Exercise habit evaluation method and motor function evaluation method

Abstract

Provided is a method for objectively evaluating the exercise habits and motor function of a subject in a simple and accurate manner. The present invention is characterized by comprising: a measurement step for measuring the RNA expression level of any one of Hif1, PHD3, GLUT1, GLUT3, and MCT4 in leukocytes in a peripheral blood sample of the subject; a cutoff value setting step for creating a receiver operating characteristic (ROC) curve and setting a predetermined cutoff value; and an evaluation step for statistically processing, by a chi-squared test, a boundary for evaluating the presence or absence of an exercise habit, and comparing the RNA expression level of the subject as measured in the measurement step and the cutoff value set in the cutoff value setting step, thereby evaluating the presence or absence of the exercise habit of the subject.

Description

Exercise habit evaluation method and motor function evaluation method

[0001] The present invention relates to an exercise habit evaluation method and a motor function evaluation method.

[0002] In an aging society, it is desirable to evaluate exercise habits and motor function in order to maintain healthy life expectancy and prevent frailty, etc. For example, the J-CHS criteria, one of the representative methods for assessing frailty, requires subjects to measure grip strength and walking speed in addition to answering a questionnaire (Patent Document 1, Non-Patent Document 1).

[0003] However, this evaluation method requires the preparation of equipment and a place for physical fitness testing, and it is not possible to easily evaluate motor function. Furthermore, the subject's motivation during the test affects the evaluation results, and if the subject is faking illness, completely incorrect measurements will be taken, making it insufficient for use as an objective evaluation method.

[0004] Furthermore, motor function is also being continuously evaluated in the living space, focusing on movements that occur in daily life. For example, a method has been proposed in which a subject is fitted with a measuring device and motor function is evaluated by focusing on standing and walking movements (Patent Documents 2 and 3).

[0005] However, this evaluation method requires subjects to wear a measuring device on a daily basis, which places a burden on the subjects and makes it difficult to easily evaluate motor function.

[0006] JP 2020-92977 A JP 2020-44295 A Japanese Patent No. 6448015 A

[0007] An attempt to assess frailty in the elderly based on a questionnaire: A preliminary analysis using item response theory, Tomoyuki Shinohara et al., Journal of Japanese Geriatrics, 2022, 59, 169-177

[0008] The present invention has been made in view of the above problems, and aims to provide a method for objectively evaluating a subject's exercise habits and motor functions in a simple and accurate manner.

[0009] The exercise habit evaluation method of the present invention is a method for evaluating whether a subject has an exercise habit, and is characterized by comprising: a measurement step of measuring the expression level of any one of RNAs Hif1, PHD3, GLUT1, GLUT3, or MCT4 in leukocytes in a peripheral blood sample of the subject; a cutoff value setting step of creating an ROC curve and setting a predetermined cutoff value; and an evaluation step of statistically processing the boundary for evaluating the presence or absence of an exercise habit using a chi-square test, and comparing the expression level of the RNA of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of an exercise habit of the subject.

[0010] The motor function evaluation method of the present invention is a motor function evaluation method for evaluating the presence or absence of a motor function disorder in a subject, wherein the motor function disorder is a swallowing disorder in the subject, and is characterized by comprising: a measurement step of measuring the expression level of any one of RNAs PHD3, GLUT1, GLUT3, MCT4, or AMPKa in leukocytes in a peripheral blood sample of the subject; a cutoff value setting step of creating an ROC curve and setting a predetermined cutoff value; and an evaluation step of statistically processing the boundary for evaluating the presence or absence of a swallowing disorder using a chi-square test, and comparing the expression level of the RNA of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of a swallowing disorder in the subject.

[0011] The motor function evaluation method of the present invention is a method for evaluating the presence or absence of a motor function disorder in a subject, wherein the motor function disorder is a masticatory disorder in the subject, and is characterized by comprising: a measurement step of measuring the RNA expression level of one of PHD3, GLUT1, GLUT3, or AMPKa in leukocytes in a peripheral blood sample from the subject; a cutoff value setting step of creating an ROC curve and setting a predetermined cutoff value; and an evaluation step of statistically processing the boundary for evaluating the presence or absence of a masticatory disorder using a chi-square test, and comparing the RNA expression level of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of a masticatory disorder in the subject.

[0012] Furthermore, the motor function evaluation method of the present invention is a method for evaluating the presence or absence of a motor function disorder in a subject, wherein the motor function disorder is the subject's risk of falling, and is characterized by comprising: a measurement step of measuring the expression level of any one of RNA PHD3, GLUT1, or AMPKa in leukocytes in a peripheral blood sample of the subject; a cutoff value setting step of creating an ROC curve and setting a predetermined cutoff value; and an evaluation step of statistically processing a boundary for evaluating the presence or absence of a fall risk using a chi-square test, and comparing the expression level of the RNA of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of a fall risk in the subject.

[0013] According to the present invention, it is possible to objectively, simply, and accurately evaluate the exercise habits and motor functions of a subject.

[0014] Shows the ROC curve for exercise habits for Hif1. Shows the ROC curve for exercise habits for PHD3. Shows the ROC curve for exercise habits for GLUT1. Shows the ROC curve for exercise habits for GLUT3. Shows the ROC curve for exercise habits for MCT4. Shows the ROC curve for dysphagia for PHD3. Shows the ROC curve for dysphagia for GLUT1. Shows the ROC curve for dysphagia for GLUT3. Shows the ROC curve for dysphagia for MCT4. Shows the ROC curve for dysphagia for AMPKa. Shows the ROC curve for masticatory disorder for PHD3. Shows the ROC curve for masticatory disorder for GLUT1. Shows the ROC curve for masticatory disorder for GLUT3. Shows the ROC curve for masticatory disorder for AMPKa. Shows the ROC curve for fall risk for PHD3. Shows the ROC curve for fall risk for GLUT1. Shows the ROC curve for fall risk for AMPKa.

[0015] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

[0016] The inventors conducted research into hematological testing methods for frailty in elderly people. Frailty refers to the stage before the need for nursing care due to weakening of the body, mind, or social network, and is a multifaceted concept proposed by the Japan Geriatrics Society in May 2014. Frailty is the Japanese translation of "Frailty," which has long been used in the field of geriatrics, and is a concept that encompasses the reversibility of returning to a healthy state with appropriate intervention.

[0017] The inventors asked the elderly subjects to fill out a frailty questionnaire, took blood samples from the subjects, and measured the RNA expression of metabolism-related genes in leukocytes in the peripheral blood samples using quantitative PCR.

[0018] As a result, we have discovered a new finding that RNA expression of metabolism-related genes differs depending on whether or not a subject has an exercise habit, and have completed the present invention.We have also discovered a new finding that RNA expression of metabolism-related genes differs between subjects with and without skeletal muscle weakness, and have completed the present invention.

[0019] The exercise habit evaluation method according to the present invention will be described. In the present invention, exercise refers to physical exercise in a broad sense, including, for example, exercise in the narrow sense, such as sports, training, and aerobic exercise, as well as work involving muscle exertion and daily activities. Exercise habits are the number of exercises, which indicates the frequency of exercise within a certain period of time, the exercise duration, which is the time spent exercising per session, the exercise intensity, which is the strain on the subject's muscles during exercise, or the exercise duration, which is the time interval between exercise at one point in time and exercise at another point in time.

[0020] In this embodiment, the exercise habit is, for example, the number of times the subject exercises. The certain period is not particularly limited, but may be, for example, one week, ten days, or one month, and is preferably one week.

[0021] The subject's exercise frequency can be evaluated by the following method: measuring the RNA expression level of any one of Hif1, PHD3, GLUT1, GLUT3, or MCT4 in leukocytes in the subject's peripheral blood sample (measurement step), creating an ROC curve and setting a predetermined cutoff value (cutoff value setting step), statistically processing the boundary for evaluating the presence or absence of exercise habits using a chi-square test, and comparing the subject's RNA expression level measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of exercise habits in the subject (evaluation step).

[0022] The RNA expression level of a subject measured to evaluate their exercise habits is calculated by measuring the expression level of a gene selected from the above gene group (Hif1, PHD3, GLUT1, GLUT3, or MCT4) in leukocytes in an obtained peripheral blood sample and normalizing the expression level by the number of cycles, etc. For example, such normalization is calculated using the following formula: Score value = Σ(ΔΔCt (genes whose expression level increases with a decrease in the number of exercises)) - Σ(ΔΔCt (genes whose expression level decreases with an increase in the number of exercises)).The internal standard gene 18S rRNA can be used as the control gene used to calculate ΔCt.

[0023] In this embodiment, the cutoff value for evaluating the number of exercises can be determined in advance by analyzing the cohort and performing statistical processing. The cutoff value can be set, for example, by receiver operating characteristic curve (ROC) analysis. The ROC curve is created by calculating the sensitivity and specificity at each cutoff value and plotting them on a coordinate system with the specificity on the horizontal axis and the sensitivity on the vertical axis.

[0024] When determining a cutoff value using an ROC curve, the cutoff value may be determined by balancing sensitivity and specificity. For example, the cutoff value may be the value of the point that is the smallest distance from the upper left corner of the ROC curve, or the value of the point that is the furthest from the dotted diagonal line where the area under the curve (AUC) = 0.500.

[0025] In this embodiment, in the evaluation step, the boundary for evaluating the presence or absence of an exercise habit is statistically processed using a chi-square test, and the RNA expression level of the subject measured in the measurement step is compared with the cutoff value set in the cutoff value setting step.If the expression level is significantly elevated, it can be evaluated that the subject does not have an exercise habit.

[0026] The motor function evaluation method according to the present invention will be described. Motor function is the ability to move a person's body, and is the ability to move by influencing tissues such as the brain, spinal cord, peripheral nerves, muscles, joints, bones, cartilage, and intervertebral discs.

[0027] In this embodiment, the motor function is the subject's swallowing function, which is the function of the human body required to complete a series of processes from recognizing food, taking it into the mouth, and then reaching the stomach.

[0028] The swallowing function of a subject can be evaluated by the following method: measuring the RNA expression level of any one of PHD3, GLUT1, GLUT3, MCT4, and AMPKa in leukocytes in a peripheral blood sample from the subject (measurement step), creating an ROC curve and setting a predetermined cutoff value (cutoff value setting step), statistically determining the boundary for assessing the presence or absence of dysphagia using a chi-square test, and comparing the RNA expression level of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of dysphagia in the subject (evaluation step).

[0029] The expression level of a subject's RNA measured to evaluate swallowing function is calculated by measuring the expression level of a gene selected from the above-mentioned group of genes (PHD3, GLUT1, GLUT3, MCT4, or AMPKa) in leukocytes in an obtained peripheral blood sample and normalizing the expression level by the number of cycles, etc. For example, such normalization is calculated using the following formula: Score value = Σ(ΔΔCt (genes whose expression level increases with reduced swallowing function)) - Σ(ΔΔCt (genes whose expression level decreases with maintained swallowing function)) In this embodiment, the cutoff value for evaluating swallowing function can be determined in advance by analyzing a cohort and performing statistical processing. The cutoff value can be set, for example, by ROC analysis.

[0030] In this embodiment, in the evaluation step, the boundary for assessing the presence or absence of swallowing disorders is statistically processed using a chi-square test, and the RNA expression level of the subject measured in the measurement step is compared with the cutoff value set in the cutoff value setting step.If the expression level is significantly elevated, it can be assessed that there is a swallowing disorder.

[0031] In this embodiment, the motor function is the subject's masticatory function, which is a function of the human body required to complete a series of processes from recognizing food, taking it into the mouth, and then transporting it to the stomach.

[0032] The masticatory function of a subject can be evaluated by the following method: measuring the RNA expression level of any one of PHD3, GLUT1, GLUT3, or AMPKa in leukocytes in a peripheral blood sample from the subject (measurement step), creating an ROC curve and setting a predetermined cutoff value (cutoff value setting step), statistically processing the boundary for evaluating the presence or absence of a masticatory disorder using a chi-square test, and comparing the RNA expression level of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of a masticatory disorder in the subject (evaluation step).

[0033] The expression level of the RNA of a subject measured to evaluate masticatory function is calculated by measuring the expression amount of a gene selected from the above-mentioned group of genes (PHD3, GLUT1, GLUT3, or AMPKa) in leukocytes in an obtained peripheral blood sample and normalizing the expression amount by the number of cycles or the like. For example, such normalization is calculated using the following formula: Score value=Σ(ΔΔCt (gene whose expression amount increases with a decrease in masticatory function))−Σ(ΔΔCt (gene whose expression amount decreases with maintenance of masticatory function)) In this embodiment, the cutoff value for evaluating masticatory function can be determined in advance by analyzing a cohort and performing statistical processing. The cutoff value can be set, for example, by ROC analysis.

[0034] In this embodiment, in the evaluation step, the boundary for assessing the presence or absence of a chewing disorder is statistically processed using a chi-square test, and the RNA expression level of the subject measured in the measurement step is compared with the cutoff value set in the cutoff value setting step.If the expression level is significantly elevated, it can be assessed that a chewing disorder is present.

[0035] In this embodiment, motor function is the subject's risk of falling. Fall risk indicates the degree of risk that the subject is likely to fall. Specifically, it refers to the high possibility that the subject will fall multiple times (e.g., two or more times) in the future over a certain period of time (e.g., one week, one month, one year, etc.).

[0036] The risk of a subject falling can be assessed by the following method: measuring the RNA expression level of one of PHD3, GLUT1, or AMPKa in leukocytes in a peripheral blood sample from the subject (measurement step), creating an ROC curve and setting a predetermined cutoff value (cutoff value setting step), statistically determining the boundary for assessing the presence or absence of a risk of falling using a chi-square test, and comparing the RNA expression level of the subject measured in the measurement step with the cutoff value set in the cutoff value setting step to assess the presence or absence of a risk of falling in the subject (assessment step).

