JP7650205B2 - Method for quantitatively determining coenzyme Q in blood - Google Patents
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本明細書は、被験体の血中の補酵素Ｑを定量する方法、被験体における血中の補酵素Ｑの量が不足しているか否かを評価するための指標を取得する方法、被験体が摂取した補酵素Ｑの生体内移行を検査するための指標を取得する方法、並びに、被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標を取得する方法を開示する。 This specification discloses a method for quantifying coenzyme Q in the blood of a subject, a method for obtaining an indicator for evaluating whether the amount of coenzyme Q in the blood of a subject is insufficient, a method for obtaining an indicator for examining the transfer of coenzyme Q ingested by a subject to the body, and a method for obtaining an indicator for examining the effect of a test substance on the synthesis or metabolism of coenzyme Q in the body of a subject.
補酵素Ｑは、細菌から哺乳動物まで広く生体に分布する必須成分であり、生体内の細胞中におけるミトコンドリアの電子伝達系構成成分として知られている。補酵素Ｑは、ミトコンドリア内で酸化と還元を繰り返すことで、電子伝達系における伝達成分としての機能を担っているほか、還元型補酵素Ｑは抗酸化作用を持つことが知られている。ヒトでは、補酵素Ｑの側鎖が繰り返し構造を１０個持つ補酵素Ｑ１０が主成分であり、生体内においては、通常、４０～９０％程度が還元型として存在している。 Coenzyme Q is an essential component that is widely distributed in living organisms, from bacteria to mammals, and is known as a component of the mitochondrial electron transport chain in living cells. Coenzyme Q functions as a transport component in the electron transport chain by repeatedly undergoing oxidation and reduction in mitochondria, and reduced coenzyme Q is known to have antioxidant properties. In humans, the main component is coenzyme Q10, a coenzyme Q side chain with 10 repeating structures, and in the body, approximately 40-90% usually exists in the reduced form.
補酵素Ｑの生理的作用としては、ミトコンドリア賦活作用によるエネルギー生産の活性化、心機能の活性化、細胞膜の安定化効果、抗酸化作用による細胞の保護効果等が挙げられている。 Physiological effects of coenzyme Q include mitochondrial activation, which stimulates energy production; cardiac activation; stabilizing cell membranes; and protecting cells through its antioxidant properties.
生体内での補酵素Ｑによる生理的作用の発現の程度は、補酵素Ｑの血中濃度と相関する。このため、被験体の生体内において、所望の生理的作用を発現できる量の補酵素Ｑが存在するのかを判断するためには、被験体の血中の補酵素Ｑを定量することが通常である。しかし、血中の補酵素Ｑの定量は、医療機関において専門の技術者（医師や看護師）が血液検体を採取することを必要とするため、簡便に実施することができない。 The degree to which the physiological action of coenzyme Q is exerted in the body correlates with the blood concentration of coenzyme Q. For this reason, in order to determine whether there is an amount of coenzyme Q present in the body of a subject that is sufficient to exert the desired physiological action, it is common to quantify the amount of coenzyme Q in the subject's blood. However, quantifying the amount of coenzyme Q in blood requires specialized technicians (doctors or nurses) to collect blood samples at a medical institution, and therefore cannot be easily performed.
特許文献１では、健康補助食品、薬剤または食物（健康補助食品等）の摂取における、健康補助食品等に含まれる栄養成分である脂溶性ビタミン及び／又は脂溶性フードファクターの生体内移行を検査する簡易な手段として、唾液中の脂溶性ビタミン及び／又は脂溶性フードファクターを分析することを特徴とする、生体内の脂溶性ビタミン及び／又は脂溶性フードファクターを検査する方法が記載されている。特許文献１では、脂溶性ビタミン及び／又は脂溶性フードファクターの具体例として補酵素Ｑ１０が記載されている。 Patent Document 1 describes a method for testing fat-soluble vitamins and/or fat-soluble food factors in the body, characterized by analyzing fat-soluble vitamins and/or fat-soluble food factors in saliva, as a simple means for testing the transfer of fat-soluble vitamins and/or fat-soluble food factors, which are nutritional components contained in dietary supplements, drugs, or foods (dietary supplements, etc.), into the body when the dietary supplements, drugs, or foods are taken. Patent Document 1 describes coenzyme Q10 as a specific example of fat-soluble vitamins and/or fat-soluble food factors.
特許文献１に記載の方法は、分析試料として唾液の採取を利用する。しかし唾液の分泌量は個人差が大きい。特に、ドライマウスと呼ばれる唾液分泌量が低下する症状を有する被験者から唾液を採取することは容易ではない。また、一般に高齢になるほど唾液の分泌量が低下する傾向がある。このため、分析試料として唾液を利用する方法は、被験体の状態や年齢によって、分析に必要な量の唾液を採取することが容易でない場合があるという課題があった。 The method described in Patent Document 1 utilizes the collection of saliva as an analytical sample. However, the amount of saliva secreted varies greatly from person to person. In particular, it is not easy to collect saliva from subjects who have a condition known as dry mouth, which reduces the amount of saliva secreted. Furthermore, there is a general tendency for the amount of saliva secreted to decrease with age. For this reason, the method of using saliva as an analytical sample has the problem that it may not be easy to collect the amount of saliva required for analysis, depending on the condition and age of the subject.
また本発明者らは、比較例において、全唾液中の補酵素Ｑ濃度は、血中の補酵素Ｑ濃度と相関せず、血中の補酵素Ｑ濃度の指標とはできないことを確認した。 The inventors also confirmed in a comparative example that the coenzyme Q concentration in whole saliva does not correlate with the coenzyme Q concentration in blood and cannot be used as an indicator of the coenzyme Q concentration in blood.
そこで本明細書は、被験体からの採取が容易な試料を用いて、被験体の血中の補酵素Ｑを定量する方法、被験体における血中の補酵素Ｑの量が不足しているか否かを評価するための指標を取得する方法、被験体が摂取した補酵素Ｑの生体内移行を検査するための指標を取得する方法、並びに、被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標を取得する方法を開示する。 This specification discloses a method for quantifying coenzyme Q in the blood of a subject using a sample that is easily collected from the subject, a method for obtaining an indicator for evaluating whether the amount of coenzyme Q in the blood of a subject is insufficient, a method for obtaining an indicator for examining the transfer of coenzyme Q ingested by a subject in the body, and a method for obtaining an indicator for examining the effect of a test substance on the synthesis or metabolism of coenzyme Q in the body of a subject.
本発明者らは、被験体に由来する角質層試料等の皮膚試料中の補酵素Ｑの量が、被験体の血中の補酵素Ｑの量と正に相関するという予想外の知見に基づき、以下の、本発明の一以上の実施形態を完成させた。
［１］被験体の血中の補酵素Ｑを定量する方法であって、
被験体に由来する皮膚試料中の補酵素Ｑを定量することを含む方法。
［２］皮膚試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定することを更に含む、［１］に記載の方法。
［３］皮膚試料が、角質層試料である、［１］又は［２］に記載の方法。
［４］角質層試料が、被験体の皮膚の表面に粘着性シートを貼付したのち剥離して粘着性シート上に採取された角質層試料である、［３］に記載の方法。
［５］補酵素Ｑが補酵素Ｑ１０である、［１］～［４］のいずれか記載の方法。
［６］被験体の血中の補酵素Ｑを定量する方法であって、
被験体の皮膚の表面に粘着性シートを貼付したのち剥離して、被験体の角質層試料を粘着性シート上に採取すること、
採取された角質層試料中の補酵素Ｑを定量すること、及び、
角質層試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること
を含む方法。
［７］被験体における血中の補酵素Ｑの量が不足しているか否かを評価するための指標を取得する方法であって、
被験体に由来する皮膚試料中の補酵素Ｑを定量すること、
皮膚試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること、及び、
被験体の血中の補酵素Ｑの量と、補酵素Ｑの推奨血中量とを比較すること
を含む方法。
［８］被験体が摂取した補酵素Ｑの生体内移行を検査するための指標を取得する方法であって、
補酵素Ｑを摂取した被験体に由来する皮膚試料中の補酵素Ｑを定量することを含む方法。
［９］皮膚試料が、角質層試料である、［８］に記載の方法。
［１０］角質層試料が、被験体の皮膚の表面に粘着性シートを貼付したのち剥離して粘着性シート上に採取された角質層試料である、［９］に記載の方法。
［１１］補酵素Ｑが補酵素Ｑ１０である、［８］～［１０］のいずれかに記載の方法。
［１２］被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標を取得する方法であって、
被験物質を摂取した被験体に由来する皮膚試料中の補酵素Ｑを定量することを含む方法。
［１３］皮膚試料が、角質層試料である、［１２］に記載の方法。
［１４］角質層試料が、被験体の皮膚の表面に粘着性シートを貼付したのち剥離して粘着性シート上に採取された角質層試料である、［１３］に記載の方法。
［１５］補酵素Ｑが補酵素Ｑ１０である、［１２］～［１４］のいずれかに記載の方法。
Based on the unexpected finding that the amount of Coenzyme Q in a skin sample, such as a stratum corneum sample, derived from a subject positively correlates with the amount of Coenzyme Q in the subject's blood, the inventors have completed one or more embodiments of the present invention, which are described below.
[1] A method for quantifying coenzyme Q in the blood of a subject, comprising the steps of:
A method comprising quantifying coenzyme Q in a skin sample from a subject.
[2] The method according to [1], further comprising determining the amount of coenzyme Q in the subject's blood based on the amount of coenzyme Q in the skin sample.
[3] The method according to [1] or [2], wherein the skin sample is a stratum corneum sample.
[4] The method according to [3], wherein the stratum corneum sample is a stratum corneum sample obtained by attaching an adhesive sheet to the surface of the skin of a subject and then peeling it off to collect the stratum corneum sample on the adhesive sheet.
[5] The method according to any one of [1] to [4], wherein the coenzyme Q is coenzyme Q10.
[6] A method for quantifying coenzyme Q in the blood of a subject, comprising the steps of:
affixing an adhesive sheet to the surface of the subject's skin and then peeling it off to collect a sample of the subject's stratum corneum on the adhesive sheet;
Quantifying the amount of coenzyme Q in the collected stratum corneum sample; and
determining the amount of Coenzyme Q in the subject's blood based on the amount of Coenzyme Q in the stratum corneum sample.
[7] A method for obtaining an indicator for evaluating whether or not a subject has a deficiency in the amount of coenzyme Q in the blood, comprising:
Quantifying coenzyme Q in a skin sample from a subject;
Determining the amount of coenzyme Q in the subject's blood based on the amount of coenzyme Q in the skin sample; and
A method comprising comparing the amount of coenzyme Q in the subject's blood with a recommended blood amount of coenzyme Q.
[8] A method for obtaining an indicator for examining the transfer of coenzyme Q into the body of a subject, comprising the steps of:
A method comprising quantifying coenzyme Q in a skin sample derived from a subject who has ingested coenzyme Q.
[9] The method according to [8], wherein the skin sample is a stratum corneum sample.
[10] The method according to [9], wherein the stratum corneum sample is a stratum corneum sample obtained by attaching an adhesive sheet to the surface of the skin of a subject and then peeling it off to collect the stratum corneum sample on the adhesive sheet.
[11] The method according to any one of [8] to [10], wherein the coenzyme Q is coenzyme Q10.
[12] A method for obtaining an indicator for examining an effect of a test substance on the synthesis or metabolism of coenzyme Q in a living body of a subject, comprising:
A method comprising quantifying coenzyme Q in a skin sample derived from a subject who has ingested a test substance.
[13] The method according to [12], wherein the skin sample is a stratum corneum sample.
[14] The method according to [13], wherein the stratum corneum sample is a stratum corneum sample obtained by attaching an adhesive sheet to the surface of the skin of a subject and then peeling it off, and collecting the stratum corneum sample on the adhesive sheet.
[15] The method according to any one of [12] to [14], wherein the coenzyme Q is coenzyme Q10.
本発明の一以上の実施形態によれば、被験体からの安定した採取が可能な皮膚試料を用いて、被験体の血中の補酵素Ｑの定量、被験体における血中の補酵素Ｑの量が不足しているか否かを評価するための指標の取得、被験体が摂取した補酵素Ｑの生体内移行を検査するための指標の取得、並びに、被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標の取得を行うことができる。 According to one or more embodiments of the present invention, a skin sample that can be reliably collected from a subject can be used to quantify the amount of coenzyme Q in the subject's blood, obtain an indicator for evaluating whether the amount of coenzyme Q in the subject's blood is insufficient, obtain an indicator for testing the transfer of coenzyme Q ingested by the subject in the body, and obtain an indicator for testing the effect of a test substance on the synthesis or metabolism of coenzyme Q in the subject's body.
１．本発明の第１の実施形態
本発明の第１の実施形態は、
被験体の血中の補酵素Ｑを定量する方法であって、
被験体に由来する皮膚試料中の補酵素Ｑを定量することを含む方法
に関する。
1. First embodiment of the present invention The first embodiment of the present invention is
A method for quantifying coenzyme Q in blood of a subject, comprising the steps of:
The present invention relates to a method comprising quantifying coenzyme Q in a skin sample from a subject.
本発明の第１の実施形態に係る方法は血液の採取の必要がない。一般家庭等の分析設備の無い環境で採取した皮膚試料は、分析設備を備えた機関に送付し、当該機関において皮膚試料中の補酵素Ｑを定量することができる。皮膚試料は、被験体の年齢や状態によらずに安定して採取することが可能である。 The method according to the first embodiment of the present invention does not require the collection of blood. Skin samples collected in an environment without analytical equipment, such as an ordinary home, can be sent to an institution with analytical equipment, where the amount of coenzyme Q in the skin sample can be quantified. Skin samples can be collected reliably regardless of the age or condition of the subject.
被験体（Ｓｕｂｊｅｃｔ）とは、検査の対象となるヒト又は非ヒト動物を指し、好ましくはヒトを指す。非ヒト動物としては、哺乳動物が好ましい。 Subject refers to a human or non-human animal that is the subject of a test, and preferably refers to a human. As a non-human animal, a mammal is preferred.
補酵素Ｑは、好ましくは補酵素Ｑ１０（ＣｏＱ１０）である。 The coenzyme Q is preferably coenzyme Q10 (CoQ10).
「血中の補酵素Ｑ」とは、被験体の血清、血漿又は全血中の補酵素Ｑを指し、好ましくは被験体の血清又は血漿中の補酵素Ｑを指す。 "Coenzyme Q in blood" refers to coenzyme Q in the serum, plasma, or whole blood of a subject, and preferably refers to coenzyme Q in the serum or plasma of a subject.
皮膚試料としては、特に、採取が容易な角質層試料であることが好ましい。角質層試料は、被験体の皮膚の表面に粘着性シートを貼付したのち剥離すると、粘着性シートの粘着性面上に粘着して残存するため採取が容易である。また、粘着性面上に角質層試料が粘着した粘着性シートは、必要に応じて粘着性面を保護したうえで、通常の封筒等によって包装することが容易である。粘着性シートとしては、市販されている角質層採取用フィルムを使用することができる。角質層試料を採取する皮膚の領域は特に限定されないが、被験体がヒトである場合、体毛の少ない領域、例えば、前腕又は上腕の内側の皮膚の一部の領域が好ましい。 As the skin sample, it is particularly preferable to use a stratum corneum sample, which is easy to collect. When an adhesive sheet is applied to the surface of the skin of a subject and then peeled off, the stratum corneum sample remains adhered to the adhesive surface of the adhesive sheet, making it easy to collect. In addition, the adhesive sheet with the stratum corneum sample adhered to its adhesive surface can be easily packaged in a normal envelope or the like, after protecting the adhesive surface as necessary. As the adhesive sheet, a commercially available film for collecting stratum corneum can be used. The area of skin from which the stratum corneum sample is collected is not particularly limited, but when the subject is a human, an area with little body hair, such as a part of the skin on the inside of the forearm or upper arm, is preferable.
皮膚試料中の補酵素Ｑの量は、採取した皮膚試料からの抽出物を、高速液体クロマトグラフィー、ガスクロマトグラフィー等の分析手段により分析して定量することができる。補酵素Ｑは脂溶性であるため、前記抽出物は疎水性有機溶媒による抽出物であることが好ましい。定量結果は、被験体の皮膚の所定面積の領域に由来する皮膚試料中の補酵素Ｑの量、或いは、被験体に由来する所定重量の皮膚試料中の補酵素Ｑの量として表すことができる。 The amount of coenzyme Q in a skin sample can be quantified by analyzing an extract from the collected skin sample using analytical means such as high performance liquid chromatography or gas chromatography. Since coenzyme Q is fat-soluble, the extract is preferably an extract using a hydrophobic organic solvent. The quantification result can be expressed as the amount of coenzyme Q in a skin sample derived from a region of a given area of the subject's skin, or the amount of coenzyme Q in a given weight of skin sample derived from the subject.
本発明の第１の実施形態に係る方法では、皮膚試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定することができる。この決定のためには、予め又はその都度作製された、被験体に由来する皮膚試料中の補酵素Ｑの量と、被験体の血中の補酵素Ｑの量との相関関係を示す検量線を用いることができる。 In the method according to the first embodiment of the present invention, the amount of coenzyme Q in the blood of a subject can be determined based on the amount of coenzyme Q in a skin sample. For this determination, a calibration curve showing the correlation between the amount of coenzyme Q in a skin sample derived from a subject and the amount of coenzyme Q in the blood of the subject, which is prepared in advance or on each occasion, can be used.
本発明の第１の実施形態の好ましい態様は、
被験体の血中の補酵素Ｑを定量する方法であって、
被験体の皮膚の表面に粘着性シートを貼付したのち剥離して、被験体の角質層試料を粘着性シート上に採取すること、
採取された角質層試料中の補酵素Ｑを定量すること、及び、
角質層試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること
を含む方法である。
A preferred aspect of the first embodiment of the present invention is
A method for quantifying coenzyme Q in blood of a subject, comprising the steps of:
affixing an adhesive sheet to the surface of the subject's skin and then peeling it off to collect a sample of the subject's stratum corneum on the adhesive sheet;
Quantifying the amount of coenzyme Q in the collected stratum corneum sample; and
The method includes determining the amount of Coenzyme Q in the subject's blood based on the amount of Coenzyme Q in the stratum corneum sample.
２．本発明の第２の実施形態
本発明の第２の実施形態は、
被験体における血中の補酵素Ｑの量が不足しているか否かを評価するための指標を取得する方法であって、
被験体に由来する皮膚試料中の補酵素Ｑを定量すること、
皮膚試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること、及び、
被験体の血中の補酵素Ｑの量と、補酵素Ｑの推奨血中量とを比較すること
を含む方法
に関する。
2. Second embodiment of the present invention The second embodiment of the present invention is
A method for obtaining an indicator for evaluating whether or not a subject has a deficiency in the amount of coenzyme Q in the blood, comprising:
Quantifying coenzyme Q in a skin sample from a subject;
Determining the amount of coenzyme Q in the subject's blood based on the amount of coenzyme Q in the skin sample; and
The method comprises comparing the amount of Coenzyme Q in the subject's blood with a recommended blood amount of Coenzyme Q.
本発明の第２の実施形態における、第１の実施形態と共通する特徴及びその好ましい態様については、第１の実施形態に関して説明した通りであり、説明を省略する。 The features and preferred aspects of the second embodiment of the present invention that are common to the first embodiment are the same as those described for the first embodiment, and therefore will not be described here.
本発明の第２の実施形態に係る方法によれば、医療機関での血液の採取を行うことなく被験体の血中の補酵素Ｑの量を決定し、それを補酵素Ｑの推奨血中量と比較することにより、被験体における血中の補酵素Ｑの量が不足しているか否かを評価するための指標を取得することが可能である。 According to the method of the second embodiment of the present invention, it is possible to determine the amount of coenzyme Q in the blood of a subject without taking blood samples at a medical institution, and by comparing this with the recommended blood amount of coenzyme Q, it is possible to obtain an index for evaluating whether the amount of coenzyme Q in the blood of the subject is insufficient.
補酵素Ｑの推奨血中量とは、補酵素Ｑが被験体内において所望の生理的作用を奏する目的で推奨される補酵素Ｑの推奨血中量を指す。決定された被験体の血中の補酵素Ｑの量が、補酵素Ｑの推奨血中量を有意に下回ることは、被験体における血中の補酵素Ｑの量が不足していることの指標となる。決定された被験体の血中の補酵素Ｑの量が、補酵素Ｑの推奨血中量以上であることは、被験体における血中の補酵素Ｑの量が不足していないことの指標となる。 The recommended blood amount of coenzyme Q refers to the recommended blood amount of coenzyme Q for the purpose of coenzyme Q exerting a desired physiological effect in a subject. If the determined amount of coenzyme Q in the subject's blood is significantly lower than the recommended blood amount of coenzyme Q, this indicates that the amount of coenzyme Q in the subject's blood is insufficient. If the determined amount of coenzyme Q in the subject's blood is equal to or greater than the recommended blood amount of coenzyme Q, this indicates that the amount of coenzyme Q in the subject's blood is not insufficient.
３．本発明の第３の実施形態
本発明の第３の実施形態は、
被験体が摂取した補酵素Ｑの生体内移行を検査するための指標を取得する方法であって、
補酵素Ｑを摂取した被験体に由来する皮膚試料中の補酵素Ｑを定量することを含む方法
に関する。
3. Third embodiment of the present invention The third embodiment of the present invention is
A method for obtaining an indicator for examining the transfer of coenzyme Q into the body of a subject, comprising the steps of:
The present invention relates to a method comprising quantifying Coenzyme Q in a skin sample derived from a subject who has ingested Coenzyme Q.
本発明の第３の実施形態における、第１の実施形態と共通する特徴及びその好ましい態様については、第１の実施形態に関して説明した通りであり、説明を省略する。 The features and preferred aspects of the third embodiment of the present invention that are common to the first embodiment are the same as those described for the first embodiment, and therefore will not be described here.
本発明の第３の実施形態に係る方法では、血液の採取を行うことなく、補酵素Ｑを摂取した被験体に由来する皮膚試料中の補酵素Ｑを定量し、その定量結果を、被験体が摂取した補酵素Ｑの、生体内移行の程度の指標とする。 In the method according to the third embodiment of the present invention, coenzyme Q is quantified in a skin sample from a subject who has ingested coenzyme Q without taking a blood sample, and the quantification result is used as an index of the degree of in vivo transfer of the coenzyme Q ingested by the subject.
本発明の第３の実施形態に係る方法において、「摂取」とは、好ましくは「経口摂取」である。 In the method according to the third embodiment of the present invention, "ingestion" preferably means "oral ingestion."
本発明の第３の実施形態に係る方法では、補酵素Ｑを摂取した被験体に由来する皮膚試料中の補酵素Ｑの量が小さいことが、被験体が摂取した補酵素Ｑの生体内移行量が小さいことの指標となり、補酵素Ｑを摂取した被験体に由来する皮膚試料中の補酵素Ｑの量が大きいことが、被験体が摂取した補酵素Ｑの生体内移行量が大きいことの指標となる。 In the method according to the third embodiment of the present invention, a small amount of coenzyme Q in a skin sample derived from a subject who ingested coenzyme Q is an indication that the amount of coenzyme Q ingested by the subject that is transferred to the body is small, and a large amount of coenzyme Q in a skin sample derived from a subject who ingested coenzyme Q is an indication that the amount of coenzyme Q ingested by the subject that is transferred to the body is large.
本発明の第３の実施形態に係る方法は、更に、皮膚試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定することを含んでもよい。この態様では、被験体の血中の補酵素Ｑの量を、被験体が摂取した補酵素Ｑの、生体内移行の程度の指標とすることができる。 The method according to the third embodiment of the present invention may further include determining the amount of coenzyme Q in the subject's blood based on the amount of coenzyme Q in the skin sample. In this aspect, the amount of coenzyme Q in the subject's blood can be used as an indicator of the degree of biotransfer of the coenzyme Q ingested by the subject.
４．本発明の第４の実施形態
本発明の第４の実施形態は、
被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標を取得する方法であって、
被験物質を摂取した被験体に由来する皮膚試料中の補酵素Ｑを定量することを含む方法に関する。
4. Fourth embodiment of the present invention The fourth embodiment of the present invention is
A method for obtaining an indicator for examining an effect of a test substance on the synthesis or metabolism of coenzyme Q in a living body of a subject, comprising:
The present invention relates to a method comprising quantifying coenzyme Q in a skin sample derived from a subject who has ingested a test substance.
本発明の第４の実施形態における、第１の実施形態と共通する特徴及びその好ましい態様については、第１の実施形態に関して説明した通りであり、説明を省略する。 The features and preferred aspects of the fourth embodiment of the present invention that are common to the first embodiment are the same as those described for the first embodiment, and therefore will not be described here.
本発明の第４の実施形態に係る方法では、血液の採取を行うことなく、被験物質を摂取した被験体に由来する皮膚試料中の補酵素Ｑを定量し、その定量結果を、被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標とする。 In the method according to the fourth embodiment of the present invention, coenzyme Q is quantified in a skin sample derived from a subject who has ingested a test substance without taking a blood sample, and the quantification result is used as an index for examining the effect of the test substance on the synthesis or metabolism of coenzyme Q in the subject's body.
本発明の第４の実施形態に係る方法において、「被験物質」とは、生体内における補酵素Ｑの合成又は代謝への影響の確認が求められる物質を指す。例えば、生体内における補酵素Ｑの合成又は代謝へ影響を与える薬剤の候補物質や、生体内における補酵素Ｑの合成又は代謝に対する毒性の確認が望まれる物質が「被験物質」である。 In the method according to the fourth embodiment of the present invention, the term "test substance" refers to a substance for which confirmation of its effect on the synthesis or metabolism of coenzyme Q in a living body is desired. For example, a "test substance" is a candidate drug that affects the synthesis or metabolism of coenzyme Q in a living body, or a substance for which confirmation of its toxicity on the synthesis or metabolism of coenzyme Q in a living body is desired.
本発明の第４の実施形態に係る方法では、被験物質を摂取した被験体に由来する皮膚試料中の補酵素Ｑの量が小さいことが、被験物質が、被験体の生体内における補酵素Ｑの合成を抑制又は代謝を促進する作用を有することの指標となり、被験物質を摂取した被験体に由来する皮膚試料中の補酵素Ｑの量が大きいとが、被験物質が、被験体の生体内における補酵素Ｑの合成を促進又は代謝を抑制する作用を有することの指標となる。 In the method according to the fourth embodiment of the present invention, a small amount of coenzyme Q in a skin sample derived from a subject that has ingested the test substance is an indication that the test substance has the effect of inhibiting the synthesis or promoting the metabolism of coenzyme Q in the subject's body, and a large amount of coenzyme Q in a skin sample derived from a subject that has ingested the test substance is an indication that the test substance has the effect of promoting the synthesis or suppressing the metabolism of coenzyme Q in the subject's body.
本発明の第４の実施形態に係る方法は、更に、皮膚試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定することを含んでもよい。この態様では、被験体の血中の補酵素Ｑの量を、被験物質の、被験体の生体内における補酵素Ｑの合成又は代謝に対する作用を検査するための指標とすることができる。 The method according to the fourth embodiment of the present invention may further include determining the amount of coenzyme Q in the subject's blood based on the amount of coenzyme Q in the skin sample. In this aspect, the amount of coenzyme Q in the subject's blood can be used as an indicator for examining the effect of a test substance on the synthesis or metabolism of coenzyme Q in the subject's body.
１．実施例
１．１．被験体
下記表に示す被験体１～６のヒトの男女から角質層試料を採取し、各被験体における、角質層試料中の補酵素Ｑ１０（ＣｏＱ１０）の量と、血清中の補酵素Ｑ１０濃度との関係を確認した。
1. Example 1.1. Subjects Stratum corneum samples were collected from human male and female subjects 1 to 6 shown in the table below, and the relationship between the amount of coenzyme Q10 (CoQ10) in the stratum corneum sample and the concentration of coenzyme Q10 in serum for each subject was confirmed.
１．２．角質層試料中のＣｏＱ１０の定量
一方の表面が粘着性を有する、直径２２．００ｍｍの円形の市販の角質層採取用フィルム（プロモツール株式会社、角質チェッカーＤ１００）を用意した。
1.2. Quantitative Determination of CoQ10 in Stratum Corneum Samples A commercially available circular film for sampling stratum corneum (Promo Tools, Inc., Keratin Checker D100) having one adhesive surface and a diameter of 22.00 mm was prepared.
各被験体の左右両腕の前腕の内側の皮膚の、後述する所定の領域に、前記角質層採取用フィルムの粘着性面を貼付し、軽く指で押さえたのち剥離して、前記粘着性面上に角質層試料を採取した。１回の採取に１枚の新しい角質層採取用フィルムを用いた。 The adhesive side of the stratum corneum sampling film was attached to the skin on the inside of the forearm of both the left and right arms of each subject in a specified area described below, and the film was lightly pressed with a finger and then peeled off to collect a stratum corneum sample on the adhesive side. One new piece of stratum corneum sampling film was used for each collection.
前記所定の領域とは、図１に示す、Ａ（右腕の前腕の内側の皮膚の、親指側の位置の直径２２．００ｍｍの円形領域）、Ｂ（右腕の前腕の内側の皮膚の、小指側の位置の直径２２．００ｍｍの円形領域）、Ｃ（左腕の前腕の内側の皮膚の、小指側の位置の直径２２．００ｍｍの円形領域）、Ｄ（左腕の前腕の内側の皮膚の、親指側の位置の直径２２．００ｍｍの円形領域）である。各被験体の領域Ａ及びＣからそれぞれ１回、領域Ｂ及びＤからそれぞれ３回、角質層試料を採取した。領域Ｂ及びＤからの３回の採取は連続して、同じ箇所から行った。 The predetermined areas are A (a circular area of 22.00 mm in diameter on the thumb side of the skin on the inside of the right forearm), B (a circular area of 22.00 mm in diameter on the little finger side of the skin on the inside of the right forearm), C (a circular area of 22.00 mm in diameter on the little finger side of the skin on the inside of the left forearm), and D (a circular area of 22.00 mm in diameter on the thumb side of the skin on the inside of the left forearm) as shown in FIG. 1. Stratum corneum samples were collected once each from areas A and C, and three times each from areas B and D for each subject. The three collections from areas B and D were performed consecutively from the same locations.
角質層採取用フィルムの粘着性面上に採取された角質層試料からの、ＣｏＱ１０の測定方法は以下の手順で行った。
角質層を採取した前記フィルムを、１－プロパノール５ｍＬに添加し、超音波抽出を１分間実施して、１－プロパノール中にＣｏＱ１０を抽出した。抽出後、ＣｏＱ１０を含む１－プロパノールをデカンテーションで回収した。その後、前記フィルムに再度１－プロパノール５ｍＬを添加し、超音波抽出・抽出液回収を２回実施した。回収した１－プロパノール抽出液を遠心濃縮機で濃縮乾固し、ＬＣ－ＭＳ／ＭＳ分析用の希釈液に残渣を溶解させた後、メンブランフィルターでろ過を行って、ＣｏＱ１０含量分析を実施した。
The method for measuring CoQ10 from the stratum corneum sample collected on the adhesive surface of the stratum corneum collecting film was carried out as follows.
The film from which the stratum corneum had been collected was added to 5 mL of 1-propanol, and ultrasonic extraction was performed for 1 minute to extract CoQ10 into the 1-propanol. After extraction, the 1-propanol containing CoQ10 was collected by decantation. Thereafter, 5 mL of 1-propanol was added again to the film, and ultrasonic extraction and extraction solution collection were performed twice. The collected 1-propanol extract was concentrated to dryness using a centrifugal concentrator, and the residue was dissolved in a diluent for LC-MS/MS analysis, followed by filtration using a membrane filter, and the CoQ10 content was analyzed.
上記のＬＣ－ＭＳ／ＭＳ分析では、還元型ＣｏＱ１０は実質的に存在せず定量限界未満であり、酸化型ＣｏＱ１０のみが定量された。上記のＬＣ－ＭＳ／ＭＳ分析により測定された酸化型ＣｏＱ１０量を、酸化型と還元型のＣｏＱ１０の総量（総ＣｏＱ１０量）とみなした。 In the above LC-MS/MS analysis, reduced CoQ10 was substantially absent and below the quantification limit, and only oxidized CoQ10 was quantified. The amount of oxidized CoQ10 measured by the above LC-MS/MS analysis was considered to be the total amount of oxidized and reduced CoQ10 (total CoQ10 amount).
各被験体の各採取領域から採取された角質層試料中のＣｏＱ１０の量（ｎｇ）を求めた。領域Ｂ及びＤから採取された角質層試料中のＣｏＱ１０の量は、それぞれ、３枚の角質層採取用フィルム上に採取された角質層試料中のＣｏＱ１０の合計量を指す。また、各被験体における、領域Ａ～Ｄの１領域あたりの角質層試料中のＣｏＱ１０量の平均値を求めた。 The amount of CoQ10 (ng) in the stratum corneum samples collected from each collection area of each subject was determined. The amount of CoQ10 in the stratum corneum samples collected from areas B and D refers to the total amount of CoQ10 in the stratum corneum samples collected on the three sheets of stratum corneum collection film. In addition, the average amount of CoQ10 in the stratum corneum samples per area A to D for each subject was determined.
１．３．血清中の総ＣｏＱ１０の定量
各被験体の血清中の総ＣｏＱ１０濃度を次の手順で測定した。
採取した血液を遠心分離し血清を取得した。得られた血清１００μＬに対して、２－プロパノール７００μＬを添加し、ボルテックスミキサーで均一になるよう混合した。混合後、遠心分離を行い、得られた上澄み２００μＬに対して、ＬＣ－ＭＳ／ＭＳ分析用の希釈液３００μＬを添加した。この溶液をボルテックスミキサーで混合し、メンブランフィルターでろ過を行って、ＣｏＱ１０含量分析を実施した。この分析では、酸化型と還元型のＣｏＱ１０をそれぞれ定量し合計して、総ＣｏＱ１０濃度を求めた。
1.3. Quantification of Total CoQ10 in Serum The total CoQ10 concentration in the serum of each subject was measured by the following procedure.
The collected blood was centrifuged to obtain serum. 700 μL of 2-propanol was added to 100 μL of the obtained serum, and mixed to homogenize using a vortex mixer. After mixing, centrifugation was performed, and 300 μL of diluent for LC-MS/MS analysis was added to 200 μL of the obtained supernatant. This solution was mixed using a vortex mixer, filtered using a membrane filter, and CoQ10 content analysis was performed. In this analysis, oxidized and reduced CoQ10 were quantified and summed to obtain the total CoQ10 concentration.
１．４．結果
結果を下記表に示す。
1.4. Results The results are shown in the table below.
更に、上記表に示した、被験体１～６の、採取領域１箇所あたりの角質層試料中の総ＣｏＱ１０量と血清中の総ＣｏＱ１０濃度とを、プロットしたグラフを図２に示す。 Furthermore, Figure 2 shows a graph plotting the total CoQ10 amount in the stratum corneum samples per collection area and the total CoQ10 concentration in the serum for subjects 1 to 6 shown in the table above.
角質層試料中の総ＣｏＱ１０量と、血清中の総ＣｏＱ１０濃度とは、正の相関関係があることが確認された。このことは、被験体の角質層試料中の総ＣｏＱ１０量に基づいて、血清中の総ＣｏＱ１０濃度を決定することが可能であることを示す。 It was confirmed that there is a positive correlation between the amount of total CoQ10 in the stratum corneum sample and the total CoQ10 concentration in serum. This indicates that it is possible to determine the total CoQ10 concentration in serum based on the amount of total CoQ10 in the stratum corneum sample of a subject.
２．比較例
２．１．被験体
実施例と同じ被験体１～６から全唾液を採取し、各被験体における、唾液試料中のＣｏＱ１０濃度と、血清中のＣｏＱ１０濃度との関係を確認した。
2. Comparative Example 2.1. Subjects Whole saliva was collected from the same subjects 1 to 6 as in the Example, and the relationship between the CoQ10 concentration in the saliva sample and the CoQ10 concentration in the serum of each subject was confirmed.
２．２．唾液試料中の総ＣｏＱ１０濃度の定量
各被験体から全唾液を採取し、以下の手順で総ＣｏＱ１０濃度を測定した。
採取した全唾液１００μＬに、２－プロパノール７００μＬとＬＣ－ＭＳ／ＭＳ分析用の希釈液７００μＬを添加し、ボルテックスミキサーで均一になるよう混合した。混合後、遠心分離を行い、得られた上澄みをメンブランフィルターでろ過して、ＣｏＱ１０含量分析を実施した。この分析では、酸化型と還元型のＣｏＱ１０をそれぞれ定量し合計して、総ＣｏＱ１０濃度を求めた。
2.2. Determination of total CoQ10 concentration in saliva samples Whole saliva was collected from each subject, and the total CoQ10 concentration was measured by the following procedure.
700 μL of 2-propanol and 700 μL of diluent for LC-MS/MS analysis were added to 100 μL of collected whole saliva, and mixed to homogenity using a vortex mixer. After mixing, the mixture was centrifuged, and the resulting supernatant was filtered through a membrane filter to perform CoQ10 content analysis. In this analysis, the oxidized and reduced forms of CoQ10 were quantified and summed to determine the total CoQ10 concentration.
２．３．結果
結果を下記表に示す。なお血清中の総ＣｏＱ１０濃度は実施例での測定値である。
2.3. Results The results are shown in the table below. The total CoQ10 concentration in serum was measured in the examples.
更に、上記表に示した、被験体１～６の、唾液中の総ＣｏＱ１０濃度と血清中の総ＣｏＱ１０濃度とを、プロットしたグラフを図３に示す。
唾液中の総ＣｏＱ１０濃度と、血清中の総ＣｏＱ１０濃度との間に相関関係は認められなかった。
Furthermore, FIG. 3 shows a graph plotting the total CoQ10 concentrations in saliva and serum for subjects 1 to 6 shown in the above table.
No correlation was found between the total CoQ10 concentration in saliva and the total CoQ10 concentration in serum.
Claims (5)
被験体に由来する角質層試料中の補酵素Ｑを定量すること、及び
角質層試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること
を含む方法。 A method for quantifying coenzyme Q in blood of a subject, comprising the steps of:
Quantifying coenzyme Q in a stratum corneum sample from a subject ; and
Determining the amount of coenzyme Q in the subject's blood based on the amount of coenzyme Q in the stratum corneum sample.
The method includes:
被験体の皮膚の表面に粘着性シートを貼付したのち剥離して、被験体の角質層試料を粘着性シート上に採取すること、
採取された角質層試料中の補酵素Ｑを定量すること、及び、
角質層試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること
を含む方法。 A method for quantifying coenzyme Q in blood of a subject, comprising the steps of:
affixing an adhesive sheet to the surface of the subject's skin and then peeling it off to collect a sample of the subject's stratum corneum on the adhesive sheet;
Quantifying the amount of coenzyme Q in the collected stratum corneum sample; and
determining the amount of Coenzyme Q in the subject's blood based on the amount of Coenzyme Q in the stratum corneum sample.
被験体に由来する角質層試料中の補酵素Ｑを定量すること、
角質層試料中の補酵素Ｑの量に基づき、被験体の血中の補酵素Ｑの量を決定すること、及び、
被験体の血中の補酵素Ｑの量と、補酵素Ｑの推奨血中量とを比較すること
を含む方法。 A method for obtaining an indicator for evaluating whether or not a subject has a deficiency in the amount of coenzyme Q in the blood, comprising:
Quantifying coenzyme Q in a stratum corneum sample from a subject;
determining the amount of Coenzyme Q in the subject's blood based on the amount of Coenzyme Q in the stratum corneum sample; and
A method comprising comparing the amount of coenzyme Q in the subject's blood with a recommended blood amount of coenzyme Q.
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| WO2005085843A1 (en) | 2004-03-03 | 2005-09-15 | Mitsubishi Kagaku Bio-Clinical Laboratories, Inc. | Method of in vivo inspection of fat soluble vitamin and/or fat soluble food factor through saliva analysis |
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| WO2006104153A1 (en) | 2005-03-29 | 2006-10-05 | Kaneka Corporation | Composition for increasing anti-oxidation activity in blood |
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| JP2016513795A (en) | 2013-03-15 | 2016-05-16 | ザ プロクター アンド ギャンブル カンパニー | Non-invasive method for measuring oxidative stress and oxidative damage from skin biomarkers |
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| US20080090265A1 (en) | 2002-05-23 | 2008-04-17 | Littarru Gian P | Method to Assay Coenzzyme Q10 in Blood Plasma or Blood Serum |
| JP2005535614A (en) | 2002-06-25 | 2005-11-24 | コズメシューティック ソリューションズ プロプライエタリー リミテッド | Topical beauty composition |
| WO2005085843A1 (en) | 2004-03-03 | 2005-09-15 | Mitsubishi Kagaku Bio-Clinical Laboratories, Inc. | Method of in vivo inspection of fat soluble vitamin and/or fat soluble food factor through saliva analysis |
| WO2006104153A1 (en) | 2005-03-29 | 2006-10-05 | Kaneka Corporation | Composition for increasing anti-oxidation activity in blood |
| JP2007114170A (en) | 2005-10-18 | 2007-05-10 | Sogo Ikagaku Kenkyusho:Kk | Degree of fatigue evaluation method, and its use |
| JP2012526824A (en) | 2009-05-11 | 2012-11-01 | バーグ バイオシステムズ，エルエルシー | Methods of treating neoplastic disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmentally affecting factors |
| JP2016513795A (en) | 2013-03-15 | 2016-05-16 | ザ プロクター アンド ギャンブル カンパニー | Non-invasive method for measuring oxidative stress and oxidative damage from skin biomarkers |
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