Stress detection method using body hair

Abstract

To provide a method capable of measuring a specific substance in body hair to check stress situation.SOLUTION: A stress detection method using body hair comprises measuring at least one of a specific substance from body hair collected from a subject; and evaluating whether there is an increase or decrease with respect to a control value. The specific substance is, for example, (1) selenium sulfide, 8-hydroxycarteolol, or the like, as a substance that increases when stressed, or (2) 2-Oxo-4-methylthiobutanoic acid as a substance that decreases when stressed.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a stress detection method using body hair collected from a subject.

Background Art

[0002] In modern society, humans have to have interpersonal relationships with others (including friends, seniors, colleagues, etc.). At this time, if mental instability such as overwork and bullying continues, stress may be applied for a long time. If stress continues, the mood will drop mentally and lead to a pathological state (such as hair loss) physically. For example, it is known that if social stress is continuously received, it also affects the intestinal flora. To determine the stress situation of a subject, a medical interview and a check test are conducted in a psychosomatic medicine department. However, since these methods often depend on the subjectivity of the subject, a more objective method has been demanded. On the other hand, research has been conducted to identify a specific state of a subject (such as a disease such as cancer, a drug intake state) by measuring the components in the body hair (hair) collected from the subject (Patent Documents 1 and 2).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Disclosure of the Invention

[0005] The inventors diligently conducted research to solve the above problems and discovered that certain substances significantly increase or decrease in the body hair of individuals exposed to stress compared to the control group, and that these substances return to control levels when the stress is avoided. This discovery essentially led to the completion of the present invention. Thus, the stress detection according to the present invention A way to provide indicators for this is, From the body hair collected from the subjects Specific substances Measure and compare with the control value The increase Whether or not it will be accepted A method for providing indicators to show and the specified substance However, stress As a substance that increases when exposed to, It is selenium sulfide. It is characterized by the following:

[0006] Furthermore, relating to another invention In body hair non-invasive stress biomarkers Its use is to measure selenium sulfide. It is characterized by the following:

[0007] Furthermore, a method for identifying stress-detecting substances according to another invention is characterized by comprising: (1) a hair component measurement step of measuring the components in each hair of control hair, which is body hair collected from a healthy person, and test hair, which is body hair collected from a subject exposed to stress; (2) a specific substance selection step of selecting candidate substances in which a statistically significant difference in concentration is observed by measuring the components in the control hair and test hair; and (3) a substance identification step of measuring the candidate substances in a second test hair collected from the subject exposed to stress after stress avoidance, and identifying substances in which no statistically significant difference is observed between the concentration of the candidate substances in the control hair and the second test hair. Human or animal body hair essentially shares the same structure with the entire epidermis and capillaries. Specifically, hair bulbs (dermal papillae and hair matrix cells) absorb nutrients from capillaries, and body hair grows as the hair matrix cells repeatedly divide. Hair is a preferred embodiment because it is relatively easy to collect among body hairs. However, hair is susceptible to damage from UV exposure and physical contact. Therefore, using body hair that is more protected from the outside (such as nasal hair, armpit hair, arm hair, lower limb hair, and pubic hair) may allow for the confirmation of pure stress response signals, enabling a more sensitive and objective assessment of the subject's stress level. [Effects of the Invention]

[0008] According to the present invention, by measuring specific substances in body hair collected from subjects, the stress level of the subjects can be determined more objectively. [Brief explanation of the drawing]

[0009] [Figure 1] This diagram shows the procedure for creating mice that have experienced social defeat (social defeat stress mice). [Figure 2] These are photographic diagrams showing the areas used in the SI test. (a) shows a 14cm x 24cm interaction zone and a 9cm x 9cm waiting zone, while (b) shows the partition tubes that prevent physical contact between B mice (mice that have experienced social defeat) and aggressor mice when they are confronted during the SI test, from the front (left side) and side (right side), respectively. [Figure 3]Representative results of examining the behavioral trajectories of mice in OF tests and SI tests are shown. (A) shows the behavioral trajectories of healthy mice that did not experience social defeat in the OF test (left), and in the SI test when the Anew mouse was absent (center) and when the Anew mouse was placed in the cage (right). (B) shows the behavioral trajectories of mice that experienced social defeat when they were given tap water in the OF test (left), and in the SI test when the Anew mouse was absent (center) and when the Anew mouse was placed in the cage (right). (C) shows the behavioral trajectories of mice that experienced social defeat when they were given theanine in the OF test (left), and in the SI test when the Anew mouse was absent (center) and when the Anew mouse was placed in the cage (right). [Figure 4] These are photographs showing the results of brain imaging measurements of control mice, stressed mice, and stressed mice with theanine intake. (A) shows the results of dopamine distribution, (B) shows the results of GABA distribution, and (C) shows the results of theanine distribution. [Figure 5] This paper presents the results of investigating the effect of theanine on the concentration of substances that significantly increase or decrease in the hair of socially defeated mice compared to the hair of control mice. (A) Corticosterone concentration, (B) Selenium sulfide concentration, and (C) Concentration of an unknown substance with (m / z) 144.866 are shown. The upper panel shows graphs illustrating the changes in the concentration of each substance, and the lower panel shows photographs of each substance in the hair. [Modes for carrying out the invention]

[0010] Next, embodiments of the present invention will be described with reference to the figures and tables. The technical scope of the present invention is not limited by these embodiments, and it can be implemented in various forms without changing the gist of the invention. Stress refers to functional changes that occur in the mind and body as a result of various external stimuli acting as a burden. Stress can be classified into acute stress and chronic stress. Acute stress refers to functional changes in the mind and body caused by short-term external stimuli, such as those resulting from continuous or intermittent exposure to external stimuli for a short period (e.g., 10 minutes to 24 hours). Examples of acute stress include short-term stress such as written tests, crowded trains, and long hours of overtime. Chronic stress refers to functional changes in the mind and body caused by long-term external stimuli. Examples of chronic stress include those resulting from continuous or intermittent exposure to external stimuli (e.g., for a period longer than 24 hours). Examples of chronic stress include interpersonal relationships at school or work. The stress of this invention includes both acute stress and / or chronic stress. Body hair refers to hair that grows on the body of a human or animal. Specifically, it includes scalp hair, nasal hair, armpit hair, arm hair, lower limb hair, and pubic hair. Of the body hairs, scalp hair is preferred because it can be collected relatively easily. Scalp hair refers to hair that grows on the head of a human or animal. Scalp hair has a triple structure, consisting of the medulla, cortex, and cuticle from the inside out. While studies have been conducted to assess health status and disease severity by analyzing body hair (especially scalp hair), the relationship between substances in body hair and stress was not well understood.

[0011] The present invention will be further explained below with reference to examples, but it is not limited to these. <Test Method and Test Results> 1. Creation of a mouse model representing a social defeat situation. Figure 1 shows an overview of the test method. We prepared 10 aggressor mice (mice that physically attack others (ICR mice): A mice) and 10 small mice that are more likely to be targeted for aggression (B mice, C57BL / 6 mice (B6 mice)), and assigned numbers from 1 to 10 to each individual (A1 to A10, B1 to B10). One A mouse and one B mouse were each housed in the same cage (D0). At this time, a small transparent hole was opened in the middle of the housing cage. Partition board was installed. Partition board Regarding Partition board , being transparent is for visual perception, and making a hole is for olfactory perception. Partition board This is to always make the other party recognize Partition board on the opposite side. Regarding the A mouse, it was housed in the same cage for 10 days during D1 to D10 below. To exist

[0012] Starting from the day before the A mouse attacked the B mouse (D0), breeding was started as described above. On the next day (D1), the B mouse was moved to the A mouse's side, and the A mouse was allowed to attack the B mouse for 5 to 10 minutes. Then, Partition board the B mouse was placed on the opposite side of Partition board . Evacuation 24 hours later (D2), the B mouse was successively moved to the cages of other A mice, and the A mouse was allowed to attack the B mouse for 5 to 10 minutes. Then, Partition board the B mouse was blamed on the opposite side of Partition board . After repeating the attack on the B mouse by the A mouse for 10 days (D1 to D10) in this way, a Social Interaction test (SI test) was conducted on the 11th day. In addition, the B mice were divided into two groups of five each. Ion-exchanged water was given to one group of B mice (control group (G1)), and water in which theanine (5%) was dissolved was given to the other group of B mice (test group (G2)) by free drinking.

[0013] 2. SI Test (1) Explanation of the test method Figure 2 shows the area diagram used in the SI test. The upper Interaction zone in Figure 2(a) indicates the contactable area, and the lower Comer zone indicates the waiting area. For the SI test, we prepared Anew mice, which were not used in the "1. Social Defeat Situation Model Mouse" described above. We newly screened CD-1 mice as aggressor mice. Using the screening criteria, we screened the target CD-1 mice and confirmed the presence of aggressive traits. (i) The B mice that were attacked in the "1. Social Defeat Situation Model Mouse" described above were placed in the test room one hour before the start of the SI test. The test was conducted in a room isolated from external sound sources and under constant red light. Two 150-second SI tests were performed with a 30-second interval in between. (ii) With the Anew mouse absent, a small cage was placed in the accessible area, and mouse B was placed at the "S" (start) position in the open field. A 150-second free exploration period was provided (acclimatization IT(mouse A-)).

[0014] (iii) Return the B mouse to its rearing cage, and within 30 seconds, place the Anew mouse in a small cage and position it within reach. (iv) Mouse B was placed in the Corner Zone and observed for 150 seconds (IT(Mouse A+)). The movement trajectory of Mouse B was recorded throughout the entire time. Details of the recording process are described below. (v) Behavioral analysis software (MATLAB).<https: / / jp.mathworks.com / products / matlab.html> The analysis was performed using (iv). From the behavioral trajectory of mouse B, the proportion of time spent in the 9cm x 9cm area in Figure 2 was converted into points. It was defined that the longer the time spent in the area farther away from mouse A (9cm x 9cm), the higher the degree of social defeat. (vi) After the test, the body hair (hair) on the back was shaved with clippers and a body hair sample was taken.

[0015] (2) Details of the shoot OF test: At the start of filming, mouse B was slowly placed into the open field (OF) with its head against the wall. A 10-second acclimatization period was observed (filming ON). Filming was stopped, and mouse B was returned to its rearing cage. Next, mouse B was placed in a tube and slowly lowered into the OF with its head against the wall. Filming was performed for 150 seconds (filming ON). Filming was stopped and mouse B was returned to its cage. Next, after inserting the Anew mouse, mouse B was slowly placed into the OF with its head against the wall. Recording was performed for 150 seconds (recording ON). After stopping the recording and returning mouse B, the OF was cleaned with alcohol. The left side of Figures 3(A) to (C) shows representative results of the OF test. In both healthy mice and socially defeated mice (tap water intake and theanine intake), the OF test showed that each mouse moved within the entire movable area.

[0016] 3. SI Test Results The center and right sides of Figures 3(A) to (C) show representative results from the study of the behavioral trajectories of mouse B. In healthy mice that had not experienced social defeat (Figure 3(A)), they showed interest in other individuals (Anew mice), and therefore spent more time around the cage and had higher sociability scores when an unfamiliar mouse was inside the cage (Figure 3(A) right side) compared to when the cage was empty (Figure 3(A) center). In contrast, in mice that had experienced social defeat and consumed tap water (Figure 3(B)), they moved further away from the cage and spent less time around the cage and more time in the corners of the box compared to when the cage was empty (Figure 3(B) center), resulting in lower sociability scores. Furthermore, in mice exhibiting social defeat, those that ingested theanine (Figure 3(C)) spent more time around the cage when an unfamiliar mouse was in the cage (Figure 3(C) right side) compared to when the cage was empty (Figure 3(C) center). sociability My score improved.

[0017] As described above, healthy mice were interested in Anew mice and often stayed near the accessible area (1.2 points), whereas tap water intake social defeat model mice initially showed interest in Anew mice, but the memory of being attacked was recalled, causing them to move away from the accessible area (0.03 points). Furthermore, mice that received free access to theanine scored significantly higher points and showed improved social defeat compared to mice that did not receive theanine, and were able to return to the control group. The SI test points for the control group (n=10) and the theanine free intake group (n=10) were 0.49±0.36 (mean ± standard deviation) and 0.88±0.24 (mean ± standard deviation), respectively. A significance test (non-related two-group t-test) showed a statistically significant difference with P<0.05. Standard deviation In contrast to the group with large standard deviations (SD) (indicating individual differences), the group with free access to theanine has small SD values ​​relative to the points, suggesting that social defeat is stably suppressed regardless of the individual.

[0018] 4. Cross-sectional photography of body hair and measurement of components in body hair (Body hair component measurement process) (1) Microscopic image of cross-section of body hair After the SI test, hair samples from the back of the animal were shaved with clippers. Cross-sectional hair sections were prepared, and then microscopic images were taken. (2) Nanoparticle spraying Next, the components of the body hair were measured. For the measurement of the components in the body hair, nanoparticles were used as an ionization support agent (matrix). A wet precipitation method was used to obtain monodisperse nanoparticles. For preparation, Macaulayite ultra-nanoparticles were prepared using FeCl2·4H2O and 3-aminopropyltriethoxysilane as materials. 2 g of FeCl2·4H2O was weighed and dissolved in 100 mL of distilled water to obtain a 10 mM aqueous solution. After thorough stirring at room temperature for approximately 10 minutes, it was mixed with 100 mL of 3-aminopropyltriethoxysilane (APTES) to complete the reaction. Subsequently, the solution was centrifuged for 60 minutes, the supernatant was discarded, and the precipitate was washed by repeatedly adding distilled water for redispersion, centrifuging, and discarding the supernatant several times. Next, the nanoparticles were suspended in methanol, and the supernatant was sprayed onto sample sections using an airbrush (GSI Creos). Since the spray was applied from a distance of 10 cm to 15 cm from the tissue, the methanol evaporated, and only the fine particle powder was sprayed onto the tissue.

[0019] (3) Imaging mass spectrometry Hair samples from C57BL / 6JJcl mice (8 weeks old; male) were used. The collected hair sections were placed on indium cinnamoxide (ITO) coated glass slides to prepare tissue sections. Imaging mass spectrometry measurements were performed using a mass spectrometer (tims-tof flex; Bruker Daltonics). The laser irradiation interval was set to 5 μm. The obtained signal was obtained from 100 laser shots. Furthermore, after the experiment was completed, the brains of each mouse were collected and subjected to imaging mass spectrometry. Figure 4 shows representative results from imaging mass spectrometry of mouse brains. Figure 4(A) shows the results of dopamine distribution, Figure 4(B) shows the results of GABA (γ-aminobutyric acid) distribution, and Figure 4(C) shows the results of theanine distribution. Dopamine was increased in the caudate nucleus (CPu) and nucleus accumbens (Nac) of the striatum in the control group (Cont.) and theanine-administered group (G2), while no clear distribution was observed in the stress-loaded group (G1). GABA was increased in the nucleus accumbens (Nac) of the striatum, hypothalamus (HY), ventral tegmental area (VTA), and cerebral cortex (motor cortex: MA) in theanine-administered group (G2). Theanine was localized in the lateral ventricle (LV) in theanine-administered group (G2).

[0020] Numerous strong signals were obtained from body hair sprayed with fine powder between 100 and 800 (m / z). Figure 5 shows (A) corticosterone and (B) corticosterone as representative signals with m / z values, which increased or decreased when social defeat occurred compared to the control (Cont.) (G1), and returned to the control state upon administration of theanine (G2). Selenium sulfide The results for (C)(m / z)144.866 (unknown substance) are summarized below. The upper panel shows graphs summarizing the changes in substance concentration in each group, and the lower panel shows imaging images of each substance in the body hair samples collected from each group. As shown in the upper panel of Figure 5(A), corticosterone signals were barely detectable in control mice (Cont.) and theanine-free drinking socially defeated mice (G2), while they were detected at more than 10 times the value in socially defeated mice (G1). Theanine-free drinking socially defeated mice (G2) had values ​​approximately 3 times lower than the control. Similarly, as shown in the lower panel of Figure 5(A), corticosterone signals were observed only in the cross-section of the body hair of socially defeated mice (G1). At this time, the location of the corticosterone signal and the rate of body hair growth indicated that the period and duration of stress coincided with those of the study. From these data, it was found that the period and intensity of stress can be evaluated by measuring the location and amount of substance signals in the cross-section of body hair. Furthermore, it was found that the social defeat status was improved in the theanine-free drinking water social defeat mice (G2). As shown in Figure 5(B) Selenium sulfide The same situation as with corticosterone was observed with (Selenium sulfide). In particular, as shown in the photograph below, Selenium sulfide So, in the cross-section of the body hair of the socially defeated mouse (G1), Selenium sulfide A strong signal (even more clearly than the corticosterone signal) was observed.

[0021] Furthermore, regarding the signal at (m / z) 144.866 shown in Figure 5(C), although the substance was not identified, it was detected in high concentrations in control mice (Cont.) and theanine-free drinking socially defeated mice (G2), while it was detected at lower levels in socially defeated mice (G1). This result also indicates that the socially defeated state improved in theanine-free drinking socially defeated mice (G2). As shown in Figures 5(A) and (B), substances that were significantly increased in the hair of socially defeated mice compared to control mice (corticosterone and selenium disulfide) were reduced to control mouse levels by theanine administration. Furthermore, as shown in Figure 5(C), a substance that was significantly decreased in the hair of socially defeated mice compared to control mice (an unknown substance with a m / z of 144.866) was increased to control mouse levels by theanine administration. These results demonstrate that theanine can objectively assess whether mice in a socially defeated state have recovered from that state and returned to a control state. Next, to search for substances that showed changes in the obtained data, we used omics analysis software (metaboscape; Bruker Daltonics). Table 1 shows a summary of substances that increased or decreased the m / z signal in socially defeated mice compared to control mice (specific substance selection step). Furthermore, regarding the substances listed in the last six lines, although their names are unknown, the m / z signals can be identified, indicating that measuring their concentration in body hair can be used to assess the stress level of the subjects.

[0022] [Table 1]

[0023] In Table 1, "ID.1" indicates substances that increase with stress and decrease with theanine intake, returning to control (inhibition); "ID.2" indicates substances that decrease with stress and increase with theanine intake, returning to control (recovery); and "ID.3" indicates substances that decrease with stress and decrease further with theanine intake. ID.1 specifically means, Selenium sulfide(Selenium Sulfide), 8-Hydroxycarteolol, 3Z,13Z-Octadecadienyl acetate, N-[2-(6-oxopyridazin-1-yl)ethyl]-6-pyrrolidin-1-ylpyridazine-3-carboxamide, 1-(3,4-dihydro-1H-isoquinolin-2-yl)-2-(1,2-dimethylindol-3-yl)ethane-1,2-dione ane-1,2-dione), corticosterone, 5-methylsulfonyl-9-(3-propan-2-yl-1,2,4-oxadiazol-5-yl)-1,5-diazacycloundecan-2-one, 2,2-bis(1,2-dimethylindol-3-yl)propanoic acid acid) (AC1L6HIG), 2-[[4-(cyclohexylcarbamoyl)piperazin-1-yl]methyl]-1-methyl-N-propan-2-ylbenzimidazole-5-carboxamide (2-[[4-(cyclohexylcarbamoyl)piperazin-1-yl]methyl]-1-methyl-N-propan-2-ylbenzimidazole-5-carboxamide), 3-[[4-[[3-(4-fluorophenyl)imidazo[4,5-b]pyridine-2-yl]methyl]piperazin-1-carbonyl]amino]propanoate ethyl (ethyl 3-[[4-[[3-(4-fluorophenyl)imidazo[4,The compounds were (5-b]pyridin-2-yl]methyl]piperazine-1-carbonyl]amino]propanoate and (4-phenyl-3,6-dihydro-2H-pyridin-1-yl)-[1-(7,8,9,10-tetrahydro-6H-purino[9,8-a]azepin-4-yl)piperidine-3-yl]methanone).

[0024] ID.2 specifically refers to 2-oxo-4-methylthiobutanoic acid, 2-N-ethyl-4-N-[(4-methylphenyl)methyl]-1,3-thiazole-2,4-dicarboxamide, 8-ethyl-N-methyl-N-[2-(methylamino)-2-oxoethyl]-4,5,6,7-tetrahydrothieno[2,3-b]azepine-2-carboxamide, Ro 19-4603, N-[[2-(1-butanoylpiperidin-3-yl)-1,3-thiazol-4-yl]methyl]-2-methylpropanamide, 1-[4-[5-(4-ethylphenyl)-4-methyl-1,1-dioxo-1,2-thiazol-3-yl]-1,4-diazepam-1-yl]-2-methylhexan-1-one, and a substance whose name is unknown but whose m / z value is 72.937. The values ​​were 105.934, 144.866, 178.920, and 285.815. Of these, corticosterone and Selenium sulfide The details were as follows: 1) It was found that corticosterone, a known stress marker, does not increase with the administration of theanine. 2) Selenium sulfide It appeared to act as an antibiotic. Although it is unknown whether it can be synthesized in the body, the raw material selenium is known to be present in the diet. If in the mouse body Selenium sulfide If it can be synthesized, it has been confirmed that it increases in situations of social defeat, so in an attempt to protect the body, Selenium sulfide It may produce [something]. Or, since excess selenium in the body promotes hair loss, to prevent that... Selenium sulfide It may be being replaced with this. In the control and theanine intake groups, Selenium sulfide No increase was observed. By detecting at least one of the substances listed in ID.1 and ID.2 in the subject's body hair, the subject's current stress level and recovery status can be objectively evaluated (substance identification step). Thus, according to this embodiment, by measuring specific substances in the body hair collected from the subject, the subject's stress level could be determined more objectively.

Claims

1. A method for measuring a specific substance from body hair collected from a subject and providing an index indicating whether or not an increase is observed between the measured value and the control value, A method for providing an index for detecting stress, characterized in that the aforementioned specific substance is selenium sulfide, which increases when subjected to stress.

2. A stress assessment method characterized by measuring selenium sulfide in body hair as a non-invasive biomarker.

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