JP3869603B2 - Homocysteine determination method and reagent - Google Patents

Description

【００１７】
上式では、ニコチンアミド補酵素として還元型ニコチンアミドアデニンジヌクレオチド、チオニコチンアミド補酵素として酸化型チオニコチンアミドアデニンジヌクレオチド、また２−ケト酪酸脱水素酵素類として２−ケト酪酸脱水素酵素を使用した例を示している。
【００１８】
本発明は、さらに精度の高い簡便かつ高感度なホモシステインの定量法を提供する。上記の酵素サイクリング法を実施する場合、最終的に酵素サイクリング反応の経過に伴って増減するニコチンアミド補酵素またはチオニコチンアミド補酵素を定量する際、条件によっては各々の補酵素がそれぞれ補酵素の定量を妨害することがある。この課題を解消するためには、ニコチンアミド補酵素濃度とチオニコチンアミド補酵素濃度の差を大きくすればこれらの妨害を少なくすることが可能となる。しかし一方で、片方の補酵素の濃度が低いことに起因する酵素サイクリング反応速度の低下や、片方の補酵素の濃度変化による一方の補酵素測定の誤差を招くことがある。これらの難点を解消する手段として、濃度を低くした補酵素の再生反応を酵素サイクリング反応に組み入れる手段が好適に使用出来る。
【００１９】
該補酵素の再生反応としては、２−ケト酪酸脱水素酵素類の酵素反応には影響を与えず、かつ対応ニコチンアミド補酵素あるいは対応チオニコチンアミド補酵素を可逆的に酸化還元する脱水素酵素反応であれば如何なるものでも良く特に限定されない。
【００２０】
この補酵素の再生反応を含む代表的ホモシステイン測定原理を下式に例示する。
【００２１】
【化２】

【００２２】
上式では、ニコチンアミド補酵素として還元型ニコチンアミドアデニンジヌクレオチド、チオニコチンアミド補酵素として酸化型チオニコチンアミドアデニンジヌクレオチド、２−ケト酪酸脱水素酵素類類として２−ケト酪酸脱水素酵素、ニコチンアミド補酵素あるいはチオニコチンアミド補酵素を可逆的に酸化還元する脱水素酵素（以下、酸化還元脱水素酵素類という）としてアルコール脱水素酵素、及びアルコール脱水素酵素の基質としてエタノールを使う例を示す。
かかる補酵素の再生反応を含む被検体中のホモシステインの高感度測定方法を詳細に説明すると以下の通りである。
【００２３】
ホモシステインを含む被検体にホモシステインデスルフラーゼを作用させ、酵素反応に伴って生成する２−ケト酪酸に、高濃度の酸化型チオニコチンアミド補酵素と低濃度の還元型ニコチンアミド補酵素の存在下で２−ケト酪酸脱水素酵素類を作用させると、可逆反応の進行に伴って還元型チオニコチンアミド補酵素が生成すると共に、還元型ニコチンアミド補酵素が消費されて行く。この還元型ニコチンアミド補酵素が消費されて行くと該可逆反応は速度が低下するか、あるいは反応が停止する。そこで該可逆反応を行わしめながら、チオニコチンアミド補酵素には作用せず、かつまた該可逆反応には影響を与えない還元型ニコチンアミド補酵素の再生反応のための酸化還元脱水素酵素類とその基質を加えると、消費された還元型ニコチンアミド補酵素が酸化型ニコチンアミド補酵素から再生され、該可逆反応が継続すると共にニコチンアミド補酵素の還元型と酸化型の濃度の割合が比較的一定になる。反応速度が減少することなく継続する該可逆反応に伴って生成して行く還元型チオニコチンアミド補酵素の濃度は、例えば吸光度測定によって還元型ニコチンアミド補酵素由来の吸光度に影響されることなく容易に測定され、最終的には被検体中のホモシステイン濃度を正確かつ高感度に定量することが可能となる。
【００２４】
本発明における被検体とは、医学や臨床検査分野等におけるホモシステインの測定対象となる検体を指し、例えば血清、血漿、細胞液、及び臓器抽出液等を言う。
【００２５】
本発明の定量法あるいは定量用試薬におけるホモシステインデスルフラーゼ（ＥＣ 4.4.1.2）は、測定対象となる被検体の種類や測定条件によって異なるが、ホモシステインを基質として２−ケト酪酸を生成する反応を触媒する酵素であれば如何なるものでも良く、かかる特性を有する公知の酵素が特に制限なく使用される。この特性を有する酵素を例示すれば、Methods in Enzymology、第２巻、318頁（1955年）及びJournal of Biological Chemistry、192巻、371頁（1951年）に記載のプロテウス・モルガニ由来のホモシステインデスルフラーゼ、Journal of Biochemistry、79巻、1263頁（1976年）、Biochemistry、16巻、100頁（1977年）、及びMethods in Enzymology、143巻、459頁（1987年）に記載のシュードモナス属由来のホモシステイナーゼ、Medical Science、第13巻、493頁（1985年）、及び国際特許WO 9807872号公報に記載の線虫トリコモナス属由来のホモシステインデスルフラーゼ等が挙げられる。
【００２６】
本発明の定量法において、被検体にホモシステインデスルフラーゼを作用させる方法は、被検体の種類や測定条件によって任意に選択することが出来る。即ち、測定の煩雑さを問わない場合は、被検体に含まれるホモシステインの殆ど全てがホモシステインデスルフラーゼの作用によって２−ケト酪酸に変換した後に２−ヒドロキシ酪酸との可逆反応あるいは該可逆反応と補酵素の再生反応を行わしめる方法を選ぶことが出来る。測定を簡便に実施しようとする場合は、被検体中のホモシステインをホモシステインデスルフラーゼの作用によって２−ケト酪酸に変換せしめつつ、２−ヒドロキシ酪酸との可逆反応あるいは該可逆反応と補酵素の再生反応を同時進行させる方法を選ぶことが可能である。
【００２７】
本発明における被検体に作用させるホモシステインデスルフラーゼの量は、被検体の種類や酵素の種類によって適宜決定されるが、一般的には0.1〜1000μmoles/min/mlの濃度範囲、特に0.2〜500μmoles/min/mlの濃度範囲が好適に使用される。ホモシステインデスルフラーゼを作用させる際のｐＨは、酵素の由来によって適宜選ばれるが、一般的にはｐＨ3.5〜10.5の範囲、特にｐＨ4.0〜10.0の範囲が好適に選ばれる。また、ホモシステインデスルフラーゼを作用させる際の反応温度は、一般的には20〜45゜Cの温度範囲、好ましくは25〜40゜Cの温度範囲が好適に選ばれる。
【００２８】
本発明の定量法あるいは定量用試薬におけるニコチンアミド補酵素としては、ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ：還元型、ＮＡＤ：酸化型、以下同様）、ニコチンアミドアデニンジヌクレオチドホスフェイト（ＮＡＤＰＨ、ＮＡＤＰ）、アセチルピリジンアデニンジヌクレオチド、アセチルピリジンアデニンジヌクレオチドホスフェイト、ニコチンアミドヒポキサンチンジヌクレオチド、あるいはニコチンアミドヒポキサンチンジヌクレオチドホスフェイト等が具体的に挙げられる。また、チオニコチンアミド補酵素としては、チオニコチンアミドアデニンジヌクレオチド（チオＮＡＤＨ、チオＮＡＤ）、チオニコチンアミドアデニンジヌクレオチドホスフェイト（チオＮＡＤＰＨ、チオＮＡＤＰ）、チオニコチンアミドヒポキサンチンジヌクレオチド、あるいはチオニコチンアミドヒポキサンチンジヌクレオチドホスフェイト等が具体的に挙げられる。
【００２９】
本発明の定量法において使用されるニコチンアミド補酵素あるいはチオニコチンアミド補酵素の濃度は、1μM〜200mMの広い濃度範囲が一般的であるが、特に5μM〜100mMの濃度範囲が好適に使用される。
【００３０】
本発明の２−ケト酪酸脱水素酵素類とは、ＮＡＤＨに代表される還元型ニコチンアミド補酵素、あるいはチオＮＡＤＨに代表される還元型チオニコチンアミド補酵素の存在下で、２−ケト酪酸を可逆的に還元して２−ヒドロキシ酪酸を生成せしめる酵素であれば特に限定されることはなく、かかる特性を有する公知の酵素が制限なく使用される。この特性を有する酵素を例示すれば、動物由来、植物由来、及び微生物由来のＬ−乳酸脱水素酵素（ＥＣ 1.1.1.27）、あるいは多くの微生物由来のＤ−乳酸脱水素酵素（ＥＣ 1.1.1.28）が挙げられるが、特にブタ心臓あるいはブタ筋肉由来のＬ−乳酸脱水素酵素、微生物Bacillus属由来のＬ−乳酸脱水素酵素、微生物Lactobacillus属由来のＬ−乳酸脱水素酵素、あるいは大腸菌由来のＤ−乳酸脱水素酵素等が入手の容易さで好適に使用出来る。
【００３１】
本発明の定量法における２−ケト酪酸脱水素酵素類の量は、使用する被検体の種類や測定条件あるいは酵素の由来によって適宜決定されるが、一般的には0.1〜1000μmoles/min/mlの濃度範囲、特に0.5〜500μmoles/min/mlの濃度範囲が好適に使用される。２−ケト酪酸脱水素酵素類の反応ｐＨは、使用する酵素によって適宜決定されるが、一般的にはｐＨ4.0〜10.5の範囲、好ましくはｐＨ4.5〜10.0の範囲が好適である。また、２−ケト酪酸脱水素酵素類の反応温度は、一般的には20〜45゜Cの温度範囲、好ましくは25〜40゜Cの温度範囲が好適に選ばれる。
