JP2564013B2 - Method and apparatus for measuring degree of nucleic acid damage due to nucleic acid constituent peroxide - Google Patents
Method and apparatus for measuring degree of nucleic acid damage due to nucleic acid constituent peroxideInfo
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- JP2564013B2 JP2564013B2 JP1314690A JP31469089A JP2564013B2 JP 2564013 B2 JP2564013 B2 JP 2564013B2 JP 1314690 A JP1314690 A JP 1314690A JP 31469089 A JP31469089 A JP 31469089A JP 2564013 B2 JP2564013 B2 JP 2564013B2
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- nucleic acid
- peroxide
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は生体の過酸化反応および発ガン、突然変異過
程で生じる核酸、ヌクレオチド、またはその構成物質で
ある塩基の過酸化物を発光試薬と反応させることにより
生じる極微弱な化学発光を検出することにより測定する
方法および装置に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention uses nucleic acid, nucleotide, or a peroxide of a base which is a constituent thereof as a luminescent reagent in a living body peroxidation reaction, carcinogenesis, and mutation process. The present invention relates to a method and apparatus for measuring by detecting extremely weak chemiluminescence generated by the reaction.
核酸は全ての生物に存在し、遺伝現象を担う物質であ
る。核酸にはDNA、RNAがあり、それぞれがヌクレオチド
が多数つながったものである。ヌクレオチドは塩基とリ
ボースまたはデオキシリボースが結合したヌクレオチド
とりん酸から構成される。塩基にはアデニン、グアニ
ン、シトシン、チミン、ウラシルがある。核酸は生物の
生命活動において、重要な役割を果たしている。Nucleic acid is a substance that exists in all living organisms and is responsible for genetic phenomena. Nucleic acid includes DNA and RNA, each of which has a large number of linked nucleotides. A nucleotide is composed of a base and a nucleotide in which ribose or deoxyribose is bound, and phosphate. Bases include adenine, guanine, cytosine, thymine, and uracil. Nucleic acid plays an important role in the life activity of living organisms.
ところで、生体は生命維持活動過程においてさまざま
な物質を代謝吸収しているが、その一方で生体内におい
て各種ラジカルを生成していることが知られている。こ
のラジカルにより生体内の脂質が影響を受け、過酸化脂
質となることが知られているが、最近これらの活性酸素
種による核酸損傷が示唆されている。活性酸素種による
遺伝子に対する障害は発ガン、制ガンあるいは老化のラ
ジカル説との関わりなどから重要な問題である。By the way, it is known that the living body metabolizes and absorbs various substances in the course of life-sustaining activity, while generating various radicals in the living body. It is known that lipids in the living body are affected by these radicals and become lipid peroxides, but recently, nucleic acid damage by these reactive oxygen species has been suggested. The damage to genes caused by reactive oxygen species is an important problem because it is related to carcinogenesis, carcinogenesis, or the radical theory of aging.
現在まで核酸損傷の度合は、核酸構成成分の酸化度、
またはその酸化によって生じる産物を測定することによ
り調べられている。核酸構成成分の過酸化物については
最近チミジンの酸化中間産物として酸化物が高速液体ク
ロマトグラフィーを用いて分離同定された。また、塩基
の１つであるチミンは酸化を受けるとチミンハイドロパ
ーオキサイド及びチミングリコールとなることが知られ
ており、チミングリコールを測定することにより、核酸
の損傷程度を知ろうとする方法がある。To date, the degree of nucleic acid damage depends on the degree of oxidation of nucleic acid constituents,
Or it has been investigated by measuring the product resulting from its oxidation. Regarding the peroxide of nucleic acid component, oxide was recently separated and identified by high performance liquid chromatography as an intermediate product of oxidation of thymidine. Further, it is known that thymine, which is one of the bases, becomes thymine hydroperoxide and thymine glycol when it is oxidized, and there is a method in which thymine glycol is measured to know the degree of damage to nucleic acid.
