. The reagent composition cannot be made by combining the compositions.

[0027] In addition, if a substance that inhibits the quantitative reaction or the same product as the product in the quantitative reaction exists in the test sample, these substances cause a positive error. A mechanism to avoid it must be prepared.

[0028] The method of the present invention is a method for quantifying uric acid using a reagent in which uric acid and peroxidase do not come into contact before uric acid and uricase react, and the reagent power first added to the test sample also excludes peroxidase. It is.

[0029] The negative effect of hemoglobin in the test sample is an adverse effect of removing the reagent force peroxidase first added to the test sample. As a result of intensive studies, the bad effects of peroxidase can be avoided by first adding sodium azide as the reagent to be added to the test sample. Therefore, in the method of the present invention, sodium azide may be contained in the reagent initially added to the test sample.

[0030] The reagent composition used in the uric acid quantitative method of the present invention satisfies the following requirements (a) to (d): You may choose.

[0031] (a) Uric acid is not contacted with peroxidase prior to degradation by uricase.

(B) Since peroxyhydrogen generated when uric acid is decomposed by uricase is decomposed over time, uricase and peroxidase are preferably included in the same reagent.

[0033] (c) Hydrogen donor compound and 4AA are not included in the same reagent to ensure storage stability.

[0034] (d) Azidosodium can also be included in the first reagent that is first added to the test sample.

[0035] Hereinafter, specific reagent configuration examples will be shown and described with the reagent composition of each reagent. In the present invention, the reagents added to the reaction system in which the reaction of the above formula occurs are referred to as the first reagent, the second reagent, and the third reagent in the order of addition. The process of mixing and reacting the first reagent and the test sample is called the first process, and the process of adding the second reagent and reacting is called the second process. When using the third reagent, the step of adding the third reagent to react is called the third step.

[0036] In the following reagent configuration examples, other hydrogen donor compounds can be used in place of TOOS, and the second reagent and the third reagent which do not need to contain sodium azide in the first reagent. One or both of the reagents may contain sodium azido. However, azido-sodium has the effect of inhibiting peroxidase action, and if these are included in the same reagent, the stability of peroxidase will deteriorate, so reagents containing peroxidase may not contain azido-sodium. preferable. Since peroxyhydrogen generated when uric acid is decomposed by uricase is decomposed over time, uricase and peroxidase are preferably included in the same reagent, but uricase and peroxidase are included in the same reagent. It is also possible to add uricase with the previous reagent and not add peroxidase with the later reagent. Further, any one of the first reagent, the second reagent, and the third reagent, and any two or three of them may contain a surfactant. Further, ascorbate oxidase may be contained in any one of the first reagent, the second reagent, and the third reagent, and any two or three of them.

[0037] (1)

First reagent: TOOS, azido sodium

Second reagent: uricase, 4-aminoantipyrine, peroxidase (2)

First reagent: 4-Aminoantipyrine, azido sodium

Second reagent: uricase, TOOS, peroxidase

(3)

First reagent: TOOS, azido sodium

Second reagent: 4-Aminoantipyrine

Third reagent: uricase, peroxidase

(Four)

First reagent: 4-Aminoantipyrine, azido sodium

Second reagent: TOOS

Third reagent: uricase, peroxidase

TOOS contained in the first reagent is a hydrogen donor compound. Among the hydrogen donor compounds, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyoxyline (HDAOS), N-ethyl-N-sulfopropyl- 3-methoxy-line (ADPS), N-ethyl-N-sulfopropyl-line (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxy-line (DAPS), N-sulfopropyl- 3,5-dimethoxy-line (HDAPS), N-ethyl-N-sulfopropyl-3,5-dimethyl-line (MAPS), N-ethyl-N-sulfopropyl-3-methyl-line (TOPS) , N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyline (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -line (ALOS) N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-line (DAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5- Dimethylaline ( MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaline (TOOS), N-sulfopropylaline (HALPS), etc., both of which provide the hydrogen donation of the present invention It can be used as a body. A phenolic hydrogen donor compound can also be used.

