JP2020180897A - Method for assisting detection of fatigue state - Google Patents
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Abstract
Description
本発明は、生体の疲労状態の検出を補助する方法に関する。 The present invention relates to a method that assists in detecting a fatigue state of a living body.
体が疲労していても疲労を感じない場合もあり、逆に、慢性疲労症候群のように、体が疲労していなくても疲労感を感じる疾患もある。このため、疲労の診断は、本人の問診のみによっては必ずしも正確に行うことができない。また、ペットや家畜等の動物に問診することは当然できない。したがって、客観的に疲労状態を検出できる方法が求められている。 Sometimes the body is tired but not tired, and conversely, there are diseases such as chronic fatigue syndrome that feel tired even if the body is not tired. Therefore, the diagnosis of fatigue cannot always be made accurately only by interviewing the person himself / herself. In addition, it is of course not possible to ask questions about animals such as pets and livestock. Therefore, there is a need for a method that can objectively detect a fatigue state.
生体中の成分をバイオマーカーとして疲労を検出する方法として、特許文献１に記載された方法が知られている。特許文献１には、このようなバイオマーカーとして、種々の物質が記載されているが、タンパク質を構成するアミノ酸であるアラニン、アルギニン、アスパラギン又はアスパラギン酸をバイオマーカーとして用いることも記載されている。しかしながら、各アミノ酸の光学異性体については言及されていない。 As a method for detecting fatigue using a component in a living body as a biomarker, the method described in Patent Document 1 is known. Patent Document 1 describes various substances as such biomarkers, but also describes that alanine, arginine, asparagine or aspartic acid, which are amino acids constituting proteins, are used as biomarkers. However, no mention is made of the optical isomers of each amino acid.
本願発明の目的は、生体内の成分濃度を指標として、客観的に疲労状態を検出することを可能にする新規な方法を提供することである。 An object of the present invention is to provide a novel method capable of objectively detecting a fatigue state by using a component concentration in a living body as an index.
本願発明者らは、鋭意研究の結果、生体内におけるＤ体アミノ酸濃度、又はＤ体アミノ酸とＬ体アミノ酸の濃度比を指標として疲労状態を検出できることを見出し、本発明を完成した。 As a result of diligent research, the inventors of the present application have found that the fatigue state can be detected using the D-amino acid concentration in the living body or the concentration ratio of the D-amino acid and the L-amino acid as an index, and completed the present invention.
すなわち、本発明は以下のものを提供する。
(1) 生体から採取された生体試料中のＤ体アミノ酸を定量することを含み、定量されたＤ体アミノ酸の濃度を指標とする、生体の疲労状態の検出を補助する方法。
(2) 前記Ｄ体アミノ酸が、プロリン、アラニン、イソロイシン、アルギニン、バリン、スレオニン及びセリンから成る群より選ばれる少なくとも１種であり、プロリン、アラニン、イソロイシン、アルギニン、バリン、スレオニン及びセリンから成る群より選ばれる少なくとも１種のＤ体アミノ酸濃度が正常群よりも有意に低い(1)記載の方法。
(3) 前記Ｄ体アミノ酸がメチオニンであり、該Ｄ体メチオニン濃度が正常群よりも有意に高い(1)記載の方法。
(4) 生体から採取された生体試料中のＤ体アミノ酸及びＬ体アミノ酸を定量することを含み、定量された各Ｄ体アミノ酸の濃度と、該各Ｄ体アミノ酸と同種の各Ｌ体アミノ酸の濃度との比を指標とする、生体の疲労状態の検出を補助する方法。
(5) 前記Ｄ体アミノ酸が、プロリン、イソロイシン、アラニン、バリン、ロイシン、アスパラギン酸及びフェニルアラニンから成る群より選ばれる少なくとも１種であり、プロリン、イソロイシン、アラニン、バリン、ロイシン、アスパラギン酸及びフェニルアラニンから成る群より選ばれる少なくとも１種の各Ｄ体アミノ酸濃度と、該各Ｄ体アミノ酸と同種の各Ｌ体アミノ酸の濃度との比が、正常群よりも有意に低い(4)記載の方法。
(6) 前記Ｄ体アミノ酸がメチオニンであり、Ｌ体メチオニンの濃度との比が、正常群よりも有意に高い(4)記載の方法。
That is, the present invention provides the following.
(1) A method for assisting the detection of a fatigue state of a living body, which comprises quantifying a D-amino acid in a biological sample collected from a living body and using the quantified concentration of the D-amino acid as an index.
(2) The D-amino acid is at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, threonine and serine, and is a group consisting of proline, alanine, isoleucine, arginine, valine, threonine and serine. The method according to (1), wherein the concentration of at least one D-amino acid selected from the above is significantly lower than that in the normal group.
(3) The method according to (1), wherein the D-amino acid is methionine and the D-methionine concentration is significantly higher than that in the normal group.
(4) Quantifying D-amino acids and L-amino acids in biological samples collected from living organisms, including the quantified concentration of each D-amino acid and each L-amino acid of the same type as each D-amino acid. A method of assisting the detection of a fatigue state of a living body using the ratio with the concentration as an index.
(5) The D-amino acid is at least one selected from the group consisting of proline, isoleucine, alanine, valine, leucine, aspartic acid and phenylalanine, from proline, isoleucine, alanine, valine, leucine, aspartic acid and phenylalanine. The method according to (4), wherein the ratio of the concentration of each D-amino acid selected from the group consisting of at least one D-amino acid to the concentration of each L-amino acid of the same species as the D-amino acid is significantly lower than that of the normal group.
(6) The method according to (4), wherein the D-amino acid is methionine and the ratio to the concentration of L-methionine is significantly higher than that in the normal group.
本発明の方法によれば、生体内に実在する成分の濃度又は濃度比を指標として、客観的に疲労状態を検出することが可能である。 According to the method of the present invention, it is possible to objectively detect a fatigue state using the concentration or concentration ratio of a component actually existing in a living body as an index.
本発明の方法において、評価対象となる生体としては、哺乳類及び鳥類が好ましく、ヒト、サル、チンパンジー等の霊長類；ネコ、イヌ、ハムスター等の食肉類；ウシ、ブタ、ウマ、ヒツジ、ヤギ等の草食類；マウス、ラット等の齧歯類；ニワトリ等の鳥類を挙げることができるがこれらに限定されるものではない。 In the method of the present invention, mammals and birds are preferable as living organisms to be evaluated, and primates such as humans, monkeys and orangutans; carnivores such as cats, dogs and hamsters; cattle, pigs, horses, sheep, goats and the like. Herbivorous animals; rodents such as mice and rats; birds such as chickens, but are not limited thereto.
本明細書では各種アミノ酸を一部略称で表記するが、それらの正式名称は以下の通りである。
（略称） （正式名称）
Ａｌａ アラニン
Ａｒｇ アルギニン
Ａｓｎ アスパラギン
Ｇｌｎ グルタミン
Ｇｌｙ グリシン
Ｈｉｓ ヒスチジン
Ｉｌｅ イソロイシン
Ｌｅｕ ロイシン
Ｌｙｓ リジン
Ｍｅｔ メチオニン
Ｐｈｅ フェニルアラニン
Ｐｒｏ プロリン
Ｓｅｒ セリン
Ｔｈｒ スレオニン
Ｔｒｐ トリプトファン
Ｔｙｒ チロシン
Ｖａｌ バリン
In this specification, various amino acids are partially abbreviated, but their official names are as follows.
(Abbreviation) (Official name)
Ala Alanin Arg Arginine Asparagine Gln Glycine Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Met Methionin Phenylalanine Pro Proline Ser Serin Thr Threonine Trp Tryptophan Tyr Tyr
生体試料としては、生体から採取された試料であれば特に限定されないが、血液、尿、唾液等の体液が好ましく、特に血液が好ましい。血液試料は、全血、血清、血漿のいずれであってもよい。 The biological sample is not particularly limited as long as it is a sample collected from a living body, but body fluids such as blood, urine, and saliva are preferable, and blood is particularly preferable. The blood sample may be whole blood, serum, or plasma.
本発明の方法により検出される「疲労状態」は、精神疲労した状態と、肉体疲労した状態の両者を包含する。下記実施例では、主に精神疲労状態が検出されていると考えられている。 The "fatigue state" detected by the method of the present invention includes both a mentally tired state and a physically tired state. In the following examples, it is considered that the mental fatigue state is mainly detected.
本発明の方法では、生体試料中の少なくともＤ体アミノ酸を定量する。また、一実施形態では、Ｌ体アミノ酸も定量する。以前は、生体のタンパク質を構成しているのはＬ体アミノ酸のみであると考えられていたが、近年、分析技術が向上し、アミノ酸をＤ体とＬ体とで区別して定量することが可能となり、生体内にＤ体アミノ酸も含まれることがわかってきた。アミノ酸をＤ体とＬ体とで区別して定量する方法は公知であり（WO 2017/057433)、下記実施例にも具体的に記載されている。 In the method of the present invention, at least D-amino acid in a biological sample is quantified. In one embodiment, L-amino acids are also quantified. Previously, it was thought that only L-form amino acids make up proteins in living organisms, but in recent years, analytical techniques have improved, and it is possible to distinguish and quantify amino acids between D-form and L-form. It has become clear that D-amino acids are also contained in the living body. A method for distinguishing and quantifying amino acids between D-form and L-form is known (WO 2017/057433), and is specifically described in the following examples.
本発明の第１の実施形態では、生体試料中のＤ体アミノ酸を定量し、定量されたＤ体アミノ酸の濃度を指標として疲労状態を検出する。定量するＤ体アミノ酸としては、プロリン、アラニン、イソロイシン、アルギニン、バリン、メチオニン、スレオニン及びセリンから成る群より選ばれる少なくとも１種であることが好ましい。下記実施例では、これらのＤ体アミノ酸は、疲労群と正常群との間で統計学的有意差が認められたものである。これらのうち、プロリン、アラニン、イソロイシン、アルギニン、バリン、スレオニン及びセリンは、疲労群において正常群よりも濃度が有意に低くなり、メチオニンは、疲労群において正常群よりも濃度が有意に高くなる。下記実施例で測定された、Ｄ体アミノ酸の濃度の疲労群の平均値と正常群の平均値との比（疲労群／正常群）を下記表１に示す。 In the first embodiment of the present invention, the D-amino acid in the biological sample is quantified, and the fatigue state is detected using the quantified concentration of the D-amino acid as an index. The D-amino acid to be quantified is preferably at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, methionine, threonine and serine. In the following examples, these D-amino acids are statistically significant differences between the fatigue group and the normal group. Of these, proline, alanine, isoleucine, arginine, valine, threonine and serine are significantly lower in the fatigue group than in the normal group, and methionine is significantly higher in the fatigue group than in the normal group. Table 1 below shows the ratio (fatigue group / normal group) of the average value of the D-amino acid concentration in the fatigue group to the average value in the normal group measured in the following examples.
表１に示す疲労群／正常群比の値又はその近傍の値をカットオフ値として設定し、そのカットオフ値よりもそのＤ体アミノ酸の濃度が低ければ（メチオニンの場合には高ければ）、「疲労状態」と判定することができる。なお、この場合、カットオフ値は、試験する群によりある程度変動するので、適宜設定することが可能であり、例えば、上記表１に示す値の±３０％程度、好ましくは±２０％程度、さらに好ましくは±１０％程度の範囲に設定することができる。 If the fatigue group / normal group ratio value shown in Table 1 or a value in the vicinity thereof is set as the cutoff value and the concentration of the D-amino acid is lower than the cutoff value (in the case of methionine, it is higher), It can be determined as "fatigue state". In this case, the cutoff value varies to some extent depending on the group to be tested, and can be appropriately set. For example, the cutoff value is about ± 30%, preferably about ± 20% of the value shown in Table 1 above, and further. It can be preferably set in the range of about ± 10%.
本発明の第２の実施形態では、生体試料中のＤ体アミノ酸及びＬ体アミノ酸を定量し、定量された各Ｄ体アミノ酸の濃度と、該各Ｄ体アミノ酸と同種の各Ｌ体アミノ酸の濃度との比（以下、便宜的に「D/L比」と呼ぶことがある）を指標とする。定量するアミノ酸としては、プロリン、イソロイシン、アラニン、バリン、メチオニン、ロイシン、アスパラギン及びフェニルアラニンから成る群より選ばれる少なくとも１種であることが好ましい。下記実施例では、これらのアミノ酸のD/L比は、疲労群と正常群との間で統計学的有意差が認められたものである。これらのうち、プロリン、イソロイシン、アラニン、バリン、ロイシン、アスパラギン及びフェニルアラニンは、疲労群においてD/L比が正常群よりも有意に低くなり、メチオニンは、疲労群においてD/L比が正常群よりも有意に高くなる。下記実施例で測定された、各アミノ酸のD/L比の疲労群の平均値と正常群の平均値との比（疲労群／正常群）を下記表２に示す。 In the second embodiment of the present invention, D-amino acids and L-amino acids in a biological sample are quantified, and the quantified concentration of each D-amino acid and the concentration of each L-amino acid of the same type as each D-amino acid. The ratio with and (hereinafter, may be referred to as "D / L ratio" for convenience) is used as an index. The amino acid to be quantified is preferably at least one selected from the group consisting of proline, isoleucine, alanine, valine, methionine, leucine, asparagine and phenylalanine. In the examples below, the D / L ratios of these amino acids were statistically significantly different between the fatigue group and the normal group. Of these, proline, isoleucine, alanine, valine, leucine, asparagine and phenylalanine had a significantly lower D / L ratio in the fatigue group than in the normal group, and methionine had a D / L ratio in the fatigue group than in the normal group. Is also significantly higher. Table 2 below shows the ratio of the average value of the D / L ratio of each amino acid in the fatigue group to the average value in the normal group (fatigue group / normal group) measured in the following examples.
表２に示す疲労群／正常群比の値又はその近傍の値をカットオフ値として設定し、そのカットオフ値よりもそのＤ／Ｌ比が低ければ（メチオニンの場合には高ければ）、「疲労状態」と判定することができる。なお、この場合、カットオフ値は、試験する群によりある程度変動するので、適宜設定することが可能であり、例えば、上記表２に示す値の±３０％程度、好ましくは±２０％程度、さらに好ましくは±１０％程度の範囲に設定することができる。 The fatigue group / normal group ratio value shown in Table 2 or a value in the vicinity thereof is set as the cutoff value, and if the D / L ratio is lower than the cutoff value (in the case of methionine, it is higher), " It can be determined as "fatigue". In this case, the cutoff value varies to some extent depending on the group to be tested and can be appropriately set. For example, the cutoff value is about ± 30%, preferably about ± 20% of the value shown in Table 2 above, and further. It can be preferably set in the range of about ± 10%.
以下、本発明を実施例に基づき具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.
実施例１
６週齢のＷｉｓｔａｒ系雄性ラット（日本エスエルシー株式会社）を購入し、予備飼育の後に７週齢で使用した。疲労群のラット６匹は、底面から１．５ｃｍの高さに２３±１℃に調整した水を張ったケージ内で自由摂餌、自由飲水条件下、Ｄａｙ１からＤａｙ６まで５日間飼育した。ケージ内の水は毎日交換した。一方、正常群のラット６匹は、床敷き（ペパークリーン、ペパーレット株式会社）を敷いたケージ内で自由摂餌、自由飲水条件下、５日間飼育した。 イソフルラン麻酔下で腹部大動脈から注射針と適量のヘパリンナトリウムを筒内に入れたシリンジを用いて全採血を行った。得られた血液は、４℃条件下、１８７０×ｇで１０分間遠心分離を行い、血漿を分離した。得られた血漿は分析まで−２０℃以下で保存した。
Example 1
A 6-week-old Wistar male rat (Nippon SLC Co., Ltd.) was purchased and used at 7 weeks of age after preliminary breeding. Six rats in the fatigue group were bred from Day 1 to Day 6 for 5 days under free feeding and free drinking conditions in a cage filled with water adjusted to a height of 23 ± 1 ° C. from the bottom surface. The water in the cage was changed daily. On the other hand, 6 rats in the normal group were bred for 5 days under free feeding and free drinking conditions in a cage laid with a floor (Pepper Clean, Pepperlet Co., Ltd.). Under isoflurane anesthesia, total blood was collected from the abdominal aorta using a needle and a syringe containing an appropriate amount of sodium heparin in a cylinder. The obtained blood was centrifuged at 1870 × g for 10 minutes under the condition of 4 ° C. to separate plasma. The resulting plasma was stored below −20 ° C. until analysis.
・試料の調製
ラット血漿２０μＬに、アミノ酸の安定同位体標識の内標準溶液２０μＬを添加後、アセトニトリル４０μＬを添加して、２０℃、２００００×ｇで１０分間遠心分離し、上清を回収した。上清１０μＬに誘導体化試薬溶液２．５ｍｇ／ｍＬの（Ｒ）−４−ニトロフェニル−Ｎ−［２’−（ジエチルアミノ）−６，６’−ジメチル−［１，１’−ビフェニル］−２−イルカルバメートヒドロクロリド［（Ｒ）−ＢｉＡｃ］］を添加し、５５℃で１０分間反応させた後、０．１％ギ酸水溶液１００μＬを添加後、撹拌をし、液体クロマトグラフィー／三連四重極質量分析に５μＬを使用した。
-Sample preparation To 20 μL of rat plasma, 20 μL of an internal standard solution labeled with a stable isotope of amino acid was added, 40 μL of acetonitrile was added, and the mixture was centrifuged at 20 ° C. and 20000 × g for 10 minutes, and the supernatant was collected. Derivatization reagent solution in 10 μL of supernatant 2.5 mg / mL (R) -4-nitrophenyl-N- [2'-(diethylamino) -6,6'-dimethyl- [1,1'-biphenyl] -2 -Ilcarbamate hydrochloride [(R) -BiAc]] was added and reacted at 55 ° C. for 10 minutes, then 100 μL of 0.1% formic acid aqueous solution was added, and the mixture was stirred and liquid chromatography / triple quadruplex. 5 μL was used for polar mass spectrometry.
・液体クロマトグラフィーによる分離
液体クロマトグラフィーシステム（ＬＣ）は島津製作所社製のＮｅｘｅｒａ（登録商標）を用いた。分析カラムの温度は４０℃に設定した。使用した分析カラム、溶離液及び流速は以下に示すとおりである。
-Separation by liquid chromatography For the liquid chromatography system (LC), Nexera (registered trademark) manufactured by Shimadzu Corporation was used. The temperature of the analysis column was set to 40 ° C. The analytical columns, eluents and flow rates used are as shown below.
（分析カラム）
ＹＭＣ社製 Ｔｒｉａｒｔ Ｐｈｅｎｙｌ（２．１ｍｍ×７５ｍｍ １．９μｍ）
（溶離液）
Ａ溶離液：１０ｍｍｏｌ／Ｌ ギ酸アンモニウム／０．１％ギ酸／水
Ｂ溶離液：９５％アセトニトリル／水
（流速）
０．４ｍＬ／ｍｉｎ
(Analysis column)
Triart Phenyl (2.1 mm x 75 mm 1.9 μm) manufactured by YMC
(Eluent)
A Eluent: 10 mmol / L Ammonium formate / 0.1% Formic acid / water B Eluent: 95% acetonitrile / water (flow rate)
0.4 mL / min
ＬＣによる分離は、Ａ溶離液８６％、Ｂ溶離液１４％で平衡化した分析カラムへ分析試料を導入した。Ｂ溶離液を３分で１６％まで、次いで１４．３分まで３３％、１７分まで４５％まで直線的に上昇させて分析をアミノ酸を溶出した。次いで、１７．１分からＢ溶離液を９０％まで上昇させ、１８分まで約１分間分析カラムを洗浄し、その後、２０分まで初期状態（Ａ溶離液８６％、Ｂ溶離液１４％）で平衡化した。 For separation by LC, the analytical sample was introduced into an analytical column equilibrated with 86% A eluent and 14% B eluent. Amino acids were eluted by linearly increasing the B eluate to 16% in 3 minutes, then 33% to 14.3 minutes and 45% to 17 minutes. Then, from 17.1 minutes, the B eluent is raised to 90%, the analytical column is washed for about 1 minute until 18 minutes, and then equilibrated in the initial state (A eluent 86%, B eluent 14%) until 20 minutes. It became.
・質量分析計による検出
質量分析計は、Ｓｃｉｅｘ社製のＴｒｉｐｌｅ Ｑｕａｄ ６５００（登録商標）を用い、エレクトロスプレーイオン化（正イオン）、ＳＲＭモードを使用した。質量分析は以下の条件で行った。
-Detection by mass spectrometer As the mass spectrometer, Triple Quad 6500 (registered trademark) manufactured by Siex was used, and electrospray ionization (positive ion) and SRM mode were used. Mass spectrometry was performed under the following conditions.
（イオンソースのパラメーター）
Ｃｕｒｔａｉｎ：４０
Ｃｏｌｌｉｓｉｏｎ Ｇａｓ Ｓｅｔｔｔｉｎｇ：８
Ｉｏｎ Ｓｐｒａｙ Ｖｏｌｔａｇｅ：４５００
Ｔｅｍｐｅｒａｔｕｒｅ：６００
Ｉｏｎ Ｓｏｕｒｃｅ Ｇａｓ １：７０
Ｉｏｎ Ｓｏｕｒｃｅ Ｇａｓ ２：７０
Ｉｎｔｅｒｆａｃｅ Ｈｅａｔｅｒ：ＯＮ
(Ion source parameters)
Curtain: 40
Collision Gas Setting: 8
Ion Spray Voltage: 4500
Temperature: 600
Ion Source Gas 1:70
Ion Source Gas 2:70
Interface Heater: ON
Ｌ体のアミノ酸は同位体由来のプロトン化分子のm+2に設定した。また、Ｌ−Ｈｉｓ，Ｌ−Ａｒｇ，Ｌ−Ｌｙｓは２価イオンで検出した。各アミノ酸のＩＳは各Ｄ体及びＬ体のアミノ酸を定量する上で使用した内標準物質を示す。 The L-form amino acid was set to m + 2, which is an isotope-derived protonated molecule. In addition, L-His, L-Arg, and L-Lys were detected as divalent ions. The IS of each amino acid indicates the internal standard substance used in quantifying the amino acids of each D-form and L-form.
・定量方法
各種アミノ酸の血中の濃度は、ＬＣから分離された各アミノ酸のクロマトグラムのピーク面積を利用し、内標準法を選択して定量した。すなわち、ｘ軸に既知濃度となるアミノ酸の濃度を、ｙ軸にピーク面積比（各種アミノ酸のピーク面積値／内標準物質のピーク面積値）をプロットし、最小二乗法により検量線を作成した。次に、正常群及び疲労群のラット試料の各アミノ酸のクロマトグラムから、同様にピーク面積比（各種アミノ酸のピーク面積値／内標準物質のピーク面積値）を算出し、検量線より各種アミノ酸の濃度を算出した。
-Quantification method The blood concentrations of various amino acids were quantified by selecting the internal standard method using the peak area of the chromatogram of each amino acid separated from LC. That is, the concentration of amino acids having a known concentration was plotted on the x-axis, and the peak area ratio (peak area value of various amino acids / peak area value of the internal standard substance) was plotted on the y-axis, and a calibration curve was prepared by the least squares method. Next, the peak area ratio (peak area value of various amino acids / peak area value of the internal standard substance) was similarly calculated from the chromatograms of each amino acid of the rat samples of the normal group and the fatigue group, and the various amino acids were calculated from the calibration curve. The concentration was calculated.
ＬＣ−ＭＳ／ＭＳのデータ取得及び解析ソフトウエアはＳｃｉｅｘ社製のＡｎａｌｙｓｔ（登録商標）を使用し、試料中の各アミノ酸のクロマトグラムから定量値を算出した。 As the LC-MS / MS data acquisition and analysis software, Analist (registered trademark) manufactured by Siex was used, and quantitative values were calculated from chromatograms of each amino acid in the sample.
統計解析は、ＳＡＳ９．４（ＳＡＳ Ｉｎｓｔｉｔｕｔｅ Ｉｎｃ．）およびその連動システムＥＸＳＵＳ ｖｅｒ．８．１（株式会社ＣＡＣクロア）を用い、ｔ検定を実施した。 Statistical analysis is performed by SAS 9.4 (SAS Institute Inc.) and its interlocking system EXSUS ver. A t-test was performed using 8.1 (CAC Croix Co., Ltd.).
実施例１の血中Ｄ体アミノ酸濃度測定の結果を表４に示す。表中の数値は、６例の平均値と標準誤差（μＭ）を示す。表中の「＊」は、正常群と比較して統計学的に有意（Ｐ＜０．０５）な差であることを示す。 Table 4 shows the results of measuring the blood D-amino acid concentration of Example 1. The numerical values in the table show the average value and standard error (μM) of the 6 cases. "*" In the table indicates that the difference is statistically significant (P <0.05) as compared with the normal group.
正常群のアルギニン、セリン、スレオニン、アラニン、プロリン、バリンおよびイソロイシン濃度は、それぞれ１．９２、１．４８、０．１０、７．２１、０．４０、０．１４および０．２１μＭであった。これに対し、疲労群のアルギニン、セリン、スレオニン、アラニン、プロリン、バリンおよびイソロイシン濃度は、それぞれ１．１４、１．１３、０．０７、２．４９、０．１０、０．０９および０．１２μＭであり、正常群と比較して、有意に減少した。一方、正常群のメチオニン濃度は、３．０２μＭであった。これに対し、疲労群では、４．６４μＭであり、正常群と比較して、有意に増加した。 The concentrations of arginine, serine, threonine, alanine, proline, valine and isoleucine in the normal group were 1.92, 1.48, 0.10, 7.21, 0.40, 0.14 and 0.21 μM, respectively. .. In contrast, the concentrations of arginine, serine, threonine, alanine, proline, valine and isoleucine in the fatigue group were 1.14, 1.13, 0.07, 2.49, 0.10, 0.09 and 0. It was 12 μM, which was significantly decreased as compared with the normal group. On the other hand, the methionine concentration in the normal group was 3.02 μM. On the other hand, in the fatigue group, it was 4.64 μM, which was significantly increased as compared with the normal group.
実施例１の血中Ｄ体アミノ酸濃度と対応するＬ体アミノ酸濃度の比を表５に示す。表中の数値は、６例の平均値と標準誤差（％）を示す。表中の「＊」は、正常群と比較して統計学的に有意（Ｐ＜０．０５）な差であることを示す。正常群のアスパラギン、アラニン、プロリン、バリン、イソロイシン、ロイシンおよびフェニルアラニンのＤ体アミノ酸濃度とそれぞれ対応するＬ体アミノ酸濃度の比は、それぞれ２．９０、２．３１、０．２６、０．０６、０．１９、０．２９および０．０８％であった。これに対し、疲労群のアスパラギン、アラニン、プロリン、バリン、イソロイシン、ロイシンおよびフェニルアラニンのＤ体アミノ酸濃度とそれぞれ対応するＬ体アミノ酸濃度の比は、それぞれ２．２６、１．０３、０．０８、０．０３、０．０８、０．１６および０．０６％であり、正常群と比較して、有意に減少した。一方、正常群のＤ体メチオニン濃度とＬ体メチオニン濃度比は、５．１６％であった。これに対し、疲労群では、９．３５％であり、正常群と比較して、有意に増加した。 Table 5 shows the ratio of the blood D-amino acid concentration of Example 1 to the corresponding L-amino acid concentration. The numerical values in the table show the average value of 6 cases and the standard error (%). "*" In the table indicates that the difference is statistically significant (P <0.05) as compared with the normal group. The ratios of the D-amino acid concentrations of asparagine, alanine, proline, valine, isoleucine, leucine and phenylalanine in the normal group to the corresponding L-amino acid concentrations were 2.90, 2.31, 0.26 and 0.06, respectively. It was 0.19, 0.29 and 0.08%. On the other hand, the ratios of the D-amino acid concentrations of asparagine, alanine, proline, valine, isoleucine, leucine and phenylalanine in the fatigue group to the corresponding L-amino acid concentrations were 2.26, 1.03 and 0.08, respectively. It was 0.03, 0.08, 0.16 and 0.06%, which were significantly decreased as compared with the normal group. On the other hand, the ratio of D-form methionine concentration to L-form methionine concentration in the normal group was 5.16%. On the other hand, in the fatigue group, it was 9.35%, which was significantly increased as compared with the normal group.
この結果から、本発明のＤ体アミノ酸又はＤ体アミノ酸濃度と対応するＬ体アミノ酸濃度の比が、疲労状態で変化することが示された。 From this result, it was shown that the ratio of the D-amino acid or the D-amino acid concentration of the present invention to the corresponding L-amino acid concentration changes in the fatigued state.
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