現在までに核酸構成成分の過酸化物の存在自体があま
り知られておらず、その測定法についても確立されてい
ない。核酸の損傷程度を知る方法としては、核酸構成成
分の酸化によって生じた安定な酸化産物を測定する方法
のみである。しかし、脂質が酸化を受けて過酸化脂質と
なるように、核酸構成成分も活性酸素によって過酸化物
を生じることが推察されるが、その有効な検出法がな
く、高感度で検出することができなかった。Up to now, the existence of peroxide, which is a component of nucleic acid, has not been known so far, and the measuring method thereof has not been established. The only method of knowing the degree of nucleic acid damage is to measure a stable oxidation product produced by the oxidation of nucleic acid constituents. However, it is inferred that nucleic acid constituents also generate peroxides due to active oxygen, so that lipids are oxidized to lipid peroxides, but there is no effective detection method, and it is possible to detect with high sensitivity. could not.
本発明は上記課題を解決するためのもので、核酸構成
成分の過酸化物が極微弱発光を生成することを利用し、
これを捉えることにより核酸構成成分の過酸化度を高感
度に測定することができる核酸構成成分過酸化物の測定
方法および測定装置を提供することを目的とする。The present invention is to solve the above problems by utilizing that the peroxide of the nucleic acid component produces extremely weak light emission,
An object of the present invention is to provide a method and an apparatus for measuring a peroxide of a nucleic acid constituent, by which the degree of peroxide of the nucleic acid constituent can be measured with high sensitivity by grasping this.
そのために本発明は、核酸構成成分の過酸化物を含む
試料を高速液体クロマトグラフィーにより分離し、過酸
化物に発光試薬を加えたときに生じる化学発光を検出し
て核酸構成成分の過酸化度を測定し、過酸化度から核酸
損傷の程度を測定する核酸構成成分過酸化物による核酸
損傷程度測定方法、および核酸構成成分の過酸化物を含
む試料から過酸化物を分離する高速液体クロマトグラフ
ィーと、分離された核酸構成成分過酸化物に発光試薬を
加えたとき生ずる化学発光を検出する光電子増倍管と、
光電子増倍管からの出力をカウントする計数手段と、計
数結果を処理するデータ処理手段と、処理結果を表示す
る表示手段とを備え、核酸構成成分の過酸化度を測定
し、過酸化度から核酸損傷の程度を測定するようにした
核酸構成成分過酸化物による核酸損傷程度測定装置を特
徴としている。Therefore, the present invention separates a sample containing a peroxide of a nucleic acid component by high performance liquid chromatography and detects chemiluminescence generated when a luminescent reagent is added to the peroxide to detect the degree of peroxide of the nucleic acid component. Method for measuring degree of nucleic acid damage from degree of peroxide, and method for measuring degree of nucleic acid damage due to peroxide of nucleic acid constituent component, and high performance liquid chromatography for separating peroxide from sample containing peroxide of nucleic acid constituent component And a photomultiplier tube for detecting chemiluminescence generated when a luminescent reagent is added to the separated nucleic acid component peroxide.
A counting means for counting the output from the photomultiplier tube, a data processing means for processing the counting result, and a display means for displaying the processing result are provided, and the degree of peroxidation of the nucleic acid constituents is measured. It is characterized by a nucleic acid damage degree measuring device for measuring the degree of nucleic acid damage, which is caused by peroxide of a nucleic acid constituent component.
本発明は、過酸化水素、塩酸混合液と核酸構成成分を
反応させることにより核酸構成成分の過酸化物を生じさ
せてこれを高速液体クロマトグラフィーで分離し、これ
に発光試薬を添加して化学発光を生じさせ、これを検出
することにより核酸構成成分の過酸化物を測定すること
ができるので、核酸の酸化による損傷程度を知る方法に
用いることが可能となる。The present invention produces a peroxide of a nucleic acid constituent by reacting a mixed solution of hydrogen peroxide and hydrochloric acid with a nucleic acid constituent and separates the peroxide by high performance liquid chromatography. Since it is possible to measure the peroxide of the nucleic acid constituent by producing luminescence and detecting it, it can be used for a method of knowing the degree of damage due to the oxidation of nucleic acid.
以下、本発明の実施例を説明する。 Examples of the present invention will be described below.
発ガンおよび突然変異メカニズムを知るうえでDNA損
傷レベルについての研究は重要であり、現在まで脂質過
酸化反応の過程で生ずる生成物および活性酸素を含むフ
リーラジカルがDNA損傷を引き起こすことが知られてい
る。Studies on the level of DNA damage are important in understanding carcinogenesis and mutation mechanisms, and it has been known to date that free radicals including products and free radicals generated during the process of lipid peroxidation cause DNA damage. There is.
また、放射線照射により、DNA損傷が引き起こされ
て、その分解物が活性酵素を生成し、他のDNA塩基の酸
化を促進すると言われている。Further, it is said that the irradiation causes DNA damage, and the decomposition product thereof produces an active enzyme to promote the oxidation of other DNA bases.
DNA塩基のうち、最も酸化を受けやすいものはThymine
（チミン）であり、DNA損傷時にはThymine hydroperoxi
deが生成されることが報告されている。しかし、その極
微弱発光現象についてはあまり知られていない。Thymine is the DNA base that is most susceptible to oxidation
Thymine hydroperoxi at the time of DNA damage
It has been reported that de is generated. However, little is known about the extremely weak light emission phenomenon.
今まで水溶系における生体関連物質の過酸化物につい
て高速クロマトグラフ化学発光検出法（CL−HPLC）を用
いた検出を試みてきたが、これらの過酸化物についても
同様の検出法が有用ではないかと考えられる。Up to now, we have tried to detect the peroxide of bio-related substances in the water-soluble system by using the high-performance chromatographic chemiluminescence detection method (CL-HPLC), but the same detection method is not useful for these peroxides. It is thought that.
そこで、核酸構成成分の塩基の１つであるチミンおよ
びデオキシリボースと結合したデオキシリボヌクレオシ
ドの１つであるチミジン（Thymidine）に着目し、その
過酸化物についてCL−HPLC法を用いた高感度検出を行っ
た。Therefore, we focused on thymine, which is one of the deoxyribonucleosides that are bound to thymine, which is one of the bases of nucleic acid constituents, and deoxyribose, and the highly sensitive detection of its peroxide using the CL-HPLC method. went.
第１図は本発明の測定装置を示す図、第２図はチミン
の反応スキームを示す図、第３図はチミジンの反応スキ
ームを示す図、第４図、第５図はCL−HPLCによる化学発
光クロマトグラムを示す図である。図中、11は移動相、
12はポンプ,13は試料、14はインジェクタ、15はHPLCカ
ラム、16はUV検出器、17は発光試薬、18はポンプ、19は
ミキシングセル、20はフローセル、21は光電子増倍管、
22はプリアンプ、23は高圧電源、24はレコーダ、25はフ
ォトンカウンタ、26はマイクロコンピュータ、27はモニ
タである。FIG. 1 is a diagram showing the measuring apparatus of the present invention, FIG. 2 is a diagram showing a reaction scheme of thymine, FIG. 3 is a diagram showing a reaction scheme of thymidine, FIGS. 4 and 5 are chemistries by CL-HPLC. It is a figure which shows an emission chromatogram. In the figure, 11 is a mobile phase,
12 is a pump, 13 is a sample, 14 is an injector, 15 is an HPLC column, 16 is a UV detector, 17 is a luminescent reagent, 18 is a pump, 19 is a mixing cell, 20 is a flow cell, 21 is a photomultiplier tube,
22 is a preamplifier, 23 is a high voltage power supply, 24 is a recorder, 25 is a photon counter, 26 is a microcomputer, and 27 is a monitor.
検出に先立ち、チミン、チミジン各10mgを30％H2O21.
5ml,concHCl25μに溶解し、室温で所定時間放置して
酸化させ、反応後アンモニア水でpH7に調製し、10倍希
釈した。HPLCの移動相11としては蒸留水を用い、HPLCポ
ンプ12で1ml/分で移動相11を送出し、インジェクター14
で10μの試料13を注入してHPLCカラム15を通し、吸収
波長210nmを検出するUV検出器36で吸着能に応じて溶出
される成分の紫外吸収を検出してレコーダ24で記録す
る。一方、pH9.3のホウ酸緩衝液に10μg/mlのチトクロ
ムｃと１μg/mlのルミノールを溶解した発光試薬17をポ
ンプ18で0.5ml/分で送出し、ミキシングセル19で混合し
て増感し、フローセル20を通し、その時生ずる化学発光
を光電子増倍管21で検出する。検出結果をフォトンカウ
ンタ25で計数し、計数結果をマイクロコンピュータで処
理して処理結果をモニタに表示する。この場合、UV検出
器による検出結果とフォトンカウンタによる化学発光検
出結果を対照することより測定結果を容易に認識するこ
とができるが、化学発光検出で精密な測定ができるので
必ずしもUV検出器を使用しなくてもよい。Prior to detection, 10 mg each of thymine and thymidine are added to 30% H 2 O 2 1.
It was dissolved in 5 ml of 25 μl of concHCl, allowed to stand at room temperature for a predetermined time to be oxidized, and after the reaction, adjusted to pH 7 with aqueous ammonia and diluted 10 times. Distilled water was used as the mobile phase 11 of the HPLC, the mobile phase 11 was delivered at 1 ml / min by the HPLC pump 12, and the injector 14
10 μm of the sample 13 is injected and passed through the HPLC column 15, and the UV absorption of the eluted component is detected by the UV detector 36 which detects the absorption wavelength of 210 nm and recorded by the recorder 24. On the other hand, the luminescent reagent 17 in which 10 μg / ml cytochrome c and 1 μg / ml luminol were dissolved in borate buffer solution of pH 9.3 was delivered at 0.5 ml / min by the pump 18, and mixed by the mixing cell 19 to sensitize. Then, through the flow cell 20, the chemiluminescence generated at that time is detected by the photomultiplier tube 21. The detection result is counted by the photon counter 25, the counting result is processed by the microcomputer, and the processing result is displayed on the monitor. In this case, you can easily recognize the measurement result by comparing the detection result by the UV detector and the chemiluminescence detection result by the photon counter, but you cannot always use the UV detector because you can perform accurate measurement by chemiluminescence detection. You don't have to.
例えば、チミンを含むサンプルの場合には、第２図に
示すような酸化過程により、チミンの過酸化物Ｉ、II、
IIIが生成され、これらをHPLCで分離して発光試薬を加
えると、チミンのヒドロペルオキシドThy OOHとチトク
ロムｃとが反応して活性酸素が生成され、これとルミノ
ールとが反応して430nmの化学発光を生ずる。For example, in the case of a sample containing thymine, the oxidization process shown in FIG.
III was generated, and when these were separated by HPLC and a luminescent reagent was added, the hydroperoxide Thy OOH of thymine and cytochrome c react to generate active oxygen, which reacts with luminol to cause chemiluminescence at 430 nm. Cause
高速クロマトグラフィにより分離されたサンプルの成
分の発光クロマトグラムは第４図のようになる。図にお
いて、クロマトグラムＡ、Ｂ、Ｃはそれぞれ酸化時間が
97hr、24hr、3hrの場合であり、ピークP1はH2O2による
発光、ピークP2、P3はチミン過酸化物による発光であ
る。チミジンの場合も、第３図に示すように同様に酸化
により過酸化物Ｘ、XI、XIIが生成され、これらをHPLC
で分離して発光試薬を加えると、チミジンのヒドロペル
オキシドThd OOHとチトクロムｃとが反応して活性酸素
が生成され、これとルミノールとが反応して430nmの化
学発光を生ずる。The emission chromatogram of the components of the sample separated by high performance chromatography is shown in FIG. In the figure, chromatograms A, B, and C show the oxidation time.
In the case of 97 hr, 24 hr, and 3 hr, the peak P1 is due to H 2 O 2 and the peaks P2 and P3 are due to thymine peroxide. In the case of thymidine, peroxides X, XI, and XII are similarly produced by oxidation as shown in FIG.
When the luminescent reagent is separated and added with thymidine, the thymidine hydroperoxide Thd OOH reacts with cytochrome c to produce active oxygen, which reacts with luminol to generate chemiluminescence at 430 nm.
このとき高速クラマトグラフィにより分離されたサン
プルの成分の発光クロマトグラムは第５図のようにな
る。図において、クロマトグラムＣ、Ｄ、Ｅはそれぞれ
酸化時間が72hr、48hr、24hrの場合であり、ピークP1は
H2O2による発光、ピークP2、P3、P4、P5はチミジン過酸
化物による発光である。At this time, the emission chromatogram of the components of the sample separated by high speed chromatography is shown in FIG. In the figure, chromatograms C, D, and E are the cases where the oxidation time is 72 hr, 48 hr, and 24 hr, respectively, and the peak P1 is
Emission due to H 2 O 2 , peaks P2, P3, P4, and P5 are due to thymidine peroxide.
第４図、第５図から分かるようにH2O2も発光試薬と反
応して発光するので、核酸の過酸化物による発光である
ことを検証するために、サンプルにH2O2消去酵素である
カタラーゼを添加し、限外ロ過フィルタでロ過したもの
もサンプルとして使用して核酸の過酸化物による発光を
検出し、さらに過酸化物の還元剤であるNaBH4を添加し
て発光が検出できないことを確認した。As can be seen from FIGS. 4 and 5, H 2 O 2 also reacts with a luminescent reagent and emits light. Therefore, in order to verify that the emission is due to the peroxide of nucleic acid, H 2 O 2 scavenging enzyme was added to the sample. Catalase is added and filtered with an ultrafiltration filter to be used as a sample to detect luminescence due to peroxide of nucleic acid, and NaBH 4 which is a reducing agent of peroxide is added to luminescence. It was confirmed that could not be detected.
以上のように本発明によれば、核酸の構成成分の過酸
化物に対して発光試薬を添加して化学発光を生じさせ、
化学発光を検出することにより過酸化物を定量測定でき
るので、化学発光検出法により核酸の酸化による損傷程
度を知ることが可能となる。As described above, according to the present invention, a chemiluminescence is produced by adding a luminescent reagent to peroxide which is a component of nucleic acid,
Since the peroxide can be quantitatively measured by detecting the chemiluminescence, it is possible to know the degree of damage due to the oxidation of the nucleic acid by the chemiluminescence detection method.
第１図は本発明の測定装置を示す図、第２図はチミンの
反応スキームを示す図、第３図はチミジンの反応スキー
ムを示す図、第４図、第５図はCL−HPLCによる化学発光
クロマトグラムを示す図である。 11……移動相、13……試料、14……インジェクタ、15…
…HPLCカラム、16……UV検出器、17……発光試薬、19…
…ミキシングセル、20……フローセル、21……光電子増
倍管、22……プリアンプ、24……レコーダ、25……フォ
トンカウンタ、26……マイクロコンピュータ。FIG. 1 is a diagram showing the measuring apparatus of the present invention, FIG. 2 is a diagram showing a reaction scheme of thymine, FIG. 3 is a diagram showing a reaction scheme of thymidine, FIGS. 4 and 5 are chemistries by CL-HPLC. It is a figure which shows an emission chromatogram. 11 ... Mobile phase, 13 ... Sample, 14 ... Injector, 15 ...
… HPLC column, 16 …… UV detector, 17 …… Luminescent reagent, 19…
Mixing cell, 20 flow cell, 21 photomultiplier tube, 22 preamplifier, 24 recorder, 25 photon counter, 26 microcomputer.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 宮澤 陽夫 宮城県仙台市泉区高森７―17―７ (72)発明者 稲場 文男 宮城県仙台市太白区八木山南１―13―１ (56)参考文献 特開 昭63−233374（ＪＰ，Ａ) 特開 昭60−49262（ＪＰ，Ａ) 特公 昭56−38000（ＪＰ，Ｂ１) ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor, Yoshio Miyazawa, 7-17-7 Takamori, Izumi-ku, Sendai-shi, Miyagi (72) Inventor, Fumio Inaba 1-1-13-1, Minami Yagiyama, Taihaku-ku, Sendai-shi, Miyagi (56) Reference References JP-A-63-233374 (JP, A) JP-A-60-49262 (JP, A) JP-B-56-38000 (JP, B1)
Claims (2)
液体クロマトグラフィーにより分離し、過酸化物に発光
試薬を加えたときに生じる化学発光を検出して核酸構成
成分の過酸化度を測定し、過酸化度から核酸損傷の程度
を測定することを特徴とする核酸構成成分過酸化物によ
る核酸損傷程度測定方法。1. A sample containing a peroxide of a nucleic acid constituent is separated by high performance liquid chromatography, and chemiluminescence generated when a luminescent reagent is added to the peroxide is detected to detect the degree of peroxide of the nucleic acid constituent. A method for measuring the degree of nucleic acid damage due to peroxide, which is a nucleic acid constituent component, which comprises measuring and measuring the degree of nucleic acid damage from the degree of peroxide.
酸化物を分離する高速液体クロマトグラフィーと、分離
された核酸構成成分過酸化物に発光試薬を加えたとき生
ずる化学発光を検出する光電子増倍管と、光電子増倍管
からの出力をカウントする計数手段と、計数結果を処理
するデータ処理手段と、処理結果を表示する表示手段と
を備え、核酸構成成分の過酸化度を測定し、過酸化度か
ら核酸損傷の程度を測定するようにしたことを特徴とす
る核酸構成成分過酸化物による核酸損傷程度測定装置。2. High performance liquid chromatography for separating peroxide from a sample containing peroxide as a nucleic acid constituent, and detecting chemiluminescence generated when a luminescent reagent is added to the separated peroxide as a nucleic acid constituent. A photomultiplier tube, a counting means for counting the output from the photomultiplier tube, a data processing means for processing the counting result, and a display means for displaying the processing result, and measure the degree of peroxidation of nucleic acid constituents. An apparatus for measuring the degree of nucleic acid damage due to peroxides of nucleic acid constituents, wherein the degree of nucleic acid damage is measured from the degree of peroxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1314690A JP2564013B2 (en) | 1989-12-04 | 1989-12-04 | Method and apparatus for measuring degree of nucleic acid damage due to nucleic acid constituent peroxide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1314690A JP2564013B2 (en) | 1989-12-04 | 1989-12-04 | Method and apparatus for measuring degree of nucleic acid damage due to nucleic acid constituent peroxide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03175356A JPH03175356A (en) | 1991-07-30 |
| JP2564013B2 true JP2564013B2 (en) | 1996-12-18 |
Family
ID=18056378
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1314690A Expired - Fee Related JP2564013B2 (en) | 1989-12-04 | 1989-12-04 | Method and apparatus for measuring degree of nucleic acid damage due to nucleic acid constituent peroxide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2564013B2 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5638000A (en) * | 1979-08-30 | 1981-04-11 | Kayaba Industry Co Ltd | Preventive controller for overtuen of height service car |
| JPS6049262A (en) * | 1983-08-30 | 1985-03-18 | Japan Spectroscopic Co | Automatic analysis device for adenine |
| JPS63233374A (en) * | 1987-03-20 | 1988-09-29 | Tohoku Denshi Sangyo Kk | Method and apparatus for measuring lipid peroxide |
-
1989
- 1989-12-04 JP JP1314690A patent/JP2564013B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03175356A (en) | 1991-07-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |