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WO2024210185A1 - Biomarqueur de sarcopénie et son utilisation - Google Patents

Biomarqueur de sarcopénie et son utilisation Download PDF

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Publication number
WO2024210185A1
WO2024210185A1 PCT/JP2024/014010 JP2024014010W WO2024210185A1 WO 2024210185 A1 WO2024210185 A1 WO 2024210185A1 JP 2024014010 W JP2024014010 W JP 2024014010W WO 2024210185 A1 WO2024210185 A1 WO 2024210185A1
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Prior art keywords
sarcopenia
marker
measurement
sample
measuring
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English (en)
Japanese (ja)
Inventor
啓子 加藤
明子 藤田
卓也 岡
修一 大渕
恒 河合
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Tokyo Metropolitan Institute For Geriatrics And Gerontology
Kyoto Sangyo University
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Tokyo Metropolitan Institute For Geriatrics And Gerontology
Kyoto Sangyo University
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Publication of WO2024210185A1 publication Critical patent/WO2024210185A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • This disclosure relates to biomarkers for sarcopenia and their uses.
  • Sarcopenia refers to a state in which muscle mass has decreased due to aging, or a state in which muscle strength has decreased due to aging.
  • frailty is generally defined as "an age-related syndrome (geriatric syndrome) in which a person is susceptible to physical functional disorders and health disorders in response to various stresses, based on a decline in physiological function across multiple organs, a decline in homeostasis, and a lack of physical activity and energy reserve capacity to maintain good health.”
  • Frailty in the elderly can be understood as a stage prior to reaching a state of needing nursing care, which is a stage prior to reaching functional disorders.
  • sarcopenia primary sarcopenia
  • secondary sarcopenia is also known to be caused by factors other than aging, such as primary sarcopenia, disuse, inflammation, malnutrition, post-surgery, cancer, and organ failure.
  • depression-anxiety disorder refers to a state in which depressive symptoms and/or anxiety disorder have developed. Strictly speaking, depression and anxiety disorders are different diseases, but since they can show similar symptoms, mainly depressive symptoms, they are sometimes collectively referred to as "depression-anxiety disorder.”
  • sarcopenia and anxiety disorder are recognized as clearly different diseases, and the treatment methods for them are also different.
  • Patent Documents 1 and 2 are known as methods for diagnosing sarcopenia and frailty.
  • Patent Document 1 discloses a device and software for evaluating sarcopenia based on the walking movement of a subject.
  • Patent Document 2 discloses a method for diagnosing frailty or pre-frailty based on the subject's answers to questions about their condition over the past two to three years, such as how many times they get up to go to the bathroom in the middle of the night and whether they have back pain.
  • the present disclosure has been made in consideration of the above-mentioned circumstances, and has as its first object to provide a VOC marker etc. that can specifically diagnose sarcopenia by identifying volatile metabolites specifically contained in samples from sarcopenia patients, in particular urinary volatile metabolites (VOCs) specifically contained in urine.
  • the second object of the present disclosure is to provide a method for measuring sarcopenia markers, a method for testing sarcopenia, and a kit for use in measuring sarcopenia markers.
  • the test method disclosed herein is a test method for obtaining data as one of the evaluation items for diagnosing sarcopenia by measuring volatile metabolites in urine obtained from a human subject, and is characterized in that the volatile metabolite is at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, acetic acid, pyrrole, gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • the total amount of the caprylic acid and pelargonic acid as medium-chain fatty acids having 8 to 12 carbon atoms.
  • the biomarker for sarcopenia is a biomarker for diagnosing sarcopenia that measures volatile metabolites in urine obtained from a human subject, and is characterized in that it is at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, acetic acid, pyrrole, gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • the test method is a test method for obtaining data as one of the evaluation items for diagnosing sarcopenia by measuring volatile metabolites in urine obtained from human subjects (subjects), in which the subjects are divided into a younger group of 79 years old or younger and an elderly group of 80 years old or older, and in the younger group, the volatile metabolite is at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, pyrrole, gamma-butyrolactone, texanol, and octanoic acid.
  • the first method of measuring a sarcopenia marker of the present disclosure includes a measurement step of measuring, as a sarcopenia marker, metabolic products of food-derived components, intestinal bacteria-derived components, or volatile components of their analogs in a subject sample.
  • the second sarcopenia marker measurement method disclosed herein includes a measurement step of contacting a subject sample with a sarcopenia marker measurement reagent to measure the sarcopenia marker in the sample, the sarcopenia marker including a food-derived component, a metabolic product of an intestinal bacteria-derived component, or a volatile component of an analog thereof.
  • the method of testing for sarcopenia or the method of testing for the risk of developing sarcopenia disclosed herein includes a measurement step of measuring a sarcopenia marker in a subject sample, and the measurement step is carried out by the first or second method of measuring a sarcopenia marker disclosed herein.
  • the kit for use in measuring the sarcopenia markers disclosed herein includes a measurement reagent for the sarcopenia marker, and the sarcopenia marker includes a food-derived component, a metabolic product of a component derived from intestinal bacteria, or a volatile component of an analog thereof.
  • a biomarker for diagnosing human sarcopenia is provided. According to the present disclosure, since it can be performed using urine as a subject (target sample), which is less invasive, it can also be used by caregivers and others. By using the present disclosure for primary screening in clinical settings, it is possible to prevent the transition from sarcopenia to frailty.
  • Non-Patent Document 1 depression-anxiety disorders can be diagnosed by measuring urinary VOCs. Since the VOCs used to diagnose depression-anxiety disorders and the VOCs used to diagnose sarcopenia as disclosed herein are different, by combining the inventors' prior literature with this disclosure, sarcopenia and depression-anxiety disorders can be diagnosed separately, making this a very useful diagnostic method.
  • biomarker data provided by this disclosure is important for supporting the diagnosis of sarcopenia, and the final diagnosis of the disease will ultimately be made by a qualified individual, such as a doctor.
  • the present disclosure also provides a method for measuring sarcopenia markers, a method for testing sarcopenia, and a kit for use in measuring sarcopenia markers.
  • This figure shows typical TIC chromatograms of urinary VOCs from sarcopenia patients (upper: black line) and healthy individuals (lower: light black line).
  • the two graphs in the figure are enlarged graphs of acetone and pelargonic acid (Nonanoic Acid), which are representative substances that differ in amount between the two groups.
  • the graph shows a comparison of m/z values between sarcopenia patients and healthy subjects for 10 VOCs identified as potential biomarkers, along with their compound structural formulas.
  • the compounds shown from the top left are p-xylene, 1-butanol, D-limonene, nonanal, and pyrrol, and from the bottom left are gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • the patient/healthy control ratios were 1.4 times for p-xylene, 0.6 times for 1-butanol, 1.5 times for limonene, 1.3 times for nonanal, 1.4 times for pyrrole, 2.0 times for gamma-butyrolactone, 1.1 times for texanol, 1.6 times for octanoic acid, 3.2 times for nonanoic acid, and 0.8 times for diisobutyl phthalate.
  • This figure shows the ROC curves for eight VOCs after ROC analysis.
  • VOCs shown are (1) p-xylene, (2) 1-butanol, (3) limonene, (4) nonanal, (5) texanol, (6) octanoic acid, (7) nonanoic acid, and (8) diisobutyl phthalate.
  • 13 is a graph showing the results of ROC analysis performed on the combinations of 2 to 10 VOCs, combining the results for men and women.
  • A 10 VOCs (p-xylene, 1-butanol, limonene, nonanal, pyrrole, gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, diisobutyl phthalate),
  • B 9 VOCs (p-xylene, 1-butanol, limonene, nonanal, pyrrole, texanol, octanoic acid, nonanoic acid, diisobutyl phthalate),
  • C 9 VOCs (p-xylene, 1-butanol, limonene, nonanal, pyrrole, texanol, octanoic acid, nonanoic acid, diisobutyl phthalate),
  • D 10 VOCs (p-xylene, 1-butanol, limonene, nonanal, pyrrole, texanol, octanoi
  • FIG. 13 is a graph showing the results of ROC analysis of combinations of 2 to 10 VOCs performed on men only.
  • the VOC combinations are the same as those in FIG. 13 is a graph showing the results of ROC analysis of combinations of 2 to 10 VOCs performed on women only.
  • the VOC combinations are the same as those in FIG. This is a graph comparing the m/z values of six VOCs between sarcopenia patients and healthy subjects in the group aged 79 years or younger ( ⁇ 79 or ⁇ 79) or 80 years or older (>80), along with the compound structures.
  • 1 is a graph showing the results of ROC analysis for ages 79 and under or 80 and over for combinations of 5 (ages 80 and over), 6, 8 and 11 VOCs (ages 79 and under).
  • This graph summarizes the indices of six VOCs for people aged 66 to 79.
  • sarcopenia refers to a state in which muscle mass is reduced due to aging, or a state in which muscle strength is reduced due to aging.
  • the sarcopenia refers to a disease classified as ICD code M6259 (sarcopenia) in the International Classification of Diseases, 10th Edition (ICD10), for example.
  • ICD10 International Classification of Diseases, 10th Edition
  • the sarcopenia can be evaluated using the subject's grip strength, walking speed, and skeletal muscle mass, for example, with reference to the 2019 diagnostic criteria of the Asian Working Group for Sarcopenia (AWGS).
  • AGS Asian Working Group for Sarcopenia
  • the subject's grip strength is less than 28 kg for men and less than 18 kg for women, the walking speed is less than 1 m/s for both men and women, and the skeletal muscle mass is less than 7.0 kg/ m2 for men and 5.7 kg/ m2 for women (in the case of bioelectrical impedance analysis)
  • the subject can be diagnosed as having low skeletal muscle mass and low muscle strength, or low skeletal muscle mass and low physical function, and can be evaluated as having sarcopenia (Chen LK, Woo J, Aimpulsachai P, et al: Asian working group for sarcopenia: 2019 consensus update on sarcopenia diagnosis and treatment. J Am Med Dir Assoc 2020; 21: 300-307.e2.).
  • sarcopenia marker refers to a substance that is an indicator of the presence and/or progression of sarcopenia in a subject.
  • the term "subject” or “test subject” refers to an animal or a cell, tissue, or organ derived from an animal, and is used in a sense including humans in particular.
  • the animal refers to humans and non-human animals.
  • the non-human animals include mammals such as mice, rats, hamsters, rabbits, goats, cows, horses, dogs, cats, pigs, monkeys, dolphins, and sea lions.
  • the subject may be, for example, a patient with sarcopenia, a person whose sarcopenia is unknown, or a person suspected of having sarcopenia.
  • the person suspected of having sarcopenia may be, for example, a person subjectively suspected by the subject, or a person who is judged to be suspected of having sarcopenia or to be possibly affected by sarcopenia as a result of a medical examination by a doctor or the like.
  • the person subjectively suspected by the subject may be, for example, a person who has some subjective symptoms, a person who wishes to undergo a preventive medical examination, etc.
  • the age of the subject may be, for example, 79 years or younger, or 80 years or older.
  • the subject's age is, for example, 40 years or older, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older, or 95 years or older.
  • affected may mean the state of having the disease, or may mean developing the disease.
  • treatment refers to therapeutic treatment and/or prophylactic treatment.
  • treatment refers to treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder.
  • prevention refers to reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology.
  • the “treatment” may be, for example, treatment of a patient who develops the target disease, or treatment of an animal model of the target disease.
  • sample may refer to a sample that contains a sarcopenia marker, may possibly contain a sarcopenia marker, or may be a sample that is unknown as to whether it contains a sarcopenia marker.
  • sample include biological samples such as biological samples or specimens.
  • biological sample include samples containing body fluids, cells, tissues, organs, etc., and specific examples include samples of urine, feces, whole blood, serum, plasma, cerebrospinal fluid, puncture fluid, bile, etc.
  • the biological sample is preferably a urine sample, or a blood sample such as whole blood, serum, or plasma.
  • the urine sample may be a sample obtained by collecting the entire amount of urine naturally discharged (whole urine), or a sample obtained by collecting a portion of urine naturally discharged (partial urine).
  • the partial urine may be urine collected at the beginning and/or end of urination, but is preferably urine collected during urination.
  • the urine sample may be collected from a used sanitary product (e.g., a diaper, a urine pad, etc.).
  • the sample may be liquid or solid. In the present disclosure, when the sample is solid, it is preferable to prepare a liquid sample by mixing the solid sample with a liquid.
  • the liquid may be, for example, water; physiological saline; or a buffer solution such as Hanks' buffer, Good's buffer (HEPES buffer, Tricine buffer, etc.), Tris buffer, phosphate buffer, or glycine buffer.
  • food means something that can be ingested by animals, particularly humans.
  • the food may be, for example, either solid or liquid.
  • examples of the food include dairy products, oils and fats, fruits, vegetables, seaweed, nuts and seeds, confectioneries, grains, beans, meat, seafood, eggs, sweeteners, spices, beverages (for example, tea, milk, fruit juice, alcoholic beverages, etc.), etc.
  • intestinal bacteria refers to bacteria present inside the intestines of animals, particularly humans.
  • the intestinal bacteria as a whole can also be referred to as the intestinal flora.
  • the intestinal bacteria can be classified, for example, as good bacteria, bad bacteria, or opportunistic bacteria based on whether they are harmful to the health of animals, particularly humans.
  • metabolite refers to an intermediate product and/or an end product of a metabolic process in an animal.
  • the metabolite may be, for example, a primary metabolite involved in the growth of an organism, or a secondary metabolite having another biological function.
  • analog refers to a compound that is similar in properties and/or structure to a given compound.
  • the analog may be, for example, a compound in which an atom or atomic group in the given compound is replaced with another atom or atomic group.
  • the atomic group may be, for example, a substituent, a characteristic group, or a functional group.
  • volatile components refers to components that have the property of volatilization.
  • the volatile components may be, for example, compounds that turn from liquid to gas under room temperature conditions.
  • the volatile components may be, for example, volatile organic compounds.
  • the volatile components may also be referred to as volatile components.
  • the volatile components refer to, for example, components that can be detected by gas chromatography (GC) described below after performing solid phase microextraction (SPME) treatment described below at 45°C for 1 hour.
  • volatile organic compounds refers to organic compounds that have the property of volatilization.
  • the volatile organic compounds may have a boiling point range of, for example, less than 0°C, between 0°C and less than 50°C, or greater than 50°C.
  • the volatile organic compounds include aromatic compounds, alcohols, terpenes, aldehydes, carboxylic acids, amines, lactones, fatty acids, and phthalate esters.
  • semiconductor sensor refers to, for example, a sensor that adsorbs a gas component to a metal oxide and detects and/or measures a change in resistance value.
  • the semiconductor sensor can measure the presence or absence or concentration of a desired gas component, for example, the volatile component or the volatile organic compound, by, for example, adsorbing the volatile component to a metal oxide and detecting and/or measuring a change in resistance value.
  • the semiconductor sensor can also be, for example, an odor sensor, an odor meter, etc.
  • the semiconductor sensor may detect and/or measure the volatile component by disposing a sensitive film that is sensitive to a specific volatile component on its surface.
  • the semiconductor sensor may be, for example, a piezoelectric or surface acoustic wave (SAW) device.
  • the volatile component can be detected and/or measured by disposing a sensitive film that is sensitive to a specific volatile component on the piezoelectric or SAW device.
  • the semiconductor sensor may also be an array. In this case, the semiconductor sensor may be configured to simultaneously detect and/or measure multiple volatile components, for example, by arranging multiple sensitive films sensitive to different volatile components in an array.
  • adsorbent means a substance that adsorbs gas components.
  • kit generally refers to a unit in which the components to be provided (e.g., test reagents, diagnostic reagents, test reagents, labels, substrates, instructions, etc.) are provided in two or more separate compartments.
  • the kit can be suitably used, for example, for providing a composition that is not provided mixed, but is preferably mixed immediately before use, for reasons of stability, etc.
  • the kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., test reagents, diagnostic reagents, test reagents, etc.), or instructions or instructions describing the processing of the components.
  • the kit when the kit is used as a reagent kit, the kit may include instructions, etc. describing how to use the test reagents, diagnostic reagents, etc.
  • the instructions include, for example, instructions regarding the measurement method, test method, or use of the kit of the present disclosure.
  • the instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor, and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, the European Medicines Agency (EMA), etc.), and may clearly state that they have been approved by the regulatory agency.
  • the instructions may be a so-called package insert, and are usually provided in paper form, but are not limited to this, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).
  • a "binding molecule” is a molecule capable of binding to a specific molecule.
  • the binding molecule include a nucleic acid molecule, a protein, a glycan, and the like capable of binding to the specific molecule.
  • Specific examples of the binding molecule include an aptamer, an antibody, a receptor, a ligand, and the like capable of binding to the specific molecule.
  • the binding molecule may be, for example, a known binding molecule capable of binding to the specific molecule, or a binding molecule newly prepared by the SELEX method, phage display method, or the like.
  • the binding molecule may be, for example, an aptamer, an antibody, a receptor, or a ligand capable of binding to a sarcopenia marker.
  • antibody refers to a protein comprising one or more polypeptides substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene.
  • examples of the antibody include polyclonal antibodies and monoclonal antibodies.
  • examples of the isotype of the antibody include IgG (e.g., IgG1, IgG2, IgG3, IgG4, etc.), IgM, IgA (e.g., IgA1, IgA2, etc.), IgE, IgD, IgY, etc.
  • the antibody may be, for example, a camelid-derived heavy chain antibody (VHH antibody), a cartilaginous fish-derived immunoglobulin new antigen receptor (IgNAR), an antibody fragment (e.g., Fab, Fab', F(ab') 2 , single domain antibody (nanobody), etc.), a recombinant antibody (e.g., scFv, disulfide-linked Fv (dsFv), diabody, minibody, etc.).
  • the antibody may be an antibody-like molecule (e.g., affibody, anticalin, DARPins, monobody, etc.) produced by molecular biology techniques such as phage display and/or by protein engineering techniques using existing protein motifs.
  • gene refers to a factor that determines a genetic trait, and “gene” can refer to "polynucleotide,” “oligonucleotide,” and “nucleic acid.”
  • protein refers to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids.
  • the polypeptide is, for example, a peptide having a length of 10 amino acids or more.
  • nucleic acid refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides.
  • the nucleic acid may be a single-stranded or double-stranded nucleic acid molecule.
  • the polynucleotide may be composed of natural nucleotides, modified or artificial nucleotides, or both.
  • label refers to a label for distinguishing a target molecule or substance from other molecules or substances.
  • fluorescent labels such as fluorescent dyes or fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine), chemiluminescent labels such as luciferin and aequorin, luminescent substances such as acridinium derivatives, enzyme labels such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase ( ⁇ -gal), glucose oxidase, and luciferase, radioisotope (RI) labels such as 3H , 14C , 32P , 35S , and 125I , and metal nanoparticles (e.g., gold nanoparticles).
  • fluorescent labels such as fluorescent dyes or fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine), chemiluminescent labels such as luci
  • substrate refers to a member having a flat surface onto which a specific molecule can be fixed.
  • the specific molecule can be, for example, a binding molecule.
  • the specific molecule can be, for example, a sarcopenia marker.
  • immunoassay reagent refers to a reagent for use in an immunoassay.
  • the immunoassay reagent include a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blot method, immunoblot method, latex agglutination method, and immunochromatography method.
  • nucleic acid sequence of the proteins described herein or the nucleic acids is available from Protein Data Bank, UniProt, GenBank, etc.
  • nucleic acid sequence of the RNA can also be obtained from the corresponding DNA base sequence using appropriate sequence conversion software, etc.
  • the present disclosure provides a sarcopenia marker.
  • the sarcopenia marker of the present disclosure includes a volatile component of a food-derived component, a metabolic product of a gut bacteria-derived component, or an analog thereof.
  • the present inventors have found that the amount of certain volatile components is increased in biological samples from sarcopenia patients.
  • the present inventors have found that the onset of sarcopenia correlates with the volatile components of food-derived components, metabolic products of intestinal bacteria-derived components, or analogs thereof in a subject's sample, and have established the present disclosure. Therefore, according to the present disclosure, the possibility of sarcopenia (risk of sarcopenia) can be evaluated, examined, or tested by measuring the volatile components in a subject's sample.
  • candidate therapeutic substances for sarcopenia can be screened by using the volatile components as indicators in screening candidate therapeutic substances for sarcopenia using a sarcopenia model.
  • the volatile components include, for example, volatile organic compounds such as aromatic compounds, alcohols, terpenes, aldehydes, carboxylic acids, amines, lactones, fatty acids, and phthalate esters.
  • the aromatic compound is, for example, a cyclic unsaturated organic compound, specific examples of which include p-xylene, caffeine, and nicotine.
  • the alcohol is, for example, a compound in which the hydrogen atom of a hydrocarbon is replaced with a hydroxyl group, and specific examples include 1-butanol, texanol, n-propanol, lauryl alcohol (dodecanol), stearyl alcohol (octadecanol), oleyl alcohol (9-octadecen-1-ol), myristyl alcohol (tetradecanol), cetyl alcohol (hexadecanol), etc.
  • the terpene is, for example, a hydrocarbon whose constituent unit is isoprene, and specific examples include limonene (e.g., d-limonene), linalool, ⁇ -pinene, myrcene, geraniol, ⁇ -phellandrene, ⁇ -terpineol, borneol, etc.
  • limonene e.g., d-limonene
  • linalool e.g., d-limonene
  • ⁇ -pinene e.g., linalool
  • myrcene geraniol
  • ⁇ -phellandrene ⁇ -terpineol
  • borneol etc.
  • the aldehyde is, for example, an organic compound having a structure in which one hydrogen atom is substituted for the carbonyl carbon, and specific examples include nonanal, octanal, decanal, and dodecanal.
  • the carboxylic acid is, for example, an organic acid having at least one carboxy group, and a specific example is acetic acid.
  • the amine is, for example, a compound in which the hydrogen atom of ammonia is replaced with a hydrocarbon group or an aromatic atomic group, and specific examples include pyrrole, dopamine, serotonin, adrenaline, histamine, tyramine, and phenethylamine.
  • the lactone is, for example, a cyclic ester produced by dehydration condensation of a hydroxyl group and a carboxyl group in a molecule.
  • Specific examples include ⁇ -butyrolactone, ⁇ -decalactone (4-decanolide), ⁇ -decalactone (5-decanolide), ⁇ -dodecalactone (4-dodecanolide), ⁇ -octalactone (4-octanolide), cyclopentadecanolide, and mevalonolactone.
  • the fatty acid is, for example, a monovalent carboxylic acid having a carboxy group on the hydrocarbon chain, specific examples of which include octanoic acid and nonanoic acid.
  • the phthalate ester is, for example, an ester of phthalic acid and alcohol, and a specific example is diisobutyl phthalate.
  • the sarcopenia marker may be used, for example, one type alone, or multiple types may be used in combination. In the latter case, the combination of the sarcopenia markers is, for example, a combination of octanoic acid and nonanoic acid.
  • the sarcopenia marker is preferably a combination of nonanol, octanoic acid, and nonanoic acid, for example, because it has high sensitivity and specificity for sarcopenia in men.
  • the sarcopenia marker is preferably a combination of gamma-butyrolactone, octanoic acid, and nonanoic acid, for example, because it has high sensitivity and specificity for sarcopenia in women.
  • the sarcopenia marker is preferably a combination of p-xylene, 1-butanol, limonene, nonanal, acetic acid, octanoic acid, nonanoic acid, and diisobutyl phthalate, or a combination of p-xylene, 1-butanol, limonene, nonanal, pyrrole, ⁇ -butyrolactone, texanol, and octanoic acid, because it has high sensitivity and specificity for sarcopenia and can be easily measured.
  • the sarcopenia marker is preferably a combination of p-xylene, limonene, nonanal, octanoic acid, nonanoic acid, and diisobutyl phthalate, because it has high sensitivity and specificity for sarcopenia and can be easily measured.
  • the sarcopenia marker disclosed herein is also contained in biological samples that can be collected non-invasively, such as urine. Therefore, the sarcopenia marker disclosed herein makes it possible to carry out non-invasive sarcopenia testing. Therefore, the sarcopenia marker disclosed herein can reduce the physical burden on a subject (e.g., elderly people) and enable early detection of frailty, for example.
  • the sarcopenia markers disclosed herein can be used, for example, as markers for diagnosing, detecting, or testing for sarcopenia, markers for predicting the prognosis of sarcopenia, and markers for predicting or determining the effectiveness of treatment for sarcopenia, as described below.
  • kits for use in measuring a sarcopenia marker of the present disclosure
  • the sarcopenia marker includes a food-derived component, a metabolic product of an intestinal bacteria-derived component, or a volatile component of an analog thereof.
  • the kit of the present disclosure can suitably measure, for example, a sarcopenia marker in the sample.
  • the kit of the present disclosure means, for example, a sarcopenia detection kit, a sarcopenia determination kit, a sarcopenia screening kit, a sarcopenia prevention effect determination kit, a sarcopenia treatment effect determination kit, a determination kit for sarcopenia patients for whom a therapeutic drug is effective, a determination kit for a therapeutic drug that is effective for an individual sarcopenia patient, a test kit for diagnosing sarcopenia, or a test kit for treating sarcopenia.
  • the "determination of sarcopenia” may be, for example, a determination, test, detection, or diagnosis of the presence or absence of sarcopenia, a determination, test, detection, or diagnosis of the possibility (risk) of sarcopenia, a prediction of the prognosis after sarcopenia treatment, or a determination of the therapeutic effect of a sarcopenia treatment drug, and may be interpreted as any of the above.
  • the measurement reagent for the sarcopenia marker is a reagent capable of specifically detecting or measuring the sarcopenia marker, and includes, for example, a first binding molecule for the sarcopenia marker.
  • the first binding molecule is preferably an antibody or an antigen-binding fragment thereof for the sarcopenia marker.
  • the sarcopenia marker to be measured may be of one type or of multiple types.
  • the first binding molecule includes, for example, a first binding molecule that specifically binds to each sarcopenia marker.
  • the antibody for the sarcopenia marker can be produced by appropriately adopting and improving a conventionally known method.
  • the antibody for the sarcopenia marker is, for example, a monoclonal antibody
  • it can be produced by a method for producing a monoclonal antibody by a hybridoma (Kohler & Milstein, Nature, 256:495, 1975).
  • the sarcopenia marker is a low molecular weight compound
  • the antibody against the sarcopenia marker may be, for example, a VHH antibody, or may be obtained by a method for producing monoclonal antibodies using rabbits (Weber J et.al, "From rabbit antibody repertoires to rabbit monoclonal antibodies," Exp Mol Med.
  • the first binding molecule may be a nucleic acid molecule.
  • the nucleic acid molecule that binds to the sarcopenia marker can be prepared, for example, by the SELEX method.
  • the measurement reagent for the sarcopenia marker includes, for example, a sarcopenia marker.
  • the amount of the sarcopenia marker is, for example, a known amount.
  • the sarcopenia marker may be immobilized on a substrate. That is, the measurement reagent for the sarcopenia marker may include a substrate on which the sarcopenia marker is immobilized. Examples of the substrate include substrates such as glass, plastic, and metal; plates such as multi-well plates; and the like.
  • the sarcopenia marker may be configured to be immobilized on the substrate when the sarcopenia marker is measured.
  • the measurement reagents for the sarcopenia marker may be configured to exist in a separate state and to coexist during measurement, so that the sarcopenia marker is immobilized on the substrate.
  • the kit of the present disclosure may, for example, bind a first component of a pair of affinity substances to a sarcopenia marker, bind a second component of the pair of affinity substances to the substrate, and immobilize the sarcopenia marker on the substrate via affinity binding between the first component and the second component.
  • the pair of affinity substances includes the first component and the second component, and the first component and the second component are a combination of substances that exhibit specific binding to each other, and may also be referred to as, for example, a tag and a binding partner.
  • the method of immobilizing the sarcopenia marker on the substrate can be appropriately selected from conventionally known methods or methods similar thereto depending on the type of the sarcopenia marker, and the sarcopenia marker may be immobilized directly or indirectly on the surface of the substrate.
  • the direct binding method includes, for example, a method in which an active group is added to the substrate, and the resulting substrate or a substrate having an active group is used, and the sarcopenia marker is bound to the substrate by a covalent bond generated by a reaction between the active group and the sarcopenia marker.
  • the indirect binding method includes, for example, a method using a pair of affinity substances.
  • the pair of affinity substances includes, for example, a combination of biotin and avidin or streptavidin, a combination of nickel and a His tag, a combination of an epitope tag such as flag (trademark)-tag, HA-tag, T7-tag, V5-peptide-tag, and/or Myc-tag, and an antibody against the tag, etc.
  • the sarcopenia marker may be immobilized on the substrate by ionic bonding with the substrate or by being adsorbed to the substrate. When the sarcopenia marker is indirectly immobilized on the substrate, for example, the sarcopenia marker and the substrate may be contained in the same container or in separate containers.
  • the measurement reagent for the sarcopenia marker includes a substrate on which the sarcopenia marker is immobilized
  • the measurement reagent for the sarcopenia marker further includes the sarcopenia marker (reference sarcopenia marker).
  • the measurement reagent for the sarcopenia marker can measure the sarcopenia marker in the sample, for example, by making the sarcopenia in the sample compete with the immobilized sarcopenia marker for a binding molecule for the sarcopenia marker, i.e., by a competitive method.
  • the first binding molecule for the sarcopenia marker may be immobilized on a substrate, for example. That is, the sarcopenia marker measurement reagent may include, for example, a substrate on which the first binding molecule for the sarcopenia marker is immobilized.
  • the method for immobilizing the first binding molecule for the sarcopenia marker on a substrate can be described with reference to the method for immobilizing the sarcopenia marker on a substrate.
  • the sarcopenia marker measurement reagent includes a substrate on which the first binding molecule is immobilized
  • the sarcopenia marker measurement reagent further includes a sarcopenia marker (reference sarcopenia marker) containing a label.
  • the sarcopenia marker measurement reagent can measure the sarcopenia marker in the sample, for example, by making the sarcopenia in the sample compete with the sarcopenia marker having the label for the immobilized first binding molecule, i.e., by a competitive method.
  • the measurement reagent for the sarcopenia marker may contain, for example, a second binding molecule for the sarcopenia marker or a complex of the sarcopenia marker and the first binding molecule.
  • the first binding molecule and the second binding molecule bind to different sites of the sarcopenia marker, for example, that is, the two binding molecules can simultaneously bind to the sarcopenia marker.
  • the second binding molecule may contain a second binding molecule that co-specifically binds to each sarcopenia marker.
  • the sarcopenia marker, the first binding molecule, and/or the second binding molecule may have a label.
  • the label can be detected to detect the sarcopenia marker via the sarcopenia marker, the first binding molecule, and/or the second binding molecule.
  • the measurement reagent for the sarcopenia marker contains the first binding molecule and the second binding molecule, it is preferable that one of the first binding molecule and the second binding molecule is immobilized on the substrate, and the other has a label. It is more preferable that the first binding molecule is immobilized on the substrate, for example, and the second binding molecule has a label.
  • the method for introducing a label into the sarcopenia marker, the first binding molecule, and/or the second binding molecule can be carried out by appropriately adopting a conventionally known method or a method similar thereto depending on the type of the first binding molecule and the second binding molecule, and the label may be directly or indirectly bound.
  • the method for binding the label can be, for example, the same method as the method for immobilizing the first binding molecule on the substrate.
  • the measurement reagent for the sarcopenia marker may be, for example, an immunological measurement reagent.
  • the immunological measurement reagent may be, for example, a reagent for use in at least one immunological measurement method selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blot method, immunoblot method, latex agglutination method, and immunochromatography method.
  • the kit of the present disclosure may have a substrate capable of reacting with the label.
  • the label is, for example, the enzyme label described above
  • the substrate is a substance capable of reacting with the enzyme label.
  • the substrate may be a solid or a liquid.
  • the substrate may also be referred to as a substrate liquid.
  • the first binding molecule and/or the second binding molecule contained in the kit of the present disclosure may be a solution (liquid or gel) dissolved or dispersed in a buffer solution or the like, or may be a solid form such as powder or granules, for example, by freeze-drying the liquid dissolved in the buffer solution.
  • the buffer solution examples include Tris buffer solutions such as Tris-HCl buffer solution, Tris-EDTA (TE) buffer solution, TAE buffer solution, TBE buffer solution, and Tris-buffered saline; phosphate buffer solutions such as phosphate-buffered saline; carbonate buffer solutions such as carbonate-sodium bicarbonate buffer solution; and GOOD buffer solutions such as MES, ADA, PIPES, TAPS, CAPS, ACES, cholamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycineamide, and bicine.
  • the pH of the solution is, for example, 4.0 to 9.5, preferably 5 to 9 or 5.5 to 8.5, and more preferably 6 to 8.
  • the pH value can be adjusted, for example, using the buffer solution, an acidic substance such as hydrochloric acid, or an alkaline substance such as sodium hydroxide.
  • the kit of the present disclosure may further include a sarcopenia marker preparation.
  • the sarcopenia marker preparation may be, for example, an aqueous solution containing one or more sarcopenia markers at a predetermined concentration, or a powder of the sarcopenia marker (e.g., a lyophilized product).
  • the sarcopenia marker preparation is useful, for example, as a control.
  • a calibration curve corresponding to the concentration of the sarcopenia marker can be created, and the concentration of the sarcopenia marker in a sample can be analyzed.
  • the sarcopenia marker may have a label.
  • the method for introducing a label into the sarcopenia marker may be the same as that described above for the method for introducing a label into the sarcopenia marker.
  • the kit of the present disclosure may include, for example, a diluent and/or a cleaning solution.
  • the diluent includes, for example, the buffer solution.
  • the cleaning solution includes, for example, the buffer solution, a non-ionic surfactant, etc.
  • the reference sarcopenia marker may be stored separately from the measurement reagent for the sarcopenia marker, or may be stored mixed with some or all of the measurement reagent for the sarcopenia marker.
  • each reagent or component may be in a solid form such as a powder or granules, or in a liquid form such as a slurry (suspension), jelly, or solution.
  • the kit of the present disclosure may further include, for example, a container for storing the components of the kit.
  • each component may be provided in a form in which it is contained in a different container (e.g., a tube, a plate, etc.).
  • each component may be contained separately, or some or all of the components may be contained in a mixed or unmixed state.
  • the kit of the present disclosure when all the reagents are contained in a single container in a mixed or unmixed state, the kit of the present disclosure can be said to be, for example, a reagent for use in measuring sarcopenia markers.
  • the kit of the present disclosure may include, for example, instructions or instructions.
  • the kit disclosed herein can be used to suitably carry out the second measurement method disclosed herein, which will be described later.
  • the present disclosure discloses a measurement method capable of measuring a sarcopenia marker.
  • the measurement method of the present disclosure (hereinafter also referred to as "first measurement method") for a sarcopenia marker includes a measurement step of measuring a food-derived component, a metabolic product of an intestinal bacteria-derived component, or a volatile component of an analog thereof in a subject sample as a sarcopenia marker.
  • the first measurement method of the present disclosure can suitably measure, for example, a sarcopenia marker in the sample.
  • the sarcopenia marker to be measured in the measurement process is a volatile component. Therefore, the measurement process can be performed, for example, by volatilizing the sarcopenia marker in the sample and measuring the volatilized component. Therefore, in the first measurement method of the present disclosure, the measurement process may include, for example, a volatilization process for volatilizing volatile components from the sample, and a component measurement process for measuring the obtained volatile components. Furthermore, in the measurement process, the sarcopenia marker to be measured may be of one type or multiple types.
  • the processing conditions for volatilization process, for example, temperature, humidity, time
  • the processing conditions are not particularly limited and can be set appropriately depending on, for example, the type of the volatile components and their vaporization temperature, as long as the conditions are such that the volatile components can be volatilized.
  • the temperature of the volatilization process is, for example, 4 to 60°C, 20 to 50°C, or 35 to 50°C.
  • the humidity of the volatilization process is, for example, 5 to 100%, 10 to 70%, 15 to 60%, or 20 to 50%.
  • the time of the volatilization is, for example, 1 minute to 12 hours, 3 minutes to 120 minutes, or 5 to 60 minutes.
  • the processing conditions are preferably such that the temperature of the volatilization process is 45°C, the time of the volatilization is 60 minutes, and the humidity of the volatilization process is 100%.
  • the volatile components volatilized in the volatilization step may be directly subjected to the component measurement step described below, or the volatile components may be recovered and then subjected to the component measurement step.
  • the first measurement method of the present disclosure may include an adsorption step of adsorbing the volatile components to an adsorbent.
  • the adsorption step may be performed after the volatilization step, may be performed simultaneously with the volatilization step, or may be performed overlapping with part or all of the volatilization step.
  • the adsorption of the volatile components can be carried out, for example, by contacting the gas (gas phase) containing the volatile components volatilized in the volatilization step with a carrier capable of retaining the volatile components, such as the adsorbent.
  • the carrier such as the adsorbent, can be appropriately selected from conventionally known carriers by a person skilled in the art, for example, depending on the properties of the gas components to be adsorbed.
  • the adsorbent may be, for example, an adsorbent that nonspecifically adsorbs components in the gas (gas phase), or an adsorbent that specifically adsorbs a desired component.
  • the type and thickness of the adsorbent can be selected, for example, depending on the molecular weight, polarity, concentration, etc. of the compound to be adsorbed.
  • the adsorbent may be one that adsorbs gas components to the surface of the adsorbent, or one that absorbs gas components into the inside of the adsorbent.
  • the adsorbent include divinylbenzene, carboxene, polydimethylsiloxane, polyacrylate, and polyethylene glycol.
  • the adsorbent can be divinylbenzene, carboxene, or polydimethylsiloxane (Fujita A. et al.
  • the thickness of the adsorbent is, for example, 5 to 300 ⁇ m, 10 to 250 ⁇ m, 20 to 200 ⁇ m, or 30 to 100 ⁇ m.
  • the processing conditions for adsorbing the volatile components to the adsorbent are not particularly limited and can be set according to the type of volatile components and adsorbent, as long as the conditions are capable of adsorbing the volatile components.
  • the temperature of the adsorption process is, for example, 4 to 60°C, 20 to 50°C, or 37 to 45°C.
  • the humidity of the adsorption process is, for example, 10 to 70%, 15 to 60%, or 20 to 50%.
  • the time of the adsorption process is, for example, 1 minute to 12 hours, 3 minutes to 120 minutes, or 5 to 60 minutes.
  • the adsorption step may be performed by solid-phase microextraction (SPME).
  • SPME solid-phase microextraction
  • a fiber coated with the adsorbent when exposed to a sample such as a gas, the volatile components in the sample are adsorbed by the adsorbent and extracted.
  • the fiber containing the extracted volatile components can be subjected to, for example, a heat treatment to dissociate the volatile components from the adsorbent, so that the volatile components can be subjected to gas chromatography or the like.
  • the volatile components subjected to the measurement may be, for example, the volatile components volatilized from the sample in the volatilization process, or the volatile components adsorbed onto a carrier such as the adsorbent in the adsorption process, or the volatile components extracted by the SPME.
  • the volatile components can be measured using a conventionally known method for measuring gas components.
  • the measurement method include gas chromatography (GC) and a measurement method using a semiconductor sensor.
  • the gas chromatography is preferably gas chromatography mass spectrometry (GC/MS) because it can perform qualitative and quantitative analysis of the volatile components contained in the sample.
  • the first measurement method of the present disclosure can easily measure the volatile components using a small measurement device by using the measurement method using a semiconductor sensor in the component measurement step, for example.
  • the measurement conditions e.g., temperature, column type, carrier gas type, carrier gas flow rate
  • the measurement conditions are not particularly limited and may be set appropriately depending on the type of the volatile component, and may be conditions that allow the volatile component to be analyzed.
  • the measurement conditions e.g., temperature, column type, carrier gas type, carrier gas flow rate, ion source temperature, ionization energy, scan frequency
  • the measurement conditions for the GC and GC/MS can refer to, for example, the analysis conditions for volatile components in urine (Fujita A. et al. "Urinary volatilome analysis in a mouse model of anxiety and depression.”, PLOS ONE 15:e0229269, 2020.).
  • the type of metal oxide used in the semiconductor sensor and the measurement conditions are not particularly limited and can be set appropriately according to the type of volatile component, as long as the conditions are such that the volatile component can be measured.
  • the metal oxide include metal oxides such as tin oxide and zinc oxide.
  • the semiconductor sensor may detect and/or measure the volatile component by disposing a sensitive film that is sensitive to a specific volatile component on its surface.
  • the semiconductor sensor can be given selectivity for different volatile components by, for example, changing the sensitive film.
  • the semiconductor sensor can be manufactured, for example, by a conventionally known semiconductor manufacturing process.
  • the first measurement method of the present disclosure can measure the volatile components as sarcopenia markers.
  • a sample that has been collected and stored may be used as the sample.
  • the storage may be, for example, frozen storage.
  • the sample can be stored and used for measurement as follows.
  • a storage container such as a glass vial in which the sample is stored is subjected to a heat treatment to remove volatile components in the storage container.
  • the heat treatment is, for example, at 120 to 240°C for 1 to 5 hours.
  • the sample is stored in the storage container after the heat treatment, and then frozen and stored.
  • the freezing and storage can be performed using, for example, liquid nitrogen.
  • the sample being stored is used, in the first measurement method of the present disclosure, for example, the sample being stored is melted, and then impurities are removed by solid-liquid separation such as centrifugation.
  • the obtained liquid fraction can be used as the sample after storage.
  • the sample may be pretreated prior to the measurement step.
  • the pretreatment may be, for example, modification of the sarcopenia marker in the sample.
  • the modification may be, for example, chemical modification of the sarcopenia marker in the sample.
  • the chemical modification may be, for example, methylation.
  • the measurement step measures, for example, a chemically modified sarcopenia marker, such as methylated.
  • the methylation may be appropriately performed by a conventionally known method using, for example, a methylation reagent (for example, TMS (tetramethylsilane)-CNH 2 ).
  • the first measurement method of the present disclosure may, for example, optionally analyze the amount of the sarcopenia marker in the measurement step.
  • a correlation between the amount of the sarcopenia marker and a measurement value obtained by measuring the sarcopenia marker may be determined in advance, and the amount of the sarcopenia marker in the sample may be analyzed from the measurement value based on the correlation.
  • the first measurement method of the present disclosure may also be referred to as, for example, an analysis method.
  • the correlation may be determined, for example, by adding an internal standard substance to the sample in advance and performing a measurement at the same time.
  • the internal standard substance may, for example, be 1-bromo-4-fluorobenzene, olefin, paraffin, etc.
  • the sarcopenia marker can be suitably measured in biological samples such as urine samples and blood samples. Therefore, the first measurement method of the present disclosure can be suitably used, for example, as a method for detecting or testing the sarcopenia marker in the biological sample.
  • the present disclosure discloses a measurement method capable of measuring a sarcopenia marker.
  • the measurement method of the sarcopenia marker of the present disclosure includes a measurement step of contacting a sample of a subject with a measurement reagent for a sarcopenia marker to measure the sarcopenia marker in the sample, the sarcopenia marker comprising a food-derived component, a metabolic product of a component derived from an intestinal bacteria, or a volatile component analogous thereto.
  • the sample of a subject is contacted with a measurement reagent for a sarcopenia marker to measure the sarcopenia marker in the sample, so that, for example, the sarcopenia marker in the sample can be suitably measured without volatilizing the volatile component. Therefore, the second measurement method of the present disclosure can easily measure, for example, the early volatile component.
  • the subject sample is contacted with a measurement reagent for a sarcopenia marker to measure the sarcopenia marker in the sample.
  • a measurement reagent for a sarcopenia marker to measure the sarcopenia marker in the sample for example, the presence or absence of a sarcopenia marker in the sample can be qualitatively measured by measuring the presence or absence of a sarcopenia marker detected by the measurement reagent.
  • the amount of a sarcopenia marker in the sample can be quantitatively measured by measuring the amount of a sarcopenia marker detected by the measurement reagent.
  • the sarcopenia marker to be measured may be one or more.
  • the measurement of the sarcopenia marker can be performed, for example, using a binding molecule for the sarcopenia marker.
  • the measurement of the sarcopenia marker may be performed by an immunological method.
  • the immunological method include direct competitive ELISA, indirect competitive ELISA, sandwich ELISA, direct competitive immunoassay, indirect competitive immunoassay, sandwich immunoassay, immunochromatography, spin immunoassay, and latex agglutination.
  • the measurement reagent for the sarcopenia marker is a reagent containing a label
  • examples of the immunological method include fluorescent immunoassay (FIA), enzyme immunoassay (EIA), chemiluminescent immunoassay, chemiluminescent enzyme immunoassay, and radioimmunoassay (RIA), depending on the type of label.
  • the measurement step includes, for example, a complex formation step of contacting the sample, a first binding molecule for the sarcopenia marker, and a reference sarcopenia marker to form a first complex between the sarcopenia marker in the sample and the first binding molecule, and a second complex between the reference sarcopenia marker and the first binding molecule, and a complex measurement step of measuring the sarcopenia marker in the sample by measuring the second complex.
  • the contact can be performed, for example, by mixing the subject sample, the first binding molecule for the sarcopenia marker, and the reference sarcopenia marker.
  • the contact is preferably performed in a liquid system containing water, physiological saline, the buffer solution, or the like.
  • the contact conditions e.g., temperature, time, pH
  • the temperature of the contact is, for example, 4 to 42°C, or 18 to 40°C.
  • the contact time is, for example, 1 minute to 12 hours, 3 minutes to 120 minutes, or 5 to 60 minutes.
  • the pH during the complex formation is, for example, 4 to 9.5, preferably 5 to 9 or 5.5 to 8.5, and more preferably 6 to 8.
  • the reference sarcopenia marker In the complex formation process, the reference sarcopenia marker must be present when the sample and the first binding molecule are brought into contact, and the order of contact is not particularly limited.
  • the reference sarcopenia marker and the sample may be brought into contact with the first binding molecule, and then the reference sarcopenia marker and the sample may be brought into contact with the first binding molecule simultaneously.
  • the sample in the complex formation process, may be diluted with a diluent containing the reference sarcopenia marker, thereby allowing the sample and the reference sarcopenia marker to coexist, and then the sample may be brought into contact with the first binding molecule.
  • the volume of the diluent used to dilute the sample can be appropriately determined depending on the volume of the sample and the purpose of using the sample (e.g., qualitative measurement or quantitative measurement, etc.), and can be set to a volume larger than the volume of the sample, for example.
  • the diluent includes, for example, a buffer solution.
  • the complex measurement process is, for example, a process of measuring a second complex between the reference sarcopenia marker and the first binding molecule, i.e., a process of measuring the binding between the reference sarcopenia marker and the first binding molecule.
  • a process of measuring the binding between the reference sarcopenia marker and the first binding molecule by detecting the presence or absence of binding between the two, for example, the presence or absence of a sarcopenia marker in the sample can be analyzed (qualitatively determined), and by detecting the degree of binding between the two (amount of binding), for example, the amount of the sarcopenia marker in the sample can be analyzed (quantitatively determined).
  • the method for measuring the binding between the reference sarcopenia marker and the first binding molecule is not particularly limited, and for example, a conventionally known method for measuring the binding between substances can be adopted, specifically, SPR, fluorescence polarization, etc. can be used. Furthermore, when the reference sarcopenia marker or the first binding molecule has the label, the binding between the reference sarcopenia marker and the first binding molecule may be measured in the complex measurement step by directly or indirectly detecting the label in the second complex. The detection of the label can be appropriately determined depending on, for example, the type of the label.
  • the measurement step includes, for example, a first complex formation step of contacting the sample with a first binding molecule for the sarcopenia marker to form a first complex between the sarcopenia marker in the sample and the first binding molecule, a second complex formation step of contacting the first complex with a second binding molecule for the sarcopenia marker to form a second complex between the first complex and the second binding molecule, and a complex measurement step of measuring the sarcopenia marker in the sample by measuring the second complex.
  • the first binding molecule is immobilized on the substrate before, during, or after the formation of the first complex.
  • the second measurement method of the present disclosure may immobilize the first binding molecule on the substrate, for example, in the second complex formation step described below.
  • the first complex formation step can be carried out in the same manner as the complex formation step in the competitive method.
  • it is preferable to separate the first complex and optionally, it is more preferable to further wash the separated first complex with a washing solution.
  • the separation of the first complex can be carried out, for example, by solid-liquid separation.
  • the separation of the first complex can be carried out, for example, by separating a solid fraction containing the substrate from a liquid fraction.
  • the first complex is contacted with the second binding molecule to form the second complex.
  • the contact between the first complex and the second binding molecule is preferably carried out in a liquid system containing, for example, water, physiological saline, the buffer solution, or the like.
  • the contact conditions between the first complex and the second binding molecule are not particularly limited as long as the second complex can be formed.
  • Specific examples of the contact temperature are, for example, 4 to 42°C, or 18 to 40°C.
  • the contact time is, for example, 1 minute to 12 hours, 3 minutes to 120 minutes, or 5 minutes to 60 minutes.
  • the measurement step since the measurement accuracy of the sarcopenia marker can be improved after the second complex formation step, it is preferable to separate the second complex, and optionally, it is more preferable to further wash the separated second complex with a washing solution.
  • the separation of the second complex can be performed, for example, in the same manner as the separation of the first complex.
  • the complex measurement step is, for example, a step of measuring a second complex of the first complex and the second binding molecule, that is, a step of measuring the binding between the first complex and the second binding molecule.
  • the complex measurement step by detecting the presence or absence of the binding between the two, for example, the presence or absence of a sarcopenia marker in the sample can be analyzed (qualitative), and by detecting the degree of binding between the two (amount of binding), for example, the amount of the sarcopenia marker in the sample can be analyzed (quantitative).
  • the method of measuring the binding between the first complex and the second binding molecule can be carried out, for example, in the same manner as the method of measuring the binding between the sarcopenia marker and the first binding molecule.
  • the label in the second complex may be directly or indirectly detected to measure the binding between the first complex and the second binding molecule.
  • the detection of the label can be appropriately determined, for example, depending on the type of the label.
  • the first complex formation step and the second complex formation step may be carried out simultaneously.
  • the first and second complex formation steps are carried out simultaneously, they can be carried out in the same manner as the complex formation step.
  • the second measurement method of the present disclosure can measure the sarcopenia marker.
  • the second measurement method of the present disclosure may, for example, optionally analyze the amount of the sarcopenia marker in the measurement step.
  • a correlation between the amount of the sarcopenia marker present and a measurement value obtained by measuring the sarcopenia marker may be determined in advance, and the amount of the sarcopenia marker in the sample may be analyzed from the measurement value based on the correlation.
  • the second measurement method of the present disclosure may also be referred to as, for example, an analysis method.
  • the sarcopenia marker can also be suitably measured in biological samples such as urine samples and blood samples. Therefore, the second measurement method of the present disclosure can be suitably used, for example, as a method for detecting or testing the sarcopenia marker in the biological sample.
  • the present disclosure provides a method for testing the risk of developing sarcopenia, comprising a step of measuring a sarcopenia marker in a sample from a subject, the step being carried out by the first or second method for measuring a sarcopenia marker of the present disclosure.
  • the testing method of the present disclosure by measuring a sarcopenia marker in a sample from the subject, the risk of developing sarcopenia in the subject can be evaluated, or the possibility of developing sarcopenia can be tested.
  • the test method disclosed herein means, for example, a method for detecting sarcopenia, a method for determining sarcopenia, a method for screening sarcopenia, a method for determining the preventive effect of sarcopenia, a method for determining the therapeutic effect of sarcopenia, a method for determining which patients with sarcopenia will respond to a therapeutic drug, a method for determining which therapeutic drug will be effective for an individual patient with sarcopenia, a test method for diagnosing sarcopenia, or a test method for treating sarcopenia.
  • the "determination of sarcopenia” may be, for example, a determination, test, detection, or diagnosis of the presence or absence of sarcopenia, a determination, test, detection, or diagnosis of the possibility (risk) of developing sarcopenia, a prediction of the prognosis after treatment for sarcopenia, or a determination of the therapeutic effect of a therapeutic drug for sarcopenia, and can be interpreted as any of the above.
  • the measurement step can be explained using the description of the measurement step in the first or second measurement method of the present disclosure.
  • the testing method of the present disclosure may include a testing (evaluation) step of testing (evaluating) the possibility of the subject having sarcopenia based on the measurement value of the sarcopenia marker.
  • the possibility of the subject having sarcopenia may be evaluated by statistically evaluating the measurement value of the sarcopenia marker, or may be evaluated by comparing the measurement value of the sarcopenia marker with a threshold value.
  • the test step can, for example, evaluate the measured values of the sarcopenia marker by combining them with the measured values of a reference sample described below, through regression analysis (e.g., simple regression analysis).
  • the test step can, for example, evaluate the measured values of the sarcopenia marker by combining them with the measured values of a reference sample described below, through discriminant analysis.
  • the test method of the present disclosure calculates a discriminant function from the distribution of the measured values of the reference sample.
  • test method of the present disclosure can, for example, evaluate the risk of sarcopenia in a subject, or test (assess) the possibility of sarcopenia, based on the measured values of the sarcopenia marker and the discriminant function.
  • the test method of the present disclosure may include, for example, a test (evaluation) step of testing (evaluating) the possibility of the subject having sarcopenia by comparing the measured value of the sarcopenia marker with a threshold value.
  • the threshold value is not particularly limited, and may be, for example, a threshold value calculated from the measured value of the sarcopenia marker in samples from healthy subjects, patients with sarcopenia, or the healthy subjects and the patients with sarcopenia.
  • the threshold value may be, for example, the measured value of the sarcopenia marker before or after treatment (for example, immediately after treatment) of the same subject.
  • the measured value of the sarcopenia marker may be, for example, a non-standardized coefficient (sarcopenia index (SI)) calculated by linear regression analysis (multiple regression analysis) using software (for example, SPSS (IBM's statistical analysis software product line), etc.) from the measured values of the multiple sarcopenia markers.
  • SI sarcopenia index
  • SPSS multiple regression analysis
  • the threshold value can be set, for example, by using the SI as the measurement value of the sarcopenia marker.
  • the threshold value can be obtained, for example, using a sample (hereinafter also referred to as a "reference sample”) isolated from a healthy subject and/or a sarcopenia patient, as described above.
  • the threshold value can also be calculated statistically based on the sarcopenia marker obtained by isolating reference samples from a plurality of healthy subjects and a plurality of sarcopenia patients, measuring the sarcopenia marker, and then performing a statistical calculation based on the sarcopenia marker obtained.
  • the threshold value can be, for example, a clinical diagnostic value such as a diagnostic threshold value (cut-off value), a treatment threshold value, and a preventive medicine threshold value.
  • a reference sample isolated from the same subject after treatment can be used.
  • the threshold value can be measured, for example, simultaneously with the test sample of the subject, or can be measured in advance. In the latter case, it is preferable because it is not necessary to obtain a threshold value each time the test sample of the subject is measured. It is preferable that the test sample of the subject and the reference sample are collected under the same conditions, and the sarcopenia marker is measured under the same conditions.
  • the method for evaluating the possibility of the subject having sarcopenia is not particularly limited and can be determined appropriately depending on the type of threshold.
  • the test process can evaluate, for example, as follows.
  • the sarcopenia marker include aromatic compounds, terpenes, aldehydes, amines, lactones, fatty acids, etc.
  • the subject when the measurement value of the sarcopenia marker in the test sample of the subject is the same as the measurement value of the sarcopenia marker in the reference sample of the healthy subject (when there is no significant difference), is significantly lower than the measurement value of the sarcopenia marker in the reference sample of the healthy subject, is significantly lower than the measurement value of the sarcopenia marker in the reference sample of the sarcopenia patient, and/or is lower than a threshold value calculated from the measurement values of the sarcopenia marker in the biological samples of the healthy subject and the sarcopenia patient, the subject can be evaluated as having no or low possibility (also referred to as "risk” or "degree of risk", the same below) of suffering from sarcopenia.
  • the subject can be evaluated as having a high possibility (risk) of suffering from sarcopenia.
  • the test process can evaluate, for example, as follows.
  • the sarcopenia marker include alcohol, carboxylic acid, phthalate ester, etc.
  • the subject when the measurement value of the sarcopenia marker in the test sample of the subject is significantly lower than the measurement value of the sarcopenia marker in the reference sample of the healthy subject, when the measurement value is significantly lower than the measurement value of the sarcopenia marker in the reference sample of the sarcopenia patient, and/or when the measurement value is lower than a threshold value calculated from the measurement values of the sarcopenia marker in the biological samples of the healthy subject and the sarcopenia patient, the subject can be evaluated as having a possibility or a high possibility (also referred to as "risk” or "degree of risk", the same applies below) of suffering from sarcopenia.
  • a possibility or a high possibility also referred to as "risk” or "degree of risk"
  • the subject can be evaluated as having no or a low possibility (risk) of suffering from sarcopenia.
  • the degree of sarcopenia can be evaluated by comparing the measurement value of the sarcopenia marker in the test sample from the subject with the measurement value of the sarcopenia marker in a reference sample from patients with sarcopenia of each degree of sarcopenia. Specifically, if the measurement value of the sarcopenia marker in the test sample from the subject is similar to that of the reference sample for sarcopenia (if there is no significant difference), the subject can be evaluated as having a possibility or high possibility of having the degree of sarcopenia.
  • the evaluation when the prognosis is evaluated, for example, the evaluation may be performed in the same manner as described above, or the measurement value of the sarcopenia marker in a reference sample after treatment of the same subject may be used as the threshold value.
  • the sarcopenia marker is an increased marker
  • the measurement value of the sarcopenia marker in the subject's test sample is the same as the threshold value (if there is no significant difference) and/or is significantly lower than the threshold value, the subject can be evaluated as having no or low possibility of recurrence or worsening after the treatment.
  • the subject can be evaluated as having a possibility or high possibility of recurrence or worsening after the treatment.
  • the sarcopenia marker is a decreased marker
  • the measurement value of the sarcopenia marker in the subject's test sample is the same as the threshold value (if there is no significant difference) and/or is significantly higher than the threshold value
  • the subject can be evaluated as having no or low possibility of recurrence or worsening after the treatment.
  • the measured value of the sarcopenia marker in the test sample of the subject is significantly lower than the threshold value, the subject can be evaluated as having a possibility or high possibility of recurrence or worsening after the treatment.
  • the first test step biological samples from the same subject may be collected over time, and the measurement values of the sarcopenia marker in the biological samples may be compared. If the sarcopenia marker is an increasing marker, in the test step, for example, if the measurement value decreases over time, it is possible to determine that the possibility of disease has decreased or that the subject has been cured, and if the measurement value increases over time, it is possible to determine that the possibility of disease has increased.
  • the sarcopenia marker is a decreasing marker, in the test step, for example, if the measurement value increases over time, it is possible to determine that the possibility of disease has decreased or that the subject has been cured, and if the measurement value increases over time, it is possible to determine that the possibility of disease has increased.
  • the testing method of the present disclosure may, for example, include a step of implementing a dietary therapy and/or an exercise therapy for a subject who is evaluated as having a high possibility of suffering from sarcopenia based on the results of the testing process, and/or a step of administering a sarcopenia therapeutic drug to the subject.
  • Conditions such as the administration form, administration timing, dosage, and administration interval of the sarcopenia therapeutic drug can be set appropriately depending on the type of sarcopenia therapeutic drug.
  • the sarcopenia therapeutic drug may be a candidate therapeutic drug obtained by a screening method described below.
  • testing method of the present disclosure may, for example, determine whether or not to administer a sarcopenia therapeutic drug, whether or not to increase or decrease the dosage of the sarcopenia therapeutic drug relative to a reference value for the dosage of the sarcopenia therapeutic drug, and/or a follow-up protocol for the subject after administration of the sarcopenia therapeutic drug based on the evaluation process.
  • test method of the present disclosure may, for example, determine whether to implement dietary therapy and/or exercise therapy, whether to increase or decrease the amount of food and/or the amount of exercise relative to the reference value of the amount of food and/or the amount of exercise for a sarcopenia patient, and/or a follow-up protocol for the subject after administration of a sarcopenia therapeutic drug, based on the evaluation step.
  • the testing method disclosed herein makes it possible to easily test the possibility of the subject suffering from sarcopenia.
  • the present disclosure discloses a method for screening a candidate substance for a therapeutic agent against sarcopenia.
  • the screening method for a candidate substance for a therapeutic agent against sarcopenia of the present disclosure includes a selection step of selecting a substance that changes a sarcopenia marker from test substances as a candidate substance for a therapeutic agent against sarcopenia.
  • a candidate substance for a therapeutic agent against sarcopenia can be screened.
  • the test substance may be, for example, a low molecular weight compound, a peptide, a protein, or a nucleic acid.
  • the low molecular weight compound may be, for example, a library of known low molecular weight compounds.
  • the peptide may be, for example, a linear, branched, or cyclic peptide, and each amino acid constituting the peptide may be a natural amino acid, a modified amino acid, an artificial amino acid, or a combination thereof.
  • the protein may be either a natural protein or an artificial protein.
  • the protein may be, for example, an antibody, a growth factor, or a modified form thereof.
  • the selection process includes, for example, a measurement process of administering the test substance to a sarcopenia model to measure the sarcopenia marker, and a selection process of selecting the test substance as the candidate therapeutic agent if the sarcopenia marker obtained in the measurement process is lower than that of a control to which the test substance is not administered.
  • the selection process includes, for example, a measurement process of administering the test substance to a sarcopenia model to measure the sarcopenia marker, and a selection process of selecting the test substance as the candidate therapeutic agent if the sarcopenia marker obtained in the measurement process is higher than that of a control to which the test substance is not administered.
  • the measurement of the sarcopenia marker can be, for example, the description of the measurement method of the sarcopenia marker described above.
  • the sarcopenia model is, for example, a sarcopenia model in a non-human animal.
  • the sarcopenia model can be, for example, a mouse sarcopenia model.
  • the mouse sarcopenia model can be, for example, a conventionally known one that is appropriately used (Xie WQ et.al., "Mouse models of sarcopenia: classification and evaluation," J Cachexia Sarcopenia Muscle. 2021 Jun;12(3):538-554.; Shin J et.al., "Ageing and rejuvenation models reveal changes in key microbial communities associated with healthy ageing," Microbiome. 2021 Dec 15;9(1):240.).
  • the sarcopenia marker used in the selection process may be one or more.
  • the selection in the selection process may be, for example, a statistical evaluation, similar to the evaluation process in the test method of the present disclosure, and a test substance in which the measured value of the sarcopenia marker has decreased or increased may be selected as the candidate therapeutic agent.
  • the present disclosure discloses a method for detecting sarcopenia that can be used to detect the possibility of sarcopenia in a subject suspected of having sarcopenia.
  • the detection method disclosed herein is a method for detecting a sarcopenia marker in a subject suspected of having sarcopenia, comprising a detection step of detecting a sarcopenia marker in a sample from the subject using a measurement kit for a sarcopenia marker.
  • the subject suspected of having sarcopenia may be, for example, a person who the subject himself/herself subjectively suspects, or a person who has been determined as being suspected of having sarcopenia or possibly suffering from sarcopenia as a result of an examination by a doctor or the like.
  • subjects who are subjectively suspected of having sarcopenia include those who have some subjective symptoms, those who wish to undergo a preventive checkup, etc.
  • VOC markers were selected using the following method. 1. Obtaining specimens Urine samples used as specimens were obtained from subjects who participated in the "Otassha Health Check” (2015) and were detoxified. The "Otassha Health Check” is one of the studies conducted at the Tokyo Metropolitan Institute of Gerontology with the aim of maintaining the health and independence of the elderly. The aim of the "Otassha Health Check” is to maintain health and independence in the elderly, especially in the late elderly stage, and to develop a new health check system to prevent geriatric syndromes.
  • the program is also intended to verify the effectiveness of the new health check system for efficiently implementing subsequent intervention programs such as exercise and nutrition for high-risk elderly people screened in this health check (see ⁇ http://square.umin.ac.jp/otassha/about/>).
  • Selection method The selection methods used were (i) 1st screening (GS-MS TIC qualitative analysis), (ii) 2nd screening (GS-MS m/z quantitative analysis), and (iii) 3rd screening (ROC analysis). After that, specificity and sensitivity were verified by combined ROC analysis.
  • SPME Solid-Phase Microextraction
  • the oven temperature was as follows: held at 40°C for 10 minutes, heated to 240°C at a rate of 5°C per minute, and then held at 240°C for 10 minutes.
  • Helium was used as the carrier gas, with a constant linear flow rate of 55cm/sec.
  • the processing parameters of the mass spectrometer were as follows: ion source temperature 200°C, ionization energy 70 eV, scan frequency 0.2 s per scan from 30 m/z to 300 m/z, and column length 65 m.
  • GC-MS raw data were converted to mzXML format using GCMSsolution ver.4.45 software (Shimadzu Corporation), and the XCMS software package ver.1.3.2 (http://masspec.scripps.edu) running in R version 3.2.3 (http://cran.r-project.org/) was used to extract the difference ion peaks (m/z) between the sarcopenia patient group and the healthy control group.
  • TICs total ion currents
  • RT retention times
  • an autosampler system (Shimadzu Multifunctional autosampler system) was used. Metabolite concentrations were determined by calculating the ratio of ion peak areas of volatile substances and peak areas of limiting diluted external standards.
  • VOCs 1 to 23 were identified from the TIC peaks.
  • VOCs were determined by using commercially available standards.
  • the areas of clearly detected m/z peaks were used to quantify VOCs in urine.
  • the Mann-Whitney U test was used.
  • VOC fragment ion m/z value with the largest area in each fragmentation pattern of the 23 VOCs was selected for comparison of the absolute area between the two groups, and 10 compounds that were significantly contained in the VOCs of the sarcopenia patient group compared to the VOCs of the healthy control group were identified using the Mann-Whitney U test.
  • the 10 compounds were p-xylene, 1-butanol, limonene, nonanal, pyrrole, gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • Figure 2 shows the structural formulas of the 10 compounds and the increase or decrease in the main m/z peak area between the two groups.
  • Table 3 shows the results of ROC analysis performed with a combination of two or more VOCs.
  • Figure 4 shows the results of ROC analysis performed on both men and women.
  • Figure 5 shows the results of ROC analysis performed on only men, and
  • Figure 6 shows the results of ROC analysis performed on only women.
  • sarcopenia could be diagnosed with a sufficiently high discrimination ability, with a sensitivity of 69.1% or more and a specificity of 80.3%.
  • Performance Comparison Table 4 shows the results of a comparison of the characteristics of the evaluation method using the VOC marker of this embodiment and the conventional evaluation method.
  • the sensitivity of the VOC marker (81%) was significantly improved compared to the sensitivity of the conventional simplified method (3.8-4.8%), and it was found to be suitable for screening for sarcopenia. Furthermore, by combining this with the simplified VOC measurement method, it is believed to be suitable for use as point-of-care testing (POCT) in nursing care settings, etc.
  • POCT point-of-care testing
  • the "Arrow Y" in Table 4 indicates that the simplified measurement method of the present embodiment can be used.
  • Examples of such simplified measurement methods include (1) a method in which gamma-butyrolactone is treated as gamma-hydroxybutyric acids and the total amount is measured using an enzymatic method, and (2) a method in which caprylic acid and pelargonic acid are measured as medium-chain fatty acids with 8 to 12 carbon atoms and the total amount is measured.
  • VOCs In a group of elderly people aged 66 to 79 years, a combination of six VOCs had a sensitivity of 82%, a specificity of 90%, and a likelihood ratio of 8.2, making it an advantageous simple diagnostic method (p-Xylene, d-limonene, nonanal, octanoic acid, nonanoic acid, and diisobutyl phthalate).
  • VOCs No. 4, 6, 9, 21, and 22 were significantly increased by 1.4 times, 1.5 times, 1.3 times, 1.3 times, and 4.0 times, respectively, compared to healthy subjects (two-tailed test).
  • VOC No. 19 was significantly increased by 1.1 times, respectively, compared to healthy subjects (one-tailed test).
  • VOCs No. 5, 10, and 23 were significantly decreased by 0.7 times, 0.6 times, and 0.7 times, respectively (two-tailed test).
  • Table 6 shows the absolute values of 23 VOCs in the urine of 30 sarcopenia patients aged 80-88 (10 severe + 20 mild) and 31 healthy individuals of the same age, extracted from the urinary VOCs of the 68 sarcopenia patients (13 severe + 55 mild) shown in Table 1 and 71 healthy individuals of the same age, and shows the VOCs that showed differences in area between healthy individuals and those with sarcopenia as relative values to healthy individuals.
  • VOCs No. 4, 6, 9, 11, 14, and 21 were significantly increased by 1.4 times, 1.5 times, 1.3 times, 1.3 times, 2.7 times, and 2.0 times, respectively, compared to healthy subjects (two-tailed test).
  • VOCs No. 19, 20, and 22 were significantly increased by 1.1 times, 1.5 times, and 2.2 times, respectively, compared to healthy subjects (one-tailed test).
  • VOC No. 5 was significantly decreased by 0.6 times (two-tailed test).
  • Table 7 Figures 7 and 8 show the results of an analysis of the analytical data for 11 VOCs between the two groups of people aged 79 or younger and between the two groups of people aged 80 or older, using the Mann-Whitney U test to examine the increase or decrease in nuclear structural formulas and main m/z peak area.
  • Figure 9 shows a summary of the results of ROC analysis for those aged 66 to 79 and for those aged 80 and over.
  • the results of the ROC analysis for those aged 66 to 79 showed that the 11 VOCs identified were p-Xylene, 1-butanol, d-limonen, nonanal, acetic acid, pyrrol, ⁇ -butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate, with a sensitivity of 82% and specificity of 90%.
  • the 8 VOCs identified were p-Xylene, 1-butanol, d-limonen, nonanal, acetic acid, octanoic acid, nonanoic acid, and diisobutyl phthalate, with a sensitivity of 82% and specificity of 93%.
  • the six VOCs were p-Xylene, d-limonene, nonanal, octanoic acid, nonanoic acid, and diisobutyl phthalate, and the sensitivity was 82% and the specificity was 90%. Numerically, the analysis results using eight VOCs were the best, but from the perspective of simplicity, six VOCs were considered to be the easiest to use.
  • VOCs namely p-Xylene, d-limonene, nonanal, ⁇ -butyrolactone, and octanoic acid.
  • the sensitivity was 80% and the specificity was 94%.
  • One method is to detect nine VOCs (p-Xylene, 1-butanol, d-limonen, nonanal, acetic acid, ⁇ -butyrolactone, octanoic acid, nonanoic acid, diisobutyl phthalate) and then analyze eight VOCs (p-Xylene, d-limonen, nonanal, octanoic acid, 1-butanol, acetic acid, nonanoic acid, diisobutyl phthalate) for those aged 66 to 79, and five VOCs (p-Xylene, d-limonen, nonanal, octanoic acid, ⁇ -butyrolactone) for those aged 80 or over.
  • VOCs four (p-Xylene, d-limonen, nonanal, octanoic acid) are common.
  • four (p-Xylene, d-limonen, nonanal, octanoic acid) are common to all VOCs.
  • Figure 10 shows a summary of the six VOC indices (40 healthy subjects, 38 sarcopenia patients) for subjects aged 66 to 79.
  • Figure 11 shows a summary of the results for four of the five VOC indices (31 healthy subjects, 30 sarcopenia patients) for subjects aged 80 or older.
  • the VOC index of eight sarcopenia biomarkers and the six VOC indexes were negatively correlated with BMI, waist-to-hip ratio (WHR), obesity degree (Obesity Degree, OD), and grip strength (paku).
  • WHR waist-to-hip ratio
  • OD obesity degree
  • CKCPK creatine kinase/creatine phosphokinase
  • Table 8 shows the results of a total of 24 cohort analysis items and frailty items.
  • the sarcopenia biomarkers 8VOCs index and 6VOCs index were negatively correlated with BMI, waist-to-hip ratio (WHR), obesity degree (Obesity Degree, OD), and grip strength (paku).
  • WHR waist-to-hip ratio
  • OD obesity degree
  • paku grip strength
  • the 6 VOCs index showed a slightly stronger correlation than the 8 VOCs index.
  • Table 9 shows the results of a comparison of the characteristics of the evaluation method using VOC markers of this embodiment and the conventional evaluation method when analyzing by age group.
  • the sensitivity of the VOC marker (80-82%) was significantly improved compared to the sensitivity of the conventional simplified method (3.8-4.8%), and it was found to be suitable for screening for sarcopenia. Furthermore, by combining this with the simplified VOC measurement method, it is believed to be suitable for use as a POCT in nursing care settings, etc.
  • the "Arrow Y" in Table 9 indicates that the simplified measurement method of the present embodiment can be used.
  • Examples of such measurement methods include (1) a method in which gamma-butyrolactone is treated as gamma-hydroxybutyric acids and the total amount is measured using an enzymatic method, and (2) a method in which caprylic acid and pelargonic acid are measured as medium-chain fatty acids with 8 to 12 carbon atoms and the total amount is measured.
  • Urinary volatile compounds including p-xylene, 1-butanol, limonene, nonanal, acetic acid, pyrrole, ⁇ -butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate were found to be biomarkers that can be used to diagnose sarcopenia.
  • the above biomarkers can be extracted from urine, which can be collected non-invasively, and do not require harm to the subject, so they can be used by caregivers and others. In addition, by using them for primary screening in clinical settings, it can be used as a diagnostic support measure to prevent the transition from sarcopenia to frailty.
  • VOCs p-xylene, 1-butanol, limonene, nonanal, pyrrole, gamma-butyrolactone, texanol, octanoic acid
  • VOCs p-xylene, 1-butanol, limonene, nonanal, pyrrole, gamma-butyrolactone, texanol, octanoic acid
  • six VOCs p-xylene, limonene, nonanal, octanoic acid, nonanoic acid, and diisobutyl phthalate
  • five VOCs p-xylene, limonene, nonanal, gamma-butyrolactone, and octanoic acid
  • the biomarker data provided by this embodiment is important for supporting the diagnosis of sarcopenia.
  • a qualified person such as a doctor will ultimately make the final diagnosis of the disease.
  • the data of this embodiment is treated as an important piece of data for the final diagnosis.
  • a biomarker for diagnosing sarcopenia that measures volatile metabolites in urine obtained from a human subject the biomarker for sarcopenia being at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, acetic acid, pyrrole, gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • a test method for obtaining data as one of the evaluation items for diagnosing sarcopenia by measuring volatile metabolites in urine obtained from a human subject comprising dividing the subjects into a younger group of subjects aged 79 or younger and an elderly group of subjects aged 80 or older, and wherein in the younger group, the volatile metabolite is at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, pyrrole, gamma-butyrolactone, texanol, and octanoic acid.
  • a method for measuring a sarcopenia marker comprising a measurement step of measuring volatile components of food-derived components, metabolic products of intestinal bacteria-derived components, or analogs thereof in a subject sample as sarcopenia markers.
  • the measuring step includes: a volatilization step of volatilizing volatile components from the sample; A component measurement step of measuring the obtained volatile components;
  • the measurement method according to claim 5, comprising: (Appendix 7) The method according to claim 6, wherein the component measuring step measures the volatile components by gas chromatography-mass spectrometry.
  • the method includes an adsorption step of adsorbing the volatile components onto an adsorbent, The component measuring step measures the volatile components adsorbed by the adsorbent.
  • the measurement method according to any one of appendix 6 to 8.
  • Appendix 11 The measurement method according to any one of appendixes 5 to 10, wherein the volatile components are volatile organic compounds.
  • the measurement method includes a measurement step of contacting a subject sample with a measurement reagent for measuring a sarcopenia marker in the sample, A method for measuring sarcopenia markers, wherein the sarcopenia markers include volatile components of food-derived components, metabolic products of intestinal bacteria-derived components, or analogs thereof.
  • the measuring step includes: a complex formation step of contacting the sample with a first binding molecule for the sarcopenia marker and a reference sarcopenia marker to form a first complex between the sarcopenia marker in the sample and the first binding molecule, and a second complex between the reference sarcopenia marker and the first binding molecule;
  • the measurement method described in Appendix 16 comprising a complex measurement step of measuring the sarcopenia marker in the sample by measuring the second complex.
  • the measuring step includes: a first complex formation step of contacting the sample with a first binding molecule for the sarcopenia marker to form a first complex between the sarcopenia marker in the sample and the first binding molecule; A second complex formation step of contacting the first complex with a second binding molecule for the sarcopenia marker to form a second complex between the first complex and the second binding molecule;
  • the measurement method described in Appendix 16 comprising a complex measurement step of measuring the sarcopenia marker in the sample by measuring the second complex.
  • (Appendix 19) The measurement method according to claim 17 or 18, wherein the first binding molecule is an aptamer, an antibody, a receptor, or a ligand capable of binding to the sarcopenia marker.
  • (Appendix 20) The measurement method according to any one of appendixes 16 to 19, wherein the volatile components are volatile organic compounds.
  • (Appendix 21) The measurement method according to claim 20, wherein the volatile organic compound is at least one selected from the group consisting of aromatic compounds, alcohols, terpenes, aldehydes, carboxylic acids, amines, lactones, fatty acids, and phthalate esters.
  • (Appendix 22) The measurement method according to claim 20 or 21, wherein the volatile organic compound is at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, acetic acid, pyrrole, ⁇ -butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • (Appendix 24) The measurement method according to claim 23, wherein the biological sample is a urine sample or a blood sample.
  • Appendix 25 A measuring step of measuring a sarcopenia marker for a subject sample, A method for testing the risk of developing sarcopenia, wherein the measuring step is carried out by a measuring method described in any one of appendices 5 to 24.
  • Appendix 26 The method according to claim 25, further comprising a step of testing the possibility of the subject suffering from sarcopenia based on the measured value of the sarcopenia marker.
  • Appendix 27 The method according to claim 25 or 26, comprising a step of testing the possibility of the subject suffering from sarcopenia by comparing the measured value of the sarcopenia marker with a threshold value.
  • the sarcopenia marker is at least one selected from the group consisting of aromatic compounds, terpenes, aldehydes, amines, lactones, and fatty acids;
  • the sarcopenia marker is at least one selected from the group consisting of alcohol, carboxylic acid, and phthalate ester;
  • the subject is deemed to have a high possibility of suffering from sarcopenia.
  • Test method described in Appendix 27 Includes a reagent for measuring sarcopenia markers, The kit is for use in measuring sarcopenia markers, the sarcopenia markers including metabolic products of food-derived components, intestinal bacteria-derived components, or volatile components of their analogs.
  • Appendix 30 The kit described in Appendix 29, wherein the measurement reagent contains a binding molecule for the sarcopenia marker.
  • Appendix 31 The kit of claim 30, wherein the binding molecule is an aptamer, antibody, receptor, or ligand capable of binding to the sarcopenia marker.
  • the measurement reagent includes a binding molecule for the sarcopenia marker, the sarcopenia marker, and a substrate, The kit described in Appendix 30 or 31, wherein the sarcopenia marker is immobilized on the substrate.
  • the kit according to claim 30 or 31, wherein the measurement reagent comprises a substrate on which a binding molecule for the sarcopenia marker is immobilized, and a sarcopenia marker containing a label.
  • Appendix 34 34.
  • Appendix 35 The kit described in Appendix 34, wherein the immunoassay reagent is a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blot method, immunoblot method, latex agglutination method, and immunochromatography method.
  • Appendix 36 The kit according to claim 35, wherein the immunoassay reagent is a reagent for use in an immunochromatography method.
  • Appendix 37 37.
  • the volatile organic compound is at least one selected from the group consisting of aromatic compounds, alcohols, terpenes, aldehydes, carboxylic acids, amines, lactones, fatty acids, and phthalate esters.
  • (Appendix 39) The kit according to claim 37 or 38, wherein the volatile organic compound is at least one selected from the group consisting of p-xylene, 1-butanol, limonene, nonanal, acetic acid, pyrrole, gamma-butyrolactone, texanol, octanoic acid, nonanoic acid, and diisobutyl phthalate.
  • Appendix 40 A screening method for candidate therapeutic agents for sarcopenia, comprising a selection step of selecting from test substances a substance that changes a sarcopenia marker as a candidate therapeutic agent for sarcopenia.
  • (Appendix 41) A measurement step of administering the test substance to a sarcopenia model and measuring the sarcopenia marker; and a selection step of selecting the test substance as the candidate therapeutic agent if the sarcopenia marker obtained in the measurement step is lower than that of a control not administered the test substance; or a measurement step of administering the test substance to a sarcopenia model and measuring the sarcopenia marker, and a selection step of selecting the test substance as the candidate therapeutic agent if the sarcopenia marker obtained in the measurement step is higher than that of a control not administered the test substance. (Appendix 42) 42.
  • test substance is at least one selected from the group consisting of a low molecular weight compound, a peptide, a protein, and a nucleic acid.
  • Appendix 43 A method for detecting a sarcopenia marker in a subject suspected of having sarcopenia, comprising a detection step of detecting a sarcopenia marker in a sample from the subject using a measurement kit for a sarcopenia marker.
  • sarcopenia marker measurement kit is a measurement kit described in any one of Appendix 29 to 39.
  • the present disclosure can provide a sarcopenia marker.
  • the present disclosure can provide a method for measuring a sarcopenia marker, a method for testing sarcopenia, and a kit for measuring a sarcopenia marker. For this reason, the present disclosure can be said to be extremely useful, for example, in the field of testing.

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Abstract

La présente divulgation concerne : un procédé de mesure d'un marqueur de sarcopénie ; une méthode de dépistage de la sarcopénie ; et une trousse à utiliser dans la mesure du marqueur de sarcopénie. Une méthode de mesure d'un marqueur de sarcopénie selon la présente divulgation comprend une étape de mesure pour mesurer, en tant que marqueur de sarcopénie, un composant volatil d'un composant dérivé d'un aliment, un métabolite d'un composant dérivé de bactéries intestinales, ou un analogue de ceux-ci dans un échantillon cible.
PCT/JP2024/014010 2023-04-07 2024-04-05 Biomarqueur de sarcopénie et son utilisation Pending WO2024210185A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
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WO2015152413A1 (fr) * 2014-04-03 2015-10-08 地方独立行政法人東京都健康長寿医療センター Biomarqueur pour diagnostic de vieillissement ou d'amyotrophie
WO2020184660A1 (fr) * 2019-03-13 2020-09-17 味の素株式会社 Procédé d'évaluation de la sarcopénie, procédé de calcul, dispositif d'évaluation, dispositif de calcul, programme d'évaluation, programme de calcul, support d'enregistrement, système d'évaluation et dispositif terminal
JP2021511497A (ja) * 2018-01-17 2021-05-06 ユニヴァーシティ・オブ・サウザンプトンＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｓｏｕｔｈａｍｐｔｏｎ サルコペニア及びｎａｄ欠乏のリスクの予測及び階層化の方法

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WO2015152413A1 (fr) * 2014-04-03 2015-10-08 地方独立行政法人東京都健康長寿医療センター Biomarqueur pour diagnostic de vieillissement ou d'amyotrophie
JP2021511497A (ja) * 2018-01-17 2021-05-06 ユニヴァーシティ・オブ・サウザンプトンＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｓｏｕｔｈａｍｐｔｏｎ サルコペニア及びｎａｄ欠乏のリスクの予測及び階層化の方法
WO2020184660A1 (fr) * 2019-03-13 2020-09-17 味の素株式会社 Procédé d'évaluation de la sarcopénie, procédé de calcul, dispositif d'évaluation, dispositif de calcul, programme d'évaluation, programme de calcul, support d'enregistrement, système d'évaluation et dispositif terminal

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