JPS6134622B2 - - Google Patents
Info
- Publication number
- JPS6134622B2 JPS6134622B2 JP8743977A JP8743977A JPS6134622B2 JP S6134622 B2 JPS6134622 B2 JP S6134622B2 JP 8743977 A JP8743977 A JP 8743977A JP 8743977 A JP8743977 A JP 8743977A JP S6134622 B2 JPS6134622 B2 JP S6134622B2
- Authority
- JP
- Japan
- Prior art keywords
- uric acid
- ions
- sample
- solution
- ferrous ions
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 claims description 26
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 claims description 26
- 229940116269 uric acid Drugs 0.000 claims description 26
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000004040 coloring Methods 0.000 claims description 8
- 229910001448 ferrous ion Inorganic materials 0.000 claims description 8
- 239000011668 ascorbic acid Substances 0.000 claims description 7
- 229960005070 ascorbic acid Drugs 0.000 claims description 7
- 235000010323 ascorbic acid Nutrition 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 6
- 229910001447 ferric ion Inorganic materials 0.000 claims description 6
- 230000001603 reducing effect Effects 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 14
- 238000002835 absorbance Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- FPFSGDXIBUDDKZ-UHFFFAOYSA-N 3-decyl-2-hydroxycyclopent-2-en-1-one Chemical compound CCCCCCCCCCC1=C(O)C(=O)CC1 FPFSGDXIBUDDKZ-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002736 nonionic surfactant Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- LJJZKMDEQVYRJX-UHFFFAOYSA-N 4,5,6-tripyridin-2-yltriazine Chemical compound N1=CC=CC=C1C1=NN=NC(C=2N=CC=CC=2)=C1C1=CC=CC=N1 LJJZKMDEQVYRJX-UHFFFAOYSA-N 0.000 description 1
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】
本発明は新規な尿酸の定量法に関する。従来、
尿酸の定量法としてはいくつかの方法が提案され
ている。それらは例えば尿酸が不溶性の塩を形成
しやすいこと、特定のpHで紫外部に極大吸収を
示すこと、特異性の高い尿酸分解酵素が存在する
こと、還元性を有すること等を利用したものであ
る。しかしながらこれらはいずれも操作が煩雑で
ある、測定に長時間を要す、精度が悪い等の欠点
を有し、改良された定量法の提案が望まれてい
る。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for quantifying uric acid. Conventionally,
Several methods have been proposed for quantifying uric acid. They take advantage of, for example, the fact that uric acid easily forms insoluble salts, shows maximum absorption in ultraviolet light at a certain pH, the presence of highly specific uric acid degrading enzymes, and has reducing properties. be. However, all of these methods have drawbacks such as being complicated to operate, requiring a long time for measurement, and having poor accuracy, and there is a need for an improved quantitative method.
本発明者らは、従来法のうち特に尿酸の還元性
を利用する方法、すなわちFe2+により発色する
発色剤の存在下、尿酸によりFe3+→Fe2+の還元
反応を行なわしめ、反応終了後生じた発色液の吸
光度を測定する方法に着目し研究を進めた。 Among conventional methods, the present inventors have developed a method that takes advantage of the reducing properties of uric acid, namely, a reduction reaction of Fe 3+ → Fe 2+ with uric acid in the presence of a coloring agent that develops color with Fe 2+ . The research focused on a method of measuring the absorbance of the coloring solution produced after the completion of the experiment.
この方法はFe3+→Fe2+の還元にたん白質の影
響が少ないので、たとえば血清中の尿酸を定量す
る場合でも除たん白操作を行なわずに直接定量が
できるので、有利な方法の一つとして知られてい
る。しかしながらこの方法にも大きな難点があつ
て、アスコルビン酸のようにFe3+→Fe2+の還元
反応を行なう物質が共存すると過大な尿酸値を与
え、特に治療にアスコルビンを用いた患者の血清
では極端な異常値を与えるので、日常検査法とし
て採用するのは危険である。 This method has little influence of proteins on the reduction of Fe 3+ →Fe 2+ , so it is one of the advantageous methods because, for example, when quantifying uric acid in serum, it can be directly quantified without removing protein. known as one. However, this method also has a major drawback; the coexistence of a substance that performs the reduction reaction of Fe 3+ → Fe 2+ , such as ascorbic acid, causes excessive uric acid levels, especially in the serum of patients treated with ascorbic acid. Since it gives extremely abnormal values, it is dangerous to use it as a routine testing method.
本発明者らはこれらの点に鑑み種々検討したと
ころつぎの新知見を得た。 The present inventors conducted various studies in view of these points and obtained the following new findings.
すなわち、適当な緩衝液中でFe2+により発色
する試薬とFe3+の適量を用いると、アスコルビ
ン酸はFe3+→Fe2+の反応を瞬時に完結して発色
するのに反し、尿酸はこの反応にある時間を必要
とし、その反応速度はFe2+によつて発色する試
薬の種類によつて多少異るが各試薬について適当
な緩衝液と適量のFe3+を用いると反応速度を測
定することができることを知つた。また、この反
応速度を発色液の吸光度の時間的変化として測定
すれば、反応速度と尿酸量とは良好な比例関係に
あることが判明した。 In other words, when using a reagent that develops color with Fe 2+ and an appropriate amount of Fe 3+ in an appropriate buffer solution, ascorbic acid instantly completes the Fe 3+ → Fe 2+ reaction and develops color, whereas uric acid This reaction requires a certain amount of time, and the reaction rate varies somewhat depending on the type of reagent that develops color with Fe 2+ , but if an appropriate buffer and appropriate amount of Fe 3+ are used for each reagent, the reaction rate will be I learned that it is possible to measure Furthermore, when this reaction rate was measured as a temporal change in the absorbance of the coloring solution, it was found that there was a good proportional relationship between the reaction rate and the amount of uric acid.
本発明はこれらの新知見に基づいて完成された
もので、第一鉄イオン（Fe2+）により発色する発
色試薬および第二鉄イオン（Fe3+）を添加した酸
性緩衝液に尿酸含有試料を加え、該試料中に存在
するアスコルビン酸により第二鉄イオンが第一鉄
イオン（Fe2+）に還元され、この第一鉄イオンの
生成に伴つて生じる発色の終了後、試料中の尿酸
の存在量に応じ、その還元作用によつて第二鉄イ
オンが還元されて生成した第一鉄イオンによつて
生ずる発色の変化を光学的に測定することを特徴
とする反応速度法による尿酸の定量法である。 The present invention was completed based on these new findings, and involves applying a uric acid-containing sample to an acidic buffer containing a coloring reagent that develops color using ferrous ions (Fe 2+ ) and ferric ions (Fe 3+ ). The ferric ions are reduced to ferrous ions (Fe 2+ ) by the ascorbic acid present in the sample, and after the color development that occurs due to the production of ferrous ions, the uric acid in the sample is reduced. uric acid by the reaction rate method, which is characterized by optically measuring the color change caused by the ferrous ions that are generated by reducing ferric ions due to their reducing action, depending on the amount of uric acid present. It is a quantitative method.
本発明を実施するに当つて、用いられる緩衝液
としては酸性緩衝液たとえば酢酸緩衝液、クエン
酸緩衝液、マレイン酸緩衝液等が好ましい。この
緩衝液に加えられるFe3+としては硫酸第２鉄ア
ンモニウム、塩化第二鉄などが適し、Fe2+によ
つて発色する試薬としてはフエロジン、０―フエ
ナントロリン、ジピリジル、トリピリジルトリア
ジンなどを用いることができる。これらを緩衝液
に溶解させる場合、通常の非イオン界面活性剤を
使用すると良い結果が得られる。 In carrying out the present invention, the buffer used is preferably an acidic buffer such as an acetate buffer, a citrate buffer, a maleate buffer, or the like. Suitable Fe 3+ to be added to this buffer solution include ferric ammonium sulfate and ferric chloride, and reagents that develop color with Fe 2+ include ferrozine, 0-phenanthroline, dipyridyl, tripyridyl triazine, etc. can be used. When dissolving these in buffers, good results are obtained using common nonionic surfactants.
本発明の方法は反応速度法であるからアスコル
ビン酸を多量に含む試料においてもアスコルビン
酸による発色は瞬時に終るのでその後の吸光度の
時間的変化を測定すればよく、尿酸値に全く影響
を与えない。ブドウ糖、グルタチオンも障害とな
らない。また血清中の尿酸を測定する場合にビリ
ルビンのごとき着色物質が存在していても影響を
受けない。本法は試薬ブランク値が変化しても反
応速度は変化しないので、用手法による尿酸の分
析のみでなく自動分析機のようにいつたん調製し
た試薬溶液を長時間用いる尿酸の分析にも適して
いる。 Since the method of the present invention is a reaction rate method, even in samples containing a large amount of ascorbic acid, the color development due to ascorbic acid ends instantly, so it is only necessary to measure the subsequent change in absorbance over time, and it does not affect the uric acid value at all. . Glucose and glutathione are also not a problem. Furthermore, the presence of colored substances such as bilirubin does not affect the measurement of uric acid in serum. Since this method does not change the reaction rate even if the reagent blank value changes, it is suitable not only for manual uric acid analysis, but also for uric acid analysis using a reagent solution prepared once using an automatic analyzer over a long period of time. There is.
