JP6598425B2 - Fatigue evaluation method - Google Patents
Fatigue evaluation method Download PDFInfo
- Publication number
- JP6598425B2 JP6598425B2 JP2014036554A JP2014036554A JP6598425B2 JP 6598425 B2 JP6598425 B2 JP 6598425B2 JP 2014036554 A JP2014036554 A JP 2014036554A JP 2014036554 A JP2014036554 A JP 2014036554A JP 6598425 B2 JP6598425 B2 JP 6598425B2
- Authority
- JP
- Japan
- Prior art keywords
- fatigue
- acid
- concentration
- subject
- biological sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Description
本発明は、疲労の評価方法及び疲労評価マーカーに関する。 The present invention relates to a fatigue evaluation method and a fatigue evaluation marker.
疲労は、現代の社会に生きる人が日常向き合っている現象である。我が国で疲労感を自覚しているヒトの割合は、就労人口の約６０％以上とも云われている。
このような状況の中、疲労の指標化が望まれ、疲労のバイオマーカーの探索研究等が精力的になされてきた。
Fatigue is a phenomenon in which people living in modern society face each other everyday. It is said that the proportion of people who are aware of fatigue in Japan is about 60% or more of the working population.
Under such circumstances, it has been desired to index fatigue, and research on biomarkers of fatigue has been energetically performed.
例えば、特許文献１には、健常者と慢性疲労症候群の患者の間にグルコース、クエン酸、ｃｉｓ−アコニット酸、イソクエン酸、コハク酸、リンゴ酸、乳酸の量的差異が見られ、被験者の生体サンプル中のこれらの健常者の測定値に対する比率を算出し、疲労を評価する方法が報告されている。
また、特許文献２には、生体個体から分離した血液中の総アミノ酸、分岐鎖アミノ酸、芳香族アミノ酸、システイン、メチオニン、リジン、アルギニン、ヒスチジン、バリン、ロイシン、及びイソロイシンからなる群より選択されるアミノ酸の濃度が所定の値より低い場合を精神疲労の疲労度が高いと評価し、グリシン、プロリン、アラニン、アスパラギン、リジン及びヒスチジンからなる群より選択されるアミノ酸の濃度が所定の値より低い場合を肉体疲労の疲労度が高いと評価し、トリプトファンの濃度が所定の値より低い場合を複合的疲労の疲労度として評価する方法が報告されている。
For example, Patent Document 1 shows quantitative differences in glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid, and lactic acid between healthy subjects and patients with chronic fatigue syndrome. A method for calculating the ratio of these healthy subjects in the sample to the measured values and evaluating fatigue has been reported.
Further, Patent Document 2 is selected from the group consisting of total amino acids in blood separated from living organisms, branched chain amino acids, aromatic amino acids, cysteine, methionine, lysine, arginine, histidine, valine, leucine, and isoleucine. When the amino acid concentration is lower than the predetermined value, the fatigue level of mental fatigue is evaluated as high, and the amino acid concentration selected from the group consisting of glycine, proline, alanine, asparagine, lysine and histidine is lower than the predetermined value Has been reported that the fatigue level of physical fatigue is evaluated as being high, and the case where the concentration of tryptophan is lower than a predetermined value is evaluated as the fatigue level of combined fatigue.
しかしながら、従来の技術は、半年以上続く慢性的な疲労を抱える者、すなわち慢性疲労症候群の病的な疲労や、一時的な肉体負荷・精神負荷を与えたときの急性の変化による疲労を評価対象としたもので、健常なヒトの、日々の生活や労働により生じる疲労を評価するものではなかった。
したがって、本発明は、健常者の日常の生活や労働で生じる疲労を客観的に評価するために有用なマーカーを特定し、当該マーカーを用いた疲労を評価するための方法を提供することに関する。
However, the conventional technology evaluates those who have chronic fatigue that lasts more than half a year, that is, morbid fatigue of chronic fatigue syndrome, and fatigue due to acute changes when temporary physical or mental stress is applied. It was not intended to evaluate the fatigue caused by daily life and labor of healthy humans.
Therefore, the present invention relates to providing a method for identifying a marker useful for objectively evaluating fatigue that occurs in the daily life and work of a healthy person and for evaluating fatigue using the marker.
本発明者らは、上記課題に鑑み、健常な労働者について、１週間分の疲労が蓄積していると認められた金曜日の血中成分と、疲労が回復していると認められた休養日後の月曜日の血中成分を比較したところ、これまで知られていなかった化合物の量的な変化が見られることを見出した。そして、当該特定の化合物が、健常者の日常の生活や労働で生じる疲労を客観的に評価するために有用なマーカーとなり得ることを見出した。 In view of the above-mentioned problems, the present inventors have found that a healthy worker has a blood component on Friday in which one week of fatigue has been accumulated, and after a rest day in which fatigue has been confirmed to have recovered. As a result of comparing the blood components on Monday, it was found that there was a quantitative change of the compound that was not known so far. And the said specific compound discovered that it could become a useful marker in order to objectively evaluate the fatigue which arises in a normal person's daily life and work.
すなわち、本発明は、次の工程（ａ）〜（ｄ）：
（ａ）被験者から採取された生体試料中のアザライン酸の濃度を測定する工程、
（ｂ）被験者から採取された生体試料中のピメリン酸の濃度を測定する工程、
（ｃ）被験者から採取された生体試料中の２−アミノ酪酸の濃度を測定する工程、
（ｄ）被験者から採取された生体試料中のシトルリンの濃度を測定する工程、
より選ばれる１又は２以上の工程を含む、疲労の評価方法を提供するものである。
また、本発明は、アザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンより選ばれる１種又は２種以上からなる疲労評価マーカーを提供するものである。
That is, the present invention includes the following steps (a) to (d):
(A) a step of measuring the concentration of azaline acid in a biological sample collected from a subject;
(B) measuring the concentration of pimelic acid in a biological sample collected from a subject;
(C) measuring the concentration of 2-aminobutyric acid in a biological sample collected from a subject;
(D) a step of measuring the concentration of citrulline in a biological sample collected from a subject;
The present invention provides a method for evaluating fatigue, including one or more steps selected from the above.
Moreover, this invention provides the fatigue evaluation marker which consists of 1 type, or 2 or more types chosen from azaline acid, pimelic acid, 2-aminobutyric acid, and citrulline.
本発明によれば、健常者の日常の生活や労働で生じる疲労を客観的に評価することができる。これにより、疲労度を的確に捉え、適切な人材を選択することや、状態に応じたアドバイスを与えることが可能である。 According to the present invention, it is possible to objectively evaluate fatigue caused by daily life and work of a healthy person. As a result, it is possible to accurately grasp the degree of fatigue, select an appropriate human resource, and give advice in accordance with the state.
以下、本発明の実施の形態を説明する。
図１は本発明の疲労の評価方法の概略フローチャートであり、当該疲労の評価方法では、被験者から採取された生体試料の分析を行う。詳細には、次の工程（ａ）〜（ｄ）：
（ａ）被験者から採取された生体試料中のアザライン酸の濃度を測定する工程、
（ｂ）被験者から採取された生体試料中のピメリン酸の濃度を測定する工程、
（ｃ）被験者から採取された生体試料中の２−アミノ酪酸の濃度を測定する工程、
（ｄ）被験者から採取された生体試料中のシトルリンの濃度を測定する工程、
より選ばれる１又は２以上の工程を行う。
本発明において、疲労とは、肉体的及び精神的活動、又は疾病によって生じた独特の不快感と休養の願望を伴う身体の活動能力の減退状態を云い、疲労度とは該疲労の度合いをいう。
疲労は、前記のとおり、病的な疲労とそれ以外の生理的な疲労に分類されるが、本発明における疲労は病的な疲労ではなく、生理的な、肉体的及び精神的な疲労を含む複合疲労であることが好ましい。
すなわち、本発明の方法が適用される被験者は、健常者、更に健常労働者、更に健常なディスクワーカーであることが好ましい。
Embodiments of the present invention will be described below.
FIG. 1 is a schematic flowchart of a fatigue evaluation method of the present invention. In the fatigue evaluation method, a biological sample collected from a subject is analyzed. Specifically, the following steps (a) to (d):
(A) a step of measuring the concentration of azaline acid in a biological sample collected from a subject;
(B) measuring the concentration of pimelic acid in a biological sample collected from a subject;
(C) measuring the concentration of 2-aminobutyric acid in a biological sample collected from a subject;
(D) a step of measuring the concentration of citrulline in a biological sample collected from a subject;
1 or 2 or more processes chosen from more are performed.
In the present invention, fatigue refers to a state of reduced physical activity with physical and mental activities, or unique discomfort caused by illness and desire for rest, and the degree of fatigue refers to the degree of fatigue. .
As described above, fatigue is classified into pathological fatigue and other physiological fatigue, but the fatigue in the present invention is not pathological fatigue but includes physiological, physical and mental fatigue. Complex fatigue is preferred.
That is, the subject to which the method of the present invention is applied is preferably a healthy person, further a healthy worker, and a more healthy disk worker.
本発明において、生体試料としては、例えば、血液（全血、血漿、血清等）、髄液、汗、尿、涙液、唾液等が挙げられる。なかでも、血液が好ましい。 In the present invention, examples of biological samples include blood (whole blood, plasma, serum, etc.), spinal fluid, sweat, urine, tears, saliva and the like. Of these, blood is preferred.
生体試料中のアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンの濃度を測定する方法は、従来公知の方法でよいが、後述する実施例に記載の方法に準ずる方法が好ましい。 Although the method of measuring the concentration of azaline acid, pimelic acid, 2-aminobutyric acid and citrulline in a biological sample may be a conventionally known method, a method according to the method described in the examples described later is preferable.
本発明では、被験者から採取された生体試料中のアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンから選ばれる少なくとも１種の濃度に基づいて疲労が評価される。好ましくは、アザライン酸、ピメリン酸、２−アミノ酪酸、シトルリンの濃度の上昇或いは低下により被験者の疲労を評価することができる。この際、アザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンに関して予め所定の閾値を設定し、被験者の生体試料中の濃度と該所定の閾値を比較する工程を含むのが好ましい。 In the present invention, fatigue is evaluated based on at least one concentration selected from azaline acid, pimelic acid, 2-aminobutyric acid and citrulline in a biological sample collected from a subject. Preferably, the subject's fatigue can be evaluated by increasing or decreasing the concentrations of azaline acid, pimelic acid, 2-aminobutyric acid, and citrulline. At this time, it is preferable to include a step of setting a predetermined threshold in advance for azaline acid, pimelic acid, 2-aminobutyric acid and citrulline, and comparing the concentration in the biological sample of the subject with the predetermined threshold.
後述するように、被験者の疲労度が高いと血中のアザライン酸、２−アミノ酪酸及びシトルリンの濃度は高くなり、一方、ピメリン酸の濃度は低くなることが見出された。したがって、アザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンの濃度は、被験者の疲労を反映する指標となり得る。
すなわち、アザライン酸、２−アミノ酪酸及びシトルリンについては、被験者から採取された生体試料中の濃度が高い場合、被験者の疲労度が高いと判定され、濃度が低い場合、被験者の疲労度が低いと判定される。
好ましくは、生体試料中のアザライン酸、２−アミノ酪酸又はシトルリンの濃度が、これらに関して予め設定した第１、第３又は第４の所定の閾値より高いと被験者の疲労度が高いと判定され、濃度が該第１、第３又は第４の所定の閾値未満であると被験者の疲労度が低いと判定される。
他方、ピメリン酸については、被験者から採取された生体試料中の濃度が低い場合、被験者の疲労度が高いと判定され、濃度が高い場合、被験者の疲労度が低いと判定される。
好ましくは、生体試料中のピメリン酸の濃度が、これに関して予め設定した第２の所定の閾値未満であると被験者の疲労度が高いと判定され、濃度が該第２の所定の閾値より高いと被験者の疲労度が低いと判定される。
閾値は、この値を超えると或いは満たないと疲労度が高い、疲労が蓄積していると判断される基準となる値である。
As will be described later, it has been found that when the subject's fatigue level is high, the concentrations of azaline acid, 2-aminobutyric acid and citrulline in the blood are high, while the concentration of pimelic acid is low. Therefore, the concentration of azaline acid, pimelic acid, 2-aminobutyric acid and citrulline can be an index reflecting the fatigue of the subject.
That is, for azaline acid, 2-aminobutyric acid and citrulline, when the concentration in the biological sample collected from the subject is high, it is determined that the subject's fatigue level is high, and when the concentration is low, the subject's fatigue level is low Determined.
Preferably, when the concentration of azaline acid, 2-aminobutyric acid, or citrulline in the biological sample is higher than the first, third, or fourth predetermined threshold set in advance with respect to these, it is determined that the fatigue level of the subject is high, If the concentration is less than the first, third, or fourth predetermined threshold, it is determined that the subject's fatigue level is low.
On the other hand, for pimelic acid, when the concentration in the biological sample collected from the subject is low, it is determined that the subject's fatigue is high, and when the concentration is high, the subject's fatigue is determined to be low.
Preferably, when the concentration of pimelic acid in the biological sample is less than a second predetermined threshold set in advance, it is determined that the subject's fatigue is high, and the concentration is higher than the second predetermined threshold. It is determined that the subject's fatigue level is low.
The threshold value is a value serving as a reference for determining that fatigue is high when this value is exceeded or not, and fatigue is high.
閾値は、例えば、後述するＶＡＳ評価で「疲労あり」の被験者、「疲労なし」の被験者毎に、生体試料中の各成分についてＲＯＣ曲線（Ｒｅｃｅｉｖｅｒ Ｏｐｅｒａｔｉｎｇ Ｃｈａｒａｃｔｅｒｉｓｔｉｃ ｃｕｒｖｅ、受信者動作特性曲線）を作成し、該ＲＯＣ曲線を用いて設定することができる。 For example, for each of the components in the biological sample, a ROC curve (Receiver Operating Characteristic curve, receiver operating characteristic curve) is created for each component in the biological sample for each subject having “fatigue” and “no fatigue” in the VAS evaluation described below. , And can be set using the ROC curve.
この際、診査の信頼性を図る尺度である感度・特異度・有効度をそれぞれ任意に設定し、その割合に応じた閾値を設定することができる。
感度は、疲労感がある被験者が陽性となる割合（真陽性率）を意味し、特異度は疲労感のない被験者が陰性となる割合（真陰性率）を意味する。特異度が高くなるように閾値を設定すると偽陰性が多くなり、感度が低くなる。一方、感度が高くなるように設定すると偽陽性が多くなり、特異度は低くなる。そこで、閾値は、目的に応じて決定されるのが好ましい。
At this time, it is possible to arbitrarily set sensitivity, specificity, and effectiveness, which are measures for improving the reliability of the examination, and to set a threshold corresponding to the ratio.
Sensitivity means the rate at which subjects with fatigue feel positive (true positive rate), and specificity means the rate at which subjects without fatigue feel negative (true negative rate). If the threshold is set so that the specificity is high, false negatives increase and sensitivity decreases. On the other hand, if the sensitivity is set to be high, false positives increase and specificity decreases. Therefore, the threshold is preferably determined according to the purpose.
閾値は、予想される疲労度に応じて変えるのが好ましい。
例えば、アザライン酸の閾値は好ましくは０．７８μｍｏｌ／Ｌ以上１．１μｍｏｌ／Ｌ以下に、更に好ましくは０．９６μｍｏｌ／Ｌに、ピメリン酸の閾値は好ましくは１．１４μｍｏｌ／Ｌ以上１．７２μｍｏｌ／Ｌ以下に、更に好ましくは１．２２μｍｏｌ／Ｌに、２−アミノ酪酸の閾値は、好ましくは２２．５μｍｏｌ／Ｌ以上２７．３μｍｏｌ／Ｌ以下に、更に好ましくは２４．９μｍｏｌ／Ｌ、シトルリンの閾値は３７．５μｍｏｌ／Ｌに設定することが好ましい。
疲労度が低いことが明らかな被験者の場合は、２−アミノ酪酸の閾値を２６．８μｍｏｌ／Ｌ、シトルリンの閾値を４３．４μｍｏｌ／Ｌに設定することがより好ましい。
また、疲労度が高いことが明らかな被験者の場合は、シトルリンの閾値を３２．７μｍｏｌ／Ｌに設定する。
例えば、後述する実施例では、アザライン酸の閾値を０．９６μｍｏｌ／Ｌとすると、感度、特異度共に１００％となり、有効度が１００％と極めて信頼性の高い評価法となる。また、ピメリン酸の閾値を１．２２μｍｏｌ／Ｌとすると、感度、特異度共に１００％となり、有効度が１００％と極めて信頼性の高い評価法となる。
The threshold is preferably changed according to the expected degree of fatigue.
For example, the threshold of azaline acid is preferably 0.78 μmol / L or more and 1.1 μmol / L or less, more preferably 0.96 μmol / L, and the threshold of pimelic acid is preferably 1.14 μmol / L or more and 1.72 μmol / L. L or less, more preferably 1.22 μmol / L, and the threshold value of 2-aminobutyric acid is preferably 22.5 μmol / L or more and 27.3 μmol / L or less, more preferably 24.9 μmol / L, a threshold value of citrulline. Is preferably set to 37.5 μmol / L.
In the case of a subject whose low degree of fatigue is apparent, it is more preferable to set the threshold value of 2-aminobutyric acid to 26.8 μmol / L and the threshold value of citrulline to 43.4 μmol / L.
In addition, in the case of a test subject whose fatigue level is clear, the citrulline threshold is set to 32.7 μmol / L.
For example, in the examples described later, when the azaline acid threshold is 0.96 μmol / L, both sensitivity and specificity are 100%, and the effectiveness is 100%, which is an extremely reliable evaluation method. Moreover, when the threshold value of pimelic acid is 1.22 μmol / L, both sensitivity and specificity are 100%, and the effectiveness is 100%, which is an extremely reliable evaluation method.
また、後述するように、被験者の疲労度が上昇すると血中のアザライン酸、２−アミノ酪酸及びシトルリンの濃度は高くなり、一方、ピメリン酸の濃度は低くなることが見出された。したがって、アザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンの濃度は、被験者の疲労の増減を判断する指標となり得る。すなわち、被験者から２回以上生体試料を採取し、これらの濃度の変化を検出することにより、被験者の疲労の上昇又は低下を判定することができる。
より詳しく述べると、被験者の生体試料をあらかじめ採取してアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンから選ばれる少なくとも１種の濃度を測定しておく。そして、疲労の増減を判断したいときに被験者から再び生体試料を採取する。ｎ回目（ｎは２以上の正の整数）に採取された生体試料中のアザライン酸、２−アミノ酪酸及びシトルリンについては、被験者から採取された生体試料中の濃度が対照とする基準時（１回目〜（ｎ−１）回目）より上昇した場合、被験者の疲労が上昇したと判定され、濃度が低下した場合、被験者の疲労が低下したと判定される。
他方、ピメリン酸については、被験者からｎ回目に採取された生体試料中の濃度が対照とする基準時より低下した場合、被験者の疲労が上昇したと判定され、濃度が上昇した場合、被験者の疲労が低下したと判定される。
Further, as described later, it was found that the concentration of azaline acid, 2-aminobutyric acid, and citrulline in the blood increased while the concentration of pimelic acid decreased as the fatigue level of the subject increased. Therefore, the concentration of azaline acid, pimelic acid, 2-aminobutyric acid, and citrulline can be an index for determining the increase or decrease in fatigue of the subject. That is, it is possible to determine an increase or decrease in the fatigue of the subject by collecting biological samples twice or more from the subject and detecting changes in these concentrations.
More specifically, a biological sample of a subject is collected in advance, and at least one concentration selected from azaline acid, pimelic acid, 2-aminobutyric acid and citrulline is measured. Then, when it is desired to determine the increase or decrease in fatigue, a biological sample is again collected from the subject. As for azaline acid, 2-aminobutyric acid and citrulline in a biological sample collected at the nth time (n is a positive integer of 2 or more), the concentration in the biological sample collected from the subject is a reference time (1 It is determined that the subject's fatigue has increased when the number of times increases from the first time to the (n-1) th), and when the concentration has decreased, it is determined that the subject's fatigue has decreased.
On the other hand, for pimelic acid, if the concentration in the biological sample collected n times from the subject is lower than the reference time as a control, it is determined that the subject's fatigue has increased, and if the concentration has increased, the subject's fatigue Is determined to have decreased.
更に、本発明の疲労評価マーカー、すなわちアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンを指標とすれば、抗疲労物質がスクリーニングできる。すなわち、ｉｎ ｖｉｔｒｏ又はｉｎ ｖｉｖｏにおいて、ある物質によりアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンから選ばれる少なくとも１種の濃度が変動すれば、その物質は抗疲労物質となり得る。 Furthermore, anti-fatigue substances can be screened using the fatigue evaluation markers of the present invention, that is, azaline acid, pimelic acid, 2-aminobutyric acid and citrulline as indices. That is, if the concentration of at least one selected from azaline acid, pimelic acid, 2-aminobutyric acid and citrulline varies depending on a certain substance in vitro or in vivo, the substance can be an anti-fatigue substance.
また、本発明の疲労評価マーカーを利用した疲労のメカニズムの解明等が期待できる。
疲労は画一の症状ではなく、疲労の原因や影響も多岐にわたることが知られている。一方、アザライン酸は、β酸化を受けてマロニルＣｏＡとアセチルＣｏＡになりＴＣＡサイクルに入ることが知られている。ピメリン酸もβ酸化に関連する。また２−アミノ酪酸も、ＴＣＡサイクルに関連する。シトルリンは、尿素サイクルで生成されるアミノ酸である。これらのことから、疲労、ことに健常者の日常の生活や労働で生じる疲労はβ酸化やＴＣＡサイクル、尿素サイクルに影響すると想像できる。
したがって、本発明の疲労評価マーカー、すなわちアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンを指標とすれば、疲労が影響している代謝系を明らかにできる。すなわち、アザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンから選ばれる少なくとも１種の濃度が変動すれば、疲労が影響する代謝系を解明できる。疲労が影響する代謝系を明らかにすることにより、その疲労のメカニズムの解明等が期待でき、更に、疲労が影響した代謝系が明らかになれば、その疲労改善に有用な代謝系、更には生成系が予測でき、疲労の種類に応じた効果的な疲労改善が期待できる。
Moreover, elucidation of the mechanism of fatigue using the fatigue evaluation marker of the present invention can be expected.
It is known that fatigue is not a uniform symptom, but causes and effects of fatigue vary. On the other hand, azaline acid is known to undergo β-oxidation to become malonyl CoA and acetyl CoA and enter the TCA cycle. Pimelic acid is also associated with β-oxidation. 2-Aminobutyric acid is also associated with the TCA cycle. Citrulline is an amino acid produced in the urea cycle. From these facts, it can be imagined that fatigue, especially fatigue caused by the daily life and work of a healthy person, affects β-oxidation, TCA cycle, and urea cycle.
Therefore, if the fatigue evaluation markers of the present invention, that is, azaline acid, pimelic acid, 2-aminobutyric acid and citrulline are used as indices, the metabolic system affected by fatigue can be clarified. That is, if the concentration of at least one selected from azaline acid, pimelic acid, 2-aminobutyric acid and citrulline varies, the metabolic system affected by fatigue can be clarified. By clarifying the metabolic system affected by fatigue, elucidation of the mechanism of the fatigue can be expected, and when the metabolic system affected by fatigue is clarified, the metabolic system useful for improving fatigue is further generated. The system can be predicted, and an effective fatigue improvement according to the type of fatigue can be expected.
最初に、閾値の設定に用いた計測、および本発明での計測における化合物の分析方法を示す。
シトルリン、２−アミノ酪酸等の血中（血漿）アミノ酸の分析は以下のアミノ酸分析方法に従った。
［試料の調製（前処理）］
生体試料（血漿）１０μＬを正確に測り、ＭａｓｓＴｒａｋ ＡＡＡ試薬１（ホウ酸バッファ）を７０μＬ添加し、攪拌後、ＭａｓｓＴｒａｋ ＡＡＡ試薬２を２０μＬ添加後に攪拌し、ヒートブロック（５５℃）で１０分加温しアミノ酸分析用試料とした。
First, the measurement used for setting the threshold and the method for analyzing the compound in the measurement according to the present invention will be described.
Analysis of blood (plasma) amino acids such as citrulline and 2-aminobutyric acid was performed according to the following amino acid analysis method.
[Preparation of sample (pretreatment)]
Accurately measure 10 μL of biological sample (plasma), add 70 μL of MassTrak AAA Reagent 1 (borate buffer), stir, then add 20 μL of MassTrak AAA Reagent 2 and stir and heat for 10 minutes with a heat block (55 ° C.) A sample for amino acid analysis was used.
［アミノ酸分析条件］
システム： Ｗａｔｅｒｓ ＡＣＱＵＩＴＹ ＵＰＬＣR システム
カラム： ＭａｓｓＴｒａｋTM Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｓｉｓカラム 内径２．１×長さ１５０ｍｍ
移動相Ａ： ＭａｓｓＴｒａｋ溶離液Ａ
移動相Ｂ： ＭａｓｓＴｒａｋ溶離液Ｂ
流量： ０．４ｍＬ／分
カラム温度： ４３℃
サンプル温度： ２０℃
サンプル注入量： １μＬ
検出： ＵＶ２６０ｎｍ
[Amino acid analysis conditions]
System: Waters ACQUITY UPLCR System column: MassTrak ™ Amino Acid Analysis column inner diameter 2.1 x length 150 mm
Mobile phase A: MassTrak eluent A
Mobile phase B: MassTrak eluent B
Flow rate: 0.4 mL / min Column temperature: 43 ° C
Sample temperature: 20 ° C
Sample injection volume: 1 μL
Detection: UV260nm
アザライン酸、ピメリン酸等のアミノ酸以外の化合物の分析は以下のメタボローム解析方法に従った。
［試料の調製（前処理）］
生体試料（血漿）４０μＬを正確に測り、これに内部標準溶液４００μＬ（メチオニンスルホン、カンファースルホン酸、２−モルホリノエタンスルホン酸をそれぞれ２０μｍｏｌ／Ｌ含む）を加える。混合後、クロロホルム４００μＬと超純水１２０μＬを加えて激しく混和後、遠心分離し水層を分取する。分取した水層を限外濾過（分画分子量 ５，０００）で除タンパク後、乾固する。乾固した試料を４０μＬの３−アミノピロリジン，トリメサート水溶液（各２００μＭ／Ｌ）に溶解し、メタボローム解析用の試料とした。
Analysis of compounds other than amino acids such as azaline acid and pimelic acid was performed according to the following metabolome analysis method.
[Preparation of sample (pretreatment)]
A biological sample (plasma) 40 μL is accurately measured, and an internal standard solution 400 μL (containing 20 μmol / L each of methionine sulfone, camphor sulfonic acid, and 2-morpholinoethane sulfonic acid) is added thereto. After mixing, add 400 μL of chloroform and 120 μL of ultrapure water, mix vigorously, and centrifuge to separate the aqueous layer. The separated aqueous layer is deproteinized by ultrafiltration (molecular weight cut off 5,000) and dried. The dried sample was dissolved in 40 μL of 3-aminopyrrolidine / trimesate aqueous solution (200 μM / L each) to prepare a sample for metabolome analysis.
［メタボローム解析（キャピラリー電気泳動／質量分析装置、ＣＥ／ＭＳ）条件］
システム： Ａｇｉｌｅｎｔ ＣＥ−ＴＯＦＭＳ Ｓｙｓｔｅｍ
陽イオン性代謝物質測定モード
高性能キャピラリー電気泳動（Ｈｉｇｈ Ｐｅｒｆｏｒｍａｎｃｅ Ｃａｐｉｌｌａｒｙ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ，ＨＰＣＥ）条件
Ｃａｐｉｌｌａｒｙ： Ｆｕｓｅｄ−Ｓｉｌｉｃａ， 内径５０μｍ×長さ１００ｃｍ
Ｂｕｆｆｅｒ： １Ｍ Ｆｏｒｍａｔｅ
Ｖｏｌｔａｇｅ： Ｐｏｓｉｔｉｖｅ， ３０ｋＶ
Ｔｅｍｐｅｒａｔｕｒｅ： ２０℃
Ｉｎｊｅｃｔｉｏｎ： Ｐｒｅｓｓｕｒｅ ｉｎｊｅｃｔｉｏｎ ５０ｍｂａｒ， ５ｓｅｃ（ａｐｐｒｏｘｉｍａｔｅｌｙ ５ｎＬ）
Ｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇ： ４ｍｉｎ ａｔ ｒｕｎ ｂｕｆｆｅｒ
[Metabolome analysis (capillary electrophoresis / mass spectrometer, CE / MS) conditions]
System: Agilent CE-TOFMS System
Cationic Metabolite Measurement Mode High Performance Capillary Electrophoresis (HPCE) Conditions Capillary: Fused-Silica, inner diameter 50 μm × length 100 cm
Buffer: 1M Format
Voltage: Positive, 30kV
Temperature: 20 ° C
Injection: Pressure injection 50mbar, 5sec (approximated 5nL)
Preconditioning: 4min at run buffer
飛行時間型質量分析（Ｔｉｍｅ−ｏｆ−Ｆｌｉｇｈｔ ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｅｒ， ＴＯＦ−ＭＳ）条件
Ｐｏｌａｒｉｔｙ： Ｐｏｓｉｔｉｖｅ
Ｃａｐｉｌｌａｒｙ ｖｏｌｔａｇｅ： ４，０００Ｖ
Ｆｒａｇｍｅｎｔｏｒ： ７５Ｖ
Ｓｋｉｍｍｅｒ： ５０Ｖ
ＯＣＴ ＲＦＶ： ５００Ｖ
Ｄｒｙｉｎｇ ｇａｓ：Ｎ2， １０Ｌ／ｍｉｎ
Ｄｒｙｉｎｇ ｇａｓ ｔｅｍｐ．：３００℃
Ｎｅｂｕｌｉｚｅｒ ｇａｓ ｐｒｅｓｓ．： ７ｐｓｉｇ
Ｓｈｅａｔｈ ｌｉｑｕｉｄ： ５０％ＭｅＯＨ ／ Ｗａｔｅｒ ｃｏｎｔａｉｎｉｎｇ ０．０１Ｍ Ｈｅｘａｋｉｓ（２，２−ｄｉｆｌｕｏｒｏｅｔｈｏｘｙ） ｐｈｏｓｐｈａｚｅｎｅ
Ｆｌｏｗ ｒａｔｅ： １０Ｌ／ｍｉｎ
Ｌｏｃｋ ｍａｓｓ： ２ＭｅＯＨ13Ｃｉｓｏｔｏｐｅ ｍ／ｚ６６．０６３０６１，
Ｈｅｘａｋｉｓ（２，２−ｄｉｆｌｕｏｒｏｅｔｈｏｘｙ）ｐｈｏｓｐｈａｚｅｎｅ ｍ／ｚ ６２２．０２８９６３
Time-of-flight mass spectrometry (Time-of-Flight mass spectrometer, TOF-MS) conditions Polarity: Positive
Capillary voltage: 4,000V
Fragmentor: 75V
Skimmer: 50V
OCT RFV: 500V
Drying gas: N 2 , 10 L / min
Drying gas temp. : 300 ° C
Nebulizer gas press. : 7 psig
Sheath liquid: 50% MeOH / Water containing 0.01M Hexakis (2,2-difluoroethoxy) phosphazene
Flow rate: 10L / min
Lock mass: 2MeOH 13 Cisotope m / z 66.063061,
Hexakis (2,2-difluoroethoxy) phosphazene m / z 622.0289963
陰イオン性代謝物質測定モード
高性能キャピラリー電気泳動（Ｈｉｇｈ Ｐｅｒｆｏｒｍａｎｃｅ Ｃａｐｉｌｌａｒｙ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ， ＨＰＣＥ）条件
Ｃａｐｉｌｌａｒｙ： ＣＯＳＭＯ（＋），内径５０μｍ×長さ１０５ｃｍ
Ｂｕｆｆｅｒ： ５０ｍＭ Ａｍｍｏｎｉｕｍ ａｃｅｔａｔｅ， ｐＨ８．５
Ｖｏｌｔａｇｅ： Ｎｅｇａｔｉｖｅ，３０ｋＶ
Ｔｅｍｐｅｒａｔｕｒｅ： ２０℃
Ｉｎｊｅｃｔｉｏｎ： Ｐｒｅｓｓｕｒｅ ｉｎｊｅｃｔｉｏｎ ５０ｍｂａｒ， ３０ｓｅｃ（ａｐｐｒｏｘｉｍａｔｅｌｙ ３０ｎＬ）
Ｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇ： ２ｍｉｎ ａｔ ５０ｍＭ Ａｍｍｏｎｉｕｍ ａｃｅｔａｔｅ， ｐＨ３．４ ａｎｄ ５ｍｉｎ ａｔ ｒｕｎ ｂｕｆｆｅｒ
Anionic Metabolite Measurement Mode High Performance Capillary Electrophoresis (HPCE) Conditions Capillary: COSMO (+), inner diameter 50 μm × length 105 cm
Buffer: 50 mM Ammonium acetate, pH 8.5
Voltage: Negative, 30kV
Temperature: 20 ° C
Injection: Pressure injection 50mbar, 30sec (approximate 30nL)
Preconditioning: 2 min at 50 mM Ammonium acetate, pH 3.4 and 5 min at run buffer
飛行時間型質量分析（Ｔｉｍｅ−ｏｆ−Ｆｌｉｇｈｔ ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｅｒ， ＴＯＦ−ＭＳ）条件
Ｐｏｌａｒｉｔｙ： Ｎｅｇａｔｉｖｅ
Ｃａｐｉｌｌａｒｙ ｖｏｌｔａｇｅ： ３，５００Ｖ
Ｆｒａｇｍｅｎｔｏｒ： １００Ｖ
Ｓｋｉｍｍｅｒ ： ５０Ｖ
ＯＣＴ ＲＦＶ： ２００Ｖ
Ｄｒｙｉｎｇ ｇａｓ：Ｎ2， １０Ｌ／ｍｉｎ
Ｄｒｙｉｎｇ ｇａｓ ｔｅｍｐ．： ３００℃
Ｎｅｂｕｌｉｚｅｒ ｇａｓ ｐｒｅｓｓ．： ７ｐｓｉｇ
Ｓｈｅａｔｈ ｌｉｑｕｉｄ：５ｍＭ Ａｍｍｏｎｉｕｍ ａｃｅｔａｔｅ ｉｎ ５０ ％ ＭｅＯＨ／Ｗａｔｅｒ ｃｏｎｔａｉｎｉｎｇ ０．１Ｍ Ｈｅｘａｋｉｓ（２，２−ｄｉｆｌｕｏｒｏｅｔｈｏｘｙ） ｐｈｏｓｐｈａｚｅｎｅ
Ｆｌｏｗ ｒａｔｅ： １０Ｌ／ｍｉｎ
Ｌｏｃｋ ｍａｓｓ：２ＣＨ3ＣＯＯＨ13Ｃｉｓｏｔｏｐｅ ｍ／ｚ １２０．０３８３３９，
Ｈｅｘａｋｉｓ（２，２−ｄｉｆｌｕｏｒｏｅｔｈｏｘｙ）ｐｈｏｓｐｈａｚｅｎｅ＋ＣＨ3ＣＯＯＨ ｍ／ｚ ６８０．０３５５４１
Time-of-flight mass spectrometry (Time-of-Flight mass spectrometer, TOF-MS) conditions Polarity: Negative
Capillary voltage: 3,500V
Fragmentor: 100V
Skimmer: 50V
OCT RFV: 200V
Drying gas: N 2 , 10 L / min
Drying gas temp. : 300 ° C
Nebulizer gas press. : 7 psig
Sheath liquid: 5 mM Ammonium acetate in 50% MeOH / Water containing 0.1M Hexakis (2,2-difluoroethoxy) phosphazene
Flow rate: 10L / min
Lock mass: 2CH 3 COOH 13 Cisotope m / z 120.038339,
Hexakis (2,2-difluoroethoxy) phosphazene + CH 3 COOH m / z 680.035541
計測１（閾値設定）
〔対象者〕
週５日（月曜日〜金曜日勤務、週休２日（土曜日と日曜日））の日勤労働者（標準労働時間８：３０〜１７：００、１時間の昼休み含む）のうち、月曜日から金曜日の５日間の労働により疲労感があり、週末の休息で疲労感がなくなっていた４名の日勤労働者（以下「疲労あり」、４３〜５４歳、平均年齢４８．５±４．９歳）と月曜日から金曜日の５日間の労働により疲労感がない５名の労働者（以下「疲労なし」、２５〜４９歳、平均年齢３６．４±１０．５歳）を対象とした。
Measurement 1 (Threshold setting)
[Target person]
5 days a week (Monday-Friday, 2 days a week (Saturday and Sunday)) of day shift workers (standard working hours 8: 30-17: 00, including 1 hour lunch break) Monday to Friday with four day shift workers who were tired from work but no longer feel tired at rest on weekends (hereinafter “fatigue”, 43-54 years old, average age 48.5 ± 4.9 years old) 5 workers who did not feel tired after 5 days of work (hereinafter “no fatigue”, 25-49 years old, average age 36.4 ± 10.5 years old) were targeted.
〔疲労評価〕
１．ＶＡＳ評価
対象者の主観的な疲労の評価は、日本疲労学会の抗疲労臨床評価ガイドライン第５版（ｈｔｔｐ：／／ｗｗｗ．ｈｉｒｏｕｇａｋｋａｉ．ｃｏｍ／ｇｕｉｄｅｌｉｎｅ．ｐｄｆ）の疲労感ＶＡＳ（Ｖｉｓｕａｌ Ａｎａｌｏｇｕｅ Ｓｃａｌｅ）検査方法（ｈｔｔｐ：／／ｗｗｗ．ｈｉｒｏｕｇａｋｋａｉ．ｃｏｍ／ＶＡＳ．ｐｄｆ）を利用した。
疲労感ＶＡＳ検査は、１００ｍｍの直線の左端（０ｍｍ）を「疲れを全く感じない」、右端（１００ｍｍ）を「何もできないほど疲れきっている」感覚として、今、感じている疲労感を、直線の左右両端に示した感覚を参考に１００ｍｍの直線上に示してもらう方法である。
ＶＡＳ検査は、試験初日の金曜日就寝時から、連続１０日間、毎日就寝時に実施し、２回実施した金曜日、土曜日、日曜日の結果は平均値で示した。
その結果、表１に示すように、「疲労あり」では、金曜日就寝時の数値が高く、金曜日に疲労が蓄積している状態と判断した。「疲労なし」では、数値の大きな上昇は認められず疲労が蓄積していない状態と判断した。
[Fatigue evaluation]
1. VAS Evaluation Subjective fatigue assessment is based on the Fatigue VAS (Visual Analogue Scale) test by the Japanese Fatigue Society Anti-Fatigue Clinical Evaluation Guidelines, 5th edition (http://www.hirogakukai.com/guideline.pdf) The method (http://www.hirogukakai.com/VAS.pdf) was used.
Fatigue feeling VAS inspection, the left edge (0mm) of the 100mm straight line "feels no fatigue at all", the right edge (100mm) as "feeling tired enough to do nothing", feel the fatigue feeling now, This is a method in which the sensation shown on the left and right ends of the straight line is shown on a 100 mm straight line.
The VAS test was carried out at bedtime every day for 10 consecutive days from bedtime on Friday the first day of the test, and the results of Friday, Saturday, and Sunday, which were carried out twice, were shown as average values.
As a result, as shown in Table 1, in the case of “with fatigue”, the numerical value at bedtime on Friday was high, and it was determined that fatigue was accumulated on Friday. In the case of “no fatigue”, a large increase in the numerical value was not recognized and it was judged that fatigue was not accumulated.
２．血中濃度の測定
月曜日と金曜日の早朝の空腹時に採血した。「疲労あり」の金曜日の血液を「疲労」、「疲労あり」の月曜日の血液と「疲労なし」の金曜日の血液を「非疲労」とし、対象者から採取した血液中の表２に示す成分の濃度を測定した。血中（血漿）アミノ酸は、上述のアミノ酸自動分析により測定し、血中（血漿）有機酸は、上述のキャピラリー電気泳動／質量分析装置（ＣＥ／ＭＳ）により測定した。
2. Measurement of blood concentration Blood samples were collected on an early hunger Monday and Friday. “Fatigue” Friday blood is “Fatigue”, “Fatigue” Monday blood and “No fatigue” Friday blood is “Non-fatigue” The concentration of was measured. Blood (plasma) amino acids were measured by the above-described automatic amino acid analysis, and blood (plasma) organic acids were measured by the above-described capillary electrophoresis / mass spectrometer (CE / MS).
その結果、表２に示すように、「疲労」の血液中では、アザライン酸、２−アミノ酪酸及びシトルリンの濃度が増加し、反対にピメリン酸の濃度が減少していることが見出された。
また、「疲労」の血液中では、分岐鎖アミノ酸、バリン、イソロイシン、ロイシン、ヒスチジン、芳香族アミノ酸、トリプトファン、フェニルアラニン、チロシン、アルギニン、アスパラギン、メチオニン、リジンの濃度と２−アミノ酪酸／ロイシン比が増加し、他方、ピルピン酸、乳酸、クエン酸、ｃｉｓ−アコニット酸、イソクエン酸、コハク酸、フマル酸、リンゴ酸、総アミノ酸、アラニン、アスパラギン酸、グルタミン酸の濃度が減少していることが見出された。
すなわち、これらの化合物は疲労の評価マーカーとして使える可能性がある。なかでもアザライン酸、ピメリン酸、２−アミノ酪酸及びシトルリンは、増加や減少の幅に比べて、測定されている疲労時あるいは非疲労時の数値の範囲が小さいので、適切な閾値を定めれば後述するようにその値をそのまま疲労の有無の判定に使うことができる。
As a result, as shown in Table 2, in the “fatigue” blood, it was found that the concentrations of azaline acid, 2-aminobutyric acid and citrulline increased, and conversely, the concentration of pimelic acid decreased. .
In “fatigue” blood, the concentration of branched chain amino acids, valine, isoleucine, leucine, histidine, aromatic amino acids, tryptophan, phenylalanine, tyrosine, arginine, asparagine, methionine, lysine and the 2-aminobutyric acid / leucine ratio On the other hand, it is found that the concentrations of pyruvic acid, lactic acid, citric acid, cis-aconitic acid, isocitric acid, succinic acid, fumaric acid, malic acid, total amino acids, alanine, aspartic acid, glutamic acid are decreased. It was done.
That is, these compounds may be used as fatigue evaluation markers. Among them, azaline acid, pimelic acid, 2-aminobutyric acid and citrulline have a smaller range of measured values during fatigue or non-fatigue than the range of increase or decrease. As will be described later, the value can be used as it is to determine the presence or absence of fatigue.
３．閾値の設定
「疲労」と「非疲労」の、それぞれの血中成分のＲＯＣ曲線（Ｒｅｃｅｉｖｅｒ Ｏｐｅｒａｔｉｎｇ Ｃｈａｒａｃｔｅｒｉｓｔｉｃ ｃｕｒｖｅ、受信者動作特性曲線）を作成し、表４に示す閾値を設定した。
この際、前述したように診査の信頼性を図る尺度である感度・特異度・有効度をそれぞれ設定し、それに応じた閾値を表４に示すように設定した。
具体的には疲労感がある被験者を確実に疲労有りとして検出するためには感度が１００％であることが好ましいので、感度１００％となる閾値を表４に示すように設定した。また、疲労感がない被験者を確実に疲労なしと判定するためには特異度が１００％であることが好ましいので特異度１００％となる閾値を２−アミノ酪酸及びシトルリンについて設定した。また、誤検出を少なくする閾値として、シトルリンについて３７．５μｍｏｌ／Ｌを設定した。
なお、感度・特異度・有効度の算出方法は表３のとおりである。
3. Setting of threshold values ROC curves (Receiver Operating Characteristic curves) of blood components of “fatigue” and “non-fatigue” were created, and the threshold values shown in Table 4 were set.
At this time, as described above, sensitivity, specificity, and effectiveness, which are measures for improving the reliability of the examination, were set, and threshold values corresponding to the sensitivity were set as shown in Table 4.
Specifically, since it is preferable that the sensitivity is 100% in order to reliably detect a subject who has a feeling of fatigue as having fatigue, a threshold value at which the sensitivity becomes 100% is set as shown in Table 4. Moreover, since it is preferable that specificity is 100% in order to reliably determine that there is no fatigue for a subject who does not feel fatigue, a threshold value for setting specificity of 100% was set for 2-aminobutyric acid and citrulline. In addition, 37.5 μmol / L was set for citrulline as a threshold for reducing erroneous detection.
The calculation method of sensitivity, specificity, and effectiveness is as shown in Table 3.
表４に示すように、本発明によれば、的確に疲労を評価できることが確認された。例えば、アザライン酸の閾値を０．９６μｍｏｌ／Ｌとすると、感度、特異度共に１００％となり、疲労度が高いヒト、或いは疲労度が低いヒトを確実に抽出できた。
図２に、表２に示した各疲労評価マーカーの代謝マップを示す。疲労評価マーカーは、解糖系、ＴＣＡサイクル、尿素サイクル、β酸化等のエネルギー代謝系と神経伝達物質のカテコールアミン生成系等に関与していた。図２中、「↑」は濃度が増加したことを示し、「↓」は濃度が減少したことを示す。
As shown in Table 4, according to the present invention, it was confirmed that fatigue can be accurately evaluated. For example, when the threshold value of azaline acid is 0.96 μmol / L, both sensitivity and specificity are 100%, and humans with high or low fatigue levels can be reliably extracted.
FIG. 2 shows a metabolic map of each fatigue evaluation marker shown in Table 2. Fatigue evaluation markers have been involved in glycolysis, TCA cycle, urea cycle, energy metabolism system such as β-oxidation, catecholamine production system of neurotransmitter, and the like. In FIG. 2, “↑” indicates that the concentration has increased, and “↓” indicates that the concentration has decreased.
実施例１
〔対象者〕
週５日（月曜日〜金曜日勤務、週休２日（土曜日と日曜日））の日勤労働者（標準労働時間８：３０〜１７：００、１時間の昼休み含む）のうち、月曜日から金曜日の５日間の労働により疲労感があり、週末の休息で疲労感がなくなっていた４名の日勤労働者（以下、「疲労あり」、４３〜５４歳、平均年齢４８．５±４．９歳）を対象とした。
Example 1
[Target person]
5 days a week (Monday-Friday, 2 days a week (Saturday and Sunday)) of day shift workers (standard working hours 8: 30-17: 00, including 1 hour lunch break) For four day shift workers who were tired from work and who had lost their fatigue after resting on weekends (hereinafter “fatigue”, 43-54 years old, average age 48.5 ± 4.9 years old) did.
〔疲労評価〕
１．ＶＡＳ評価
対象者の主観的な疲労の評価は、計測１と同様にＶＡＳ評価により行った。
その結果、表５に示すように、「疲労あり」の金曜日就寝時の数値が高く、金曜日に疲労が蓄積している状態と判断した。
[Fatigue evaluation]
1. VAS Evaluation Subjective subject's subjective fatigue was evaluated by VAS evaluation as in Measurement 1.
As a result, as shown in Table 5, the value of “with fatigue” on Friday night was high, and it was determined that fatigue was accumulated on Friday.
２．血中濃度の測定
計測１と同様にして、金曜日の早朝の空腹時に対象者から血液を採取し、計測１で定めた閾値に基づく、疲労評価の感度、特異度、有効度を求め、表６に示した。なお、本発明で用いない化合物についても、表中に、適宜閾値を設定して求めた感度、特異度、有効度を示す。
2. Measurement of blood concentration In the same manner as Measurement 1, blood was collected from the subject on an early hungry Friday morning, and the sensitivity, specificity, and effectiveness of fatigue evaluation based on the threshold values determined in Measurement 1 were determined. It was shown to. For the compounds that are not used in the present invention, the sensitivity, specificity, and effectiveness obtained by setting appropriate threshold values are shown in the table.
その結果、良好な感度、特異度、有効度が示され、本発明の頑健性、汎用性が確認された。 As a result, good sensitivity, specificity, and effectiveness were shown, and the robustness and versatility of the present invention were confirmed.
実施例２
〔対象者〕
週５日勤務（月〜金）、週休２日（土日休日）の日勤労働者（標準労働時間８：３０〜１７：００、１時間の昼休み含む）のうち、月曜日から金曜日の労働により疲労感がある４名の労働者（以下「疲労あり」、４３〜５４歳、平均年齢４８．５±４．９歳）と、疲労感がない５名の労働者（以下「疲労なし」、２５〜４９際、平均年齢３６．４±１０．５歳）を対象とした。
Example 2
[Target person]
Among the day shift workers who work 5 days a week (Monday to Friday) and 2 days a week (Saturdays, Sundays, and holidays) (standard working hours 8:30 to 17:00, including 1 hour lunch break), they feel tired due to work from Monday to Friday There are four workers (hereinafter “fatigue”, 43-54 years old, average age 48.5 ± 4.9 years old) and five workers who have no feeling of fatigue (hereinafter “no fatigue”, 25-25 The average age was 36.4 ± 10.5 years).
〔疲労評価〕
１．ＶＡＳ評価
対象者の主観的な疲労の評価は、計測１と同様にＶＡＳ評価により行った。
その結果、表７に示すように、「疲労あり」の金曜日就寝時の数値が高く、金曜日に疲労が蓄積している状態と判断した。
[Fatigue evaluation]
1. VAS Evaluation Subjective subject's subjective fatigue was evaluated by VAS evaluation as in Measurement 1.
As a result, as shown in Table 7, it was determined that fatigue was accumulated on Friday because the numerical value at the time of bedtime on “Fatigue” was high.
２．血中濃度の測定
計測１と同様にして、金曜日の早朝の空腹時に対象者から血液を採取し、計測１で定めた閾値に基づく、疲労評価の感度、特異度、有効度を求め、表８に示した。なお、本発明で用いない化合物についても、表中に、適宜閾値を設定して求めた感度、特異度、有効度を示す。
2. Measurement of blood concentration As in Measurement 1, blood was collected from the subject on an early hungry Friday morning, and the sensitivity, specificity, and effectiveness of fatigue evaluation based on the threshold values determined in Measurement 1 were determined. It was shown to. For the compounds that are not used in the present invention, the sensitivity, specificity, and effectiveness obtained by setting appropriate threshold values are shown in the table.
その結果、良好な感度、特異度、有効度が示され、本発明の頑健性、汎用性が確認された。 As a result, good sensitivity, specificity, and effectiveness were shown, and the robustness and versatility of the present invention were confirmed.
Claims (2)
（ａ）被験者から採取された生体試料中のアザライン酸の濃度を測定し、当該生体試料中のアザライン酸の濃度と０．７８〜０．９６μｍｏｌ／Ｌである第１の閾値を比較し、アザライン酸の濃度が該閾値より高いと疲労度が高いと判定され、アザライン酸の濃度が該閾値未満であると疲労度が低いと判定される工程、
（ｂ）被験者から採取された生体試料中のピメリン酸の濃度を測定し、当該生体試料中のピメリン酸の濃度と１．１４〜１．２２μｍｏｌ／Ｌである第２の閾値を比較し、ピメリン酸の濃度が該閾値未満であると疲労度が高いと判定され、ピメリン酸の濃度が該閾値より高いと疲労度が低いと判定される工程、
（ｃ）被験者から採取された生体試料中の２−アミノ酪酸の濃度を測定し、当該生体試料中の２−アミノ酪酸の濃度と２２．５〜２７．３μｍｏｌ／Ｌである第３の閾値を比較し、２−アミノ酪酸の濃度が該閾値より高いと疲労度が高いと判定され、２−アミノ酪酸の濃度が該閾値未満であると疲労度が低いと判定される工程、
（ｄ）被験者から採取された生体試料中のシトルリンの濃度を測定し、当該生体試料中のシトルリンの濃度と３７．５μｍｏｌ／Ｌである第４の閾値を比較し、シトルリンの濃度が該閾値より高いと疲労度が高いと判定され、シトルリンの濃度が該閾値未満であると疲労度が低いと判定される工程、
から選択される１又は２以上の工程を含む、疲労の評価方法。 Next steps (a) to (d):
(A) The concentration of azaline acid in a biological sample collected from a subject is measured, the concentration of azaline acid in the biological sample is compared with the first threshold value of 0.78 to 0.96 μmol / L, and azaline When the acid concentration is higher than the threshold, it is determined that the degree of fatigue is high, and when the azaline acid concentration is less than the threshold, the fatigue level is determined to be low.
(B) measuring the concentration of pimelic acid in a biological sample collected from a subject, comparing the concentration of pimelic acid in the biological sample with a second threshold value of 1.14 to 1.22 μmol / L, and pimelin A step of determining that the degree of fatigue is high when the acid concentration is less than the threshold, and a step of determining that the degree of fatigue is low when the concentration of pimelic acid is higher than the threshold;
(C) The concentration of 2-aminobutyric acid in the biological sample collected from the subject is measured, and the concentration of 2-aminobutyric acid in the biological sample and a third threshold value that is 22.5 to 27.3 μmol / L are set. In comparison, when the concentration of 2-aminobutyric acid is higher than the threshold, it is determined that the degree of fatigue is high, and when the concentration of 2-aminobutyric acid is less than the threshold, the degree of fatigue is determined to be low,
(D) The citrulline concentration in the biological sample collected from the subject is measured, the citrulline concentration in the biological sample is compared with the fourth threshold value of 37.5 μmol / L, and the citrulline concentration is greater than the threshold value. A process in which it is determined that the degree of fatigue is high when it is high, and the degree of fatigue is determined to be low if the concentration of citrulline is less than the threshold;
1 or a more two or more engineering evaluation method of fatigue selected from.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014036554A JP6598425B2 (en) | 2014-02-27 | 2014-02-27 | Fatigue evaluation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014036554A JP6598425B2 (en) | 2014-02-27 | 2014-02-27 | Fatigue evaluation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015159942A JP2015159942A (en) | 2015-09-07 |
| JP6598425B2 true JP6598425B2 (en) | 2019-10-30 |
Family
ID=54183475
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014036554A Active JP6598425B2 (en) | 2014-02-27 | 2014-02-27 | Fatigue evaluation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6598425B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11266331B2 (en) | 2016-05-16 | 2022-03-08 | Sony Corporation | Optical apparatus and information processing method |
| CN109425669B (en) * | 2017-09-01 | 2022-09-16 | 中国民用航空局民用航空医学中心 | Method for screening biomarkers related to fatigue degree in human body fluid by liquid chromatography-mass spectrometry |
| CN109425670B (en) * | 2017-09-01 | 2022-09-16 | 中国民用航空局民用航空医学中心 | Method for detecting fatigue degree of team based on human urine |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030105104A1 (en) * | 2001-11-27 | 2003-06-05 | Burzynski Stanislaw R. | Formulation of amino acids and riboflavin useful to reduce toxic effects of cytotoxic chemotherapy |
| WO2008016101A1 (en) * | 2006-08-04 | 2008-02-07 | Ajinomoto Co., Inc. | Method for evaluation of stress, stress evaluation apparatus, stress evaluation method, stress evaluation system, stress evaluation program, and recording medium |
| WO2015030211A1 (en) * | 2013-08-30 | 2015-03-05 | 独立行政法人理化学研究所 | Fatigue biomarker and use thereof |
-
2014
- 2014-02-27 JP JP2014036554A patent/JP6598425B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2015159942A (en) | 2015-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Giacomello et al. | Current methods for stress marker detection in saliva | |
| US8951739B2 (en) | Biomarker for depression, method for measuring a biomarker for depression, computer program, and recording medium | |
| Okuma et al. | Effect of masticatory stimulation on the quantity and quality of saliva and the salivary metabolomic profile | |
| JP6598425B2 (en) | Fatigue evaluation method | |
| US20180080946A1 (en) | Method for diagnosing diseases through oligomer analysis of abnormally aggregated proteins | |
| Feres et al. | Implications for the use of acid preservatives in 24-hour urine for measurements of high demand biochemical analytes in clinical laboratories | |
| Parmar et al. | Estimation of postmortem interval through albumin in CSF by simple dye binding method | |
| Heywood et al. | The development of a peptide SRM-based tandem mass spectrometry assay for prenatal screening of Down syndrome | |
| US20090286324A1 (en) | Serum markers for type ii diabetes mellitus | |
| Kummer et al. | A fully validated method for the quantification of ethyl glucuronide and ethyl sulphate in urine by UPLC–ESI-MS/MS applied in a prospective alcohol self-monitoring study | |
| Burling et al. | Analysis of molecular forms of urine Retinol-Binding Protein in Fanconi Syndrome and design of an accurate immunoassay | |
| Yang et al. | An alpha‐synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression | |
| Janzen et al. | UPLC–MS/MS analysis of C5-acylcarnitines in dried blood spots | |
| CA2586654A1 (en) | Use of asc as a marker for colorectal cancer | |
| Jiang et al. | Quantitation of phenylbutyrate metabolites by UPLC-MS/MS demonstrates inverse correlation of phenylacetate: phenylacetylglutamine ratio with plasma glutamine levels | |
| Wu et al. | Identification and validation of argininosuccinate synthase as a candidate urinary biomarker for major depressive disorder | |
| Liu et al. | Chiral amino acid profiling in serum reveals potential biomarkers for Alzheimer’s disease | |
| Goyal et al. | Increasing glucose concentrations interfere with estimation of electrolytes by indirect ion selective electrode method | |
| Conde-Sánchez et al. | Analytical performance evaluation of a particle-enhanced turbidimetric cystatin C assay on the Roche COBAS 6000 analyzer | |
| JP2018124239A (en) | Method for evaluating the state of health of the mouth | |
| JP5832429B2 (en) | Method or apparatus for determining the stage of chronic kidney disease or method for operating the same | |
| Bargnoux et al. | Glomerular filtration rate as a determinant of free light chains in renal transplantation | |
| Yu et al. | A liquid chromatography–tandem mass spectrometry method for the determination of nodularin-R in human plasma and its preliminary clinical application | |
| EP2594938B1 (en) | Ex-vivo method for the early diagnosis of minimal hepatic encephalopathy by means of the determination of 3-nitrotyrosine in serum | |
| Nam et al. | Evaluation of critical factors in the preparation of saliva sample from healthy subjects for metabolomics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20170216 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20170217 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170216 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20171222 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20180116 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20180312 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20180904 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20181030 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20181120 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190121 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20190625 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190702 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190903 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20191001 |
|
| R151 | Written notification of patent or utility model registration |
Ref document number: 6598425 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |