JP5522665B2 - Nucleotide extraction method - Google Patents
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Description
本発明は、試料中に含有されるヌクレオチドの抽出方法に関し、具体的にはアデノシン三リン酸（ＡＴＰ）、グアノシン三リン酸（ＧＴＰ）及び／又はウリジン三リン酸（ＵＴＰ）等のヌクレオチドの抽出方法に関する。さらには、本発明はヌクレオチド抽出用試薬及びヌクレオチド測定用試薬キットに関する。 The present invention relates to a method for extracting nucleotides contained in a sample, specifically, extraction of nucleotides such as adenosine triphosphate (ATP), guanosine triphosphate (GTP) and / or uridine triphosphate (UTP). Regarding the method. Furthermore, the present invention relates to a nucleotide extraction reagent and a nucleotide measurement reagent kit.
本出願は、参照によりここに援用されるところの日本出願特願２００８−０１７８６３号優先権を請求する。 This application claims the priority of Japanese Patent Application No. 2008-017863, which is incorporated herein by reference.
ＡＴＰは、すべての生物の細胞内に含まれるヌクレオチドである。試料中に含まれるＡＴＰの測定は、各種工業分野において広く行なわれている。特に、食品衛生、バイオ、臨床検査、医学等の現場では、試料中の微生物の有無の判定、該微生物の細胞数の計数等を目的として、細胞内ＡＴＰの測定が行なわれている。細胞内ＡＴＰを測定する場合、細胞を含む試料に界面活性剤等を添加して細胞内ＡＴＰを抽出した後、抽出されたＡＴＰを測定する方法が多用されている。 ATP is a nucleotide contained in the cells of all living organisms. Measurement of ATP contained in a sample is widely performed in various industrial fields. In particular, in the field of food sanitation, biotechnology, clinical examination, medicine, etc., intracellular ATP is measured for the purpose of determining the presence or absence of microorganisms in a sample, counting the number of cells of the microorganisms, and the like. When measuring intracellular ATP, a method of measuring the extracted ATP after adding a surfactant or the like to a sample containing cells and extracting the intracellular ATP is frequently used.
近年、ヒトで起こる様々な疾患が、組織のエネルギー産生の低下によって起こることが明らかにされている。組織のエネルギー産生の低下とは、具体的には、組織を構成する個々の細胞のＡＴＰレベルの減少を示しており、先天的なミトコンドリア遺伝子の変異によって起こる場合と、後天的な遺伝子変異やウイルス感染等によって起こる場合がある。先天性の疾患としてはミトコンドリア脳筋症(CPEO: chronic progressive external ophthalmoplegia, MELAS: mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, MERRF: myoclonus epilepsy associated with ragged red fibers)等が知られており、後天性の疾患としては脳梗塞、脳の神経変性疾患(Alzheimer's 病、Parkinson's病、Huntington's病、ウイルス性の脳症)等が報告されている。また、これらの疾患では、痙攣、てんかん、精神運動発達遅延、筋力低下、肝機能障害、感音性難聴等の全身での臨床症状が認められることが特徴である。 In recent years, it has been clarified that various diseases occurring in humans are caused by a decrease in tissue energy production. Specifically, the decrease in tissue energy production indicates a decrease in the ATP level of individual cells constituting the tissue, and it is caused by congenital mitochondrial gene mutations, acquired gene mutations and viruses. It may be caused by infection. Known congenital diseases include mitochondrial encephalomyopathy (CPEO: chronic progressive external ophthalmoplegia, MELAS: mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, MERRF: myoclonus epilepsy associated with ragged red fibers) As acquired diseases, cerebral infarction, neurodegenerative diseases of the brain (Alzheimer's disease, Parkinson's disease, Huntington's disease, viral encephalopathy) and the like have been reported. In addition, these diseases are characterized by general clinical symptoms such as convulsions, epilepsy, psychomotor development delay, muscle weakness, liver dysfunction, and sensorineural hearing loss.
このような事実から、生体内の様々な組織のＡＴＰレベルを正確に測定する技術は、1)組織におけるミトコンドリアの病理学の研究、2)遺伝子疾患の診断・予防という観点から、臨床医学的にも重要である。もし、この技術が確立されれば、各組織のエネルギー状態、代謝機能の状態、生理学的ストレスの状態を調べることが可能となる。このような細胞内ＡＴＰレベルの減少・枯渇によって発症する多様な疾患の研究は、その大半が培養細胞を用いた人工的環境下での研究であり、細胞のミトコンドリアの膜電位を指標とした蛍光プローブ(MitoTracker(R)、JC-1(R)：Invitrogen社)等を用いた間接的半定量法による細胞外からのＡＴＰレベルのモニターか、細胞を破壊して含まれるＡＴＰを測定しているのが現状である。From these facts, the technology to accurately measure the ATP level of various tissues in the living body is clinically and medically from the viewpoints of 1) research of mitochondrial pathology in tissues and 2) diagnosis and prevention of genetic diseases. It is also important. If this technology is established, it will be possible to examine the energy status, metabolic function status, and physiological stress status of each tissue. Most of the studies on various diseases that develop due to the decrease and depletion of intracellular ATP level are studies in an artificial environment using cultured cells, and fluorescence using the membrane potential of mitochondria of cells as an index. Monitors the ATP level from the outside by an indirect semi-quantitative method using probes (MitoTracker (R) , JC-1 (R) : Invitrogen), or measures ATP contained by destroying cells. is the current situation.
ＡＴＰの測定法としては、例えば生物発光反応、具体的にはルシフェリン−ルシフェラーゼ発光反応を利用して測定するキット(ENLITEN(R) ATP Assay System：プロメガ社)がすでに開発されており、市販されている。特に、食品衛生、バイオ、臨床検査、医学等の現場では、試料中の微生物の有無の判定、該微生物の細胞数の計数等を目的として、細胞内ＡＴＰレベルの測定が行なわれている。試料中のＡＴＰレベルを測定する場合、試料からＡＴＰを抽出してＡＴＰを定量的に測定することが必要である。As a method for measuring ATP, for example, a kit (ENLITEN® ATP Assay System: Promega ) that uses a bioluminescence reaction, specifically a luciferin-luciferase luminescence reaction, has already been developed and is commercially available. Yes. In particular, in the field of food hygiene, biotechnology, clinical examination, medicine, etc., intracellular ATP levels are measured for the purpose of determining the presence or absence of microorganisms in a sample, counting the number of cells of the microorganisms, and the like. When measuring the ATP level in a sample, it is necessary to extract ATP from the sample and quantitatively measure ATP.
試料からＡＴＰを抽出する方法として、１）過塩素酸をベースにした抽出法(ＰＣＡ抽出法)（非特許文献１）、２）蒸留水中で加熱する方法(Boiling抽出法)（非特許文献２）が報告されている。その後、ＡＴＰ抽出効率の高い方法として、プロテインキナーゼＫで処理をする改良法(Proteinase K抽出法)が報告された（非特許文献３）。また、培養細胞からＡＴＰを抽出する方法として、トリクロロ酢酸をベースにした抽出法（ＴＣＡ抽出法）が報告されている（非特許文献４，５）。さらに、最近では、動物組織のＡＴＰレベルを測定するためのキット(『組織の』ＡＴＰ測定キットTM：東洋ビーネット社)が市販されており、ＰＥＧ含有抽出用溶液がＡＴＰ抽出用試薬として添付されている。As a method for extracting ATP from a sample, 1) an extraction method based on perchloric acid (PCA extraction method) (Non-Patent Document 1), 2) a method of heating in distilled water (Boiling extraction method) (Non-Patent Document 2) ) Has been reported. Thereafter, an improved method of treating with protein kinase K (Proteinase K extraction method) was reported as a method with high ATP extraction efficiency (Non-patent Document 3). As a method for extracting ATP from cultured cells, an extraction method based on trichloroacetic acid (TCA extraction method) has been reported (Non-patent Documents 4 and 5). Furthermore, recently, kits for measuring ATP levels in animal tissues ("Tissue" ATP Measurement Kit TM : Toyo B-Net Co., Ltd.) are commercially available, and a PEG-containing extraction solution is attached as an ATP extraction reagent. ing.
このように様々な改良方法を考案しなければならないように、従来の方法は操作が煩雑であったり、試料中のタンパク質変性凝集塊中にＡＴＰが混入して抽出効率が下がる、用いるタンパク質変性剤の影響によるＡＴＰ測定の阻害、等の問題があった。例えば、ＡＴＰの測定法がルシフェリン−ルシフェラーゼ発光反応を利用する方法である場合、上記タンパク質変性剤としてＴＣＡを用いると、測定系が酸性となって発光反応が阻害されるという問題、試料中のタンパク質濃度が高い場合に、変性タンパク質に覆われたＡＴＰが抽出されない問題、等がある。そのため、従来の測定方法では、培養細胞等比較的タンパク質濃度の低い条件ではほぼ正確にＡＴＰレベルを測定することは可能であるが、タンパク質濃度の高い生体組織中のＡＴＰを測定する場合、ＡＴＰの抽出効率が急激に低下してＡＴＰレベルを正確に測定しているとはいえない。 As described above, it is necessary to devise various improved methods. Conventional methods are complicated in operation, or ATP is mixed in protein-denatured agglomerates in a sample to reduce extraction efficiency. There was a problem such as inhibition of ATP measurement due to the influence of. For example, when the method for measuring ATP is a method using luciferin-luciferase luminescence reaction, if TCA is used as the protein denaturing agent, the measurement system becomes acidic and the luminescence reaction is inhibited. When the concentration is high, there is a problem that ATP covered with the denatured protein is not extracted. Therefore, in the conventional measurement method, it is possible to measure the ATP level almost accurately under conditions of relatively low protein concentration such as cultured cells. However, when measuring ATP in a biological tissue having a high protein concentration, It cannot be said that the extraction efficiency decreases rapidly and the ATP level is accurately measured.
さらには、生物の細胞内には、ＡＴＰ以外にもＧＴＰ、ＵＴＰ、ＣＴＰや各種デオキシヌクレオチド等多種のヌクレオチドが存在している。これらのヌクレオチドも、生体を維持するために必要であるが、効果的な抽出方法や測定方法が十分に解明されているとはいえない。各種ヌクレオチドが効果的に抽出され、各ヌクレオチド特異的な測定方法が開発されれば、生物中における各ヌクレオチドの存在や機能について更に解明されることになり医療、食品、各種工業分野において有用と考えられるが、これらのヌクレオチドの抽出効率もいまだ十分とはいえない。 Furthermore, in addition to ATP, various nucleotides such as GTP, UTP, CTP, and various deoxynucleotides exist in living cells. These nucleotides are also necessary for maintaining the living body, but it cannot be said that effective extraction methods and measurement methods have been sufficiently elucidated. If various nucleotides are effectively extracted and each nucleotide-specific measurement method is developed, the existence and function of each nucleotide in living organisms will be further elucidated, and it will be useful in medical, food and various industrial fields. However, the extraction efficiency of these nucleotides is still not sufficient.
本発明は、簡便に効率よく試料中のヌクレオチドを抽出する方法を提供することを課題とし、具体的にＡＴＰ、ＧＴＰ及び／又はＵＴＰ等のヌクレオチドの抽出方法を提供することを課題とする。さらにはヌクレオチド測定のために使用するヌクレオチド抽出用試薬及びヌクレオチド測定用試薬キットを提供することを課題とする。 An object of the present invention is to provide a method for easily and efficiently extracting nucleotides in a sample, and specifically to provide a method for extracting nucleotides such as ATP, GTP and / or UTP. It is another object of the present invention to provide a nucleotide extraction reagent and a nucleotide measurement reagent kit used for nucleotide measurement.
本発明者らは上記課題を解決するために鋭意研究を重ねた結果、フェノール化合物を含む溶液を用いることで、タンパク質変性凝集塊へのＡＴＰ混入による抽出効率の低下を生じることなく、試料から効率よくヌクレオチドを抽出できることを見出し、本発明を完成した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have used a solution containing a phenol compound, so that the efficiency from a sample can be reduced without causing a decrease in extraction efficiency due to ATP mixing into a protein-denatured aggregate. The inventors have found that nucleotides can be extracted well and completed the present invention.
すなわち本発明は、以下よりなる。
［１］試料中に含有されるヌクレオチド三リン酸の測定のための、ヌクレオチド三リン酸の抽出方法であって、以下の工程（ａ）及び（ｂ）からなり、かつ工程（ａ）及び（ｂ）を順次備えたことを特徴とする抽出方法。
（ａ）試料にフェノール化合物を含む溶液を混合する工程；
（ｂ）前記工程（ａ）で得られた混合液を遠心処理して、上清を抽出液として回収する工程；
［２］ヌクレオチド三リン酸が、アデノシン三リン酸（ＡＴＰ）、グアノシン三リン酸（ＧＴＰ）及び／又はウリジン三リン酸（ＵＴＰ）である前記［１］に記載の抽出方法。
［３］フェノール化合物を含む溶液が、さらにタンパク質変性剤を含むことを特徴とする前記［１］又は［２］に記載の抽出方法。
［４］フェノール化合物がフェノールである前記［１］〜［３］のいずれか１に記載の抽出方法。
［５］試料中に含有されるヌクレオチド三リン酸の測定方法であって、以下の工程（Ａ）〜（Ｃ）を順次備えたことを特徴とする測定方法。
（Ａ）試料にフェノール化合物を含む溶液を混合する工程；
（Ｂ）前記工程（Ａ）で得られた混合液を遠心処理して、フェノール化合物を含む上清を抽出液として回収する工程；
（Ｃ）前記工程（Ｂ）で得られたフェノール化合物を含む抽出液中のヌクレオチド三リン酸を、ヌクレオチド三リン酸測定用試薬を用いて定量する工程；
［６］測定用試薬が、ルシフェラーゼを含むＡＴＰ測定用試薬であって、ルシフェラーゼの発光度を測定することによりＡＴＰを定量することを特徴とする前記［５］に記載の測定方法。
［７］試料からヌクレオチド三リン酸を抽出するためのフェノール化合物を含む溶液からなる抽出用試薬と、ヌクレオチド三リン酸測定用試薬と、前記抽出用試薬を前記［１］〜［４］のいずれかに記載の方法によるヌクレオチド三リン酸の抽出に使用するための説明書とを含むヌクレオチド三リン酸測定用試薬キット。
［８］ヌクレオチド三リン酸がＡＴＰであり、ヌクレオチド三リン酸測定用試薬がＡＴＰ測定用試薬であることを特徴とする前記［７］記載の測定用試薬キット。
［９］ＡＴＰ測定用試薬が、ルシフェラーゼを含むことを特徴とする前記８記載の測定用試薬キット。
［１０］抽出用試薬を調製するための、０．１重量％の８−ヒドロキシキノリンを含むフェノールＴＥ飽和溶液からなる抽出試薬Ａ及びクロロホルムからなる抽出試薬Ｂ、並びに、ルシフェラーゼを含むＡＴＰ測定用試薬とを備えたことを特徴とする、前記［７］〜［９］に記載の測定用試薬キット。
That is, this invention consists of the following.
[1] A method for extracting nucleotide triphosphates for measurement of nucleotide triphosphates contained in a sample, comprising the following steps (a) and (b), and steps (a) and ( b) an extraction method characterized by comprising sequentially.
(A) mixing a solution containing a phenol compound with a sample;
(B) centrifuging the mixed solution obtained in the step (a) and recovering the supernatant as an extract;
[2] The extraction method according to [1], wherein the nucleotide triphosphate is adenosine triphosphate (ATP), guanosine triphosphate (GTP) and / or uridine triphosphate (UTP).
[3] The extraction method according to [1] or [2], wherein the solution containing a phenol compound further contains a protein denaturant.
[4] The extraction method according to any one of [1] to [3], wherein the phenol compound is phenol.
[5] A method for measuring nucleotide triphosphate contained in a sample, comprising the following steps (A) to (C) in sequence.
(A) A step of mixing a solution containing a phenol compound with a sample;
(B) A step of centrifuging the mixed solution obtained in the step (A) and recovering a supernatant containing a phenol compound as an extract;
(C) a step of quantifying the nucleotide triphosphate in the extract containing the phenol compound obtained in the step (B) using a nucleotide triphosphate measurement reagent;
[6] The measurement method according to [5], wherein the measurement reagent is an ATP measurement reagent containing luciferase, and ATP is quantified by measuring the luminescence of luciferase .
[7 ] Any of [1] to [4], wherein the extraction reagent is a solution containing a phenol compound for extracting nucleotide triphosphate from a sample , the nucleotide triphosphate measurement reagent, and the extraction reagent. A reagent kit for measuring nucleotide triphosphate, comprising a manual for use in extraction of nucleotide triphosphate by the method according to claim 1 .
[8] The reagent kit for measurement according to [7], wherein the nucleotide triphosphate is ATP, and the reagent for measuring nucleotide triphosphate is a reagent for measuring ATP.
[9] ATP measuring reagent, the 8 Symbol mounting reagent kit for measuring, characterized in that it comprises a luciferase.
[1 0 ] For preparing an extraction reagent, extraction reagent A consisting of a phenol TE saturated solution containing 0.1% by weight of 8-hydroxyquinoline, extraction reagent B consisting of chloroform, and ATP measurement containing luciferase The reagent kit for measurement according to any one of [ 7 ] to [ 9 ] above, comprising a reagent.
本発明のヌクレオチド抽出方法によれば、生体等の試料から効果的にヌクレオチド、具体的にはＡＴＰ、ＧＴＰ及び／又はＵＴＰを抽出することができる。従来では、培養細胞や血液等、タンパク質濃度の低い試料についてはＡＴＰ等所望のヌクレオチドを抽出し、測定することが可能であったが、生体組織等タンパク質濃度の高い試料では、正確な測定結果が得られているとはいえない状況であった。本発明の抽出方法によれば、従来困難であった試料中のヌクレオチドを効果的に抽出することができる。また、本発明の抽出方法により、ＡＴＰ等のヌクレオチドを抽出したのち、ＡＴＰ特異的に反応するルシフェラーゼを含むＡＴＰ測定試薬を用いて測定することにより、ＡＴＰを定量的に、より正確に測定することができる。 According to the nucleotide extraction method of the present invention, nucleotides, specifically ATP, GTP and / or UTP can be extracted effectively from a sample such as a living body. Conventionally, it was possible to extract and measure desired nucleotides such as ATP for samples with low protein concentration, such as cultured cells and blood, but for samples with high protein concentration such as biological tissue, accurate measurement results were obtained. The situation was not obtained. According to the extraction method of the present invention, nucleotides in a sample, which has been difficult in the past, can be extracted effectively. Moreover, after extracting nucleotides such as ATP by the extraction method of the present invention, ATP is quantitatively and more accurately measured by using an ATP measurement reagent containing luciferase that reacts specifically with ATP. Can do.
本発明のヌクレオチド抽出方法におけるヌクレオチドとは、ＤＮＡやＲＮＡ等のポリヌクレオチドとは区別される核酸であり、具体的にはＡＴＰ、ＧＴＰ及び／又はＵＴＰ等のヌクレオチドをいう。以下、本発明のヌクレオチド、即ちポリヌクレオチドでないヌクレオチドについて、単に核酸という場合もある。 The nucleotide in the nucleotide extraction method of the present invention is a nucleic acid that is distinguished from a polynucleotide such as DNA or RNA, and specifically refers to a nucleotide such as ATP, GTP and / or UTP. Hereinafter, the nucleotide of the present invention, that is, a nucleotide that is not a polynucleotide may be simply referred to as a nucleic acid.
本発明において、試料とは上記ヌクレオチドを含有可能性のある試料であれば良く、生体由来の試料であってもよいし、生体外に存在する試料であってもよい。また、生体は、ヒト及びヒトをのぞく脊柱動物であっても良い。試料として、例えば生体組織、尿、糞便、血液、喀痰、膿汁、細胞、細胞の培養液、飲食物・医薬品・化粧品等の各工業製品・その半製品・その原料、海水、河川水、工業用水、下水、土壌等を挙げることができる。また、細菌汚染検査、清浄度検査、拭き取り検査等の各種検査における検体も、本発明における試料とすることができる。さらには、上記試料を適当な溶媒（例えば、蒸留水、生理的食塩水、リン酸緩衝液、トリス緩衝液、酢酸ナトリウム緩衝液等）に懸濁した溶液を試料としてもよい。試料が固形分を含む場合には、該試料を上記溶媒に懸濁するか、ミキサー等でホモジナイズすれば溶液状のものと同様に扱うことができる。 In the present invention, the sample may be a sample that may contain the nucleotide, and may be a sample derived from a living body or a sample that exists outside a living body. Further, the living body may be a vertebrate animal other than humans and humans. Samples include, for example, biological tissues, urine, feces, blood, sputum, pus, cells, cell cultures, industrial products such as food, medicine, cosmetics, semi-finished products, raw materials, seawater, river water, industrial water Sewage, soil and the like. Samples in various tests such as a bacterial contamination test, a cleanliness test, and a wiping test can also be used as samples in the present invention. Furthermore, a solution obtained by suspending the sample in an appropriate solvent (for example, distilled water, physiological saline, phosphate buffer, Tris buffer, sodium acetate buffer, or the like) may be used as the sample. When the sample contains a solid content, the sample can be treated in the same manner as a solution by suspending the sample in the solvent or homogenizing with a mixer or the like.
本発明のヌクレオチド抽出方法に使用されるフェノール化合物は、フェノール基を有する化合物であって、試料中のヌクレオチドを抽出しうる化合物であれば良く、特に限定されない。特に好適にはフェノールである。フェノール化合物を含む溶液には、クロロホルムを含んでいても良い。また、フェノール化合物を含む溶液には、タンパク質変性剤を含んでいても良い。ここにおいて、タンパク質変性剤とは、自体公知のものが挙げられ、特に従来のＡＴＰ抽出方法に使用可能であったタンパク質変性剤が好適である。そのようなタンパク質変性剤としては、グアニジンイソチアネート、過塩素酸、ＴＣＡ、プロテインキナーゼＫ等が挙げられる。しかしながら、試料中に、ＡＴＰ分解酵素以外のタンパク質が多く含まれる場合には、タンパク質変性剤を加えることでタンパク質が変性して凝集する場合がある。このような場合には測定対象物であるＡＴＰ等の核酸が変性により凝集したタンパク質中に埋没して抽出が困難になってしまう場合もあり、正確な測定ができるとはいえない。また、従来のＡＴＰ抽出方法である加熱処理方法も、本発明のヌクレオチド抽出方法に組み合わせて処理することができるが、タンパク質が多く含まれる試料の場合には、同様に加熱により変性したタンパク質中に核酸が取り込まれる場合もある。従って、タンパク変性剤や加熱処理を組み合わせないでフェノール化合物を含む溶液で処理することが最も効率的にヌクレオチドを抽出することができるものと考えられる。 The phenol compound used in the nucleotide extraction method of the present invention is not particularly limited as long as it is a compound having a phenol group and can extract a nucleotide in a sample. Particularly preferred is phenol. The solution containing the phenol compound may contain chloroform. Further, the solution containing the phenol compound may contain a protein denaturant. Here, examples of the protein denaturing agent include those known per se, and protein denaturing agents that can be used in conventional ATP extraction methods are particularly suitable. Examples of such protein denaturing agents include guanidine isothiocyanate, perchloric acid, TCA, protein kinase K, and the like. However, if the sample contains a large amount of protein other than ATP-degrading enzyme, the protein may be denatured and aggregated by adding a protein denaturant. In such a case, a nucleic acid such as ATP, which is a measurement object, may be buried in a protein aggregated due to denaturation and extraction may be difficult, and it cannot be said that accurate measurement can be performed. In addition, the heat treatment method, which is a conventional ATP extraction method, can also be treated in combination with the nucleotide extraction method of the present invention. Nucleic acids may be incorporated. Therefore, it is considered that nucleotides can be extracted most efficiently by treatment with a solution containing a phenol compound without combining a protein denaturant or heat treatment.
本発明のフェノール化合物を含む溶液のpHは特に限定されないが、最も感度良く測定を行うためには、ルシフェラーゼ測定試薬等、核酸測定試薬において最も効率的に反応しうるpHに合わせることが適当と考えられる。本発明のフェノール化合物を含む溶液を用いて試料を処理した場合には、例えばpH 4〜10等どのようなpHの溶液で処理しても、従来の抽出方法に比べて効果的にヌクレオチドが抽出される。例えばルシフェラーゼ試薬を用いてＡＴＰを測定する場合には、ルシフェラーゼの特質によりpH 7〜9付近で効果的な測定を行うことができるため、本発明のフェノール化合物を含む溶液は、これらのpHとすることができ、より好適にはpH 8付近とすることができる。上記のヌクレオチド抽出方法に使用されるフェノール化合物を含む溶液は、試料からのヌクレオチド抽出用試薬として利用することができる。 The pH of the solution containing the phenolic compound of the present invention is not particularly limited, but in order to perform the measurement with the highest sensitivity, it is considered appropriate to adjust to the pH at which the nucleic acid measurement reagent can react most efficiently, such as a luciferase measurement reagent. It is done. When a sample is treated with a solution containing the phenolic compound of the present invention, nucleotides can be extracted more effectively than with conventional extraction methods, regardless of the pH, such as pH 4-10. Is done. For example, when ATP is measured using a luciferase reagent, effective measurement can be performed in the vicinity of pH 7 to 9 due to the characteristics of luciferase, so the solution containing the phenol compound of the present invention has these pHs. More preferably, the pH can be around 8. The solution containing the phenol compound used in the above-described nucleotide extraction method can be used as a reagent for nucleotide extraction from a sample.
本発明のヌクレオチド抽出用試薬におけるフェノール化合物の溶媒は、特に限定されないが、例えばTE(10mM Tris-HCl pH8.0, 1mM EDTA)を用いることができる。さらに、必要に応じて安定化剤を加えても良い。 The solvent of the phenol compound in the reagent for nucleotide extraction of the present invention is not particularly limited, and for example, TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) can be used. Furthermore, you may add a stabilizer as needed.
本発明のヌクレオチド抽出方法によると、試料に各種ヌクレオチドが存在する場合は、ＡＴＰのみならず、ＧＴＰやＵＴＰ等も効果的に抽出される。本発明のヌクレオチドの測定は、測定目的対象物に応じて適宜測定方法を選択することができ、自体公知の方法、または今後開発されるあらゆる測定方法を含めることができる。今後開発される方法については、例えばＡＴＰ、ＧＴＰ又はＵＴＰ等の測定用試薬であって、目的に応じて、各々の核酸特異的に測定可能な試薬を用いて測定することが好ましい。 According to the nucleotide extraction method of the present invention, when various nucleotides are present in a sample, not only ATP but also GTP and UTP are effectively extracted. For the measurement of the nucleotide of the present invention, a measurement method can be appropriately selected according to the measurement target, and a method known per se or any measurement method developed in the future can be included. As for a method to be developed in the future, for example, it is preferable to measure using a reagent for measurement such as ATP, GTP, UTP, etc., and depending on the purpose, it can be measured specifically for each nucleic acid.
自体公知の方法として、例えばＡＴＰの測定についてはルシフェリン−ルシフェラーゼ発光反応を利用する方法、ＡＴＰ変換反応を利用する方法等を適用することができる。ルシフェリン−ルシフェラーゼ発光反応を利用するＡＴＰの測定方法としては、ルシフェリンおよびルシフェラーゼを含む発光試薬と、標的ＡＴＰとを、金属イオン（マグネシウムイオン等）の存在下で接触させ、生成した光の発光量を測定する方法が挙げられる。例えば、市販のＡＴＰ測定用キットとして、ENLITEN(R) ATP Assay System：プロメガ社、「細胞の」ＡＴＰ測定キットTM：東洋ビーネット社、「組織の」ＡＴＰ測定キットTM：東洋ビーネット社、ルシフェール250プラスTM：キッコーマン、等を用いることができる。これらのＡＴＰ測定用試薬を用いて測定すると、本発明のヌクレオチド抽出方法によりヌクレオチドを抽出して得た試料について、例えばＧＴＰやＵＴＰ等のＡＴＰを除くヌクレオチドが含まれていても、ＡＴＰ特異的に測定することができる。As a method known per se, for example, for the measurement of ATP, a method using a luciferin-luciferase luminescence reaction, a method using an ATP conversion reaction, or the like can be applied. As a method for measuring ATP using a luciferin-luciferase luminescence reaction, a luminescence reagent containing luciferin and luciferase is brought into contact with a target ATP in the presence of a metal ion (magnesium ion, etc.), and the amount of light emitted is determined. The method of measuring is mentioned. For example, as a commercially available ATP measurement kit, ENLITEN (R) ATP Assay System: Promega, "Cellular" ATP measurement kit TM : Toyo Benet, "Tissue" ATP measurement kit TM : Toyo Benet, Lucifer 250 Plus TM : Kikkoman, etc. can be used. When measured using these reagents for ATP measurement, the sample obtained by extracting nucleotides by the nucleotide extraction method of the present invention is ATP-specific even if nucleotides other than ATP such as GTP and UTP are included. Can be measured.
今後開発されるヌクレオチド測定方法、例えば新たなＡＴＰ測定方法、その他ＧＴＰやＵＴＰ特異的な新規測定方法が開発される場合にも本発明のヌクレオチド抽出方法を適用することができる。 The nucleotide extraction method of the present invention can also be applied when a nucleotide measurement method to be developed in the future, for example, a new ATP measurement method or a new measurement method specific to GTP or UTP is developed.
本発明は、フェノール化合物を含む溶液からなる、試料からヌクレオチドを抽出するためのヌクレオチド抽出用試薬にも及ぶ。ヌクレオチド抽出用試薬には、フェノール化合物のほか、さらにクロロホルムを含有していても良い。また、本発明は、ヌクレオチド抽出用試薬及びヌクレオチド測定用試薬を含むヌクレオチド測定用試薬キットにも及ぶ。具体的には、フェノール化合物を含むヌクレオチド抽出用試薬と、ルシフェラーゼを含むＡＴＰ測定用試薬を含むヌクレオチド測定用試薬キットにも及ぶ。 The present invention also extends to a nucleotide extraction reagent for extracting nucleotides from a sample, which comprises a solution containing a phenol compound. In addition to the phenol compound, the nucleotide extraction reagent may further contain chloroform. The present invention also extends to a nucleotide measurement reagent kit including a nucleotide extraction reagent and a nucleotide measurement reagent. Specifically, it extends to a nucleotide measurement reagent kit including a nucleotide extraction reagent containing a phenol compound and an ATP measurement reagent containing luciferase.
本発明の理解を助けるために、以下に実施例を示して具体的に本発明を説明するが、本発明は本実施例に限定されるものでないことはいうまでもない。 In order to help understanding of the present invention, the present invention will be specifically described with reference to the following examples. However, it is needless to say that the present invention is not limited to these examples.
（実施例１）トリゾル（TRIzol(R)）試薬を用いたヌクレオチド抽出方法
３週齢のマウス(C57BL/6J)の各組織（心臓、肝臓、脾臓、筋肉、血液）を摘出し、各組織0.05〜0.4 gに、本実施例のヌクレオチド抽出用溶液3.0 mLを加え、ホモジナイズした。
本実施例のヌクレオチド抽出用溶液として、フェノール、グアニジンイソチオシアネートを含む市販のＲＮＡ抽出用試薬であるTRIzol(R)試薬(pH 5.0：Invitrogen社）2.8 mLにクロロホルム200 μLを加えたものを用いた。
上記ホモジナイズを４℃で12,000 rpm×１０分間遠心処理し、上清200 μLを保存用チューブに入れ、−８０℃にて保存した。(Example 1) Trizol (TRIzol (R)) each tissue nucleotide extraction method using the reagent 3-week-old mice (C57BL / 6J) were removed (heart, liver, spleen, muscle, blood), and each tissue 0.05 To ~ 0.4 g, 3.0 mL of the nucleotide extraction solution of this example was added and homogenized.
As the nucleotide extraction solution of this example, TRIzol (R) reagent (pH 5.0: Invitrogen ), which is a commercially available RNA extraction reagent containing phenol and guanidine isothiocyanate, was added with 200 μL of chloroform. .
The homogenization was centrifuged at 12,000 rpm × 10 minutes at 4 ° C., and 200 μL of the supernatant was placed in a storage tube and stored at −80 ° C.
（実施例２）フェノールＴＥ飽和溶液を用いたヌクレオチド抽出方法
本実施例のヌクレオチド抽出用溶液として、市販のフェノールＴＥ飽和溶液(TE (10mM Tris-HCl pH8.0, 1mM EDTA：WAKO社）2.8 mLにクロロホルム200 μLを加えたものを用いた他は、実施例１と同様の処理を行った。本抽出用溶液は、安定化剤として0.1% (w/v)の8-ヒドロキシキノリンを含み、pH8.0に調整されたものである。(Example 2) Nucleotide extraction method using phenol TE saturated solution As a nucleotide extraction solution of this example, commercially available phenol TE saturated solution (TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA: WAKO) 2.8 mL) The same treatment as in Example 1 was performed except that 200 μL of chloroform was added to the solution, and this extraction solution contained 0.1% (w / v) of 8-hydroxyquinoline as a stabilizer. The pH is adjusted to 8.0.
（実施例３）フェノールＴＥ飽和溶液(mod.)を用いたヌクレオチド抽出方法
本実施例のヌクレオチド抽出用溶液として、フェノール、グアニジンイソチオシアネートを含むフェノールＴＥ飽和溶液（TE (10 mM Tris-HCl pH8.0, 1 mM EDTA）2.8 mLにクロロホルム200 μLを加えたものを用いた他は、実施例１と同様の処理を行った。本抽出用溶液は、pH 8.0に調整されたものである。(Example 3) Nucleotide extraction method using phenol TE saturated solution (mod.) As the nucleotide extraction solution of this example, phenol TE saturated solution containing phenol and guanidine isothiocyanate (TE (10 mM Tris-HCl pH8. (0, 1 mM EDTA) 2.8 mL was used except that 200 μL of chloroform was used, and the same treatment was performed as in Example 1. The solution for extraction was adjusted to pH 8.0.
（比較例１）ＴＣＡを用いたヌクレオチド抽出方法
本比較例のヌクレオチド抽出用溶液として、市販のＡＴＰ測定用試薬（ENLITEN(R) rLuciferase/Luciferin Reagent：プロメガ社）が推奨しているＡＴＰ抽出用試薬（ＴＣＡ含有）を用いた。
実施例１と同様に３週齢のマウス(C57BL/6J)の各組織（心臓、肝臓、脾臓、筋肉、血液）を摘出した。その後各組織0.05〜0.4 gあたりに0.25 M スクロース溶液（10 mM HEPES-NaOH、pH 7.4）を3.0 mL加え、ホモジナイズした。上清500 μLをチューブに入れ、更に10％ＴＣＡを含む抽出用溶液500 μLをチューブに加えた。上記抽出用溶液を含む試料液を４℃で12,000 rpm×１０分間遠心処理し、上清400 μLを新たなチューブに分注した。1.0 M Tris-Acetate（pH 7.75）を200 μL加えて攪拌した後、−８０℃にて保存した。(Comparative Example 1) a nucleotide extraction method nucleotide extracting solution of this comparative example using the TCA, commercial ATP measuring reagent (ENLITEN (R) rLuciferase / Luciferin Reagent: Promega) ATP extraction reagent is recommended (Containing TCA) was used.
In the same manner as in Example 1, each tissue (heart, liver, spleen, muscle, blood) of a 3-week-old mouse (C57BL / 6J) was extracted. Thereafter, 3.0 mL of a 0.25 M sucrose solution (10 mM HEPES-NaOH, pH 7.4) was added to 0.05 to 0.4 g of each tissue and homogenized. 500 μL of the supernatant was placed in a tube, and 500 μL of an extraction solution containing 10% TCA was further added to the tube. The sample solution containing the extraction solution was centrifuged at 12,000 rpm × 10 minutes at 4 ° C., and 400 μL of the supernatant was dispensed into a new tube. 200 μL of 1.0 M Tris-Acetate (pH 7.75) was added and stirred, and then stored at −80 ° C.
（比較例２）ポリエチレングリコール（ＰＥＧ）を用いたヌクレオチド抽出方法
本比較例のヌクレオチド抽出用溶液として、市販のＡＴＰ測定用試薬（『組織の』ＡＴＰ測定キットTM：東洋ビーネット社）に添付のＡＴＰ抽出用試薬（ＰＥＧ含有）を用いた。
比較例１と同様に３週齢のマウス(C57BL/6J)の各組織（心臓、肝臓、脾臓、筋肉、血液）を摘出し、各組織0.05〜0.4 gあたりに0.25 M スクロース溶液（10 mM HEPES-NaOH、pH 7.4）を3.0 mL加え、ホモジナイズした。ホモジェネートを４℃で12,000 rpm×１０分間遠心処理し、上清100 μLを新たなチューブに分注した。本比較例のＰＥＧ含有ＡＴＰ抽出用溶液100 μLを加えて攪拌した後、室温にて３０分放置した後、−８０℃にて保存した。(Comparative Example 2) Nucleotide Extraction Method Using Polyethylene Glycol (PEG) As a nucleotide extraction solution of this Comparative Example, a commercially available ATP measurement reagent ("Tissue" ATP Measurement Kit TM : Toyo B-Net Co., Ltd.) ATP extraction reagent (containing PEG) was used.
As in Comparative Example 1, each tissue (heart, liver, spleen, muscle, blood) of a 3-week-old mouse (C57BL / 6J) was removed and 0.25 M sucrose solution (10 mM HEPES) per 0.05 to 0.4 g of each tissue. -3.0 mL of -NaOH, pH 7.4) was added and homogenized. The homogenate was centrifuged at 12,000 rpm × 10 minutes at 4 ° C., and 100 μL of the supernatant was dispensed into a new tube. After adding 100 μL of the PEG-containing ATP extraction solution of this comparative example and stirring, the mixture was allowed to stand at room temperature for 30 minutes and then stored at −80 ° C.
（実施例４）改良型ＡＴＰ測定用試薬（rLuciferase/Luciferin Reagent mod.）
改良型ＡＴＰ測定用試薬の組成を以下に示す。
D-ルシフェリンカリウム (D-Luciferin Potassium Salt) 0.8 mg
ホタル由来遺伝子組換ルシフェラーゼ(Luciferase Recombinant) 1 mg/mL(緩衝液)
緩衝液組成
20 mM トリシン(Tricine: (HOCH2)3C-NHCH2CO2H)
1.05 mM Mg-Carbonate Hydroxide Pentahydrate
2.7 mM 硫酸マグネシウム(Magnesium Sulfate)
33.3 mM ジチオスレイトール(Dithiothreitol)(Example 4) Improved ATP measurement reagent (rLuciferase / Luciferin Reagent mod.)
The composition of the improved ATP measurement reagent is shown below.
D-Luciferin Potassium Salt 0.8 mg
Firefly-derived recombinant luciferase (Luciferase Recombinant) 1 mg / mL (buffer)
Buffer composition
20 mM Tricine (Tricine: (HOCH 2 ) 3 C-NHCH 2 CO 2 H)
1.05 mM Mg-Carbonate Hydroxide Pentahydrate
2.7 mM Magnesium Sulfate
33.3 mM Dithiothreitol
（実験例１）ＨＰＬＣによる抽出産物の解析
実施例２、比較例１及び２の、各ヌクレオチド抽出方法で得られた肝組織の抽出液中のヌクレオチド（ＡＴＰ、ＵＴＰ、ＧＴＰ、ＣＴＰ、ＩＴＰ）の抽出について調べた。
各抽出物の分析は、東ソー株式会社のＨＰＬＣ分析情報(Data code: G00707)に準じて行った。本分析は溶離液としてアセトニトリル/75 mM カリウム2水素リン酸水溶液(70:30, v/v)、ＨＰＬＣカラムとしてTSK-gel(R) Amide-80(5 μm、I.D. 4.6×250 mm)(東ソー株式会社)およびＨＰＬＣ装置として日立高速液体クロマトグラフィー LaChromTM ELUTEシリーズ：日立ハイテクノロジーズ社を使用して実施した。抽出液は希釈せず0.025 mLをカラムに供し、カラム温度３０℃、1.0 mL/minの流速、260 nmの波長でモニターした。なお、標準核酸物質としてＡＴＰ、ＵＴＰ、ＧＴＰ、ＣＴＰ（シグマ社）を使用した。(Experimental Example 1) Analysis of Extraction Product by HPLC The nucleotides (ATP, UTP, GTP, CTP, ITP) in the extract of liver tissue obtained by each nucleotide extraction method of Example 2 and Comparative Examples 1 and 2 The extraction was examined.
Each extract was analyzed according to HPLC analysis information (Data code: G00707) of Tosoh Corporation. In this analysis, acetonitrile / 75 mM potassium dihydrogen phosphate aqueous solution (70:30, v / v) was used as the eluent, and TSK-gel (R) Amide-80 (5 μm, ID 4.6 × 250 mm) (Tosoh) was used as the HPLC column. Hitachi High-Performance Liquid Chromatography LaChrom ™ ELUTE Series: Hitachi High-Technologies Corporation as HPLC equipment. The extract was not diluted, but 0.025 mL was applied to the column, and monitored at a column temperature of 30 ° C., a flow rate of 1.0 mL / min, and a wavelength of 260 nm. In addition, ATP, UTP, GTP, and CTP (Sigma) were used as standard nucleic acid substances.
上記の分析結果を、図１〜４に示した。図１は、標準核酸物質の抽出パターンであり、図２は実施例２のフェノールＴＥ飽和溶液（pH8.0）を用いたヌクレオチド抽出方法により得られた抽出物の抽出パターンであり、図３は同様に比較例１のＴＣＡを用いたヌクレオチド抽出方法によるものであり、図４は比較例２のＰＥＧを用いたヌクレオチド抽出方法によるものである。これらの結果より、実施例２の抽出方法により得られた抽出物中には、比較例１、２の抽出方法により得られた抽出物に比べ、ＡＴＰ、ＧＴＰ及びＵＴＰがより多く抽出されることが確認された。 The above analysis results are shown in FIGS. FIG. 1 is an extraction pattern of a standard nucleic acid substance, FIG. 2 is an extraction pattern of an extract obtained by a nucleotide extraction method using a phenol TE saturated solution (pH 8.0) in Example 2, and FIG. Similarly, this is based on the nucleotide extraction method using TCA of Comparative Example 1, and FIG. 4 is based on the nucleotide extraction method using PEG of Comparative Example 2. From these results, more ATP, GTP and UTP are extracted in the extract obtained by the extraction method of Example 2 than in the extract obtained by the extraction method of Comparative Examples 1 and 2. Was confirmed.
（実験例２）各組織中のヌクレオチド量について
実施例２、比較例１及び２の、各ヌクレオチド抽出方法で得られた脳、心臓、肝臓、筋肉の各組織の抽出液中のヌクレオチド（ＡＴＰ、ＵＴＰ、ＧＴＰ）の抽出について、ＨＰＬＣにより調べた。ＨＰＬＣの測定条件は、実験例１と同様に行った。(Experimental example 2) About the amount of nucleotides in each tissue Nucleotides (ATP, ATP) in the extracts of brain, heart, liver and muscle tissues obtained by the nucleotide extraction methods of Example 2 and Comparative Examples 1 and 2 The extraction of UTP, GTP) was examined by HPLC. The HPLC measurement conditions were the same as in Experimental Example 1.
上記の分析の結果、実施例２の方法で抽出した場合には、比較例１及び２の方法で抽出した場合に比べて、各ヌクレオチドについて高い抽出効果が認められた（図５）。 As a result of the above analysis, when extracted by the method of Example 2, a higher extraction effect was recognized for each nucleotide as compared to the case of extraction by the methods of Comparative Examples 1 and 2 (FIG. 5).
（実験例３）各組織のＡＴＰ抽出について
実施例１〜３、及び比較例１、２の各抽出方法を行った希釈試料10μLを、ENLITEN(R) rLuciferase/Luciferin Reagent：プロメガ社90μLで、1/10倍に希釈し、ＡＴＰの測定を行った。タンパク質量は、ＢＣＡTMタンパク定量アッセイキット（BCATM Protein Assay Kit, PIERCE社）により測定した。(Experimental Example 3) ATP Extraction of Each Tissue 10 μL of the diluted sample subjected to each extraction method of Examples 1 to 3 and Comparative Examples 1 and 2 was added to ENLITEN (R) rLuciferase / Luciferin Reagent: 90 μL of Promega. The ATP was measured after diluting to 10 times. The amount of protein was measured with a BCA ™ protein assay kit (BCA ™ Protein Assay Kit, PIERCE).
その結果、タンパク質あたりのＡＴＰ量は、比較例１及び２の方法で抽出した場合に比べて、実施例１〜３の方法で抽出した場合のほうが、高い結果が得られ、効果的にＡＴＰ量を測定できたことが示唆された（図６）。 As a result, the amount of ATP per protein was higher when extracted by the methods of Examples 1 to 3 than when extracted by the methods of Comparative Examples 1 and 2, and the amount of ATP was effectively increased. It was suggested that was able to be measured (FIG. 6).
（実験例４）遺伝性カルニチン欠乏ＪＶＳマウスのＡＴＰ量の測定
本発明のヌクレオチド抽出方法により抽出した場合のＡＴＰ測定値について、動物組織でのＡＴＰの低下を、全身性カルニチン欠損症、即ちミトコンドリアの脂肪酸代謝障害の疾患モデルとして確立しているＪＶＳ(juvenile visceral steatosis) マウスを用いて調べた。ＪＶＳマウスは、sodium-dependent carnitine cotransporter (OCTN2)遺伝子のミスセンス変異を伴ったマウスで、先天性長鎖脂肪酸代謝障害を示す (Lu K, Nishimori H, Nakamura Y, Shima K, Kuwajima M. Biochem Biophys Res Commun 252:290-594, 1998)。このマウス に、インフルエンザウイルス(Influenza A/PR/8/38株）を感染させた場合の、血液、肝臓、心臓及び脾臓でのＡＴＰレベルを測定し、多臓器不全の程度を判定した。(Experimental example 4) Measurement of ATP amount in hereditary carnitine-deficient JVS mice Regarding the ATP measurement value when extracted by the nucleotide extraction method of the present invention, the decrease in ATP in animal tissues was expressed as systemic carnitine deficiency, that is, mitochondrial It investigated using the JVS (juvenile visceral steatosis) mouse established as a disease model of fatty-acid-metabolism disorder. The JVS mouse is a mouse with a missense mutation in the sodium-dependent carnitine cotransporter (OCTN2) gene and has a congenital long-chain fatty acid metabolism disorder (Lu K, Nishimori H, Nakamura Y, Shima K, Kuwajima M. Biochem Biophys Res. Commun 252: 290-594, 1998). When this mouse was infected with influenza virus (Influenza A / PR / 8/38 strain), ATP levels in blood, liver, heart and spleen were measured to determine the degree of multiple organ failure.
７日齢の野生型マウス(C57BL/6J)、OCTN2遺伝子のヘテロ型欠損マウスとホモ型欠損マウス（ＪＶＳマウス）に、インフルエンザウイルスを感染させ、３日放置した。１０日齢の野生型及びＪＶＳマウスについて、インフルエンザウイルス感染なしのマウスと３日間感染させた各マウスについて、実施例１と同様に各組織を摘出し、実施例２の方法に従いヌクレオチド抽出を行い、ＡＴＰの定量を行った。ＡＴＰの定量は、実験例３の方法に従った。 Seven-day-old wild type mice (C57BL / 6J), OCTN2 gene heterozygous and homozygous mice (JVS mice) were infected with influenza virus and left for 3 days. For 10-day-old wild-type and JVS mice, for each mouse infected for 3 days with mice without influenza virus infection, each tissue was extracted in the same manner as in Example 1, and nucleotide extraction was performed according to the method of Example 2, ATP was quantified. ATP was quantified according to the method of Experimental Example 3.
各組織におけるＡＴＰレベルを、図７〜１０に示した。各図において、+/+は野生型マウスを、+/-はヘテロ型欠損マウスを、-/-はＪＶＳマウスを示す。各組織について、インフルエンザに感染したマウスでは、非感染マウスに比べて野生型、ヘテロ型欠損、ＪＶＳマウスのいずれでもＡＴＰ量は低下傾向にあるが、中でもＪＶＳマウスの場合ではＡＴＰ量が著名に低値を示した事実から、多臓器不全を来していることが確認された。 The ATP level in each tissue is shown in FIGS. In each figure, + / + indicates a wild type mouse, +/− indicates a heterozygous mouse, and − / − indicates a JVS mouse. For each tissue, the ATP level in mice infected with influenza is lower in wild type, heterozygous and JVS mice than in non-infected mice, but the ATP level is particularly low in JVS mice. From the facts showing the value, it was confirmed that multiple organ failure occurred.
（実験例５）ルシフェラーゼを用いたＡＴＰ測定試薬のＡＴＰ特異性の確認
以上の実験例の結果より、本発明のヌクレオチド抽出法により動物の組織から効率良くヌクレオチドを抽出することが可能であることが明らかとなった。しかし、実験例１及び２に示したように、動物の組織にはＡＴＰの他にも様々なヌクレオチドが存在する。本実験例では、各抽出液に含まれるＡＴＰ以外のヌクレオチドがルシフェラーゼ発光反応に影響するか否かを検討した。ＡＴＰ各希釈溶液に各濃度のＧＴＰ又はＡＭＰを混合した溶液を調製し、これらの溶液について実験例３の方法に従い、ルシフェラーゼ発光反応を行った。(Experimental Example 5) Confirmation of ATP Specificity of ATP Measurement Reagent Using Luciferase From the results of the above experimental examples, it is possible to extract nucleotides from animal tissues efficiently by the nucleotide extraction method of the present invention. It became clear. However, as shown in Experimental Examples 1 and 2, there are various nucleotides in animal tissues in addition to ATP. In this experimental example, it was examined whether nucleotides other than ATP contained in each extract affect the luciferase luminescence reaction. Solutions in which each concentration of GTP or AMP was mixed with each diluted solution of ATP were prepared, and luciferase luminescence reaction was performed on these solutions according to the method of Experimental Example 3.
その結果、ルシフェラーゼ発光反応によるＡＴＰ測定には、組織中に存在すると考えられるＧＴＰ、ＡＭＰ量、さらにそのＡＴＰ濃度の106〜109倍のＧＴＰ、ＡＭＰ濃度存在下においても影響のないことが確認された（図１１）。As a result, it was confirmed that the ATP measurement by the luciferase luminescence reaction had no effect even in the presence of GTP and AMP that are considered to be present in the tissue, and GTP and AMP concentrations 10 6 to 10 9 times the ATP concentration. (FIG. 11).
（実験例６） ヒトの血液試料のＡＴＰの測定
健常者（被験者）の末梢血を試料とし、実施例１〜３に示すヌクレオチド抽出方法により抽出したＡＴＰ測定値と、比較例１及び２の、各ヌクレオチド抽出方法で測定したＡＴＰ測定値を比較した。被験者（３名）の末梢血試料は、インフォームド・コンセントを書面で行い、徳島大学倫理審査委員会の承認を得て、採血を行ったものを用いた。ＡＴＰの定量は、実験例３に従った。(Experimental example 6) ATP measurement of human blood sample ATP measurement value extracted by the nucleotide extraction method shown in Examples 1 to 3, using the peripheral blood of a healthy person (subject) as a sample, and Comparative Examples 1 and 2 The ATP measurement value measured by each nucleotide extraction method was compared. Peripheral blood samples from the subjects (3 subjects) were collected from blood after informed consent was obtained in writing and approved by the Tokushima University Ethics Review Board. ATP was quantified according to Experimental Example 3.
上記の測定の結果、実施例１〜３の方法で抽出した場合には、比較例１及び２の各抽出法を用いて抽出した場合に比べて、ＡＴＰの高い抽出効果が認められた（図１２参照）。 As a result of the above measurement, when extracted by the methods of Examples 1 to 3, a high extraction effect of ATP was recognized as compared with the case of extracting using the extraction methods of Comparative Examples 1 and 2 (Fig. 12).
（実験例７）ヒト患者の血液試料のＡＴＰの測定
各種疾患で入院したヒト患者（被験者）の末梢血を試料とし、実施例２の方法で抽出した場合のＡＴＰ測定値について、健常者の末梢血を試料に比べてＡＴＰレベルが低下しているかどうかについて調べた。被験者の末梢血試料は、インフォームド・コンセントを書面で行い、徳島大学の倫理審査委員会の承認を得て、採血を行ったものを用いた。被験者は、脳症患者６名、ミトコンドリア脳筋症の患者５名、そして、脳症の回復期の患者３名であった。(Experimental example 7) Measurement of ATP of blood sample of human patient ATP measurement value when extracted by the method of Example 2 using peripheral blood of a human patient (subject) hospitalized for various diseases as a sample. The blood was examined for reduced ATP levels compared to the sample. Peripheral blood samples from the subjects were used after informed consent was written and blood was collected with the approval of the Ethics Review Committee of Tokushima University. The subjects were 6 patients with encephalopathy, 5 patients with mitochondrial encephalomyopathy, and 3 patients in the recovery period of encephalopathy.
上記の測定の結果、脳症（急性期）の患者、ミトコンドリア脳筋症の患者においては、末梢血のＡＴＰレベルは低値（1 mM以下）を示したが、脳症の回復期の患者では、末梢血中のＡＴＰレベルが1.2 mM以上であり、ＡＴＰレベルが回復していることが確認された（図１３参照）。 As a result of the above measurement, ATP levels in peripheral blood showed a low value (1 mM or less) in patients with encephalopathy (acute stage) and patients with mitochondrial encephalomyopathy, but in patients in the recovery stage of encephalopathy, peripheral blood The ATP level in the blood was 1.2 mM or more, and it was confirmed that the ATP level was recovered (see FIG. 13).
（実験例８）各組織のＡＴＰ抽出量の測定について
脳症で入院したヒト患者（脳症の回復期の患者（健常者）５名、脳症（急性期）の患者４名）の末梢血を試料とし、実施例２の方法で抽出した。既存の市販品及び実施例４の改良型ＡＴＰ測定用試薬を用いたＡＴＰ測定方法の比較試験を行った。
実施例２の方法で抽出した希釈試料（1/10,000倍希釈）10μLを、市販のＡＴＰ試薬（ENLITEN(R) rLuciferase/Luciferin Reagent：プロメガ社）90μLで、1/10倍に希釈し、ルミノメーター（TD-20/2：TURNER DESIGN社）でルシフェラーゼの発光量を測定した。同時に、ＡＴＰ標準溶液（10-7 M）を1/10倍〜1/10,000,000倍まで段階的に希釈した溶液を調製し、ＡＴＰ濃度とルシフェラーゼの発光量との相関関係を表す標準曲線を作成した。この標準曲線をもとに、ルシフェラーゼの発光量から試料中に存在するＡＴＰ濃度を算出した。(Experimental example 8) Measurement of ATP extract amount of each tissue Using peripheral blood of human patients hospitalized with encephalopathy (5 patients with encephalopathy recovery (healthy), 4 patients with encephalopathy (acute)) Extracted by the method of Example 2. A comparative test of the ATP measurement method using the existing commercial product and the improved ATP measurement reagent of Example 4 was performed.
The diluted sample (1 / 10,000 dilution) 10 [mu] L extracted by the method of Example 2, a commercially available ATP reagent (ENLITEN (R) rLuciferase / Luciferin Reagent: Promega) in 90 [mu] L, was diluted to 1/10-fold, luminometer (TD-20 / 2: TURNER DESIGN) was used to measure the amount of luciferase luminescence. At the same time, a solution in which an ATP standard solution (10 −7 M) was diluted stepwise from 1/10 to 1 / 10,000,000 was prepared, and a standard curve representing the correlation between the ATP concentration and the amount of luciferase luminescence was prepared. . Based on this standard curve, the ATP concentration present in the sample was calculated from the amount of luciferase luminescence.
同様に実施例２の方法で抽出した希釈試料10μLを、実施例４の改良型ＡＴＰ測定用試薬90μLで希釈した。他は、上記市販の試薬を用いた方法と同様の方法で試料中に存在するＡＴＰ濃度を算出した。 Similarly, 10 μL of the diluted sample extracted by the method of Example 2 was diluted with 90 μL of the improved ATP measurement reagent of Example 4. Other than that, the ATP concentration present in the sample was calculated by the same method as that using the commercially available reagent.
上記の比較試験の結果、市販のＡＴＰ試薬又は実施例４の改良型ＡＴＰ試薬で測定した場合では、ほぼ同様の結果が得られることが確認された（図１４参照）。 As a result of the above comparative test, it was confirmed that almost the same result was obtained when measured with a commercially available ATP reagent or the improved ATP reagent of Example 4 (see FIG. 14).
以上の結果から、本発明のフェノール化合物を含む溶液を用いたヌクレオチドの抽出法によると、動物組織中のヌクレオチドを効率良く抽出可能なことが確認され、さらに本発明のＡＴＰの測定方法に使用したルシフェラーゼ発光を指標とする試薬では、ルシフェラーゼ発光は抽出した核酸のＡＴＰのみに反応することが再確認された。さらに、改良型ＡＴＰ測定用試薬（rLuciferase/Luciferin Reagent mod.）を用いることで、従来の方法と比較して、より安価に測定試料中のＡＴＰ量の測定を実現可能なことが明らかになった。 From the above results, it was confirmed that the nucleotide extraction method using the solution containing the phenolic compound of the present invention can efficiently extract nucleotides in animal tissues, and further used for the ATP measurement method of the present invention. With the reagent using luciferase luminescence as an index, it was reconfirmed that luciferase luminescence reacts only with ATP of the extracted nucleic acid. Furthermore, it has been clarified that by using an improved ATP measurement reagent (rLuciferase / Luciferin Reagent mod.), It is possible to measure the ATP content in the measurement sample at a lower cost compared to the conventional method. .
以上詳述したように、本発明のヌクレオチドの抽出方法により、効果的にＡＴＰ、ＧＴＰ又はＵＴＰを抽出可能なことが確認された。従って、効果的にこれらのヌクレオチドの測定を行うことができる。そのため、本発明のヌクレオチド抽出方法に使用する抽出用溶液を、ヌクレオチド抽出用試薬とすることができ、ＡＴＰ、ＧＴＰ又はＵＴＰの測定用試薬キットに含めることができる。特に、現実に市販されているＡＴＰ測定用試薬を用いて測定する場合にも、本発明の抽出方法は有効に利用できることから、既存のＡＴＰ測定用試薬に添付するＡＴＰ抽出用試薬としても用いることができ、このような抽出用試薬を含むＡＴＰ測定用試薬キットとして提供することができる。 As described in detail above, it was confirmed that ATP, GTP or UTP can be extracted effectively by the nucleotide extraction method of the present invention. Therefore, these nucleotides can be measured effectively. Therefore, the extraction solution used in the nucleotide extraction method of the present invention can be used as a nucleotide extraction reagent, and can be included in a reagent kit for measuring ATP, GTP, or UTP. In particular, even when measuring using a commercially available ATP measurement reagent, since the extraction method of the present invention can be used effectively, it can also be used as an ATP extraction reagent attached to an existing ATP measurement reagent. And can be provided as a reagent kit for ATP measurement containing such an extraction reagent.
本発明のヌクレオチドの抽出方法は、各種工業分野、特に、バイオ・臨床検査・医学等の現場における標的ＡＴＰの測定法として好適に使用できる。なかでも、患者の末梢血のＡＴＰレベルを測定する技術は、患者の容態をモニタリングする一つの指標として、医療機関に波及する可能性がある。 The nucleotide extraction method of the present invention can be suitably used as a method for measuring target ATP in various industrial fields, particularly in fields such as biotechnology, clinical examination, and medicine. Especially, the technique which measures the ATP level of a patient's peripheral blood may spread to a medical institution as one parameter | index which monitors a patient's condition.
Claims (10)
（ａ）試料にフェノール化合物を含む溶液を混合する工程；
（ｂ）前記工程（ａ）で得られた混合液を遠心処理して、上清を抽出液として回収する工程； A method for extracting nucleotide triphosphates for measurement of nucleotide triphosphates contained in a sample, comprising the following steps (a) and (b), and comprising steps (a) and (b): An extraction method characterized by being sequentially provided.
(A) mixing a solution containing a phenol compound with a sample;
(B) centrifuging the mixed solution obtained in the step (a) and recovering the supernatant as an extract;
（Ａ）試料にフェノール化合物を含む溶液を混合する工程；
（Ｂ）前記工程（Ａ）で得られた混合液を遠心処理して、フェノール化合物を含む上清を抽出液として回収する工程；
（Ｃ）前記工程（Ｂ）で得られたフェノール化合物を含む抽出液中のヌクレオチド三リン酸を、ヌクレオチド三リン酸測定用試薬を用いて定量する工程； A method for measuring a nucleotide triphosphate contained in a sample, comprising the following steps (A) to (C) in sequence.
(A) A step of mixing a solution containing a phenol compound with a sample;
(B) A step of centrifuging the mixed solution obtained in the step (A) and recovering a supernatant containing a phenol compound as an extract;
(C) a step of quantifying the nucleotide triphosphate in the extract containing the phenol compound obtained in the step (B) using a nucleotide triphosphate measurement reagent;
An extraction reagent A composed of a saturated solution of phenol TE containing 0.1% by weight of 8-hydroxyquinoline, an extraction reagent B composed of chloroform, and a reagent for ATP measurement including luciferase for preparing an extraction reagent The reagent kit for measurement according to any one of claims 7 to 9, wherein
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