[0038] Azidosodium that can be contained in the first reagent is added to avoid the influence of hemoglobin in the test sample. In order to avoid the effects of hemoglobin after adding the first reagent to the sample, it is desirable to react for a certain period of time.

[0039] Test samples used in the measurement method of the present invention are serum, plasma, and urine. Automatic analyzer (Hitachi, Model 7150), add 270 μL of the first reagent to 3 to 20 μL of the test sample. The amount of test sample and reagent used can be changed as appropriate depending on the analyzer used.

[0040] The concentration of sodium azide in the reaction solution in the first step is preferably 0.1 to 2 g / L (0.01% to 0.2% (w / v)).

[0041] The first step is preferably carried out in a pH 6.0 to pH 8.0 buffer solution, preferably a phosphate buffer solution or a Good buffer solution. Especially N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), Ν, Ν-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), phosphate buffer and Good buffer, 2- [4- (2-Hydroxyethyl) -1-piperaduryl] ethanesulfonic acid (ΗEPES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO) Buffer concentrations preferred by piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) and N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) are 10-200 mM. The degree is preferred. Further, the first step preferably contains a surfactant. Examples of the surfactant used in the first step include non-ionic surfactants such as polyoxyethylene alkyl ether and polyoxyethylene alkyl phenyl ether. The concentration of the surfactant in the reaction solution in the first step is preferably about 0.1 to 10 g / L (0.01 to 1% (w / v)), more preferably 0.5 to 5 _§ / y (0.05 to 0.5). % (w / v)). The buffer solution and surfactant used in the first step should be contained in the first reagent! However, after mixing the sample with the above buffer solution and / or surfactant-containing solution The first reagent may be added.

[0042] When the reagent configuration is two reagents, after the first reagent is added, the reaction is performed for a certain period of time, and then the second reagent is added to perform the reaction in the second step. At this time, the reaction time (the time from the addition of the first reagent to the addition of the second reagent) is preferably about 10 minutes and 5 minutes.

[0043] When the reagent configuration is three reagents, after the first reagent is added and reacted for the above time, the second reagent and the third reagent are added in order, and the reactions in the second and third steps are performed. Just do it. The second reagent and the third reagent may be added simultaneously, or the third reagent may be added 1 to 10 minutes after the addition of the second reagent.

[0044] As shown in the first reaction of the above formula by the uricase contained in the second reagent or the third reagent. In addition, uric acid in the test sample is decomposed into allantoin, carbon dioxide and hydrogen peroxide by the action of uricase.

[0045] The uric acid was quantified by converting the hydrogen peroxide produced in the first reaction into a colored quinone by peroxidase by an acid-acid condensation reaction of 4-aminoantipyrine with an α-phosphorus hydrogen donor compound. The measurement is performed at a wavelength of 400 to 700 nm.

[0046] The second step and the third step preferably include a surfactant. Examples of the surfactant used in the second step and the third step include nonionic surfactant polyoxyethylene alkyl ether and polyoxyethylene alkyl ether. The concentration of the surfactant in the reaction solution of the second step and the third step is preferably about 0.1 to 10 g / L (0.0 l to l% (w / v)), more preferably 0.5 to 5 ₈ / (0.05 to 0.5%).

[0047] The concentration of peroxidase when converting hydrogen peroxide to quinone dye in the reaction solution of the second step or the third step is preferably 0.5-10 IU / mL. The concentration of 4-aminoantipyrine The concentration of hydrogen donating compounds such as aniline-based hydrogen donor compounds is preferably 0.1 to 5 mmol / L.

[0048] The concentration of uricase is preferably 50 to 500 IU / L. Other preferable reaction conditions in the second step and the third step are the same as the preferable reaction conditions in the first step.

[0049] When the reagent configuration is 2 reagents, after the end of the second step, and when the reagent configuration is 3 reagents, after the end of the 3rd step, the amount of the produced quinone dye is measured by the absorbance at 550 to 650 nm in the sample. Measure by measuring. Absorbance may be measured using an absorptiometer.

In the present invention, “reagent” includes a reagent composition, and “reagent composition” refers to an enzyme, a surfactant, or the like constituting the reagent. Say the substance.

[0051] In the description of the present invention, for convenience, the steps such as "first step", "second step", "third step", "first reagent", "second reagent", "third reagent", etc. Names and reagent names will be explained using arithmetic numbers 1, 2, and 3, which indicate the order of the steps and reagents, and are not limited to the number of steps and the number of reagents limited to 2 or 3. The number of steps and reagents can be increased.

Example [0052] Hereinafter, specific description will be given based on examples of the present invention. However, the present invention is not limited to the following examples.

[0053] Reagent compositions used in the first step and the second step (the first reagent composition and the first step, respectively)

2 reagent composition) was prepared so as to have the following composition, and used as the first reagent and the second reagent.

[Example 1]

First reagent

Phosphate buffer solution pH 7.0 0 50 mmol / L

T00S 1. mmol / L

Nonionic surfactant polyethylene glycol mono-p-isooctylphenyl ester (manufactured by Nacalai Testa) 0.1% (w / v) ascorbate oxidase 3000 IU / L

Second reagent

Phosphate buffer solution pH 7.0 0 50 mmol / L

Nonionic surfactant ¾ agent Polyethylene glycol mono-p-isococ / Refeninore Itel (Nacalai Tesque) 0. l¾ (w / v) 4-aminoantipyrine 2.0 mmol / L

POD (Peroxidase) 9 units / mL

URICASE 700 IU / L

(Example 2)

First reagent

Phosphate buffer solution pH 7.0 0 50 mmol / L

T00S 1.0 mmol / L

Nonionic surfactant Polyol mono-p isococtyl fe / Rhe Itel (Nacalai Tesque) 0.1% (w / v) ascorbate oxidase 3000 IU / L

Sodium azide 0. 05% (w / v)

¾ 2 drugs

Phosphate buffer solution pH 7.0 0 50 mmol / L

Nonionic surfactant Poly-Mono-p-isooctylphenol Itel (manufactured by Nacalai Tesque) 0.1% (w / v) 4-aminoantipyrine 2.0 mmol / L

POD (Peroxidase) 9 units / mL

URICASE 700 IU / L

[Example 3] First reagent

Phosphate buffer PH7. 0 50 mmol / L

4-Aminoantipyrine 0. 67 mmol / L Nonionic surfactant Poly-mono-p-isooctylphenyl ester (Nacalai Testa) 0. l% (w / v) ascorbate oxidase 3000IU / L

Sodium azide 0. 05% (w / v) Second reagent

Phosphate buffer pH 7.0 0 50 mmol / L Non-ionic surfactant Polyol Mono-p-isooctylphenol / Rye Itel (manufactured by Nacalai Tesque) 0. l% (w / v) T00S 4.5 mmol / L

POD (Peroxidase) 9 IU / mL

URICASE 700 IU / L Samples with uric acid concentrations of 0.84, 1.68, 2.52, 3.36, 4.20, 8.40, 12.60, and 16.80 mg / dL were prepared by diluting a control sample with a uric acid concentration of 16.80 mg / dL in physiological saline.

[0055] For the measurement, a 7150 type automatic analyzer manufactured by Hitachi, Ltd. was used.

[0056] Add 270 μL of the first reagent composition to 6 μL of the sample, heat at 37 ° C for 5 minutes, and then react 90 L of the second reagent composition at 37 ° C for 5 minutes. Measure the absorbance at 600-nm and 700-nm wavelength using the reagent blank as a control, and calculate the absorbance obtained by subtracting the sub-wavelength from the main wavelength in order to correct the effect of absorbance on the sample-derived absorbance and power fluctuation. The uric acid concentration in the sample was determined from the calibration curve.

[0057] At this time, the uric acid concentration in the sample was determined by the method using the first reagent containing peroxidase, which was conventionally used as a control. The reagent compositions of the first reagent and the second reagent used were as follows.

