JPH0698030B2 - Determination method of NADPH by chemiluminescence method - Google Patents
Determination method of NADPH by chemiluminescence methodInfo
- Publication number
- JPH0698030B2 JPH0698030B2 JP216587A JP216587A JPH0698030B2 JP H0698030 B2 JPH0698030 B2 JP H0698030B2 JP 216587 A JP216587 A JP 216587A JP 216587 A JP216587 A JP 216587A JP H0698030 B2 JPH0698030 B2 JP H0698030B2
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- Prior art keywords
- nadph
- fluorescent substance
- reaction
- oxalic acid
- solution
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は，化学発光法によるNADPHの定量法に関する。The present invention relates to a method for quantifying NADPH by a chemiluminescence method.
（従来の技術） グルタミン酸，グルコース−６−リン酸などは生体成分
として重要であり，例えば生化学的検査や病理学的研究
において血清や尿などの試料中のグルタミン酸，グルコ
ース−６−リン酸などをNADPHに変換し，このNADPHを測
定することにより，上記生体成分の定量が広く行われて
いる。NADPHは，例えば，グルタミン酸とNADP+とからグ
ルタメートデヒドロゲナーゼの働きにより，また，グル
コース−６−リン酸NADP+とからグルコース−６−リン
酸デヒドロゲナーゼの働きにより生成する。NADPHの定
量は，通常，その物質が光照射されたときに示す紫外部
の吸収（340nm）を測定することによりなされる。しか
し，血清や尿などの試料が濁度成分を含有する場合，例
えば脂肪粒を含む血清であるような場合は,NADPHが正確
に測定されにくい。NADPHの紫外部の吸光度を測定する
場合の検出限界濃度は10-6Mであり，感度が充分に高い
とはいえない。(Prior Art) Glutamic acid, glucose-6-phosphate and the like are important as biological components, and for example, glutamic acid, glucose-6-phosphate and the like in samples such as serum and urine in biochemical tests and pathological studies. Is converted into NADPH, and the NADPH is measured to quantify the biological components. NADPH is produced, for example, from glutamate and NADP + by the action of glutamate dehydrogenase and from glucose-6-phosphate NADP + by the action of glucose-6-phosphate dehydrogenase. NADPH is usually quantified by measuring the ultraviolet absorption (340 nm) of the substance when it is exposed to light. However, when a sample such as serum or urine contains a turbidity component, for example, serum containing fat particles, NADPH is difficult to be accurately measured. When the absorbance of NADPH in the ultraviolet is measured, the detection limit concentration is 10 -6 M, and the sensitivity cannot be said to be sufficiently high.
（発明が解決しようとする問題点） 本発明は，上記従来の欠点を解決するものであり，その
目的とするところは，試料中のNADPHを感度よく，かつ
簡単に定量する方法を提供することにある。本発明の他
の目的は，化学発光法を用いてNADPHを定量する方法を
提供することにある。(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and an object of the present invention is to provide a method for sensitively and easily quantifying NADPH in a sample. It is in. Another object of the present invention is to provide a method for quantifying NADPH using a chemiluminescence method.
（問題点を解決するための手段） 本発明方法は，旧黄色酵素を利用した化学発光法による
NADPHの定量法であり，その手法は次の２種に大別され
る。(Means for Solving Problems) The method of the present invention is based on chemiluminescence method using old yellow enzyme.
It is a quantitative method of NADPH, and the method is roughly classified into the following two types.
本発明のNADPHの定量法は,NADPHを含む試料に旧黄色酵
素（old yellow enzyme）を作用させ，生成する過酸化
水素を蓚酸ジエステルおよび螢光物質と反応させ，生成
する発光量を測定することを特徴とするNADPHの化学発
光法による定量法である。In the method for quantifying NADPH of the present invention, an old yellow enzyme is allowed to act on a sample containing NADPH, hydrogen peroxide produced is reacted with an oxalic acid diester and a fluorescent substance, and the amount of luminescence produced is measured. Is a method for quantifying NADPH by a chemiluminescence method.
本発明のNADPHの定量法は,NADPHを含む試料に旧黄色酵
素（old yellow enzyme）を作用させ，生成する過酸化
水素を酸化触媒の存在下で螢光物質前駆体と反応させ，
生成する螢光物質を酸化触媒の阻害条件下に蓚酸ジエス
テルおよび新たに加えた過酸化水素と反応させ，生成す
る発光量を測定することを特徴とするNADPHの化学発光
法による定量法である。The method for quantifying NADPH of the present invention comprises reacting a sample containing NADPH with an old yellow enzyme to react the produced hydrogen peroxide with a fluorescent substance precursor in the presence of an oxidation catalyst,
A method for the determination of NADPH by chemiluminescence method, which comprises reacting the generated fluorescent substance with oxalic acid diester and newly added hydrogen peroxide under the condition of inhibiting the oxidation catalyst, and measuring the amount of luminescence generated.
本発明の第１の方法は，次式により説明される。The first method of the present invention is described by the following equation.
H2O2＋蓚酸ジエステル＋螢光物質 →ｈν＋螢光物質＋蓚酸ジエステル分解物 （２） この方法により試料中のNADPHを定量するには，例え
ば，まずNADPHを含む試料を適当な緩衝液で希釈し，水
系の試料溶液を調製する。これに旧黄色酵素を加えて，
酵素反応を行う。本発明におけるNADPHを含む試料と
は，血清または尿などの試料中に含まれる生体成分，例
えばグルタミン酸，グルコース−６−リン酸などから由
来したNADPHを含む溶液を包含する。本発明に用いる旧
黄色酵素（old yellow enzyme）とは，別名NADPHジアホ
ラーゼ,NADPH−フラビンレダクターゼであり,NADPH脱水
素酵素の一種である（E.C.1.6.99.1）。この酵素の存在
下で,NADPHは酸素と反応してNADP+と過酸化水素を生成
する（（１）式）。この酵素は，例えば，酵母（Yeas
t）などが産生し,E.Haasらの方法（Methods in Enzymol
ogy,Vol.2,712〜719）により精製される。本発明におい
て該酵素は反応液約1mlあたり,1〜50Uの濃度で使用され
る。酵素反応時のpHは,6.5〜8.5が適当であり，緩衝液
としては25〜100mMのリン酸緩衝液やトリス緩衝液が好
適に使用される。 H 2 O 2 + oxalic acid diester + fluorescent substance → hν + fluorescent substance + oxalic acid diester decomposition product (2) To quantify NADPH in a sample by this method, for example, first, a sample containing NADPH is washed with an appropriate buffer solution. Dilute and prepare an aqueous sample solution. Add the old yellow enzyme to this,
Carry out an enzymatic reaction. The sample containing NADPH in the present invention includes a solution containing NADPH derived from biological components contained in a sample such as serum or urine, for example, glutamic acid, glucose-6-phosphate and the like. The old yellow enzyme used in the present invention is also known as NADPH diaphorase or NADPH-flavin reductase, which is a kind of NADPH dehydrogenase (EC 1.6.99.1). In the presence of this enzyme, NADPH reacts with oxygen to produce NADP + and hydrogen peroxide (equation (1)). This enzyme is used, for example, in yeast (Yeas
t) etc., and the method of E. Haas et al. (Methods in Enzymol
Ogy, Vol.2, 712-719). In the present invention, the enzyme is used at a concentration of 1 to 50 U per about 1 ml of the reaction solution. The pH during the enzyme reaction is suitably 6.5 to 8.5, and a 25 to 100 mM phosphate buffer or Tris buffer is preferably used as the buffer.
次に，有機溶媒，好ましくは水と混合しうる有機溶媒
（例えばアセトニトリルなど），蓚酸ジエステルおよび
螢光物質が上記酵素反応が終了した溶液に加えられる。
蓚酸ジエステルとしては，ビス（2,4,6−トリクロロフ
ェニル）オキザレート（TCPOと略する），ビス（2,4−
ジニトロフェニル）オキザレートなどが挙げられる。こ
の蓚酸ジエステルの反応液中の濃度は,0.1〜50mMであ
る。上記螢光物質としては，フルオレセイン，ペリレ
ン,8−アニリノ−１−ナフタレンスルホン酸などが挙げ
られる。（１）式の反応によって生じた過酸化水素は，
蓚酸ジエステルと反応し，その結果，螢光物質は励起さ
れて発光する。発光後はももとの螢光物質に戻る。蓚酸
ジエステルは酸化されて分解し，例えば,TCPOからは2,
4,6−トリクロロフェノールが生成する。この（２）式
の反応において生じた光を測定することによってNADPH
の定量がなされる。光の測定は，通常の発光測定装置で
なされ得る。例えば，既知量のNADPHを用いて反応を行
い，適当なラグタイム（例えば15秒間）の後，所定時間
（例えば10秒間）の発光量を測定し，これを積分すると
直線性のよい検量線が得られる。光の測定は発光強度の
ピーク値であってもよい。Next, an organic solvent, preferably an organic solvent that is miscible with water (such as acetonitrile), an oxalic acid diester and a fluorescent substance are added to the solution in which the enzymatic reaction has been completed.
As oxalic acid diesters, bis (2,4,6-trichlorophenyl) oxalate (abbreviated as TCPO), bis (2,4-
And dinitrophenyl) oxalate. The concentration of this oxalic acid diester in the reaction solution is 0.1 to 50 mM. Examples of the fluorescent substance include fluorescein, perylene, 8-anilino-1-naphthalenesulfonic acid and the like. Hydrogen peroxide generated by the reaction of the formula (1) is
Reacts with oxalic acid diester, which results in the fluorescent material being excited to emit light. After the light emission, it returns to the original fluorescent substance. Oxalic acid diester is oxidized and decomposed, for example, from TCPO 2,
4,6-Trichlorophenol is produced. By measuring the light generated in the reaction of this equation (2), NADPH
Is quantified. The measurement of light can be done with a conventional luminescence measuring device. For example, the reaction is performed using a known amount of NADPH, and after a suitable lag time (for example, 15 seconds), the amount of light emission for a predetermined time (for example, 10 seconds) is measured and integrated to obtain a calibration curve with good linearity. can get. The light measurement may be a peak value of emission intensity.
本発明の第２の方法は，次式により説明される。The second method of the present invention is described by the following equation.
螢光物質＋蓚酸ジエステル＋H2O2 →ｈν＋螢光物質＋蓚酸ジエステル分解物 （３） この方法における第１段階の反応（（１）式）は，前記
第１の方法と同様である。ここで発生した過酸化水素は
（２）式において酸化触媒の存在下で螢光物質前駆体と
反応し，螢光物質を生じる。本発明に使用する螢光物質
前駆体とは，それ自体は螢光を発しない被酸化性の物質
であり，酸化を受けると変化して螢光を発するようにな
る化合物をさしていう。このような化合物としては，ロ
イコフルオレセイン（フルオレシン）,2′,7′−ジクロ
ロフルオレシンなどが挙げられる。螢光物質前駆体は，
反応液中に0.001〜5mMとなるように加えられる。このと
きにβ−シクロデキストリンが存在すると螢光物質前駆
体が自然酸化を受けることなく安定化されるため好まし
い。酸化触媒としては，ペルオキシダーゼ，ミクロペル
オキシダーゼ，ミエロペルオキシダーゼ，ヘミン，ヘマ
チンなどが挙げられる。このような酸化触媒の使用量
は，通常，反応液約1mlあたり１〜100Uである。この
（１）式および（２）式の反応は，同一系内で同時に行
われることが好ましい。例えば,2′,7′−ジクロロフル
オレシンジアセテートをアルカリ水溶液により脱アセチ
ル化して螢光物質前駆体である２′,7′−ジクロロフル
オレシンを調製し，これに適当な緩衝液を加えてpH6.5
〜7.5に調整する。緩衝液としては,25〜100mMのリン酸
緩衝液やトリス緩衝液が使用される。螢光物質前駆体を
含む緩衝液に旧黄色酵素，酸化触媒および試料を加えて
30℃前後で反応させると（２）式までの反応が完了し，
螢光物質前駆体は酸化されて螢光物質を生じる。ここま
では上記のように水系の溶媒中で行われる。（１）式お
よび（２）式の反応に関与する物質の添加順序には特に
制限はない。 Fluorescent substance + oxalic acid diester + H 2 O 2 → hν + fluorescent substance + oxalic acid diester decomposition product (3) The reaction of the first step (equation (1)) in this method is the same as in the first method. The hydrogen peroxide generated here reacts with the fluorescent substance precursor in the presence of an oxidation catalyst in the formula (2) to generate a fluorescent substance. The fluorescent substance precursor used in the present invention refers to a compound which itself does not emit fluorescence and is oxidizable, and changes upon being oxidized to emit fluorescence. Examples of such compounds include leucofluorescein (fluorescein), 2 ', 7'-dichlorofluorescein and the like. The fluorescent substance precursor is
It is added to the reaction solution at a concentration of 0.001 to 5 mM. At this time, the presence of β-cyclodextrin is preferable because the fluorescent substance precursor is stabilized without being subjected to spontaneous oxidation. Examples of the oxidation catalyst include peroxidase, microperoxidase, myeloperoxidase, hemin, hematin and the like. The amount of such an oxidation catalyst used is usually 1 to 100 U per 1 ml of the reaction solution. The reactions of formulas (1) and (2) are preferably carried out simultaneously in the same system. For example, 2 ', 7'-dichlorofluorescein diacetate is deacetylated with an aqueous alkaline solution to prepare a fluorescent substance precursor, 2', 7'-dichlorofluorescein, to which an appropriate buffer solution is added. PH 6.5
Adjust to ~ 7.5. As the buffer, 25 to 100 mM phosphate buffer or Tris buffer is used. Add the old yellow enzyme, oxidation catalyst and sample to the buffer containing the fluorescent precursor
The reaction up to the equation (2) is completed when the reaction is performed at around 30 ° C,
The fluorescent material precursor is oxidized to produce a fluorescent material. Up to this point, it is performed in an aqueous solvent as described above. There is no particular limitation on the order of addition of the substances involved in the reactions of formulas (1) and (2).
次に有機溶媒系において（３）式の反応が行われる。有
機溶媒としては，前記第１の方法と同様に有機溶媒，好
ましくは水と混合しうる有機溶媒，例えばアセトニトリ
ルなどが用いられる。（２）式の反応が完了した系に前
記有機溶媒，蓚酸ジエステルおよび過酸化水素が加えら
れる。有機溶媒中では酸化触媒，例えばペルオキシダー
ゼなどは阻害される。蓚酸ジエステルとしては前記第１
の方法で使用される化合物がいずれも使用可能である。
過酸化水素は，通常，過酸化水素水として添加される。
（３）式の反応の原理は第１の方法（２）式と同様であ
り，ここでは新たに加えられた過酸化水素および蓚酸ジ
エステルが反応して，その結果，螢光物質が励起されて
発光する。この反応は有機溶媒系で行われるため，
（２）式の酸化触媒は失活し，そのため，新たに加えら
れた過酸化水素と螢光物質前駆体とが反応することはな
い。この（３）式の反応において生じた光（ｈν）を測
定することによりNADPHの定量がなされる。光の測定に
は，通常の発光測定装置が用いられる。例えば，発光ピ
ークの強度を測定することにより精度よく定量がなされ
る。既知濃度のNADPHを含む試料をこの第２の方法で測
定するとNADPH濃度と発光強度のピーク値とは直線性を
示す。光の測定は所定時間の発光量の積分値を測定して
もよい。Next, the reaction of the formula (3) is carried out in the organic solvent system. As the organic solvent, an organic solvent, preferably an organic solvent miscible with water, such as acetonitrile, is used as in the first method. The organic solvent, oxalic acid diester and hydrogen peroxide are added to the system where the reaction of the formula (2) is completed. Oxidation catalysts such as peroxidase are inhibited in organic solvents. As the oxalic acid diester, the first
Any of the compounds used in the above method can be used.
Hydrogen peroxide is usually added as hydrogen peroxide solution.
The reaction principle of the equation (3) is similar to that of the first method (2), in which newly added hydrogen peroxide and oxalic acid diester are reacted, and as a result, the fluorescent substance is excited. It emits light. Since this reaction is carried out in an organic solvent system,
The oxidation catalyst of the formula (2) is deactivated, so that the newly added hydrogen peroxide does not react with the fluorescent substance precursor. NADPH is quantified by measuring the light (hν) generated in the reaction of the equation (3). A usual luminescence measuring device is used for measuring the light. For example, quantification can be performed accurately by measuring the intensity of the emission peak. When a sample containing a known concentration of NADPH is measured by this second method, the NADPH concentration and the peak value of the emission intensity show linearity. The light may be measured by measuring the integrated value of the light emission amount for a predetermined time.
（作用） 本発明によれば、旧黄色酵素をNADPHを含む試料中のNAD
PHに作用させ，生じたH2O2を蓚酸ジエステルを用いる化
学発光法で測定することにより，該NADPHを定量するこ
とができる。化学発光による光を測定するため，従来の
NADPHの紫外部吸収を測定する方法に比べて，血清また
は尿などの試料中の濁質成分の影響を受けることなく高
感度でNADPHを定量できる。従来の紫外部吸収法によるN
ADPHの検出限界濃度が10-6Mであるのに対して，本法の
検出限界濃度は10-8Mである。本法では，特別の装置を
必要とせず汎用の発光測定装置を使用するため，安価に
定量がなされる。本発明の方法を用いて，生体由来の試
料（例えば血液や尿）中のNADPHを誘導する生体成分
（例えば，グルタミン酸，グルコース−６−リン酸な
ど）を定量することも可能である。(Effect) According to the present invention, the old yellow enzyme is added to NAD in a sample containing NADPH.
The NADPH can be quantified by acting on PH and measuring the produced H 2 O 2 by a chemiluminescence method using an oxalic acid diester. Since light from chemiluminescence is measured,
Compared with the method of measuring the ultraviolet absorption of NADPH, NADPH can be quantified with high sensitivity without being affected by suspended matter in samples such as serum or urine. N by conventional ultraviolet absorption method
The detection limit of ADPH is 10 -6 M, whereas the detection limit of this method is 10 -8 M. In this method, a general-purpose luminescence measuring device is used without the need for a special device, and therefore quantitative determination can be made at low cost. The method of the present invention can be used to quantify a biological component (eg, glutamic acid, glucose-6-phosphate, etc.) that induces NADPH in a biological sample (eg, blood or urine).
（実施例） 以下に本発明を実施例により説明する。(Example) Hereinafter, the present invention will be described with reference to an example.
実施例１ 0.1Mリン酸緩衝液（pH7.0）3.0mlに旧黄色酵素溶液（1U
/ml）1mlを加え,25℃にて５分間加温した。これに既知
量のNADPHを含有する試料溶液1mlを加えて，さらに15分
間加温しH2O2を発生させた。この反応液0.5mlを発光測
定用バイアルに移し，これに,TCPOを0.1mMの割合で，そ
して，フルオレセインを１μＭの割合で含有する溶液0.
5mlを加えた。添加後15秒経てから反応液の発光を発光
測定装置で10秒間にわたり測定した。NADPHの最終濃度
が10-8〜10-4Mとなるような上記試料溶液を用いて，そ
れぞれ測定を行なった。NADPH濃度と発光量（積分値）
との関係を第１図に示す。第１図からNADPHの終濃度10
-8〜10-5Mの範囲において直線的な関係が得られ，この
範囲において定量が可能であることがわかる。Example 1 3.0 ml of 0.1 M phosphate buffer (pH 7.0) was added to the old yellow enzyme solution (1 U
/ ml) 1 ml, and heated at 25 ° C for 5 minutes. To this, 1 ml of a sample solution containing a known amount of NADPH was added and heated for an additional 15 minutes to generate H 2 O 2 . 0.5 ml of this reaction solution was transferred to a luminescence measurement vial, and a solution containing TCPO at a rate of 0.1 mM and fluorescein at a rate of 1 μM was added to this solution.
5 ml was added. After 15 seconds from the addition, the luminescence of the reaction solution was measured with a luminescence measuring device for 10 seconds. The measurements were carried out using the above sample solutions such that the final concentration of NADPH was 10 −8 to 10 −4 M. NADPH concentration and luminescence (integrated value)
The relationship with is shown in FIG. From Fig. 1 NADPH final concentration 10
A linear relationship is obtained in the range of -8 to 10 -5 M, indicating that quantification is possible in this range.
実施例２ ２′,7′−ジクロロフルオレシンジアセチートのエタノ
ール溶液（5mg/10ml）10mlに0.01N NaOH水溶液40mlを加
えて脱アセチル化反応を行った。これに１％のβ−シク
ロデキストリンを含有する25mMリン酸バッファー（pH7.
0）150mlを加え，螢光物質前駆体を含む試薬（LDCF）溶
液を得た。このLDCF溶液3mlに，旧黄色酵素およびペル
オキシダーゼをそれぞれ5U/mlの濃度で含有する溶液1ml
を加えて，全容を4mlとした。これを30℃で５分間加温
した後，既知量のNADPHを含む試料溶液1mlを加えて30℃
にして１時間インキュベートして反応させた。LDCFはペ
ルオキシダーゼの存在下で酸化され，螢光を発するジク
ロロフルオレセイン（DCF）が反応液中に生成する。こ
の反応液0.5mlを採取しバイアルに移し，さらに,TCPOを
0.1mMの濃度で，そてH2O2を0.4mMの濃度で含有するアセ
トニトリル溶液0.5mlを加えた。反応液の発光強度のピ
ーク値を発光測定装置にて測定した。上記ペルオキシダ
ーゼ存在下の反応における反応液中のNADPH濃度をNADPH
終濃度とし,NADPH終濃度10-8〜10-5Mの範囲で上記反応
を行なった。NADPH終濃度と発光強度（ピーク値）との
関係を第２図に示す。第２図においては、NADPH量がOM
の場合の値がブランクとして差し引かれている。第２図
から,NADPH濃度10-8〜10-5Mの範囲で直線的な関係が得
られ，この範囲において定量が可能であることがわか
る。Example 2 A deacetylation reaction was carried out by adding 40 ml of 0.01N NaOH aqueous solution to 10 ml of an ethanol solution (5 mg / 10 ml) of 2 ', 7'-dichlorofluorescein diacetate. 25 mM phosphate buffer containing 1% β-cyclodextrin (pH 7.
0) 150 ml was added to obtain a reagent (LDCF) solution containing a fluorescent substance precursor. 1 ml of a solution containing the old yellow enzyme and peroxidase at a concentration of 5 U / ml in 3 ml of this LDCF solution
Was added to bring the total volume to 4 ml. After heating this at 30 ℃ for 5 minutes, add 1 ml of sample solution containing known amount of NADPH to 30 ℃.
And incubated for 1 hour to react. LDCF is oxidized in the presence of peroxidase, and fluorescent dichlorofluorescein (DCF) is produced in the reaction solution. Collect 0.5 ml of this reaction solution and transfer it to a vial.
0.5 ml of acetonitrile solution containing H 2 O 2 at a concentration of 0.1 mM and 0.4 mM of H 2 O 2 was added. The peak value of the luminescence intensity of the reaction solution was measured by a luminescence measuring device. The NADPH concentration in the reaction solution in the reaction in the presence of the above-mentioned peroxidase is changed to NADPH.
The above reaction was carried out at the final concentration of NADPH in the range of 10 -8 to 10 -5 M. The relationship between the final NADPH concentration and the emission intensity (peak value) is shown in FIG. In Figure 2, the NADPH amount is OM.
The value for is subtracted as a blank. From Fig. 2, it is found that a linear relationship is obtained in the NADPH concentration range of 10 -8 to 10 -5 M, and quantification is possible in this range.
（発明の効果） 本発明によれば，試料中のNADPHが濁質成分の影響を受
けることなく高感度で定量される。本法によりNADPHの
定量,NADPHを生じる生体成分（グルタミン酸，グルコー
ス−６−リン酸など）の定量などが極めて効果的になさ
れ得る。(Effect of the Invention) According to the present invention, NADPH in a sample can be quantified with high sensitivity without being affected by turbid components. By this method, quantification of NADPH and biological components (glutamic acid, glucose-6-phosphate, etc.) that produce NADPH can be quantified very effectively.
【図面の簡単な説明】 第１図は，本発明方法により試料溶液中のNADPHを測定
したときのNADPH濃度と発光量（積分値）との関係を示
すグラフ，そして第２図は本発明の方法により試料溶液
中のNADPHを測定したときのNADPH濃度と発光強度との関
係を示すグラフである。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the relationship between NADPH concentration and luminescence amount (integral value) when NADPH in a sample solution was measured by the method of the present invention, and FIG. 2 is a graph of the present invention. 3 is a graph showing the relationship between NADPH concentration and luminescence intensity when NADPH in a sample solution is measured by the method.
Claims (2)
w enzyme）を作用させ，生成する過酸化水素を蓚酸ジエ
ステルおよび螢光物質と反応させ，生成する発光量を測
定することを特徴とするNADPHの化学発光法による定量
法。1. A sample containing NADPH containing an old yellow enzyme (old yello
A method for quantifying NADPH by a chemiluminescence method, which comprises reacting the produced hydrogen peroxide with an oxalic acid diester and a fluorescent substance, and measuring the amount of luminescence produced.
w enzyme）を作用させ，生成する過酸化水素を酸化触媒
の存在下で螢光物質前駆体と反応させ，生成する螢光物
質を酸化触媒の阻害条件下に蓚酸ジエステルおよび新た
に加えた過酸化水素と反応させ，生成する発光量を測定
することを特徴とするNADPHの化学発光法による定量
法。2. A sample containing NADPH contains an old yellow enzyme (old yello
The resulting hydrogen peroxide reacts with a fluorescent substance precursor in the presence of an oxidation catalyst, and the generated fluorescent substance undergoes oxalic acid diester and newly added peroxidation under conditions that inhibit the oxidation catalyst. A method for quantifying NADPH by a chemiluminescence method, which comprises reacting with hydrogen and measuring the amount of luminescence produced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP216587A JPH0698030B2 (en) | 1987-01-08 | 1987-01-08 | Determination method of NADPH by chemiluminescence method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP216587A JPH0698030B2 (en) | 1987-01-08 | 1987-01-08 | Determination method of NADPH by chemiluminescence method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63169998A JPS63169998A (en) | 1988-07-13 |
| JPH0698030B2 true JPH0698030B2 (en) | 1994-12-07 |
Family
ID=11521747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP216587A Expired - Lifetime JPH0698030B2 (en) | 1987-01-08 | 1987-01-08 | Determination method of NADPH by chemiluminescence method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0698030B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018143106A1 (en) * | 2017-02-03 | 2018-08-09 | 国立大学法人大阪大学 | Device and determination system using same |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948906B (en) * | 2004-08-05 | 2013-02-27 | 旭化成制药株式会社 | Reagent with protease reaction promoter and/or pigment stabilizing agent |
| CN110132945B (en) * | 2019-06-10 | 2021-09-03 | 天津市宝坻区人民医院 | Chemiluminescence method kit formula for eliminating urea interference |
-
1987
- 1987-01-08 JP JP216587A patent/JPH0698030B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018143106A1 (en) * | 2017-02-03 | 2018-08-09 | 国立大学法人大阪大学 | Device and determination system using same |
| US11619628B2 (en) | 2017-02-03 | 2023-04-04 | Osaka University | Device and determination system using same |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63169998A (en) | 1988-07-13 |
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