JPH03175356A - Method and apparatus for measuring peroxide of nucleic acid constituent - Google Patents
Method and apparatus for measuring peroxide of nucleic acid constituentInfo
- Publication number
- JPH03175356A JPH03175356A JP31469089A JP31469089A JPH03175356A JP H03175356 A JPH03175356 A JP H03175356A JP 31469089 A JP31469089 A JP 31469089A JP 31469089 A JP31469089 A JP 31469089A JP H03175356 A JPH03175356 A JP H03175356A
- Authority
- JP
- Japan
- Prior art keywords
- peroxide
- nucleic acid
- chemiluminescence
- acid component
- detected
- 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.)
- Granted
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 36
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 36
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 35
- 150000002978 peroxides Chemical class 0.000 title claims abstract description 35
- 239000000470 constituent Substances 0.000 title abstract description 10
- 238000000034 method Methods 0.000 title description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 15
- 238000012545 processing Methods 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 239000012153 distilled water Substances 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 17
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 15
- 230000003647 oxidation Effects 0.000 description 14
- 238000007254 oxidation reaction Methods 0.000 description 14
- 238000004020 luminiscence type Methods 0.000 description 13
- 229940113082 thymine Drugs 0.000 description 9
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 8
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 229940104230 thymidine Drugs 0.000 description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 230000006378 damage Effects 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 150000003254 radicals Chemical class 0.000 description 4
- -1 thymine peroxides Chemical class 0.000 description 4
- 208000005623 Carcinogenesis Diseases 0.000 description 3
- 108010052832 Cytochromes Proteins 0.000 description 3
- 102000018832 Cytochromes Human genes 0.000 description 3
- 230000005778 DNA damage Effects 0.000 description 3
- 231100000277 DNA damage Toxicity 0.000 description 3
- 230000036952 cancer formation Effects 0.000 description 3
- 231100000504 carcinogenesis Toxicity 0.000 description 3
- 238000002038 chemiluminescence detection Methods 0.000 description 3
- SGQMFJVGYHLIMJ-UHFFFAOYSA-N hydrogen peroxide 5-methyl-1H-pyrimidine-2,4-dione Chemical compound OO.CC1=CNC(=O)NC1=O SGQMFJVGYHLIMJ-UHFFFAOYSA-N 0.000 description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 3
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 210000003323 beak Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000005502 peroxidation Methods 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GUKSGXOLJNWRLZ-UHFFFAOYSA-N thymine glycol Chemical compound CC1(O)C(O)NC(=O)NC1=O GUKSGXOLJNWRLZ-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 238000000504 luminescence detection Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000003546 nucleic acid damage Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
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 peroxides of nucleic acids, nucleotides, or their constituent bases, which are produced during peroxidation reactions, carcinogenesis, and mutation processes in living organisms, as a luminescent reagent. The present invention relates to a method and apparatus for measuring by detecting extremely weak chemiluminescence generated by a 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。Nucleic acids are present in all living things and are responsible for genetic phenomena. Nucleic acids include DNA%RNA, each consisting of a large number of nucleotides linked together.
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。Nucleotides are composed of nucleotides and phosphoric acid with a base and ribose or deoxyribose bonded together. Bases include adenine, guanine, cytosine, thymine, and uracil. Nucleic acids play an important role in the life activities of 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, living organisms metabolize and absorb various substances in the process of life-sustaining activities, but at the same time, it is known that various radicals are generated within the living body. It is known that these radicals affect lipids in living organisms and turn them into peroxidized lipids, and recently it has been suggested that these reactive oxygen species can damage nucleic acids. Damage to genes caused by reactive oxygen species is an important issue because of its relationship to carcinogenesis, cancer prevention, and 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 has been investigated by measuring the degree of oxidation of nucleic acid components or the products resulting from this oxidation. Regarding peroxides, which are constituents of nucleic acids, oxides have recently been isolated and identified as oxidation intermediate products of thymidine using high performance liquid chromatography. Furthermore, it is known that thymine, which is one of the bases, becomes thymine hydroperoxide and thymine glycol when oxidized, and there is a method of determining the degree of damage to nucleic acids by measuring thymine glycol.
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。Until now, the existence of peroxide, a nucleic acid constituent, is not well known, and a method for measuring it has not been established. The only method for determining the degree of damage to nucleic acids is to measure stable oxidation products produced by oxidation of nucleic acid constituents. However, just as lipids undergo oxidation and become peroxidized lipids, nucleic acid constituents are also presumed to produce peroxides due to active oxygen, but there is no effective method for detecting this.
It could not be detected with high sensitivity.
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 aimed at solving the above-mentioned problems, and utilizes the fact that peroxide, which is a nucleic acid component, generates extremely weak luminescence, and by capturing this, the degree of peroxidation of a nucleic acid component can be measured with high sensitivity. An object of the present invention is to provide a method and apparatus for measuring peroxide, a nucleic acid component, which can be used to measure peroxide.
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する
ことを特徴とする核酸構成成分過酸化物の測定方法、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に生ずる化
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
えた核酸構成成分過酸化物の測定装置を特徴としている
。To this end, the present invention is characterized in that a sample containing a peroxide of a nucleic acid component is separated by high performance liquid chromatography, and chemiluminescence generated when a luminescent reagent is added to the peroxide is detected. A method for measuring peroxide, high-performance liquid chromatography to separate peroxide from a sample containing peroxide, a nucleic acid component, and the chemistry that occurs when a luminescent reagent is added to the separated peroxide, a nucleic acid component. A nucleic acid component comprising a photomultiplier tube for detecting luminescence, a counting means for counting the output from the photomultiplier tube, a data processing means for processing the counting results, and a display means for displaying the processing results. Features a peroxide measuring device.
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 involves reacting a mixed solution of hydrogen peroxide and hydrochloric acid with a nucleic acid component to produce peroxide of a nucleic acid component, which is separated by high performance liquid chromatography, and then a luminescent reagent is added to the resulting product. By generating and detecting luminescence, peroxides in nucleic acid constituents can be measured, so it can be used as a method for determining the degree of damage caused by oxidation of nucleic acids.
以下、本発明の詳細な説明する。 The present invention will be explained in detail 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。Research on the level of DNA damage is important in understanding carcinogenesis and mutation mechanisms, and until now it is known that products generated in the process of lipid peroxidation and free radicals including active oxygen 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
、その分解物が活性酸素を生成し、他のＤＮＡ塩基の酸
化を促進すると言われている。It is also said that radiation irradiation causes DNA damage, and its decomposition products generate active oxygen, which promotes 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Ｔｈｙ
ｍｉｎｅ （チミン）であり、Ｄ Ｎ Ａ損傷時には
Ｔｈｙｍｉｎｅ ｈｙｄｒｏｐｅｒｏｘｉｄｅ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。Among DNA bases, the one most susceptible to oxidation is Thy
It has been reported that thymine hydroperoxide is produced when DNA is damaged. However, little is known about this extremely weak luminescence 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 until now, high-performance chromatographic chemiluminescence detection method (CL-HPLC) has been used for peroxides of biologically related substances in aqueous systems.
We have attempted to detect these peroxides, but it is thought that a similar detection method would be useful for these peroxides as well.
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ポヌクレオシド
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 thymidine, a deoxyliponucleoside bonded to thymine, which is one of the bases of nucleic acid constituents, and deoxyribose, and detected its peroxide with high sensitivity using the CL-HPLC method. I did it.
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。Figure 1 shows the measuring device of the present invention, Figure 2 shows the reaction scheme of thymine, Figure 3 shows the reaction scheme of thymidine, and Figures 4 and 5 show chemistry by CL-HPLC. FIG. 3 is a diagram showing a luminescence chromatogram. In the figure, 11 is a mobile phase and 12 is a pump.
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、２ｏ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。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, 2o is a flow cell, 21 is a photomultiplier tube, 22 is a preamplifier, 23 is 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％８
２０２１．５ｍ１． ｃｏｎｄＨｃｊ！ ２５μｆ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１ｍ１１分で移動相１１を送出し、インジェクター１
４で１０μｌ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／ｍβのチトクロムＣと１μｇ ／ ｍ
１のルミノールを溶解した発光試薬１７をポンプ１８
で０．５ｒｒｌ／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７オトンカウンタによ
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, add 10 mg each of thymine and thymidine to 30% 8
2021.5m1. condHcj! The solution was dissolved in an aqueous solution of 25 μf and left to oxidize at room temperature for a predetermined time. After the reaction, the pH was adjusted to 7 with aqueous ammonia and diluted 10 times. Distilled water is used as the HPLC mobile phase 11, and the HPLC pump 12
The mobile phase 11 was delivered in 1 m 11 minutes using the injector 1.
4, inject 10 μl of sample 13 and transfer it to HPLC column 15.
A UV detector 36 detects an absorption wavelength of 210 nm through
The ultraviolet absorption of the components eluted according to the adsorption capacity is detected and recorded by the recorder 24. On the other hand, 10 μg/m β of cytochrome C and 1 μg/m
Pump 18 a luminescent reagent 17 in which luminol of 1 is dissolved.
The light is sent out at 0.5 rrl/min, mixed and sensitized in a mixing cell 19, passed through a flow cell 20, and the chemiluminescence generated at that time is detected with a photomultiplier tube 21. The detection results are counted by a photon counter 25, the counted results are processed by a microcomputer, and the processed results are displayed on a monitor. In this case, the measurement results can be easily recognized by comparing the detection results by the UV detector and the chemiluminescence detection results by the 7-oton counter, but since chemiluminescence detection allows precise measurements, it is not necessary to use the UV detector. Does not need to be used.
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、■、■
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４３０ ｎｍの化学発光を生
ずる。For example, in the case of a sample containing thymine, the oxidation process shown in Figure 2 produces thymine peroxides I, ■, ■.
are produced, and when these are separated by HPLC and a luminescent reagent is added, thymine hydroperoxide Thy OOH
Active oxygen is produced by the reaction between the active oxygen and cytochrome C, and this reacts with luminol to produce chemiluminescence at 430 nm.
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ＡＳＢ、Ｃはそれぞれ酸化時間が９
７ ｈ ｒ ｓ ２４ ｈ ｒ ｓ ３ ｈ ｒの場合
であり、ビークＰ１はＨ３Ｏ２による発光、ビーク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、Ｘ■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ｒｈｄ００Ｈ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-speed chromatography is shown in FIG. In the figure, chromatograms ASB and C each have an oxidation time of 9
7 h r s 24 h r s 3 h r, beak P1 is light emission due to H3O2, beak P2
, P3 is luminescence due to thymine peroxide. In the case of thymidine, as shown in Figure 3, peroxides X, XI, and X■ are similarly produced by oxidation, and when these are separated by HPLC and a luminescent reagent is added, thymidine hydroperoxide rhd00H and cytochrome C are produced. The reaction between the two reacts to produce active oxygen, which reacts with luminol to produce chemiluminescence at 430 nm.
このとき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５図のようになる。At this time, the emission chromatogram of the components of the sample separated by high-speed chromatography is as shown in FIG.
図において、クロマトグラムＣ１Ｄ、Ｅはそれぞれ酸化
時間が７２ ｈ ｒ、 ４８ ｈ ｒ。In the figure, chromatograms C1D and E have oxidation times of 72 hr and 48 hr, respectively.
２４ｈｒの場合であり、ビークＰ１はＨ２Ｏ２による発
光、ビークＰ２、Ｐ３、Ｐ４、Ｐ５はチミジン過酸化物
による発光である。This is the case of 24 hours, and the peak P1 is luminescence due to H2O2, and the peaks P2, P3, P4, and P5 are luminescence 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ｔに、サンプルにＨ２Ｏ２消去酵素
であるカタラーゼを添加し１、限外口過フィルタで口過
しだ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Ｎ ａ
Ｂ Ｈ４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 Figures 4 and 5, H2O2 also reacts with the luminescent reagent and emits light, so catalase, an H2O2 scavenging enzyme, was added to the sample to verify that the luminescence was caused by peroxides of nucleic acids. 1. The material passed through the ultrafilter was also used as a sample to detect luminescence due to nucleic acid peroxide, and furthermore, Na, which is a reducing agent for peroxide, was detected.
It was confirmed that no luminescence could be detected by adding B H4.
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, peroxide can be quantitatively measured by adding a luminescent reagent to peroxide, which is a component of a nucleic acid, to generate chemiluminescence, and detecting the chemiluminescence. The luminescence detection method makes it possible to determine the degree of damage caused by oxidation of nucleic acids.
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・・・７０−セル、２１・・・光電子増倍管、２
２・・・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。Figure 1 shows the measuring device of the present invention, Figure 2 shows the reaction scheme of thymine, Figure 3 shows the reaction scheme of thymidine, and Figures 4 and 5 show chemistry by CL-HPLC. FIG. 3 is a diagram showing a luminescence chromatogram. DESCRIPTION OF SYMBOLS 11... Mobile phase, 13... Sample, 14... Injector, 15... HPLC column, 16... UV detector, 17... Luminescence reagent, 19... Mixing cell, 20... ...70-cell, 21...photomultiplier tube, 2
2...Preamplifier, 24...Recorder, 25...
Photon counter, 26...microcomputer.
Claims (2)
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することを特徴とす
る核酸構成成分過酸化物の測定方法。(1) Nucleic acid component peroxide, which is characterized in that a sample containing peroxide, a nucleic acid component, is separated by high-performance liquid chromatography, and chemiluminescence generated when a luminescent reagent is added to the peroxide is detected. How to measure.
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
たことを特徴とする核酸構成成分過酸化物の測定装置。(2) High-performance liquid chromatography to separate peroxide from a sample containing peroxide, a nucleic acid component, and photoelectron enhancement to detect chemiluminescence generated when a luminescent reagent is added to the separated peroxide, a nucleic acid component. A nucleic acid component peroxide comprising a multiplier tube, a counting means for counting the output from the photomultiplier tube, a data processing means for processing the counting results, and a display means for displaying the processing results. measuring device.
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 true JPH03175356A (en) | 1991-07-30 |
| JP2564013B2 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) |
Citations (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
Patent Citations (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 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2564013B2 (en) | 1996-12-18 |
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