[0037] The expression level of a subject's RNA to be measured for assessing fall risk is calculated by measuring the expression level of a gene selected from the above-mentioned group of genes (PHD3, GLUT1, or AMPKa) in leukocytes in an obtained peripheral blood sample and normalizing the expression level by the number of cycles or the like. For example, such normalization is calculated using the following formula: Score value = Σ(ΔΔCt (genes whose expression level increases with an increase in fall risk)) - Σ(ΔΔCt (genes whose expression level decreases with a decrease in fall risk)) In this embodiment, the cutoff value for assessing fall risk can be determined in advance by analyzing a cohort and performing statistical processing. The cutoff value can be set, for example, by ROC analysis.

[0038] In this embodiment, in the evaluation step, the boundary for assessing the presence or absence of a risk of falling is statistically processed using a chi-square test, and the expression level of the subject's RNA measured in the measurement step is compared with the cutoff value set in the cutoff value setting step.If the expression level is significantly higher or lower, it can be assessed that there is no risk of falling.

[0039] In the present invention, leukocytes from a peripheral blood sample are obtained from a subject. The inventors collected blood from the subject and performed genetic analysis of the whole leukocytes and the monocytes. They found that, unlike the genetic analysis of the monocytes, the genetic analysis of the whole leukocytes accurately indicates the subject's exercise habits and motor function.

[0040] Genetic analysis of whole white blood cells can be performed using, for example, Taqman®, Midori Green, SYBR® Green I, SYBR® Green II, SYBR® Gold, etc., but in this embodiment, the SYBR® Green I method is used. SYBR® Green I is an asymmetric cyanine dye available from Life Technologies, and its structure was revealed by Zipper H et al. ("Nucleic Acids Research," 2004, Vol. 32, No. 12, e103).

[0041] 1. Evaluation of exercise habits Nineteen elderly subjects were asked, "Do you walk at least once a week?" The 19 elderly subjects were aged between 60 and 90 years old. Subjects who answered "yes" to the question, "Do you walk at least once a week?" were considered to have an exercise habit, i.e., exercise frequently, while subjects who answered "no" to the same question were considered to have an exercise habit, i.e., exercise infrequently, and the subjects were divided into two groups.

[0042] Blood was collected from each subject, and quantitative PCR (qPCR) analysis of peripheral blood leukocytes and mononuclear cells was performed. The analysis method was as follows: fasting blood was collected, and RNA expression in mononuclear cells and total leukocytes was evaluated. Mononuclear cells were isolated using mononuclear cell preparation tubes (BD Vacutainer #362760, BD Bioscience, NJ, USA). RNA from mononuclear cells and total leukocytes was stabilized using PAXgene Blood RNA Tubes (#762165, BD Bioscience, NJ). Total RNA was isolated using the RNeasy Plus Universal Mini Kit (Qiagen, CA, USA) according to the manufacturer's instructions. PrimeScript TM cDNA was synthesized from 1 μg of total RNA using the II 1st strand cDNA Synthesis Kit (TAKARA, Kyoto, Japan). TM SYBR TMAnalysis was performed using Agilent AriaMx Real Time Quantitative PCR System with Green Master Mix (Applied Biosystems, CA, USA). 18S ribosomal RNA was used as the reference gene. The list of target genes and primer sequences are shown in Table 13, and the amplification protocol is shown in Table 14. Analysis was performed using the SYBR Green I method with each primer.

[0043] The analysis results for leukocytes in peripheral blood are shown in Table 1.

[0044]

[0045] The analysis results for mononuclear cells are shown in Table 2.

[0046]

[0047] Statistical analysis was performed using the delta-delta Ct method (ΔΔCt method) using the internal standard gene 18S rRNA. The results in Table 1 show that the expression levels of Hif1, PHD3, GLUT1, GLUT3, and MCT4 in leukocytes in peripheral blood samples were significantly higher in subjects with no exercise habits than in subjects with exercise habits.

[0048] On the other hand, according to the results in Table 2, there was no significant difference in mononuclear cell Hif1, PHD3, PDK1, GLUT1, GLUT3, or MCT4 between subjects with and without exercise habits.

[0049] Thus, it was found that the RNA expression level of any one of Hif1, PHD3, GLUT1, GLUT3, or MCT4 in leukocytes in the subjects' peripheral blood samples was related to whether or not the subjects had an exercise habit.

[0050] This allows the expression level of any one of Hif1, PHD3, GLUT1, GLUT3, or MCT4 RNA in leukocytes in a peripheral blood sample from the subject to be measured (measurement step), an ROC curve to set a predetermined cutoff value (cutoff value setting step), a boundary for evaluating whether or not the subject has an exercise habit to be statistically processed using a chi-square test, and the RNA expression level of the subject measured in the measurement step to be compared with the cutoff value set in the cutoff value setting step to evaluate whether or not the subject has an exercise habit (evaluation step).

[0051] In addition to the 19 subjects mentioned above, Subject A, an elderly individual, was asked the same question as above: "Do you walk at least once a week?" As before, blood was drawn from Subject A and quantitative PCR (qPCR) analysis was performed on the leukocytes in the peripheral blood. The results are shown in Table 3 below.

[0052]

[0053] For example, for Hif1, an ROC curve was drawn using the data in Table 1 (Figure 1). The cutoff value for reduced exercise frequency was calculated based on this ROC curve and was found to be 1.200. The Hif1 expression level in leukocytes in a peripheral blood sample from subject A was measured and found to be 3.56199 (measurement step). When the RNA expression level of subject A measured in the measurement step was compared with a predetermined cutoff value, it was found to be higher than the cutoff value, and this subject was assessed as having a low exercise frequency (no exercise habit) (evaluation step). In fact, this subject exercised infrequently, and the evaluation method according to the present invention matched this fact.

[0054] For example, an ROC curve for PHD3 was drawn using the data in Table 1 (Figure 2). The cutoff value for reduced exercise frequency was calculated based on this ROC curve and was found to be 2.200. The PHD3 expression level in leukocytes in a peripheral blood sample from subject A was measured and found to be 5.02573 (measurement step). When the RNA expression level of subject A measured in the measurement step was compared with a predetermined cutoff value, it was found to be higher than the cutoff value, and this subject was assessed as having a low exercise frequency (no exercise habit) (evaluation step). In fact, this subject exercised infrequently, and the evaluation method according to the present invention matched this fact.

[0055] For example, an ROC curve for GLUT1 was drawn using the data in Table 1 (Figure 3). The cutoff value for reduced exercise frequency was calculated based on this ROC curve and was found to be 1.010. The GLUT1 expression level in leukocytes in a peripheral blood sample from subject A was measured and found to be 1.75642 (measurement step). When the RNA expression level of subject A measured in the measurement step was compared with a predetermined cutoff value, it was found to be higher than the cutoff value, and this subject was assessed as having a low exercise frequency (no exercise habit) (evaluation step). In fact, this subject exercised infrequently, and the evaluation method according to the present invention matched this fact.

[0056] For example, an ROC curve for GLUT3 was drawn using the data in Table 1 (Figure 4). The cutoff value for reduced exercise frequency was calculated based on this ROC curve and was found to be 0.800. The GLUT3 expression level in leukocytes in a peripheral blood sample from subject A was measured and found to be 2.19858 (measurement step). When the RNA expression level of subject A measured in the measurement step was compared with a predetermined cutoff value, it was found to be higher than the cutoff value, and this subject was assessed as having a low exercise frequency (no exercise habit) (evaluation step). In fact, this subject exercised infrequently, and the evaluation method according to the present invention matched this fact.

[0057] For example, for MCT4, an ROC curve was drawn using the data in Table 1 (Figure 5). The cutoff value for reduced exercise frequency was calculated based on this ROC curve and was found to be 0.870. The MCT4 expression level in leukocytes in the peripheral blood sample of subject A was measured and found to be 3.19724 (measurement step). When the RNA expression level of subject A measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having a low exercise frequency (no exercise habit) (evaluation step). In fact, this subject's exercise frequency was low, and the evaluation method according to the present invention matched this fact.

[0058] 2. Evaluation of swallowing function Nineteen elderly subjects were asked, "Do you ever choke on tea or soup?" Subjects who answered "yes" to the question, "Do you ever choke on tea or soup?" were considered to have a swallowing disorder, and subjects who answered "no" to the question were considered to have a swallowing disorder, and the subjects were divided into two groups.

[0059] Blood was collected from each subject, and leukocytes and mononuclear cells in the peripheral blood were analyzed using primers and the SYBR Green I method in the same manner as described above.

[0060] The analysis results for leukocytes in peripheral blood are shown in Table 4.

[0061]

[0062] The analysis results for mononuclear cells are shown in Table 5.

[0063]

[0064] Statistical analysis was performed using the delta-delta Ct method (ΔΔCt method) using the internal standard gene 18S rRNA. The results in Table 4 show that the expression levels of PHD3, GLUT1, GLUT3, MCT4, and AMPKa in leukocytes in peripheral blood samples were significantly higher in subjects with dysphagia than in subjects without dysphagia.

[0065] On the other hand, according to the results in Table 5, there was no significant difference in mononuclear cell PHD3, GLUT1, GLUT3, MCT4, or AMPKa between subjects with dysphagia and subjects without dysphagia.

[0066] Thus, it was found that the RNA expression level of any one of PHD3, GLUT1, GLUT3, MCT4 or AMPKa in leukocytes in the peripheral blood samples of subjects was associated with the presence or absence of dysphagia in the subjects.

[0067] This allows the expression level of any one of the RNAs PHD3, GLUT1, GLUT3, MCT4, or AMPKa in leukocytes in a peripheral blood sample from the subject to be measured (measurement step), an ROC curve to set a predetermined cutoff value (cutoff value setting step), a boundary for assessing the presence or absence of dysphagia to be statistically processed using a chi-square test, and the expression level of the subject's RNA measured in the measurement step to be compared with the cutoff value set in the cutoff value setting step to assess the presence or absence of dysphagia in the subject (evaluation step).

[0068] In addition to the 19 subjects mentioned above, Subject B, an elderly individual, was asked the same question as above: "Do you ever choke on tea or soup?" As before, blood was drawn from Subject B, and quantitative PCR (qPCR) analysis of peripheral blood leukocytes was performed. The results are shown in Table 6 below.

[0069]

[0070] For example, for PHD3, an ROC curve was drawn using the data in Table 4 (FIG. 6). The cutoff value for dysphagia was calculated based on this ROC curve and was found to be 3.300. The PHD3 expression level in leukocytes in the peripheral blood sample of subject B was measured and found to be 7.09304 (measurement step). When the RNA expression level of subject B measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having dysphagia (evaluation step). In fact, this subject had dysphagia, and the evaluation method according to the present invention matched this fact.

[0071] For example, for GLUT1, an ROC curve was drawn using the data in Table 4 ( FIG. 7 ). The cutoff value for dysphagia was calculated based on this ROC curve and was found to be 1.112. The GLUT1 expression level in leukocytes in the peripheral blood sample of subject B was measured and found to be 2.72749 (measurement step). When the RNA expression level of subject B measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having dysphagia (evaluation step). In fact, this subject had dysphagia, and the evaluation method according to the present invention matched this fact.

[0072] For example, for GLUT3, an ROC curve was drawn using the data in Table 4 ( FIG. 8 ). The cutoff value for dysphagia was calculated based on this ROC curve and was found to be 1.331. The GLUT3 expression level in leukocytes in a peripheral blood sample from subject B was measured and found to be 2.75165 (measurement step). When the RNA expression level of subject B measured in the measurement step was compared with a predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be assessed as having dysphagia (evaluation step). In fact, this subject had dysphagia, and the evaluation method according to the present invention matched this fact.

[0073] For example, for MCT4, an ROC curve was drawn using the data in Table 4 (FIG. 9). The cutoff value for dysphagia was calculated based on this ROC curve and was found to be 1.439. The MCT4 expression level in leukocytes in the peripheral blood sample of subject B was measured and found to be 2.80538 (measurement step). When the RNA expression level of subject B measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having dysphagia (evaluation step). In fact, this subject had dysphagia, and the evaluation method according to the present invention matched this fact.

[0074] For example, for AMPKa, an ROC curve was drawn using the data in Table 4 (FIG. 10). The cutoff value for dysphagia was calculated based on this ROC curve and was found to be 1.935. The AMPKa expression level of leukocytes in the peripheral blood sample of subject B was measured and found to be 3.39923 (measurement step). When the RNA expression level of subject B measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having dysphagia (evaluation step). In fact, this subject had dysphagia, and the evaluation method according to the present invention matched this fact.

[0075] 3. Evaluation of masticatory function Nineteen elderly subjects were asked, "Is it more difficult for you to eat hard foods compared to six months ago?" Subjects who answered "yes" to the question, "Is it more difficult for you to eat hard foods compared to six months ago?" were classified as having a "decline" in masticatory function, and subjects who answered "no" to the question were classified as having an "unchanged" masticatory function, and the subjects were divided into two groups.

[0076] Blood was collected from each subject, and leukocytes and mononuclear cells in the peripheral blood were analyzed using the SYBR Green I method with primers.

[0077] The analysis results for leukocytes in peripheral blood are shown in Table 7.

[0078]

[0079] The analysis results for mononuclear cells are shown in Table 8.

[0080]

[0081] Statistical analysis was performed using the ΔΔCt method (delta-delta Ct method) using the internal standard gene 18S rRNA. The results in Table 7 show that the expression levels of PHD3, GLUT1, GLUT3, and AMPKa in leukocytes in peripheral blood samples were significantly higher in subjects with masticatory disorders than in subjects without masticatory disorders.

[0082] On the other hand, according to the results in Table 8, there was no significant difference in mononuclear cell PHD3, GLUT1, GLUT3, or AMPKa between subjects with and without masticatory disorders.

[0083] Thus, it was found that the RNA expression level of any one of PHD3, GLUT1, GLUT3 or AMPKa in leukocytes in the peripheral blood samples of subjects was related to the presence or absence of masticatory disorders in the subjects.

[0084] This allows the expression level of any one of the RNAs PHD3, GLUT1, GLUT3, or AMPKa in leukocytes in the subject's peripheral blood sample to be measured (measurement step), an ROC curve to be created and a predetermined cutoff value to be set (cutoff value setting step), a boundary for evaluating the presence or absence of a masticatory disorder to be statistically processed using a chi-square test, and the expression level of the subject's RNA measured in the measurement step to be compared with the cutoff value set in the cutoff value setting step to evaluate the presence or absence of a masticatory disorder in the subject (evaluation step).

[0085] In addition to the 19 subjects mentioned above, Subject C, an elderly subject, was asked the same question as above: "Is it more difficult for you to eat solid foods compared to six months ago?" As before, blood was drawn from Subject C, and quantitative PCR (qPCR) analysis of peripheral blood leukocytes was performed. The results are shown in Table 9 below.

[0086]

[0087] For example, for PHD3, an ROC curve was drawn using the data in Table 7 (FIG. 11). The cutoff value for masticatory disorder was calculated based on this ROC curve and was found to be 1.923. The PHD3 expression level in leukocytes in the peripheral blood sample of subject C was measured and found to be 5.95531 (measurement step). When the RNA expression level of subject C measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having a masticatory disorder (evaluation step). In fact, this subject had a masticatory disorder, and the evaluation method according to the present invention matched this fact.

[0088] For example, for GLUT1, an ROC curve was drawn using the data in Table 7 (FIG. 12). The cutoff value for masticatory disorder was determined based on this ROC curve and was found to be 1.703. The GLUT1 expression level in leukocytes in the peripheral blood sample of subject C was measured and found to be 3.20965 (measurement step). When the RNA expression level of subject C measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having a masticatory disorder (evaluation step). In fact, this subject had a masticatory disorder, and the evaluation method according to the present invention matched this fact.

[0089] For example, for GLUT3, an ROC curve was drawn using the data in Table 7 (FIG. 13). The cutoff value for masticatory disorder was determined based on this ROC curve and was found to be 1.502. The GLUT3 expression level in leukocytes in the peripheral blood sample of subject C was measured and found to be 2.19743 (measurement step). When the RNA expression level of subject C measured in the measurement step was compared with the predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having a masticatory disorder (evaluation step). In fact, this subject had a masticatory disorder, and the evaluation method according to the present invention matched this fact.

[0090] For example, for AMPKa, an ROC curve was drawn using the data in Table 7 (FIG. 14). The cutoff value for masticatory disorder was determined based on this ROC curve and was found to be 1.800. The expression level of AMPKa in leukocytes in a peripheral blood sample from subject C was measured and found to be 3.03309 (measurement step). When the RNA expression level of subject C measured in the measurement step was compared with a predetermined cutoff value, it was found to be higher than the cutoff value, and this subject could be evaluated as having a masticatory disorder (evaluation step). In fact, this subject had a masticatory disorder, and the evaluation method according to the present invention matched this fact.

[0091] 4. Assessment of Fall Risk Nineteen elderly subjects were asked, "Have you fallen in the past year?" Subjects who answered "yes" to the question "Have you fallen in the past year?" were considered to be at risk of falling, while subjects who answered "no" to the question were considered to be at risk of falling, and the subjects were divided into two groups.

[0092] Blood was collected from each subject, and leukocytes and mononuclear cells in the peripheral blood were analyzed using the SYBR Green I method with primers.

[0093] The analysis results for leukocytes in peripheral blood are shown in Table 10.

[0094]

[0095] The analysis results for mononuclear cells are shown in Table 11.

[0096]

[0097] Statistical analysis was performed using the delta-delta Ct method (ΔΔCt method) using the internal standard gene 18S rRNA. The results in Table 10 show that the expression levels of PHD3, GLUT1, and AMPKa in leukocytes in peripheral blood samples were significantly higher in subjects with a risk of falling than in subjects without a risk of falling.

[0098] On the other hand, according to the results in Table 11, there was no significant difference in mononuclear cell PHD3, GLUT1, or AMPKa between subjects with a "yes" risk of falling and subjects with a "no" risk of falling.

[0099] Thus, it was found that the RNA expression level of any one of PHD3, GLUT1, or AMPKa in leukocytes in a subject's peripheral blood sample was associated with whether or not the subject was at risk of falling.

[0100] This allows the expression level of one of the RNAs PHD3, GLUT1, or AMPKa in leukocytes in a peripheral blood sample from the subject to be measured (measurement step), an ROC curve to set a predetermined cutoff value (cutoff value setting step), a boundary for assessing the presence or absence of a fall risk to be statistically processed using a chi-square test, and the RNA expression level of the subject measured in the measurement step to be compared with the cutoff value set in the cutoff value setting step to assess the presence or absence of a fall risk in the subject (evaluation step).

[0101] In addition to the 19 subjects mentioned above, Subject D, an elderly individual, was asked the same question as above: "Have you fallen in the past year?" As before, blood was drawn from Subject D, and quantitative PCR (qPCR) analysis of peripheral blood leukocytes was performed. The results are shown in Table 12 below.

[0102]

[0103] For example, for PHD3, an ROC curve was drawn using the data in Table 10 ( FIG. 15 ). The cutoff value for fall risk was calculated based on this ROC curve and was found to be 2.188. The PHD3 expression level in leukocytes in a peripheral blood sample from subject D was measured and found to be 1.76117 (measurement step). When the RNA expression level of subject D measured in the measurement step was compared with a predetermined cutoff value, it was found to be lower than the cutoff value, and this subject could be assessed as not having a fall risk (evaluation step). In fact, this subject did not have a fall risk, and the evaluation method according to the present invention matched this fact.

[0104] For example, for GLUT1, an ROC curve was drawn using the data in Table 10 ( FIG. 16 ). The cutoff value for fall risk was determined based on this ROC curve and was found to be 1.582. The GLUT1 expression level in leukocytes in the peripheral blood sample of subject D was measured and found to be 1.14321 (measurement step). When the RNA expression level of subject D measured in the measurement step was compared with the predetermined cutoff value, it was found to be lower than the cutoff value, and this subject could be assessed as not having a fall risk (evaluation step). In fact, this subject did not have a fall risk, and the evaluation method according to the present invention matched this fact.

[0105] For example, for AMPKa, an ROC curve was drawn using the data in Table 10 ( FIG. 17 ). The cutoff value for fall risk was determined based on this ROC curve and was found to be 1.835. The AMPKa expression level of leukocytes in the peripheral blood sample of subject D was measured and found to be 1.13472 (measurement step). When the RNA expression level of subject D measured in the measurement step was compared with the predetermined cutoff value, it was found to be lower than the cutoff value, and this subject could be assessed as not having a fall risk (evaluation step). In fact, this subject did not have a fall risk, and the evaluation method according to the present invention matched this fact.

[0106]

[0107]

[0108] It can be used to evaluate exercise habits and motor function.

Claims

1. A method for evaluating the presence or absence of a subject's exercise habit, comprising: a measurement step of measuring the expression level of RNA of any one of Hif1, PHD3, GLUT1, GLUT3, or MCT4 in leukocytes in the peripheral blood sample of the subject; a cut-off value setting step of creating an ROC curve and setting a predetermined cut-off value; and an evaluation step of statistically processing the boundary for evaluating the presence or absence of an exercise habit by a chi-square test, comparing the expression level of the RNA of the subject measured in the measurement step with the cut-off value set in the cut-off value setting step, and evaluating the presence or absence of the exercise habit of the subject.

2. A method for evaluating motor function for evaluating the presence or absence of motor dysfunction in a subject, wherein the motor dysfunction is dysphagia in the subject, comprising: a measurement step of measuring the expression level of RNA of any one of PHD3, GLUT1, GLUT3, MCT4, or AMPKa in leukocytes in the peripheral blood sample of the subject; a cut-off value setting step of creating an ROC curve and setting a predetermined cut-off value; and an evaluation step of statistically processing the boundary for evaluating the presence or absence of dysphagia by a chi-square test, comparing the expression level of the RNA of the subject measured in the measurement step with the cut-off value set in the cut-off value setting step, and evaluating the presence or absence of dysphagia in the subject.

3. A method for evaluating motor function for evaluating the presence or absence of motor dysfunction in a subject, wherein the motor dysfunction is mastication disorder in the subject, comprising: a measurement step of measuring the expression level of RNA of any one of PHD3, GLUT1, GLUT3, or AMPKa in leukocytes in the peripheral blood sample of the subject; a cut-off value setting step of creating an ROC curve and setting a predetermined cut-off value; and an evaluation step of statistically processing the boundary for evaluating the presence or absence of mastication disorder by a chi-square test, comparing the expression level of the RNA of the subject measured in the measurement step with the cut-off value set in the cut-off value setting step, and evaluating the presence or absence of mastication disorder in the subject.

4. A motor function evaluation method for evaluating the presence or absence of motor function impairment in a subject, wherein the motor function impairment is the risk of falling for the subject, the method comprising: a measurement step of measuring the expression level of RNA of any one of PHD3, GLUT1, or AMPKa of white blood cells in a peripheral blood sample of the subject; a cut-off value setting step of creating an ROC curve and setting a pre-determined cut-off value; and an evaluation step of statistically processing a boundary for evaluating the presence or absence of the risk of falling by a chi-square test, comparing the expression level of the RNA of the subject measured in the measurement step with the cut-off value set in the cut-off value setting step, and evaluating the presence or absence of the risk of falling for the subject. A motor function evaluation method characterized by comprising the above steps.

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