JP5751109B2 - Method for measuring the concentrations of hippuric acid and methylhippuric acid in biological samples - Google Patents
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- UAQVHNZEONHPQG-ZETCQYMHSA-N N-benzoyl-L-alanine Chemical compound OC(=O)[C@H](C)NC(=O)C1=CC=CC=C1 UAQVHNZEONHPQG-ZETCQYMHSA-N 0.000 title claims description 46
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Description
本発明は、馬尿酸およびメチル馬尿酸のそれぞれの濃度を液状試薬にて測定する方法ならびにキットに関する。 The present invention relates to a method and a kit for measuring respective concentrations of hippuric acid and methylhippuric acid with a liquid reagent.
馬尿酸はトルエンの尿中代謝物として、メチル馬尿酸はキシレンの尿中代謝物として、特殊健康診断での測定項目のひとつになっている。 Hippuric acid is a urinary metabolite of toluene, and methylhippuric acid is a urinary metabolite of xylene.
しかしながら、これらの測定は高速液体クロマトグラフィー（ＨＰＬＣ）によるものであり（例えば、特許文献１および２参照）、測定が煩雑である。また、測定を簡便に行うには、液状試薬にすることが挙げられるが、馬尿酸およびメチル馬尿酸はメチル基の有無しか化学構造の差異がないため、これらを区別して測定することは困難とされている。
However, these measurements are based on high performance liquid chromatography (HPLC) (see, for example,
本発明は、より簡便に馬尿酸とメチル馬尿酸のそれぞれの濃度を測定するための方法ならびにキットを提供することを目的とする。 An object of the present invention is to provide a method and a kit for more easily measuring the respective concentrations of hippuric acid and methylhippuric acid.
本発明者らは、馬尿酸の加水分解物である安息香酸およびメチル馬尿酸の加水分解物であるメチル安息香酸がＤ−アミノ酸オキシダーゼによるアミノ酸の分解反応を阻害するが、メチル安息香酸の酸化物であるフタル酸は阻害しないことに着目し、本発明を完成させた。 The present inventors have disclosed that benzoic acid, which is a hydrolyzate of hippuric acid, and methylbenzoic acid, which is a hydrolyzate of methylhippuric acid, inhibit the degradation reaction of amino acids by D-amino acid oxidase. Focusing on the fact that phthalic acid is not hindered, the present invention was completed.
本発明は、生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を測定する方法を提供し、該方法は、
（１）生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させる工程、
（２）（１）の工程で得た反応液を酸化する工程、
（３）（２）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程、
（５）（２）〜（４）の工程とは別に、（１）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程、および
（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程
を含む。
The present invention provides a method for measuring the respective concentrations of hippuric acid and methylhippuric acid in a biological sample, the method comprising:
(1) a step of causing an enzyme that hydrolyzes hippuric acid to benzoic acid and glycine and hydrolyzes methylhippuric acid to methylbenzoic acid (toluic acid) and glycine on a biological sample collected from a living body;
(2) a step of oxidizing the reaction solution obtained in the step (1),
(3) a step of mixing the reaction solution obtained in the step (2), D-amino acid and D-amino acid oxidase to generate hydrogen peroxide,
(4) A step of specifying the concentration of the hydrogen peroxide solution generated in the step (3),
(5) Separately from the steps (2) to (4), the reaction solution obtained in the step (1), D-amino acid and D-amino acid oxidase are mixed to generate hydrogen peroxide solution,
(6) The step of specifying the concentration of the hydrogen peroxide solution generated in the step (5), the concentration of the hydrogen peroxide solution obtained in the steps (7) and (4), and the step (6) A step of calculating respective concentrations of hippuric acid and methylhippuric acid in the biological sample from the concentration of the hydrogen peroxide solution.
１つの実施態様では、前記工程（１）と前記工程（２）の反応順を逆転させてもよい。具体的には、生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を測定する方法を提供し、該方法は、
（１’）生体から採取された生体試料を酸化する工程、
（２’）（１’）の工程で得た反応液に、馬尿酸を安息香酸とグリシンに加水分解し、かつＮ−カルボキシベンゾイルグリシンをフタル酸とグリシンに加水分解する酵素を作用させる工程、
（３）（２’）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程、
（５’）（２）〜（４）の工程とは別に、生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させて得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程、および
（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程
を含む。
In one embodiment, the reaction order of the step (1) and the step (2) may be reversed. Specifically, a method for measuring the respective concentrations of hippuric acid and methylhippuric acid in a biological sample is provided.
