Quantitative methods for mental stress

Abstract

To quantify mental stress of a healthy person.SOLUTION: A method for quantifying mental stress of a healthy person includes the following steps: (1) a step which measures one kind or two or more kinds of neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxy indole acetic acid present in urine sampled from a healthy subject; and (2) a step which calculates a stress index on the basis of a stress index calculation formula combining measured concentration of the neurotransmitters and coefficients obtained from stress items corresponding to the neurotransmitters.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying mental stress. [Background technology]

[0002] More than one million people in Japan develop depression each year, causing immeasurable economic losses not only to individuals and families but also to companies and society. The relationship between mental stress and depression is still unclear, and medically they are different conditions. However, given the fact that people under greater stress are more likely to develop depression, there is no doubt that detecting stress early and eliminating its causes before it occurs will help prevent the onset of depression.

[0003] In light of this situation, the Ministry of Health, Labour and Welfare has made it mandatory for companies with 50 or more employees to conduct a 57-item "Stress Check" (hereafter referred to as BJSQ). The BJSQ includes questions about the subject's (employee's) physical condition and their working environment, and is considered more effective in assessing potential mental stress than other depression assessment tests used in clinical settings (such as CES-D).

[0004] By conducting the BJSQ once a year, companies can identify employees who are experiencing stress and take measures to address that stress. However, the following problems have been identified with the BJSQ:

[0005] One problem is that because it is a self-assessment method, there are many cases where the individual is not properly aware of their stress, or conversely, exaggerates their stress levels despite being under low stress. Also, it is difficult to reflect differences in individual sensitivity to stress. For example, people who answer "yes" to the question "I have more work than I can handle" do not necessarily all feel the same level of stress.

[0006] In light of this situation, many objective diagnostic indicators using non-invasive human body fluids (blood, serum, plasma, saliva, urine, etc.) have been developed in the past.

[0007] Riederer et al. found that homovanillic acid (HVA) and vanillylmandelic acid (VMA) decreased in the urine of depressed patients, demonstrating the possibility of applying this to depression testing (Non-Patent Document 1). Tomei et al. When comparing plasma dopamine, they found that the plasma dopamine of police officers was significantly increased (Non-Patent Document 2). On the other hand, Chen et al. analyzed the serum dopamine of 35 depressed patients and 33 healthy individuals, but could not find any difference between the two (Non-Patent Document 3). Xie et al. also analyzed the plasma dopamine of 42 postpartum depression patients and 42 healthy individuals, but found no difference between the two. However, this could not be confirmed (Non-Patent Document 4).

[0008] Xie et al. analyzed plasma serotonin levels in 42 patients with postpartum depression and 42 healthy individuals. Furthermore, Colle et al. analyzed the plasma serotonin levels of 173 depressed patients and 214 healthy individuals and found that the plasma serotonin levels were significantly reduced in depressed patients (Non-Patent Document 3). It was found that plasma serotonin levels in these patients were reduced (Non-Patent Document 5).

[0009] Hashimoto et al. discovered that the proteins MICB and PDGF-BB in the blood are effective markers for diagnosing patients with major depressive disorder and bipolar disorder (Patent Document 1). Fukuda et al. developed a method for measuring the expression levels of IGHA1, THEM4, and TNFRSF25 in the blood to determine whether or not depression has occurred (Patent Document 2). Mikuni et al. furthermore discovered that the expression levels of STYXL1, SLC36A1, RNASE1, ARFRP1, BCL11B, SLC35F2, BANP, RAB11FIP4, FYCO1, NIPAL3, RP1 in the blood are effective markers for diagnosing patients with major depressive disorder and bipolar disorder (Patent Document 1). A method for assessing the severity of depression was developed by measuring the expression levels of one or more of the following genes: L23A, RPS2, and SIGIRR (Patent Document 3). Onishi et al. developed a method for assessing psychiatric and neurological disorders using free immunoglobulin kappa chain, free immunoglobulin lambda chain, and their fragments in blood samples from patients with psychiatric and neurological disorders (Patent Document 4). Morinobu et al. discovered that depression and schizophrenia can be diagnosed by examining the methylation status of the CpG island upstream of exon I of the BDNF gene (Patent Document 5). Bilello et al. developed a panel for assessing depression by combining brain-derived neurotrophic factor (BDNF), interleukins, fatty acid-binding protein (FABP), and cortisol (Patent Document 6). Kawamura et al. invented a test method for assessing depression by combining fibrinogen Aα chain, fibrinogen Bβ chain, and inter-α-trypsin inhibitor heavy chain in biological samples (Patent Document 7). Furthermore, Mutsutani et al. have discovered a method for determining whether a subject suffers from depression by measuring the expression levels of 18 genes using whole peripheral blood from the subject (Patent Document 8). Furthermore, Kawamura et al. have discovered that phosphoethanolamine is useful as a biomarker for depression (Patent Document 9).

[0010] Although numerous studies have been conducted to date with the aim of diagnosing depression, none of them have been sufficiently clinically evaluated, and none have the accuracy to be put to practical use and are capable of quantifying stress in healthy individuals.

[0011] To begin with, all research to date has focused on mental illness (depression), and no attempts have been made to quantify stress in healthy people. This is because, until the BJSQ became widespread, there was no reliable method for defining mental stress in healthy people. Furthermore, as mentioned above, because stress checks are self-checks, it is impossible to eliminate the possibility that they may be influenced by differences in individual sensitivity, and there is a need for a method of quantifying stress based on objective indicators. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Patent application 2018-23766 [Patent Document 2] Patent Publication No. 2017-63 [Patent Document 3] Patent application 2015-562880 [Patent Document 4] Patent application 2013-104124 [Patent Document 5] WO2012 / 017867 [Patent Document 6] Patent application 2010-549863 [Patent Document 7] Patent application 2009-551587 [Patent Document 8] Patent application 2008-65185 [Patent Document 9] WO2011 / 019072 [Non-patent literature]

[0013] [Non-Patent Document 1] Journal of neural transmission (1997)35(1),23-45. [Non-patent document 2] Toxicol. Ind. Health. (2007)23(7):421-7. [Non-patent document 3] J Pharm Biomed Anal(2021)doi: 10.1016 / j.jpba.2020.113773. [Non-patent document 4] Zhong Nan Da Xue Xue Bao Yi Xue Ban. (2018)43(3):274-281. [Non-patent document 5] Psychiatry Clin. Neurosci. (2020)74(2):112-117. Summary of the Invention [Problem to be solved by the invention]

[0014] Stress checks, in which differences in sensitivity affect the results, are effective in encouraging workers to become aware of their own stress levels and reducing the risk of mental health problems. However, since it is easy to identify people who are highly self-conscious, a method is needed to quantify stress while also taking into account objective indicators. If more accurate stress quantification becomes possible, This will enable use not only for the purpose of primary prevention, but also as a screening method for diagnosing depression and the like.

[0015] The present inventors set out to quantify latent mental stress in healthy individuals using urine samples from the subjects. [Means for solving the problem]

[0016] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that in urine samples collected from subjects suffering from high levels of mental stress, the urinary levels of dopamine, homovanillylmandelic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid change, and that these levels correlate with the stress of the subjects.

[0017] In other words, the present invention relates to a technology for quantifying one or more, preferably all, of six neurotransmitters in the urine of healthy individuals, calculating a stress index by combining the resulting value with a coefficient obtained from a stress item corresponding to the neurotransmitter, and determining the type and degree of stress of the subject. That is, the gist of the present invention is as follows.

[0018] [1] A method for quantifying mental stress in a healthy human subject, comprising the steps of: (1) A process for measuring one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA (gamma-aminobutyric acid), and 5-hydroxyindoleacetic acid present in urine collected from a healthy subject. (2) A step of calculating a stress index based on a stress index calculation formula that combines the measured concentration of the neurotransmitter and coefficients obtained from stress items corresponding to the neurotransmitter. [2] The method according to [1], wherein the coefficient is calculated so that the correlation between the concentration of the neurotransmitter and the stress item is significant. [3] The method according to [1] or [2], wherein the method for calculating the coefficients obtained from the stress items corresponding to the neurotransmitters is the logistic regression method or the linear regression method. [4] A method according to any one of [1] to [3], in which the coefficients obtained from the stress items corresponding to the neurotransmitters are calculated based on the scores of the answers to the questions in the stress check. [5] The method described in [4], in which the value obtained by scoring the answers to the stress check questions to calculate the coefficient is the value obtained by scoring the answers to all questions in the stress check. [6] The method described in [4] uses one or more of the following as the score values ​​for the answers to the stress check questions to calculate the coefficient: Coefficient A: Scored value of answers to questions about "life and work satisfaction" and "physical stress"; Coefficient B: Score of answers to questions about "my surrounding environment"; Coefficient C: score of answers to questions about the "digestive system"; Coefficient D: a score calculated from responses to questions about "worry" and "anxiety"; Coefficient E: A score calculated from the answers to the question about "environments that are beyond one's control"; Coefficient F: A score obtained by calculating the answers to questions about "anxiety about the future." [7] A combination of coefficients calculated based on the scores of neurotransmitter concentrations and responses to stress check questions, selected from one or two of the following (a) to (f): The method according to [4], using more than one species: (a) Scores for answers to questions about "life and work satisfaction" and "physical stress" the combination of the product of coefficient A and dopamine concentration calculated based on the calculated values; (b) The combination of the product of coefficient B, calculated based on the scored answers to the question about “my surrounding environment,” and the homovanillic acid concentration; (c) a combination of the product of coefficient C calculated based on the scored answers to questions about the “digestive system” and GABA concentration; (d) a combination of the product of coefficient D, calculated based on the scored answers to questions about “worry” and “anxiety,” and serotonin concentration; (e) The combination of the product of the coefficient E calculated based on the scored answers to the question about "environments beyond one's control" and the concentration of vanillylmandelic acid; (f) A combination of the product of the coefficient F calculated based on the scored answers to the question about “anxiety about the future” and the concentration of serotonin and / or 5-hydroxyindoleacetic acid. [8] The method described in [7] uses the sum of one or more combinations selected from (a) to (f) as a stress index calculation formula that combines coefficients calculated based on the scores of neurotransmitter concentrations and answers to stress check questions. [9] The method described in [7] uses the sum of all combinations of (a) to (f) as a stress index calculation formula that combines coefficients calculated based on the scores of neurotransmitter concentrations and responses to stress check questions.

[10] A method according to any one of [4] to [9], in which the stress index calculation formula is expressed as the following formula 1, which combines coefficients calculated based on the neurotransmitter concentration and the scores of the answers to the stress check questions. Stress index = A × (dopamine concentration) + B × (homovanillic acid concentration) + C×(GABA concentration)+ D × (serotonin concentration) + E × (concentration of vanillylmandelic acid) + F × (5-hydroxyindoleacetic acid concentration) (Equation 1) (In the formula, A, B, C, D, E, and F have the same meanings as defined above.)

[11] A method for determining recovery of a patient with depression using the method described in any one of [1] to

[10] .

[12] A method according to any one of [1] to

[11] , in which the concentrations of neurotransmitters are simultaneously analyzed using a liquid chromatography-mass spectrometry (LC / MS) device.

[13] The method according to any one of [1] to

[12] , wherein the ratio of the neurotransmitter concentration to the urinary creatinine concentration is used as the neurotransmitter concentration.

[14] An acquisition unit that acquires concentration data of one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid present in urine collected from a healthy subject; a calculation unit that calculates a stress index, which is an index related to mental stress of the subject, based on the concentrations of the neurotransmitters acquired by the acquisition unit and coefficients obtained from stress items corresponding to the neurotransmitters; an output unit that outputs the stress index calculated by the calculation unit, Stress index calculation device.

[15] The computer A compound selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid present in urine collected from healthy subjects. Obtain concentration data for one or more neurotransmitters, calculating a stress index, which is an index related to the mental stress of the subject, based on the acquired concentrations of the neurotransmitters and coefficients obtained from stress items corresponding to the neurotransmitters; a stress index processor for outputting the calculated stress index; X calculation program. [Effects of the Invention]

[0019] The method of the present invention allows companies to more accurately understand the type and level of stress in their employees. Specifically, by combining the values ​​of urinary substances selected from dopamine, homovanillylmandelic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid in urine samples with coefficients obtained from stress items in stress checks such as the BJSQ, a more accurate and comprehensive assessment can be made. Furthermore, employees who are judged to be "high risk" based on both the results of stress checks such as the BJSQ and the results of this quantitative test can be given stronger guidance than ever before for work improvement.

[0020] Furthermore, the timing for an employee with depression to return to work has traditionally been determined by interviewing the employee with their doctor or industrial physician, but the influence of the doctor's subjective opinion cannot always be eliminated, and employees who return early may repeatedly relapse, making it extremely difficult to determine the timing for return. However, by implementing the present invention, the attending physician and industrial physician can make a more accurate judgment. [Brief explanation of the drawings]

[0021] [Figure 1] This is a correlation diagram (scatter plot) between the total score of the BJSQ and the total score of the CES-D for healthy individuals. [Figure 2] FIG. 1 shows the chemical structural formulas of six neurotransmitters used in the present invention and their metabolic routes. [Figure 3] The subjects were classified into high-stress and low-stress groups based on the BJSQ, and the urinary concentrations, medians, and quartiles of six neurotransmitters were calculated and box-and-whisker plots were created. Box-and-whisker plots are shown for A) 5-HIAA, B) DA, C) GABA, D) HVA, E) 5-HT, and F) VMA, respectively. [Figure 4] This diagram shows a correlation diagram (scatter plot) of all combinations (15 patterns) of two neurotransmitters selected from six. [Figure 5]The urinary concentrations, medians, and quartiles of six neurotransmitters were calculated when subjects were classified into high-stress and low-stress groups according to the BJSQ. Box-and-whisker plots were then created for (G) the training set, (H) the test set, and (I) all subjects). The creatinine-corrected values ​​for the six neurotransmitters were logarithmically transformed and then normalized (mean zero, variance 1). Each value was then multiplied by a coefficient (weight) to create a linear combination equation. The resulting value is called the stress index. Using Excel's Solver (a nonlinear optimization method), the coefficients (weights) were adjusted to minimize the p-value of the Student t-test for the stress index of the two groups (high-stress and low-stress groups). The adjusted coefficients for the stress index of the six neurotransmitters are shown in the bottom of the figure. [Figure 6] FIG. 1 illustrates an example of the configuration of a system according to an embodiment. [Figure 7] FIG. 2 is a diagram illustrating an example of a hardware configuration of an information processing device. [Figure 8] FIG. 10 is a diagram illustrating an example of an operation flow of the stress index calculation device. DETAILED DESCRIPTION OF THE INVENTION

[0022] An embodiment of the present invention for evaluating the type and severity of stress using the method for quantifying stress in healthy individuals will be described in detail below, but the mode of use is not limited to this.

[0023] As used herein, the term "healthy individual" refers to an individual who is at least free of psychiatric or neurological disorders, and preferably a healthy individual who is free of any serious disorders.

[0024] <Method for quantifying mental stress in healthy individuals> The present invention provides a method for quantifying mental stress in healthy individuals, comprising the steps of: (1) measuring the concentrations of neurotransmitters contained in the urine of a subject as markers; and (2) calculating a stress index based on a predetermined discriminant that uses at least one of the measured marker concentrations as a variable and combines coefficients obtained from at least one of the markers and a stress item corresponding to the marker. The method of the present invention enables the type and severity of stress to be evaluated simply and accurately.

[0025] The present invention is a method for determining the type and degree of mental stress in a healthy individual based on the concentration of a marker containing at least one of serotonin, dopamine, GABA, HVA, VMA, and 5-HIAA present in urine collected from the subject. Another embodiment of the present invention is a method for determining that the lower the concentrations of serotonin, dopamine, GABA, HVA, VMA, and 5-HIAA in urine collected from a subject, the higher the stress level.

[0026] The coefficients obtained from the stress items corresponding to the markers are not limited, but for example, values ​​calculated based on the scores for all questions in the stress check, scores for answers to a specific group of questions in the stress check, etc. can be used as coefficients calculated in association with the marker values. The stress check score may be a score obtained from accumulated data from the stress check, a score obtained from the results of the stress check for the group to which the subject belongs, etc. From the perspective of quantifying the subject's mental stress more accurately, the scores of the stress items corresponding to each marker obtained from the results of the stress check for the group to which the subject belongs can be used.

[0027] [Stress check implementation process] In the present invention, the answers to accumulated stress check questions can be scored, or a stress check can be conducted and the answers to the questions scored, and these can be used to calculate the stress index in the present invention. For example, the Ministry of Health, Labor and Welfare's mandatory "BJSQ" stress check, consisting of 57 questions, can be used for stress checks. Because the BJSQ includes questions about the subject's physical condition and work environment, its use is also preferable for understanding the subject's background and assessing potential mental stress. In addition to the BJSQ, other options include depression tests using the Hamilton Rating Scale for Depression (HAMD), self-administered questionnaires (e.g., Patient Health Questionnaire (PHQ)-9, Beck Depression Inventory-II (BDI-2)), depression tests using genes, proteins, and compounds correlated with depression as indicators, and the CES-D, a depression assessment test used in clinical settings. The BJSQ is preferred because it is considered effective for assessing potential mental stress. Stress checks can be performed using one or a combination of two or more types.

[0028] The questions in the BJSQ are divided into "questions about work," "questions about current status," "questions about the surrounding environment," and "questions about satisfaction," and specifically include the following:

[0029] For example, questions about work include having to do a lot of work, not being able to complete all the work in time, having to work very hard, needing to concentrate a lot, it is a difficult job that requires advanced knowledge and skills, having to think about work all the time during work hours, it is a job that requires a lot of physical exertion, being able to work at one's own pace, etc. I can do it, I can decide the order and method of work by myself, I can reflect my opinions on the work policy at work, I rarely use my skills and knowledge at work, there are differences of opinion within my department, my department does not get along with other departments, the atmosphere at my workplace is friendly, the working environment at my workplace (noise, lighting, temperature, ventilation, etc.) is not good, the work content suits me, it is a rewarding job, etc.

[0030] For example, questions about current conditions (or questions about the subject's condition over the past month) include feeling energized, full of energy, lively, angry, feeling resentful, irritated, extremely tired, worn out, lethargic, on edge, anxious, restless, depressed, finding everything a hassle, unable to concentrate, feeling depressed, unable to concentrate on work, feeling sad, dizzy, joint pain, heavy head or headache, stiff neck or shoulders, lower back pain, tired eyes, palpitations or shortness of breath, stomach and intestinal problems, loss of appetite, constipation or diarrhea, and difficulty sleeping.

[0031] For example, "questions about the subject's surroundings" include how easily the subject can talk to their superiors, coworkers, spouse, family, friends, etc.; how reliable they are when they are in trouble; and how well they listen when they consult with them about personal problems.

[0032] For example, questions about satisfaction include whether you are satisfied with your job, satisfied with your family life, etc.

[0033] These questions may be arbitrarily categorized by content to facilitate association with markers.

[0034] Examples of classified questions include "questions to assess life and work satisfaction," "questions to assess physical stress levels," "questions to assess the environment around oneself," "questions to assess the state of the digestive system," "questions to assess worries and anxiety levels," "questions to assess the environment outside one's control," and "questions to assess anxiety about the future."

[0035] For example, as a "group of questions to measure satisfaction with life and work," it is possible to select and use from a group of questions categorized as "this job is not suitable for me" or "this job is depressing," etc.

[0036] For example, as a "group of questions to check the state of physical stress," questions can be selected and used from a group of questions categorized as "very tired," "my joints hurt," etc.

[0037] For example, as a "group of questions to examine one's surrounding environment," questions can be selected from a group of questions categorized as "My work environment is bad," "I can't sleep well," "When I encounter trouble, my family doesn't help me," etc.

[0038] For example, a "group of questions to check the state of the digestive system" can be selected from a group of questions categorized into gastrointestinal problems, loss of appetite, and the like.

[0039] For example, a "group of questions to assess the state of worry and anxiety" can be selected from a group of questions categorized as "anxiety" and the like.

[0040] For example, a "group of questions to look at circumstances that are beyond one's control" can be selected from a group of questions categorized as not being able to finish work within the required time, etc.

[0041] For example, as a "group of questions to check the state of anxiety about the future," questions classified as joint pain, stomach problems, anxiety, etc. can be selected and used.

[0042] Examples of differently classified question groups include "questions to find out dissatisfaction with life and work," "questions to find out the state of mental exhaustion," "questions to find out the state of the work environment and insomnia," "questions to find out the state of loneliness," "questions to find out the state of loss of appetite and gastrointestinal problems," "questions to find out the state of excessive workload," and "questions to find out the state of anxiety about the future."

[0043] For example, a "group of questions to assess dissatisfaction with work and life" could be selected from a group of questions categorized as follows: I have to do an enormous amount of work; I can't finish all my work in the allotted time; I have to work very hard; I need to concentrate a lot; it is a difficult job that requires advanced knowledge and skills; I have to think about work all the time during working hours; it is a job that requires a lot of physical activity; I can work at my own pace; I can decide the order and method of my work; I can reflect my opinions on work policies at work; I rarely use my skills or knowledge at work; there are differences of opinion within my department; my department does not get along with other departments; the atmosphere in my workplace is friendly; the working environment in my workplace (noise, lighting, temperature, ventilation, etc.) is not good; the work content suits me; it is a rewarding job.

[0044] For example, a "group of questions to assess mental exhaustion" can be selected from a group of questions categorized into categories such as having to do an enormous amount of work, not being able to complete all the work in the allotted time, having to work very hard, needing to concentrate a great deal of attention, having a difficult job that requires advanced knowledge and skills, having to think about work all the time during work hours, etc.

[0045] For example, a "group of questions to assess the working environment and insomnia" can be selected from a group of questions categorized as follows: "I have to think about work all the time during working hours," "I can work at my own pace," "I can decide the order and method of work by myself," "I can reflect my opinion on the work policy at work," "I rarely use my skills and knowledge at work," "There are differences of opinion within my department," "My department does not get along with other departments," "The atmosphere at my workplace is friendly," "The working environment at my workplace (noise, lighting, temperature, ventilation, etc.) is not good," "I can't sleep well," etc.

[0046] For example, a set of questions to assess the state of loneliness could be selected from a set of questions categorized as follows: Are there differences of opinion within my department? Do my department and other departments not get along? Is the atmosphere in my department friendly? How easily can I talk to my boss, coworkers, spouse, family, friends, etc.? How reliable are they when I'm in trouble? How well do they listen when I consult them about personal problems?

[0047] For example, a "group of questions to check the state of appetite loss and gastrointestinal problems" can be selected from a group of questions classified into gastrointestinal problems, loss of appetite, constipation or diarrhea, etc.

[0048] For example, a set of questions to check for excessive workload can be selected from a set of questions categorized into having to do too much work, not being able to complete work in time, having to work too hard, etc.

[0049] For example, the "question group to assess the state of anxiety about the future" includes questions such as "feeling energized," "full of energy," "lively," "angry," "inner anger," and "irritated." The questionnaire can be selected from a group of questions categorized into the following: feeling tired, extremely tired, worn out, listless, tense, anxious, restless, depressed, finding everything a hassle, unable to concentrate on things, feeling gloomy, unable to concentrate on work, feeling sad, how easily you can talk to your boss, colleagues at work, spouse, family, friends, etc., how reliable you are when you are in trouble, how well they listen when you consult them about personal problems, etc.

[0050] These questions can be used by classifying them so that similar questions form the same group based on their intent, based on the content of the questions. Scoring similar questions together is preferable because it makes the correlation with the markers clearer.

[0051] Since all of these questions are answered in advance as a score indicating whether or not they apply, these scores can be used as they are when calculating the coefficients. In the present invention, rather than using the answers to the stress check as they are, which may still be subjective, for stress quantification, substances in the body are measured and combined as objective values ​​for use in stress quantification, which is preferable because it allows for more accurate stress quantification in healthy individuals.

[0052] [Measurement process] In the method of the present invention, one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid are measured in a sample from a healthy subject.

[0053] Urine is used as the sample to be measured. The urine used in the present invention includes not only urine collected from a subject, but also urine obtained by pretreating the urine. Examples of urine obtained by processing include urine that has been pretreated by standing or centrifugation. These pretreatments may be performed using known methods. Furthermore, the urine sample may be diluted or concentrated as appropriate before use. Sampling is preferably performed in the morning, but may be performed at a time other than the morning. Fasting from the night before is desirable, but not necessarily required.

[0054] The test values ​​may be the urinary concentrations of the six markers or may be corrected by the value of a substance in a biological sample, such as creatinine. When creatinine concentration is used, it can be measured using commercially available assay reagents. The correction value may be any numerical value, such as a reference value or the amount of a specific substance, added, subtracted, multiplied, or divided (ratio value). In urine, the amount of a marker per unit volume may depend on the subject's renal function, etc. Therefore, when urine production is reduced, substances in the urine are concentrated, resulting in an artificially high measured value of accumulation per unit volume. Such correction is preferable because it can reduce such artifacts. Conversely, when urine production is increased, such as during diuresis, the marker may be diluted, producing an artificially low measured value of accumulation. However, such correction is preferable because it can correct for the effects of diuretic use, water intake, and other factors that may produce fluctuations in marker accumulation that are not related to the true accumulation or mass of the marker in the sample. The urine used may be a single urine sample or a 24-hour urine collection.

[0055] First, the concentrations of the above-mentioned markers in urine collected from a subject are measured. In the present invention, the six markers may be used individually or in combination of two or more. It is preferable to measure at least two or more of the creatinine-corrected concentrations of serotonin, dopamine, GABA, HVA, VMA, and 5-HIAA, and it is even more preferable to measure all of them.

[0056] Combining multiple markers to assess the type and severity of stress Accuracy can be improved.

[0057] As a method for measuring the concentration of various markers, a known method can be appropriately selected depending on the type of marker. For example, the concentration of a marker can be measured by selecting and combining a quantification method appropriate for the marker to be measured from among quantification by nuclear magnetic resonance (NMR), quantification by acid-alkali neutralization titration, quantification by an amino acid analyzer, quantification by enzymatic methods, quantification using aptamers such as nucleic acid aptamers and peptide aptamers, colorimetric quantification, capillary electrophoresis, liquid chromatography, gas chromatography, mass spectrometry, etc. The concentration of the marker may also be measured using a commercially available quantification kit appropriate for the marker to be measured. The use of liquid chromatography-mass spectrometry is preferred because it allows accurate measurement and can rapidly process a large number of samples.

[0058] The concentration of dopamine in the urine of a healthy subject is not limited, but may be, for example, 0.674 to 0.847 μg / mL. The homovanillic acid concentration is not limited, but may be, for example, 12.0 to 14.6 μg / mL. The serotonin concentration is not limited, but may be, for example, 0.242 to 0.289 μg / mL. The concentration of vanillylmandelic acid is not limited, but may be, for example, 6.76 to 8.50 μg / mL. The GABA concentration is not limited, but may be, for example, 0.302 to 0.412 μg / mL.

[0059] [Stress index value calculation process] Next, a stress index is calculated based on the concentration of at least one of the measured markers and a stress index calculation formula that combines a predetermined discriminant that uses the concentration of the marker as a variable and coefficients obtained from stress items corresponding to the marker. Here, if the degree of stress can be evaluated more accurately, data such as missing values ​​and outliers may be removed from the data on the measured marker concentrations before calculating the stress index.

[0060] To derive the stress index calculation formula, the method for calculating the concentration of each neurotransmitter and the coefficient obtained from the stress items corresponding to that neurotransmitter may be logistic regression, linear regression, principal component analysis, OPLS-DA, machine learning, etc. Data preprocessing may involve using the data as is, or may involve logarithmic transformation, normalization, or a combination of these. Here, in one embodiment, the coefficient is calculated so that the correlation between the concentration of the neurotransmitter and the stress item is significant. When calculating the coefficient, "significance" means, for example, that the p-value is < 0.05, preferably a p-value of <0.01. Specifically, a method for calculating coefficients obtained from stress items corresponding to neurotransmitters can be, for example, to set a high-stress group and a low-stress group based on the scores of answers to questions in a stress check (such as the BJSQ), and set a coefficient that maximizes the difference in stress index between the high-stress group and the low-stress group (so that the p-value of the Student T-test is minimized). More specifically, this can be done based on the method described in the Examples below.

[0061] The stress index may be calculated by selecting any one of six types of markers, and it is possible to use only one type or a combination of several types. When calculating the stress index using one type of marker, for example, when dopamine concentration is used, the stress of a healthy person can be quantified and used for assessment in the form of stress index = A x (dopamine concentration). When homovanillic acid is used as a marker, When GABA is used as a marker, the stress index is calculated as B x (homovanillic acid concentration). When GABA is used as a marker, the stress index is calculated as C x (GABA concentration). When serotonin is used as a marker, the stress index is calculated as D x (serotonin concentration). When vanillylmandelic acid is used as a marker, the stress index is Stress index = E × (vanillylmandelic acid concentration), and when 5-hydroxyindoleacetic acid is used as a marker, stress index = F × (5-hydroxyindoleacetic acid concentration). The stress index can be calculated as the concentration of By using a stress index value calculated by applying the coefficient and the concentrations of various measured markers, stress can be quantified with high accuracy, which is preferable. When multiple types are used in combination, these single stress indices can be added together for calculation. By using multiple types, preferably four or more types, more preferably five or more types, and particularly preferably all six types in combination, stress can be quantified more accurately, which is preferable. When combining multiple types of indexes, one possible embodiment is to combine substances on the same metabolic pathway. For example, 5-HIAA and serotonin are metabolites that begin with tryptophan, and therefore are thought to be closely related. Therefore, the ratio (balance) of 5-HIAA to serotonin may enable more accurate assessment of a subject's stress. This combination is merely an example, and is not limited to the combination of 5-HIAA and serotonin. Those skilled in the art can determine and use these combinations with reference to known knowledge.

[0062] For example, when calculating the stress index using all markers, the discriminant may be specifically represented by the following formula 1.

[0063] Stress index = A × (dopamine concentration) + B × (homovanillic acid concentration) + C×(GABA concentration)+ D × (serotonin concentration) + E × (concentration of vanillylmandelic acid) + F × (5-hydroxyindoleacetic acid concentration) (Equation 1)

[0064] (In the formula, A, B, C, D, E, and F each independently represent a coefficient, and are calculated based on the scores of answers to questions in a stress check (such as the BJSQ).) The concentrations of various markers are independently multiplied by coefficients calculated based on the scores of responses to stress check questions such as the BJSQ.

[0065] The value obtained by scoring the answers to the stress check questions can be a value obtained by scoring the answers to all questions in the stress check, or a value obtained by scoring the answers to specific questions in the stress check. In one embodiment, the scores obtained in the above-mentioned stress check implementation process can be used as individual stress factors and the numbers answered by the subject from the BJSQ questions can be used as a score as a means of correlating them with fluctuations in neurotransmitters alone.

[0066] For example, A can be classified into questions related to life and work satisfaction, such as "This job is not suitable for me" or "I feel depressed," and questions related to physical stress, such as "I am very tired" or "My joints hurt," and a coefficient calculated based on the score obtained for the answers can be multiplied by dopamine. B is categorized into questions related to one's surrounding environment, such as "My work environment is bad," "I can't sleep well," and "When I have trouble, my family doesn't help me," and the score is calculated based on the answers obtained. The coefficient calculated based on the above can be used by multiplying it by the HVA. C can be classified into questions related to the digestive system, such as "I have stomach problems" or "I have no appetite," and a coefficient calculated based on the score obtained for the answers can be multiplied by GABA. A coefficient calculated based on the score obtained by classifying D into questions related to worry and anxiety, such as "I feel anxious," can be used by multiplying it by serotonin. E can be classified into questions related to environments that cannot be controlled by the employee, such as "I can't finish my work within the required time," and a coefficient calculated based on the score obtained for the answers can be multiplied by VMA. F can be classified into questions related to anxiety about the future, such as "I have joint pain," "I have stomach problems," and "I'm anxious," and a coefficient calculated based on the scores obtained for the answers can be used by multiplying it by serotonin and / or 5-hydroxyindoleacetic acid.

[0067] The value of A is not limited, but may be, for example, 0.5 to 1.0. The value of B is not limited, but may be, for example, 0.1 to 0.6. The value of C is not limited, but may be, for example, −0.1 to 0.4. The value of D is not limited, but may be, for example, −0.4 to 0.1. The value of E is not limited, but may be, for example, −0.8 to −0.3. The value of F is not limited, but may be, for example, −0.2 to 0.2. In one embodiment, A, B, and C are positive, and D, E, and F are negative.

[0068] Furthermore, in addition to the markers and stress check scores, other information on the subject (for example, biological metabolites such as minerals and hormones; sex, age, eating habits, drinking habits, exercise habits, obesity level, disease history, interview data, etc.) may be used as variables in the multivariate discriminant. May be used.

[0069] [Evaluation process] The subject's mental stress can be quantified based on the calculated stress index. The type and severity of the subject's stress may also be evaluated. In this case, the type of stress of the subject can be determined by comparing the stress index values ​​of each neurotransmitter. Furthermore, the severity of stress can be determined by comparing the stress index value with a preset threshold (cutoff value). For example, the greater the stress index value is above the cutoff value, the higher the severity of stress, and the closer the stress index value is to the cutoff value, the lower the severity of stress. Alternatively, a method of classifying stress into multiple stages to evaluate the severity of stress may be used.

[0070] Such cutoff values ​​can be appropriately determined and used by those skilled in the art. The cutoff value is preferably determined based on the stress index values ​​calculated by measuring the markers in both case and control subjects. The control subjects are preferably free from diseases (psychiatric or neurological disorders) that correlate with the measured markers.

[0071] For example, in the case of a marker in which stress severity is high when the measured value is equal to or greater than the cutoff value, the cutoff value can be determined so that a predetermined proportion (which may be 100%) of case subjects are at or above the cutoff value, and a predetermined proportion (which may be 100%) of control subjects are below the cutoff value. In the case of a marker in which stress severity is high when the measured value is equal to or less than the cutoff value, the cutoff value can be determined so that a predetermined proportion (which may be 100%) of case subjects are at or below the cutoff value, and a predetermined proportion (which may be 100%) of control subjects are above the cutoff value. The cutoff value may also be a previous value in the subject. It is possible to determine changes in the condition (worsening, recovery, etc.).

[0072] The method of the present invention allows companies to more accurately understand the type and degree of stress in their employees. Specifically, by combining the results of the BJSQ and urine tests, more accurate judgments can be made. Employees who are judged to be "high risk" based on both the BJSQ and urine tests can be given stronger guidance than ever before to improve their work.

[0073] Furthermore, until now, the timing for an employee with depression to return to work has mainly been determined by interviewing the employee with their personal doctor or industrial physician, but because this was based on the doctor's subjective opinion, it was extremely difficult to determine the timing of return, as employees who returned early often relapsed. However, by combining this test, personal doctors and industrial physicians can make more accurate judgments. For example, by calculating a stress index from the results of stress checks such as the BJSQ and the concentration of urinary markers in depressed patients, and comparing it with the cutoff value, recovery from depression can be more accurately determined. Furthermore, by performing the method of quantifying a subject's stress of the present invention multiple times, the subject's stress can be recorded over time. For example, by performing the method multiple times and tracking the quantified stress amount, it is expected that the result will be used as an aid in determining the timing of recovery, which is preferable. By recording the subject's stress over time, it is not only possible to simply grasp the amount of stress, but it is also expected to contribute to the investigation of underlying causes and to reveal characteristics such as differences in individual susceptibility to stress that differ from subject to subject. In this case, a method of quantifying the subject's stress over time may be used in combination with setting the above-mentioned cutoff value.

[0074] [Stress index calculation device and system] FIG. 6 is a diagram showing an example of the configuration of the system of this embodiment. The system of this embodiment includes a neurotransmitter concentration measuring device 10 and a stress index calculation device 20. The neurotransmitter concentration measuring device 10 measures the concentration of one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid present in urine collected from a healthy subject. Stress The index calculation device 20 calculates a stress index and the like based on the measurement results of the neurotransmitter concentration measurement device 10. Here, the neurotransmitter concentration measurement device 10 and the stress index calculation device 20 are separate entities, but they may be integrated to operate as a single neurotransmitter concentration measurement device.

[0075] The neurotransmitter concentration measuring device 10 is not particularly limited, and the above-mentioned measuring devices can be used. The neurotransmitter concentration measuring device 10 may be controlled by the stress index calculation device 20.

[0076] The stress index calculation device 20 includes an acquisition unit 21, a calculation unit 22, and an output unit 23. The stress index calculation device 20 calculates a stress index based on the neurotransmitter concentration calculated by the neurotransmitter concentration measurement device 10 and a coefficient obtained from a stress item corresponding to the neurotransmitter. The acquisition unit 21 acquires the neurotransmitter concentration in the subject's urine from the neurotransmitter concentration measurement device 10. The calculation unit 22 calculates a stress index based on the neurotransmitter concentration acquired by the acquisition unit 21 and a coefficient obtained from a stress item corresponding to the neurotransmitter. The output unit 23 outputs the stress index calculated by the calculation unit 22. Here, a preferred embodiment of the stress index calculation formula for calculating the stress index is the stress index calculation formula shown in Equation 1 above.

[0077] [Stress index calculation program] The present invention includes a computer program for executing the calculation of the stress index described above, and a computer-readable recording medium having the program recorded thereon. The recording medium having the program recorded thereon makes it possible to calculate the stress index described above by causing a computer to execute the program.

[0078] FIG. 7 is a diagram showing an example of the hardware configuration of an information processing device. The information processing device 90 shown in FIG. 7 has the configuration of a general computer. The stress index calculation device 20 is realized by using the information processing device 90 shown in FIG. 7. The information processing device 90 in FIG. 7 has a processor 91, a memory 92, a storage unit 93, an input unit 94, an output unit 95, and a communication control unit 96. These are connected to each other by a bus. The memory 92 and the storage unit 93 are computer-readable recording media. The hardware configuration of the information processing device is not limited to the example shown in FIG. 7, and components may be omitted, replaced, or added as appropriate.

[0079] The information processing device 90 has a processor 91 that loads a program stored on a recording medium into a working area of ​​a memory 92 and executes it, and each component is controlled through the execution of the program, thereby realizing functions that meet a specified purpose.

[0080] The processor 91 is, for example, a CPU (Central Processing Unit) or a DSP (Digital Signal Processor).

[0081] The memory 92 includes, for example, a random access memory (RAM) and a read only memory (ROM). The memory 92 is also called a main storage device.

[0082] The storage unit 93 is, for example, an EPROM (Erasable Programmable ROM), a hard disk, The storage unit 93 is a removable media drive (HDD, Hard Disk Drive). The storage unit 93 may include a removable storage medium, such as a USB (Universal Serial Bus) memory or a disk storage medium such as a CD (Compact Disc) or a DVD (Digital Versatile Disc). The storage unit 93 is also called a secondary storage device.

[0083] The storage unit 93 stores various programs, various data, and various tables used in the information processing device 90 on a readable and writable recording medium. The storage unit 93 stores an operating system (OS), various programs, various tables, etc. The information stored in the storage unit 93 may be stored in the memory 92. Furthermore, the information stored in the memory 92 may be stored in the storage unit 93.

[0084] The operating system is software that mediates between software and hardware, manages memory space, manages files, and manages processes and tasks. The operating system also includes a communication interface. The communication interface is a program that exchanges data with other external devices connected via the communication control unit 96. Examples of external devices include other information processing devices and external storage devices.

[0085] The input unit 94 includes a keyboard, a pointing device, a wireless remote control, a touch panel, etc. The input unit 94 may also include an input device for video or images such as a camera, and an input device for audio such as a microphone.

[0086] The output unit 95 is a liquid crystal display (LCD), an electroluminescence (EL) panel, The output unit 95 includes display devices such as a display panel, a CRT (Cathode Ray Tube) display, a PDP (Plasma Display Panel), and an output device such as a printer. An output device may be included.

[0087] The communication control unit 96 connects to other devices and controls communication between the information processing device 90 and the other devices. The communication control unit 96 is, for example, a LAN (Local Area Network) interface board, a wireless communication circuit for wireless communication, or a communication circuit for wired communication. The LAN interface board and the wireless communication circuit are connected to a network such as the Internet.

[0088] In the information processing device 90, the processor deploys a program stored in the auxiliary storage unit in an executable manner in the work area of ​​the main storage unit, and controls peripheral devices and the like through the execution of the program. This allows the information processing device to realize functions that meet a predetermined purpose. The main storage unit and the auxiliary storage unit are recording media that can be read by the information processing device.

[0089] (Example of operation) Here, an example of the operation of the stress index calculation device 20 will be described. 8 is a diagram showing an example of the operation flow of the stress index calculation device. The stress index calculation device 20 calculates a stress index based on the neurotransmitter concentration calculated by the neurotransmitter concentration measurement device 10. The neurotransmitter concentration measurement device 10 calculates the neurotransmitter concentration in the urine of the subject in advance.

[0090] In S101, the acquisition unit 21 of the stress index calculation device 20 acquires concentration data of a neurotransmitter in the subject's urine from the neurotransmitter concentration measurement device 10. The acquisition unit 21 may acquire the neurotransmitter concentration of the subject by having a user input the data using an input means or the like, by extracting the data from a memory means where the data is stored in advance, by acquiring the data from another information processing device, or the like. The acquisition unit 21 may acquire a parameter value equivalent to the neurotransmitter concentration.

[0091] In S102, the calculation unit 22 of the stress index calculation device 20 calculates a stress index based on the information acquired by the acquisition unit 21 and a coefficient obtained from the stress item corresponding to the neurotransmitter. The stress index is calculated, for example, as described above, by multiplying the concentration of the neurotransmitter by the coefficient (and the sum thereof) obtained from the stress item corresponding to the neurotransmitter.

[0092] In S103, the output unit 23 of the stress index calculation device 20 outputs the stress index calculated by the calculation unit 22 to an output device such as a display. The output unit 23 may also output the stress index to another information processing device, etc. The user can recognize the type and severity of the subject's stress from the output stress index.

[0093] A program that causes a computer or other machine or device (hereinafter referred to as a computer, etc.) to realize any of the above functions can be recorded on a computer-readable recording medium. Then, by having the computer, etc. read and execute the program from this recording medium, the function can be provided.

[0094] Here, a computer-readable recording medium refers to a recording medium that stores information such as data and programs electrically, magnetically, optically, mechanically, or chemically and can be read by a computer, etc. Such a recording medium may be provided with elements that constitute a computer, such as a CPU and memory, and the CPU may be made to execute the program.

[0095] Among such recording media, those that can be removed from a computer or the like include, for example, flexible disks, optical magnetic disks, CD-ROMs, CD-R / Ws, DVDs, and DVDs. AT, 8mm tape, memory card, etc.

[0096] Furthermore, examples of recording media fixed to computers include hard disks and ROMs. [Example]

[0097] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0098] Example 1: Method for determining total stress (1) Sample preparation

[0099] A physician interviewed 100 healthy volunteers, and those with serious illnesses (such as severe kidney, liver, lung, or heart dysfunction) were excluded from the study. 10 mL of urine, 1 mL of saliva, and 5 mL of serum were collected from the subjects, and informed consent was obtained from all patients.

[0100] In addition, all subjects completed the 57-item "Stress Check" (BJSQ) issued by the Ministry of Health, Labor and Welfare and the CES-D (The Center for Epidemiologic Studies Dementia Scale), which is used to assess depression. The participants answered 20 items of the Stress Check and CES-D. The items mainly involve stress, such as "I have to do a lot of work," but also include items that are inversely proportional to stress, such as "I feel full of energy." When summing all the stress items, the scores of items that are inversely proportional to stress were reversed beforehand.

[0101] The scores for the 57 stress check items and the 20 CES-D items were added up to obtain a total value for each. The total values ​​for each subject were plotted on a scatter diagram (Figure 1). Because the stress check is a questionnaire in which subjects fill out their own subjective opinions, it is possible that they may not fill out the questionnaire accurately or may not correctly understand their own stress levels. Because the stress check and CES-D contain many similar questions, if subjects answer the questionnaire sincerely, the two should show a positive correlation. Looking at the scatter diagram in Figure 1, the correlation coefficient was 0.69, and no outliers were found. Based on these results, we determined that the stress check responses were reliable enough to be analyzed.

[0102] All subjects underwent biochemical tests covering 36 items, as shown in Table 1. The 100 subjects were divided into a high-stress group (50 subjects) and a low-stress group (50 subjects) based on their overall stress check scores, and the average values ​​of the 36 biochemical test items for the high-stress and low-stress groups, as well as their significant differences (Student T-test p-values), were examined. The results are shown in Table 1. As a result, it was confirmed that there were no differences between the subjects in the high-stress and low-stress groups except for the overall stress check score, meaning that there were no differences in their health status other than stress.

[0103] [Table 1]

[0104] (2) Analysis of neurotransmitters using a mass spectrometer Figure 2 shows the six test substance components and their relationships (metabolism).

[0105] (3) Pretreatment Urine (10 μL), water (230 μL), and internal standard solution (10 μL) were placed in a 1.5 mL microtube and mixed thoroughly by vortexing. When creating a calibration curve, add the working solution (10 μL). The working solution consisted of six neurotransmitters (dopamine (DA), serotonin (5-HT), gamma-aminobutyric acid (GABA), vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA)) dissolved in solution A (50% methanol, 3% acetic acid, and 47% water (v / v)) at a concentration of 100 μg / mL. The concentrations were then adjusted to 30, 10, 1, 0.3, 0.1, 0.03, and 0.01 μg / mL. The internal standard solutions were dopamine-d4, gamma-aminobutyric acid-d6, 5-hydroxtryptamine-d4, homovanillic acid-d3, 4-hydroxy-3-methoxymandelic acid-d3, and creatinine-d3, each dissolved in solution A to a concentration of 10 μg / mL. The internal standard for 5-HIAA was 5-hydroxtryptamine-d4, as hydrogen-labeled compounds were not available for purchase. was used.

[0106] The mixed solution was then centrifuged using an ultrafiltration membrane (Amicon 30K, Millipore) at 10,000 g for 30 minutes to remove macromolecules such as proteins. The supernatant was transferred to an LC-MS vial and analyzed by LC-MS.

[0107] (4)LC-MS analysis The analytical equipment used was an Agilent Technologies ULTIVO. The liquid chromatography separation column was a Shimadzu Shim-pack MAqC-ODS I column (2.1 mm × 150 mm, 2.7 μm). The mobile phases were A buffer: 0.1% formic acid in water, and B buffer: 0.05% formic acid in methanol. The liquid chromatography flow rate was 0.2 mL / min, and the column oven temperature was set to 40°C. The gradient was set as follows: 1% B buffer (0 to 0.5 min); 1% to 10% B buffer (0.5 to 4.0 min); 10% to 50% B buffer (4.0 to 10.0 min); 3 min hold at 100% B buffer. The injection volume was 5.0 μL, and the autosampler temperature was set to 4°C. The mass spectrometer used electrospray ionization, and the MRM conditions were as shown in Table 2. The quality control (QC) was performed by pooling urine from 18 healthy volunteers and aliquoting 10 μL of the pooled urine. Each sample was dispensed into a 1.5 mL microtube and stored at -80°C until analysis. When the QC values ​​were within 15% of the theoretical values, the test within the batch was considered valid.

[0108] [Table 2]

[0109] Urinary concentrations, medians, and quartiles of six neurotransmitters were calculated, and box-and-whisker plots were created for each group. (Figure 3) 100 subjects were divided into a high-stress group (50 subjects) and a low-stress group (50 subjects) based on their total BJSQ scores. A Student T-test was then conducted between the two groups, and if the p-value was less than 0.05, the value was plotted on a graph. As a result, it was confirmed that the urinary concentrations of dopamine (DA) and HVA were significantly lower in the high stress group compared to the low stress group.

[0110] Two components were extracted from the six neurotransmitters, and their respective concentrations were plotted on a scatter plot. All 15 combinations were plotted on a graph (Figure 4). As a result, although a high correlation (>0.7) was observed in some combinations, the correlation coefficient for most was below 0.7. This means that the expression of each component is somewhat independent, and their relationship is complementary. This is because each component increases or decreases due to different factors, and therefore a comprehensive assessment of the six components may enable more accurate assessment of stress than using each component individually.

[0111] The urinary concentration of six neurotransmitters versus creatinine ratios were transformed into common logarithms and then normalized (mean zero, variance 1). These values ​​were multiplied by coefficients (weights) and linearly combined to create a "stress index." Here, the total values ​​of the stress check answers (scores) of the 100 subjects were sorted in descending order, and the top 50 were defined as the high stress group and the bottom 50 as the low stress group. The 100 subjects were then randomly divided into a training set of 66 subjects and a test set of 34 subjects. Each set contained an equal number of high-stress and low-stress subjects. Using Excel's Solver (a nonlinear constrained optimization method), we optimized the coefficients (A-F) that would maximize the difference in stress index between high-stress and low-stress subjects in the training set (minimizing the p-value of the Student's T-test). We imposed a constraint on each coefficient that the sum of squares must be 1. The coefficients obtained using the training set were then used in the test set to determine whether the calculated stress index of the test set showed a significant difference.

[0112] As a result, the coefficients for each were as shown in Figure 5, with a p-value of 0.012 for the training set and 0.016 for the test set. It was found that combining the stress index of six neurotransmitters was more effective in determining stress than using a single neurotransmitter.

[0113] Example 2: Correlation between fluctuations in neurotransmitters alone and individual stress factors To clarify the correlation between urinary neurotransmitters and stress factors, participants were classified into "high stressors" (those who answered 3 or 4) and "low stressors" (those who answered 1 or 2) for each question, and correlations between the two groups were examined using Student's t-tests. Table 3 lists the questions with p values ​​below 0.01. Results indicated that dopamine responded to life and work satisfaction, such as "This job isn't for me" and "I feel depressed." It also responded to physical stress, such as "I'm very tired" and "My joints hurt." On the other hand, GABA responded to digestive problems, such as "I have stomach problems" and "I have no appetite." HVA responded to the environment surrounding one's work, such as "My work environment is bad," "I can't sleep well," and "When I encounter problems, my family doesn't help me." VMA responded to uncontrollable circumstances, such as "I can't finish my work within the required time." Serotonin responded to worries, anxiety, and fears about the future, such as "I'm anxious," "my joints hurt," and "I have stomach problems." In Table 3, all questions have been converted into responses that express stress. From the above results, (a) the combination of coefficient A calculated based on the scores of answers to questions about "life and work satisfaction" and "physical stress" and dopamine concentration; (b) the combination of coefficient B calculated based on the scores of answers to questions about "your surrounding environment" and homovanillic acid concentration; (c) the combination of coefficient C calculated based on the scores of answers to questions about the "digestive system" and GABA concentration; (d) the combination of coefficient C calculated based on the scores of answers to questions about "worry" and "anxiety" It was shown that stress can be quantified more accurately using a stress index calculation formula using (a) a combination of coefficient D and serotonin concentration; (b) a combination of coefficient E, calculated based on the scored answers to a question about “environments that you cannot control,” and vanillylmandelic acid concentration; and (c) a combination of coefficient F, calculated based on the scored answers to a question about “anxiety about the future,” and serotonin concentration and / or 5-hydroxyindoleacetic acid concentration.

[0114] [Table 3] [Industrial Applicability]

[0115] The present invention provides a method for quantitatively measuring mental stress in a subject.

Claims

1. A method for quantifying mental stress in a healthy human subject, comprising the following steps (1) and (2): (1) measuring the concentration of one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA (gamma-aminobutyric acid), and 5-hydroxyindoleacetic acid present in urine collected from a healthy subject; (2) calculating a stress index based on a stress index calculation formula that combines the measured concentrations of the neurotransmitters and coefficients obtained from stress items corresponding to the neurotransmitters; The method, wherein the coefficient obtained from the stress item corresponding to the neurotransmitter is calculated based on a score obtained by answering one or more questions in a stress check selected from the following: Coefficient A: Scored value of answers to questions regarding "life / job satisfaction" and "physical stress"; Coefficient B: A score obtained by answering questions about one's surrounding environment; Coefficient C: A score obtained by answering questions about the "digestive system"; Coefficient D: A score obtained by calculating the responses to questions about "worry" and "anxiety"; Coefficient E: A score obtained by answering questions about the environment that one cannot control; Coefficient F: A score obtained by calculating the answers to questions about "anxiety about the future."

2. The method according to claim 1 , wherein the coefficients are calculated so that the correlation between the neurotransmitter concentration and the stress item is significant.

3. The method according to claim 1 or 2, wherein the method for calculating coefficients obtained from stress items corresponding to neurotransmitters is logistic regression or linear regression.

4. The method according to any one of claims 1 to 3, wherein the value obtained by scoring the answers to the questions in the stress check to calculate the coefficient is a value obtained by scoring the answers to all questions in the stress check.

5. The method according to any one of claims 1 to 4, wherein one or more of the following (a) to (f) are used as a combination of coefficients calculated based on the scores of neurotransmitter concentrations and answers to questions in a stress check: (a) A combination of the product of coefficient A calculated based on the scores of answers to questions about “life / job satisfaction” and “physical stress” and dopamine concentration; (b) a combination of the product of coefficient B calculated based on the score of the answer to the question about “your surrounding environment” and the homovanillic acid concentration; (c) a combination of the product of a coefficient C calculated based on the scored answers to questions about the “digestive system” and the GABA concentration; (d) a combination of the product of coefficient D calculated based on the scores of answers to questions about “worry” and “anxiety” and serotonin concentration; (e) A combination of the product of the coefficient E calculated based on the scored value of the answer to the question about “environment that cannot be controlled by oneself” and the concentration of vanillylmandelic acid; (f) A combination of the product of a coefficient F calculated based on the scored value of the answers to the question about "anxiety about the future" and the concentration of serotonin and / or 5-hydroxyindoleacetic acid.

6. The method according to claim 5, wherein the stress index calculation formula, which combines coefficients calculated based on the scores of neurotransmitter concentrations and answers to questions in a stress check, uses the sum of two or more combinations selected from (a) to (f).

7. The method according to claim 5, wherein the stress index calculation formula combines coefficients calculated based on the scores of neurotransmitter concentrations and answers to questions in a stress check, and uses the sum of all combinations of (a) to (f).

8. The stress index calculation formula combining a coefficient calculated based on the concentration of a neurotransmitter and a score of the answers to the questions in the stress check is represented by the following formula 1. The method according to any one of claims 1 to 7. Stress index = A x (dopamine concentration) + B x (homovanillic acid concentration) + C × (GABA concentration) + D x (serotonin concentration) + E x (concentration of vanillylmandelic acid) + F × (5-hydroxyindoleacetic acid concentration) (Equation 1) (In the formula, A, B, C, D, E, and F have the same meanings as defined above.)

9. A method described in any one of claims 1 to 8, further comprising a step of comparing the stress index of the healthy subject calculated in step (2) with a predetermined cutoff value.

10. A method according to any one of claims 1 to 9, in which the quantification of mental stress in healthy subjects is carried out over time.

11. The method according to any one of claims 1 to 10, wherein the concentrations of neurotransmitters are analyzed simultaneously using a liquid chromatography-mass spectrometry (LC / MS) apparatus.

12. The method according to any one of claims 1 to 11, wherein the ratio of the neurotransmitter concentration to the urinary creatinine concentration is used as the neurotransmitter concentration.

13. A method for assisting in determining recovery of a depressed patient or a subject after the onset of depression, comprising: The method comprises steps (1) and (2): (1) measuring the concentration of one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA (gamma-aminobutyric acid), and 5-hydroxyindoleacetic acid present in urine collected from a subject; (2) calculating a stress index based on a stress index calculation formula that combines the measured concentrations of the neurotransmitters and coefficients obtained from stress items corresponding to the neurotransmitters; The method, wherein the coefficient obtained from the stress item corresponding to the neurotransmitter is calculated based on a score obtained by answering one or more questions in a stress check selected from the following: Coefficient A: Scored value of answers to questions regarding "life / job satisfaction" and "physical stress"; Coefficient B: A score obtained by answering questions about one's surrounding environment; Coefficient C: A score obtained by answering questions about the "digestive system"; Coefficient D: A score obtained by calculating the responses to questions about "worry" and "anxiety"; Coefficient E: A score obtained by answering questions about the environment that one cannot control; Coefficient F: A score obtained by calculating the answers to questions about "anxiety about the future."

14. an acquisition unit that acquires concentration data of one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid present in urine collected from a healthy subject; a calculation unit that calculates a stress index, which is an index related to mental stress of the subject, based on the concentrations of the neurotransmitters acquired by the acquisition unit and coefficients obtained from stress items corresponding to the neurotransmitters; an output unit that outputs the stress index calculated by the calculation unit, A stress index calculation device, the stress index calculation device, wherein the coefficient obtained from the stress item corresponding to the neurotransmitter is calculated based on a value obtained by scoring answers to one or more questions in a stress check selected from the following: Coefficient A: Scored value of answers to questions regarding "life / job satisfaction" and "physical stress"; Coefficient B: A score obtained by answering questions about one's surrounding environment; Coefficient C: A score obtained by answering questions about the "digestive system"; Coefficient D: A score obtained by calculating the responses to questions about "worry" and "anxiety"; Coefficient E: A score obtained by answering questions about the environment that one cannot control; Coefficient F: A score obtained by calculating the answers to questions about "anxiety about the future."

15. The computer obtaining concentration data of one or more neurotransmitters selected from dopamine, homovanillic acid, serotonin, vanillylmandelic acid, GABA, and 5-hydroxyindoleacetic acid present in urine collected from a healthy subject; calculating a stress index, which is an index related to the mental stress of the subject, based on the acquired concentrations of the neurotransmitters and coefficients obtained from stress items corresponding to the neurotransmitters; A stress index calculation program for executing a step of outputting the calculated stress index, The coefficient obtained from the stress item corresponding to the neurotransmitter is calculated based on the score of the answers to one or more of the following stress check questions: The stress index calculation program: Coefficient A: Scored value of answers to questions regarding "life / job satisfaction" and "physical stress"; Coefficient B: A score obtained by answering questions about one's surrounding environment; Coefficient C: A score obtained by answering questions about the "digestive system"; Coefficient D: A score obtained by calculating the responses to questions about "worry" and "anxiety"; Coefficient E: A score obtained by answering questions about the environment that one cannot control; Coefficient F: A score obtained by calculating the answers to questions about "anxiety about the future."

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