Chronic fatigue marker, method for determining chronic fatigue level, and determination reagent

Abstract

To provide a chronic fatigue marker capable of noninvasively performing inspection, and a method for determining a chronic fatigue level.SOLUTION: A chronic fatigue marker including both sIgA and cortisol is the index of a human chronic fatigue level. A method for determining a chronic fatigue level comprises: a measuring step of measuring the amount of the chronic fatigue marker including both sIgA and cortisol in the biological specimen of a subject at a timing before performing work with suffering from stress in the daytime; and a determination step of determining the chronic fatigue level of the subject on the basis of the measured amount of the chronic fatigue marker.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a chronic fatigue marker, a method for determining a chronic fatigue level, and a determination reagent. [Background technology]

[0002] Conventionally, a method for evaluating stress using α-amylase in saliva as a marker has been known (Non-Patent Document 1). By using a substance contained in saliva as a marker, the test can be performed non-invasively without the need for invasive tests such as blood sampling. [Prior art documents] [Patent documents]

[0003] [Non-Patent Document 1] "Salivary Alpha-Amylase as a Biomarker of Stress in Behavioral Medicine." Ali N, Nater UM. Int J Behav Med. 2020 Jan 3 Summary of the Invention [Problem to be solved by the invention]

[0004] However, there is a demand for a chronic fatigue marker that can determine a subject's chronic fatigue level.

[0005] Therefore, an object of the present invention is to provide a chronic fatigue marker that can be tested non-invasively. [Means for solving the problem]

[0006] To achieve the above object, the chronic fatigue marker of the present invention includes both sIgA and cortisol, and serves as an indicator of a human chronic fatigue level.

[0007] The method for determining a chronic fatigue level of the present invention comprises the steps of: The method includes a measuring step and a determining step, The measuring step includes measuring the amount of a chronic fatigue marker in a biological sample of the subject before performing a stressful task during the day; The chronic fatigue markers include both sIgA and cortisol; In the determination step, a chronic fatigue level of the subject is determined based on the measured amount of the chronic fatigue marker.

[0008] The determination reagent of the present invention contains a binding substance that specifically binds to sIgA and a binding substance that specifically binds to cortisol, and is used in the above-mentioned method for determining a level of chronic fatigue. Effect of the Invention

[0009] According to the present invention, a chronic fatigue marker that can be tested non-invasively can be provided. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 is the structural formula of cortisol. [Diagram 2] FIG. 2 is a diagram showing an example of a questionnaire used for measuring acute stress. [Diagram 3] FIG. 3 is a diagram showing the experimental schedule in Example 1. [Figure 4] FIG. 4 is a graph showing saliva marker concentrations in Example 1. [Diagram 5] FIG. 5 is a graph showing saliva marker concentrations in Example 1. [Figure 6] FIG. 6 is a diagram showing an example of a questionnaire used for measuring accumulated fatigue. [Figure 7] FIG. 7 is a CSFI radar chart in the second embodiment. [Figure 8] FIG. 8 is a CSFI radar chart in the second embodiment. [Figure 9] FIG. 9 is a CSFI radar chart in the third embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The chronic fatigue marker of the present invention may be in an embodiment in which the sIgA and cortisol are sIgA and cortisol in saliva.

[0012] In the chronic fatigue level determination method of the present invention, the measurement step may further include measuring the amount of sIgA in the subject's biological sample before performing stressful work at night, and determining in the determination step that the subject's level of chronic fatigue is high if the amount of sIgA measured before the daytime work is the same as or lower than the amount of sIgA measured before the nighttime work, and if the amounts of sIgA measured before the daytime work and before the nighttime work remain at high concentrations.

[0013] The chronic fatigue level assessment method of the present invention may have an aspect in which, in the assessment step, the subject's chronic fatigue level is assessed to be high if the amount of cortisol measured before daytime work over multiple days remains at a low concentration.

[0014] In the chronic fatigue level determination method of the present invention, the measurement step may further include measuring the amount of sIgA in the subject's biological sample before performing stressful work at night, and determining in the determination step that the subject's chronic fatigue level is high if the amounts of sIgA measured before the daytime work and before the nighttime work are high and the amounts of cortisol measured over multiple days before the daytime work are low.

[0015] The chronic fatigue level determination method of the present invention may be configured such that the biological sample is saliva.

[0016] The determination reagent of the present invention may be in an embodiment in which the binding substance is at least one of an antibody and an aptamer.

[0017] Terms used in this specification can be used in the same manner as commonly used in the art unless otherwise specified.

[0018] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiment.

[0019] (marker) The chronic fatigue marker of the present invention contains at least one of sIgA (Secretory Immunoglobulin A) and cortisol, and serves as an indicator of a human chronic fatigue level.

[0020] Cortisol is an adrenal cortical hormone represented by the structural formula shown in Figure 1.

[0021] The sIgA and cortisol can be obtained, for example, from a biological sample or by chemical synthesis.

[0022] The sIgA and cortisol are, for example, sIgA and cortisol, respectively, in non-blood components such as saliva, tears, nasal secretions, urine, etc. The biological sample is not particularly limited and is, for example, a human biological sample.

[0023] The chronic fatigue marker of the present invention serves as an index of the level of chronic fatigue.

[0024] The chronic fatigue is not particularly limited. The chronic fatigue can be, for example, rephrased as accumulated fatigue and cumulative fatigue. The chronic fatigue can be measured, for example, by using the questionnaire shown in Fig. 6 described later, by cumulative fatigue symptom index.

[0025] The marker of the present invention may include, for example, both sIgA and cortisol. This makes it possible to determine the chronic fatigue level according to, for example, a combination of the detection results of sIgA and cortisol.

[0026] The marker of the present invention may further include, for example, a substance other than sIgA and cortisol. The substance other than sIgA and cortisol is not particularly limited and may be a marker other than the marker of the present invention.

[0027] The marker of the present invention may be used, for example, as a subject to be quantified in the chronic fatigue level determination method of the present invention described below.

[0028] For methods for detecting the markers of the present invention and methods for determining a chronic fatigue level using the markers of the present invention, reference can be made to the description of the chronic fatigue level determination method of the present invention described below.

[0029] The marker of the present invention, for example, enables earlier diagnosis and diagnosis before burnout, whereas in the past, chronic fatigue level could not be known by anyone other than the person himself / herself without a medical interview or the person's complaint. Therefore, since the intervention period is short, it is possible to prevent the occurrence of mental disorders such as mental disorders, reduce turnover rates, and prevent burnout. Furthermore, the marker of the present invention can grasp the condition of the person more accurately by performing a test based on biological information in addition to a chronic fatigue level evaluation other than the chronic fatigue level determination method of the present invention, such as a subjective evaluation.

[0030] (How to determine chronic fatigue level) The chronic fatigue level determination method of the present invention includes a measurement step and a determination step, in which the measurement step measures the amount of a chronic fatigue marker in a biological sample of a subject before performing stressful daytime work, the chronic fatigue marker being at least one of sIgA (Secretory Immunoglobulin A) and cortisol, and in the determination step, the chronic fatigue level of the subject is determined based on the measured amount of the chronic fatigue marker.

[0031] The biological sample is, for example, a non-blood component such as saliva, tears, nasal discharge, urine, etc. The biological sample is not particularly limited and is, for example, a human biological sample.

[0032] The stressful work is not particularly limited. The stress includes mental stress and physical stress. For example, it also includes physiological fatigue, peripheral fatigue, and physical fatigue caused by physical work. For example, the stressful work includes day shifts and night shifts.

[0033] The stressful work may be preset based on, for example, a predetermined condition. The predetermined condition may be based on a known stress assessment method, may be based on the magnitude of physical or mental stress in the work, or may be calculated from the number of steps, the amount of activity, and calories burned. Specific examples of the known stress assessment method include the Simple Occupational Stress Questionnaire, K6, and CES-D (the Center for Epidemiologic Studies - Depression Scale). Also, as shown in FIG. 2, a questionnaire such as "Subjective Symptom Survey" (measurement of acute stress) may be used.

[0034] The time period for "stressful daytime work" is not particularly limited, and specifically, for example, work from 08:30 to 17:00. The time period for "stressful nighttime work" described below is not particularly limited, and specifically, for example, work from 16:30 to 09:00. "Daytime work" and "nighttime work" can also be referred to as, for example, the day shift and the night shift, respectively.

[0035] The "timing before performing stressful work" is not particularly limited, and examples thereof include 0 to 60 minutes and 15 to 30 minutes before performing the stressful work. It may also be 0 to 30 minutes after the start of the work. In the present invention, "measurement before daytime work" means, for example, measurement between 1 hour before the start of daytime work and 30 minutes after the start of work, specifically, for example, measurement between 7:30 and 9:00. In the present invention, "measurement before nighttime work" means, for example, measurement between 1 hour and 30 minutes before the start of nighttime work and 30 minutes after the start of work, specifically, for example, measurement between 15:00 and 17:00.

[0036] The amount of sIgA and cortisol, which are the chronic fatigue markers, may be measured directly, or may be calculated based on the indirect amount of mRNA, precursor, etc. The amount of sIgA and cortisol may be a combination of multiple measurement results, for example, an average value of multiple measurement results. The number of times is not particularly limited, and may be, for example, 2 to 10 times, 2 times, and 4 times.

[0037] The amount of sIgA and cortisol can be, for example, the concentration of sIgA and cortisol in a biological sample. The concentration of sIgA and cortisol in the biological sample can be measured, for example, by detecting sIgA and cortisol using an ELISA kit (trade name: ELISA Kit for Secretory Immunoglobulin A (sIgA), Cloud-clone, product number: #SEA641HU, sandwich method; and trade name: YK241 Cortisol (Saliva) EIA kit, Yanaihara Laboratories, product number: #YK241, competitive method) on a saliva sample collected using a saliva collection kit (trade name: Salisoft, Sarstedt Co., Ltd.).

[0038] In the determination step, the chronic fatigue level of the subject is determined based on the measured amounts of sIgA and cortisol.

[0039] The measuring step may further include measuring the amount of the chronic fatigue marker in the biological sample of the subject at a timing before the subject performs stressful work at night.

[0040] If the chronic fatigue marker is sIgA, for example, if in the determination step the amount of sIgA during the day is the same as or less than the amount of sIgA at night, and if the amounts of sIgA measured during the day and at night over multiple days remain at high concentrations, the subject's level of chronic fatigue can be determined to be high.

[0041] The term "multiple days" is not particularly limited and may refer to two or more days.

[0042] The determination of whether "the amount of sIgA during the day is the same as or less than the amount of sIgA during the night" is not particularly limited, and may be, for example, whether the amount of sIgA during the day is equal to or less than the amount of sIgA during the night, or whether the amount of sIgA during the day is less than the amount of sIgA during the night by more than a predetermined reference value. The reference value may be set as appropriate, and may be set in advance, for example, based on measurements using a biological sample from a subject with a known chronic fatigue level (the same applies below).

[0043] The determination of whether "the amount of sIgA during the day and during the night measured over a plurality of days is changing at a high concentration" is not particularly limited, and may be determined, for example, by whether the amount of sIgA during the day and during the night measured over a plurality of days is higher than a predetermined reference value, or may be determined based on the average value and variance of the amount of sIgA, etc. The reference value is, for example, 8000, 9000, or 10000 ng / ml.

[0044] Furthermore, when the chronic fatigue marker is cortisol, for example, when the amount of cortisol measured during the day over multiple days in the determination step remains at a low concentration, the subject's chronic fatigue level can be determined to be high.

[0045] The determination of whether "the amount of cortisol during the day measured over a plurality of days is changing at a low concentration" is not particularly limited, and may be determined, for example, by whether the amount of cortisol during the day measured over a plurality of days is lower than a predetermined reference value, or may be determined based on the average value and variance of the amount of cortisol, etc. The reference value is, for example, 0.5, 0.6, 0.7, or 0.8 μg / dl.

[0046] In the chronic fatigue level determination method of the present invention, for example, the amount of both sIgA and cortisol in the biological sample of the subject may be measured in the measuring step, and the chronic fatigue level of the subject may be determined based on the measured amounts of both sIgA and cortisol in the determining step.

[0047] In this case, for example, if the amount of sIgA measured during the day and at night over multiple days in the determination process remains at a high concentration, and the amount of cortisol measured during the day over multiple days remains at a low concentration, the subject's chronic fatigue level can be determined to be high.

[0048] In the chronic fatigue level determination method of the present invention, for example, in the measuring step and the determining step, a substance other than sIgA and cortisol may be further measured, and the chronic fatigue level may be determined based on the measured amount.

[0049] (Determination Reagent) The determination reagent of the present invention contains a binding substance that specifically binds to at least one of sIgA and cortisol, and is used in the chronic fatigue level determination method of the present invention. The binding substance can be, for example, an antibody or an aptamer that specifically binds to sIgA and cortisol, respectively.

[0050] The determination reagent of the present invention may contain, for example, a binding substance that specifically binds to both sIgA and cortisol, respectively, so that, for example, the chronic fatigue level can be determined according to the combination of the detection results of sIgA and cortisol. EXAMPLES

[0051] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise indicated.

[0052] [Example 1] The amounts of sIgA and cortisol in the biological samples of the subjects were measured before they performed the stressful task, and the subjects were divided into groups based on the measured amounts of sIgA and cortisol.

[0053] (Saliva marker measurement) For subjects (ages 20-30; 46 people), who were nurses, saliva was collected four times over two days before work (indicated by black circles in the figure) during the day shift (8:30-17:00) and the night shift (16:30-9:00 the next day) according to the schedule shown in Figure 3. The saliva was collected from 7:30 to 9:00 for the day shift and from 15:00 to 17:00 for the night shift. The saliva was collected using a saliva collection kit (product name: Salisoft, manufactured by Sarstedt Co., Ltd.) according to the attached manual. The four samples were designated as night shift_Day1, day shift_Day1, day shift_Day2, and night shift_Day2, respectively.

[0054] The sIgA and cortisol concentrations (saliva marker concentrations) in the collected saliva were measured by ELISA. The measurements were performed using ELISA kits (product name: ELISA Kit for Secretory Immunoglobulin A (sIgA), Cloud-clone, product number: #SEA641HU, sandwich method; product name: YK070 Human Chromogranin A EIA kit, Yanaihara Laboratory, product number: #YK070, competitive method; product name: YK241 Cortisol (Saliva) EIA kit, Yanaihara Laboratory, product number: #YK241, competitive method) according to the attached manual.

[0055] In the measurement of sIgA, the saliva was diluted to a concentration of 1 / 1000 to prepare a sample of 100 μl. The sample was reacted with the reagent attached to the kit, and then the absorbance at a wavelength of 450 nm was measured using a microplate reader (Infinite M1000Pro, manufactured by TECAN). In the measurement of cortisol, the saliva was diluted to a concentration of 1 / 2 to prepare a sample of 50 μl, and the absorbance at a wavelength of 450 nm was measured in the same manner.

[0056] The saliva marker concentration was calculated from the absorbance value. The saliva marker concentration was calculated by creating a standard curve from four-parameter logistic regression (4PL). Two samples were prepared for each condition, and the absorbance was measured. The average of the absorbance measurements was calculated, and this value was used to calculate the concentration.

[0057] (Cluster analysis) Based on the results of the salivary marker concentrations before and after work, the subjects were divided into groups using hierarchical cluster analysis (Ward method).

[0058] (Data Analysis) In this Example and Example 2 described later, data analysis was performed using JMP Pro software, ver.14.0 (SAS Institute Inc, Cary, NC, USA) with a significance level set at 5%. Unless otherwise specified, continuous variables were expressed as medians (interquartile ranges) and categorical variables as frequencies (%).

[0059] As a result, as shown in Figure 4, the sIgA concentrations could be classified into three clusters: the high level group (19 people), in which the concentrations remained high before shifts all four times; the high concentration group before night shifts (Day ≦ Night group) (12 people), in which the concentrations before the day shift and the night shift were similar or slightly higher; and the high concentration group before day shifts (Day > Night group) (11 people), in which the concentrations before the day shift were higher than before the night shift.

[0060] Furthermore, as shown in Figure 5, the subjects were able to be classified into two clusters in terms of cortisol concentrations: a high concentration trend group (25 subjects) whose concentrations remained high before the day shift, and a low concentration trend group (19 subjects) whose concentrations remained low before the day shift.

[0061] As described above, the amounts of sIgA and cortisol in the subjects' biological samples were measured before they performed stressful tasks, and the subjects were divided into groups based on the measured amounts of sIgA and cortisol.

[0062] [Example 2] It was confirmed that the amounts of sIgA and cortisol measured in Example 1 correlated with the chronic fatigue level of the subject.

[0063] (Measurement of accumulated fatigue) The subjects in Example 1 were subjected to a measurement of accumulated fatigue using the cumulative fatigue symptom index before working a night shift (night shift_Day1) as shown in the schedule in FIG. 3. The measurement of accumulated fatigue was performed using a questionnaire shown in FIG. 6. Specifically, the responses to all 81 items (questionnaire items 1 to 81) were classified into characteristic item groups (categories) shown in FIG. 6, and the average complaint rate for each characteristic item group was calculated using the formula: "average complaint rate = (total number of complaints for the item group) / (number of items in the item group × number of target nurses) × 100". In FIG. 6, of the characteristic item groups, NF5-2, NF5-1, and NF1 are indexes representing mental burden, NF2-1, NF2-2, and NF6 are indexes representing physical burden, and NF3 and NF4 are indexes representing social burden.

[0064] A CSFI (Cumulative Fatigue Symptom Index) radar chart was created for each of the clusters grouped in Example 1 for the calculated average complaint rates for each of the characteristic item groups. In addition, the average complaint rates for each of the characteristic item groups were compared between the clusters. The comparison was performed using the Mann-Whitney U test or the Steel-Dwass test. Furthermore, the association between the clusters was analyzed using Fisher's exact test.

[0065] These results are shown in Figures 7 and 8. Figures 7 and 8 are CSFI radar charts for each cluster, with Figure 7 showing the results of the clusters grouped based on the amount of sIgA, and Figure 8 showing the results of the clusters grouped based on the amount of cortisol. The CSFI radar charts show the results of NF1 (loss of energy), NF2-1 (general fatigue), NF2-2 (physical discomfort), NF3 (irritability), NF4 (loss of motivation to work), NF5-1 (anxiety), NF5-2 (depression (depressed state)), and NF6 (chronic fatigue symptoms).

[0066] As shown in Figure 7, the high sIgA concentration trend group (outer solid line chart in the figure) had significantly higher rates of complaints of "irritability" (p = 0.036), "loss of motivation to work" (p = 0.020), "depression" (p = 0.010), and "loss of energy" (p = 0.028) compared to the high daytime pre-work concentration group (inner chart in the figure).

[0067] In addition, the group of characteristic items was divided into indices representing the mental burden, the physical burden, and the social burden, and the analysis showed that the high concentration transition group (outer solid line chart in the figure) had a significantly higher complaint rate in the mental aspects (p = 0.028) and the social aspects (p = 0.008) compared to the daytime pre-work high concentration group (inner chart in the figure).

[0068] Next, the group with high sIgA concentrations before nighttime work (outer dotted line chart in the figure) had significantly higher rates of complaining of "decreased motivation to work" (p = 0.037) and "physical discomfort" (p = 0.042) compared to the group with high concentrations before daytime work (inner chart in the figure).

[0069] In addition, the high concentration group before nighttime work (outer dotted line chart in the figure) had a significantly higher complaint rate in the social aspect (p = 0.026) compared to the high concentration group before daytime work (inner chart in the figure).

[0070] In addition, when comparing the high concentration transition group (the outer solid line chart in the figure) with the high concentration group before nighttime work (the outer dotted line chart in the figure), no statistically significant differences were found for any item.

[0071] As shown in Figure 8, the low cortisol concentration trend group (outer chart in the figure) had significantly higher rates of complaints of "irritability" (p = 0.030) and "decreased motivation to work" (p = 0.040) compared to the high cortisol concentration trend group (inner chart in the figure).

[0072] Furthermore, the low concentration transition group (outer chart in the figure) had a significantly higher complaint rate in the social aspect (p=0.018) compared to the high concentration transition group (inner chart in the figure).

[0073] As described above, it was confirmed that there is a relationship between the amounts of sIgA and cortisol measured in Example 1 and the chronic fatigue level of the subject.

[0074] [Example 3] It was confirmed that the combination of the amounts of sIgA and cortisol measured in Example 1 correlated with the subject's chronic fatigue level.

[0075] The distribution of the subjects into the sIgA and cortisol clusters grouped in Example 1 was analyzed.

[0076] The results are shown in Table 1. Table 1 shows the distribution of the subjects, and the numbers in the table show the number of people in each group and their percentage (%). As shown in Table 1, there was a high percentage of subjects classified into the high sIgA concentration transition group and the low cortisol concentration transition group (n=13, 68.4%) (high sIgA concentration-low cortisol concentration group). [Table 1]

[0077] For the high sIgA / low cortisol group and the other subject groups, CSFI radar charts were created and inter-cluster comparisons were performed in the same manner as in Example 2.

[0078] The results are shown in Figure 9. Figure 9 is a CSFI radar chart for each cluster.

[0079] As shown in Figure 9, the high sIgA / low cortisol group (outer chart in the figure) had a significantly higher rate of complaints of "chronic fatigue symptoms" (p=0.034) and "irritability" (p=0.009) compared to the other groups (inner chart in the figure).

[0080] In addition, the high sIgA / low cortisol group (outer chart in the figure) had a significantly higher complaint rate in the social aspect (p=0.012) compared to the other groups (inner chart in the figure).

[0081] As described above, it was confirmed that there is a relationship between the combination of the amounts of sIgA and cortisol measured in Example 1 and the chronic fatigue level of the subject.

[0082] Although the present invention has been described above with reference to the embodiment and examples, the present invention is not limited to the above embodiment. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention. [Industrial Applicability]

[0083] According to the present invention, a chronic fatigue marker that can be tested non-invasively can be provided.

Claims

1. The method includes a measuring step and a determining step, The measuring step includes measuring the amount of a chronic fatigue marker in a biological sample of the subject before performing a stressful task during the day; The measuring step further includes measuring the amount of sIgA in the biological sample of the subject at a timing before performing the stressful work at night, The daytime hours are from 8:30 to 17:00, The nighttime period is from 16:30 to 9:00, The timing before performing the stressful task is 0 to 60 minutes before performing the stressful task, The chronic fatigue marker includes both sIgA and cortisol, and in the determining step, a chronic fatigue level of the subject is determined based on the amount of the chronic fatigue marker measured; In the determination step, if the amount of sIgA measured before the daytime work is comparable to or less than the amount of sIgA measured before the nighttime work, and if the amounts of sIgA measured before the daytime work and before the nighttime work remain at higher concentrations than a predetermined reference value, the subject is determined to have a high level of chronic fatigue. How to determine chronic fatigue level.

2. 2. The method for determining a chronic fatigue level according to claim 1, wherein in the determination step, the subject's chronic fatigue level is determined to be high if the amount of cortisol measured before daytime work over multiple days remains at a lower concentration than a predetermined standard value.

3. The measuring step further includes measuring the amount of sIgA in the biological sample of the subject at a timing before the subject performs the stressful work at night; 3. The method for determining a level of chronic fatigue according to claim 1 or 2, wherein, in the determination step, the subject's chronic fatigue level is determined to be high if the amount of sIgA measured before the daytime work and before the nighttime work remains at a higher concentration than a predetermined standard value, and the amount of cortisol measured before the daytime work over multiple days remains at a lower concentration than a predetermined standard value.

4. The method for determining a chronic fatigue level according to claim 1 , wherein the biological sample is saliva.

5. A reagent for use in the method for determining a chronic fatigue level according to any one of claims 1 to 4, comprising a binding substance that specifically binds to sIgA and a binding substance that specifically binds to cortisol.

6. The determination reagent according to claim 5 , wherein the binding substance is at least one of an antibody and an aptamer.

Images