【００３２】
本発明の定量法において、反応の経過に伴って消費または生成するニコチンアミド補酵素、チオニコチンアミド補酵素、対応ニコチンアミド補酵素、若しくは対応チオニコチンアミド補酵素を定量する方法としては、これら何れの補酵素の増減を定量しても良いが、一般的には、還元型ニコチンアミド補酵素あるいは還元型チオニコチンアミド補酵素が有する極大分子吸光係数を示す波長における吸光度分析が好適に採用される。還元ニコチンアミド補酵素の場合は、340nm付近における吸光度の増減で定量を行い、還元チオニコチンアミド補酵素の場合は、400nm付近における吸光度の増減で容易に定量が可能である。
【００３３】
本発明の定量法において、補酵素の再生反応を組み合わせることで更なる高感度化を実現することが出来る。最終的な測定対象補酵素が対応チオニコチンアミド補酵素である場合は対応ニコチンアミド補酵素の再生反応を、最終的な測定対象補酵素が対応ニコチンアミド補酵素である場合は、対応チオニコチンアミド補酵素の再生反応を適宜組み合わせることによって、ホモシステインの高感度定量法を更に精度の高いものとすることが可能となる。補酵素の再生反応を行わしめる酵素とその基質は、２−ケト酪酸と２−ヒドロキシ酪酸の可逆反応に影響を及ぼさないものであれば如何なるもので良く、かかる条件を満たす再生反応を進行させる酵素とその基質を例示すればアルコール脱水素酵素（ＥＣ 1.1.1.1、またはＥＣ 1.1.1.2）とエタノールあるいはアセトアルデヒド；グリセロール脱水素酵素（ＥＣ 1.1.1.6）とグリセロールあるいはジヒドロキシアセトン；アラビニトール脱水素酵素（ＥＣ 1.1.1.12、あるいはＥＣ 1.1.1.14）とアラビニトール、キシルロースあるいはリブロース；グリセリン酸脱水素酵素（ＥＣ 1.1.1.29）とグリセリン酸あるいはヒドロキシピルビン酸；リンゴ酸脱水素酵素（ＥＣ 1.1.1.37、あるいはＥＣ 1.1.1.82）とリンゴ酸あるいはオギザロ酢酸；アリールアルコール脱水素酵素（ＥＣ 1.1.1.90）とベンジルアルコールあるいはベンズアルデヒド；酒石酸脱水素酵素（ＥＣ 1.1.1.93）と酒石酸あるいはオギザログリコール酸等が挙げられる。これらの補酵素の再生反応に使用される酸化還元脱水素酵素類の濃度は、使用される補酵素の種類や濃度によって異なるが、一般的には0.1〜1000μmoles/min/mlの濃度範囲、特に0.5〜500μmoles/min/mlの範囲が好適に使用される。
【００３４】
本発明によるホモシステインの測定方法は酵素サイクリング法を使用する高感度測定方法であるため、本方法を実施する際、以下に示す実施方法が測定の精度を上げるために好適である。
【００３５】
本発明の測定方法における温度範囲は、使用する各酵素成分の平均的至適温度範囲、すなわち20〜45゜Cの温度範囲、好ましくは25〜40゜Cの温度範囲が好適である。測定精度に最も影響する温度因子は各被検体あるいは検量線を作成するための標準ホモシステイン標準液を測定する際の測定温度である。測定精度を上げるためには酵素反応セルあるいはキュベットの温度調整誤差範囲が、±0.5゜C以下の範囲、好ましくは±0.2゜C以下の範囲が好適である。
【００３６】
また、セルあるいはキュベットに分注する被検体、ホモシステイン標準液、及び使用する酵素溶液の量は、極めて再現精度の高いピペットあるいは分注ポンプを使うことが必要となる。本発明の測定方法に使用するピペットあるいは分注ポンプの再現精度は、±0.3%以下の精度範囲、好ましくは±0.1%以下の精度範囲が好適である。
【００３７】
さらに、測定ごとの反応時間の精度も測定精度に大きく影響されため、被検体あるいはホモシステイン標準液における反応時間を一定にする必要がある。各測定における反応時間の精度は、±2秒以下の時間範囲、好ましくは±1秒以下の温度範囲が精度を上げるために好適である。
【００３８】
種々の被検体中のホモシステイン濃度を測定する際には、高感度測定法の常法である検量線作成のために予めホモシステイン標準液の測定を行う。この検量線作成は、測定精度を高くするためには種々の濃度の標準液の測定することが好ましいが、被検体中のホモシステイン濃度の上限に近い濃度の標準液とブランク（ホモシステイン濃度がゼロ）の２点検量線法に依っても高い精度が得られる。検量線作成のための測定頻度は、精度を上げるためには多くすることが好ましいが、通常50回の被検体測定あたり1回以上、好ましくは20回の測定あたり1回以上の検量線作成測定が好適である。
【００３９】
本発明の定量用試薬は、前述の高感度定量法に使用される各種酵素及び補酵素、更に必要に応じて酵素が作用する基質を主成分とし、通常緩衝液中に溶液状態で存在させるものが一般的であるが、使用直前に緩衝液で溶解させる凍結乾燥状態の試薬とすることも可能である。
【００４０】
具体的な本発明の定量用試薬の主構成は、ホモシステインデスルフラーゼ、酸化型または還元型ニコチンアミド補酵素、該ニコチンアミド補酵素と異なる型のチオニコチンアミド補酵素、及び２−ケト酪酸を基質として２−ヒドロキシ酪酸を可逆的に生成させる反応を触媒する脱水素酵素から成る。
【００４１】
また、補酵素の再生反応を利用する場合の定量用試薬の主構成は、ホモシステインデスルフラーゼ、酸化型または還元型ニコチンアミド補酵素、該ニコチンアミド補酵素と異なる型のチオニコチンアミド補酵素、２−ケト酪酸を基質として２−ヒドロキシ酪酸を可逆的に生成しうる反応を触媒する脱水素酵素、対応ニコチンアミド補酵素あるいは対応チオニコチンアミド補酵素を可逆的に酸化還元する脱水素酵素、及び対応ニコチンアミド補酵素あるいは対応チオニコチンアミド補酵素を可逆的に酸化還元する脱水素酵素が作用する基質からなる。
【００４２】
上記定量用試薬は構成する成分をすべて含んだ一液の試薬とした場合でも、定量の基礎となる各反応は逐次的に進行するので定量が可能となる。必要に応じて、各反応毎に必要な成分から成る複数の試薬としても構わない。
【００４３】
上記構成成分を緩衝液中に溶解させる場合の緩衝液としては、各酵素が有効に作用するｐＨを維持できるものであれば特に制限はなく、トリス−塩酸緩衝液、リン酸緩衝液、グッド緩衝液などが使用できる。
【００４４】
当該試薬中の各成分の濃度や試薬のｐＨ等の諸条件は、測定時に該試薬と被検体とを混合した際に、前述の高感度定量法の項で述べた諸条件を満足するように調製すればよい。また、当該試薬中には、試薬を安定化させるためのシュークロースなどの糖類、アジ化ナトリウム、抗菌剤などの防腐剤等々必要に応じて公知の成分を含有させることができる。
【００４５】
【実施例】
次に実施例を挙げて本発明を更に具体的に説明するが、本発明はこれらの実施例に限定されるものではない。尚、酵素量は37゜C下で1.0μmole/minの反応を触媒する酵素量を1.0Uと示す。
【００４６】
実施例１ ホモシステイン水溶液の測定
以下に示す組成の測定用試液Ａ、測定試液Ｂ及びホモシステイン溶液を調製した。
［測定用試液Ａ］
50mM トリス−塩酸緩衝液（pH 8.0）
10U/ml ホモシステインデスルフラーゼ
(Proteus morganii IFO-3168由来)
［測定用試液Ｂ］
50mM トリス−塩酸緩衝液（pH 8.0）
5mM チオＮＡＤ（シグマ社製）
1mM ＮＡＤＨ（シグマ社製）
200U/ml L-乳酸脱水素酵素
(オリエンタル酵母社製、ブタ筋肉由来)
［ホモシステイン溶液］
50μM ホモシステイン標準水溶液
【００４７】
上記ホモシステイン標準水溶液を精製水にて希釈して、10μM,20μM,30μM, 40μM濃度の希釈系列のホモシステイン希釈溶液を調製した。20μlのホモシステイン標準水溶液、それぞれのホモシステイン希釈溶液、及び精製水を各々のキュベットに分注し、さらに0.5mlのあらかじめ37゜Cに加温した測定用試液Ａを各々のキュベットに分注し混和後、37゜C下で10分間反応させた。反応後、各々のキュベットにあらかじめ37゜Cに加温した測定試液Ｂを0.5mlずつ、各々のキュベットに分注混和後、3分間後と13分間後の400nmの吸光度を測定し、それぞれの吸光度の差をΔAbsとした。ホモシステイン濃度とΔAbsとの関係を図１に示す。この結果から水溶液中の微量のホモシステインを正確に定量できることが判る。
【００４８】
実施例２ ホモシステインデスルフラーゼの反応方式
以下に示す組成の測定用試液Ａ、測定試液Ｂ及びホモシステイン溶液を調製した。
［測定用試液Ａ］
50mM トリス−塩酸緩衝液（pH 8.0）
10U/ml ホモシステインデスルフラーゼ
(Proteus morganii IFO-3168由来)
［測定用試液Ｂ］
