JPH07102155B2 - Method for removing reducing substances in biological samples - Google Patents
Method for removing reducing substances in biological samplesInfo
- Publication number
- JPH07102155B2 JPH07102155B2 JP21421893A JP21421893A JPH07102155B2 JP H07102155 B2 JPH07102155 B2 JP H07102155B2 JP 21421893 A JP21421893 A JP 21421893A JP 21421893 A JP21421893 A JP 21421893A JP H07102155 B2 JPH07102155 B2 JP H07102155B2
- Authority
- JP
- Japan
- Prior art keywords
- biological sample
- reaction
- peroxidase
- reducing substance
- component
- 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 - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は生体試料成分の光学的測
定において、妨害となる還元物質を除去し、測定を正確
に行う方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for removing an interfering reducing substance in an optical measurement of a biological sample component and accurately performing the measurement.
【０００２】[0002]
【従来の技術】生体試料成分の分析は臨床検査診断の分
野において重要であり、血液、尿、唾液、涙液等が検査
試料となる。しかしながら、これら生体試料には代謝物
としての還元物質が多く含まれ、酸化及び還元反応を利
用した光学的測定法において、酸化反応の抑制、もしく
は過剰な還元反応として、測定を妨げる場合がある。2. Description of the Related Art The analysis of biological sample components is important in the field of clinical laboratory diagnosis, and blood, urine, saliva, tears, etc. are used as test samples. However, these biological samples contain a large amount of reducing substances as metabolites, and in an optical measurement method utilizing oxidation and reduction reactions, there are cases where the measurement is hindered due to suppression of oxidation reactions or excessive reduction reactions.
【０００３】典型的な例は、アスコルビン酸であるが、
このアスコルビン酸を除去するには、アスコルビン酸オ
キシダーゼにより酸化する方法が有利である。またビリ
ルビンについてはビリルビンオキシダーゼを用いて消去
するなどの工夫がなされている。しかしながら、血清中
のこれらの還元物質については比較的よく研究されてい
るが、尿については、還元物質の実体も不明であり、ま
してその消去法について十分に検討されていない。A typical example is ascorbic acid,
In order to remove this ascorbic acid, a method of oxidizing with ascorbate oxidase is advantageous. In addition, as for bilirubin, measures such as elimination using bilirubin oxidase have been made. However, although these reducing substances in serum have been relatively well studied, the substance of the reducing substances in urine is unknown, let alone the elimination method thereof has not been sufficiently studied.
【０００４】測定対象成分が二電子還元をうけて受容体
により酸化される場合にも、生体成分中に存在する一電
子還元性成分が、妨害を与え、特異性の高い酵素を使用
しても、酸化発色の抑制もしくは過剰な還元反応が出現
する。しかも、これらの還元成分は検体により組成・濃
度が異なり、検体ブランクを用いて補正する必要があ
る。しかし、あまりに大きい妨害発色の場合に補正は困
難であり、逆に酸化発色の抑制については検体ブランク
による補正そのものが不可能となる。Even when the component to be measured undergoes a two-electron reduction and is oxidized by the receptor, the one-electron reducing component present in the biological component interferes with the use of a highly specific enzyme. , Suppression of oxidative coloring or excessive reduction reaction appears. Moreover, these reducing components have different compositions and concentrations depending on the sample, and it is necessary to correct them using a sample blank. However, it is difficult to correct when the interference color is too large, and conversely, it is impossible to correct the oxidative color by the sample blank itself.
【０００５】[0005]
【発明が解決しようとする課題】一般に生体試料として
血液や尿を対象とするが、測定対象成分がこれらの試料
中に少ない場合、多量の試料を反応系に加えて測定する
ために、生じる還元成分による影響の回避が困難であ
る。すなわち生体試料中の成分をペルオキシダーゼの酸
化発色反応により光学的に測定する系において、生体成
分中の還元物質の影響を除去し、測定を精度よく行う方
法を提供することが本発明の課題である。Generally, blood or urine is used as a biological sample, but when the components to be measured are small in these samples, the reduction caused by adding a large amount of sample to the reaction system for measurement. It is difficult to avoid the effects of the components. That is, it is an object of the present invention to provide a method for accurately measuring a component in a biological sample by an oxidative coloring reaction of peroxidase, which eliminates the influence of a reducing substance in the biological component. .