実施例 １
緩衝液は酢酸15mlを水約900ml中に加え、水酸
化ナトリウム５ｇを加えて溶かし、さらにブリジ
―35（米国Atlas Powder Co.製非イオン界面活
性剤）２ｇを加えて溶かし、水を追加して1000ml
とする。この液30mlに６％硫酸第二鉄アンモニウ
ム液１mlおよび２％フエロジン液１mlを加え発色
液とする。Example 1 For the buffer solution, 15 ml of acetic acid was added to about 900 ml of water, 5 g of sodium hydroxide was added and dissolved, and 2 g of Brizy-35 (nonionic surfactant manufactured by Atlas Powder Co., USA) was added and dissolved. Add 1000ml
shall be. Add 1 ml of 6% ferric ammonium sulfate solution and 1 ml of 2% ferrozine solution to 30 ml of this solution to obtain a coloring solution.
発色液３mlを37℃で５分間加温したのち尿酸を
含む試料液0.05mlを加え混和し、37℃，560mmに
おいて混和の１分後から４分後までの３分間の吸
光度の増加を測定する。試料液100ml中に尿酸を
それぞれ５mg，10mg，15mg，20mgを含む液につい
ての吸光度の増加はそれぞれ0.051，0.102，
0.152，0.200であつて反応速度と尿酸量は直線関
係にある。 After heating 3 ml of coloring solution at 37°C for 5 minutes, add 0.05 ml of sample solution containing uric acid and mix. Measure the increase in absorbance for 3 minutes from 1 minute to 4 minutes after mixing at 37°C and 560 mm. . The increases in absorbance for solutions containing 5 mg, 10 mg, 15 mg, and 20 mg of uric acid in 100 ml of sample solution were 0.051, 0.102, and 0.102, respectively.
0.152, 0.200, and there is a linear relationship between the reaction rate and the amount of uric acid.
実施例 ２
緩衝液はマレイン酸11.6ｇを水約900mlに溶か
した液に水酸化ナトリウム5.5ｇを加えて溶か
し、さらにブリジ―352ｇを加えて溶かし、水を
追加して1000mlとして製する。この液30mlに2.5
％硫酸第二鉄アンモニウム液１mlおよび２％０―
フエナントロリン液１mlを加え発色液とする。Example 2 A buffer solution is prepared by adding 5.5 g of sodium hydroxide to a solution of 11.6 g of maleic acid dissolved in about 900 ml of water, then adding and dissolving 352 g of Brizy, and adding water to make 1000 ml. 2.5 per 30ml of this liquid
% ferric ammonium sulfate solution 1 ml and 2% 0-
Add 1 ml of phenanthroline solution to make a coloring solution.
発色液３mlをとり実施例１と同様に操作し、試
料液混和の30秒後から１分30秒後までの１分間の
吸光度の増加を500mmで測定する。実施例１と同
じ量の尿酸を含む試料液についての吸光度の増加
はそれぞれ0.030，0.059，0.090，0.121であつて
反応速度と尿酸量とは直線関係にある。 Take 3 ml of the coloring solution and operate in the same manner as in Example 1, and measure the increase in absorbance for 1 minute from 30 seconds after mixing the sample solution to 1 minute and 30 seconds at 500 mm. The increases in absorbance for sample solutions containing the same amount of uric acid as in Example 1 were 0.030, 0.059, 0.090, and 0.121, respectively, and there was a linear relationship between the reaction rate and the amount of uric acid.
Claims (1)
薬および第二鉄イオン（Fe3+）を添加した酸性緩
衝液に尿酸含有試料を加え、該試料中に存在する
アスコルビン酸により第二鉄イオンが、第一鉄イ
オンに還元され、この第一鉄イオンの生成に伴つ
て生じる発色の終了後、試料中の尿酸の存在量に
応じ、その還元作用によつて第二鉄イオンが還元
されて生成した第一鉄イオンによつて生ずる発色
の変化を光学的に測定することを特徴とする反応
速度法による尿酸の定量法。1 A uric acid-containing sample is added to an acidic buffer containing a coloring reagent that develops color with ferrous ions (Fe 2+ ) and ferric ions (Fe 3+ ), and ferric ions are generated by ascorbic acid present in the sample. is reduced to ferrous ions, and after the color development that occurs with the production of ferrous ions is completed, ferric ions are reduced by its reducing action, depending on the amount of uric acid present in the sample. A method for quantifying uric acid using a reaction rate method, which is characterized by optically measuring changes in color caused by ferrous ions produced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8743977A JPS5422896A (en) | 1977-07-22 | 1977-07-22 | Determination of uric acid by using reaction rate system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8743977A JPS5422896A (en) | 1977-07-22 | 1977-07-22 | Determination of uric acid by using reaction rate system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5422896A JPS5422896A (en) | 1979-02-21 |
| JPS6134622B2 true JPS6134622B2 (en) | 1986-08-08 |
Family
ID=13914880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8743977A Granted JPS5422896A (en) | 1977-07-22 | 1977-07-22 | Determination of uric acid by using reaction rate system |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5422896A (en) |
-
1977
- 1977-07-22 JP JP8743977A patent/JPS5422896A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5422896A (en) | 1979-02-21 |
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