(1 ′) a step of oxidizing a biological sample collected from a living body,
(2 ′) a step of allowing the reaction solution obtained in the step (1 ′) to act on an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine and hydrolyzes N-carboxybenzoylglycine into phthalic acid and glycine;
(3) A step of mixing the reaction solution obtained in the step (2 ′), D-amino acid and D-amino acid oxidase to generate hydrogen peroxide solution,
(4) A step of specifying the concentration of the hydrogen peroxide solution generated in the step (3),
(5 ′) Separately from the steps (2) to (4), hippuric acid is hydrolyzed into benzoic acid and glycine, and methylhippuric acid is converted to methylbenzoic acid (toluic acid). And a reaction solution obtained by acting an enzyme that hydrolyzes glycine, D-amino acid and D-amino acid oxidase are mixed to generate hydrogen peroxide solution,
(6) The step of specifying the concentration of the hydrogen peroxide solution generated in the step (5), the concentration of the hydrogen peroxide solution obtained in the steps (7) and (4), and the step (6) A step of calculating respective concentrations of hippuric acid and methylhippuric acid in the biological sample from the concentration of the hydrogen peroxide solution.
本発明は、また、馬尿酸を安息香酸とグリシンに加水分解する酵素、アミノ酸、Ｄ−アミノ酸オキシダーゼ、過酸化水素水の濃度を特定するための試薬を含む、生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を測定するキットを提供する。 The present invention also provides an enzyme for hydrolyzing hippuric acid into benzoic acid and glycine, an amino acid, a D-amino acid oxidase, a reagent for specifying the concentration of hydrogen peroxide solution, and hippuric acid and methyl horse in a biological sample. A kit for measuring each concentration of uric acid is provided.
本発明によれば、より簡便に馬尿酸とメチル馬尿酸のそれぞれの濃度を測定することができる。 According to the present invention, the respective concentrations of hippuric acid and methylhippuric acid can be measured more easily.
本発明は、馬尿酸およびメチル馬尿酸のそれぞれの濃度を液状試薬にて測定する方法ならびにキットに関する。 The present invention relates to a method and a kit for measuring respective concentrations of hippuric acid and methylhippuric acid with a liquid reagent.
本発明における「馬尿酸」とは、何も修飾されていない馬尿酸そのものをいい、別名でＮ−ベンゾイルグリシンをいう。馬尿酸は、トルエンの代謝物質である。 In the present invention, “hippuric acid” refers to hippuric acid itself which is not modified at all, and also refers to N-benzoylglycine. Hippuric acid is a metabolite of toluene.
本発明における「メチル馬尿酸」とは、馬尿酸のベンゼン環構造のｏ−位、ｍ−位、ｐ−位のいずれか１つにメチル基が修飾されたものをいい、別名でＮ−トルオイルグリシンをいう。メチル馬尿酸は、キシレンの代謝物質である。なお、「メチル馬尿酸」は、馬尿酸の不斉炭素にメチル基を有するもの（Ｎ−ベンゾイルアラニン）の別名としても用いられることがあるが、本発明における「メチル馬尿酸」としては、Ｎ−ベンゾイルアラニンは除外される。 The term “methyl hippuric acid” in the present invention refers to a methyl group modified at any one of the o-position, m-position and p-position of the benzene ring structure of hippuric acid. Oil glycine. Methyl hippuric acid is a metabolite of xylene. “Methylhippuric acid” is sometimes used as an alias for those having a methyl group on the asymmetric carbon of hippuric acid (N-benzoylalanine). As “methylhippuric acid” in the present invention, N -Benzoylalanine is excluded.
本発明の第１の態様による方法は、以下の工程を含む。
（１）生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させる工程
（２）（１）の工程で得た反応液を酸化する工程
（３）（２）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程
（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程
（５）（２）〜（４）の工程とは別に、（１）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程
（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程
（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程
The method according to the first aspect of the present invention includes the following steps.
(1) A step of causing an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine and hydrolyzes methylhippuric acid into methylbenzoic acid (toluic acid) and glycine is allowed to act on a biological sample collected from a living body (2) Step (3) for oxidizing the reaction solution obtained in step (1) (3) Step for generating hydrogen peroxide solution by mixing the reaction solution obtained in steps (2), D-amino acid and D-amino acid oxidase (4) ) (5) Separately from the steps (2) to (4), the reaction solution obtained in the step (1), D-amino acid and Peroxidation obtained in the steps (7) and (4) of mixing the D-amino acid oxidase and specifying the concentration of the hydrogen peroxide solution generated in the steps (6) and (5) of generating the hydrogen peroxide solution. Concentration of hydrogen water and the concentration of hydrogen peroxide water obtained in step (6) Step of calculating the concentrations of hippuric acid and methyl hippuric acid Luo biological sample
まず、本発明は（１）生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させる工程を実施する。 First, the present invention (1) acts on a biological sample collected from a living body with an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine and hydrolyzes methylhippuric acid into methylbenzoic acid (toluic acid) and glycine. The process to make is performed.
生体としては、本発明が有機溶媒を取り扱うヒトの特殊健康診断に主に用いられる観点から、主にヒトであるが、必ずしもこれに限定されるものではなく、例えば、イヌ、ネコ、ブタおよびウシ等の動物も含まれる。 The living body is mainly a human from the viewpoint that the present invention is mainly used for a special health diagnosis of a human who handles an organic solvent, but is not necessarily limited to this. For example, dogs, cats, pigs and cattle Etc. are also included.
生体試料としては、本発明が有機溶媒を取り扱うヒトの特殊健康診断に主に用いられる観点から、主に尿であるが、必ずしもこれに限定されるものではなく、例えば、血液、唾液、骨髄液および細胞間質液等も含まれる。 The biological sample is mainly urine from the viewpoint that the present invention is mainly used for special health diagnosis of humans handling organic solvents, but is not necessarily limited to this, for example, blood, saliva, bone marrow fluid And cell interstitial fluid.
「馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素」としては、例えば、アミノアシラーゼ、および馬尿酸加水分解酵素等が挙げられる。特に安価に入手できる観点から、馬尿酸を安息香酸とグリシンに加水分解する酵素としては、アミノアシラーゼが好ましい。生体試料に当該酵素を作用させることにより、生体試料中の馬尿酸は安息香酸とグリシンに、生体試料中のメチル馬尿酸はメチル安息香酸（トルイル酸）とグリシンに、それぞれ加水分解される。 Examples of “enzymes that hydrolyze hippuric acid into benzoic acid and glycine and hydrolyze methylhippuric acid into methylbenzoic acid (toluic acid) and glycine” include aminoacylase and hippuric acid hydrolase, etc. It is done. In particular, aminoacylase is preferable as an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine from the viewpoint of availability at low cost. By causing the enzyme to act on the biological sample, hippuric acid in the biological sample is hydrolyzed to benzoic acid and glycine, and methylhippuric acid in the biological sample is hydrolyzed to methylbenzoic acid (toluic acid) and glycine, respectively.
アミノアシラーゼ（ＥＣ番号：ＥＣ３．５．１．１４）は、別名として、アシラーゼ、Ｎ−アシル−Ｌ−アミノ酸アミドヒドロラーゼ、Ｎ−アシルアミノ酸アミドヒドロラーゼ、Ｌ−アミノ酸アシラーゼ、アミノ酸デアシラーゼ、またはベンズアミダーゼとも呼ばれている。 Aminoacylase (EC number: EC 3.5.1.14) is also known as acylase, N-acyl-L-amino acid amide hydrolase, N-acyl amino acid amide hydrolase, L-amino acid acylase, amino acid deacylase, or benzamidase. being called.
なお、本発明における「安息香酸」とは、馬尿酸の分解物であり、何も修飾されていない安息香酸そのものをいう。 The “benzoic acid” in the present invention is a decomposition product of hippuric acid and refers to benzoic acid itself that is not modified at all.
また、本発明における「メチル安息香酸」とは、メチル馬尿酸の分解物であり、安息香酸のベンゼン環構造のｏ−位、ｍ−位、ｐ−位のいずれか１つにメチル基が修飾されたものをいう。 The “methylbenzoic acid” in the present invention is a decomposition product of methylhippuric acid, and a methyl group is modified at any one of the o-position, m-position and p-position of the benzene ring structure of benzoic acid. It means what was done.
次いで、（２）（１）の工程で得た反応液を酸化する工程を実施する。つまり、メチル安息香酸を酸化させ、フタル酸にする。一方、安息香酸は酸化されない。 Next, the step (2) of oxidizing the reaction solution obtained in the step (1) is performed. That is, methylbenzoic acid is oxidized to phthalic acid. On the other hand, benzoic acid is not oxidized.
酸化に用いる酸化剤としては、例えば、過マンガン酸カリウム、二クロム酸カリウム塩化クロミル、過硫酸カリウムが挙げられる。特に安価に入手でき、温和な条件で反応を進行できる観点から、酸化剤としては、過硫酸カリウムが好ましい。 Examples of the oxidizing agent used for oxidation include potassium permanganate, potassium dichromate chromyl chloride, and potassium persulfate. In particular, potassium persulfate is preferable as the oxidizing agent from the viewpoint of being available at low cost and allowing the reaction to proceed under mild conditions.
次いで、（３）（２）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程を実施する。 Next, the step of (3) mixing the reaction solution obtained in the step (2), D-amino acid and D-amino acid oxidase to generate a hydrogen peroxide solution is performed.
ここで、Ｄ−アミノ酸オキシダーゼとは、補因子としてフラビンアデニンジヌクレオチド（ＦＡＤ）を含むペルオキシソーム酵素の一つであり、Ｄ−アミノ酸を相当するイミノ酸に変換し、同時にアンモニアと過酸化水素を合成する酵素をいう。 Here, D-amino acid oxidase is a peroxisomal enzyme containing flavin adenine dinucleotide (FAD) as a cofactor, which converts D-amino acid into the corresponding imino acid and simultaneously synthesizes ammonia and hydrogen peroxide. An enzyme that does.
また、Ｄ−アミノ酸としては、例えば、アラニン、ロイシン、イソロイシン、リジン、バリン、グルタミン酸、プロリンおよびチロシン等のα−アミノ酸だけでなく、β−アミノ酸およびγ−アミノ酸等も挙げられ、特に限定されるものではない。また、Ｄ−アミノ酸自体が高価であるため、本発明におけるＤ−アミノ酸としては、Ｄ−アミノ酸のみの純粋な物質のみならず、当該Ｄ−アミノ酸も含むラセミ体も含まれる。これらのことをふまえ、安価に入手できる観点から、Ｄ−アミノ酸としては、アラニン（ラセミ体）が好ましい。 Examples of D-amino acids include not only α-amino acids such as alanine, leucine, isoleucine, lysine, valine, glutamic acid, proline and tyrosine, but also β-amino acids and γ-amino acids, and the like. It is not a thing. Moreover, since D-amino acid itself is expensive, the D-amino acid in the present invention includes not only a pure substance containing only D-amino acid but also a racemate containing the D-amino acid. Based on these facts, alanine (racemic) is preferred as the D-amino acid from the viewpoint of availability at low cost.
例えば、Ｄ−アミノ酸としてアラニン（ラセミ体）を用いた場合、本工程によりピルビン酸、アンモニア、そして過酸化水素水が発生する反応が実施される。 For example, when alanine (racemate) is used as the D-amino acid, a reaction in which pyruvic acid, ammonia, and aqueous hydrogen peroxide are generated is performed in this step.
工程（３）の反応時、（２）の工程で得た反応液に安息香酸が含まれる場合、当該反応は当該安息香酸によって阻害される。ただし、（２）の工程で得た反応液には、メチル安息香酸は存在せず（フタル酸に変化している）、当該メチル安息香酸が当該反応を阻害することはない。 When benzoic acid is contained in the reaction solution obtained in the step (2) during the reaction in the step (3), the reaction is inhibited by the benzoic acid. However, in the reaction solution obtained in the step (2), methylbenzoic acid does not exist (changes to phthalic acid), and the methylbenzoic acid does not inhibit the reaction.
表１は、安息香酸、メチル安息香酸、フタル酸それぞれにおいて、Ｄ−アミノ酸オキシダーゼの酵素活性を５０％阻害させるために必要な量を示している。それぞれのメチル安息香酸のＤ−アミノ酸オキシダーゼの酵素活性を５０％阻害させるために必要な量は、その酸化物であるフタル酸になることにより、多くなることがわかる。 Table 1 shows the amounts necessary to inhibit the enzyme activity of D-amino acid oxidase by 50% in each of benzoic acid, methylbenzoic acid, and phthalic acid. It can be seen that the amount necessary to inhibit the enzyme activity of the D-amino acid oxidase of each methylbenzoic acid by 50% increases with the phthalic acid that is the oxide.
そして、（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程を実施する。過酸化水素水の濃度を特定する方法としては公知の液状試薬を用いた方法を採用することができる。例えば、４−アミノアンチピリン（４−ＡＡ）および／またはＮ−エチル−Ｎ−（３−スルホプロピル）−ｍ−アニシジン（ＡＤＰＳ）を基質とするペルオキシダーゼを用いた酵素発色法および過酸化水素による鉄イオンの酸化に伴う色素の呈色反応を利用する方法等が挙げられる。 And the process of specifying the density | concentration of the hydrogen-peroxide solution generated at the process of (4) and (3) is implemented. As a method for specifying the concentration of the hydrogen peroxide solution, a method using a known liquid reagent can be employed. For example, an enzyme coloring method using peroxidase with 4-aminoantipyrine (4-AA) and / or N-ethyl-N- (3-sulfopropyl) -m-anisidine (ADPS) as a substrate and iron with hydrogen peroxide Examples thereof include a method utilizing a coloring reaction of a dye accompanying oxidation of ions.
本発明は、（５）（２）〜（４）の工程とは別に、（１）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程を実施する。つまり、安息香酸だけでなくメチル安息香酸もＤ−アミノ酸オキシダーゼによるＤ−アミノ酸の反応も阻害することになる。 In the present invention, apart from the steps (5) (2) to (4), the reaction solution obtained in the step (1), D-amino acid and D-amino acid oxidase are mixed to generate hydrogen peroxide. Perform the process. That is, not only benzoic acid but also methyl benzoic acid inhibits D-amino acid reaction by D-amino acid oxidase.
次いで、（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程を実施する。本工程は、（４）の工程と相違するものではない。いうまでもないが、（４）に用いる試薬と同じ試薬を採用することが好ましい。 Next, a step of specifying the concentration of the hydrogen peroxide solution generated in the steps (6) and (5) is performed. This step is not different from the step (4). Needless to say, it is preferable to employ the same reagent as that used in (4).
最後に、（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程を実施する。（４）の工程で得た過酸化水素水の濃度は、馬尿酸の濃度に関連し、一方、（６）の工程で得た過酸化水素水の濃度は、馬尿酸およびメチル馬尿酸の総濃度に関連する。したがって、馬尿酸およびメチル馬尿酸の総濃度から馬尿酸の濃度を差し引くことにより、メチル馬尿酸の濃度も算定することできる。 Finally, from the concentration of the hydrogen peroxide solution obtained in the steps (7) and (4) and the concentration of the hydrogen peroxide solution obtained in the step (6), each of hippuric acid and methyl hippuric acid in the biological sample is obtained. A step of calculating the concentration is performed. The concentration of the hydrogen peroxide solution obtained in the step (4) is related to the concentration of hippuric acid, while the concentration of the hydrogen peroxide solution obtained in the step (6) is the total of hippuric acid and methyl hippuric acid. Related to concentration. Therefore, the concentration of methyl hippuric acid can also be calculated by subtracting the concentration of hippuric acid from the total concentration of hippuric acid and methyl hippuric acid.
図１は、上述した本発明の工程（１）〜（７）における反応系を示す図である。 FIG. 1 is a diagram showing a reaction system in the above-described steps (1) to (7) of the present invention.
本発明の第２の態様による方法は、前記工程（１）と前記工程（２）の反応順を逆転させた発想に基づく。具体的には、以下の工程を含む、生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を測定する方法を提供する。
（１’）生体から採取された生体試料を酸化する工程
（２’）（１’）の工程で得た反応液に、馬尿酸を安息香酸とグリシンに加水分解し、かつＮ−カルボキシベンゾイルグリシンをフタル酸とグリシンに加水分解する酵素を作用させる工程
（３）（２’）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程
（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程
（５’）（２）〜（４）の工程とは別に、生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させて得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程
（６）（５’）の工程で発生した過酸化水素水の濃度を特定する工程
（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程
The method according to the second aspect of the present invention is based on the idea of reversing the reaction order of the step (1) and the step (2). Specifically, a method for measuring the respective concentrations of hippuric acid and methylhippuric acid in a biological sample, including the following steps, is provided.
(1 ′) Step of oxidizing a biological sample collected from a living body (2 ′) Hydrolyzing hippuric acid into benzoic acid and glycine into the reaction solution obtained in the steps of (1 ′) and N-carboxybenzoylglycine A step (3) in which an enzyme that hydrolyzes phthalic acid and glycine is allowed to act (3) and a step (2 ′) in which the reaction solution, D-amino acid and D-amino acid oxidase are mixed to generate hydrogen peroxide ( 4) Separately from the steps (5 ′) and (2) to (4) for determining the concentration of the hydrogen peroxide solution generated in the step (3), benzoic acid is added to hippuric acid to the biological sample collected from the living body. A reaction solution obtained by the action of an enzyme that hydrolyzes acid to glycine and hydrolyzes methylhippuric acid to methylbenzoic acid (toluic acid) and glycine, D-amino acid and D-amino acid oxidase are mixed and peroxidized Generate hydrogen water Steps (6), (7) Identifying the concentration of the hydrogen peroxide solution generated in the step (5 ′), (7) The concentration of the hydrogen peroxide solution obtained in the step (4), and the step (6) A step of calculating each concentration of hippuric acid and methylhippuric acid in a biological sample from the concentration of hydrogen peroxide water
まず、（１’）生体から採取された生体試料を酸化する工程を実施する。つまり、メチル馬尿酸を酸化させ、Ｎ−カルボキシベンゾイルグリシンにする。一方、馬尿酸は酸化されない。 First, (1 ′) a step of oxidizing a biological sample collected from a living body is performed. That is, methyl hippuric acid is oxidized to N-carboxybenzoylglycine. On the other hand, hippuric acid is not oxidized.
次いで、（２’）（１’）の工程で得た反応液に、馬尿酸を安息香酸とグリシンに加水分解し、かつＮ−カルボキシベンゾイルグリシンをフタル酸とグリシンに加水分解する酵素を作用させる工程を実施する。なお、「馬尿酸を安息香酸とグリシンに加水分解し、かつＮ−カルボキシベンゾイルグリシンをフタル酸とグリシンに加水分解する酵素」とは、上述の「馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素」と同じ酵素をいう。 Next, an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine and hydrolyzes N-carboxybenzoylglycine into phthalic acid and glycine is allowed to act on the reaction solution obtained in steps (2 ′) and (1 ′). Perform the process. In addition, “an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine and hydrolyzes N-carboxybenzoylglycine into phthalic acid and glycine” means “hydrolyzing hippuric acid into benzoic acid and glycine, And the same enzyme as “an enzyme that hydrolyzes methylhippuric acid into methylbenzoic acid (toluic acid) and glycine”.
次いで、（３）（２’）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程を実施する。用いるＤ−アミノ酸およびＤ−アミノ酸オキシダーゼは上述の通りである。 Next, the step (3) of mixing the reaction solution obtained in the step (2 '), D-amino acid and D-amino acid oxidase to generate a hydrogen peroxide solution is performed. The D-amino acid and D-amino acid oxidase used are as described above.
そして、（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程を実施する。過酸化水素水の濃度の特定については上述の通りである。 And the process of specifying the density | concentration of the hydrogen-peroxide solution generated at the process of (4) and (3) is implemented. The specification of the concentration of the hydrogen peroxide solution is as described above.
本発明は、（５’）（２）〜（４）の工程とは別に、生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させて得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程を実施する。ここで注意しなければならないのは、（１’）の工程を実施するのではなく、生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させる工程、つまり、第１の態様における（１）の工程と同じ工程を実施することに注意する。また、用いるＤ−アミノ酸およびＤ−アミノ酸オキシダーゼは上述の通りである。 In the present invention, apart from the steps (5 ′) (2) to (4), hippuric acid is hydrolyzed into benzoic acid and glycine on a biological sample collected from a living body, and methylhippuric acid is converted to methylbenzoic acid. (Toluic acid) and a reaction solution obtained by acting an enzyme that hydrolyzes glycine, D-amino acid and D-amino acid oxidase are mixed to carry out a step of generating hydrogen peroxide. It should be noted here that the step (1 ′) is not performed, but hippuric acid is hydrolyzed into benzoic acid and glycine, and methylhippuric acid is converted into methyl. Note that the step of allowing an enzyme that hydrolyzes benzoic acid (toluic acid) and glycine to act, that is, the same step as the step (1) in the first embodiment is performed. The D-amino acid and D-amino acid oxidase used are as described above.
次いで、（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程を実施し、最後に、（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程を実施する。これらの工程は上述の通りである。 Next, a step of specifying the concentration of the hydrogen peroxide solution generated in the steps (6) and (5) is performed. Finally, the concentration of the hydrogen peroxide solution obtained in the steps of (7) and (4), and ( A step of calculating respective concentrations of hippuric acid and methylhippuric acid in the biological sample from the concentration of the hydrogen peroxide solution obtained in the step 6) is performed. These steps are as described above.
図２は、上述した本発明の工程（１’）〜（７）における反応系を示す図である。 FIG. 2 is a diagram showing a reaction system in the above-described steps (1 ′) to (7) of the present invention.
また、本発明は、これらの方法を実施するためのキットを含む。キットは、馬尿酸を安息香酸とグリシンに加水分解する酵素、アミノ酸、Ｄ−アミノ酸オキシダーゼ、過酸化水素水の濃度を特定するための試薬を含む。 The present invention also includes kits for performing these methods. The kit includes an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine, an amino acid, D-amino acid oxidase, and a reagent for specifying the concentration of hydrogen peroxide.
以下、実施例により本発明をより具体的に説明するが、本発明はこれらの実施例により限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited by these Examples.
（Ａ）ヒトから採取した０．２ｍＬの血液に、アミノアシラーゼ（５０００ｋＵ／Ｌ）水溶液を０．８ｍＬ添加し、５分間反応させる。次いで、得られた反応液に、過硫酸カリウム［酸化剤］（２０ｍＭ）水溶液を０．２ｍＬ添加し、５分間反応させる。さらに、得られた反応液に、アラニン（ラセミ体）（１５ｍＭ）およびＤ−アミノ酸オキシダーゼ（２０ｋＵ／Ｌ）の水溶液を０．４ｍＬ添加し、５分間反応させる。これにより発生した過酸化水素水の濃度について、４−アミノアンチピリン（４−ＡＡ）およびＮ−エチル−Ｎ−（３−スルホプロピル）−ｍ−アニシジン（ＡＤＰＳ）を基質とするペルオキシダーゼを用いた酵素発色法を用いて特定する。 (A) 0.8 mL of an aminoacylase (5000 kU / L) aqueous solution is added to 0.2 mL of blood collected from a human and allowed to react for 5 minutes. Subsequently, 0.2 mL of potassium persulfate [oxidant] (20 mM) aqueous solution is added to the obtained reaction liquid, and it is made to react for 5 minutes. Furthermore, 0.4 mL of an aqueous solution of alanine (racemate) (15 mM) and D-amino acid oxidase (20 kU / L) is added to the obtained reaction solution and reacted for 5 minutes. An enzyme using peroxidase having 4-aminoantipyrine (4-AA) and N-ethyl-N- (3-sulfopropyl) -m-anisidine (ADPS) as substrates for the concentration of the hydrogen peroxide solution generated thereby. Identifies using color development.
（Ｂ）上記（Ａ）とは別にヒトから採取した０．２ｍＬの血液に、アミノアシラーゼ（５０００ｋＵ／Ｌ）の水溶液を０．８ｍＬ添加し、５分間反応させた反応液に、アラニン（ラセミ体）（１５ｍＭ）およびＤ−アミノ酸オキシダーゼ（２０ｋＵ／Ｌ）の水溶液を添加し、５分間反応させる。これにより発生した過酸化水素水の濃度について、４−アミノアンチピリン（４−ＡＡ）およびＮ−エチル−Ｎ−（３−スルホプロピル）−ｍ−アニシジン（ＡＤＰＳ）を基質とするペルオキシダーゼを用いた酵素発色法を用いて特定する。 (B) Aside from the above (A), 0.8 mL of an aminoacylase (5000 kU / L) aqueous solution was added to 0.2 mL of blood collected from a human, and the reaction solution was allowed to react for 5 minutes. ) (15 mM) and an aqueous solution of D-amino acid oxidase (20 kU / L) are added and allowed to react for 5 minutes. An enzyme using peroxidase having 4-aminoantipyrine (4-AA) and N-ethyl-N- (3-sulfopropyl) -m-anisidine (ADPS) as substrates for the concentration of the hydrogen peroxide solution generated thereby. Identifies using color development.
上記（Ａ）および（Ｂ）より求まる過酸化水素水の濃度から、馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する。 The respective concentrations of hippuric acid and methylhippuric acid are calculated from the concentration of the hydrogen peroxide solution obtained from the above (A) and (B).
本発明は、特殊健康診断の分野において利用され得る。 The present invention can be used in the field of special health examinations.
Claims (3)
（１）生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させる工程、
（２）（１）の工程で得た反応液を酸化する工程、
（３）（２）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程、
（５）（２）〜（４）の工程とは別に、（１）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程、および
（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程
を含む、方法。 A method for measuring the respective concentrations of hippuric acid and methylhippuric acid in a biological sample,
(1) a step of causing an enzyme that hydrolyzes hippuric acid to benzoic acid and glycine and hydrolyzes methylhippuric acid to methylbenzoic acid (toluic acid) and glycine on a biological sample collected from a living body;
(2) a step of oxidizing the reaction solution obtained in the step (1),
(3) a step of mixing the reaction solution obtained in the step (2), D-amino acid and D-amino acid oxidase to generate hydrogen peroxide,
(4) A step of specifying the concentration of the hydrogen peroxide solution generated in the step (3),
(5) Separately from the steps (2) to (4), the reaction solution obtained in the step (1), D-amino acid and D-amino acid oxidase are mixed to generate hydrogen peroxide solution,
(6) The step of specifying the concentration of the hydrogen peroxide solution generated in the step (5), the concentration of the hydrogen peroxide solution obtained in the steps (7) and (4), and the step (6) A method comprising a step of calculating respective concentrations of hippuric acid and methylhippuric acid in a biological sample from the concentration of hydrogen peroxide water.
（１’）生体から採取された生体試料を酸化する工程、
（２’）（１’）の工程で得た反応液に、馬尿酸を安息香酸とグリシンに加水分解し、かつＮ−カルボキシベンゾイルグリシンをフタル酸とグリシンに加水分解する酵素を作用させる工程、
（３）（２’）の工程で得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（４）（３）の工程で発生した過酸化水素水の濃度を特定する工程、
（５’）（２）〜（４）の工程とは別に、生体から採取された生体試料に、馬尿酸を安息香酸とグリシンに加水分解し、かつメチル馬尿酸をメチル安息香酸（トルイル酸）とグリシンに加水分解する酵素を作用させて得た反応液、Ｄ−アミノ酸およびＤ−アミノ酸オキシダーゼを混合し、過酸化水素水を発生させる工程、
（６）（５）の工程で発生した過酸化水素水の濃度を特定する工程、および
（７）（４）の工程で得た過酸化水素水の濃度と、（６）の工程で得た過酸化水素水の濃度から生体試料中の馬尿酸およびメチル馬尿酸のそれぞれの濃度を算出する工程
を含む、方法。 A method for measuring the respective concentrations of hippuric acid and methylhippuric acid in a biological sample,
(1 ′) a step of oxidizing a biological sample collected from a living body,
(2 ′) a step of allowing the reaction solution obtained in the step (1 ′) to act on an enzyme that hydrolyzes hippuric acid into benzoic acid and glycine and hydrolyzes N-carboxybenzoylglycine into phthalic acid and glycine;
(3) A step of mixing the reaction solution obtained in the step (2 ′), D-amino acid and D-amino acid oxidase to generate hydrogen peroxide solution,
(4) A step of specifying the concentration of the hydrogen peroxide solution generated in the step (3),
(5 ′) Separately from the steps (2) to (4), hippuric acid is hydrolyzed into benzoic acid and glycine, and methylhippuric acid is converted to methylbenzoic acid (toluic acid). And a reaction solution obtained by acting an enzyme that hydrolyzes glycine, D-amino acid and D-amino acid oxidase are mixed to generate hydrogen peroxide solution,
(6) The step of specifying the concentration of the hydrogen peroxide solution generated in the step (5), the concentration of the hydrogen peroxide solution obtained in the steps (7) and (4), and the step (6) A method comprising a step of calculating respective concentrations of hippuric acid and methylhippuric acid in a biological sample from the concentration of hydrogen peroxide water.
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