【０００６】[0006]
【課題を解決するための手段】本発明は生体試料中の成
分をペルオキシダーゼの酸化発色反応により光学的に測
定する系において、反応を妨害する還元物質を安息香
酸、ハラヒドロキシ安息香酸、パラアミノ安息香酸およ
びジブロモフェノールからなる群から選ばれた一電子受
容体を用いて除去することを特徴とする生体試料中の還
元物質の除去法である。The present invention is a system for optically measuring components in a biological sample by an oxidative coloring reaction of peroxidase, and a reducing substance which interferes with the reaction is benzoic acid, halohydroxybenzoic acid or para-aminobenzoic acid. And a one-electron acceptor selected from the group consisting of dibromophenol, and a method of removing a reducing substance in a biological sample.
【０００７】未同定の還元成分の多い試料として尿があ
るので、次に尿について説明する。尿中のアスコルビン
酸をアスコルビン酸オキシダーゼにより消去することは
公知であるが、他の成分については、同じ反応組成液に
より消去系を組み、主反応をあとから行う方法がとられ
ている。しかしながら、尿の還元成分は、この方法でも
消去するのに時間がかかりすぎる場合がある。Since urine is an unidentified sample containing a large amount of reducing components, urine will be described below. It is known to eliminate ascorbic acid in urine by ascorbate oxidase, but for other components, a method of forming an elimination system with the same reaction composition liquid and performing the main reaction later is adopted. However, the reducing components of urine may take too long to be eliminated by this method as well.
【０００８】例えば、フエナジンメトソルフエート（Ｐ
ＭＳ）、１−メトキシーＰＭＳ，ＦＭＮ，ＦＡＤなどの
前処理剤として加えて還元物質を酸素との反応により過
酸化水素に転換して消去する方法が考えられる。この場
合はカタラーゼの添加が反応を促進するが、尿を対象と
した場合、消去に時間を要し、実用性があるとはいえな
い方法である。上記方法も一電子還元物質を除去する方
法であるが、反応系に共存させると、かえって還元発色
もしくは酸化の抑制を促進する。主反応における酵素反
応においては更に妨害となる還元成分が生成することが
ある。理由は反応に必要な酵素が非特異的なためであ
る。For example, phenazine methosulfate (P
MS), 1-methoxy-PMS, FMN, FAD and the like, and a method of erasing by converting the reducing substance to hydrogen peroxide by the reaction with oxygen can be considered. In this case, the addition of catalase accelerates the reaction, but when urine is used as a target, it takes time to eliminate the urine, and this is not a method having practical utility. The above method is also a method of removing the one-electron reducing substance, but when it is made to coexist in the reaction system, the reduction coloring or the suppression of oxidation is promoted. In the enzyme reaction in the main reaction, a reducing component that further interferes may be produced. The reason is that the enzyme required for the reaction is non-specific.
【０００９】本発明の酸化発色の場合は、Ｈ2Ｏ2生成反
応を用いてペルオキシダーゼによる色原体の酸化発色を
行う。この場合、一電子還元性成分をＨ2Ｏ2を発生しな
い形で消去すればよい。In the case of the oxidative color development of the present invention, the oxidative color development of the chromogen by peroxidase is carried out using the H 2 O 2 forming reaction. In this case, the one-electron reducing component may be erased without generating H 2 O 2 .
【００１０】本法の適用例として、尿中ポリアミンの測
定の場合を挙げることができる。本発明では生体試料中
の成分をペルオキシダーゼの酸化発色反応により光学的
に測定する系は、生体試料中の成分にアシルポリアミン
アミドヒドロラーゼ、４−アミノアンチピリン、ペルオ
キシダーゼ、ポリアミンオキシダーゼ、例えばプトレシ
ンオキシダーゼおよび色原体を作用させ、その発色を吸
光度測定する系である。色原体としては、ＥＨＳＰＴな
どが挙げられる。An example of application of this method is the case of measuring urinary polyamines. In the present invention, a system for optically measuring a component in a biological sample by an oxidative coloring reaction of peroxidase is a system in which a component in the biological sample is acyl polyamine amide hydrolase, 4-aminoantipyrine, peroxidase, polyamine oxidase such as putrescine oxidase and chromogen. It is a system in which the body is allowed to act and the color development is measured by absorbance. EHSPT etc. are mentioned as a chromogen.
【００１１】[0011]
【実施例】以下に本発明を実施例により説明する。 実施例１ 尿中の還元物質の影響を回避してポリアミンの測定をペ
ルオキシダーゼの酸化発色系について行った。下記反応
ではプトレシンオキシダーゼ作用により生成するＨ2Ｏ2
をペルオキシダーゼの酸化発色系にて測定した。EXAMPLES The present invention will be described below with reference to examples. Example 1 The measurement of polyamine was carried out on the peroxidase oxidative coloring system while avoiding the influence of reducing substances in urine. In the following reaction, H 2 O 2 produced by the action of putrescine oxidase
Was measured by an oxidative coloring system of peroxidase.
【００１２】 試液１ リン酸バッフアー，ｐＨ７．０ １００ｍＭ アシルポリアミンアミドヒドロラーゼ ２５Ｕ／ml アスコルビン酸オキシダーゼ ４Ｕ／ml 安息香酸 ６０ｍＭ ４−アミノアンチピリン ０．０４％ ペルオキシダーゼ ３Ｕ／mlTest solution 1 Phosphate buffer, pH 7.0 100 mM Acyl polyamine amide hydrolase 25 U / ml Ascorbate oxidase 4 U / ml Benzoic acid 60 mM 4-Aminoantipyrine 0.04% Peroxidase 3 U / ml
【００１３】 試液２ リン酸バッフアー，ｐＨ７.０ １００ｍＭ プトレシンオキシダーゼ ５Ｕ／ml ＥＨＳＰＴ ０．０２％Test solution 2 Phosphate buffer, pH 7.0 100 mM putrescine oxidase 5 U / ml EHSPT 0.02%
【００１４】該反応は３７℃、５５３ｎｍの吸光度を測
定した。図１に、反応の時間経過を示す。試液１に２．
７mlの尿検体を加え、５分間反応後０．１mlの試液２を
加えた。反応後の発色は極めて安定であり、図中、Ｂは
プトレシンオキシダーゼにより生成するＨ2Ｏ2を測定し
た場合を示す。安息香酸に代えて、パラヒドロキシ安息
香酸、パラアミノ安息香酸およびジブロモフェノールを
用いても同様の効果が得られた。In the reaction, the absorbance at 37 ° C. and 553 nm was measured. FIG. 1 shows the time course of the reaction. Test solution 1 to 2.
7 ml of urine sample was added, and after reacting for 5 minutes, 0.1 ml of the test solution 2 was added. The color development after the reaction is extremely stable, and B in the figure shows the case where H 2 O 2 produced by putrescine oxidase was measured. Similar effects were obtained by using parahydroxybenzoic acid, paraaminobenzoic acid and dibromophenol instead of benzoic acid.
【００１５】[0015]
【発明の効果】本発明では生体試料中の成分をペルオキ
シダーゼの酸化発色反応により光学的に測定する系にお
いて、反応を妨害する還元物質を安息香酸、ハラヒドロ
キシ安息香酸、パラアミノ安息香酸およびジブロモフェ
ノールからなる群から選ばれた一電子受容体を用いて除
去することにより、尿中の還元物質の影響を受けず、反
応後の発色は安定であり、正確に生体試料中の成分を測
定できる。INDUSTRIAL APPLICABILITY In the present invention, in a system for optically measuring components in a biological sample by an oxidative coloring reaction of peroxidase, a reducing substance which interferes with the reaction is selected from benzoic acid, halahydroxybenzoic acid, para-aminobenzoic acid and dibromophenol. By removing it by using the one-electron acceptor selected from the group consisting of, the color development after the reaction is stable without being affected by the reducing substance in urine, and the component in the biological sample can be accurately measured.
【図１】尿中ポリアミンのペルオキシダーゼによる酸化
発色反応の時間経過を示す。FIG. 1 shows the time course of oxidative coloring reaction of urinary polyamine by peroxidase.
Claims (4)
酸化発色反応により光学的に測定する系において、反応
を妨害する還元物質を安息香酸、パラヒドロキシ安息香
酸、パラアミノ安息香酸およびジブロモフェノールから
なる群から選ばれた一電子受容体を用いて除去すること
を特徴とする生体試料中の還元物質の除去法。1. A system for optically measuring a component in a biological sample by an oxidative coloring reaction of peroxidase, wherein a reducing substance which interferes with the reaction is selected from the group consisting of benzoic acid, parahydroxybenzoic acid, paraaminobenzoic acid and dibromophenol. A method for removing a reducing substance in a biological sample, which comprises removing the electron using a selected one-electron acceptor.
とを特徴とする請求項第１項記載の生体試料中の還元物
質の除去法。2. The method for removing a reducing substance in a biological sample according to claim 1, wherein the component in the biological sample is polyamine.
酸化発色反応により光学的に測定する系が、生体試料中
の成分にアシルポリアミンアミドヒドロラーゼ、４−ア
ミノアンチピリン、ペルオキシダーゼ、ポリアミンオキ
シダーゼおよび色原体を作用させ、その発色を吸光度測
定する系であることを特徴とする請求項２記載の生体試
料中の還元物質の除去法。3. A system for optically measuring components in a biological sample by an oxidative coloring reaction of peroxidase, wherein acylpolyamine amide hydrolase, 4-aminoantipyrine, peroxidase, polyamine oxidase and a chromogen are added to the components in the biological sample. The method for removing a reducing substance in a biological sample according to claim 2, which is a system in which the color is developed and the absorbance is measured.
酸化発色反応により光学的に測定する系が、生体試料中
の成分にアシルポリアミンアミドヒドロラーゼ、４−ア
ミノアンチピリン、ペルオキシダーゼ、プトレシンオキ
シダーゼおよび色原体を作用させ、その発色を吸光度測
定する系であることを特徴とする請求項２記載の生体試
料中の還元物質の除去法。4. A system for optically measuring a component in a biological sample by an oxidative coloring reaction of peroxidase comprises an acylpolyamine amide hydrolase, 4-aminoantipyrine, peroxidase, putrescine oxidase and a chromogen as components in the biological sample. The method for removing a reducing substance in a biological sample according to claim 2, which is a system in which the color is developed and the absorbance is measured.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21421893A JPH07102155B2 (en) | 1993-08-30 | 1993-08-30 | Method for removing reducing substances in biological samples |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21421893A JPH07102155B2 (en) | 1993-08-30 | 1993-08-30 | Method for removing reducing substances in biological samples |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62035191A Division JPH0668490B2 (en) | 1987-02-17 | 1987-02-17 | Method for removing reducing substances in biological samples |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06339397A JPH06339397A (en) | 1994-12-13 |
| JPH07102155B2 true JPH07102155B2 (en) | 1995-11-08 |
Family
ID=16652174
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21421893A Expired - Fee Related JPH07102155B2 (en) | 1993-08-30 | 1993-08-30 | Method for removing reducing substances in biological samples |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07102155B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6913956B2 (en) * | 2016-09-26 | 2021-08-04 | 池田食研株式会社 | L-Kynurenine measurement method and measurement kit |
-
1993
- 1993-08-30 JP JP21421893A patent/JPH07102155B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH06339397A (en) | 1994-12-13 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |