JP3602956B2 - Method for measuring superoxide dismutase activity and active oxygen - Google Patents

Description

但し、式（１）中、Ｒ^１ ，Ｒ^２ ，Ｒ^３ およびＲ^４ は、それぞれ独立して、水素原子、ニトロ基、アルコキシル基またはハロゲン原子を示し、Ｍはアルカリ金属またはアンモニウムを示す。
【００１０】
式（１）のテトラゾリウム化合物は、ＳＯＤ（スーパーオキシドジスムターゼ）活性を測定するのに特に適しているが、活性酸素そのものを測定するのにも適用することができる。すなわち、本発明は、別の態様として、活性酸素発生系に、下記の式（１）で表されるテトラゾリウム化合物を添加して、該テトラゾリウム化合物が活性酸素により還元されて生成したホルマザンの吸光度を測定することにより該活性酸素を定量することを特徴とする活性酸素の測定法も提出する。
【００１１】
【発明の実施の形態】
本発明のＳＯＤ活性測定法および活性酸素測定法は、上記式（１）で表されるテトラゾリウム化合物の特性を利用することによって実施される。式（１）で表される化合物のうち、特に好ましいものは、Ｒ^１ 〜Ｒ^４ のうち少なくとも１つがニトロ基であるものであり、例えば、Ｒ^１ およびＲ^４ が水素、Ｒ^２ がニトロ基、Ｒ^３ がヨウ素であるテトラゾリウム化合物（以下、本明細書においてはＷＳＴ−１と略称する）、または、Ｒ^１ が水素、Ｒ^２ およびＲ^３ がニトロ基、Ｒ^４ がメトキシ基であるテトラゾリウム化合物（以下、ＷＳＴ−８と略称する）等がある。
【００１２】
本発明に従いＳＯＤ活性を測定するには、適当な緩衝液中でスーパーオキシドアニオン発生系に、式（１）で表されるテトラゾリウム化合物とスーパーオキシドジスムターゼ（ＳＯＤ）酵素を添加する。スーパーオキシドジスムターゼ発生系としては、キサンチン酸化酵素（ＸＯＤ）を用いるキサンチン（またはヒポキサンチン）−キサンチン酸化酵素系が一般的であるが、その他の化学反応によるスーパーオキシドアニオン発生系も適用可能である。
【００１３】
後述するように式（１）の化合物の特性の１つは、スーパーオキシドアニオンのような活性酸素により還元されて吸光光度分析の可能な還元型ホルマザンを生成することにある。したがって、スーパーオキシドアニオン発生系において、スーパーオキシドアニオンに対するテトラゾリウム化合物の反応（スーパーオキシドアニオンによる還元型ホルマザンの生成反応）と、スーパーオキシドアニオンに対するＳＯＤの反応（ＳＯＤによるスーパーオキシドアニオンの不均化反応）とが競合し、ＳＯＤの活性が高い程、前者の反応が阻害されホルマザンの吸光度変化は小さくなる。かくして、還元型ホルマザンの吸光度を測定することによりＳＯＤの活性を求めることができる。
【００１４】
活性酸素によって還元されて水溶性ホルマザンを生成する式（１）のテトラゾリウム化合物を使用すれば、ＳＯＤ活性を測定できるのみならず、活性酸素そのものを測定することもできる。この場合には、活性酸素発生系（インビトロ系のみならずインビボ系も含む）に、式（１）で表されるテトラゾリウム化合物を添加して、該テトラゾリウム化合物が活性酸素により還元されて生成した還元型ホルマザンの吸光度を測定することにより活性酸素を定量することができる。なお、ここで、活性酸素とは、スーパーオキシドアニオンを意味する。
【００１５】
本発明者らは、先に、式（１）に重複する一連のテトラゾリウム化合物の創製に成功し、そして、該化合物が乳酸脱水素酵素等の脱水素酵素の定量に使用し得ることを見出している（特許第２５９２４３６ 号および特願平８−１２１１３４）。本発明者は、この種のテトラゾリウム化合物について更に研究を重ねた結果、式（１）で表される化合物が、スーパーオキシドアニオンのような活性酸素によって還元される性質を有し、しかも、以下に記すように、ＳＯＤ活性測定に従来より採用されているＮＢＴおよびＸＴＴのそれぞれの欠点を解消して両化合物の長所を兼備し、ＳＯＤ活性測定、さらには活性酸素測定における理想的な試薬となり得ることを見出し、本発明の測定法を案出した。
【００１６】
１．先ず、式（１）の化合物は、スーパーオキシドアニオンにより還元されて、ＸＴＴと同様に水溶性のホルマザンを生成する。したがって、該化合物を用いる測定系においては測定器具への固形分の吸着という問題は生じない。
【００１７】
２．また、式（１）の化合物は、常温で溶解するので、ＳＯＤ活性や活性酸素の測定における測定液の調製が容易である。例えば、ＳＯＤ活性測定においては、テトラゾリウム化合物を０．７５ｍＭ程度の濃度に設定するのが一般的であるが、ＸＴＴは加熱しなければその濃度に調製できないのに対して、式（１）の化合物は常温でも簡単に該濃度に調製できる。
【００１８】
３．さらに、式（１）のテトラゾリウム化合物（酸化型）は高濃度の溶液でも殆ど無色に近く、吸光度がきわめて低い。例えば、同じ０．２ｍＭ の終濃度で比較したとき、ＸＴＴは約０．０４ＡＵを示したのに対して、ＷＳＴ−１やＷＳＴ−８のような式（１）の化合物は殆ど無視し得る程度の吸光度しか示さない。したがって、式（１）のテトラゾリウム化合物の被還元性を利用してＳＯＤ活性や活性酸素を測定すると、ローブランクで測定を行うことができるという利点がある。
【００１９】
４．式（１）のテトラゾリウム化合物を用いるＳＯＤ活性測定は、従来のテトラゾリウム化合物と同等以上の感度を示し、しかも感度のｐＨ依存性がきわめて小さい。例えば、ＳＯＤに対する検量線を作成し、５０％の阻害を示す濃度の比較から感度の比較を行ったところ、ｐＨ１０．２において式（１）に属するＷＳＴ−１は、ＸＴＴとほぼ同じ感度、またＮＢＴの約２倍の感度を示した。しかも、ＸＴＴはｐＨの低下とともに感度が大きく減少するが、式（１）の化合物ではこのようなｐＨ依存性は見られず、ｐＨ８ではＷＳＴ−１の方がＸＴＴよりも３倍程度、感度が高くなることも見出された。
【００２０】
５．式（１）の化合物は測定系に含まれる酵素類と直接的な相互作用を示さないので、測定結果の解釈が容易になる。例えば、式（１）に属するＷＳＴ−１やＷＳＴ−８を用いたＳＯＤ活性測定においても、ＸＴＴを用いる場合と同様に反応系へのＳＯＤの添加により１００ ％の阻害または１００ ％に近い阻害が認められ、ＮＢＴを用いる場合の問題点であったＸＯＤとの直接的な相互作用は存しないことが明らかにされている。
【００２１】
本発明のＳＯＤ活性測定法および活性酸素測定法において用いられる式（１）のテトラゾリウム化合物は、特許第２５９２４３６ 号および特願平８−１２１１３４に記載しているように常法に従って合成することができる。すなわち、例えば、官能基Ｒ^１ およびＲ^２ を有するフェニルヒドラジンにアルデヒド化合物をアルコール溶媒中で反応させることにより、対応するヒドラゾンを合成する。次いで、このヒドラゾンに、官能基Ｒ^３ およびＲ^４ を有するフェニルジアゾニウム塩を水溶媒中塩基性条件下で反応させて、式（１）に対応するホルマザンを得る。ここで、塩基性化剤としては水酸化ナトリウム、水酸化カリウムなどが用いられる。次に、得られたホルマザンを亜硝酸ブチルまたは次亜塩素酸ナトリウム等の酸化剤を用いてアルコール溶媒中で酸化することにより式（１）のテトラゾリウム化合物を得ることができる。
【００２２】
【実施例】
本発明の特徴を更に明らかにするため、式（１）で表されるテトラゾリウム化合物をＳＯＤ活性測定に適用した実施例を以下に示す。
式（１）に属するテトラゾリウム化合物としてＷＳＴ−１（Ｒ^１ ＝Ｒ^４ ＝Ｈ；Ｒ^２ ＝ＮＯ_２ ；Ｒ^３ ＝Ｉ）およびＷＳＴ−８（Ｒ^１ ＝Ｈ；Ｒ^２ ＝Ｒ^３ ＝ＮＯ_２ ；Ｒ^４ ＝ＯＣＨ_３ ）を合成して、その特性を評価し、必要に応じてＸＴＴおよびＮＢＴとの比較を行った。活性酸素（スーパーオキシドアニオン）発生系として、キサンチン酸化酵素（ＸＯＤ）によるキサンチン−キサンチン酸化酵素系を採用した。なお、実験に用いた試薬の入手源は次のとおりである：ＳＯＤ（ウシ赤血球由来；ＥＣ １． １５． １． １． ；４０００Ｕ／ｍｇタンパク質；Ｓｉｇｍａ 社製）、ＸＯＤ（バターミルク由来；ＥＣ １． ２． ３． ２ ；０．３Ｕ／ｍｇ ；オリエンタル酵母（株）製）、ＮＢＴ（和光純薬（株）製）、ＸＴＴ（米国Ｐｏｌｙｓｃｉｅｎｃｅ 社製）、ＧＯ（グルコースオキシダーゼ）（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来；ＥＣ １． １． ３． ４ ；１１０Ｕ／ｍｇ ；天野製薬（株）製）。
【００２３】
ＳＯＤ活性測定は次のように行った：５０ｍＭの炭酸ナトリウム緩衝液（ｐＨ９．４ および１０．２の場合）またはリン酸ナトリウム緩衝液（ｐＨ７．０ 、７．５ および８．０ の場合）の２．５ｍｌ に、３ｍＭのキサンチン、３ｍＭのＥＤＴＡ、ＷＳＴ溶液、およびＳＯＤ含有溶液または水を、各０．１ｍｌ ずつ添加した。ＸＯＤ溶液（０．１ｍｌ）を添加することにより反応を開始させた。分光光度計（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ Ｕｌｔｒｏｓｐｅｃ ３０００）を用い２５℃で、ＷＳＴ−１については４３８ｎｍ 、ＷＳＴ−８については４６０ｎｍ における吸光度変化を測定することによりＳＯＤ活性を求めた。ＸＴＴおよびＮＢＴを用いる場合も同様であるが、吸光度変化の測定は、それぞれ、４７０ｎｍ および５６０ｎｍ において行った。
【００２４】
ＳＯＤ活性測定への適用性の検討：
図１に、ｐＨ１０．２、キサンチン濃度３ｍＭおよびＷＳＴ−１またはＷＳＴ−８の濃度０．７５ｍＭにおけるＸＯＤ濃度の変化に対する吸光度変化を示す。ＸＯＤ濃度（ＸＯＤ活性）が約１５５ｍＵ／ｍｌに達するまで、ＸＯＤ濃度の上昇に従って、吸光度変化がほぼ直線的に上昇していることが認められる。また、図２に、ｐＨ１０．２、ＸＯＤ濃度５８ｍＵ／ｍｌ 、ＷＳＴ−１またはＷＳＴ−８の濃度０．７５ｍＭにおけるキサンチン濃度に対する吸光度変化を示すが、キサンチン濃度の上昇に伴い吸光度変化がほぼ直線的に上昇している。
【００２５】
さらに、図３に、ｐＨ１０．２、キサンチン濃度３ｍＭおよびＸＯＤ濃度５８ｍＵ／ｍｌ におけるＷＳＴ−１またはＷＳＴ−８の濃度変化に対する吸光度変化を示している。図に示されるように、ＷＳＴ−１または−８の濃度が一定値に到達するまでＷＳＴ−１または−８の濃度の上昇に伴い吸光度変化がほぼ直線的に上昇している。
【００２６】
以上の結果から示されるように、ＷＳＴ−１やＷＳＴ−８のようなテトラゾリウム化合物は、スーパーオキシドアニオン発生系においてその生成量に比例する吸光度変化を発現し、ＳＯＤ活性の測定に適用できることが明らかである。
【００２７】
ｐＨの影響および酵素との相互作用の検討：
図４に、キサンチン濃度３ｍＭ、ＸＯＤ濃度５８ｍＵ／ｍｌ 、ＷＳＴ−１またはＷＳＴ−８の濃度０．７５ｍＭにおける吸光度変化に対するｐＨの影響を示す。ＷＳＴ−１およびＷＳＴ−８のいずれについても、ｐＨ９．４ において最大の吸光度変化が認められる。
【００２８】
さらに、上述の最適測定条件、すなわち、キサンチン濃度３ｍＭ、ＸＯＤ濃度５８ｍＵ／ｍｌ 、およびＷＳＴ−１またはＷＳＴ−８の濃度０．７５ｍＭにおけるＳＯＤの阻害を測定した。ＷＳＴ−１によるＳＯＤ阻害曲線を図５に、ＷＳＴ−８によるＳＯＤ阻害曲線を図６にそれぞれ示す。ＷＳＴ−１およびＷＳＴ−８のいずれを用いる系においてもＳＯＤ濃度が高くなると１００ ％または１００ ％に近い阻害が得られ、これらのテトラゾリウム化合物がスーパーオキシドアニオン発生に用いられる酵素（ＸＯＤ）と相互作用しないことが示唆される。
【００２９】
特に注目すべきは、ＷＳＴ−１を用いる場合であり、図５に示されるように、ｐＨ８．０ 、９．４ および１０．２のいずれにおいてもＳＯＤ添加による１００ ％の阻害が認められ、しかも、ほぼ同一のＳＯＤ阻害曲線が得られており、ＷＳＴ−１を用いる系の感度がｐＨに依存していないことを示している。ちなみに図７はｐＨ１０．２における阻害をＸＴＴおよびＮＢＴと比較して示すものであり（キサンチン濃度３ｍＭ；ＸＯＤ濃度５７ｍＵ／ｍｌ）、ＷＳＴ−１はＸＴＴを用いる測定系と同様に１００ ％阻害が認められる。但し、前述したように、ＸＴＴはｐＨの低下とともに感度が大きく減少しｐＨ８ではＷＳＴ−１の方が感度が高くなった。
【００３０】
本発明において用いるＷＳＴ−１およびＷＳＴ−８が酵素と相互作用しないことを確認するため、ＧＯによるグルコース酸化反応系にＷＳＴ−１およびＷＳＴ−８を添加して還元型ホルマザンが生成するか否かを試験した。ｐＨ９．５ における結果を図８に示すが、吸光度の変化は見られずＷＳＴ−１およびＷＳＴ−８はＧＯとも相互作用しないことが明らかである。
【００３１】
生体サンプルのＳＯＤ活性の実測
上述の最適条件下にＷＳＴ−１およびＷＳＴ−８、さらに、比較のためにＸＴＴおよびＮＢＴを用いてラットの血液サンプル中のＳＯＤ活性を測定した。なお、ラットの血液サンプルは次のように調製した：ラット（体重約５００ｇ）の血液に冷却した０．９％ＮａＣｌ溶液を加え攪拌、遠沈を繰り返して得た赤血球に冷却蒸留水を加えて溶血赤血球を得た。これにエタノール／クロロホルムを加えて遠沈して得た上清を１０ｍＭリン酸緩衝液（ｐＨ７．８）を用いて４℃で透析し、これを遠心分離により沈殿を除去した上清をサンプルとした。
【００３２】
ＳＯＤ活性測定の結果を、各テトラゾリウム化合物を用いる方法について対比させて図９のＡ〜Ｃに示す。これらの図から理解されるように、ＷＳＴ−１を用いる方法は従来のＮＢＴを用いる方法よりも高感度であり（図９のＡ）、ＸＴＴとほぼ同等の感度を有し（図９のＢ）、さらに、ＷＳＴ−１の方がＷＳＴ−８よりも高い感度を有する（図９のＣ）。
【００３３】
また、ＷＳＴ−１、ＷＳＴ−８、ＮＢＴ、ＸＴＴのそれぞれを用いるＳＯＤ活性の値には良好な直線関係があり（図９のＡ、ＢおよびＣにおけるｒ＝０．９９９ 、０．９６８ および０．９５０）、本発明に従うＷＳＴ−１、ＷＳＴ−８のような化合物は、従来よりＳＯＤ測定に一般に使用されているＮＢＴのような化合物に代わるＳＯＤ測定用試薬として適していることが明らかである。
【００３４】
【発明の効果】
式（１）のテトラゾリウム化合物を利用する本発明のＳＯＤ活性測定法および活性酸素測定法は、測定液の調製が容易であり、分光光度計のセルなど器具類に固形分が吸着することもないので、簡便で迅速性を要求される連続流れ分析システムに適している。
特に、本発明のＳＯＤ活性測定法は、広範囲のｐＨ領域にわたってデータ解析が容易で高感度の分析を行うことができるので、医療、臨床検査、機能性食品開発などにおいてＳＯＤまたは活性酸素と健康との関わりを探究する研究分野の発展に資するものと考えられる。
【図面の簡単な説明】
【図１】本発明に従うＳＯＤ活性測定法におけるＸＯＤ濃度と吸光度変化の関係の１例を示す。
【図２】本発明に従うＳＯＤ活性測定法におけるキサンチン濃度と吸光度変化の関係の１例を示す。
【図３】本発明に従うＳＯＤ活性測定法におけるテトラゾリウム化合物の濃度と吸光度変化の関係の１例を示す。
【図４】本発明に従うＳＯＤ活性測定法における吸光度変化に対するｐＨの影響の１例を示す。
【図５】本発明に従うＳＯＤ活性測定法におけるＳＯＤ阻害曲線の１例を示す。
【図６】本発明に従うＳＯＤ活性測定法におけるＳＯＤ阻害曲線の別の例を示す。
【図７】本発明に従うＳＯＤ活性測定法におけるＳＯＤ阻害曲線を従来より使用されていたテトラゾリウム化合物と比較して示す。
【図８】本発明において用いられるテトラゾリウム化合物のグルコース酸化反応系における吸光度変化の１例を示す。
【図９】本発明に従うテトラゾリウム化合物を用いて測定したラットの血液サンプルのＳＯＤ活性を従来の化合物を用いた場合と対比して示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel method for measuring the activity and active oxygen of superoxide dismutase.
[0002]
[Conventional technology and its problems]
All the cells that make up the body of animals and plants are breathing oxygen, and without oxygen the cells can choke and cannot survive. However, oxygen can sometimes change to a form that is extremely harmful to living organisms. For example, among the harmful oxygen species, a superoxide anion (O ₂ ⁻ ), which is one of active oxygens and is generated by triplet oxygen undergoing one-electron reduction, is an enzyme such as xanthine oxidase (XOD) and NADH oxidase. It is known that it is produced by a reaction or autoxidation of a reducing substance and is directly involved in the development of arteriosclerosis, emphysema, Fanconi's disease and the like. Furthermore, active oxygen is involved in many physiological phenomena such as bactericidal action of leukocytes. For example, if the functions of leukocytes such as granulocytes and macrophages are normal, superoxide anions and hydroxyl radicals (.OH ) Is also known to be released.
[0003]
On the other hand, the living body has various control mechanisms for reactive oxygen species including O ₂ ⁻ . For example, we have provided superoxide dismutase (SOD) enzymes as a mechanism for detoxifying harmful active oxygen. This enzyme catalyzes a reaction that disproportionates two molecules of O ₂ ⁻ to convert it into oxygen and hydrogen peroxide, thereby reducing the reactivity of O ₂ ⁻ , thereby reducing its toxicity. So far, a high linear relationship has been established between the activity of SOD (the value obtained by dividing the liver SOD activity by the basal metabolic rate) and the maximum life span of the animal species, and the longevity of Drosophila genetically deficient in SOD It has been revealed that the mutant nematode having high SOD activity has a long life. This strongly suggests that SOD activity is one of the factors that determine the lifespan of oxygen-respiring organisms.
[0004]
Therefore, measuring the activity of active oxygen and the enzymes involved in its regulation is extremely important in research for examining the function and condition of living organisms, diagnosis of diseases and clinical tests for analyzing biological components, etc. Various methods have been devised. In particular, regarding SOD, which is being clarified to have a deep relationship with aging and longevity, not only in the medical field as described above, but also in foods to which components having SOD-like activity are added from the viewpoint of aging prevention and health promotion. In terms of development, the establishment of a practical measurement technique for its activity is desired also in the food field.
[0005]
Conventionally, as a method for measuring the activity of SOD, a method of measuring the ability to capture O ₂ ⁻ generated by an enzyme reaction (XOD reaction) or a chemical reaction by competing with a tetrazolium compound (tetrazolium salt) is generally used. It's being used. However, nitro blue tetrazolium (NBT), which is a commonly used tetrazolium compound, is reduced to water-insoluble formazan when reduced, and is adsorbed to various instruments including a spectrophotometer cell, making it extremely difficult to handle. . Therefore, it was not suitable for a continuous flow analysis system widely used in clinical tests. The method also O ₂ ^- it from causing a direct redox reaction and reduction of XOD generation system, the addition of high concentrations of SOD to the reaction system to completely inhibit the production of NBT formazan However, there is a defect that the interpretation of the activity measurement becomes very complicated.
[0006]
The present inventors have previously proposed a new tetrazolium compound XTT (3 ′-{1-[(phenylamino) -carbonyl] -3,4-tetrazolium} -bis (4-methoxy-) in which reduced formazane is water-soluble. A method for measuring SOD activity using (6-nitro) benzenesulfonic acid hydrate) was proposed (Analytical Biochemistry, 251, 206-209 (1997)). In this method, since the reduced formazane of XTT has a property of being easily dissolved in water, the phenomenon of adsorption to the measuring instrument was improved. In addition, XTT did not show a direct interaction with XOD, indicating that the addition of SOD to the reaction resulted in 100% inhibition of XTT. However, this method using XTT has a disadvantage that the detection sensitivity of SOD activity is extremely lowered at a low pH as compared with the method using NBT. Further, in order to prepare an XTT solution having an optimum concentration for measurement, the XTT must be dissolved by heating. Therefore, the method using XTT is still not ideal for measuring SOD activity.
[0007]
[Means for Solving the Problems]
An object of the present invention is to develop a new technique that can easily and quickly measure active oxygen and SOD. The present inventor has now found that the use of a specific tetrazolium compound can solve the above-mentioned drawbacks of the conventional SOD activity measurement method, and has completed the present invention.
[0008]
That is, in the present invention, a tetrazolium compound represented by the following formula (1) and a superoxide dismutase enzyme are added to a superoxide anion generating system to compete the reaction of the tetrazolium compound and the enzyme for superoxide anion. A method for measuring the activity of the superoxide dismutase enzyme, which comprises determining the activity of the superoxide dismutase enzyme by measuring the absorbance of formazan generated by reducing the tetrazolium compound with a superoxide anion. .
[0009]
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However, in the formula (1), R ¹ , R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a nitro group, an alkoxyl group or a halogen atom, and M represents an alkali metal or ammonium.
[0010]
The tetrazolium compound of the formula (1) is particularly suitable for measuring SOD (superoxide dismutase) activity, but can also be used for measuring active oxygen itself. That is, in another aspect of the present invention, a tetrazolium compound represented by the following formula (1) is added to an active oxygen generation system, and the absorbance of formazan generated by reduction of the tetrazolium compound by active oxygen is determined. A method for measuring active oxygen, characterized in that the active oxygen is quantified by measuring, is also submitted.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
The SOD activity measurement method and the active oxygen measurement method of the present invention are carried out by utilizing the characteristics of the tetrazolium compound represented by the above formula (1). Among the compounds represented by the formula (1), particularly preferred are compounds in which at least one of R ^{1 to} R ⁴ is a nitro group. For example, R ¹ and R ⁴ are hydrogen and R ² is a nitro group. , A tetrazolium compound in which R ³ is iodine (hereinafter abbreviated as WST-1 in the present specification), or a tetrazolium compound in which R ¹ is hydrogen, R ² and R ³ are nitro groups, and R ⁴ is a methoxy group (Hereinafter abbreviated as WST-8).
[0012]
In order to measure the SOD activity according to the present invention, a tetrazolium compound represented by the formula (1) and a superoxide dismutase (SOD) enzyme are added to a superoxide anion generating system in a suitable buffer. As a superoxide dismutase generation system, a xanthine (or hypoxanthine) -xanthine oxidase system using xanthine oxidase (XOD) is generally used, but a superoxide anion generation system by other chemical reaction is also applicable.
[0013]
As described below, one of the characteristics of the compound of the formula (1) is that it is reduced by active oxygen such as superoxide anion to produce a reduced formazane that can be analyzed by absorptiometry. Therefore, in the superoxide anion generating system, the reaction of the tetrazolium compound with the superoxide anion (reduction of formazan by the superoxide anion) and the reaction of SOD with the superoxide anion (the disproportionation reaction of the superoxide anion with SOD) And the higher the activity of SOD, the more the former reaction is inhibited and the change in absorbance of formazan becomes smaller. Thus, the activity of SOD can be determined by measuring the absorbance of reduced formazan.
[0014]
The use of the tetrazolium compound of the formula (1), which is reduced by active oxygen to produce a water-soluble formazan, enables not only measurement of SOD activity but also measurement of active oxygen itself. In this case, a tetrazolium compound represented by the formula (1) is added to an active oxygen generating system (including not only an in vitro system but also an in vivo system), and the tetrazolium compound is reduced by active oxygen to form a reduced product. Active oxygen can be quantified by measuring the absorbance of type formazan. Here, the active oxygen means a superoxide anion.
[0015]
The present inventors have previously succeeded in creating a series of tetrazolium compounds overlapping formula (1), and have found that the compounds can be used for the quantification of dehydrogenases such as lactate dehydrogenase. (Japanese Patent No. 2592436 and Japanese Patent Application No. 8-121134). As a result of further studies on this kind of tetrazolium compound, the present inventor has found that the compound represented by the formula (1) has a property of being reduced by active oxygen such as a superoxide anion. As described above, it is possible to solve the respective disadvantages of NBT and XTT conventionally used for SOD activity measurement and to combine the advantages of both compounds, and to be an ideal reagent for SOD activity measurement and furthermore for active oxygen measurement. And devised a measuring method of the present invention.
[0016]
1. First, the compound of formula (1) is reduced by a superoxide anion to produce water-soluble formazan, similar to XTT. Therefore, in the measurement system using the compound, the problem of adsorption of the solid content to the measurement instrument does not occur.
[0017]
2. Further, since the compound of the formula (1) dissolves at room temperature, it is easy to prepare a measurement solution for measuring SOD activity and active oxygen. For example, in the measurement of SOD activity, the tetrazolium compound is generally set to a concentration of about 0.75 mM, but the XTT cannot be adjusted to that concentration without heating, whereas the compound of the formula (1) Can be easily adjusted to this concentration even at room temperature.
[0018]
3. Further, the tetrazolium compound (oxidized form) of the formula (1) is almost colorless even in a high-concentration solution, and has an extremely low absorbance. For example, when compared at the same final concentration of 0.2 mM, XTT showed about 0.04 AU, whereas compounds of formula (1) such as WST-1 and WST-8 were almost negligible. Shows only the absorbance of Therefore, when the SOD activity or active oxygen is measured by utilizing the reducibility of the tetrazolium compound of the formula (1), there is an advantage that the measurement can be performed with a raw blank.
[0019]
4. The SOD activity measurement using the tetrazolium compound of the formula (1) shows sensitivity equal to or higher than that of the conventional tetrazolium compound, and the sensitivity has very small pH dependence. For example, when a calibration curve for SOD was prepared and the sensitivity was compared from the comparison of the concentration showing 50% inhibition, at pH 10.2, WST-1 belonging to the formula (1) showed almost the same sensitivity as XTT, The sensitivity was about twice that of NBT. Moreover, although the sensitivity of XTT greatly decreases with a decrease in pH, such a pH dependence is not observed in the compound of the formula (1). At pH 8, WST-1 is about three times more sensitive than XTT. It was also found to be higher.
[0020]
5. Since the compound of the formula (1) does not directly interact with the enzymes contained in the measurement system, the interpretation of the measurement results is facilitated. For example, in the measurement of SOD activity using WST-1 or WST-8 belonging to the formula (1), similarly to the case of using XTT, the addition of SOD to the reaction system causes 100% inhibition or nearly 100% inhibition. It was confirmed that there was no direct interaction with XOD, which was a problem when using NBT.
[0021]
The tetrazolium compound of the formula (1) used in the SOD activity measurement method and the active oxygen measurement method of the present invention can be synthesized according to a conventional method as described in Japanese Patent No. 2592436 and Japanese Patent Application No. 8-121134. . That is, for example, the corresponding hydrazone is synthesized by reacting an aldehyde compound with phenylhydrazine having functional groups R ¹ and R ² in an alcohol solvent. Next, the hydrazone is reacted with a phenyldiazonium salt having functional groups R ³ and R ⁴ in an aqueous solvent under basic conditions to obtain formazan corresponding to the formula (1). Here, sodium hydroxide, potassium hydroxide, or the like is used as the basifying agent. Next, the obtained formazan is oxidized in an alcohol solvent using an oxidizing agent such as butyl nitrite or sodium hypochlorite to obtain a tetrazolium compound of the formula (1).
[0022]
【Example】
In order to further clarify the features of the present invention, an example in which the tetrazolium compound represented by the formula (1) is applied to SOD activity measurement will be described below.
As the tetrazolium compounds belonging to the formula (1), WST-1 (R ¹ = R ⁴ = H; R ² = NO ₂ ; R ³ = I) and WST-8 (R ¹ = H; R ² = R ³ = NO ₂₎ R ⁴ ＯOCH ₃ ), its properties were evaluated, and compared with XTT and NBT as needed. As an active oxygen (superoxide anion) generating system, a xanthine-xanthine oxidase system using xanthine oxidase (XOD) was employed. The reagents used in the experiments were obtained from the following sources: SOD (derived from bovine erythrocyte; EC 1.15.1.1.1; 4000 U / mg protein; manufactured by Sigma), XOD (derived from buttermilk; EC) 1.2.3.2; 0.3 U / mg; manufactured by Oriental Yeast Co., Ltd.), NBT (manufactured by Wako Pure Chemical Industries, Ltd.), XTT (manufactured by Polyscience, USA), GO (glucose oxidase) ( Aspergillus niger ) Origin: EC 1.1.3.4; 110 U / mg; manufactured by Amano Pharmaceutical Co., Ltd.).
[0023]
SOD activity measurements were performed as follows: 50 mM sodium carbonate buffer (at pH 9.4 and 10.2) or sodium phosphate buffer (at pH 7.0, 7.5 and 8.0). To 2.5 ml, 0.1 mM each of 3 mM xanthine, 3 mM EDTA, WST solution, and SOD-containing solution or water were added. The reaction was started by adding the XOD solution (0.1 ml). The SOD activity was determined by measuring changes in absorbance at 438 nm for WST-1 and 460 nm for WST-8 at 25 ° C. using a spectrophotometer (Pharmacia Biotech Ultraspec 3000). The same applies to the case where XTT and NBT are used, but the measurement of absorbance change was performed at 470 nm and 560 nm, respectively.
[0024]
Examination of applicability to SOD activity measurement:
FIG. 1 shows the change in absorbance with respect to the change in XOD concentration at pH 10.2, xanthine concentration of 3 mM, and WST-1 or WST-8 concentration of 0.75 mM. It can be seen that the absorbance change increases almost linearly with increasing XOD concentration until the XOD concentration (XOD activity) reaches about 155 mU / ml. FIG. 2 shows the absorbance change with respect to the xanthine concentration at a pH of 10.2, an XOD concentration of 58 mU / ml, and a concentration of WST-1 or WST-8 of 0.75 mM. Has risen.
[0025]
FIG. 3 shows the change in absorbance with respect to the change in the concentration of WST-1 or WST-8 at a pH of 10.2, a xanthine concentration of 3 mM, and an XOD concentration of 58 mU / ml. As shown in the figure, the absorbance change increases almost linearly with the increase in the concentration of WST-1 or -8 until the concentration of WST-1 or -8 reaches a constant value.
[0026]
As shown from the above results, it is clear that tetrazolium compounds such as WST-1 and WST-8 exhibit a change in absorbance in proportion to the amount generated in a superoxide anion generating system, and can be applied to the measurement of SOD activity. It is.
[0027]
Examination of the effect of pH and interaction with enzymes:
FIG. 4 shows the effect of pH on the change in absorbance at a xanthine concentration of 3 mM, an XOD concentration of 58 mU / ml, and a WST-1 or WST-8 concentration of 0.75 mM. For both WST-1 and WST-8, the largest change in absorbance is observed at pH 9.4.
[0028]
Furthermore, inhibition of SOD was measured under the above-mentioned optimal measurement conditions, that is, a xanthine concentration of 3 mM, an XOD concentration of 58 mU / ml, and a WST-1 or WST-8 concentration of 0.75 mM. The SOD inhibition curve by WST-1 is shown in FIG. 5, and the SOD inhibition curve by WST-8 is shown in FIG. In systems using both WST-1 and WST-8, higher SOD concentrations provide 100% or near 100% inhibition, and these tetrazolium compounds interact with the enzyme (XOD) used to generate superoxide anions. Not suggested.
[0029]
Particularly noteworthy is the case where WST-1 is used. As shown in FIG. 5, 100% inhibition by the addition of SOD was observed at any of pH 8.0, 9.4 and 10.2, and , Almost the same SOD inhibition curve was obtained, indicating that the sensitivity of the system using WST-1 was not dependent on pH. FIG. 7 shows the inhibition at pH 10.2 in comparison with XTT and NBT (xanthine concentration 3 mM; XOD concentration 57 mU / ml). WST-1 showed 100% inhibition similarly to the measurement system using XTT. Can be However, as described above, the sensitivity of XTT greatly decreased with the decrease in pH, and the sensitivity of WST-1 was higher at pH 8 at pH 8.
[0030]
In order to confirm that WST-1 and WST-8 used in the present invention do not interact with the enzyme, whether WST-1 and WST-8 are added to the glucose oxidation reaction system by GO to form reduced formazane Was tested. The results at pH 9.5 are shown in FIG. 8, where no change in absorbance was observed, and it is clear that WST-1 and WST-8 do not interact with GO.
[0031]
Actual measurement of SOD activity of biological sample The SOD activity in a blood sample of a rat was measured using WST-1 and WST-8 under the above-mentioned optimum conditions, and for comparison, XTT and NBT. A rat blood sample was prepared as follows: a cold 0.9% NaCl solution was added to the blood of a rat (body weight: about 500 g), and the mixture was stirred and centrifuged repeatedly. Hemolysed red blood cells were obtained. The supernatant obtained by adding ethanol / chloroform thereto and centrifuging was dialyzed at 4 ° C. using 10 mM phosphate buffer (pH 7.8), and the supernatant obtained by removing the precipitate by centrifugation was used as a sample. did.
[0032]
The results of the SOD activity measurement are shown in FIGS. 9A to 9C in comparison with the method using each tetrazolium compound. As understood from these figures, the method using WST-1 has higher sensitivity than the method using the conventional NBT (FIG. 9A), and has almost the same sensitivity as XTT (FIG. 9B). ) Furthermore, WST-1 has higher sensitivity than WST-8 (C in FIG. 9).
[0033]
Also, there is a good linear relationship between the values of SOD activity using WST-1, WST-8, NBT and XTT (r = 0.999, 0.968 and 0 in FIGS. 9A, 9B and 9C). .950), and compounds such as WST-1 and WST-8 according to the present invention are apparently suitable as reagents for SOD measurement in place of compounds such as NBT conventionally conventionally used for SOD measurement. .
[0034]
【The invention's effect】
In the SOD activity measurement method and the active oxygen measurement method of the present invention using the tetrazolium compound of the formula (1), the measurement solution is easily prepared, and the solid content is not adsorbed on instruments such as a spectrophotometer cell. Therefore, it is suitable for a continuous flow analysis system that requires simple and quick operation.
In particular, the method for measuring SOD activity of the present invention can easily perform data analysis over a wide range of pH and can perform highly sensitive analysis. It is thought to contribute to the development of the research field that explores the involvement of the university.
[Brief description of the drawings]
FIG. 1 shows an example of the relationship between XOD concentration and change in absorbance in the SOD activity measurement method according to the present invention.
FIG. 2 shows an example of the relationship between xanthine concentration and change in absorbance in the SOD activity measurement method according to the present invention.
FIG. 3 shows an example of the relationship between the concentration of a tetrazolium compound and a change in absorbance in the SOD activity measurement method according to the present invention.
FIG. 4 shows an example of the effect of pH on a change in absorbance in the SOD activity measurement method according to the present invention.
FIG. 5 shows an example of an SOD inhibition curve in the SOD activity measurement method according to the present invention.
FIG. 6 shows another example of the SOD inhibition curve in the SOD activity measurement method according to the present invention.
FIG. 7 shows an SOD inhibition curve in the SOD activity measurement method according to the present invention, in comparison with a conventionally used tetrazolium compound.
FIG. 8 shows an example of a change in absorbance of a tetrazolium compound used in the present invention in a glucose oxidation reaction system.
FIG. 9 shows the SOD activity of a blood sample of a rat measured using a tetrazolium compound according to the present invention, as compared to the case using a conventional compound.

Claims (2)

A tetrazolium compound represented by the following formula (1) and a superoxide dismutase enzyme are added to the superoxide anion generating system to compete with the tetrazolium compound for the superoxide anion and the reaction of the enzyme. A method for measuring the activity of a superoxide dismutase enzyme, wherein the activity of the superoxide dismutase enzyme is determined by measuring the absorbance of formazan produced by reduction with an oxide anion.

(In the formula (1), R ¹ , R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a nitro group, an alkoxyl group or a halogen atom, and M represents an alkali metal or ammonium.) Quantifying the active oxygen by adding a tetrazolium compound represented by the following formula (1) to the active oxygen generating system and measuring the absorbance of formazan generated by reduction of the tetrazolium compound by active oxygen. A method for measuring active oxygen, characterized in that:

(In the formula (1), R ¹ , R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a nitro group, an alkoxyl group or a halogen atom, and M represents an alkali metal or ammonium.)

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