[0058] First reagent

Phosphate buffer pH 7.0 0 50 mmo l / L

TOOS 1.5 匪 o l / L

Nonionic surfactant polyethylene glycol mono-P-isooctylphenyl ester (manufactured by Nacalai Tesque) 0. (w / v) ascorbate oxidase 3000 IU / L

POD (Peroxidase) 3 units / mL

Second reagent

Phosphate buffer PH7. 0 50 ol ol / L

Nonionic Surfactant Polyethylene Daricol Mono-P-Isooctylphenyl

—Tell (Nacalai Tesque) 0. l w / v)

4 Aminoantipyrine 2. 0 匪 ol / L

URICASE 700 IU / L The results are shown in Table 1. The table shows the measured values of the method of the present invention (Example 1, Example 2, Example 3) and the sample diluted with the control sample in the conventional method.

[table 1] Theoretical value Example 1 Example 2 Example 3 Follow fe method

(mg / dL) Measured value Measured value / Measured value Measured value / Measured value Measured value / Measured value Measured value /

(rag / dL) Theoretical value Theoretical value (mg / dL) Theoretical value (mg / dL) Theoretical value

(¾) (%) (%) (%)

0. OU 0. 00 One 0. 00 —― 0. 00 —— 0. 00

0. 84 0. 82 98% 0. 86 102¾ 0. 84 100¾ 0. 72 86¾

1. 68 1. 63 97¾ 1. 64 98% 1. 63 97¾ 1. 9 89¾

I. 52 2. 45 97¾ 2. 45 97¾ 2. 41 96¾ 2. 25 89¾

3.36 3.29 98¾ 3.31 99¾ 3.28 98¾ 3.09 92%

4. 20 4. 12 4. 13 98¾ 4. 1 1 98% 3. 91 93¾

8. 40 8. 38 Stroke 8. 33 99¾ 8. 26 98¾ S.】 0 96¾

12. 60 12. 51 12. 56 100¾ 12.41 98¾ 12.39 98¾

16. 80 17. 05 101¾ 16. 86 100¾ 16. 72 100¾ 16. 75 100¾

[0059] As shown in Table 1, this method is superior to the conventional method in accuracy in the low value range.

[0060] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

[1] Uric acid in the test sample is decomposed into peroxyhydrogen and allantoin by uricase enzyme, and the peracid hydrogen is quantified using peroxidase to quantify uric acid in the test sample. A method for quantifying uric acid, characterized in that a low uric acid level can be accurately quantified by not contacting uric acid with peroxidase before decomposition of uric acid with a uricase enzyme.

[2] The uric acid according to claim 1, wherein a first reagent containing a hydrogen donor compound is added to a test sample, and then a second reagent containing uricase, 4-aminoantipyrine, and peroxidase is added. Quantitation method.

[3] The method for quantifying uric acid according to claim 1 or 2, wherein the second reagent is added 1 to 10 minutes after the addition of the first reagent.

[4] The uric acid according to claim 1, wherein the first reagent containing 4-aminoantipyrine is added to the test sample, and then the second reagent containing hydrogen donor, uricase, and peroxidase is added. Quantification method.

[5] Add a first reagent containing a hydrogen donor compound to the test sample, then add a second reagent containing 4-amaminoantipyrine, and then add a third reagent containing uricase and peroxidase. The method for quantifying uric acid according to claim 1, characterized in that:

[6] Add the first reagent containing 4-aminoantipyrine to the test sample, then add the second reagent containing the hydrogen donor compound, and then add the third reagent containing uricase and peroxidase. The method for quantifying uric acid according to claim 1, characterized in that:

[7] The method for quantifying uric acid according to any one of [2] to [6], wherein the first reagent further contains sodium azide.

[8] A uric acid determination reagent comprising a first reagent containing a hydrogen donor and a second reagent containing peroxidase and uricase, wherein the first reagent does not contain peroxidase! /, A uric acid determination reagent.

[9] The uric acid quantitative reagent according to claim 8, wherein the first reagent further contains sodium azide.