WO2011161544A2

WO2011161544A2 - Biomarker for fatigue, and use thereof

Info

Publication number: WO2011161544A2
Application number: PCT/IB2011/001917
Authority: WO
Inventors: 弘彦倉恒; 洋祐片岡; 光華金; 恭良渡辺; 世貴田島; 朋義曽我; 恵美山野
Original assignee: 独立行政法人理化学研究所; 公立大学法人大阪市立大学
Priority date: 2010-06-24
Filing date: 2011-08-22
Publication date: 2011-12-29
Also published as: JP2012024555A; WO2011161544A3; WO2011161544A8; JP5811545B2

Abstract

The ratio of measurement values of glucose, citric acid, and cis-aconitic acid in a biological sample of a subject to the measurement values of healthy subjects is calculated, and the glucose ratio, citric acid ratio, and cis-aconitic acid ratio are used to assess and/or determine fatigue. In the same way, the isocitric acid ratio, succinic acid ratio, malic acid ratio, and lactic acid ratio are calculated, combined with the three abovementioned ratios, and used to assess and/or determine fatigue. Thus enabled is the objective and simple diagnosis and assessment of fatigue.

Description

Fatigue biomarkers and their use

The present invention relates to a biomarker for evaluating fatigue and evaluation and diagnosis of fatigue using the biomarker.

According to an epidemiological survey in Japan, about 60% of the general public is aware of fatigue, and more than half of them are suffering from fatigue that lasts more than 6 months (ie, chronic fatigue). Also, about half of those who feel chronic fatigue complain of reduced work or school efficiency. The economic loss due to chronic fatigue is estimated to be 1.2 trillion yen. Further, among those who feel chronic fatigue, there are patients with chronic fatigue syndrome whose cause is strong fatigue and whose cause has not been fully elucidated.
Since many of the conventional fatigue diagnoses are based on the subjectivity of patients, establishment of an index for objectively diagnosing fatigue is desired. Patent Document 1 discloses a technique for evaluating viral infection as an indicator of fatigue level, focusing on viral infections listed as one of the causes of immunity decline. Non-Patent Document 1 discloses an attempt to objectively measure / evaluate fatigue using an acceleration pulse wave.

Japanese Patent Publication “JP 2007-330263” (released on December 27, 2007) Japanese Patent Publication “JP 9-77688 A” (published on March 25, 1997) Japanese Patent Publication “JP 9-59161 A” (published March 4, 1997)

A mechanism for maintaining homeostasis (homeostasis), that is, a mechanism for restoring disordered homeostasis is inherently provided in the living body. Development of a technique for objectively evaluating fatigue in accordance with the mechanism inherent to the living body is desired. Specifically, it is expected to identify a biological index (biomarker) that can quantify or quantify biological information based on the concentration of a chemical substance contained in a biological sample.
The technique described in Patent Document 1 collects the body fluid of a subject, measures the amount of human herpesvirus in the body fluid, and evaluates the relationship with the degree of fatigue. However, this technique observes the behavior of a virus that infects a human (host), and does not measure a biomarker that reflects the mechanism of fatigue in humans. For this reason, the technique described in Patent Document 1 cannot evaluate the fatigue state of an individual subject or provide a treatment or prevention method. The technique described in Non-Patent Document 1 has an advantage that it can be implemented non-invasively, but requires measurement of fingertip volume pulse waves by a special device and data processing based on a special principle. Poor sex. Further, Non-Patent Document 2 lists fatigue biochemical marker candidates that can be evaluated in blood, saliva, and urine, and describes that these factors are closely related to fatigue and fatigue. Yes. Non-Patent Document 3 describes that several factors in various biological samples change their amounts in relation to mental fatigue and physical fatigue. However, many of the factors described in Non-Patent Documents 2 and 3 reflect fatigue due to temporary load, and in particular, diagnosis and evaluation of fatigue that has been sustained for a long time in daily life has not yet been realized. Not.
The present invention has been made in view of the above problems, and an object thereof is to provide a biomarker that enables objective and simple diagnosis and evaluation of fatigue.

The first fatigue evaluation method according to the present invention includes (1) a step of obtaining a first ratio of a measured value (first measured value) of a glucose concentration in a biological sample obtained from a subject to a first reference value. (2) A step of obtaining a second ratio of the measured value (second measured value) of the citric acid concentration in the biological sample obtained from the subject to the second reference value, (3) the biological sample obtained from the subject A step of obtaining a third ratio of a measured value (third measured value) of the cis-aconitic acid concentration in the sample to a third reference value, (4) a measured value of the isocitrate concentration in the biological sample obtained from the subject A step of obtaining a fourth ratio of the (fourth measurement value) to the fourth reference value; (5) a measurement value (fifth measurement value) of the succinic acid concentration in the biological sample obtained from the subject; Obtaining a fifth ratio to a reference value of 5, (6) raw obtained from the subject Obtaining a sixth ratio of the measured value of the malic acid concentration in the sample (sixth measured value) to the sixth reference value; and (7) the measured value of the lactic acid concentration in the biological sample obtained from the subject. It includes at least two steps selected from the group consisting of a step of obtaining a seventh ratio of (seventh measurement value) to a seventh reference value.
In the diagnosis of chronic fatigue syndrome, there are problems such as the number of measurement items until diagnosis, long-term observation (measurement) time, economic burden, and individual differences in doctor's judgment criteria. By having the above configuration, the method according to the present invention can provide data for making an objective diagnosis only by measuring a specific metabolite in a biological sample. Enables costly and objective diagnosis. That is, the method according to the present invention may be a method for providing a diagnostic criterion or determination criterion for fatigue. In addition, since glucose, citric acid and cis-aconitic acid are deeply related to energy production, the method according to the present invention is also used for diagnosis of general fatigue (including chronic fatigue, cumulative fatigue, and chronic fatigue syndrome). Is possible.
As used herein, the term “biological sample” is intended to be any tissue (including body fluids such as blood) or cells taken from a subject, and tissue sections or cells prepared therefrom. Cell lysates can also be included in the biological sample. Preferred biological samples for use in the present invention include, but are not limited to, blood, saliva, urine, interstitial fluid, sweat, and preparations thereof (eg, serum, plasma, etc.). It should be noted that the step of directly removing the tissue or cells from the human subject as the first stage of sample acquisition is performed by a doctor and is outside the scope of the present invention. In addition, the step in which the doctor determines whether or not the patient is fatigue (including chronic fatigue, cumulative fatigue, and chronic fatigue syndrome) using the result obtained by the method of the present invention is also outside the scope of the present invention. It is.
As used herein, the first to seventh reference values are the average glucose concentration, average citric acid concentration, average cis-aconitic acid concentration in biological samples obtained from a plurality of healthy subjects, respectively. The average isocitrate concentration, the average succinic acid concentration, the average malic acid concentration, and the average lactic acid concentration are preferable, but are not limited to the average value, and may be a mode value obtained from a binomial distribution or the like. In addition, even if the first to seventh reference values are predetermined values, the healthy person is sampled at the same time as the sampling from the subject, and based on the concentration of each metabolite in the obtained biological sample. It may be a calculated value. The first to seventh reference values are preferably obtained by the same measurement method as that used when obtaining the first to seventh measurement values.
The first fatigue evaluation method according to the present invention may further include a step of comparing at least a pair in at least two ratios obtained by the at least two steps, preferably, the first fatigue evaluation method according to the present invention. The fatigue evaluation method includes at least two steps selected from the group consisting of the steps (1) to (5). The comparison in the step of comparing at least a pair may obtain a difference between two values or a ratio.
In one aspect, the first fatigue evaluation method according to the present invention includes (a) a step of comparing the first ratio and the second ratio, and (b) comparing the second ratio and the third ratio. And (c) comparing at least one of the first ratio and the third ratio, (I) the first ratio is greater than the second ratio, (II) The method further includes the step of determining whether or not at least one of the second ratio is greater than the third ratio and (III) the first ratio is greater than the third ratio is satisfied. Also good.
In a further aspect, (d) comparing the first ratio and the fourth ratio, (e) comparing the second ratio and the fourth ratio, and (f) the third ratio and the second ratio. And (IV) the first ratio is greater than the fourth ratio, and (V) the second ratio is greater than the fourth ratio. (VI) You may further include the process of determining whether at least 1 condition that a 4th ratio is larger than a 3rd ratio is satisfied. When the step (4) is included, the first fatigue evaluation method according to the present invention preferably further includes the step (5). In this case, the fatigue evaluation method according to the present invention includes ( g) comparing the first ratio and the fifth ratio, (h) comparing the second ratio and the fifth ratio, (i) comparing the third ratio and the fifth ratio. And (j) at least one of comparing the fourth ratio and the fifth ratio, (VII) the first ratio is greater than the second ratio, (VIII) The method may further include a step of determining whether or not at least one condition of (IX) the fifth ratio is greater than the fourth ratio is satisfied. .
Succinic acid is also as closely related to energy production as glucose, citric acid and cis-aconitic acid, and can be used to diagnose fatigue overall. Since cis-aconitic acid is unstable, it is not easy to measure the concentration in the sample. However, if the concentration of succinic acid in the sample is used, the present invention can be carried out more easily.
When the ratio is obtained in the above steps (a) to (j), the first fatigue evaluation method according to the present invention uses discriminant analysis, Partial Last Square, Support using at least one of the obtained ratios. You may further include the process of performing analysis, such as Vector Machine. Further, the sixth ratio and the seventh ratio obtained by (6) and (7) can be used in the same manner as the above-described first to fifth ratios. Malic acid and lactic acid are also as closely related to energy production as glucose, citric acid, cis-aconitic acid and succinic acid, and can be used for diagnosis of general fatigue.
By having the said structure, the method concerning this invention can provide the objective diagnosis about chronic fatigue rapidly and at low cost. Furthermore, since the diagnosis result according to the present invention represents the disease state itself, it is possible to make a treatment policy by using the present invention. That is, according to the present invention, it is determined not only whether or not the subject is in a fatigue state but also whether or not the subject is suffering from chronic fatigue, and further whether or not the patient belongs to chronic fatigue syndrome (or Provide data for determination).
The second fatigue evaluation method according to the present invention includes at least one step selected from the group consisting of the steps (2) to (7). The second fatigue evaluation method according to the present invention may further include, for the second ratio to the seventh ratio, a step of comparing the obtained ratio with a reference value corresponding to the ratio. Corresponding to the above steps (2) to (7), (k) comparing the second ratio with a reference value corresponding to the ratio, and (l) a reference corresponding to the third ratio corresponding to the ratio. (M) comparing the fourth ratio with a reference value corresponding to the ratio; (n) comparing the fifth ratio with a reference value corresponding to the ratio; It may further include at least one of comparing a ratio of 6 with a reference value corresponding to the ratio, and (p) comparing a seventh ratio with a reference value corresponding to the ratio. The reference value is obtained as a ratio between a threshold value obtained by analysis based on a decision tree mathematical model using measured values and a reference value, and specifically, (threshold value / reference value) × 100 ( %). In this case, corresponding to the above steps (k) to (p), (X) the second ratio is smaller than the reference value corresponding to the second ratio, (XI) the third ratio is the third (XII) the fourth ratio is smaller than the reference value corresponding to the fourth ratio, (XIII) the fifth ratio corresponds to the fifth ratio, which is smaller than the reference value corresponding to the ratio of (XIV) the sixth ratio is smaller than the reference value corresponding to the sixth ratio, (XV) the seventh ratio is smaller than the reference value corresponding to the seventh ratio, It is preferable to further include a step of determining whether or not at least one condition is satisfied.
The doctor can determine that the subject who provided the biological sample may belong to chronic fatigue syndrome when at least one of the conditions (X) to (XV) is satisfied.
Moreover, in the fatigue evaluation system mentioned later, the determination part can perform the same determination. In the second fatigue evaluation method according to the present invention, there is no substantial difference between the first measured value and the first reference value (that is, the first obtained in the step (1)). It is preferably used when the ratio is substantially equal to 1. In this specification, it is also possible to objectively determine that there is no substantial difference between the first measured value and the first reference value without using the step (1). ) To determine that the first ratio is substantially equal to 1.
In one aspect, the second fatigue evaluation method according to the present invention includes at least one of a step of obtaining a third ratio, a step of obtaining a fourth ratio, and a step of obtaining a fifth ratio. Is preferred. As shown in FIGS. 3 and 5, the third to fifth ratios are clearly different between healthy individuals and patients with chronic fatigue syndrome. Moreover, as shown in the Example mentioned later, even if cis-aconitic acid, isocitric acid, and succinic acid are independent, the marker which determines whether a test subject is a chronic fatigue syndrome patient with a 95% probability. Can be. Of these, succinic acid is most preferred as a marker, followed by isocitrate and cis-aconitic acid in this order. That is, in the second fatigue evaluation method according to the present invention, the step of obtaining the fifth ratio is preferable, the step of obtaining the fourth ratio is more preferably performed following the step of obtaining the fifth ratio, Most preferably, the step of obtaining a ratio of 3 is performed subsequent to the step of obtaining a fourth ratio.
The fatigue evaluation method according to the present invention includes (1 ′) a step of comparing a measured value of glucose concentration in a biological sample obtained from a subject with a first threshold, and (2 ′) in a biological sample obtained from the subject. (3 ′) comparing the measured value of the cis-aconitic acid concentration in the biological sample obtained from the subject with the third threshold value (3 ′). 4 ′) a step of comparing the measured value of the isocitrate concentration in the biological sample obtained from the subject with the fourth threshold; (5 ′) the measured value of the succinic acid concentration in the biological sample obtained from the subject and the fifth A step of comparing with a threshold, (6 ′) a step of comparing with a sixth threshold of the measured value of malic acid concentration in the biological sample obtained from the subject, and (7 ′) lactic acid in the biological sample obtained from the subject. Comparing the seventh measured value of the concentration with the seventh threshold value. It is characterized by including at least one, and preferably includes at least one of the steps (3 ′) to (5 ′), and more preferably includes at least (5 ′). More preferably, the step (4 ′) is performed after the step (5 ′), and the step (3 ′) is further preferably performed subsequently. The fatigue evaluation method according to the present embodiment may further include a step of determining whether or not the condition that the measurement value is smaller than the corresponding threshold value is satisfied in (1 ′) to (7 ′), Whether or not the condition that the measured value is smaller than the corresponding threshold value is satisfied in (3 ′) to (5 ′) from the viewpoint of determining whether or not the subject has chronic fatigue syndrome with a probability of 95% or more. It is preferable to further include a step of determining whether or not. In this case, you may further include the process of determining whether the conditions that at least 1 is smaller than the threshold value corresponding to each are satisfied. The doctor can determine that the subject who provided the biological sample may belong to chronic fatigue syndrome when at least one of the above conditions is satisfied. Moreover, in the fatigue evaluation system mentioned later, the determination part can perform the same determination.
In the third fatigue evaluation method according to the present invention, in at least two selected from the group consisting of the first measurement value to the fifth measurement value in the biological sample obtained from the subject without using the reference value. And at least a step of obtaining a pair of ratios. It is preferable that the method further includes a step of subjecting the obtained at least one pair of ratios to analysis of discriminant analysis, Partial Last Square, or Support Vector Machine. The fatigue evaluation method according to the present invention includes, for example, a ratio of a second measurement value to a first measurement value, a ratio of a third measurement value to a first measurement value, and a third measurement value in a biological sample obtained from a subject. The ratio of the measured value to the second measured value, the ratio of the fourth measured value to the first measured value, the ratio of the fourth measured value to the second measured value, and the third of the fourth measured value The analysis is preferably performed using at least one of the ratios to the measured values. By having the above-described configuration, the method according to the present invention can discriminate patients with chronic fatigue syndrome with an accuracy of 90% or more. Also, for example, in a biological sample obtained from a subject, the ratio of the second measurement value to the first measurement value, the ratio of the fourth measurement value to the first measurement value, the second of the fourth measurement value At least one of a ratio to the measurement value, a ratio of the fifth measurement value to the first measurement value, a ratio of the fifth measurement value to the second measurement value, and a ratio of the fifth measurement value to the fourth measurement value; It is more preferable that the analysis method is used, and by having the above configuration, the method according to the present invention can discriminate chronic fatigue syndrome patients with 90% accuracy and 95% sensitivity / specificity. it can.
In the present invention, the step of measuring the concentrations of glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid and lactic acid in the biological sample (steps for obtaining the first to seventh measurement values) Although the “measurement value” to be obtained is not essential, the value obtained and / or calculated by a third party is provided to the practitioner of the present invention, even if the practitioner of the present invention obtains and / or calculates the “measurement value”. May be.
The first kit according to the present invention is characterized by including a fifth reagent for measuring a succinic acid concentration in order to evaluate fatigue. The first kit according to the present invention may further include a fourth reagent for measuring isocitrate concentration, and further measures the first reagent for measuring glucose concentration, citric acid concentration. A second reagent for measuring, a third reagent for measuring cis-aconitic acid concentration, a sixth reagent for measuring malic acid concentration, and a seventh reagent for measuring lactic acid concentration At least one reagent selected from the group may be provided. It is preferable that the kit according to the present invention further includes an instruction sheet displaying the respective reference values of the concentrations of glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid and lactic acid.
In order to evaluate fatigue, the second kit according to the present invention is characterized by including a fifth presenting portion showing a fifth ratio. The 2nd kit concerning the present invention may further be provided with the 4th presentation part which shows the 4th ratio, and also the 1st presentation part which shows the 1st ratio, and the 2nd presentation which shows the 2nd ratio At least one of a presentation unit selected from the group consisting of: a third presentation unit showing a third ratio; a sixth presentation unit showing a sixth ratio; and a seventh presentation unit showing a seventh ratio. You may have.
By having the above-described configuration, the kit according to the present invention can be easily used in an actual medical field, so that a faster, cheaper, objective fatigue evaluation method and chronic fatigue syndrome diagnosis method can be provided. Can be provided.
A first fatigue evaluation system according to the present invention includes a measurement value receiving unit for receiving at least two measurement values selected from the group consisting of first to seventh measurement values, and first to seventh reference values. A reference value storage unit storing at least two reference values selected from the group, an operation for receiving the measurement value from the measurement value receiving unit and the reference value from the reference value storage unit, and generating information for evaluating fatigue And an evaluation unit that receives evaluation information from the calculation unit and evaluates fatigue, and the calculation unit is selected from the group consisting of the first ratio to the seventh ratio. It is characterized by calculating a ratio.
In the first fatigue evaluation system according to the present invention, the calculation unit may execute at least a pair of comparisons in the at least two ratios. Preferably, in the first fatigue evaluation system according to the present invention, the measurement value receiving unit receives at least two measurement values selected from the group consisting of the first to fifth measurement values, and the reference value storage unit Stores at least two reference values selected from the group consisting of the first to fifth reference values, and the arithmetic unit is at least two selected from the group consisting of the first ratio to the fifth ratio A ratio is further calculated and at least a pair of comparisons are performed on the at least two ratios. The at least one pair of comparisons may obtain a difference between two values or may obtain a ratio. For example, in the first fatigue evaluation system according to the present invention, the evaluation unit (I) the first ratio is larger than the second ratio, (II) the second ratio is larger than the third ratio, (III) the first ratio is greater than the third ratio, (IV) the first ratio is greater than the fourth ratio, (V) the second ratio is greater than the fourth ratio, (VI ) The fourth ratio is greater than the third ratio, (VII) the first ratio is greater than the second ratio, (VIII) the second ratio is greater than the fourth ratio, (IX) th It is preferable to determine whether or not at least one condition that the ratio of 5 is larger than the fourth ratio is satisfied. When a ratio is obtained in the at least one pair of comparisons, the calculation unit may perform a discriminant analysis, a Partial Last Square, or an analysis of a support vector machine based on the obtained ratio. Further, the obtained sixth ratio and seventh ratio can be processed by the first fatigue evaluation system according to the present invention in the same manner as the first to fifth ratios described above.
In the second fatigue evaluation system according to the present invention, the calculation unit calculates the first ratio and calculates at least one ratio selected from the group consisting of the second to seventh ratios. It is said. In the second fatigue evaluation system according to the present invention, the reference value storage unit stores at least one reference value selected from the group consisting of reference values corresponding to the second ratio to the seventh ratio. In this case, it is preferable that the arithmetic unit receives the reference value from the reference value storage unit and compares the calculated ratio with the reference value corresponding to the ratio. The reference value is obtained as a ratio between a threshold value obtained by analysis based on a decision tree mathematical model using measured values and a reference value, and specifically, (threshold value / reference value) × 100 ( %). In this case, the evaluation unit has a fourth ratio in which the second ratio is smaller than the reference value corresponding to the second ratio, the third ratio is smaller than the reference value corresponding to the third ratio. Is smaller than the reference value corresponding to the fourth ratio, the fifth ratio is smaller than the reference value corresponding to the fifth ratio, and the sixth ratio is more than the reference value corresponding to the sixth ratio. If the at least one condition that the seventh ratio is smaller than the reference value corresponding to the seventh ratio is satisfied, it is determined that the subject who provided the biological sample belongs to the chronic fatigue syndrome. Preferably, in the second fatigue evaluation system according to the present invention, the calculation unit calculates at least one of a third ratio to a fifth ratio. In this case, it is preferable that the calculation unit calculates the fourth ratio subsequent to the fifth ratio, and more preferably calculates the third ratio subsequent to the fourth ratio. In the second fatigue evaluation system according to the present invention, it is preferable that the determination unit determines whether or not the first ratio is substantially 1. By having the said structure, the system concerning this invention can discriminate | determine a chronic fatigue syndrome patient with 90% or more of accuracy, and 95% or more of sensitivity / specificity.
The fatigue evaluation system according to the present invention is characterized in that the calculation unit compares the first to seventh measured values with thresholds corresponding to the first to seventh measured values. In the fatigue evaluation system according to the present invention, the reference value storage unit may store at least one threshold value selected from the group consisting of threshold values corresponding to each of the first to seventh measurement values. In this case, it is preferable that the arithmetic unit receives the threshold value from the reference value storage unit and compares the measured value with the threshold value corresponding to the measured value. In particular, it is preferable to perform the comparison for the third to fifth measurement values, more preferably to perform the comparison for the fifth measurement value, and then to perform the comparison for the fourth measurement value, It is further preferred to make a comparison for the third measurement value. From the viewpoint that the determination unit can determine whether or not the subject has chronic fatigue syndrome with a probability of 95% or more, in (3 ′) to (5 ′), the condition that the measured value is smaller than the corresponding threshold value It is preferable to determine whether or not is established. In this case, when the condition that at least one is smaller than the corresponding threshold value is satisfied, it is determined that the subject who provided the biological sample belongs to chronic fatigue syndrome.
A third fatigue evaluation system according to the present invention receives a measurement value receiving unit for receiving at least two measurement values selected from the group consisting of first to fifth measurement values, and a measurement value from the measurement value receiving unit. A calculation unit that generates information for evaluating fatigue, and an evaluation unit that receives evaluation information from the calculation unit and evaluates fatigue, wherein the calculation unit includes the at least two measured values. The method is characterized in that at least a pair of comparisons is performed to obtain at least a pair of ratios. In the third fatigue evaluation system according to the present invention, it is preferable that the calculation unit performs a discriminant analysis, a Partial Last Square, or a Support Vector Machine analysis based on the obtained at least one pair of ratios. By having the above configuration, the system according to the present invention can be determined with an accuracy of 90% or more.
In a preferred aspect, the third fatigue evaluation system according to the present invention evaluates fatigue using at least one of the third, fourth, and fifth measurement values in a biological sample obtained from a subject. The fatigue evaluation system according to the present invention corresponds to each of a measurement value receiving unit that receives at least one of the third, fourth, and fifth measurement values, a cis-aconitic acid concentration, an isocitrate concentration, and a succinic acid concentration. A reference value storage unit storing at least one of the third, fourth and fifth reference values, a measurement value from the measurement value receiving unit and a reference value from the reference value storage unit for receiving and evaluating fatigue An arithmetic unit that generates information, and an evaluation unit that receives evaluation information from the arithmetic unit and evaluates fatigue, and the arithmetic unit includes a third ratio, a fourth ratio, and a fifth ratio. At least one of the following is calculated. In this case, the calculation unit preferably calculates the fifth ratio, more preferably calculates the fourth ratio following the fifth ratio, and further, the third ratio continues after the fourth ratio. Is most preferably calculated. In the third fatigue evaluation system according to the present invention, the reference value storage unit may store at least one of reference values corresponding to each of cis-aconitic acid concentration, isocitrate concentration, and succinic acid concentration. In this case, it is preferable that the arithmetic unit receives the reference value from the reference value storage unit and compares the calculated ratio with the reference value corresponding to the ratio. In addition, the calculation unit may perform analysis using at least one of the third ratio, the fourth ratio, and the fifth ratio, for example.
The fatigue evaluation system according to the present invention includes first to seventh measuring units for measuring glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitric acid concentration, succinic acid concentration, malic acid concentration, and lactic acid concentration, respectively. In this case, the values obtained by the first to seventh measurement units are input to the measurement value receiving unit.
By having the above-described configuration, the system according to the present invention processes measurement values that can be obtained by a conventionally known simple technique according to a unique procedure, and therefore, objective and highly reliable determination that could not be obtained conventionally. Results can be presented quickly and at low cost.
The compound preferably used in the present invention is glucose, and at least one selected from the group consisting of citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid and lactic acid is used in combination with glucose. Is preferred. In addition, since the concentration of citric acid, succinic acid, malic acid or lactic acid is relatively easy to measure rather than the concentration of cis-aconitic acid or isocitric acid, it is from the group consisting of citric acid, succinic acid, malic acid and lactic acid. More preferably, at least one selected is used in combination with glucose, and at least two selected from the group consisting of citric acid, succinic acid, malic acid and lactic acid are also preferably used in combination with glucose.
Thus, in the present invention, when two biomarkers are used, the target compound combination is selected from the group consisting of glucose and citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid and lactic acid. Preferably in combination with one selected, and when three biomarkers are used, the compound combination of interest is glucose, citric acid and succinic acid, glucose, citric acid and malic acid, glucose, citric acid And lactic acid, glucose, succinic acid and malic acid, glucose, succinic acid and lactic acid, or glucose, malic acid and lactic acid, and when four or more biomarkers are used, the combination of compounds of interest is: Glucose, citric acid, succinic acid and malic acid, glucose, Phosphate, succinic acid and lactic acid, glucose, citric acid, malic acid and lactic acid, glucose, succinic acid, malic acid and lactic acid or, glucose, citric acid, succinic acid is preferably malic acid and lactic acid.

The present invention enables objective and simple evaluation of fatigue and diagnosis of chronic fatigue syndrome. The present invention may further provide techniques useful for treating fatigue.

FIG. 1 is a diagram showing the concentrations of various metabolites contained in the plasma of healthy subjects and patients with chronic fatigue syndrome (CFS).
FIG. 2 is a diagram showing the results of examining the correlation between a metabolite and a metabolite and the correlation between the metabolite and performance status (PS) for a metabolite forming a glycolytic system and a citric acid cycle.
FIG. 3 is a graph showing the results of the amount of candidate substances contained in the plasma of healthy subjects and patients with chronic fatigue syndrome (CFS). The average value of the amount of each candidate substance of a healthy person was set to 100, and the ratio was shown as a ratio.
FIG. 4 is a graph showing the ratios of glucose, citric acid, cis-aconitic acid and isocitric acid contained in the plasma of healthy subjects and patients with chronic fatigue syndrome (CFS) on three axes. It is.
FIG. 5 is a graph showing the results of the amount of candidate substances contained in the plasma of healthy subjects and patients with chronic fatigue syndrome (CFS). The average value of the amount of each candidate substance of a healthy person was set to 100, and the ratio was shown as a ratio.
FIG. 6 is a graph showing the ratios of glucose, citric acid, isocitrate and succinic acid on the three axes included in the plasma of healthy subjects and patients with chronic fatigue syndrome (CFS). .
FIG. 7 is a diagram illustrating a result of executing a random forest program using all parameters including measured values of metabolites in a plasma sample.

[1] Fatigue
Currently, the number of patients who visit a medical institution with fatigue and fatigue as the main symptom is the second largest after the number of patients with pain as the main symptom. However, no method for objectively evaluating fatigue has been developed so far. Fatigue and fatigue are sensations that humans experience on a daily basis, and are important biological signals that inform the disturbance of homeostasis in the living body. However, the feeling of fatigue is subjective only and does not objectively indicate the degree of fatigue. Although there were times when lactic acid was considered to be a causative agent of fatigue, it has also been found that fluctuations in lactic acid level can be an indicator of exercise, but not an indicator of fatigue. Furthermore, no effective treatment for fatigue has been found, and only caffeine, taurine, ascorbic acid and the like are known as anti-fatigue components.
The US Center for Disease Control and Prevention (CDC) reported a chronic fatigue syndrome called “chronic fatigue syndrome” in 1988, elucidating the mechanism, searching for biomarkers, and developing treatment prevention methods. Various researches have been made with a focus on. However, the onset mechanism of chronic fatigue syndrome has not yet been elucidated, and no objective biomarker has been developed. Even now, the diagnosis of chronic fatigue syndrome is made using the criteria of symptoms and physical findings published by CDC in 1994.
On the other hand, fatigue biomarkers using virus activation or autonomic abnormality as an index have been proposed. However, since these do not conform to the cause of human fatigue and its mechanism, they are not biomarkers specific to fatigue (particularly chronic fatigue, chronic fatigue syndrome). Furthermore, since these are indirect indicators of the maintenance / recovery mechanism of homeostasis, they are not direct indicators, and it is necessary to trial and error to develop them to provide specific treatments.
In the fatigue-bearing model animals, changes in blood amino acid levels (especially increases in branched chain amino acid levels) were found. Therefore, the present inventors conducted a similar test using the blood of a fatigue patient, but could not find a similar result. In order to verify this result, the present inventors performed metabolomic analysis that can comprehensively analyze various metabolites using plasma of patients with chronic fatigue syndrome. However, regarding the above-mentioned branched chain amino acids, no quantitative difference was found between healthy subjects and patients with chronic fatigue syndrome.
In studies using model animals, ATP reduction in liver and muscle tissue, activation of cytokines in the brain, etc. have been shown so far, but such findings are useful for diagnosis and / or treatment in humans. It is difficult to apply from an ethical point of view. Thus, the knowledge obtained from model animals cannot be applied to fatigue patients, and the cause of fatigue and the mechanism by which fatigue is generated have not yet been elucidated.
[2] Biomarker of the present invention
The present inventors further analyzed changes in metabolites in the plasma of healthy subjects and patients with chronic fatigue syndrome, and found a biomarker of fatigue by combining the original measurement with the obtained measurement values, The present invention has been completed.
As shown in the examples described later, the biomarker according to the present invention can be used for the concentration of glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, cis-aconitic acid, malic acid, and lactic acid in a biological sample. Is based. In recent years, techniques for exhaustively analyzing various samples for various items have been developed. Metabolome analysis adopted by the present inventors is one of them. However, as shown in FIG. 1, there are many compounds (metabolites) whose concentrations in the biological sample are significantly different between those of the subject and those of healthy subjects, and at least two of the above (necessary) There is no knowledge in the field that clearly motivates the selection of all types). The present inventors narrowed down the metabolites whose concentrations in the biological sample are significantly different between those of the subject and those of the healthy subject to those showing a significant correlation with PS, and further to various mathematical model analysis methods. Provided. Performing such a procedure is also a viewpoint unique to the present inventors. In addition, it is the inventors' original viewpoint that glucose was selected as a compound to be used for path analysis in addition to those obtained from metabolome analysis and that glucose and other metabolites were associated. Furthermore, when using the above metabolite (that is, when using at least two biomarkers), it is possible to distinguish between a healthy person and a patient with chronic fatigue syndrome with an accuracy exceeding 90%. provide.
In addition, there have been reports so far that citric acid is associated with fatigue (see, for example, Patent Documents 2 and 3). However, those skilled in the art cannot conceive of the present invention based on any report.
In the present invention, the ratio of the measured value of the subject to the measured value of the healthy subject of the metabolite (glucose, citric acid, cis-aconitic acid, isocitrate, succinic acid) in the biological sample is calculated. The difference between the ratio of the metabolite and one of the other metabolites is used as a biomarker. For example, in the present invention, the glucose concentration in the biological sample obtained from the subject (measured value M₁) And a reference value for glucose (first reference value B)₁) Ratio (first ratio R)₁) And citric acid concentration (measured value M) in the biological sample obtained from the subject.₂) And a reference value for citric acid (second reference value B)₂) Ratio (second ratio R)₂), And the cis-aconitic acid concentration (measured value M) in the biological sample obtained from the subject.₃) And a reference value for the cis-aconitic acid (third reference value B)₃) (The third ratio R)₃) To calculate these R₁~ R₃Is obtained, the biomarker of the present invention is obtained.
In the first embodiment, the biomarker of the present invention is R₁And R₂Difference Q from₁, R₂And R₃Difference Q from₂It can be.
Biomarker Q₁= R₁-R₂
Biomarker Q₂= R₂-R₃
Biomarker Q obtained as₁And Q₂Is positive (Q₁> 0 or Q₂> 0) is evaluated as “fatigue” and biomarker Q₁And Q₂Are both negative (Q₁<0 and Q₂If <0), it is evaluated as “no fatigue”. It should be noted that the ratio of the measured value of the subject to the measured value of the healthy person for isocitrate in the biological sample may be calculated, and the difference between the ratio of isocitrate and the ratio of cis-aconitic acid may be used as a biomarker. That is, the isocitrate concentration in the biological sample obtained from the subject (measured value M₄) And a reference value for isocitrate (fourth reference value B)₄) And the ratio (fourth ratio R)₄) And based on these,
Biomarker Q₃= R₄-R₃
Biomarker Q obtained as₃Also, biomarker Q₁And Q₂In this case, the biomarker Q₁~ Q₃Is positive (Q₁> 0, Q₂> 0 or Q₃> 0) is evaluated as “fatigue” and biomarker Q₁~ Q₃Are both negative (Q₁<0 and Q₂<0 and Q₃If <0), it is evaluated as “no fatigue”.
Such an embodiment is demonstrated in an example described later (for example, FIG. 3). Where biomarker Q₁~ Q₃alike,
Biomarker Q₄= R₁-R₃
Biomarker Q₅= R₁-R₄
Biomarker Q₆= R₂-R₄
Is also a biomarker of the present invention, and biomarker Q₁~ Q₃Those of ordinary skill in the art who have read this specification will readily appreciate that it can be used for fatigue assessment as well.
Since cis-aconitic acid is unstable, it is not easy to measure the concentration in the sample. In such a case, succinic acid may be used together with glucose, citric acid and isocitric acid. Succinic acid is also deeply involved in energy production, as is glucose, citric acid and cis-aconitic acid. If the concentration of succinic acid in the sample is used, the present invention can be carried out more easily. That is, in the first embodiment, the glucose concentration in the biological sample obtained from the subject (measured value M₁) And a reference value for glucose (first reference value B)₁) Ratio (first ratio R)₁) And citric acid concentration (measured value M) in the biological sample obtained from the subject.₂) And a reference value for citric acid (second B₂) Ratio (second ratio R)₂) And isocitrate concentration (measured value M) in the biological sample obtained from the subject.₄) And a reference value for isocitrate (fourth reference value B)₄) And the ratio (fourth ratio R)₄), And the succinic acid concentration (measured value M) in the biological sample obtained from the subject.₅) And a reference value for succinic acid (fifth reference value B)₅) (The fifth ratio R)₅) To calculate these R₁, R₂, R₄And R₅It is preferable to obtain the biomarker of the present invention by comparing. In this case, the biomarker of this embodiment is R₁And R₂Difference Q from_1a, R₂And R₄Difference Q from_2a, R₄And R₅Difference Q from_3aIt can be.
Biomarker Q_1a= R₁-R₂
Biomarker Q_2a= R₂-R₄
Biomarker Q_3a= R₅-R₄
At least one of the biomarkers obtained as_1a> 0, Q_2a> 0 or Q_3a> 0), it is evaluated as “fatigue” and both biomarkers are negative (Q_1a<0 and Q_2a<0 and Q_3aIf <0), it is evaluated as “no fatigue”.
Such an embodiment is demonstrated in an example described later (for example, FIG. 5). Where biomarker Q_1a, Q_2a, Q_3aalike,
Biomarker Q_4a= R₁-R₄
Biomarker Q_5a= R₁-R₅
Biomarker Q_6a= R₂-R₅
Is also a biomarker of the present invention, and biomarker Q_1a, Q_2a, Q_3aThose of ordinary skill in the art who have read this specification will readily appreciate that it can be used for fatigue assessment as well.
When there is no substantial difference between the measured value of the glucose concentration in the biological sample obtained from the subject and the reference value (ie R₁≒ 1), R₂~ R₅Can be a biomarker. In addition, malic acid concentration in the biological sample obtained from the subject (measured value M₆) And a reference value for malic acid (sixth reference value B)₆) Ratio (sixth ratio R)₆), And the lactic acid concentration (measured value M) in the biological sample obtained from the subject.₇) And a reference value for lactic acid (seventh reference value B)₇) And the ratio (seventh ratio R)₇) To calculate these R₆~ R₇R₁~ R₅May be used together. That is, in the second embodiment, the biomarker of the present invention is R₂~ R₇But it can be. Further biomarkers of the invention include
Biomarker Q₇= Second ratio R₂
Biomarker Q₈= Third ratio R₃
Biomarker Q₉= 4th ratio R₄
Biomarker Q₁₀= 5th ratio R₅
Biomarker Q₁₁= 6th ratio R₆
Biomarker Q₁₂= Seventh ratio R₇
Offered as. Biomarker (Q₇~ Q₁₂) Shows a sensitivity and specificity of 90% or more as a result of analysis by a decision tree mathematical model. As shown in the examples described later, when analyzed using the items listed in FIG.₇~ Q₁₂At least one of the conditions (R₂<79.1%, R₃<69.3%, R₄<66.0%, R₅<66.6%, R₆<83.7% and R₇<74.3%) is evaluated as “fatigue” and biomarker Q₇~ Q₁₂If all of the above do not satisfy the above conditions, it is evaluated as “no fatigue”.
As shown in the examples described later, cis-aconitic acid, isocitric acid, and succinic acid can be used alone without being combined with other factors when distinguishing healthy subjects from patients. Such a very good function is particularly remarkable and cannot be predicted by a person skilled in the art.
Based on the findings obtained by the biomarkers described above, the measured values of glucose, citric acid, cis-aconitic acid, isocitrate and succinic acid in biological samples themselves can also be used as a diagnostic / criterion for fatigue (eg, Data for diagnosing or determining fatigue) and a measure M of the concentration of a metabolite in a biological sample obtained from a subject₂~ M₇Can also be a biomarker. That is, the further biomarker of the present invention is:
Biomarker Q_7a= Second measured value M₂
Biomarker Q_8a= Third measured value M₃
Biomarker Q_9a= Fourth measured value M₄
Biomarker Q_10a= 5th measured value M₅
Biomarker Q_11a= 6th measured value M₆
Biomarker Q_12a= Seventh measured value M₇
Offered as. Biomarker (Q_7a~ Q_12a) Shows a sensitivity and specificity of 90% or more as a result of analysis by a decision tree mathematical model. In particular, Q_8a~ Q_10aIs preferred, biomarker Q_8a~ Q_10aIf at least one of the conditions is smaller than the corresponding threshold value, it is evaluated as “fatigue” and the biomarker Q_8a~ Q_10aIf all of the above do not satisfy the above conditions, it may be evaluated as “no fatigue”. Biomarker Q_8a~ Q_10aQ_9aIs preferably used in preference, then Q_8aAnd Q_10aIs preferably used. Accordingly, it can be determined with a probability of 95% or more whether or not the subject has chronic fatigue syndrome.
As shown in the examples described later, cis-aconitic acid, isocitric acid, and succinic acid can be used alone without being combined with other factors when distinguishing healthy subjects from patients. Such a very good function is particularly remarkable and cannot be predicted by a person skilled in the art.
Furthermore, in the present invention, the glucose concentration in the biological sample obtained from the subject (measured value M₁) And citric acid concentration (measured value M₂) And cis-aconitic acid concentration (measured value M₃) And isocitrate concentration (measured value M₄) And succinic acid concentration (measured value M₅) To obtain the biomarker of the present invention. In a third embodiment, the biomarker of the present invention is
Biomarker Q₁₃= M₂/ M₁
Biomarker Q₁₄= M₃/ M₂
Biomarker Q₁₅= M₄/ M₃
Biomarker Q₁₆= M₃/ M₁
Biomarker Q₁₇= M₄/ M₁
Biomarker Q₁₈= M₄/ M₂
Can be used as well. Such an embodiment is demonstrated in an example described later (for example, FIG. 4). When each is shown on three axes as shown in FIG. 4, the normal group and the chronic fatigue syndrome can be differentiated. For example, as shown in the examples described later, Q₁₃~ Q₁₅Using,
(A * Q₁₃+ B * Q₁₄+ C * Q₁₅) + D (A)
When the numerical values a, b, c, and d were calculated by discriminant analysis, it was possible to discriminate chronic fatigue syndrome patients with an accuracy of 90% or more. Here, the case where d> 0 indicates the normal group, and the case where d <0 indicates the chronic fatigue syndrome. Similar results were obtained even when analysis of Partial Last Square, Support Vector Machine or the like was performed instead of discriminant analysis.
In this embodiment, when isocitrate is used together with glucose, citric acid and succinic acid, the biomarker of the present invention is
Biomarker Q_13a= M₂/ M₁
Biomarker Q_14a= M₄/ M₂
Biomarker Q_15a= M₅/ M₄
Biomarker Q_16a= M₄/ M₁
Biomarker Q_17a= M₅/ M₁
Biomarker Q_18a= M₅/ M₂
As shown in FIG. 6, when each is shown on three axes, the normal group and the chronic fatigue syndrome can be differentiated. For example, as shown in the examples described later, Q_13a~ Q_15aUsing,
(A * Q_13a+ B * Q_14a+ C * Q_15a) + D (A ')
When the numerical values a, b, c, and d were calculated by discriminant analysis, it was possible to discriminate chronic fatigue syndrome patients with 90% accuracy and 95% sensitivity / specificity. Here, the case where d> 0 indicates the normal group, and the case where d <0 indicates the chronic fatigue syndrome. Similar results were obtained even when analysis of Partial Last Square, Support Vector Machine or the like was performed instead of discriminant analysis.
It is fully supported by the completion of the present invention that glucose, citric acid, cis-aconitic acid, isocitric acid and succinic acid are very useful factors in distinguishing healthy subjects from patients. It is a fact. That is, it has not been easy for those skilled in the art to find out based on the knowledge that has been known so far that it is a factor that can be used to distinguish between a healthy person and a patient.
Thus, the biomarker of the present invention enables objective and simple fatigue evaluation and diagnosis. Furthermore, by using the biomarker of the present invention, diagnostic methods and treatment methods for chronic fatigue syndrome can be developed.
[3] Use of the biomarker of the present invention
The present invention provides a method, kit and system for assessing fatigue based on the biomarkers described above.
[3-1] Fatigue evaluation method
Biomarker Q in the first embodiment described above₁~ Q₆At least two (or biomarkers Q)_1a~ Q_6aCan be used to evaluate the fatigue state of the subject. For example, biomarker Q₁And Q₂Is
Q₁= First ratio R₁-Second ratio R₂
Q₂= Second ratio R₂-Third ratio R₃
The evaluation of “with fatigue” is provided as
Q₁> 0 or Q₂> 0
The evaluation of “no fatigue” is provided as
Q₁<0 and Q₂<0
As provided
R₁= M₁/ B₁
R₂= M₂/ B₂
R₃= M₃/ B₃
The first to third ratios (R₁~ R₃) Based on Q₁And Q₂By calculating the fatigue state of the subject can be evaluated. Q₁And Q₂Since it is sufficient that at least one of them is positive, there is a case where the fatigue state of the subject can be evaluated only by calculating one. That is, R₁And R₂Or R₂And R₃In some cases, the fatigue state of the subject can be evaluated simply by comparing the two.
From such a viewpoint, in one embodiment, the fatigue evaluation method according to the present invention (1) obtains a first ratio of a measured value of glucose concentration in a biological sample obtained from a subject to a first reference value. (2) obtaining a second ratio of the measured value of the citric acid concentration in the biological sample obtained from the subject to the second reference value; and (3) cis-aconite in the biological sample obtained from the subject. Obtaining a third ratio of the measured value of the acid concentration with respect to the third reference value, comparing the first ratio and the second ratio, and the second ratio and the third ratio The method further includes at least one of the steps of comparing. In the fatigue evaluation method according to the present embodiment, (I) the first ratio is larger than the second ratio, (II) the second ratio is larger than the third ratio, and (III) the first ratio. A step of determining whether or not at least one of the following conditions is satisfied may be further included.
In addition, by using the fatigue evaluation method according to the present invention, not only can fatigue be evaluated and a treatment method can be proposed, but also a criterion for determination of fatigue (including chronic fatigue and chronic fatigue syndrome) can be provided. Therefore, it becomes easy to diagnose whether or not the state is fatigued. That is, the fatigue evaluation method according to the present invention diagnoses fatigue (including chronic fatigue and chronic fatigue syndrome). Or a method of acquiring data for determination).
As mentioned above, biomarker Q₃Is also useful for assessing a subject's fatigue status. Biomarker Q₃Is
Biomarker Q₃= 4th ratio R₄-Third ratio R₃
Biomarker Q₁And Q₂The evaluation of “with fatigue” in combination with
Q₁> 0, Q₂> 0 or Q₃> 0
Biomarker Q₁And Q₂The evaluation of “no fatigue” in combination with
Q₁<0 and Q₂<0 and Q₃<0
As provided
R₄= M₄/ B₄
The fourth ratio (R₄) And the third ratio already obtained (R₃) Q calculated based on₃By using Q₁And Q₂As with the case, the fatigue state of the subject can be evaluated. That is, the fatigue evaluation method according to the present invention may further include (4) a step of obtaining a fourth ratio of the measured value of isocitrate concentration in the biological sample obtained from the subject to the fourth reference value. In this case, the step of comparing the first ratio and the fourth ratio, the step of comparing the second ratio and the fourth ratio, and the step of comparing the third ratio and the fourth ratio , For example, a ratio of the second ratio to the first ratio, a third ratio to the second ratio, and a fourth ratio of the third ratio. More preferably, the method further includes a step of performing discriminant analysis, analysis of Partial Last Square, Support Vector Machine, or the like using at least one of the ratios to the above ratio.
In this embodiment, the fourth ratio R related to isocitrate₄Is used, as described above, the first ratio R for glucose R₁, Second ratio R for citric acid₂And a fifth ratio R for succinic acid₅It is preferable to be used together. That is,
Biomarker Q_1a= First ratio R₁-Second ratio R₂
Biomarker Q_2a= Second ratio R₂-Fourth ratio R₄
Biomarker Q_3a= 5th ratio R₅-Fourth ratio R₄
The evaluation of “with fatigue” in this case is
Q_1a> 0, Q_2a> 0 and Q_3aSatisfy at least one of> 0,
The evaluation of “no fatigue” is provided as
Q_1a<0 and Q_2a<0 and Q_3a<0
As provided
R₁= M₁/ B₁
R₂= M₂/ B₂
R₄= M₄/ B₄
R₅= M₅/ B₅
The first, second, fourth and fifth ratios (R₁, R₂, R₄And R₅) Based on Q_1a~ Q_3aBy calculating the fatigue state of the subject can be evaluated. Q_1a~ Q_3aSince at least one of them may be positive, the fatigue state of the subject may be evaluated only by calculating one of them.
From such a viewpoint, in a preferred embodiment, the fatigue evaluation method according to the present invention (1) obtains a first ratio of a measured value of glucose concentration in a biological sample obtained from a subject to a first reference value. A step, (2) obtaining a second ratio of the measured value of the citric acid concentration in the biological sample obtained from the subject to the second reference value, and (3) the isocitrate concentration in the biological sample obtained from the subject. A step of obtaining a fourth ratio of the measured value to the fourth reference value, and (4) a step of obtaining a fifth ratio of the measured value of the succinic acid concentration in the biological sample obtained from the subject to the fifth reference value. , And comparing the first ratio and the second ratio, comparing the second ratio and the fourth ratio, and the fourth ratio and the fifth ratio. Further wrap at least one of the comparing steps It is characterized in that. In the fatigue evaluation method according to the present embodiment, (I) the first ratio is larger than the second ratio, (II) the second ratio is larger than the third ratio, (III) the fourth ratio is The method may further include a step of determining whether or not at least one condition of greater than the third ratio is satisfied, (IV) the first ratio is greater than the second ratio, (V) The method may further include a step of determining whether or not at least one of the second ratio is larger than the fourth ratio and (VI) the fourth ratio is smaller than the fifth ratio is satisfied. Good.
Also, the biomarker Q in the second embodiment described above₇~ Q₁₂Can be used to evaluate (diagnose or determine) whether or not the patient has chronic fatigue syndrome. As mentioned above, biomarker Q₇~ Q₁₂As a result of analysis by a decision tree mathematical model, both show sensitivity and specificity of 90% or more. When the measured value of the glucose concentration in the biological sample obtained from the subject is substantially equal to the reference value, as shown in the examples described later, when the analysis is performed using the items listed in FIG.₇~ Q₁₂At least one of the conditions (R₂<79.1%, R₃<69.3%, R₄<66.0%, R₅<66.6%, R₆<83.7% and R₇<74.3%) is evaluated as “fatigue” and biomarker Q₂₁~ Q₂₆If all of the above do not satisfy the above conditions, it is evaluated as “no fatigue”.
From such a viewpoint, in a preferred embodiment, the fatigue evaluation method according to the present invention includes (1) a step of comparing a measured value of a glucose concentration in a biological sample obtained from a subject with a first reference value. (2) obtaining a second ratio of the measured value of the citric acid concentration in the biological sample obtained from the subject to the second reference value, and (3) cis-aconitic acid in the biological sample obtained from the subject. A step of obtaining a third ratio of the measured concentration value to the third reference value; (4) a fourth ratio of the measured value of isocitrate concentration in the biological sample obtained from the subject to the fourth reference value; A step of obtaining, (5) a step of obtaining a fifth ratio of the measured value of the succinic acid concentration in the biological sample obtained from the subject to the fifth reference value, and (6) the malic acid concentration in the biological sample obtained from the subject. Of the measured value of the sixth At least one of obtaining a sixth ratio with respect to the reference value, and (7) obtaining a seventh ratio with respect to the seventh reference value of the measured value of the lactic acid concentration in the biological sample obtained from the subject. It is characterized by inclusion. In step (1), when there is no substantial difference between the measured value of glucose concentration and the first reference value, the fatigue evaluation method according to the present embodiment is obtained in (2) to (7). A step of determining whether or not the condition that the ratio is smaller than the predetermined ratio is satisfied may be further included. In addition, as shown in the Example mentioned later, when analyzing using the item quoted in FIG. 7, the said predetermined ratio is 79.1%, 69.3% about (2)-(7), respectively. 66.0%, 66.6%, 83.7% and 74.3%. Accordingly, it can be determined with a probability of 95% or more whether or not the subject has chronic fatigue syndrome.
Furthermore, the above-mentioned biomarker Q_7a~ Q_12aCan be used to evaluate (diagnose or determine) whether or not the patient has chronic fatigue syndrome. As mentioned above, biomarker Q_7a~ Q_12aAs a result of analysis by a decision tree mathematical model, both show sensitivity and specificity of 90% or more. In particular, Q_8a~ Q_10aIt is preferable to use biomarker Q_8a~ Q_10aIf at least one of the conditions is smaller than the corresponding threshold value, it is evaluated as “fatigue” and the biomarker Q_8a~ Q_10aIf all of the above do not satisfy the above conditions, it may be evaluated as “no fatigue”.
From such a viewpoint, in a preferred embodiment, the fatigue evaluation method according to the present invention includes (1 ′) a step of comparing a measured value of glucose concentration in a biological sample obtained from a subject with a first threshold value. And (2 ′) a step of comparing the measured value of the citric acid concentration in the biological sample obtained from the subject with the second threshold, and (3 ′) the cis-aconitic acid concentration in the biological sample obtained from the subject. A step of comparing the measured value with a third threshold, (4 ′) a step of comparing the measured value of the isocitrate concentration in the biological sample obtained from the subject with the fourth threshold, and (5 ′) from the subject. A step of comparing the measured value of the succinic acid concentration in the obtained biological sample with the fifth threshold value, (6 ′) a sixth threshold value of the measured value of the malic acid concentration in the biological sample obtained from the subject. Comparing, and (7 ') subject The method further comprises at least one of a step of comparing the measured value of the lactic acid concentration in the obtained biological sample with a seventh threshold value, preferably (3 ′) to (5 ′) above. More preferably, the method includes at least one of the steps (5 ′), and more preferably includes the step (4 ′) after the step (5 ′). More preferably, step 3 ′) is performed. The fatigue evaluation method according to the present embodiment may further include a step of determining whether or not the condition that the measured value is smaller than the corresponding threshold value is satisfied in (1 ′) to (7 ′). Accordingly, it can be determined with a probability of 95% or more whether or not the subject has chronic fatigue syndrome.
Also, the biomarker Q in the third embodiment described above₁₃~ Q₁₈At least one of (or biomarker Q_13a~ Q_18aBy using discriminant analysis, Partial Last Square, or Support Vector Machine analysis, etc., it is evaluated (diagnosis or determination) without using a reference value. )can do. For example, biomarker Q₁₃~ Q₁₈Any three (for example, Q₁₃~ Q₁₅) For discriminant analysis, analysis of Partial Last Square, or Support Vector Machine, etc., it is possible to perform more advanced discrimination. In particular, a representative discriminant analysis is performed, and as described above, the formula (B)
(A * M₂/ M₁+ B * M₃/ M₂+ C * M₄/ M₃) + D (B)
M₁~ M₄It is preferable to determine whether or not the patient has chronic fatigue syndrome based on the result obtained by inputting all of the above. Biomarker Q_13a~ Q_18aAny three (for example, Q_13a~ Q_15a) For discriminant analysis, analysis of Partial Last Square, or Support Vector Machine, etc., it is possible to perform more advanced discrimination. In particular, a representative discriminant analysis is performed and, as described above, the formula (B ′)
(A * M_2a/ M_1a+ B * M_4a/ M_2a+ C * M_5a/ M_4a) + D
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Com, or,,,, this, we will be confused with the following:..
M_1a, M_2a, M_4aAnd M_5aIt is preferable to determine whether or not the patient has chronic fatigue syndrome based on the result obtained by inputting all of the above.
From such a viewpoint, in a preferred embodiment, the fatigue evaluation method according to the present invention includes (1) a step of obtaining a measured value of glucose concentration in a biological sample obtained from a subject, and (2) in a biological sample obtained from the subject. (3) a step of obtaining a measured value of the cis-aconitic acid concentration in the biological sample obtained from the subject, (4) a measurement of the isocitrate concentration in the biological sample obtained from the subject. A step of obtaining a value, and (5) a step of obtaining a measurement value of succinic acid concentration in a biological sample obtained from a subject, and further comprising the step of analyzing the obtained measurement value It is preferable to include.
Although the fatigue evaluation method according to the present invention has been described according to the first and second embodiments of the biomarker of the present invention, the fatigue evaluation method according to the present invention is not limited to these, for example, without using a reference value. In the biological sample obtained from the subject, the measured value of glucose concentration, the measured value of citric acid concentration, and the measured value of cis-aconitic acid concentration (if necessary, the measured value of isocitrate concentration, malic acid concentration The method of evaluating fatigue using the measured value of lactic acid and the measured value of lactic acid concentration), and the method of evaluating fatigue by comparing the measured value in the biological sample obtained from the subject with each reference value, Those skilled in the art who have read this specification will easily understand that it is within the scope of the fatigue evaluation method according to the present invention.
The fatigue evaluation method according to the present invention may further include a step of measuring a glucose concentration, a citric acid concentration, and a cis-aconitic acid concentration in a biological sample obtained from a subject. You may further include the process of measuring malic acid concentration and lactic acid concentration. That is, the fatigue evaluation method according to the present invention is executed based on the glucose concentration, citric acid concentration, and cis-aconitic acid concentration (and isocitrate concentration, malic acid concentration, and lactic acid concentration as necessary) measured in advance. Alternatively, the glucose concentration, citric acid concentration and cis-aconitic acid concentration (and optionally the isocitrate concentration, malic acid concentration and lactic acid concentration) may be measured from a biological sample obtained from the subject.
In the fatigue evaluation method according to the present invention, the first to third reference values are an average glucose concentration, an average citric acid concentration, and an average cis-aconitic acid concentration in biological samples obtained from a plurality of healthy subjects, respectively. However, it is not limited to an average value, and may be a mode value obtained from a binomial distribution or the like. Of course, the fourth to seventh reference values are also preferably the average isocitrate concentration, average succinic acid concentration, average malic acid concentration, and average lactic acid concentration in biological samples obtained from a plurality of healthy subjects, It is not limited to an average value, and may be a mode value obtained from a binomial distribution or the like. In addition, even if these values are pre-defined values, the healthy subjects are sampled simultaneously with the sampling from the subject, and the values calculated based on the concentration of each metabolite in the obtained biological sample. There may be.
In carrying out the present invention, it is necessary to measure the concentrations of metabolites (glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration and lactic acid concentration) in a biological sample. . In the art, enzymatic reactions using glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid or lactic acid as substrates are well known. This is also clear from the fact that measurement kits using such a technique are commercially available. In other words, those skilled in the art can successfully measure the concentration of a metabolite in a biological sample by arbitrarily using such various techniques.
[3-2] Fatigue evaluation kit
A kit having a reagent used for carrying out the fatigue evaluation method as described above is also within the scope of the present invention. That is, the first kit according to the present invention is characterized by including a fifth reagent for measuring the succinic acid concentration in order to evaluate fatigue. A fourth reagent for measuring may further be provided. Further, the first reagent for measuring glucose concentration, the second reagent for measuring citric acid concentration, and cis-aconitic acid concentration are measured. And at least one selected from the group consisting of a third reagent for measuring, a sixth reagent for measuring malic acid concentration, and a seventh reagent for measuring lactic acid concentration May be.
Preferably, the first kit further comprises instructions displaying a reference value for the concentration of succinic acid, optionally including isocitrate, further cis-aconitic acid, and even more. May be displayed with reference values for the concentrations of glucose, citric acid, malic acid and lactic acid.
In this field, enzyme reactions using glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid or lactic acid as a substrate are well known in the field. That is, those skilled in the art can successfully measure the concentration in a biological sample by arbitrarily using such various techniques. For example, the first reagent may be an enzyme Glucose oxidase or peroxidase using glucose as a substrate, and in this case, a color developing agent by a redox reaction (for example, o-Dianiside) as a suitable color developing reagent. The second reagent may be an enzyme Citrate Lyase or Malic dehydrogenase using citric acid as a substrate. In this case, β-NADH is provided in the kit of the present invention as a suitable coloring reagent. The third reagent may be an enzyme Aconitase or Citrate Lyase using cis-aconitic acid as a substrate. In this case, β-NADH, phenylhydrazine is a suitable coloring reagent. In Preferably, the fourth reagent may be an enzyme Isocitrate lyase using isocitrate as a substrate, and in that case, phenylhydrazine is preferably provided in the kit of the present invention as a suitable coloring reagent. preferable. Furthermore, the fifth reagent may be an enzyme Succinyl CoA Synthetase, Pyruvate Kinase, or Lactate Dehydrogenase using succinic acid as a substrate. In this case, β-NADH is provided in the kit of the present invention as a suitable coloring reagent. The sixth reagent may be the enzyme Malate Dehydrogenase using malic acid as a substrate. In this case, it is preferable that NAD + is provided in the kit of the present invention as a suitable coloring reagent. The reagent of No. 7 may be an enzyme Lactate Dehydrogenase or Glutamic Pyrovic Transaminese using lactic acid as a substrate. In this case, NAD + is provided in the kit of the present invention as a suitable coloring reagent. It is preferred that the.
Also, biomarker Q obtained by the fatigue evaluation method as described above₁~ Q₇A kit having a configuration for visually detecting is also within the scope of the present invention. In other words, the second kit according to the present invention is characterized by including a fifth presenting portion showing a fifth ratio in order to evaluate fatigue, and a fourth presenting portion showing the fourth ratio is provided. Furthermore, you may provide further, the 1st presentation part which shows the 1st ratio, the 2nd presentation part which shows the 2nd ratio, the 3rd presentation part which shows the 3rd ratio, and the 6th which shows the 6th ratio You may further provide at least 1 of the presentation part selected from the group which consists of a presentation part and the 7th presentation part which shows a 7th ratio. The second kit according to the present invention may include a separate member provided with each of the first to seventh presentation parts, or a single member provided with all of the first to seventh presentation parts. May be.
Each of the first to seventh presenting parts in the second kit preferably has a configuration that presents a visible color tone according to the measured value, and a reference to be compared with the presented color tone is described. It is preferable to further include instructions. R in the first presentation part₁Based on the first color C₁Is presented, and in the second presentation part, R₂Based on the second color C₂Is presented and C₁And C₂As well as biomarker Q by comparing references₁(Ie C₁And C₂Value based on the difference between the In the third presentation part, R₃Based on the third color C₃Is presented and C₂And C₃As well as biomarker Q by comparing references₂(Ie C₂And C₃Value based on the difference between the Thus, the biomarker (Q obtained using the second kit according to the present invention)₁And Q₂) Can be used to evaluate the fatigue state of the subject. Moreover, in the 4th presentation part, it is R as needed.₄4th color C based on₄Is presented and C₃And C₄As well as biomarker Q by comparing references₃(Ie C₃And C₄Value based on the difference between the biomarker Q and the₃Biomarker Q₁And biomarker Q₂It can also be used together. Those skilled in the art who have read the present specification can similarly provide and use the fifth to seventh presenting units. For the first to seventh presentation parts, an enzyme-coloring method using an enzyme with glucose, citric acid, cis-aconitic acid, isocitric acid, succinic acid, malic acid, or lactic acid as a substrate may be used. it can.
As used herein, the term “kit” is intended as a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material, but as a composition. Forms containing the material in the substance are also encompassed by the term “kit”. The kit preferably includes instructions for using each material. As used herein, in the aspect of a kit, “comprising” is intended to mean being contained in any of the individual containers that make up the kit. Moreover, the kit which concerns on this invention may be the packaging which packed several different compositions in one, and in the case of a solution form, you may enclose in the container. The kit according to the present invention may be provided with a plurality of components mixed in the same container or in separate containers. The “instructions” may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, etc. . The kit according to the present invention may also include a container containing a diluent, a solvent, a washing solution or other reagent. Furthermore, the kit according to the present invention may include instruments and reagents necessary for collecting a biological sample. In addition, the kit according to the present invention may be provided with instruments and reagents necessary for preparing a target preparation from a biological sample.
By having the above-described configuration, the kit according to the present invention can be used easily in an actual medical field, so that it is possible to provide an objective diagnosis with faster and cheaper fatigue.
The kit according to the present invention not only can evaluate fatigue and propose a treatment method, but can also provide a criterion for determining fatigue (including chronic fatigue and chronic fatigue syndrome). It becomes easy to diagnose whether or not That is, the kit according to the present invention diagnoses a kit (for example, fatigue (including chronic fatigue and chronic fatigue syndrome)) for providing a diagnostic standard or a criterion for fatigue (including chronic fatigue and chronic fatigue syndrome). Or a kit for acquiring data for determination).
[3-3] Fatigue evaluation system
The system used to perform the fatigue assessment method as described above is also within the scope of the present invention. In the following embodiment, each member constituting the fatigue evaluation system according to the present invention is a functional block realized by executing a program code stored in a recording medium such as a ROM or a RAM by a calculation means such as a CPU. A case of “some” will be described as an example, but it may be realized by hardware that performs the same processing. Moreover, it is also possible to realize a combination of hardware that performs a part of the processing and the above arithmetic unit that executes program code for controlling the hardware and the remaining processing. Further, even among the members described above as hardware, the hardware for performing a part of the processing and the arithmetic means for executing the program code for performing the control of the hardware and the remaining processing It can also be realized in combination. The arithmetic means may be a single unit, or a plurality of arithmetic means connected via a bus inside the apparatus or various communication paths may execute the program code jointly.
The fatigue evaluation system according to the present invention includes a measurement unit 11, a storage unit 12, a CPU 13, and a display unit 14 as functional blocks. The measuring unit 11 has functions as measuring units 11a to 11g for measuring glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitric acid concentration, succinic acid concentration, malic acid concentration, and lactic acid concentration. Part 12 is a measured value M of glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitric acid concentration, succinic acid concentration, malic acid concentration and lactic acid concentration.₁~ M₇And a reference value B for glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitric acid concentration, succinic acid concentration, malic acid concentration, and lactic acid concentration₁~ B₇The CPU 13 functions as a reference value storage unit 12b, and the CPU 13 receives the evaluation information from the calculation unit 13a that generates information for evaluating fatigue and the calculation unit 13a, and evaluates fatigue. The display part 14 has a function as the evaluation result display part 14 which displays the evaluation result by the evaluation part 13b. This functional block is realized by the CPU 13 executing a program stored in the storage unit 12 and controlling peripheral circuits such as an input / output circuit (not shown).
In the fatigue evaluation system according to the first embodiment, the following steps 11 to 16 are performed for glucose. The measurement unit 11a measures the glucose concentration in the sample, and the glucose concentration measurement value M₁Is acquired (S11). The measurement unit 11a acquires the measured glucose concentration value M.₁Is output to the measured value receiving unit 12a (S12). The calculation unit 13a receives the glucose concentration measurement value M stored in the measurement value reception unit 12a.₁Is read (S13). In addition, the calculation unit 13a has a glucose concentration reference value (first reference value) B stored in the reference value storage unit 12b.₁Is read (S14). The calculation unit 13a is M₁B₁Ratio to (first ratio) R₁Is calculated (S15). The calculation unit 13a uses the first ratio R₁Is output to the evaluation unit 13b (S16).
In the fatigue evaluation system according to the first embodiment, steps 21 to 26 corresponding to the above steps 11 to 16 are executed for citric acid, and the calculation unit 13a performs the second ratio R.₂Is output to the evaluation unit 13b, and steps 31 to 36 corresponding to the above steps 11 to 16 are executed for the cis-aconitic acid, and the calculation unit 13a performs the third ratio R.₃Is output to the evaluation unit 13b, and steps 41 to 46 corresponding to the above steps 11 to 16 are executed for isocitrate, and the calculation unit 13a performs the fourth ratio R.₄Is output to the evaluation unit 13b.
Subsequently, the evaluation unit 13b outputs the first to fourth ratios R output by the calculation unit 13a.₁~ R₄The first ratio R₁To the second ratio R₂Are compared (S51). The evaluation unit 13b uses the first ratio R₁Is the second ratio R₂Greater than (R₁> R₂), It is evaluated as “with fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S52). The evaluation unit 13b uses the first ratio R₁Is the second ratio R₂Less than (R₁<R₂), The second ratio R₂To the third ratio R₃Are compared (S53). The evaluation unit 13b uses the second ratio R₂Is the third ratio R₃Greater than (R₂> R₃), It is evaluated as “with fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S54). The evaluation unit 13b uses the second ratio R₂Is the third ratio R₃Less than (R₂<R₃), The third ratio R₃To the fourth ratio R₄Are compared (S55). The evaluation unit 13b uses the fourth ratio R₄Is the third ratio R₃Greater than (R₄> R₃), It is evaluated as “with fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S56). The evaluation unit 13b uses the fourth ratio R₄Is the third ratio R₃Less than (R₄<R₃), It is evaluated as “no fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S57).
In such a first embodiment, steps 41 to 46 may or may not be executed, and R₄May not be used for fatigue evaluation. In this case, after step 54, the evaluation unit 13b determines that the second ratio R₂Is the third ratio R₃Less than (R₂<R₃), It is sufficient to evaluate “no fatigue” and output the evaluation result to the evaluation result display unit 14 (S58).
For steps 51 to 58 described above, R₁And R₂As an example, a mode of preferentially comparing with the above has been described.₂And R₃Or R₃And R₄Comparison may be made with priority. In addition, four step groups (that is, steps 11 to 16, steps 21 to 26, steps 31 to 36, and steps 41 to 46) are executed simultaneously, so that R₁~ R₄The case where all of the above has been acquired has been described as an example, but R₁~ R₄May not be performed simultaneously. Further, although the present embodiment has been described by taking the glucose concentration, citric acid concentration, cis-aconitic acid concentration and isocitrate concentration as examples, the same processing is performed when the succinic acid concentration, malic acid concentration and lactic acid concentration are used. Is called.
In the fatigue evaluation system according to the present invention, R₁~ R₇Rather than evaluating fatigue using the difference, the following processing may be preferentially executed (second embodiment). In the second embodiment, the storage unit 12 includes the measurement value receiving unit 12a, the reference value storage unit 12b, and the glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration, and lactic acid. Reference value t of concentration₁~ t₇As a reference value storage unit 12c. The calculation unit 13a uses the first ratio R₁Is output to the evaluation unit 13b (S16), and the reference value (first reference value) t of the glucose concentration stored in the measurement value receiving unit 12c.₁Is output to the evaluation unit 13b (S116). The evaluation unit 13b uses the first ratio R₁And the first reference value t₁And compare the two (S150). The evaluation unit 13b uses the first ratio R₁Is the first reference value t₁Is almost equal to (R₁≒ t₁), The evaluation unit 13b subsequently determines that the fifth ratio R₅And the fifth reference value t₅And compare the two (S151). The evaluation unit 13b uses the fifth ratio R₅Is the fifth reference value t₅Less than (R₅<T₅), It is evaluated as “with fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S152). The evaluation unit 13b uses the fifth ratio R₅Is the fifth reference value t₅Greater than (R₅≧ t₅), The evaluation unit 13b subsequently determines that the fourth ratio R₄And the fourth reference value t₄And compare the two (S153). The evaluation unit 13b uses the fourth ratio R₄Is the fourth reference value t₄Less than (R₄<T₄), It is evaluated as “with fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S154). The evaluation unit 13b uses the fourth ratio R₄Is the fourth reference value t₄Greater than (R₄≧ t₄), The evaluation unit 13b subsequently determines that the third ratio R₃And the third reference value t₃And compare the two (S155). The evaluation unit 13b uses the third ratio R₃Is the third reference value t₃Less than (R₃<T₃), It is evaluated as “with fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S156). The evaluation unit 13b uses the third ratio R₃Is the third reference value t₃Greater than (R₃≧ t₃), It is evaluated as “no fatigue”, and the evaluation result is output to the evaluation result display unit 14 (S157). The present embodiment has been described by taking the succinic acid concentration, isocitric acid concentration, and cis-aconitic acid concentration as examples, but the same processing is performed when the glucose concentration, citric acid concentration, malic acid concentration, and lactic acid concentration are further used. . Of course, fatigue can be assessed based solely on the high probability cis-aconitic acid concentration, isocitrate concentration and / or succinic acid concentration.
In the fatigue evaluation system according to the present invention, the following processing may be preferentially executed. In the present embodiment, the storage unit 12 includes the measurement value receiving unit 12a, the reference value storage unit 12b, and the glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration, and lactic acid concentration. Threshold T₁~ T₇As a threshold storage unit 12d. The calculation unit 13a calculates the fifth measurement value M₄Is output to the evaluation unit 13b, and the succinic acid concentration threshold (fifth threshold) T stored in the measurement value receiving unit 12d₅Is output to the evaluation unit 13b. The evaluation unit 13b receives the fifth measurement value M₅And the fifth threshold T₅And compare the two. The evaluation unit 13b receives the fifth measurement value M₅Is the fifth threshold T₅Smaller than (M₅<T₅) Is evaluated, “it is fatigued”, and the evaluation result is output to the evaluation result display unit 14. The evaluation unit 13b receives the fifth measurement value M₅Is the fifth threshold T₅Larger than (M₅≧ T₅), The evaluation unit 13b subsequently determines that the fourth measurement value M₄And the fourth threshold T₄And compare the two. The evaluation unit 13b receives the fourth measurement value M₄Is the fourth threshold T₄Smaller than (M₄<T₄) Is evaluated, “it is fatigued”, and the evaluation result is output to the evaluation result display unit 14. The evaluation unit 13b receives the fourth measurement value M₄Is the fourth threshold T₄Larger than (M₄≧ T₄), The evaluation unit 13b continues to determine the third measurement value M.₃And the third threshold T₃And compare the two. The evaluation unit 13b receives the third measurement value M₃Is the third threshold T₃Smaller than (M₃<T₃) Is evaluated, “it is fatigued”, and the evaluation result is output to the evaluation result display unit 14. The evaluation unit 13b receives the third measurement value M₃Is the third threshold T₃Larger than (M₃≧ T₃) Is evaluated, “no fatigue” is evaluated, and the evaluation result is output to the evaluation result display unit 14.
In addition, when the reference value storage unit 12b does not exist or when the reference value storage unit 12b has the first to seventh reference values B₁~ B₇In the case where is not stored, the fatigue evaluation system according to the present invention can also evaluate the presence or absence of fatigue (third embodiment). The calculation unit 13a uses the measurement value M stored in the measurement value receiving unit 12a.₁~ M₇Is output to the evaluation unit 13b. The calculation unit 13a uses the measurement value M stored in the measurement value receiving unit 12a.₁~ M₇Is output to the evaluation unit 13b. Subsequently, the evaluation unit 13b first to seventh measurement values M output from the calculation unit 13a.₁~ M₇Receive M₁~ M7₄Are compared (S61). For example, the evaluation unit 13b receives the received M₁~ M₄M obtained from₂/ M₁, M₃/ M₂, M₄/ M₃, M₃/ M₁, M₄/ M₁, M₄/ M₂Is again output to the calculation unit 13a (S62). In the calculation unit 13a, for example, the input M₂/ M₁, M₃/ M₂, M₄/ M₃(For example, discriminant analysis, Partial Last Square, Support Vector Machine, etc.) is executed (S63). The evaluation unit 13b that has received the analysis result output from the calculation unit 13a outputs a determination result as to whether or not the patient is a chronic fatigue syndrome patient to the evaluation result display unit 14 based on the cutoff value (S64). Although the present embodiment has been described by taking the glucose concentration, citric acid concentration, cis-aconitic acid concentration and isocitrate concentration as examples, the same processing is performed when the succinic acid concentration, malic acid concentration and lactic acid concentration are further used. .
The measurement unit 11 measures the glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration, and lactic acid concentration, and the value obtained by the measurement unit 11 is the measured value receiving unit 12a. Although the present invention has been described using the embodiment inputted in the above, the previously obtained glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration and lactic acid concentration are directly The aspect inputted to the measured value reception part 12a may be sufficient, and the fatigue evaluation system concerning this invention does not need to be provided with the measurement part 11 in this case.
In addition, if the system according to the present invention is used, not only can fatigue be evaluated and a treatment method can be proposed, but also a judgment criterion for fatigue (including chronic fatigue and chronic fatigue syndrome) can be provided. It becomes easy to diagnose whether or not That is, the system according to the present invention diagnoses a system (for example, fatigue (including chronic fatigue, chronic fatigue syndrome)) for providing a diagnosis standard or determination standard for fatigue (including chronic fatigue, chronic fatigue syndrome). Or a system for obtaining data for determination).
The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

20 healthy persons (male / female ratio = 1: 1, average age = 36.10 years; provided by Osaka City University Hospital), 20 chronic fatigue syndrome (male / female ratio = 1: 1, average age = 36.15 years; (Provided by Osaka City University Hospital). A capillary electrophoresis-mass spectrometer was used to measure metabolites in plasma. Glucose analysis kit II (Biovision) was used for blood glucose level measurement. For statistical analysis, significance test, correlation test, and path analysis using SPSS 17.0 and Amos 18.0 were adopted.
Plasma samples were prepared from blood collected from healthy individuals and patients with chronic fatigue syndrome (CFS). We asked Keio University Institute for Advanced Life Sciences to measure metabolites in pretreated plasma samples. As a result, in the majority of metabolites containing branched chain amino acids, no quantitative difference was found between healthy subjects and CFS patients, but the amount of some metabolites was significantly different in the CFS patient group. I found that it was falling. In FIG. 1, metabolites that form a glycolytic system and a citric acid cycle, in which a quantitative difference was observed between healthy subjects and CFS patients, were listed.
Next, the correlation between metabolite whose amount in plasma significantly changed corresponding to the presence of fatigue and performance status (PS) was examined. PS is a measure of the subjective severity of fatigue, with higher numbers indicating more severe. The results are shown in FIG. It was found that some of the metabolites examined showed a significant correlation with PS.
Furthermore, the metabolite whose amount in plasma significantly changed based on the presence or absence of fatigue and showed a significant correlation with PS was analyzed in detail by a path analysis for predicting a causal relationship. Based on the results thus far, it was estimated that abnormalities occurred in the glycolysis system and citrate circuit in CFS patients, and the blood glucose level in the same sample was separately measured and similarly subjected to path analysis. As a result, it was found that CFS patients can be distinguished from healthy individuals based on the plasma levels of three types of metabolites (glucose, citric acid, cis-aconitic acid) (FIG. 3). FIG. 3 shows a metabolite comparison between healthy and CFS patients. In the figure, the average value for healthy individuals is 100. It was also found that the use of another kind of plasma (isocitrate) in plasma makes it easier to distinguish CFS patients from healthy individuals (FIG. 3).
In addition, based on discriminant analysis, the measured values of these metabolites are used to simultaneously evaluate the citric acid / glucose ratio, cis-aconitic acid / citric acid ratio, and isocitrate / cis-aconitic acid ratio on three axes. As a result, the normal group and the chronic fatigue syndrome patient were successfully separated almost completely (FIG. 4). Compared with the citric acid / glucose gradient and the cis-aconitic acid / citric acid gradient shown in FIG. Although it was thought that isocitrate may be less important than the other three metabolites, the results shown in FIG. It was found that this analysis can discriminate CFS patients with an accuracy of 90% or more even in the case of using not only discriminant analysis but also Partial Least Square, Support Vector Machine, etc. (data not shown) ).
When examined for additional metabolites, succinic acid was also found to be useful in distinguishing CFS patients from healthy individuals based on plasma levels (FIG. 5). Since cis-aconitic acid is unstable, it is not easy to measure the concentration in the sample. However, if the concentration of succinic acid in the sample is used, the present invention can be carried out more easily. Also, as a result of discriminant analysis, the normal group and the chronic fatigue syndrome patient can be almost completely separated by simultaneously evaluating the citric acid / glucose ratio, isocitrate / citric acid ratio, and succinic acid / isocitrate ratio on three axes. (Fig. 6). In addition, it was found that this analysis can discriminate CFS patients with 90% or more accuracy even when using the Partial Last Square, Support Vector Machine, etc. as well as the discriminant analysis (data shown) )
The measured value of the metabolite in the plasma sample was input to analysis software R (free software, version 2.11.1), and a random forest (Random Forest) program was executed. Random Forest is an algorithm used for identification, regression, and clustering. As shown in FIG. 7, isocitrate, succinic acid and Cis-aconitic acid are very important among a plurality of measurement items when classifying healthy subjects and chronic fatigue patients, and isocitrate It has been found that measuring one of succinic acid and Cis-aconitic acid can determine whether a subject has chronic fatigue syndrome.
The measured value of the metabolite (the compound listed in FIG. 7) in the plasma sample was input to the analysis software R to execute a tree-based model program. This program is used in the field of decision theory to plan and reach goals. As shown in Table 1, when classifying healthy subjects and chronic fatigue patients, succinic acid is the most important factor among the plurality of measurement items, and the succinic acid measurement value is a threshold T2 (14 When it is lower than .56 μM), it has been found that it can be determined with a probability of 95% that the subject has chronic fatigue syndrome. Next, the remaining measurement values excluding the succinic acid measurement values were input to the analysis software R to execute the tree model program. As a result, as shown in Table 1, isocitrate is the most important factor among a plurality of measurement items excluding succinic acid when classifying healthy subjects and chronic fatigue patients, and isocitrate When the measured value was lower than the threshold value T1 (7.46 μM), it was found that it can be determined with a probability of 95% that the subject has chronic fatigue syndrome. Further, the remaining measurement values excluding the succinic acid measurement value and the isocitrate measurement value were input to the analysis software R to execute the tree model program.
As a result, as shown in Table 1, cis-aconitic acid is the most important factor among a plurality of measurement items excluding succinic acid and isocitrate when classifying healthy subjects and chronic fatigue patients. And when the measured value of cis-aconitic acid was lower than the threshold value T3 (10.66 μM), it was found that the test subject can be determined with a probability of 95% as having chronic fatigue syndrome.
Probability of determining that a subject has chronic fatigue syndrome when the remaining measurement values excluding succinic acid measurement value, isocitric acid measurement value and cis-aconitic acid measurement value are input to analysis software R and the tree model program is executed Was less than 80%. By measuring malic acid with glutamic acid and isoleucine, the probability that the subject can be determined to have chronic fatigue syndrome is greater than 95%, and by measuring lactic acid with citric acid and creatine, the subject has chronic fatigue syndrome Is 95% or more. However, since it is necessary to measure the amount of a plurality of substances in plasma, it can be said that it is not preferable.
Thus, by measuring any one of isocitrate, succinic acid and cis-aconitic acid in the plasma of the subject, it was found that the subject can be determined with a 95% probability that the subject has chronic fatigue syndrome. . In particular, succinic acid is the most important, followed by isocitrate and cis-aconitic acid in this order. Therefore, when determining chronic fatigue syndrome using a single metabolite, succinic acid is measured. It is most preferable that when determining the chronic fatigue syndrome using a plurality of metabolites, it is most preferable to perform the measurement in the order of succinic acid and isocitric acid, or in the order of succinic acid, isocitric acid and cis-aconitic acid. .
Note that the calculated threshold value may differ depending on the method of obtaining the measurement value. In consideration of this possibility, it is preferable to determine that the patient has chronic fatigue syndrome when the condition that any one of t1, t2, and t3 is smaller than the minimum value is satisfied. In the examples, t1 to t3 are ratios between the measured values of isocitric acid, succinic acid and cis-aconitic acid from subjects and the average values of healthy individuals of isocitric acid, succinic acid and cis-aconitic acid ( %). The reference value is a value obtained by “t = (threshold value T / average value of healthy subjects) × 100%”. In the analysis target compound and analysis software used in this example, isocitrate and succinic acid were used. And the reference values for cis-aconitic acid are 66.0%, 66.6% and 69.3%, respectively.

If the present invention is used, fatigue diagnosis with a simple kit becomes possible. Furthermore, there is a possibility of finding an effective treatment based on the result of diagnosis using the present invention. For example, by applying the present invention to a patient who complains of a strong feeling of fatigue or a long-term feeling of fatigue, an objective evaluation of the degree of fatigue can be performed, and further, it can be determined whether or not the patient has chronic fatigue syndrome. The evaluation of the degree of fatigue according to the present invention does not require a high level of knowledge about fatigue, and therefore can be performed in general medical facilities. In addition, by using the present invention, it is possible to infer which part of the in vivo energy (ATP) -producing metabolic system is abnormal, so that not only can it be used for the patient's life guidance, but also the possible metabolism It becomes possible to provide a food that promotes the system or a food that can promote energy production even under abnormal metabolism.
The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

The use of the present invention makes it possible to objectively and easily perform fatigue evaluation and diagnosis. The present invention may further provide techniques useful for treating fatigue. Because fatigue is a very significant health problem, the realization of fatigue assessment, diagnosis and treatment contributes greatly across all industries.

Claims

Obtaining a first ratio of a measured value of glucose concentration in a biological sample obtained from a subject to a first reference value;
Obtaining a second ratio of a measured value of citrate concentration in a biological sample obtained from a subject to a second reference value;
Obtaining a third ratio of a measured value of cis-aconitic acid concentration in a biological sample obtained from a subject to a third reference value;
Obtaining a fourth ratio of a measured value of isocitrate concentration in a biological sample obtained from a subject to a fourth reference value;
Obtaining a fifth ratio of the measured value of the succinic acid concentration in the biological sample obtained from the subject to the fifth reference value;
Obtaining a sixth ratio of a measured value of malic acid concentration in a biological sample obtained from a subject to a sixth reference value; and a seventh reference of a measured value of lactic acid concentration in the biological sample obtained from the subject A method for assessing fatigue comprising at least two steps selected from the group consisting of obtaining a seventh ratio to value.
The method according to claim 1, further comprising the step of comparing at least one pair in at least two ratios obtained by the at least two steps.
Selected from the group consisting of obtaining the first ratio, obtaining the second ratio, obtaining the third ratio, obtaining the fourth ratio, and obtaining the fifth ratio. The method of claim 2 comprising at least two steps.
The method according to claim 1, comprising at least one step selected from the group consisting of a step of obtaining a first ratio and a step of obtaining a second ratio to a step of obtaining a seventh ratio.
5. The method according to claim 4, further comprising, for the second ratio to the seventh ratio, a step of comparing the obtained ratio with a threshold corresponding to the ratio.
6. The method according to claim 4, comprising at least one of a step of obtaining a third ratio to a step of obtaining a fifth ratio.
The method according to claim 6, wherein the step of obtaining the fourth ratio is performed subsequent to the step of obtaining the fifth ratio.
The method according to claim 7, wherein the step of obtaining the third ratio is performed subsequent to the step of obtaining the fourth ratio.
The method according to any one of claims 4 to 8, which is carried out when the first ratio is substantially 1.
Comparing a first measured value of a glucose concentration in a biological sample obtained from a subject with a first threshold;
Comparing the second measured value of the citric acid concentration in the biological sample obtained from the subject with the second threshold,
Comparing a third measured value of a cis-aconitic acid concentration in a biological sample obtained from a subject with a third threshold;
Comparing the fourth measured value of the isocitrate concentration in the biological sample obtained from the subject with a fourth threshold;
Comparing the fifth measured value of the succinic acid concentration in the biological sample obtained from the subject and the fifth threshold value,
Comparing the sixth threshold value of the sixth measurement value of the malic acid concentration in the biological sample obtained from the subject, and the seventh threshold value of the seventh measurement value of the lactic acid concentration in the biological sample obtained from the subject. A method for assessing fatigue, comprising at least one of the steps of:
Comparing the third measurement with a third threshold;
11. The method of claim 10, comprising at least one of comparing a fourth measurement value with a fourth threshold value and comparing a fifth measurement value with a fifth threshold value.
The method according to claim 11, wherein the step of comparing the fourth measured value with a fourth threshold value is performed subsequent to the step of comparing the fifth measured value with a fifth threshold value.
The method according to claim 12, wherein the step of comparing the third measurement value and the third threshold value is performed subsequent to the step of comparing the fifth measurement value and the fifth threshold value.
Selected from the group consisting of measured glucose concentration, measured citric acid concentration, measured cis-aconitic acid concentration, measured isocitrate concentration, and measured succinic acid concentration in a biological sample obtained from a subject A method of assessing fatigue, comprising the step of obtaining at least a pair of ratios in at least two.
15. The method according to claim 14, further comprising a step of subjecting the obtained at least a pair of ratios to analysis of discriminant analysis, Partial Last Square, or Support Vector Machine.
A kit for evaluating fatigue, comprising a fifth reagent for measuring succinic acid concentration.
The kit according to claim 16, further comprising a fourth reagent for measuring the isocitrate concentration.
A first reagent for measuring glucose concentration;
A second reagent for measuring citric acid concentration,
a third reagent for measuring the cis-aconitic acid concentration;
The kit according to claim 17, further comprising at least one of a sixth reagent for measuring malic acid concentration and a seventh reagent for measuring lactic acid concentration.
A kit for evaluating fatigue, comprising a fifth presenting portion showing a fifth ratio of the measured value of the succinic acid concentration to the fifth reference value.
The kit according to claim 19, further comprising a fourth presentation unit indicating a fourth ratio of the measured value of the isocitrate concentration to the fourth reference value.
A first presentation unit that indicates a first ratio of a measured value of glucose concentration to a first reference value;
A second presentation unit showing a second ratio of the measured value of the citric acid concentration to the second reference value;
a third presentation unit showing a third ratio of the measured value of the cis-aconitic acid concentration to the third reference value;
A sixth presenting unit indicating a sixth ratio of the measured value of malic acid concentration to a sixth reference value; and a seventh presenting unit indicating a seventh ratio of the measured value of lactic acid concentration to the seventh reference value. 21. The kit according to claim 20, further comprising at least one.
A measurement value receiving unit for receiving at least two measurement values selected from the group consisting of measurement values of glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration and lactic acid concentration;
At least two selected from the group consisting of first to seventh reference values corresponding to each of glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration and lactic acid concentration A reference value storage unit that stores reference values,
An evaluation unit that receives the measurement value from the measurement value receiving unit and the reference value from the reference value storage unit and generates information for evaluating fatigue, and an evaluation unit that receives evaluation information from the calculation unit and evaluates fatigue A fatigue evaluation system comprising a part,
The calculation unit
A first ratio of a measured glucose concentration value to a first reference value;
A second ratio of a measured value of citric acid concentration to a second reference value;
a third ratio of the measured value of cis-aconitic acid concentration to a third reference value;
A fourth ratio of the measured value of isocitrate concentration to a fourth reference value;
A fifth ratio of the measured value of succinic acid concentration to a fifth reference value;
Calculate at least two ratios selected from the group consisting of the sixth ratio of the measured value of malic acid concentration to the sixth reference value and the seventh ratio of the measured value of lactate concentration to the seventh reference value A fatigue evaluation system.
The fatigue evaluation system according to claim 22, wherein the calculation unit performs at least a pair of comparisons in the at least two ratios.
The measurement value receiving unit receives at least two measurement values selected from the group consisting of measurement values of glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration and succinic acid concentration;
The reference value storage unit is at least two selected from the group consisting of first to fifth reference values corresponding to glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, and succinic acid concentration, respectively. Store the reference value,
The arithmetic unit is
A first ratio of a measured glucose concentration value to a first reference value;
A second ratio of a measured value of citric acid concentration to a second reference value;
a third ratio of the measured value of cis-aconitic acid concentration to a third reference value;
At least two ratios selected from the group consisting of a fourth ratio of a measured value of isocitrate concentration to a fourth reference value and a fifth ratio of a measured value of succinic acid concentration to a fifth reference value; Calculate further,
24. The fatigue assessment system of claim 23, wherein at least a pair of comparisons are performed at the at least two ratios.
The calculation unit calculates the first ratio,
A second ratio of a measured value of citric acid concentration to a second reference value;
a third ratio of the measured value of cis-aconitic acid concentration to a third reference value;
A fourth ratio of the measured value of isocitrate concentration to a fourth reference value;
A fifth ratio of the measured value of succinic acid concentration to a fifth reference value;
Calculate at least one ratio selected from the group consisting of the sixth ratio of the measured value of malic acid concentration to the sixth reference value and the seventh ratio of the measured value of lactic acid concentration to the seventh reference value The fatigue evaluation system according to claim 24.
The reference value storage unit stores at least one threshold selected from the group consisting of thresholds corresponding to the second ratio to the seventh ratio;
26. The system according to claim 25, wherein the arithmetic unit receives the threshold value from the reference value storage unit, and compares the calculated ratio with a threshold value corresponding to the ratio.
27. The fatigue evaluation system according to claim 25, wherein the calculation unit calculates at least one of a third ratio to a fifth ratio.
The fatigue evaluation system according to claim 27, wherein the calculation unit calculates the fifth ratio subsequent to the fourth ratio.
The fatigue evaluation system according to claim 28, wherein the calculation unit calculates the third ratio subsequent to the fifth ratio.
30. The fatigue evaluation system according to any one of claims 25 to 29, wherein the determination unit determines whether or not the first ratio is substantially 1.
A measurement value receiver that receives at least one measurement value selected from the group consisting of a measurement value of glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration, and lactic acid concentration;
At least one threshold selected from the group consisting of first to seventh thresholds corresponding to each of glucose concentration, citric acid concentration, cis-aconitic acid concentration, isocitrate concentration, succinic acid concentration, malic acid concentration and lactic acid concentration Threshold storage unit storing
A calculation unit that receives the measurement value from the measurement value receiving unit and the threshold value from the threshold storage unit and generates information for evaluating fatigue, and an evaluation unit that receives evaluation information from the calculation unit and evaluates fatigue A fatigue evaluation system comprising:
The calculation unit
A first comparison between a measured glucose concentration and a first threshold;
A second comparison of the measured citric acid concentration with a second threshold;
a third comparison of the measured cis-aconitic acid concentration with a third threshold;
A fourth comparison of the measured value of isocitrate concentration with a fourth threshold;
A fifth comparison of the measured succinic acid concentration with a fifth threshold;
Performing at least one selected from the group consisting of a sixth comparison of the measured value of malic acid concentration with a sixth threshold, and a seventh comparison of the measured value of lactate concentration with a seventh threshold. Evaluation system.
32. The fatigue evaluation system according to claim 31, wherein at least one selected from the group consisting of the third to fifth comparisons is executed.
The fatigue evaluation system according to claim 32, wherein the fourth comparison is performed subsequent to the fifth comparison.
The fatigue evaluation system according to claim 33, wherein the third comparison is performed subsequent to the fifth comparison.
A measurement value receiving unit for receiving at least two measurement values selected from the group consisting of a glucose concentration, a citric acid concentration, a cis-aconitic acid concentration measurement value, an isocitrate concentration measurement value, and a succinic acid concentration measurement value;
This is a fatigue evaluation system including a calculation unit that receives measurement values from a measurement value receiving unit and generates information for evaluating fatigue, and an evaluation unit that receives evaluation information from the calculation unit and evaluates fatigue. And
The fatigue evaluation system in which the arithmetic unit performs at least a pair of comparisons in the at least two measured values to obtain at least a pair of ratios.
36. The fatigue evaluation system according to claim 35, wherein the calculation unit executes discriminant analysis, Partial Last Square, or Support Vector Machine analysis based on the obtained at least a pair of ratios.
First to third measuring units for measuring glucose concentration, citric acid concentration and cis-aconitic acid concentration, and a fourth measuring unit for measuring isocitrate concentration, if necessary, for measuring succinic acid concentration A fifth measuring unit, a sixth measuring unit for measuring malic acid concentration, and a seventh measuring unit for measuring lactic acid concentration,
37. The fatigue evaluation system according to claim 22, wherein values obtained by the first to seventh measurement units are input to the measurement value receiving unit.