50mM トリス−塩酸緩衝液（pH 8.0）
5mM チオＮＡＤ（シグマ社製）
1mM ＮＡＤＨ（シグマ社製）
200U/ml L-乳酸脱水素酵素
(オリエンタル酵母社製、ブタ筋肉由来)
［ホモシステイン溶液］
50μM ホモシステイン標準水溶液
【００４９】
上記ホモシステイン標準水溶液を精製水にて希釈して、10μM,20μM,30μM, 40μM濃度の希釈系列のホモシステイン希釈溶液を調製した。50μlのホモシステイン標準水溶液、各ホモシステイン希釈溶液、及び精製水をそれぞれのキュベットに分注し、あらかじめ37゜Cに加温した0.5mlの測定用試液Ａと0.5mlの測定用試液Ｂを各々のキュベットに分注し混和し、3分間後と13分間後の400nmの吸光度を測定した。13分後と3分後のそれぞれの吸光度の差をΔAbsとし、ホモシステイン濃度とΔAbsとの関係を図２に示す。この結果からホモシステインデスルフラーゼ反応を完結することなく酵素サイクリング反応を同時進行させることによってもホモシステンの高感度測定が可能であることが判る。
【００５０】
実施例３ 補酵素の再生反応の付加した測定
以下に示す組成の測定用試液Ａ、測定試液Ｂ及びホモシステイン溶液を調製した。
［測定用試液Ａ］
50mM トリス−塩酸緩衝液（pH 8.0）
10U/ml ホモシステインデスルフラーゼ
(Proteus morganii由来 IFO-3168より調製)
200mM エタノール
［測定用試液Ｂ］
50mM トリス−塩酸緩衝液（pH 8.0）
5mM チオＮＡＤ（シグマ社製）
0.2mM ＮＡＤＨ（シグマ社製）
300U/ml L-乳酸脱水素酵素
(オリエンタル酵母社製、ブタ筋肉由来)
50U/ml アルコール脱水素酵素
（オリエンタル酵母社製、酵母由来）
［ホモシステイン溶液］
50μM ホモシステイン標準水溶液
【００５１】
上記ホモシステイン標準水溶液を精製水にて希釈して、10μM,20μM,30μM, 40μM濃度の希釈系列のホモシステイン希釈溶液を調製した。20μlのホモシステイン標準水溶液、各ホモシステイン希釈溶液、及び精製水をそれぞれのキュベットに分注し、あらかじめ37゜Cに加温した0.5mlの測定用試液Ａと0.5mlの測定用試液Ｂを各々のキュベットに分注し混和し、3分間後と13分間後の400nmの吸光度を測定した。13分後と3分後のそれぞれの吸光度の差をΔAbsとし、ホモシステイン濃度とΔAbsとの関係を図３に示す。この結果から補酵素の再生反応を組み合わせることによってホモシステイン測定の更なる高感度化を達成出来ることが明らかになった。
【００５２】
実施例４ 血清サンプルの測定
以下に示す組成の測定用試液Ａ、測定用試液Ｂを調製した。
［測定用試液Ａ］
50mM トリス−塩酸緩衝液（pH 8.0）
10mM ジチオスレイトール（DTT）
10U/ml ホモシステインデスルフラーゼ
(Proteus morganii IFO-3168由来)
［測定用試液Ｂ］
50mM トリス−塩酸緩衝液（pH 8.0）
5mM チオＮＡＤ（シグマ社製）
1mM ＮＡＤＨ（シグマ社製）
200U/ml L-乳酸脱水素酵素
(オリエンタル酵母社製、ブタ筋肉由来)
［ホモシステイン溶液］
50μM ホモシステイン標準水溶液
【００５３】
あらかじめ37゜Cに加温した0.5mlの測定用試液Ａをキュベットに分注し、
20μlの５種類の血清サンプルをそれぞれのキュベットに添加混和して、37゜C下で反応させた。10分後、あらかじめ37゜Cに加温しておいた測定試液Ｂを0.5mlずつ、各々のキュベットに分注混和後、3分間後と13分間後の400nmの吸光度を測定し、それぞれの吸光度の差をΔAbsとした。血清サンプルの代わりにホモシステイン標準水溶液を使用したものを標準として同様の操作を行いΔAbsを測定した。標準のΔAbsから５種類の血清サンプルのホモシステイン濃度を算出した結果を表１に示す。
【００５４】
【表１】

【００５５】
【発明の効果】
本発明のホモシステインの高感度定量法及び定量用試薬は、簡便な操作で被検体中の極微量のホモシステインを正確に定量することができる方法を提供するものであり、臨床検査などの医療の分野に大きく貢献することが期待される。例えば、血清中のホモシステイン定量分析のための自動分析装置への応用等を可能にする。
【図面の簡単な説明】
【図１】 実施例１の測定法でホモシステイン標準希釈水溶液を測定した場合のホモシステイン濃度と吸光度との関係を示す。
【図２】 実施例２の測定法でホモシステイン標準希釈水溶液を測定した場合のホモシステイン濃度と吸光度との関係を示す。
【図３】 実施例３の測定法でホモシステイン標準希釈水溶液を測定した場合のホモシステイン濃度と吸光度との関係を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel method for quantifying homocysteine in a specimen with extremely high sensitivity. In particular, it is effective for quantifying homocysteine in each organ and body fluid in a living body, and can be widely used in the medical field such as clinical examination.
[0002]
[Prior art]
Conventional methods for quantifying homocysteine include prelabel HPLC, in which homocysteine in a sample is labeled using a fluorescent labeling reagent that reacts with SH groups, and then separated and quantified by high performance liquid chromatography. Post-label HPLC method in which detection and quantification is performed by reacting with a fluorescent labeling reagent that reacts with SH groups after separation by chromatography, and an enzyme that specifically acts on homocysteine is allowed to act, and the enzyme reaction product is detected and quantified by an antibody method. There have been proposed an enzyme immunization method, an enzyme method in which an enzyme that specifically acts on homocysteine is allowed to act, and then the product is detected and quantified by the enzyme method.
[0003]
None of these conventional quantification techniques are capable of quantitatively quantifying homocysteine in a simple and accurate manner. As a quantification method using high performance liquid chromatography among conventional techniques, for example, the method described in Methods in Enzymology, 143, 67 (1987) is a highly sensitive fluorescent labeling reagent for thiol compounds in a specimen. In this method, the labeled homocysteine is separated and quantified by high performance liquid chromatography after the prelabeling reaction. Although this quantification method has been improved in sensitivity, it has drawbacks such as a complicated prelabeling reaction and a low sample throughput.
[0004]
As a method for solving the complexity of such pre-labeling in the high performance liquid chromatography method, for example, after separating homocysteine by high performance liquid chromatography described in Analytical Biochemistry, Vol. 227, page 14 (1995), a fluorescent reagent A so-called post-labeling method has been proposed. However, even such a post-label high-performance liquid chromatography method still has the problem that the problem of low sample throughput is not solved, and a large amount of expensive fluorescent reagent is used for post-labeling.
[0005]
As a method for solving the problems of the high performance liquid chromatography method, a quantification method using an enzyme and an antibody has been proposed. For example, the method for quantifying homocysteine described in JP-T-8-50648 and Clinical Chemistry, Vol. 44, p. 311 (1998) uses homocysteine in a specimen as adenosine and S-adenosyl homocysteine synthase. In this method, the reaction is carried out in the presence, and the produced S-adenosylhomocysteine is quantified by an enzyme immunoassay using an antibody that specifically acts on the compound. The enzyme immunization method is advantageous in terms of the throughput of many specimens as compared with the high performance liquid chromatography method, but has the disadvantage that the complicated reaction combining the enzyme reaction and the enzyme immunization method and the use of an expensive antibody. Have.
[0006]
As a technique for improving the problem of the enzyme immunization method, a quantitative method for enzymatic homocysteine has been proposed. For example, in the method described in International Patent Publication No. WO-9905311, homocysteine in a sample is reacted with an enzyme called homocysteinase, and the resulting hydrogen sulfide is introduced into a dye, followed by colorimetric analysis. It is a method of quantification. In addition, the method described in International Patent Publication No. WO-9807872 allows homocysteine in a sample to react with an enzyme called homocysteine desulfurase, and the resulting 2-ketobutyric acid is reduced to nicotinamide adenine dinucleotide (hereinafter referred to as NADH). Quantification of homocysteine by quantifying the oxidized nicotinamide adenine dinucleotide (hereinafter referred to as NAD) produced by acting with lactate dehydrogenase or pyruvate dehydrogenase in the presence of It is.
However, among these enzymatic quantification methods, the method of quantifying homocysteine by reacting homocysteine in a sample with an enzyme called homocysteinase and colorimetrically analyzing the produced hydrogen sulfide is a dye method. Therefore, the sensitivity is low.
[0007]
In addition, homocysteine in the sample is allowed to act with an enzyme called homocysteine desulfurase, and the produced 2-ketobutyric acid is allowed to act with lactate dehydrogenase or pyruvate dehydrogenase in the presence of NADH to quantify the produced NAD. The method of quantifying homocysteine by this method has the advantage of high sensitivity, but is a method in which enzyme cycling is performed after a complicated process of degrading NADH in the middle of the quantification process. It has the difficulty of being difficult to do.
[0008]
[Problems to be solved by the invention]
The present invention provides a simple and highly sensitive method for quantifying homocysteine in a sample that solves the problems of the prior art. In other words, the present invention has the convenience that only the enzymatic reaction is used as a quantification step when quantifying homocysteine in a sample having a low homocysteine content such as serum or plasma, and is also applied to an automatic analyzer with high sensitivity. A homocysteine quantification method and a reagent composition for quantification are provided.
[0009]
[Means for Solving the Problems]
The present invention relates to the amount of 2-ketobutyric acid produced with the progress of the enzymatic reaction after reacting homocysteine desulfurase with / or reacting with a subject containing homocysteine. In the presence of nicotinamide coenzyme in the presence of nicotinamide coenzyme, it acts in the presence of dehydrogenase, nicotinamide coenzyme, and thionicotinamide coenzyme that catalyze the reaction to reversibly generate 2-hydroxybutyric acid. By quantifying the amount of the corresponding nicotinamide coenzyme or the corresponding thionicotinamide coenzyme produced along with the amount of the pre-added nicotinamide coenzyme and thionicotinamide coenzyme that decreases with the progress of the reaction. This is a simple and highly sensitive method for quantifying the amount of homocysteine in a sample.
[0010]
That is, the present invention allows homocysteine desulfurase to act on a subject to produce 2-keto acid, and then an oxidized or reduced nicotinamide coenzyme and a thionicotinamide of a type different from the nicotinamide coenzyme. In the presence of a coenzyme, a dehydrogenase that catalyzes a reaction that reversibly produces 2-hydroxybutyric acid using the 2-ketobutyric acid as a substrate is used to perform an enzymatic reaction, which is consumed as the enzymatic reaction proceeds. Quantifying the amount of the nicotinamide coenzyme or thionicotinamide coenzyme produced or the amount of the corresponding nicotinamide coenzyme or the corresponding thionicotinamide coenzyme produced as the enzyme reaction proceeds. A method for quantifying homocysteine characterized by determining the amount of cysteine.
[0011]
Other inventions include homocysteine desulfurase, oxidized or reduced nicotinamide dinucleotide phosphate, reduced or oxidized thionicotinamide adenine dinucleotide phosphate, and 2-ketobutyric acid as a substrate. A reagent for quantifying homocysteine, comprising a dehydrogenase that catalyzes a reaction that can be generated reversibly.
[0012]
Still another invention relates to homocysteine desulfurase, oxidized or reduced nicotinamide dinucleotide phosphate, reduced or oxidized thionicotinamide adenine dinucleotide phosphate, and 2-ketobutyric acid as a substrate. A dehydrogenase that catalyzes a reversible reaction, a corresponding nicotinamide coenzyme or a corresponding thionicotinamide coenzyme, a dehydrogenase that reversibly oxidizes and reduces, a corresponding nicotinamide coenzyme or a corresponding thionicotinamide coenzyme A reagent for quantifying homocysteine, comprising a substrate on which a dehydrogenase that reversibly oxidizes and reduces acts.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
There are two types of nicotinamide coenzyme and thionicotinamide coenzyme used in carrying out the enzyme cycling method of the present invention, reduced and oxidized, respectively. When an oxidized form is used as a thionicotinamide coenzyme and an oxidized form is used as a nicotinamide coenzyme, it is necessary to use a reduced form in combination as a thionicotinamide coenzyme.
[0014]
In the present invention, 2-hydroxybutyric acid is reversibly produced using 2-ketobutyric acid as a substrate in the presence of an oxidized or reduced nicotinamide coenzyme and a thionicotinamide coenzyme of a different type from the nicotinamide coenzyme. When a dehydrogenase that catalyzes the reaction (hereinafter referred to as 2-ketobutyrate dehydrogenase) is allowed to act, 2-hydroxybutyric acid is produced and used as the enzyme reaction proceeds. The coenzyme is converted to the reduced form if the coenzyme is in the oxidized form, and is converted to the oxidized form if the coenzyme is in the reduced form. Therefore, in the present invention, the corresponding nicotinamide coenzyme and the corresponding thionicotinamide coenzyme refer to a coenzyme converted by the enzymatic reaction resulting from the action of 2-ketobutyrate dehydrogenase on 2-ketoacetic acid. If the coenzyme before the reaction is an oxidized form, it means a reduced form, and if it is a reduced form, it means an oxidized form.
[0015]
When quantifying the amount of the corresponding nicotinamide coenzyme or the corresponding thionicotinamide coenzyme produced over the course of the reaction, or the amount of the nicotinamide coenzyme or the corresponding thionicotinamide coenzyme consumed over the course of the reaction As a specific method for quantifying the amount of reduced thionicotinamide coenzyme, the amount of reduced thionicotinamide coenzyme increases with the progress of the reaction. In addition, when the reaction is carried out by combining oxidized nicotinamide coenzyme and reduced thionicotinamide coenzyme, the method of quantifying the amount of reduced thionicotinamide coenzyme that decreases with the progress of the reaction is generally used. Is.
[0016]
The typical measurement principle of the present invention is illustrated in the following equation.
[Chemical 1]

[0017]
In the above formula, reduced nicotinamide adenine dinucleotide as nicotinamide coenzyme, oxidized thionicotinamide adenine dinucleotide as thionicotinamide coenzyme, and 2-ketobutyrate dehydrogenase as 2-ketobutyrate dehydrogenases The example used is shown.
[0018]
The present invention provides a simple and highly sensitive method for determining homocysteine with higher accuracy. When carrying out the above enzyme cycling method, when quantifying nicotinamide coenzyme or thionicotinamide coenzyme that eventually increases or decreases with the progress of the enzyme cycling reaction, depending on the conditions, each coenzyme may be a coenzyme. May interfere with quantification. In order to solve this problem, it is possible to reduce these interferences by increasing the difference between the nicotinamide coenzyme concentration and the thionicotinamide coenzyme concentration. However, on the other hand, there may be a decrease in the enzyme cycling reaction rate due to the low concentration of one coenzyme, or an error in measurement of one coenzyme due to a change in the concentration of one coenzyme. As a means for eliminating these difficulties, a means for incorporating a coenzyme regeneration reaction at a low concentration into the enzyme cycling reaction can be suitably used.
[0019]
As the coenzyme regeneration reaction, a dehydrogenase that does not affect the enzyme reaction of 2-ketobutyrate dehydrogenases and reversibly oxidizes or reduces the corresponding nicotinamide coenzyme or the corresponding thionicotinamide coenzyme. Any reaction can be used without any particular limitation.
[0020]
A typical homocysteine measurement principle including the coenzyme regeneration reaction is illustrated in the following formula.
[0021]
[Chemical 2]

[0022]
In the above formula, reduced nicotinamide adenine dinucleotide as nicotinamide coenzyme, oxidized thionicotinamide adenine dinucleotide as thionicotinamide coenzyme, 2-ketobutyrate dehydrogenase as 2-ketobutyrate dehydrogenases, An example of using alcohol as a substrate for alcohol dehydrogenase and a substrate for alcohol dehydrogenase as a dehydrogenase that reversibly oxidizes or reduces nicotinamide coenzyme or thionicotinamide coenzyme (hereinafter referred to as redox dehydrogenase) Show.
The method for highly sensitive measurement of homocysteine in a specimen including such a coenzyme regeneration reaction will be described in detail as follows.
[0023]
Homocysteine desulfurase is allowed to act on a subject containing homocysteine, and 2-ketobutyric acid produced by the enzymatic reaction is combined with a high concentration of oxidized thionicotinamide coenzyme and a low concentration of reduced nicotinamide coenzyme. When 2-ketobutyrate dehydrogenase is allowed to act in the presence, reduced thionicotinamide coenzyme is produced and reduced nicotinamide coenzyme is consumed as the reversible reaction proceeds. As the reduced nicotinamide coenzyme is consumed, the rate of the reversible reaction decreases or the reaction stops. Therefore, while performing the reversible reaction, redox dehydrogenases for the regenerative reaction of reduced nicotinamide coenzyme that does not act on thionicotinamide coenzyme and does not affect the reversible reaction; When the substrate is added, the consumed reduced nicotinamide coenzyme is regenerated from the oxidized nicotinamide coenzyme, and the reversible reaction continues and the ratio between the reduced and oxidized nicotinamide coenzyme concentrations is relatively high. It becomes constant. The concentration of the reduced thionicotinamide coenzyme that is generated along with the reversible reaction that continues without decreasing the reaction rate is easily affected by the absorbance derived from the reduced nicotinamide coenzyme by, for example, absorbance measurement. Finally, the homocysteine concentration in the specimen can be quantified accurately and with high sensitivity.
[0024]
The subject in the present invention refers to a sample to be measured for homocysteine in the medical or clinical laboratory fields, and includes, for example, serum, plasma, cell fluid, and organ extract.
[0025]
Homocysteine desulfurase (EC 4.4.1.2) in the quantification method or quantification reagent of the present invention varies depending on the type of analyte to be measured and the measurement conditions, but produces 2-ketobutyric acid using homocysteine as a substrate. Any enzyme can be used as long as it catalyzes the reaction, and a known enzyme having such characteristics is used without particular limitation. An example of an enzyme having this property is the homocysteine death derived from Proteus morgani described in Methods in Enzymology, Vol. 2, 318 (1955) and Journal of Biological Chemistry, 192, 371 (1951). Ruflase, Journal of Biochemistry, 79, 1263 (1976), Biochemistry, 16, 100 (1977), and Methods in Enzymology, 143, 459 (1987) Examples include homocysteinase, Medical Science, Vol. 13, 493 (1985), and homocysteine desulfurase derived from the genus Trichomonas described in International Patent Publication WO 9807872.
[0026]
In the quantification method of the present invention, the method for allowing homocysteine desulfurase to act on the analyte can be arbitrarily selected depending on the type of analyte and the measurement conditions. That is, when the measurement is not complicated, almost all homocysteine contained in the subject is converted into 2-ketobutyric acid by the action of homocysteine desulfurase, and then reversible reaction with 2-hydroxybutyric acid or the reversible The method of performing the reaction and the regeneration reaction of the coenzyme can be selected. When the measurement is to be carried out simply, the homocysteine in the subject is converted to 2-ketobutyric acid by the action of homocysteine desulfurase, and the reversible reaction with 2-hydroxybutyric acid or the reversible reaction and coenzyme is performed. It is possible to select a method for simultaneously proceeding the regeneration reactions.
[0027]
The amount of homocysteine desulfurase to act on the subject in the present invention is appropriately determined depending on the type of the subject and the type of the enzyme, but generally a concentration range of 0.1 to 1000 μmoles / min / ml, particularly 0.2 to A concentration range of 500 μmoles / min / ml is preferably used. The pH at which the homocysteine desulfurase is allowed to act is appropriately selected depending on the origin of the enzyme, but in general, the range of pH 3.5 to 10.5, particularly the range of pH 4.0 to 10.0 is preferably selected. In addition, the reaction temperature at the time of allowing homocysteine desulfurase to act is generally selected from a temperature range of 20 to 45 ° C, preferably a temperature range of 25 to 40 ° C.
[0028]
As the nicotinamide coenzyme in the quantitative method or quantitative reagent of the present invention, nicotinamide adenine dinucleotide (NADH: reduced form, NAD: oxidized form, the same shall apply hereinafter), nicotinamide adenine dinucleotide phosphate (NADPH, NADP), Specific examples include acetylpyridine adenine dinucleotide, acetylpyridine adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, nicotinamide hypoxanthine dinucleotide phosphate, and the like. Examples of thionicotinamide coenzymes include thionicotinamide adenine dinucleotide (thioNADH, thioNAD), thionicotinamide adenine dinucleotide phosphate (thioNADPH, thioNADP), thionicotinamide hypoxanthine dinucleotide, Specific examples include nicotinamide hypoxanthine dinucleotide phosphate and the like.
[0029]
The concentration of nicotinamide coenzyme or thionicotinamide coenzyme used in the quantification method of the present invention is generally in a wide concentration range of 1 μM to 200 mM, but in particular, a concentration range of 5 μM to 100 mM is preferably used. .
[0030]
The 2-ketobutyrate dehydrogenase of the present invention is a compound obtained by converting 2-ketobutyric acid in the presence of a reduced nicotinamide coenzyme represented by NADH or a reduced thionicotinamide coenzyme represented by thioNADH. The enzyme is not particularly limited as long as it is reversibly reduced to produce 2-hydroxybutyric acid, and a known enzyme having such characteristics can be used without limitation. Examples of enzymes having this property include animal-derived, plant-derived, and microorganism-derived L-lactate dehydrogenases (EC 1.1.1.27), or many microorganism-derived D-lactate dehydrogenases (EC 1.1.1.28). In particular, L-lactate dehydrogenase derived from porcine heart or porcine muscle, L-lactate dehydrogenase derived from microorganism Bacillus, L-lactate dehydrogenase derived from microorganism Lactobacillus, or D derived from E. coli -Lactate dehydrogenase and the like can be suitably used because of their availability.
[0031]
The amount of 2-ketobutyrate dehydrogenase in the quantification method of the present invention is appropriately determined depending on the type of analyte to be used, the measurement conditions, or the origin of the enzyme, but is generally 0.1 to 1000 μmoles / min / ml. A concentration range, particularly a concentration range of 0.5 to 500 μmoles / min / ml, is preferably used. The reaction pH of 2-ketobutyrate dehydrogenase is appropriately determined depending on the enzyme used, but generally it is in the range of pH 4.0 to 10.5, preferably in the range of pH 4.5 to 10.0. Further, the reaction temperature of 2-ketobutyrate dehydrogenase is generally selected from a temperature range of 20 to 45 ° C, preferably a temperature range of 25 to 40 ° C.
[0032]
In the quantification method of the present invention, any of these methods may be used to quantify nicotinamide coenzyme, thionicotinamide coenzyme, corresponding nicotinamide coenzyme, or corresponding thionicotinamide coenzyme consumed or generated with the progress of the reaction. The amount of coenzyme increase / decrease may be quantified, but in general, absorbance analysis at a wavelength exhibiting the maximum molecular extinction coefficient of reduced nicotinamide coenzyme or reduced thionicotinamide coenzyme is preferably employed. . In the case of reduced nicotinamide coenzyme, quantification is performed by increasing or decreasing the absorbance around 340 nm, and in the case of reduced thionicotinamide coenzyme, it can be easily quantified by increasing or decreasing the absorbance around 400 nm.
[0033]
In the quantification method of the present invention, further enhancement of sensitivity can be realized by combining the coenzyme regeneration reaction. When the final measurement target coenzyme is a corresponding thionicotinamide coenzyme, the corresponding nicotinamide coenzyme is regenerated. When the final measurement target coenzyme is a corresponding nicotinamide coenzyme, the corresponding thionicotinamide coenzyme is used. By combining the coenzyme regeneration reaction as appropriate, it is possible to make the highly sensitive quantitative method for homocysteine more accurate. The enzyme that performs the regeneration reaction of the coenzyme and its substrate are not particularly limited as long as they do not affect the reversible reaction between 2-ketobutyric acid and 2-hydroxybutyric acid. For example, alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2) and ethanol or acetaldehyde; glycerol dehydrogenase (EC 1.1.1.6) and glycerol or dihydroxyacetone; arabinitol dehydrogenase (EC 1.1.1.12 or EC 1.1.1.14) and arabinitol, xylulose or ribulose; glycerate dehydrogenase (EC 1.1.1.29) and glycerate or hydroxypyruvate; malate dehydrogenase (EC 1.1.1.37 or EC 1.1 .1.82) and malic acid or oxaloacetic acid; aryl alcohol dehydrogenation Containing (EC 1.1.1.90) and benzyl alcohol or benzaldehyde; and the like tartrate or o Gizaro glycolic acid tartrate dehydrogenase (EC 1.1.1.93) and the like. The concentration of redox dehydrogenases used in the regeneration reaction of these coenzymes varies depending on the type and concentration of the coenzyme used, but is generally in the concentration range of 0.1 to 1000 μmoles / min / ml, especially A range of 0.5 to 500 μmoles / min / ml is preferably used.
[0034]
Since the homocysteine measurement method according to the present invention is a highly sensitive measurement method using an enzyme cycling method, the following implementation method is suitable for increasing the measurement accuracy when performing this method.
[0035]
The temperature range in the measurement method of the present invention is preferably an average optimum temperature range of each enzyme component used, that is, a temperature range of 20 to 45 ° C, preferably a temperature range of 25 to 40 ° C. The temperature factor that has the greatest influence on the measurement accuracy is the measurement temperature at the time of measuring the standard homocysteine standard solution for preparing each analyte or calibration curve. In order to increase the measurement accuracy, the temperature adjustment error range of the enzyme reaction cell or cuvette is within a range of ± 0.5 ° C. or less, preferably within a range of ± 0.2 ° C. or less.
[0036]
In addition, it is necessary to use a pipette or a dispensing pump with extremely high reproducibility for the amount of the specimen to be dispensed into the cell or cuvette, the homocysteine standard solution, and the enzyme solution to be used. The reproducibility of the pipette or dispensing pump used in the measurement method of the present invention is within an accuracy range of ± 0.3% or less, preferably ± 0.1% or less.
[0037]
Furthermore, since the accuracy of the reaction time for each measurement is greatly affected by the measurement accuracy, it is necessary to make the reaction time in the subject or the homocysteine standard solution constant. As the accuracy of the reaction time in each measurement, a time range of ± 2 seconds or less, preferably a temperature range of ± 1 seconds or less is suitable for increasing the accuracy.
[0038]
When measuring the homocysteine concentration in various specimens, a homocysteine standard solution is measured in advance in order to prepare a calibration curve, which is an ordinary method of a highly sensitive measurement method. In order to increase the measurement accuracy, it is preferable to measure standard solutions of various concentrations in this calibration curve. However, a standard solution and a blank having a concentration close to the upper limit of the homocysteine concentration in the test sample (the homocysteine concentration is High accuracy can be obtained even by using the zero inspection 2 curve method. The measurement frequency for creating a calibration curve is preferably increased in order to increase the accuracy, but usually at least once per 50 sample measurements, preferably at least once per 20 measurements. Is preferred.
[0039]
The quantification reagent of the present invention is mainly composed of various enzymes and coenzymes used in the above-mentioned high-sensitivity quantification method, and further, if necessary, a substrate on which the enzyme acts, and is usually present in solution in a buffer solution. However, it is also possible to use a lyophilized reagent that is dissolved in a buffer immediately before use.
[0040]
Specific main components of the quantification reagent of the present invention are homocysteine desulfurase, oxidized or reduced nicotinamide coenzyme, thionicotinamide coenzyme of a type different from the nicotinamide coenzyme, and 2-ketobutyric acid It consists of dehydrogenase that catalyzes the reaction of reversibly producing 2-hydroxybutyric acid using as a substrate.
[0041]
In addition, the main components of the quantification reagent when using the coenzyme regeneration reaction are homocysteine desulfurase, oxidized or reduced nicotinamide coenzyme, and thionicotinamide coenzyme of a type different from the nicotinamide coenzyme. , A dehydrogenase that catalyzes a reaction that can reversibly produce 2-hydroxybutyric acid using 2-ketobutyric acid as a substrate, a dehydrogenase that reversibly oxidizes and reduces a corresponding nicotinamide coenzyme or a corresponding thionicotinamide coenzyme, And a substrate on which a dehydrogenase that reversibly oxidizes and reduces the corresponding nicotinamide coenzyme or the corresponding thionicotinamide coenzyme acts.
[0042]
Even when the quantification reagent is a one-component reagent containing all of the constituent components, each reaction that is the basis of quantification proceeds sequentially, so that quantification is possible. If necessary, a plurality of reagents comprising necessary components for each reaction may be used.
[0043]
The buffer for dissolving the above components in the buffer is not particularly limited as long as it can maintain the pH at which each enzyme acts effectively. Tris-HCl buffer, phosphate buffer, Good buffer Liquid etc. can be used.
[0044]
Various conditions such as the concentration of each component in the reagent and the pH of the reagent should satisfy the conditions described in the section of the above-mentioned high-sensitivity quantification method when the reagent and the sample are mixed during measurement. What is necessary is just to prepare. The reagent may contain known components such as sugars such as sucrose for stabilizing the reagent, preservatives such as sodium azide, antibacterial agents, and the like as necessary.
[0045]
【Example】
EXAMPLES Next, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples. In addition, the enzyme amount shows 1.0 U of the enzyme amount which catalyzes the reaction of 1.0 micromol / min at 37 degreeC.
[0046]
Example 1 Measurement of aqueous homocysteine solution
A measurement reagent A, a measurement reagent B, and a homocysteine solution having the following compositions were prepared.
[Measurement solution A]
50 mM Tris-HCl buffer (pH 8.0)
10U / ml homocysteine desulfurase
(From Proteus morganii IFO-3168)
[Measurement solution B]
50 mM Tris-HCl buffer (pH 8.0)
5mM Thio NAD (Sigma)
1mM NADH (manufactured by Sigma)
200 U / ml L-lactate dehydrogenase
(Oriental Yeast, pork muscle origin)
[Homocysteine solution]
50μM homocysteine standard aqueous solution
[0047]
The homocysteine standard aqueous solution was diluted with purified water to prepare a diluted series of homocysteine diluted solutions having concentrations of 10 μM, 20 μM, 30 μM, and 40 μM. Dispense 20 μl of homocysteine standard aqueous solution, each homocysteine diluted solution, and purified water into each cuvette, and then dispense 0.5 ml of the test solution A pre-warmed to 37 ° C into each cuvette. After mixing, the mixture was reacted at 37 ° C for 10 minutes. After the reaction, 0.5 ml each of the test solution B pre-warmed to 37 ° C in each cuvette is dispensed and mixed in each cuvette, and then the absorbance at 400 nm is measured 3 minutes and 13 minutes later. The difference was ΔAbs. The relationship between the homocysteine concentration and ΔAbs is shown in FIG. From this result, it is understood that a trace amount of homocysteine in the aqueous solution can be accurately quantified.
[0048]
Example 2 Reaction method of homocysteine desulfurase
A measurement reagent A, a measurement reagent B, and a homocysteine solution having the following compositions were prepared.
[Measurement solution A]
50 mM Tris-HCl buffer (pH 8.0)
10U / ml homocysteine desulfurase
(From Proteus morganii IFO-3168)
[Measurement solution B]
50 mM Tris-HCl buffer (pH 8.0)
5mM Thio NAD (Sigma)
1mM NADH (manufactured by Sigma)
200 U / ml L-lactate dehydrogenase
(Oriental Yeast, pork muscle origin)
[Homocysteine solution]
50μM homocysteine standard aqueous solution
[0049]
The homocysteine standard aqueous solution was diluted with purified water to prepare a diluted series of homocysteine diluted solutions having concentrations of 10 μM, 20 μM, 30 μM, and 40 μM. Dispense 50 µl of homocysteine standard aqueous solution, each homocysteine diluted solution, and purified water into each cuvette, and heat 0.5 ml of measuring solution A and 0.5 ml of measuring solution B that are preheated to 37 ° C. The mixture was dispensed and mixed in a cuvette, and the absorbance at 400 nm was measured after 3 minutes and 13 minutes. The difference in absorbance after 13 minutes and 3 minutes is ΔAbs, and the relationship between homocysteine concentration and ΔAbs is shown in FIG. From this result, it can be seen that high-sensitivity measurement of homocystene is also possible by simultaneously proceeding the enzyme cycling reaction without completing the homocysteine desulfurase reaction.
[0050]
Example 3 Additional measurement of coenzyme regeneration reaction
A measurement reagent A, a measurement reagent B, and a homocysteine solution having the following compositions were prepared.
[Measurement solution A]
50 mM Tris-HCl buffer (pH 8.0)
10U / ml homocysteine desulfurase
(Prepared from IFO-3168 from Proteus morganii)
200 mM ethanol
[Measurement solution B]
50 mM Tris-HCl buffer (pH 8.0)
5mM Thio NAD (Sigma)
0.2mM NADH (manufactured by Sigma)
300 U / ml L-lactate dehydrogenase
(Oriental Yeast, pork muscle origin)
50U / ml alcohol dehydrogenase
(Oriental yeast, yeast-derived)
[Homocysteine solution]
50μM homocysteine standard aqueous solution
[0051]
The homocysteine standard aqueous solution was diluted with purified water to prepare a diluted series of homocysteine diluted solutions having concentrations of 10 μM, 20 μM, 30 μM, and 40 μM. Dispense 20 μl of homocysteine standard aqueous solution, each homocysteine diluted solution, and purified water into each cuvette, and heat 0.5 ml of measuring solution A and 0.5 ml of measuring solution B that have been heated to 37 ° C in advance. The mixture was dispensed and mixed in a cuvette, and the absorbance at 400 nm was measured after 3 minutes and 13 minutes. The difference in absorbance between 13 minutes and 3 minutes is ΔAbs, and the relationship between homocysteine concentration and ΔAbs is shown in FIG. From this result, it became clear that the sensitivity of homocysteine measurement can be further increased by combining the coenzyme regeneration reaction.
[0052]
Example 4 Measurement of serum sample
A measurement reagent A and a measurement reagent B having the compositions shown below were prepared.
[Measurement solution A]
50 mM Tris-HCl buffer (pH 8.0)
10mM dithiothreitol (DTT)
10U / ml homocysteine desulfurase
(From Proteus morganii IFO-3168)
[Measurement solution B]
50 mM Tris-HCl buffer (pH 8.0)
5mM Thio NAD (Sigma)
1mM NADH (manufactured by Sigma)
200 U / ml L-lactate dehydrogenase
(Oriental Yeast, pork muscle origin)
[Homocysteine solution]
50μM homocysteine standard aqueous solution
[0053]
Dispense 0.5 ml of measuring solution A, preheated to 37 ° C, into the cuvette,
20 μl of five kinds of serum samples were added to each cuvette and mixed, and reacted at 37 ° C. Ten minutes later, 0.5 ml each of the test solution B preheated to 37 ° C was dispensed and mixed in each cuvette, and the absorbance at 400 nm was measured after 3 and 13 minutes. The difference was ΔAbs. The same procedure was performed using a homocysteine standard aqueous solution instead of a serum sample as a standard, and ΔAbs was measured. Table 1 shows the results of calculating the homocysteine concentrations of five types of serum samples from the standard ΔAbs.
[0054]
[Table 1]

[0055]
【The invention's effect】
The high-sensitivity quantification method and quantification reagent for homocysteine of the present invention provide a method capable of accurately quantifying a very small amount of homocysteine in a sample by a simple operation, and can be used for medical tests such as clinical examinations. It is expected to contribute greatly to these fields. For example, it enables application to an automatic analyzer for homocysteine quantitative analysis in serum.
[Brief description of the drawings]
1 shows the relationship between homocysteine concentration and absorbance when a homocysteine standard diluted aqueous solution is measured by the measurement method of Example 1. FIG.
FIG. 2 shows the relationship between homocysteine concentration and absorbance when a homocysteine standard diluted aqueous solution is measured by the measurement method of Example 2.
FIG. 3 shows the relationship between homocysteine concentration and absorbance when a homocysteine standard diluted aqueous solution is measured by the measurement method of Example 3.

Claims (5)

Homocysteine desulfurase is allowed to act on the subject to produce 2-keto acid, and then in the coexistence of oxidized or reduced nicotinamide coenzyme and a thionicotinamide coenzyme of a type different from the nicotinamide coenzyme. The nicotinamide complement consumed as the enzyme reaction proceeds by carrying out an enzyme reaction by acting a dehydrogenase that catalyzes a reaction that reversibly produces 2-hydroxybutyric acid using the 2-ketobutyric acid as a substrate. Determine the amount of homocysteine in the sample by quantifying the amount of the enzyme or thionicotinamide coenzyme, or the amount of the corresponding nicotinamide coenzyme or the corresponding thionicotinamide coenzyme produced as the enzyme reaction proceeds A method for quantitatively determining homocysteine. The method for quantifying homocysteine according to claim 1, wherein a dehydrogenase that catalyzes a reaction for reversibly generating 2-hydroxybutyric acid using 2-ketobutyric acid as a substrate is allowed to act on 2-keto acid. A method for quantifying homocysteine, comprising coexisting a dehydrogenase that reversibly oxidizes and reduces a coenzyme or a corresponding thionicotinamide coenzyme, and a substrate on which the dehydrogenase acts. The nicotinamide coenzyme is nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, and the thionicotinamide coenzyme is thionicotinamide adenine dinucleotide or thionicotinamide adenine dinucleotide phosphate. Method for quantifying homocysteine. Reversibly produce 2-hydroxybutyric acid using homocysteine desulfurase, oxidized or reduced nicotinamide dinucleotide phosphate, reduced or oxidized thionicotinamide adenine dinucleotide phosphate, and 2-ketobutyric acid as a substrate A reagent for quantifying homocysteine, comprising a dehydrogenase that catalyzes a possible reaction. Homocysteine desulfurase, oxidized or reduced nicotinamide dinucleotide phosphate, reduced or oxidized thionicotinamide adenine dinucleotide phosphate, and 2-ketobutyric acid as a substrate can reversibly produce 2-hydroxybutyric acid Dehydrogenase that catalyzes reaction, corresponding nicotinamide coenzyme or corresponding thionicotinamide coenzyme reversibly redox, corresponding nicotinamide coenzyme or corresponding thionicotinamide coenzyme reversibly redox A reagent for quantifying homocysteine, comprising a substrate on which a dehydrogenase acts.

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