Biomarkers and uses thereof

Abstract

The purpose of the present invention is to provide a method for evaluating the intestinal barrier function of a subject, the method comprising the steps of: (1a) measuring a biomarker in a subject derived from the subject, and acquiring a measured value of the biomarker; (1b) comparing the measured value obtained in step 1a with a reference value to obtain a comparison result; and (1c) a step for evaluating the intestinal barrier function of the subject on the basis of the comparison result obtained in step 1b, the subject being urine, and the biomarker comprising one or more substances selected from the group of substances according to the present invention.

Description

Technical Field

[0001] This invention relates to biomarkers and their uses.

[0002] Specifically, the present invention relates to a method for evaluating the intestinal barrier function of a subject, a biomarker for said evaluation, the use of said biomarker for said evaluation, a kit for said evaluation, and a computer program for causing a computer to perform said method.

[0003] In addition, the present invention relates to a method for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said diagnosis, the use of said biomarker for said diagnosis, a reagent kit for said diagnosis, and a computer program for causing a computer to perform said method.

[0004] In addition, the present invention relates to a method for treating diseases or symptoms caused by a decrease in intestinal barrier function in a subject.

[0005] In addition, the present invention relates to a method for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function or the likelihood that the subject has already developed said disease or symptoms, a biomarker for said evaluation, the use of said biomarker for said evaluation, a kit for said evaluation, and a computer program for causing a computer to perform said method.

[0006] In addition, the present invention relates to a method for obtaining data on diseases or symptoms caused by a decrease in intestinal barrier function in a diagnostic subject, a biomarker for obtaining the biomarker, a reagent kit for obtaining the biomarker, and a computer program for causing a computer to perform the method.

[0007] In addition, the present invention relates to a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said screening, the use of said biomarker for said screening, a kit for said screening, and a computer program for causing a computer to perform said method.

[0008] In addition, the present invention relates to a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said evaluation, the use of said biomarker for said evaluation, a kit for said evaluation, and a computer program for causing a computer to perform said method.

[0009] In addition, the present invention relates to a method for manufacturing food or pharmaceutical compositions. Background Technology

[0010] The intestinal lumen is a space surrounded by a layer of mucosal epithelial cells, which divide the intestinal lumen (outside the organism) from the body. The intestine is exposed to foreign substances (such as harmful microorganisms, toxins, and allergens) entering from the outside. Therefore, from the perspective of both nutrient absorption and the organism's defense mechanisms, the intestine is a vital organ. The intestinal barrier function is responsible for preventing foreign substances (such as harmful microorganisms, toxins, and allergens) from entering the intestinal lumen from the outside into the body.

[0011] The intestinal barrier function involves physical and chemical barriers. The physical barrier, as the name suggests, is a barrier that prevents the invasion of foreign substances. Physical barriers include the mucus layer covering intestinal epithelial cells, the glycocalyx (an aggregate of sugar chains on the surface of intestinal epithelial cells), and tight junctions (TJs) that act as adhesion devices between intestinal epithelial cells. The chemical barrier involves a group of molecules that induce chemical changes in foreign substances, inhibiting their invasion through antibacterial or neutralizing activities. This group includes, for example, antimicrobial peptides produced by intestinal epithelial cells such as Paneth cells (e.g., defensin family molecules, Reg3 family molecules, lactoferrin, lysozyme, etc.). M cells, which take up antigens and present them to dendritic cells, and immunoglobulin A (IgA), which exerts neutralizing activity by being secreted into the intestine to prevent foreign invasion, are also types of chemical barriers.

[0012] Decreased intestinal barrier function can be a cause of various diseases or symptoms (e.g., enteritis, allergic diseases, mental illness, non-alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome, obesity, etc.).

[0013] As a method for assessing intestinal barrier function, the lactulose-mannitol test (glucose tolerance test) is known. The lactulose-mannitol test examines intestinal permeability; increased intestinal permeability is an indicator of deterioration in intestinal barrier function (especially physical barrier functions such as tight junction structures). The lactulose-mannitol test has drawbacks, including requiring prolonged restraint and fasting periods for the subject, and placing a burden on the digestive tract from the ingested sugar alcohol.

[0014] The concentration of zonulin in the blood, which is known to be associated with intestinal barrier function (Non-Patent Literature 1), is sometimes used in examination methods for evaluating intestinal barrier function. However, there are issues such as diurnal variation in zonulin concentration and low reproducibility (Non-Patent Literature 2).

[0015] Previously, biomarkers that could easily and accurately assess intestinal barrier function were unknown.

[0016] Existing technical documents

[0017] Non-patent literature

[0018] Non-patent literature 1: Fasana A et al., Zonulin, "a newly discovered modulator of intestinal permeability, and its expression in coeliac disease.", THE LANCET., 2000; 355: pp.1518-1519

[0019] Non-patent literature 2: Aristo Vojdani et al., “Fluctuation of zonulin levels in blood vs stability of antibodies.”, World Journal of Gastroenterology., 2017; 23(31):pp.5669-5679 Summary of the Invention

[0020] The problem the invention aims to solve

[0021] The object of the present invention is to provide a method for evaluating the intestinal barrier function of a subject, a biomarker for the evaluation, the use of the biomarker for the evaluation, a kit for the evaluation, and a computer program for causing a computer to perform the method.

[0022] In addition, the present invention aims to provide a method for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said diagnosis, the use of said biomarker for said diagnosis, a reagent kit for said diagnosis, and a computer program for causing a computer to perform said method.

[0023] In addition, the present invention aims to provide a method for treating diseases or symptoms caused by a decrease in intestinal barrier function in a subject.

[0024] In addition, the present invention aims to provide a method for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function or the possibility of having already developed said disease or symptoms, a biomarker for said evaluation, the use of said biomarker for said evaluation, a kit for said evaluation, and a computer program for causing a computer to perform said method.

[0025] In addition, the present invention aims to provide a method for obtaining data on diseases or symptoms caused by a decrease in intestinal barrier function in a diagnostic subject, a biomarker for obtaining the biomarker, a reagent kit for obtaining the biomarker, and a computer program for causing a computer to perform the method.

[0026] In addition, the present invention aims to provide a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said screening, the use of said biomarker for said screening, a kit for said screening, and a computer program for causing a computer to perform said method.

[0027] In addition, the present invention aims to provide a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said evaluation, the use of said biomarker for said evaluation, a kit for said evaluation, and a computer program for causing a computer to perform said method.

[0028] In addition, the present invention aims to provide a method for manufacturing food or pharmaceutical compositions.

[0029] Solution for solving the problem

[0030] In this invention, the group of substances composed of the substances shown in Table 1 is referred to as the "group of substances of this invention".

[0031] [Table 1-1]

[0032] Table 1 (Material composition of the present invention)

[0033] No. substance name 1 4-Acetamidobutanoic acid 2 N-Acetylputrescine 3 3-Amino-2-piperidone 4 O-Succinylhomoserine 5 Sulfotyrosine 6 Malic acid 7 Tyrosine 8 N-Carbamoylaspartic acid 9 Muscimol 10 Mucic acid 11 N-acetylaspartic acid 12 Argininosuccinic acid 13 Adipic acid 14 2-Oxoadipic acid 15 Phenylalanine 16 Succinic acid 17 N-Acetylglutamic acid 18 Glycylaspartic acid (Gly-Asp) 19 N-Acetylomithine 20 Ascorbate 2-sulfate 21 Asymmetric dimethylarginine (ADMA) 22 N-Acetylneuraminic acid 23 2-Hydroxyglutaric acid 24 Cyclohexanecarboxylic acid (Cvclohexanecarboxylic acid) 25 5-Methylcytosine 26 cis-aconitic acid 27 Glycine 28 Tryptophan 29 Tyramine (Tvramine) 30 Glycerol 2-phosphate 31 Citracolic acid 32 Octopamine and Dopamine 33 Syringic acid 34 2'-Deoxyadenosine and 5'-Deoxyadenosine 35 Adenosine diphosphate (ADP) 36 Glutathione (GSH) 37 2-Aminoisobutyric acid and 2-Aminobutyric acid 38 Sinapic acid

[0034] [Table 1-2]

[0035] Table 1 (continued)

[0036]

[0037] The present invention includes the following technical solutions.

[0038] [1] A method for evaluating the intestinal barrier function of a subject, the method comprising the following steps:

[0039] (1a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0040] (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain a comparison result; and

[0041] (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0042] The sample to be tested was urine.

[0043] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0044] [2] A method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps:

[0045] (2a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0046] (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain a comparison result; and

[0047] (2c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 2b.

[0048] The sample to be tested was urine.

[0049] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0050] [3] A method for treating a disease or symptoms caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps:

[0051] (3a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0052] (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result;

[0053] (3c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 3b; and

[0054] (3d) In the event that the subject is diagnosed with the disease or symptoms, the step of administering to the subject a substance or composition for treating the disease or symptoms.

[0055] The sample to be tested was urine.

[0056] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0057] [4] A method for evaluating the risk of a subject developing a disease or symptoms caused by a decrease in intestinal barrier function, or the likelihood that a subject has already developed said disease or symptoms, the method comprising the following steps:

[0058] (4a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0059] (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain a comparison result; and

[0060] (4c) The step of evaluating the risk or the likelihood based on the comparison results obtained in step 4b.

[0061] The sample to be tested was urine.

[0062] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0063] [5] A method for obtaining data on disease or symptoms caused by reduced intestinal barrier function in a diagnostic subject, the method comprising the steps of:

[0064] (5a) The step of measuring biomarkers in a sample from the subject and obtaining the measured values ​​of the biomarkers; and

[0065] (5b) The step of comparing the measured value obtained in step 5a with the reference value and obtaining the comparison result as the data.

[0066] The sample to be tested was urine.

[0067] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0068] [6] A method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, the method comprising the following steps:

[0069] (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0070] (6b) The step of determining a biomarker in a test body obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the biomarker;

[0071] (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain a comparison result; and

[0072] (6d) Based on the comparison results obtained in step 6c, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms.

[0073] The sample to be tested was urine.

[0074] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0075] [7] A method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, the method comprising the following steps:

[0076] (7a) The step of administering the candidate substance or the candidate composition to a subject whose intestinal barrier function has been reduced;

[0077] (7b) The step of determining a biomarker in a test sample obtained from the subject after administration of the candidate substance or the candidate composition, and obtaining a measured value of the biomarker;

[0078] (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain a comparison result; and

[0079] (7d) The step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms, based on the comparison results obtained in step 7c.

[0080] The sample to be tested was urine.

[0081] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0082] [8] The method according to any one of [1] to [7], wherein the intestinal barrier function is the barrier function of the small intestine.

[0083] [9] The method according to any one of [1] to [8], wherein the biomarker comprises two, three or more substances selected from the group of substances of the present invention.

[0084]

[10] The method according to any one of [1] to [9], wherein the biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone and O-succinylhomoserine.

[0085]

[11] According to the method of

[10] , wherein the biomarker in the analyte is determined by capillary electrophoresis-mass spectrometry.

[0086]

[12] The method according to any one of [1] to [9], wherein the biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine and bis(methylbenzylidene)sorbitol.

[0087]

[13] According to the method of

[12] , wherein the biomarker in the analyte is determined by liquid chromatography-mass spectrometry.

[0088]

[14] A biomarker, which is a biomarker derived from the subject's analyte for evaluating the intestinal barrier function of the subject.

[0089] The sample to be tested was urine.

[0090] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0091]

[15] A biomarker, which is a biomarker derived from the subject's body for diagnosing disease or symptoms caused by a decrease in intestinal barrier function in a subject.

[0092] The sample to be tested was urine.

[0093] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0094]

[16] A biomarker, which is a biomarker from the subject's body used to evaluate the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the likelihood that the subject has already developed said disease or symptom.

[0095] The sample to be tested was urine.

[0096] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0097]

[17] A biomarker, which is a biomarker from the subject of a test sample used to obtain data on disease or symptoms caused by a decrease in intestinal barrier function in a subject for diagnosis.

[0098] The sample to be tested was urine.

[0099] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0100]

[18] A biomarker for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, and is a biomarker in a test sample obtained from a subject with decreased intestinal barrier function after administration of the candidate substance or candidate composition to said subject.

[0101] The sample to be tested was urine.

[0102] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0103]

[19] A biomarker for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms caused by a decrease in intestinal barrier function, and is a biomarker in a test sample obtained from a subject with decreased intestinal barrier function after administration of the candidate substance or candidate composition to the subject.

[0104] The sample to be tested was urine.

[0105] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0106]

[20] The biomarker according to any one of

[14] to

[19] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0107]

[21] The biomarker according to any one of

[14] to

[20] , wherein the biomarker comprises two, three or more substances selected from the group of substances of the present invention.

[0108]

[22] The biomarker according to any one of

[14] to

[21] , wherein the biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetyl putrescine, 3-amino-2-piperidone and O-succinylhomoserine.

[0109]

[23] According to the biomarker described in

[22] , wherein the biomarker in the analyte is determined by capillary electrophoresis-mass spectrometry.

[0110]

[24] The biomarker according to any one of

[14] to

[21] , wherein the biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine and bis(methylbenzylidene)sorbitol.

[0111]

[25] According to the biomarker described in

[24] , wherein the biomarker in the analyte is determined by liquid chromatography-mass spectrometry.

[0112]

[26] Use of a biomarker from a subject in the test sample for evaluating the intestinal barrier function of said subject.

[0113] The sample to be tested was urine.

[0114] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0115]

[27] Use of a biomarker from a subject in the test sample for the diagnosis of diseases or symptoms in the subject caused by a decrease in intestinal barrier function.

[0116] The sample to be tested was urine.

[0117] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0118]

[28] Use of a biomarker from a subject in a test sample for evaluating the risk of said subject developing or having already developed a disease or symptoms caused by a reduction in intestinal barrier function.

[0119] The sample to be tested was urine.

[0120] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0121]

[29] Use of a biomarker from a subject for obtaining data for diagnosing diseases or symptoms caused by reduced intestinal barrier function in the subject.

[0122] The sample to be tested was urine.

[0123] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0124]

[30] Use of a biomarker, wherein the biomarker is a biomarker in a test sample obtained after administration of a candidate substance or candidate composition to a subject with reduced intestinal barrier function, the use being for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function.

[0125] The sample to be tested was urine.

[0126] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0127]

[31] Use of a biomarker, wherein the biomarker is a biomarker in a test sample obtained after administration of a candidate substance or candidate composition to a subject with reduced intestinal barrier function, the use being for evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function.

[0128] The sample to be tested was urine.

[0129] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0130]

[32] The use according to any one of

[26] to

[31] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0131]

[33] The use according to any one of

[26] to

[32] , wherein the biomarker comprises two, three or four or more substances selected from the group of substances of the present invention.

[0132]

[34] The use according to any one of

[26] to

[33] , wherein the biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetyl-putrescine, 3-amino-2-piperidone and O-succinylhomoserine.

[0133]

[35] According to the use described in

[34] , wherein the biomarker in the analyte is determined by capillary electrophoresis-mass spectrometry.

[0134]

[36] The use according to any one of

[26] to

[33] , wherein the biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine and bis(methylbenzylidene)sorbitol.

[0135]

[37] According to the use described in

[36] , wherein the biomarker in the analyte is determined by liquid chromatography-mass spectrometry.

[0136]

[38] A kit for evaluating intestinal barrier function in subjects.

[0137] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0138] The sample to be tested was urine.

[0139] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0140]

[39] A kit for diagnosing diseases or symptoms caused by decreased intestinal barrier function in subjects.

[0141] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0142] The sample to be tested was urine.

[0143] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0144]

[40] A kit for evaluating the risk of a subject developing a disease or symptoms caused by a decrease in intestinal barrier function, or the likelihood that a subject already has said disease or symptoms.

[0145] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0146] The sample to be tested was urine.

[0147] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0148]

[41] A kit for obtaining data on diseases or symptoms caused by decreased intestinal barrier function in a diagnostic subject.

[0149] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0150] The sample to be tested was urine.

[0151] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0152]

[42] A kit for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function.

[0153] The kit contains one or more reagents for determining biomarkers in analytes obtained after administration of candidate substances or candidate compositions to subjects with impaired intestinal barrier function.

[0154] The sample to be tested was urine.

[0155] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0156]

[43] A kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function.

[0157] The kit contains one or more reagents for determining biomarkers in analytes obtained from subjects with impaired intestinal barrier function after administration of the candidate substance or the candidate composition to the subjects.

[0158] The sample to be tested was urine.

[0159] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0160]

[44] The kit according to any one of

[38] to

[43] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0161]

[45] The kit according to any one of

[38] to

[44] , wherein the biomarker comprises two, three or more substances selected from the group of substances of the present invention.

[0162]

[46] The kit according to any one of

[38] to

[45] , wherein the biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetyl putrescine, 3-amino-2-piperidone and O-succinylhomoserine.

[0163]

[47] According to the kit described in

[46] , the biomarkers in the analyte are determined by capillary electrophoresis-mass spectrometry.

[0164]

[48] ​​The kit according to any one of

[38] to

[45] , wherein the biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine and bis(methylbenzylidene)sorbitol.

[0165]

[49] According to the kit described in

[48] , the biomarker in the analyte is determined by liquid chromatography-mass spectrometry.

[0166]

[50] A method for manufacturing a food or pharmaceutical composition, comprising the step of mixing a substance or composition selected by the method described in [6] with one or more components constituting the food or pharmaceutical composition.

[0167]

[51] A computer program for causing a computer to perform a method for evaluating the intestinal barrier function of a subject.

[0168] The method includes the following steps:

[0169] (S101) Step of obtaining the measurement value of biomarkers from the test body of the object;

[0170] (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain a comparison result; and

[0171] (S103) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step S102.

[0172] The sample to be tested was urine.

[0173] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0174]

[52] A computer program for causing a computer to perform a method for diagnosing a disease or symptoms caused by a decrease in intestinal barrier function in a subject.

[0175] The method includes the following steps:

[0176] (S201) The step of obtaining the measurement value of the biomarker in the test body of the object;

[0177] (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result;

[0178] (S203) A step of diagnosing the disease or symptom based on the comparison results obtained in step S202.

[0179] The sample to be tested was urine.

[0180] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0181]

[53] A computer program for causing a computer to perform a method for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function, or the likelihood that the subject has already developed said disease or symptoms.

[0182] The method includes the following steps:

[0183] (S301) The step of obtaining the measurement value of the biomarker in the test body from the object;

[0184] (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain a comparison result; and

[0185] (S303) The step of evaluating the risk or the likelihood based on the comparison results obtained in step S302.

[0186] The sample to be tested was urine.

[0187] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0188]

[54] A computer program for causing a computer to perform a method for acquiring data used to diagnose a disease or symptoms caused by a decrease in intestinal barrier function in a subject.

[0189] The method includes the following steps:

[0190] (S401) The step of obtaining the measurement value of the biomarker from the test sample of the subject; and

[0191] (S402) The step of comparing the measured value obtained in step S401 with the reference value and obtaining the comparison result as the data.

[0192] The sample to be tested was urine.

[0193] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0194]

[55] A computer program for causing a computer to perform a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a reduction in intestinal barrier function.

[0195] The method includes the following steps:

[0196] (S501) A step of obtaining the determination value of the biomarker in the test body obtained after administering the candidate substance or candidate composition to the subject from a subject with reduced intestinal barrier function.

[0197] (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain a comparison result; and

[0198] (S503) Based on the comparison results obtained in step S502, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms.

[0199] The sample to be tested was urine.

[0200] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0201]

[56] A computer program for causing a computer to perform a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms caused by a decrease in intestinal barrier function.

[0202] The method includes the following steps:

[0203] (S601) A step of obtaining the determination value of the biomarker in the test body obtained after administering the candidate substance or candidate composition to the subject from a subject with reduced intestinal barrier function.

[0204] (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain a comparison result; and

[0205] (S603) Based on the comparison results obtained in step S602, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms.

[0206] The sample to be tested was urine.

[0207] The biomarker comprises one or more substances selected from the group of substances of the present invention.

[0208]

[57] The computer program according to any one of

[51] to

[56] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0209]

[58] The computer program according to any one of

[51] to

[57] , wherein the biomarker comprises two, three or more substances selected from the group of substances of the present invention.

[0210]

[59] The computer program according to any one of

[51] to

[58] , wherein the biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidone and O-succinylhomoserine.

[0211]

[60] According to the computer program described in

[59] , the biomarker in the test sample is determined by capillary electrophoresis-mass spectrometry.

[0212]

[61] The computer program according to any one of

[51] to

[58] , wherein the biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine and bis(methylbenzylidene)sorbitol.

[0213]

[62] According to the computer program described in

[61] , the biomarker in the analyte is determined by liquid chromatography-mass spectrometry.

[0214]

[63] A computer-readable recording medium having recorded any of the computer programs described in any one of

[51] to

[62] .

[0215] The effects of the invention

[0216] According to the present invention, a method for evaluating the intestinal barrier function of a subject, a biomarker for the evaluation, the use of the biomarker for the evaluation, a kit for the evaluation, and a computer program for causing a computer to perform the method are provided.

[0217] In addition, the present invention provides a method for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said diagnosis, use of said biomarker for said diagnosis, a reagent kit for said diagnosis, and a computer program for causing a computer to perform said method.

[0218] In addition, the present invention provides a method for treating diseases or symptoms caused by a decrease in intestinal barrier function in a subject.

[0219] In addition, this invention provides a method for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function or the likelihood that the subject has already developed the disease or symptoms, a biomarker for the evaluation, the use of the biomarker for the evaluation, a kit for the evaluation, and a computer program for causing a computer to execute the method.

[0220] In addition, the present invention provides a method for obtaining data on diseases or symptoms caused by a decrease in intestinal barrier function in a diagnostic subject, a biomarker for obtaining the biomarker, a reagent kit for obtaining the biomarker, and a computer program for causing a computer to perform the method.

[0221] In addition, the present invention provides a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said screening, use of said biomarker for said screening, a kit for said screening, and a computer program for causing a computer to perform said method.

[0222] In addition, the present invention provides a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a biomarker for said evaluation, the use of said biomarker for said evaluation, a kit for said evaluation, and a computer program for causing a computer to perform said method.

[0223] In addition, the present invention provides a method for manufacturing food or pharmaceutical compositions. Attached Figure Description

[0224] Figure 1 This is a schematic diagram of an apparatus according to one embodiment of the present invention.

[0225] Figure 2 This is a block diagram illustrating the hardware structure of an apparatus according to one embodiment of the present invention.

[0226] Figure 3 This is a flowchart illustrating the processing sequence of an apparatus according to one embodiment of the present invention.

[0227] Figure 4 This is a flowchart illustrating the processing sequence of an apparatus according to one embodiment of the present invention.

[0228] Figure 5 This is a flowchart illustrating the processing sequence of an apparatus according to one embodiment of the present invention. Detailed Implementation

[0229] The present invention will now be described. In the embodiments described in this specification, two or more embodiments may be combined, and these combinations are also included in the present invention. In this specification, "EX" refers to ×10 -X "E+Y" refers to ×10 Y .

[0230] Option 1

[0231] Scheme 1 of the present invention relates to a method for evaluating the intestinal barrier function of a subject.

[0232] In the method of scheme 1, biomarkers from the test subjects are used as indicators to evaluate the intestinal barrier function of the subjects.

[0233] The subject can be any animal that excretes urine; there are no particular limitations. Animals can include vertebrates such as mammals, reptiles, birds, amphibians, and fish, with mammals and birds being preferred, and mammals being more preferred. Mammals can include, for example, primates (e.g., humans, gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), livestock (e.g., cattle, pigs, sheep, goats, horses), and pets (e.g., dogs, cats), with humans being preferred. Birds can include, for example, poultry (e.g., chickens, wild ducks, domestic ducks).

[0234] The urine from the subject can be obtained from the subject's excretion using conventional methods, and the obtained urine can be used as the test sample.

[0235] The urine can be fresh urine from the subject, or urine obtained by freezing and thawing fresh urine from the subject. The freezing temperature is preferably -100°C to -10°C, more preferably -80°C to -15°C, and even more preferably -80°C to -60°C. Freezing can be performed, for example, by storing fresh urine from the subject in a container. The freezing period is preferably within 180 days from the date of urine collection, more preferably within 60 days, and even more preferably within 30 days. That is, urine collected within 180 days from the date of collection is preferably used for biomarker determination, more preferably within 60 days, and even more preferably within 30 days.

[0236] Biomarkers are substances or groups of substances present in an organism. The increase or decrease in their concentration in the organism reflects the presence, severity, risk of occurrence, risk of disease, preventative effect, therapeutic effect, or improvement effect of a specified state, symptom, or disease. A biomarker is a combination of one or more substances, preferably two or more. In this invention, a biomarker is the substance or group of substances that is the object of measurement and is used as an indicator for evaluating the intestinal barrier function of the subject.

[0237] The test sample from the subject is urine. Using urine as the test sample allows for a simple evaluation of the subject's intestinal barrier function. The urine used as the test sample is preferably the first urine excreted in the morning.

[0238] When the test sample from the subject is urine, one or more substances selected from the group of substances of the present invention are used as biomarkers for evaluating the subject's intestinal barrier function. That is, when the test sample from the subject is urine, the biomarker in the test sample from the subject contains one or more substances selected from the group of substances of the present invention.

[0239] The biomarkers from the test sample of the subject may contain one substance selected from the group of substances of the present invention, or two or more substances selected from the group of substances of the present invention. From the viewpoint of improving the accuracy of evaluating the intestinal barrier function of the subject, the biomarkers from the test sample of the subject preferably contain two, three or four or more substances selected from the group of substances of the present invention.

[0240] When the test sample is urine from the subject, the intestinal barrier function of the subject can be evaluated with high accuracy by using one or more substances selected from the material group of the present invention as biomarkers. Substances No. 1 to No. 18 and No. 39 to No. 49 in the material group of the present invention have higher accuracy in evaluating intestinal barrier function compared to other substances in the material group of the present invention, and substances No. 1 to No. 4 and No. 39 to No. 42 in the material group of the present invention have particularly high accuracy in evaluating intestinal barrier function compared to other substances in the material group of the present invention.

[0241] In one embodiment, the test sample from the subject is urine, and the biomarker in the test sample from the subject includes one or more substances selected from No. 1 to No. 4 of the substance group of the present invention (4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine). This improves the accuracy of evaluating the subject's intestinal barrier function. In addition to one or more substances selected from these four substances, the biomarker in the test sample from the subject may also include one or more other substances selected from the substance group of the present invention (e.g., one or more substances selected from No. 5 to No. 38 of the substance group of the present invention).

[0242] In another embodiment, the test sample from the subject is urine, and the biomarker in the test sample from the subject includes one or more substances selected from No. 39 to No. 42 of the substance group of the present invention (cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzyl)sorbitol). This improves the accuracy of evaluating the intestinal barrier function of the subject. In addition to one or more substances selected from these four substances, the biomarker in the test sample from the subject may also include one or more other substances selected from the substance group of the present invention (e.g., one or more substances selected from No. 43 to No. 61 of the substance group of the present invention).

[0243] In another embodiment, the test sample from the subject is urine, and the biomarker in the test sample from the subject includes substance No. 1 of the substance group of the present invention (4-acetaminophen). This improves the accuracy of evaluating the subject's intestinal barrier function. In addition to this single substance, the biomarker in the test sample from the subject may also include one or more other substances selected from the substance group of the present invention (e.g., one or more substances selected from substances No. 2 to No. 38 of the substance group of the present invention).

[0244] In another embodiment, the test sample from the subject is urine, and the biomarker in the test sample from the subject includes substance No. 2 (N-acetylputrescine) from the substance group of the present invention. This improves the accuracy of evaluating the subject's intestinal barrier function. In addition to this single substance, the biomarker in the test sample from the subject may also include one or more other substances selected from the substance group of the present invention (e.g., one or more substances selected from substances No. 1 and No. 3 to No. 38 from the substance group of the present invention).

[0245] In another embodiment, the test sample from the subject is urine, and the biomarker in the test sample from the subject includes substance No. 3 (3-amino-2-piperidinone) from the substance group of the present invention. This improves the accuracy of evaluating the subject's intestinal barrier function. In addition to this single substance, the biomarker in the test sample from the subject may also include one or more other substances selected from the substance group of the present invention (e.g., one or more substances selected from substances No. 1, No. 2, and No. 4 to No. 38 of the substance group of the present invention).

[0246] In another embodiment, the test sample from the subject is urine, and the biomarker in the test sample from the subject includes substance No. 5 (sulfotyrosine) from the substance group of the present invention. This improves the accuracy of evaluating the subject's intestinal barrier function. In addition to this single substance, the biomarker in the test sample from the subject may also include one or more other substances selected from the substance group of the present invention (e.g., one or more substances selected from substances No. 1 to No. 4 and No. 6 to No. 38 from the substance group of the present invention).

[0247] Option 1 includes the following steps:

[0248] (1a) The step of measuring biomarkers from the test sample of the subject and obtaining the measured values ​​of the biomarkers; and

[0249] (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain the comparison result; and

[0250] (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0251] <Step 1a>

[0252] Step 1a is the step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers.

[0253] The sample from the subject is urine.

[0254] When the test sample from the subject is urine, one or more substances selected from the group of substances of the present invention are used as biomarkers for evaluating the subject's intestinal barrier function. That is, when the test sample from the subject is urine, the biomarker measured in step 1a contains one or more substances selected from the group of substances of the present invention.

[0255] If the biomarker measured in step 1a contains one substance selected from the group of substances of the present invention, then that one substance is measured in step 1a. If the biomarker measured in step 1a contains two or more substances selected from the group of substances of the present invention, then those two or more substances are measured in step 1a. From the viewpoint of improving the accuracy of evaluating the intestinal barrier function of the subject, the biomarker measured in step 1a preferably contains two, three, or four or more substances selected from the group of substances of the present invention (i.e., in step 1a, two, three, or four or more substances selected from the group of substances of the present invention are measured).

[0256] "Determining one substance" means determining the concentration of that one substance; "determining two or more substances" means determining the individual concentrations of the two or more substances, or a composite variable whose statistical analysis value is derived from the concentrations of the two or more substances. In this specification, the substance being measured (biomarker) is sometimes referred to as the "target substance".

[0257] Concentration can be relative or absolute, but is preferably absolute.

[0258] The relative concentration of the target substance includes a relative value related to the absolute concentration of the target substance. The relative concentration of the target substance is, for example, the ratio of the measured value of the target substance to the measured value of the internal standard (e.g., relative area value CE or relative area value LC, described later). The relative concentration of the target substance can be this ratio obtained from one sample or the average of this ratio obtained from two or more samples. It should be noted that two or more samples were prepared from the same analyte.

[0259] Substance No. 32 in the substance group of this invention consists of two substances (octanamine and dopamine). Regarding substance No. 32 in the substance group of this invention, these two substances are treated as a whole, as one substance. Therefore, the concentration of substance No. 32 in the substance group of this invention refers to the combined concentration of these two substances.

[0260] The substance No. 34 in the material group of this invention consists of two substances (2'-deoxyadenosine and 5'-deoxyadenosine). Regarding the substance No. 34 in the material group of this invention, these two substances are treated as a whole, as one substance. Therefore, the concentration of the substance No. 34 in the material group of this invention refers to the combined concentration of these two substances.

[0261] The substance No. 37 in the group of substances of this invention consists of two substances (2-aminoisobutyric acid and 2-aminobutyric acid). Regarding the substance No. 37 in the group of substances of this invention, these two substances are treated as a whole, as one substance. Therefore, the concentration of the substance No. 37 in the group of substances of this invention refers to the combined concentration of these two substances.

[0262] In this invention, "glutathione (GSH)" (see No. 36) refers to the reduced form.

[0263] In this invention, "DL" (refer to No. 54) refers to a mixture of D-body and L-body.

[0264] Methods for determining the target substance include, for example, liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), gas chromatography-mass spectrometry (GC-MS), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), among which CE-MS or LC-MS are preferred. CE-MS is suitable for the determination of water-soluble substances, while LC-MS is suitable for the determination of lipid-soluble substances. Among CE-MS methods, capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) is particularly preferred. Among LC-MS methods, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is particularly preferred.

[0265] The sample used in the method for determining the target substance can be prepared from the analyte using conventional methods. The sample used in the method for determining the target substance may contain an internal standard. The relative concentration of the target substance can be determined, for example, as the ratio of the measured value of the target substance to the measured value of the internal standard. The absolute concentration of the target substance can be determined, for example, by pre-preparing a standard curve showing the relationship between the absolute concentration and the relative concentration of the target substance, and then using the relative concentration of the target substance.

[0266] In one embodiment, the method for determining the target substance is CE-MS (preferably CE-FTMS). The sample used in CE-MS can be prepared from the analyte using conventional methods, but is preferably prepared from the analyte according to the methods described in the examples. The determination using CE-MS can be performed under normal conditions, preferably under the conditions described in the examples. The sample used in CE-MS may contain an internal standard. Examples of internal standards include compounds obtained by labeling the target substance with a stable isotope such as DL-phenyl-D5-alanine, fatty acid methyl esters (e.g., methyl myristate), ribitol, and internal standards from Human Metabolome Technologies (HMT) (H3304-1002; HMT).

[0267] In embodiments where the target substance is determined using CE-MS (preferably CE-FTMS), the relative concentration of the target substance can be determined, for example, as the ratio of the peak area of ​​the target substance obtained by CE-MS to the peak area of ​​the internal standard obtained by CE-MS (referred to as the "relative area value CE"). The relative concentration of the target substance can be the relative area value CE obtained from one sample, or the average of the relative area values ​​CE obtained from two or more samples. The peak area of ​​the internal standard can be determined, for example, as the peak area of ​​a compound having a specified m / z value (which corresponds to the internal standard). The peak area of ​​the target substance can be determined, for example, as the peak area of ​​a compound having a specified m / z value (which corresponds to the target substance). The absolute concentration of the target substance can be determined, for example, by pre-preparing a standard curve showing the relationship between the absolute concentration and the relative concentration of the target substance, and then using the relative concentration of the target substance.

[0268] In another embodiment, the method for determining the target substance is LC-MS (preferably LC-MS / MS). The sample used in LC-MS can be prepared from the analyte using conventional methods, preferably according to the method described in the examples. The determination using LC-MS can be performed under normal conditions, preferably according to the conditions described in the examples. The sample used in LC-MS may contain an internal standard. Examples of internal standards include compounds obtained by labeling the target substance with a stable isotope such as DL-phenyl-D5-alanine, fatty acid methyl esters (e.g., methyl myristate), ribitol, and internal standards from Human Metabolome Technologies (HMT) (H3304-1002; HMT).

[0269] In embodiments where the target substance is determined by LC-MS (preferably LC-MS / MS), the relative concentration of the target substance can be determined, for example, as the ratio of the peak area of ​​the target substance obtained by LC-MS to the peak area of ​​the internal standard obtained by LC-MS (hereinafter referred to as "relative area value LC"). The relative concentration of the target substance can be the relative area value LC obtained from one sample, or the average of the relative area values ​​LC obtained from two or more samples. The peak area of ​​the internal standard can be determined, for example, as the peak area of ​​a compound having a specified m / z value (which corresponds to the internal standard). The peak area of ​​the target substance can be determined, for example, as the peak area of ​​a compound having a specified m / z value (which corresponds to the target substance). The absolute concentration of the target substance can be determined from the relative concentration of the target substance by pre-preparing a standard curve showing the relationship between the absolute concentration and the relative concentration of the target substance.

[0270] The determination of substances No. 1 to No. 38 in the substance group of the present invention is preferably performed by CE-MS (especially CE-FTMS), and the determination of substances No. 39 to No. 61 in the substance group of the present invention is preferably performed by LC-MS (especially LC-MS / MS). The m / z values ​​of each substance are shown in Table 9.

[0271] When measuring two or more substances (substance 1, substance 2, ..., substance n (n is an integer of 2 or more)) in the test sample, and calculating the composite variable as the statistical analysis value based on the concentration of the two or more substances in the test sample, the composite variable is preferably calculated according to the following formula (1).

[0272] Composite variable = (coefficient 1 × (concentration of the first substance)) + (coefficient 2 × (concentration of the second substance)) + ... + (coefficient n × (concentration of the nth substance)) + constant term ... (1)

[0273] In formula (1), "concentration" can be a relative concentration or an absolute concentration, preferably an absolute concentration. When the determination method for each substance is CE-MS (preferably CE-FTMS), the relative concentration of each substance is, for example, a relative area value CE. The relative concentration of each substance can be a relative area value CE obtained from one measured sample, or the average of relative area values ​​CE obtained from two or more measured samples. When the determination method for each substance is LC-MS (preferably LC-MS / MS), the relative concentration of each substance is, for example, a relative area value LC. The relative concentration of each substance can be a relative area value LC obtained from one measured sample, or the average of relative area values ​​LC obtained from two or more measured samples.

[0274] There are no special restrictions on n as long as it is an integer greater than or equal to 2. For example, it can be an integer greater than or equal to 2 and less than or equal to 61 (e.g., 3, 4, 6, 10, 20, 30, 40, 50, 61).

[0275] The coefficients and constants vary depending on the method of substance determination, the type of substance concentration (absolute or relative), and the quantity and type of substances in the combination. The coefficients and constants can be obtained through multinomial logistic regression analysis. The coefficients are partial regression coefficients obtained through multinomial logistic regression analysis. The method for obtaining the coefficients and constants through multinomial logistic regression analysis is well-known to those skilled in the art. Regarding this method, for example, one can refer to Metab BrainDis, DOI 10.1007 / s11011-017-0029-x, Published online: 11 May 2017, PNAS, October 14, 2003, vol.100, no.21, pp.12343-12348 (www.pnas.org / cgi / doi / 10.1073 / pnas.2033602100, AnnTransl Med, 2019, 7(16):388, p.1-10 (http: / / dx.doi.org / 10.21037 / atm.2019.07.102), etc. Regarding the coefficients used when calculating the composite variable, they can be derived from the measurement data in a way that makes the composite variable close to 1 in the group with reduced intestinal barrier function and close to 0 in the group with normal intestinal barrier function.

[0276] The test sample obtained from the subject is urine, and the biomarker measured in step 1a includes two or more substances selected from No. 1 to No. 4 of the substance group of the present invention (4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine). When the relative area value (relative concentration) of each of the two or more substances is determined by CE-FTMS according to the conditions described in the examples, for example, the coefficients and constants shown in Table 2 can be used to calculate the composite variable from the concentration of the two or more substances based on the formula (1). In Table 2, "CU_A" represents 4-acetaminophen, "CU_B" represents N-acetylputrescine, "CU_C" represents 3-amino-2-piperidinone, and "CU_D" represents O-succinylhomoserine.

[0277] [Table 2]

[0278]

[0279] The test sample from the subject is urine, and the biomarker measured in step 1a includes two or more substances selected from No. 39 to No. 42 of the present invention (cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzyl)sorbitol). When the relative area value (relative concentration) of each of the two or more substances is determined by LC-MS / MS under the conditions described in the examples, for example, the coefficients and constants shown in Table 3 can be used to determine the composite variable from the concentration of the two or more substances based on the formula (1). In Table 3, "LU_A" represents cyclopentaacetic acid, "LU_B" represents DK cypermethrin, "LU_C" represents paraxanthine, and "LU_D" represents bis(methylbenzyl)sorbitol.

[0280] [Table 3]

[0281]

[0282] <Step 1b>

[0283] Step 1b is to compare the measured value obtained in step 1a with the benchmark value to obtain the comparison result.

[0284] When measuring one substance from the analyte in step 1a, the measured value obtained in step 1a is the relative concentration or absolute concentration of that substance, preferably the absolute concentration. When the method for measuring the substance is CE-MS (preferably CE-FTMS), the relative concentration of the substance is, for example, the relative area value CE. The relative concentration of the substance can be the relative area value CE obtained from one sample, or the average of the relative area values ​​CE obtained from two or more samples. When the method for measuring the substance is LC-MS (preferably LC-MS / MS), the relative concentration of the substance is, for example, the relative area value LC. The relative concentration of the substance can be the relative area value LC obtained from one sample, or the average of the relative area values ​​LC obtained from two or more samples.

[0285] When two or more substances from the analyte are measured in step 1a, the measured values ​​obtained in step 1a are, for example, the relative concentrations or absolute concentrations of the two or more substances, preferably the absolute concentrations of the two or more substances. When the method for measuring each substance is CE-MS (preferably CE-FTMS), the relative concentration of each substance is, for example, the relative area value CE. The relative concentration of each substance can be the relative area value CE obtained from one measured sample, or the average of the relative area values ​​CE obtained from two or more measured samples. When the method for measuring each substance is LC-MS (preferably LC-MS / MS), the relative concentration of each substance is, for example, the relative area value LC. The relative concentration of each substance can be the relative area value LC obtained from one measured sample, or the average of the relative area values ​​LC obtained from two or more measured samples.

[0286] When measuring two or more substances from the analyte of the object in step 1a, the measured value obtained in step 1a is, for example, a composite variable derived from the concentrations of the two or more substances, preferably a composite variable derived from the concentrations of the two or more substances based on the formula (1). The concentration used to derive the composite variable can be a relative concentration or an absolute concentration, but an absolute concentration is preferred.

[0287] In step 1a, if one substance in the test sample from the object is measured, in step 1b, the measured value of the one substance is compared with the reference value of the one substance to obtain a comparison result.

[0288] When the measured value of a substance is a relative concentration, the reference value for that substance is also a relative concentration. The relative concentration of the latter is synonymous with the relative concentration of the former, and is calculated in the same way. When the measured value of a substance is a relative area value (CE), the reference value for that substance is also a relative area value (CE). When the measured value of a substance is a relative area value (LC), the reference value for that substance is also a relative area value (LC).

[0289] When the measured value of a substance is an absolute concentration, the reference value of that substance is also an absolute concentration. The absolute concentration of the latter is synonymous with the absolute concentration of the former, and is calculated in the same way.

[0290] In step 1a, when two or more substances from the test sample are measured, in step 1b, the measured values ​​of each of the two or more substances are compared with their respective reference values ​​to obtain a comparison result. For example, in step 1a, when substances No. 1 and No. 2 in the substance group of the present invention are measured, in step 1b, the measured value of substance No. 1 is compared with its reference value, and the measured value of substance No. 2 is compared with its reference value to obtain a comparison result.

[0291] When the measured values ​​of two or more substances are relative concentrations, the reference values ​​for each substance are also relative concentrations. The relative concentration of the latter is synonymous with the relative concentration of the former, and is calculated in the same way. When the measured values ​​of each substance are relative area values ​​(CE), the reference values ​​for each substance are also relative area values ​​(CE). When the measured values ​​of each substance are relative area values ​​(LC), the reference values ​​for each substance are also relative area values ​​(LC).

[0292] When the measured values ​​of two or more substances are absolute concentrations, the measured values ​​of the two or more substances are also absolute concentrations. The absolute relative concentration of the latter is synonymous with the relative concentration of the former, and is calculated in the same way.

[0293] When the measured values ​​of two or more substances are composite variables derived from the concentrations of the two or more substances (preferably based on the composite variable derived from the concentrations of the two or more substances using equation (1)), the reference values ​​of the two or more substances are also composite variables derived from the concentrations of the two or more substances (preferably based on the composite variable derived from the concentrations of the two or more substances using equation (1)). The latter composite variable is synonymous with the former composite variable and is derived in the same way.

[0294] <Step 1c>

[0295] Step 1c is a step to evaluate the intestinal barrier function of the subjects based on the comparison results obtained in step 1b.

[0296] In Scheme 1, "positive" means that the subject's intestinal barrier function is reduced, and "negative" means that the subject's intestinal barrier function is not reduced.

[0297] The intestinal barrier function to be evaluated can be the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, with the small intestine being the preferred candidate.

[0298] The intestinal barrier function, particularly the physical barrier function such as tight junction structures, is the subject of evaluation.

[0299] Regarding each substance selected from the group of substances of this invention, the intestinal barrier function of the subject can be evaluated based on whether the comparison results obtained in step 1b meet the criteria shown in Table 4. Specifically, for each substance whose criterion is "high" as shown in Table 4, if the measured value obtained in step 1a is higher than the criterion value (i.e., the comparison results obtained in step 1b meet the criteria shown in Table 4), it can be evaluated as positive; if the measured value obtained in step 1a is lower than the criterion value (i.e., the comparison results obtained in step 1b do not meet the criteria shown in Table 4), it can be evaluated as negative. Furthermore, for substances whose criterion is "low" as shown in Table 4, if the measured value obtained in step 1a is lower than the criterion value (i.e., the comparison results obtained in step 1b meet the criteria shown in Table 4), it can be evaluated as positive; if the measured value obtained in step 1a is higher than the criterion value (i.e., the comparison results obtained in step 1b do not meet the criteria shown in Table 4), it can be evaluated as negative. It should be noted that the criteria shown in Table 4 can also be applied to schemes other than Scheme 1.

[0300] In step 1a, if one substance selected from the group of substances of the present invention is measured, the result of the comparison of the substance can be evaluated as positive if it meets the criteria shown in Table 4, and the result of the comparison of the substance can be evaluated as negative if it does not meet the criteria shown in Table 4.

[0301] In step 1a, if two or more substances selected from the group of substances of the present invention are measured, a positive result can be evaluated if the comparison result of at least one of the two or more substances meets the criteria shown in Table 4; a negative result can be evaluated if the comparison result of any of the two or more substances does not meet the criteria shown in Table 4. The more substances whose comparison results meet the criteria shown in Table 4, the higher the probability of a positive result. Therefore, in step 1a, by measuring two or more substances selected from the group of substances of the present invention (e.g., two, three, or four or more), the accuracy of evaluating the intestinal barrier function of the subject can be improved.

[0302] [Table 4-1]

[0303] Table 2 (Benchmark)

[0304] No. substance name benchmark 1 4-Acetaminobutyric acid Low 2 N-acetylputrescine Low 3 3-Amino-2-piperidinone Low 4 O-Succinylhomoserine Low 5 sulfotyrosine Low 6 malic acid Low 7 Tyrosine Low 8 N-Carbamoyl Aspartic Acid Low 9 Musciol Low 10 mucic acid Low 11 N-acetylaspartic acid Low 12 Arginine succinic acid Low 13 adipic acid Low 14 2-Oxadipic acid Low 15 Phenylalanine Low 16 Succinic acid Low 17 N-acetylglutamic acid Low 18 Glycyl aspartic acid Low 19 N-acetylornithine Low 20 Ascorbic acid 2-sulfate Low 21 Asymmetric dimethylarginine Low 22 N-acetylneuraminic acid Low 23 2-Hydroxyglutaric acid Low 24 Cyclohexane carboxylic acid Low 25 5-Methylcytosine Low 26 cis-aconitine Low 27 glycine Low 28 Tryptophan Low 29 Tyramine Low 30 Glycerol 2-phosphate Low 31 Citric acid Low 32 Octylamine and dopamine Low 33 clove acid Low 34 2'-Deoxyadenosine and 5'-Deoxyadenosine Low 35 Adenosine diphosphate Low 36 Glutathione Low 37 2-Aminoisobutyric acid and 2-aminobutyric acid Low 38 Sinapic acid Low 39 Cyclopentaacetic acid Low 40 DK methamidophos Low

[0305] [Table 4-2]

[0306] Table 4 (continued)

[0307] No. substance name benchmark 41 paraxanthine Low 42 Bis(methylbenzylidene)sorbitol high 43 caffeine Low 44 2-Aminooctanoic acid high 45 (R)-Equivalerate high 46 Theophylline Low 47 Guanidinosuccinic acid high 48 Uracil high 49 1-Phenylacetyl-3-methyl-5-pyrazolone Low 50 5α-Androstane-3,6,17-trione Low 51 Methylmalonic acid Low 52 N-acetyl-L-cysteine high 53 Menaquinone Low 54 DL-stachyine high 55 adenosine high 56 arecoline high 57 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione Low 58 Pyrazinamide Low 59 Prolylleucine high 60 Norethindrone high 61 2-Methylbenzoic acid high

[0308] The reference values ​​for each substance selected from the group of substances of this invention can be set using conventional methods.

[0309] In one embodiment, for each substance selected from the group of substances of the present invention, the average value of the measured values ​​of each substance in the test subjects obtained from a negative group (a group of individuals whose intestinal barrier function is not reduced) consisting of individuals of the same species as the subject (e.g., if the subject is a human, then a human) will be used as the reference value for each substance. Hereinafter, this embodiment (hereinafter referred to as "Embodiment 1-1") will be described.

[0310] When forming a negative group, the determination of negative and positive can be made using well-known methods for examining intestinal barrier function (e.g., lactulose-mannitol test).

[0311] If the test sample used in step 1a is urine, the test sample obtained from the negative group is also urine.

[0312] The measured values ​​of each substance in the test sample obtained from the negative group are synonymous with the measured values ​​obtained in step 1a, and are calculated in the same way as in step 1a.

[0313] When the measured value obtained in step 1a is the relative concentration of one substance selected from the group of substances of the present invention, the reference value of that one substance used in step 1b is the average of the relative concentrations of that one substance in the test sample obtained from the negative group. The relative concentration of the latter is synonymous with the relative concentration of the former and is calculated in the same way. When the relative concentration of the former is the relative area value CE, the relative concentration of the latter is also the relative area value CE. When the relative concentration of the former is the relative area value LC, the relative concentration of the latter is also the relative area value LC.

[0314] If the measured value obtained in step 1a is the absolute concentration of one substance selected from the group of substances of the present invention, the reference value of that one substance used in step 1b is the average absolute concentration of that one substance in the test sample obtained from the negative group. The absolute concentration of the latter is synonymous with the absolute concentration of the former and is calculated in the same way as the absolute concentration of the former.

[0315] When the measured value obtained in step 1a is the relative concentration of two or more substances selected from the material group of the present invention, the reference value for each substance used in step 1b is the average of the relative concentrations of each substance in the analyte obtained from the negative group. The relative concentration of the latter is synonymous with the relative concentration of the former and is calculated in the same way. When the relative concentration of the former is the relative area value CE, the relative concentration of the latter is also the relative area value CE. When the relative concentration of the former is the relative area value LC, the relative concentration of the latter is also the relative area value LC.

[0316] When the measured value obtained in step 1a is the absolute concentration of two or more substances selected from the material group of the present invention, the reference value of each substance used in step 1b is the average value of the absolute concentration of each substance in the test sample obtained from the negative group.

[0317] Regarding each substance selected from the group of substances of the present invention, it is preferable to evaluate the intestinal barrier function of the subject based on whether the comparison results obtained in step 1b meet the criteria shown in Table 5.

[0318] Specifically, in Table 5, for substances with a benchmark of "high," a preferred benchmark of x times or more, a more preferred benchmark of y times or more, and a further preferred benchmark of z times or more, if the measured value obtained in step 1a is x times or more of the average value, it is preferably evaluated as positive; if the measured value obtained in step 1a is y times or more of the average value, it is more preferably evaluated as positive; and if the measured value obtained in step 1a is z times or more of the average value, it is even more preferably evaluated as positive. Furthermore, in Table 5, for substances with a benchmark of "low," a preferred benchmark of x times or less, a more preferred benchmark of y times or less, and a further preferred benchmark of z times or less, if the measured value obtained in step 1a is x times or less of the average value, it is preferably evaluated as positive; if the measured value obtained in step 1a is y times or less of the average value, it is more preferably evaluated as positive; and if the measured value obtained in step 1a is z times or less of the average value, it is even more preferably evaluated as positive.

[0319] Taking the No.1 substance in the material group of the present invention as an example, if the measured value obtained in step 1a is less than 0.9 times the average value, it is preferably evaluated as positive; if the measured value obtained in step 1a is less than 0.8 times the average value, it is more preferably evaluated as positive; and if the measured value obtained in step 1a is less than 0.6 times the average value, it is even more preferably evaluated as positive.

[0320] Taking substance No. 42 in the material group of the present invention as an example, if the measured value obtained in step 1a is more than 1.5 times the average value, it is preferably evaluated as positive; if the measured value obtained in step 1a is more than 2.0 times the average value, it is more preferably evaluated as positive; and if the measured value obtained in step 1a is more than 3.0 times the average value, it is even more preferably evaluated as positive.

[0321] [Table 5-1]

[0322]

[0323] [Table 5-2]

[0324]

[0325] [Table 5-3]

[0326]

[0327] In another embodiment, for each substance selected from the group of substances of the present invention, the median value of the measured values ​​of each substance in the test subjects obtained from a negative group (a group of individuals whose intestinal barrier function is not reduced) consisting of individuals of the same species as the subject (e.g., if the subject is a human, then a human) will be used as the reference value for each substance. Hereinafter, this embodiment (hereinafter referred to as "Embodiment 1-2") will be described.

[0328] The description of Implementation 1-1 also applies to Implementation 1-2, unless otherwise specified. In application, "average value" is replaced with "median value".

[0329] In yet another embodiment, for each substance selected from the group of substances of the present invention, the cutoff value obtained from the ROC (Receiver Operating Characteristic) curve of each substance can be used as the reference value for each substance. Hereinafter, this embodiment (hereinafter referred to as "Embodiments 1-3") will be described.

[0330] ROC curves for each substance can be constructed using conventional methods based on the concentrations of each substance in the analyte obtained from individuals of the same species as the subject (e.g., humans if the subject is human). The negative group (individuals with unreduced intestinal barrier function) and the positive group (individuals with reduced intestinal barrier function) are composed of individuals with the same species as the subject (e.g., humans). In the ROC curves, the vertical axis represents sensitivity, and the horizontal axis represents (1-specificity). The determination of negative and positive groups can be made using well-known methods for assessing intestinal barrier function (e.g., the lactulose-mannitol test).

[0331] If the test sample used in step 1a is urine, the test samples obtained from the negative group and the positive group are also urine.

[0332] When the measured value obtained in step 1a is the relative concentration of one substance selected from the group of substances of the present invention, the concentration of that substance used in the preparation of the ROC curve of that substance is also a relative concentration. The relative concentration of the latter is synonymous with the relative concentration of the former, and is calculated in the same way as the relative concentration of the latter. When the relative concentration of the former is the relative area value CE, the relative concentration of the latter is also the relative area value CE. When the relative concentration of the former is the relative area value LC, the relative concentration of the latter is also the relative area value LC. When the concentration used in the preparation of the ROC curve of that substance is a relative concentration, the cutoff value obtained from the ROC curve of that substance is also a relative concentration.

[0333] When the measured value obtained in step 1a is the absolute concentration of one substance selected from the group of substances of the present invention, the concentration of that substance used in the preparation of the ROC curve of that substance is also an absolute concentration. The absolute concentration of the latter is calculated in the same way as the absolute concentration of the former. When the concentration used in the preparation of the ROC curve of that substance is an absolute concentration, the cutoff value obtained from the ROC curve of that substance is also an absolute concentration.

[0334] When the measured values ​​obtained in step 1a are relative concentrations of two or more substances selected from the group of substances of this invention, the concentrations of each substance used in the preparation of the ROC curves for each substance are also relative concentrations. The latter relative concentration is synonymous with the former relative concentration and is calculated in the same manner. If the relative concentration of the former is a relative area value CE, the relative concentration of the latter is also a relative area value CE. If the relative concentration of the former is a relative area value LC, the relative concentration of the latter is also a relative area value LC. When the concentrations of each substance used to prepare the ROC curves for each substance are relative concentrations, the cutoff values ​​obtained from the ROC curves of each substance are also relative concentrations.

[0335] When the measured values ​​obtained in step 1a are absolute concentrations of two or more substances selected from the group of substances of this invention, the concentrations of each substance used to prepare the ROC curves for each substance are also absolute concentrations. The absolute concentrations of the latter are calculated in the same way as the absolute concentrations of the former. When the concentrations used to prepare the ROC curves for each substance are absolute concentrations, the cutoff values ​​obtained from the ROC curves of each substance are also absolute concentrations.

[0336] The cutoff value is a value that satisfies both high sensitivity and high specificity when evaluated against a certain benchmark. The cutoff value can be appropriately set considering the balance between sensitivity and specificity. For example, a cutoff value can be set as the value that shows a high positive rate in the positive group and a high negative rate in the negative group. Methods for setting the cutoff value are well known to those skilled in the art. For example, methods such as using the point on the ROC curve with the smallest distance from the upper left corner of the ROC curve as the cutoff value, or using the point on the ROC curve where the value of (sensitivity + specificity - 1) becomes the maximum value as the cutoff value, are known. It should be noted that the point on the ROC curve where the value of (sensitivity + specificity - 1) becomes the maximum value is called the Younden index.

[0337] Specific examples of cutoff values ​​for each substance selected from the material group of the present invention are shown in Table 9.

[0338] The cutoff values ​​for each substance selected from the group of substances of the present invention are preferably values ​​within the range of above the value shown in P1 of Table A and below the value shown in P3 of Table A. For example, the cutoff values ​​for each substance selected from the group of substances of the present invention may be values ​​within the range of above the value shown in P1 of Table A and below the value shown in P2 of Table A, or values ​​within the range of above the value shown in P2 of Table A and below the value shown in P3 of Table A. The cutoff values ​​for each substance selected from the group of substances of the present invention may be values ​​shown in P1 of Table A, values ​​shown in P2 of Table A, or values ​​shown in P3 of Table A.

[0339] [Table A-1]

[0340]

[0341] [Table A-2]

[0342]

[0343] [Table A-3]

[0344]

[0345] In another embodiment, regarding two or more substances selected from the group of substances of the present invention, the concentrations of the two or more substances in the test sample obtained from a negative group (a group of individuals whose intestinal barrier function is not reduced) and a positive group (a group of individuals whose intestinal barrier function is reduced) consisting of individuals of the same species as the subject (e.g., if the subject is a human, then a human). Based on the above formula (1), a composite variable is obtained from the concentrations of the two or more substances, and an ROC curve is generated based on the obtained composite variable. The cutoff value obtained from the generated ROC curve is used as the reference value for the two or more substances. Hereinafter, this embodiment (hereinafter referred to as "Embodiments 1-4") will be described.

[0346] Embodiments 1-4 are applicable to cases where the measured value obtained in step 1a is a composite variable derived from the concentrations of two or more substances selected from the group of substances of the present invention based on the formula (1).

[0347] When the measured value obtained in step 1a is a composite variable derived from the relative concentrations of two or more substances selected from the group of substances of the present invention based on Equation (1), the concentration used when determining the cutoff value is also a relative concentration. The relative concentration of the latter is synonymous with the relative concentration of the former, and is determined in the same way as the relative concentration of the former. When the relative concentration of the former is the relative area value CE, the relative concentration of the latter is also the relative area value CE. When the relative concentration of the former is the relative area value LC, the relative concentration of the latter is also the relative area value LC.

[0348] When the measured value obtained in step 1a is a composite variable derived from the absolute concentrations of two or more substances selected from the group of substances of the present invention based on the formula (1), the concentration used when determining the cutoff value is also an absolute concentration. The absolute concentration of the latter is synonymous with the absolute concentration of the former, and is determined in the same way as the absolute concentration of the former.

[0349] When forming negative and positive groups, the determination of negative and positive can be performed using known methods for examining intestinal barrier function (e.g., lactulose-mannitol test). ROC curves can be constructed using conventional methods based on composite variables obtained from the negative and positive groups. In the ROC curve, the vertical axis represents sensitivity, and the horizontal axis represents (1-specificity). When forming negative and positive groups, the determination of negative and positive can be performed using known methods for examining intestinal barrier function (e.g., lactulose-mannitol test). The explanation of cutoff values ​​is the same as in embodiments 1-3.

[0350] The test sample is urine, and the biomarker measured in step 1a includes two or more substances selected from No. 1 to No. 4 of the substance group of the present invention (4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine). When the relative area values ​​(relative concentrations) of each of the two or more substances are determined by CE-FTMS under the conditions described in the examples, the specific examples of the coefficients and constants are shown in Table 2, and the specific examples of the cutoff values ​​are shown in Table 6. In Table 6, "CU_A", "CU_B", "CU_C", and "CU_D" are synonymous with those in Table 2.

[0351] [Table 6]

[0352] Table 6 (Materials No. 1 to No. 4 of the Invention)

[0353]

[0354] The cutoff values ​​for No. 1 to No. 11 in Table 6 are preferably values ​​within the range of above the value shown in Q1 of Table B and below the value shown in Q3 of Table B. For example, the cutoff values ​​for No. 1 to No. 11 in Table 6 can be values ​​within the range of above the value shown in Q1 of Table B and below the value shown in Q2 of Table B, or values ​​within the range of above the value shown in Q2 of Table B and below the value shown in Q3 of Table B. The cutoff values ​​for No. 1 to No. 11 in Table 6 can be values ​​shown in Q1 of Table B, values ​​shown in Q2 of Table B, or values ​​shown in Q3 of Table B.

[0355] [Table B]

[0356] Table B

[0357] No. Q1 Q2 Q3 1 0.0407 0.0582 0.0757 2 0.0183 0.0262 0.0341 3 -0.3767 -0.2897 -0.2028 4 -0.4291 -0.3301 -0.2311 5 -0.0631 -0.0485 -0.0340 6 -0.5815 -0.4473 -0.3131 7 -0.1028 -0.0791 -0.0554 8 -0.4928 -0.3791 -0.2654 9 -0.2204 -0.1695 -0.1187 10 -0.8132 -0.6255 -0.4379 11 -0.2791 -0.2147 -0.1503

[0358] The test sample is urine, and the biomarkers measured in step 1a include two or more substances selected from No. 39 to No. 42 of the present invention (cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzyl)sorbitol). When the relative area values ​​(relative concentrations) of each of the two or more substances are determined by LC-MS / MS under the conditions described in the examples, the specific examples of coefficients and constants are shown in Table 3, and the specific examples of cutoff values ​​are shown in Table 7. In Table 7, "LU_A", "LU_B", "LU_C", and "LU_D" are synonymous with those in Table 3.

[0359] [Table 7]

[0360] Table 7 (Materials No. 39 to No. 42 of the present invention)

[0361]

[0362] The cutoff values ​​for No. 1 to No. 11 in Table 7 are preferably values ​​within the range of above the value shown in R1 of Table C and below the value shown in R3 of Table C. For example, the cutoff values ​​for No. 1 to No. 11 in Table 7 can be values ​​within the range of above the value shown in R1 of Table C and below the value shown in R2 of Table C, or values ​​within the range of above the value shown in R2 of Table C and below the value shown in R3 of Table C. The cutoff values ​​for No. 1 to No. 11 in Table 7 can be values ​​shown in R1 of Table C, values ​​shown in R2 of Table C, or values ​​shown in R3 of Table C.

[0363] [Table C]

[0364] Table C

[0365] No. R1 R2 R3 1 -0.2014 -0.1549 -0.1084 2 -0.2500 -0.1923 -0.1346 3 0.0774 0.1105 0.1437 4 -0.5295 -0.4073 -0.2851 5 -0.1491 -0.1147 -0.0803 6 0.0232 0.0332 0.0432 7 -0.1988 -0.1529 -0.1070 8 -0.1780 -0.1369 -0.0958 9 0.0088 0.0126 0.0164 10 -0.2302 -0.1771 -0.1240 11 0.2694 0.3848 0.5002

[0366] Option 2

[0367] Scheme 2 of the present invention relates to a method for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function in a subject. Hereinafter, "diseases or symptoms caused by a decrease in intestinal barrier function" will sometimes be referred to as "diseases or symptoms".

[0368] The method of Scheme 2 of the present invention includes the following steps:

[0369] (2a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0370] (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain the comparison result; and

[0371] (2c) Based on the comparison results obtained in step 2b, the steps for diagnosing the disease or symptoms caused by decreased intestinal barrier function in the subject.

[0372] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0373] The explanation of Scheme 1 also applies to Scheme 2, unless otherwise specified. When applying, replace "Step 1a" with "Step 2a", "Step 1b" with "Step 2b", "Step 1c" with "Step 2c", "evaluate the intestinal barrier function of the subject" with "diagnose the disease or symptoms of the subject", "accuracy of evaluating the intestinal barrier function of the subject" with "accuracy of diagnosing the disease or symptoms of the subject", "positive evaluation" with "positive diagnosis", and "negative evaluation" with "negative diagnosis".

[0374] In Option 2, "positive" means that the subject has contracted the disease or symptoms, and "negative" means that the subject has not contracted the disease or symptoms.

[0375] The disease or symptom being diagnosed can be caused by a decrease in the intestinal barrier function of the small intestine or the large intestine, preferably caused by a decrease in the intestinal barrier function of the small intestine.

[0376] Diseases or symptoms caused by a decrease in intestinal barrier function include, for example, enteritis, allergic diseases, mental illness, non-alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome, obesity, etc.

[0377] Examples of enteritis include irritable bowel syndrome, ulcerative colitis, and Crohn's disease. Examples of allergic diseases include food allergies, atopic dermatitis, and asthma. Examples of mental illnesses include anxiety disorders and depression. Examples of non-alcoholic fatty liver disease include non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0378] Diagnosis is typically performed by a physician. Based on the comparison results obtained in step 2b, the physician diagnoses whether the subject has developed a disease or symptoms caused by reduced intestinal barrier function. If the subject has developed a disease or symptoms caused by reduced intestinal barrier function, the physician can determine whether prevention or treatment is necessary, select preventive or therapeutic methods, and implement preventive or therapeutic measures. "Prevention" includes preventing, suppressing, and delaying the occurrence of disease or symptoms. "Treatment" includes suppressing the development or worsening of disease or symptoms, delaying the development or worsening of disease or symptoms, and alleviating, reducing, improving, and curing disease or symptoms.

[0379] The method of Scheme 2 of the present invention can also be used to assist in the diagnosis of diseases or symptoms caused by a decrease in intestinal barrier function. The act of assisting in the diagnosis of diseases or symptoms is the act of providing the comparison results obtained in step 2b as data useful for the diagnosis of the disease or symptoms; it can be a medical act or a non-medical act, but is usually a non-medical act. In addition to the data provided by the acts of assisting in the diagnosis of diseases or symptoms, a doctor's diagnosis can also be based on one or more other pieces of information. Furthermore, the doctor's experience, know-how, etc., can also participate in the doctor's diagnosis.

[0380] Option 3

[0381] Solution 3 of the present invention relates to a method for treating a disease or symptoms caused by a decrease in intestinal barrier function in a subject. Hereinafter, "disease or symptoms caused by a decrease in intestinal barrier function" will sometimes be referred to as "disease or symptoms".

[0382] The method of Scheme 3 of the present invention includes the following steps:

[0383] (3a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0384] (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result;

[0385] (3c) Based on the comparison results obtained in step 3b, the step of diagnosing the disease or symptoms caused by decreased intestinal barrier function in the subject; and

[0386] (3d) In cases where a subject is diagnosed with a disease or symptoms caused by decreased intestinal barrier function, the procedure of administering to the subject a substance or composition for treating the disease or symptoms caused by decreased intestinal barrier function.

[0387] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0388] The explanation of Scheme 2 also applies to Scheme 3, unless otherwise specified. When applying, "Step 2a" should be replaced with "Step 3a", "Step 2b" with "Step 3b", and "Step 2c" with "Step 3c".

[0389] A substance or composition for treating a disease or symptom caused by a decrease in intestinal barrier function, administered to a subject diagnosed with such a disease or symptom, may be appropriately selected based on the type of disease or symptom caused by the decrease in intestinal barrier function that the subject has. “Treatment” includes the suppression of the development or worsening of the disease or symptom, the delay of the development or worsening of the disease or symptom, and the relief, reduction, improvement, and cure of the disease or symptom.

[0390] Examples of substances or compositions for treating irritable bowel syndrome include, for example, high molecular weight polymers and serotonin receptor antagonists. Examples of substances or compositions for treating ulcerative colitis or Crohn's disease include, for example, 5-aminosalicylic acid preparations and corticosteroids. Examples of substances or compositions for treating allergic diseases include, for example, antihistamines. Examples of substances or compositions for treating anxiety disorders or depression include, for example, selective serotonin reuptake inhibitors and norepinephrine-stimulating antidepressants. Examples of substances or compositions for treating non-alcoholic fatty liver disease include, for example, antioxidants, diabetes medications, and medications for treating lipid metabolism disorders.

[0391] Substances or compositions used to treat diseases or symptoms may be administered in the form of food compositions containing such substances or compositions. Examples of food compositions include yogurt, probiotics, prebiotics, and supplements. Food compositions may also be, for example, foods for specific health purposes, functionally labeled foods, or foods for patients.

[0392] Routes of administration for substances or compositions used to treat diseases or symptoms may include, for example, oral, non-oral (e.g., intranasal, ophthalmic, ocular, dermal, respiratory, rectal, urinary, subcutaneous, intramuscular, intravenous), etc. Substances or compositions used to treat diseases or symptoms may be formulated into administration forms suitable for the route of administration. Administration forms may include, for example, tablets, injections, etc. During formulation, additives suitable for oral administration (e.g., excipients, disintegrants, lubricants, binders, etc.) or additives suitable for non-oral administration (e.g., diluents, solvents, etc.) may be used. The dosage and frequency of administration of substances or compositions used to treat diseases or symptoms may be appropriately adjusted considering the administration form, the age and weight of the recipient, etc. Administration may be once a day or more than twice a day. Administration may be administered for one day or more than two days (e.g., more than one week, more than two weeks, more than three weeks, or more than four weeks).

[0393] Option 4

[0394] Scheme 4 of the present invention relates to a method for evaluating the risk of a subject developing a disease or symptoms caused by a decrease in intestinal barrier function, or the likelihood that a subject has already developed said disease or symptoms. Hereinafter, the risk of a subject developing a disease or symptoms caused by a decrease in intestinal barrier function is sometimes referred to as "occurrence risk," and the likelihood that a subject has already developed a disease or symptoms caused by a decrease in intestinal barrier function is sometimes referred to as "potential risk."

[0395] Option 4 includes the following steps:

[0396] (4a) The step of measuring biomarkers from the test sample of the subject and obtaining the measured values ​​of the biomarkers; and

[0397] (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain the comparison result; and

[0398] (4c) Based on the comparison results obtained in step 4b, the step of evaluating the risk or likelihood of occurrence,

[0399] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0400] The explanation of Scheme 1 also applies to Scheme 4, unless otherwise specified. When applying, "Step 1a" should be replaced with "Step 4a", "Step 1b" with "Step 4b", "Step 1c" with "Step 4c", "the intestinal barrier function of the evaluated subject" should be replaced with "the risk or likelihood of occurrence", and "the accuracy of the intestinal barrier function of the evaluated subject" should be replaced with "the accuracy of the risk or likelihood of occurrence".

[0401] In scheme 4, "positive" means that the risk of infection or the possibility of contracting the disease exists or is high, and "negative" means that the risk of infection or the possibility of contracting the disease does not exist or is low.

[0402] The risk of occurrence for the evaluated subject can be the risk of the subject developing diseases or symptoms caused by a decrease in the intestinal barrier function of the small intestine, or the risk of the subject developing diseases or symptoms caused by a decrease in the intestinal barrier function of the large intestine, preferably the risk of the subject developing diseases or symptoms caused by a decrease in the intestinal barrier function of the small intestine.

[0403] The likelihood of a subject having a disease can be the likelihood that the subject has already contracted a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or the likelihood that the subject has already contracted a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, preferably the likelihood that the subject has already contracted a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0404] The description of diseases or symptoms caused by decreased intestinal barrier function is the same as in Scheme 2.

[0405] Option 5

[0406] Solution 5 of the present invention relates to a method for obtaining data on diseases or symptoms caused by a decrease in intestinal barrier function in a diagnostic subject. Hereinafter, "diseases or symptoms caused by a decrease in intestinal barrier function" will sometimes be referred to as "diseases or symptoms".

[0407] Option 5 includes the following steps:

[0408] (5a) The step of measuring biomarkers from the test sample of the subject and obtaining the measured values ​​of the biomarkers; and

[0409] (5b) The step of comparing the measured values ​​obtained in step 5a with the baseline values ​​and obtaining the comparison results as data for diagnosing the disease or symptoms caused by the reduction of intestinal barrier function in the subject.

[0410] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0411] The explanation of Scheme 1 also applies to Scheme 5, unless otherwise specified. When applying, "Step 1a" should be replaced with "Step 5a", "Step 1b" should be replaced with "Step 5b", "evaluate the intestinal barrier function of the subject" should be replaced with "obtain data for diagnosing the disease or symptoms of the subject", and "accuracy of evaluating the intestinal barrier function of the subject" should be replaced with "accuracy of diagnosing the disease or symptoms of the subject".

[0412] In scheme 5, "positive" means that the subject has contracted the disease or symptoms, and "negative" means that the subject has not contracted the disease or symptoms.

[0413] The disease or symptom being diagnosed can be caused by a decrease in the intestinal barrier function of the small intestine or the large intestine, preferably caused by a decrease in the intestinal barrier function of the small intestine.

[0414] The description of diseases or symptoms caused by decreased intestinal barrier function is the same as in Scheme 2.

[0415] Regarding each substance selected from the group of substances of this invention, comparison results that meet the criteria shown in Table 4 can be used as data for diagnosing a positive result, and comparison results that do not meet the criteria shown in Table 4 can be used as data for diagnosing a negative result. Specifically, for each substance for which the criteria shown in Table 4 is "high," a comparison result where the measured value obtained in step 5a is higher than the reference value can be used as data for diagnosing a positive result, and a comparison result where the measured value obtained in step 5a is lower than the reference value can be used as data for diagnosing a negative result. Furthermore, for substances for which the criteria shown in Table 4 is "low," a comparison result where the measured value obtained in step 5a is lower than the reference value can be used as data for diagnosing a positive result, and a comparison result where the measured value obtained in step 5a is higher than the reference value can be used as data for diagnosing a negative result.

[0416] In step 5a, if one substance selected from the group of substances of the present invention is measured, and the comparison result of the one substance meets the criteria shown in Table 4, the comparison result can be obtained as data for diagnosis of negative. If the comparison result of the one substance does not meet the criteria shown in Table 4, the comparison result can be obtained as data for diagnosis of negative.

[0417] In step 5a, when two or more substances selected from the group of substances of the present invention are measured, if the comparison result of at least one of the two or more substances meets the criteria shown in Table 4, the comparison result can be obtained as data for a positive diagnosis; if the comparison result of any of the two or more substances does not meet the criteria shown in Table 4, the comparison result can be obtained as data for a negative diagnosis. The more substances whose comparison results meet the criteria shown in Table 4, the higher the probability of a positive result. Therefore, in step 5a, by measuring two or more substances selected from the group of substances of the present invention (e.g., two, three, or four or more), the accuracy of diagnosing the disease or symptoms of the subject can be improved.

[0418] When Scheme 1, embodiment 1-1 is applied to Scheme 5, for each substance selected from the group of substances of the present invention, it is preferable to obtain comparison results based on the benchmarks shown in Table 5 as data for diagnosis.

[0419] Specifically, in Table 5, for substances with a benchmark of "high," a preferred benchmark of x times or more, a more preferred benchmark of y times or more, and a further preferred benchmark of z times or more, it is preferable to obtain a comparison result where the measured value obtained in step 5a is x times or more of the average value as data for diagnosing a positive result; more preferably, it is preferable to obtain a comparison result where the measured value obtained in step 5a is y times or more of the average value as data for diagnosing a positive result; and even more preferably, it is preferable to obtain a comparison result where the measured value obtained in step 5a is z times or more of the average value as data for diagnosing a positive result. Furthermore, in Table 5, for substances with a benchmark of "low," a preferred benchmark of x times or less, a more preferred benchmark of y times or less, and a further preferred benchmark of z times or less, it is preferable to obtain a comparison result where the measured value obtained in step 5a is x times or less of the average value as data for diagnosing a positive result; more preferably, it is preferable to obtain a comparison result where the measured value obtained in step 5a is y times or less of the average value as data for diagnosing a positive result; and even more preferably, it is preferable to obtain a comparison result where the measured value obtained in step 5a is z times or less of the average value as data for diagnosing a positive result.

[0420] Taking the No.1 substance in the substance group of the present invention as an example, it is preferable to obtain a comparison result of less than 0.9 times the average value of the measurement obtained in step 5a as the data for diagnosing a positive result, more preferably to obtain a comparison result of less than 0.8 times the average value of the measurement obtained in step 5a as the data for diagnosing a positive result, and even more preferably to obtain a comparison result of less than 0.6 times the average value of the measurement obtained in step 5a as the data for diagnosing a positive result.

[0421] Taking substance No. 42 in the material group of the present invention as an example, it is preferable to obtain a comparison result that is more than 1.5 times the average value of the measurement obtained in step 5a as data for diagnosing a positive result, more preferably to obtain a comparison result that is more than 2.0 times the average value of the measurement obtained in step 5a as data for diagnosing a positive result, and even more preferably to obtain a comparison result that is more than 3.0 times the average value of the measurement obtained in step 5a as data for diagnosing a positive result.

[0422] The data obtained in step 5b can be used to diagnose diseases or symptoms caused by decreased intestinal barrier function in the subject, or to assist in the diagnosis of diseases or symptoms caused by decreased intestinal barrier function in the subject.

[0423] Diagnosis is typically performed by a physician. Based on the data obtained in step 5b, the physician diagnoses whether the subject has developed a disease or symptoms caused by reduced intestinal barrier function. If the subject has developed a disease or symptoms caused by reduced intestinal barrier function, the physician can determine whether the subject needs prevention or treatment of the disease or symptoms, select methods for prevention or treatment, and implement prevention or treatment. "Prevention" includes preventing, suppressing, and delaying the occurrence of disease or symptoms. "Treatment" includes suppressing the development or worsening of disease or symptoms, delaying the development or worsening of disease or symptoms, and alleviating, reducing, improving, and curing disease or symptoms.

[0424] Actions that assist in the diagnosis of a disease or symptom are those that provide data obtained in step 5b as data for the diagnosis of a disease or symptom. These actions can be medical or non-medical, but are usually non-medical. A doctor's diagnosis may be based on one or more other types of data in addition to the data provided through actions that assist in the diagnosis of a disease or symptom.

[0425] Option 6

[0426] Scheme 6 of the present invention relates to a method for screening substances or compositions that have an effect on improving intestinal barrier function or have a preventive or ameliorative effect on diseases or symptoms caused by a decrease in intestinal barrier function. Hereinafter, "the preventive or ameliorative effect on diseases or symptoms caused by a decrease in intestinal barrier function" is sometimes referred to as "the preventive or ameliorative effect on diseases or symptoms".

[0427] Option 6 includes the following steps:

[0428] (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0429] (6b) The step of determining a biomarker in an analyte obtained from a subject after administration of a candidate substance or candidate composition, and obtaining a measured value of the biomarker; and

[0430] (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain the comparison result; and

[0431] (6d) Based on the comparison results obtained in step 6c, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0432] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0433] The explanation of Scheme 1 also applies to Scheme 6, unless otherwise specified. When applying, replace "Step 1a" with "Step 6b", "Step 1b" with "Step 6c", "Step 1c" with "Step 6d", "evaluate the intestinal barrier function of the subject" with "evaluate the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms", and "evaluate the accuracy of the intestinal barrier function of the subject" with "evaluate the accuracy of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms".

[0434] In scheme 6, "positive" means that the candidate substance or composition has an effect on improving intestinal barrier function or on preventing or improving disease or symptoms, while "negative" means that the candidate substance or composition does not have an effect on improving intestinal barrier function or on preventing or improving disease or symptoms. "Prevention" includes preventing, inhibiting, and delaying the occurrence of disease or symptoms. "Improvement" includes inhibiting the development or worsening of disease or symptoms, delaying the development or worsening of disease or symptoms, and alleviating, reducing, improving, and curing disease or symptoms.

[0435] The improvement effect on intestinal barrier function used for evaluation can be the improvement effect on the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, with the improvement effect on the intestinal barrier function of the small intestine being preferred.

[0436] The preventive or ameliorative effect of the disease or symptom being evaluated can be the preventive or ameliorative effect of the disease or symptom caused by the decrease in the intestinal barrier function of the small intestine, or the preventive or ameliorative effect of the disease or symptom caused by the decrease in the intestinal barrier function of the large intestine, preferably the preventive or ameliorative effect of the disease or symptom caused by the decrease in the intestinal barrier function of the small intestine.

[0437] The description of diseases or symptoms caused by decreased intestinal barrier function is the same as in Scheme 2.

[0438] In the method of scheme 6, candidate substances or compositions that are evaluated as having an effect on improving intestinal barrier function or preventing or improving disease or symptoms can be screened as substances or compositions that have an effect on improving intestinal barrier function or preventing or improving disease or symptoms.

[0439] Step 6a is the step of administering the candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced.

[0440] A decline in the intestinal barrier function of an individual can be confirmed by well-known methods for examining intestinal barrier function (e.g., lactulose-mannitol test) or by method 1.

[0441] The method of Scheme 6 may also include, prior to step 6a, a step of evaluating the subject's intestinal barrier function using the method of Scheme 1 and confirming that the subject's intestinal barrier function has been reduced. When the method of Scheme 6 includes this step, the types of biomarkers measured in step 1a of Scheme 1 may be the same as or different from the types of biomarkers measured in step 6b. From the viewpoint of improving the accuracy of evaluating the improvement effect of candidate substances or candidate compositions on intestinal barrier function or the prevention or improvement effect on disease or symptoms, the same types are preferred.

[0442] The method of Scheme 6 may also include a step of screening subjects with reduced intestinal barrier function using the method of Scheme 1 before step 6a. When the method of Scheme 6 includes this step, the types of biomarkers measured in step 1a of Scheme 1 may be the same as or different from the types of biomarkers measured in step 6b. From the viewpoint of improving the accuracy of evaluating the improvement effect of candidate substances or candidate compositions on intestinal barrier function or the prevention or improvement effect on disease or symptoms, the same types are preferred.

[0443] The recipient of the candidate substance or candidate composition is not particularly limited as long as the intestinal barrier function is reduced. It can be a person with reduced intestinal barrier function or a model animal (excluding humans) with reduced intestinal barrier function, preferably a model animal (excluding humans) with reduced intestinal barrier function. Examples of model animals include vertebrates such as mammals, reptiles, birds, amphibians, and fish, but mammals and birds are preferred, and mammals are more preferred. Examples of mammals include primates (e.g., gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), livestock (e.g., cattle, pigs, sheep, goats, horses), and pets (e.g., dogs, cats). Examples of birds include poultry (e.g., chickens, wild ducks, domestic ducks).

[0444] There are no particular limitations on the candidate substances or candidate combinations given to subjects with impaired intestinal barrier function. For example, they can be selected from high molecular weight compounds, low molecular weight compounds, cell cultures, cell extracts, antibodies, proteins, peptides, nucleic acids, sugars, inorganic salts, metal complexes, microorganisms (bacteria (probiotics), yeast, etc.), dietary fiber, indigestible sugars, indigestible proteins, fermented products (including fermented foods), prebiotics, and combinations of two or more of them.

[0445] Routes of administration for the candidate substance or candidate composition may include, for example, oral, non-oral (e.g., intranasal, ophthalmic, ocular, transdermal, intrarespiratory, rectal, urinary tract, subcutaneous, intramuscular, intravenous), etc. The candidate substance or candidate composition may be formulated into a suitable administration form. Examples of administration forms include tablets and injections. During formulation, additives suitable for oral administration (e.g., excipients, disintegrants, lubricants, binders, etc.) or additives suitable for non-oral administration (e.g., diluents, solvents, etc.) may be used. The dosage and frequency of administration of the candidate substance or candidate composition may be appropriately adjusted considering the administration form, the age and weight of the subject, etc. Administration may be once or more than once per day. Administration may be administered for one day or more than two days (e.g., more than one week, more than two weeks, more than three weeks, or more than four weeks).

[0446] Step 6b is the step of determining the biomarker in the analyte obtained from the subject after administration of the candidate substance or candidate composition, and obtaining the measured value of the biomarker.

[0447] Step 6b can be performed in the same manner as step 1a of scheme 1, except that it uses the test sample obtained from the subject after administering the candidate substance or candidate composition to the subject whose intestinal barrier function has been reduced.

[0448] The test sample used in step 6b is obtained from the subject after administering the candidate substance or candidate composition to a subject with reduced intestinal barrier function. The test sample can be obtained from the subject at multiple time points after administration of the candidate substance or candidate composition. The time point at which the test sample is obtained can be appropriately adjusted considering factors such as administration morphology, the subject's age, and weight. In one embodiment, the time point at which the test sample is obtained is, for example, any time point between 0.5 hours and 24 hours after administration, or any time point between 0.5 hours and 12 hours after administration, or any time point between 1 hour and 10 hours after administration. Here, "after administration" can refer to a single administration or to two or more consecutive administrations. Two or more consecutive administrations could be, for example, the last administration in a series of administrations lasting four weeks or more, or the last administration in a series of administrations lasting from 1 day to 4 weeks, or the last administration in a series of administrations lasting from 3 days to 3 weeks, or the last administration in a series of administrations lasting from 1 week to 2 weeks.

[0449] Step 6c is to compare the measured value obtained in step 6b with the benchmark value to obtain the comparison result.

[0450] Step 6c can be performed in the same way as step 1b of scheme 1, except that it uses the measured value obtained in step 6b.

[0451] Step 6d is a step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms, based on the comparison results obtained in step 6c.

[0452] Step 6d can be performed in the same manner as step 1c of scheme 1, except for evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0453] Regarding each substance selected from the group of substances of the present invention, the effect of the candidate substance or candidate composition on improving intestinal barrier function or preventing or improving disease or symptoms can be evaluated based on whether the comparison results obtained in step 6c meet the criteria shown in Table 4. Specifically, for each substance for which the criterion shown in Table 4 is "high", if the measured value obtained in step 6b is higher than the criterion value (i.e., the comparison results obtained in step 6c meet the criteria shown in Table 4), it can be evaluated as negative; if the measured value obtained in step 6b is lower than the criterion value (i.e., the comparison results obtained in step 6c do not meet the criteria shown in Table 4), it can be evaluated as positive. Furthermore, for substances for which the criterion shown in Table 4 is "low", if the measured value obtained in step 6b is lower than the criterion value (i.e., the comparison results obtained in step 6c meet the criteria shown in Table 4), it can be evaluated as negative; if the measured value obtained in step 6b is higher than the criterion value (i.e., the comparison results obtained in step 6c do not meet the criteria shown in Table 4), it can be evaluated as positive.

[0454] In step 6b, if one substance selected from the group of substances of the present invention is measured, it can be evaluated as negative if the comparison result of the one substance meets the criteria shown in Table 4, and as positive if the comparison result of the one substance does not meet the criteria shown in Table 4.

[0455] In step 6b, if two or more substances selected from the group of substances of the present invention are measured, a negative result can be evaluated if the comparison result of any one of the two or more substances meets the criteria shown in Table 4, and a positive result can be evaluated if the comparison result of at least one of the two or more substances does not meet the criteria shown in Table 4. The more substances whose comparison results do not meet the criteria shown in Table 4, the higher the probability of a positive result. Therefore, in step 6b, by measuring two or more substances selected from the group of substances of the present invention (e.g., two, three, or four or more), the accuracy of evaluating the effect of candidate substances or candidate compositions on improving intestinal barrier function or on preventing or improving diseases or symptoms can be improved.

[0456] Plan 7

[0457] Scheme 7 of the present invention relates to a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function. Hereinafter, the "prevention or improvement effect on diseases or symptoms caused by a decrease in intestinal barrier function" will sometimes be referred to as the "prevention or improvement effect on diseases or symptoms".

[0458] The method in Scheme 7 includes the following steps:

[0459] (7a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0460] (7b) The step of determining a biomarker in an analyte obtained from a subject after administration of a candidate substance or candidate composition, and obtaining a measured value of the biomarker; and

[0461] (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain the comparison result; and

[0462] (7d) Based on the comparison results obtained in step 7c, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms, wherein the test sample is urine and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0463] The explanation regarding Scheme 6 also applies to Scheme 7, unless otherwise specified. When applying, "Step 6a" should be replaced with "Step 7a", "Step 6b" with "Step 7b", "Step 6c" with "Step 7c", and "Step 6d" with "Step 7d".

[0464] Option 8

[0465] Scheme 8 of the present invention relates to a biomarker, which is a biomarker from a test sample of a subject for evaluating the intestinal barrier function of the subject, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0466] The explanation of Option 1 also applies to Option 8, unless otherwise specified.

[0467] Plan 9

[0468] Scheme 9 of the present invention relates to a biomarker, which is a biomarker from a test sample of a subject for diagnosing a disease or symptom caused by a decrease in intestinal barrier function, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0469] The explanation of Option 2 also applies to Option 9, unless otherwise specified.

[0470] Option 10

[0471] Scheme 10 of the present invention relates to a biomarker, which is a biomarker from a test sample of a subject for evaluating the risk of a subject developing a disease or symptom caused by a reduction in intestinal barrier function or the possibility of having already developed said disease or symptom, said test sample being urine, said biomarker comprising one or more substances selected from the group of substances of the present invention.

[0472] The explanation of Option 4 also applies to Option 10, unless otherwise specified.

[0473] Option 11

[0474] Scheme 11 of the present invention relates to a biomarker, which is a biomarker from a test sample of a subject for obtaining data on a disease or symptom caused by a decrease in intestinal barrier function for diagnosing the subject, the test sample being urine, the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0475] The explanation of Option 5 also applies to Option 11, unless otherwise specified.

[0476] Option 12

[0477] Scheme 12 of the present invention relates to a biomarker, which is a biomarker for screening substances or compositions that have an effect of improving intestinal barrier function or preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, and is a biomarker in a test sample obtained after administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0478] The explanation of Option 6 also applies to Option 12, unless otherwise specified.

[0479] Option 13

[0480] Scheme 13 of the present invention relates to a biomarker for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, and is a biomarker in a test sample obtained from a subject with decreased intestinal barrier function after the subject is given the candidate substance or candidate composition, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0481] The explanation of Scheme 7 also applies to Scheme 13, unless otherwise specified.

[0482] Option 14

[0483] Scheme 14 of the present invention relates to the use of a biomarker from a test sample of a subject for evaluating the intestinal barrier function of the subject, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0484] The explanation of Option 1 also applies to Option 14, unless otherwise specified.

[0485] Plan 15

[0486] Scheme 15 of the present invention relates to the use of a biomarker from a test sample of a subject for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function of the subject, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0487] The explanation of Option 2 also applies to Option 15, unless otherwise specified.

[0488] Option 16

[0489] Scheme 16 of the present invention relates to the use of a biomarker from a test sample of a subject for evaluating the risk of the subject developing or the likelihood of having developed a disease or symptoms caused by a reduction in intestinal barrier function, wherein the test sample is urine, and the biomarker comprises one or more substances selected from the group of substances of the present invention.

[0490] The explanation of Option 4 also applies to Option 16, unless otherwise specified.

[0491] Plan 17

[0492] Scheme 17 of the present invention relates to the use of a biomarker from a test sample of a subject for obtaining data for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function in the subject, the test sample being urine, and the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0493] The explanation of Option 5 also applies to Option 17, unless otherwise specified.

[0494] Option 18

[0495] Scheme 18 of the present invention relates to the use of biomarkers, wherein the biomarkers are biomarkers in a test sample obtained after administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced, and the use is for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the test sample is urine, and the biomarkers contain one or more substances selected from the group of substances of the present invention.

[0496] The explanation of Option 6 also applies to Option 18, unless otherwise specified.

[0497] Plan 19

[0498] Scheme 19 of the present invention relates to the use of a biomarker, said biomarker being a biomarker in a test sample obtained after administering a candidate substance or candidate composition to a subject with reduced intestinal barrier function, said use being for evaluating the effect of said candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, said test sample being urine, said biomarker comprising one or more substances selected from the group of substances of the present invention.

[0499] The explanation of Scheme 7 also applies to Scheme 19, unless otherwise specified.

[0500] Plan 20

[0501] Scheme 20 of the present invention relates to a kit for evaluating the intestinal barrier function of a subject, the kit comprising one or more reagents for measuring biomarkers in a test sample from the subject, the test sample being urine, the biomarkers comprising one or more substances selected from the group of substances of the present invention.

[0502] The explanation of Option 1 also applies to Option 20, unless otherwise specified.

[0503] The kit may contain one reagent or two or more reagents.

[0504] The type of reagent can be appropriately selected based on the type of determination method for the target substance. Examples of determination methods for the target substance include liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), gas chromatography-mass spectrometry (GC-MS), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), among which CE-MS or LC-MS are preferred. CE-MS is suitable for the determination of water-soluble substances, while LC-MS is suitable for the determination of lipid-soluble substances. Among CE-MS methods, capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) is particularly preferred. Among LC-MS methods, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is particularly preferred.

[0505] In one embodiment, the kit contains one or more reagents for determining the concentration of the target substance by CE-MS. The concentration can be a relative concentration or an absolute concentration, preferably an absolute concentration. A relative concentration is, for example, the relative area value (CE). As reagents for determining the concentration of the target substance by CE-MS, examples include standards of one or more substances selected from the group of substances of this invention.

[0506] In another embodiment, the kit contains one or more reagents for determining the concentration of the target substance by LC-MS. The concentration can be a relative concentration or an absolute concentration, preferably an absolute concentration. A relative concentration is, for example, a relative area value (LC). As reagents for determining the concentration of the target substance by LC-MS, examples include standards or other substances selected from the group of substances of this invention.

[0507] Option 21

[0508] Scheme 21 of the present invention relates to a kit for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function in a subject, the kit comprising one or more reagents for measuring a biomarker in a test sample from the subject, the test sample being urine, the biomarker comprising one or more substances selected from the group of substances of the present invention.

[0509] The explanation of Option 2 also applies to Option 21, unless otherwise specified.

[0510] The instructions regarding the reagents are the same as in Scheme 20.

[0511] Plan 22

[0512] Scheme 22 of the present invention relates to a kit for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function or the likelihood that the subject has already developed said disease or symptoms, the kit comprising one or more reagents for measuring a biomarker in a sample from said subject, said sample being urine, said biomarker comprising one or more substances selected from the group of substances of the present invention.

[0513] The explanation of Option 4 also applies to Option 22, unless otherwise specified.

[0514] The instructions regarding the reagents are the same as in Scheme 20.

[0515] Plan 23

[0516] Scheme 23 of the present invention relates to a kit for obtaining data on diseases or symptoms caused by reduced intestinal barrier function in a subject for diagnosis, the kit comprising one or more reagents for measuring biomarkers in a sample from the subject, the sample being urine, the biomarkers comprising one or more substances selected from the group of substances of the present invention.

[0517] The explanation of Option 5 also applies to Option 23, unless otherwise specified.

[0518] The instructions regarding the reagents are the same as in Scheme 20.

[0519] Option 24

[0520] Scheme 24 of the present invention relates to a kit for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function. The kit contains reagents for measuring one or more biomarkers in a test sample obtained after administering a candidate substance or candidate composition to a subject with decreased intestinal barrier function. The test sample is urine. The biomarkers contain one or more substances selected from the group of substances of the present invention.

[0521] The explanation of Option 6 also applies to Option 24, unless otherwise specified.

[0522] The instructions regarding the reagents are the same as in Scheme 20.

[0523] Plan 25

[0524] Scheme 25 of the present invention relates to a kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function. The kit comprises one or more reagents for measuring biomarkers in a test sample obtained after administration of the candidate substance or candidate composition to a subject with decreased intestinal barrier function. The test sample is urine, and the biomarkers comprise one or more substances selected from the group of substances of the present invention.

[0525] The explanation of Scheme 7 also applies to Scheme 25, unless otherwise specified.

[0526] The instructions regarding the reagents are the same as in Scheme 20.

[0527] Plan 26

[0528] Scheme 26 of the present invention relates to a method for manufacturing a food or pharmaceutical composition, comprising the step of mixing a substance or composition selected by the method of Scheme 6 with one or more components constituting the food or pharmaceutical composition.

[0529] There are no particular limitations on one or more components that can be mixed with the substances or compositions screened through method 6, as long as they are permitted to be components of the food or pharmaceutical composition. They can be appropriately selected according to the type of food or pharmaceutical composition to be manufactured.

[0530] The method of Scheme 26 may include the step of screening substances or compositions by the method of Scheme 6.

[0531] Plan 27

[0532] Scheme 27 of the present invention relates to a computer program for causing a computer to perform a method for evaluating the intestinal barrier function of a subject.

[0533] The method for enabling a computer to execute a computer program according to scheme 27 includes the following steps:

[0534] (S101) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0535] (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain the comparison result; and

[0536] (S103) Based on the comparison results obtained in step S102, the step of evaluating the intestinal barrier function of the subject is as follows.

[0537] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0538] The explanation of Option 1 also applies to Option 27, unless otherwise specified.

[0539] For the computer program of Scheme 27, when executed by a computer, the computer performs a method for evaluating the intestinal barrier function of a subject. The computer program of Scheme 27 contains instructions for causing the computer to perform a method for evaluating the intestinal barrier function of a subject.

[0540] Hereinafter, an embodiment of scheme 27 will be described with reference to the accompanying drawings. Figure 1 This is a schematic diagram of the apparatus used in this embodiment. Figure 2 This is a block diagram illustrating the hardware structure of the device according to this embodiment. Figures 3-5 This is a flowchart illustrating the processing sequence of the apparatus in this embodiment.

[0541] like Figure 1 As shown, the device 10 includes a computer system 30. (As indicated...) Figure 1 As shown, the device 10 may include a measuring device 20 connected to the computer system 30. The computer system 30 may be a device different from the measuring device 20, or it may be a device with the measuring device 20 built into it. The device 10 may also be a device in which the measuring device 20 and the computer system 30 are integrated.

[0542] The measuring device 20 performs the measurement of biomarkers from the analyte of the subject, obtains the measured value of the biomarker (e.g., absolute concentration, relative concentration, or composite variable) or the measured value required to calculate the measured value of the biomarker (e.g., the measured value required to calculate the relative concentration or composite variable), and sends it to the computer system 30. The description of the analyte from the subject, the measurement of the biomarker, and the measured value of the biomarker are the same as in Scheme 1.

[0543] The measuring device 20 is, for example, an automated measuring device that performs the measurement of biomarkers using CE-MS (especially CE-FTMS) or LC-MS (especially LC-MS / MS) to obtain the measured value of the biomarker (e.g., relative area value CE, relative area value LC, or composite variable) or to calculate the measured value of the biomarker (e.g., relative area value CE, relative area value LC, or the measured value required to calculate the composite variable).

[0544] The assay apparatus 20 need only be capable of performing the determination of biomarkers in a assay sample (e.g., an assay sample for CE-MS (especially CE-FTMS) or LC-MS (especially LC-MS / MS)) and subsequent processing. Processing preceding the determination of biomarkers in the assay sample, such as the collection of urine from the subject (i.e., collecting urine from the subject's excretion for use as the analyte), the preparation of the assay sample from the subject's urine, and the setting of the assay sample in the assay apparatus 20, can be performed by a human or by the assay apparatus 20. In one embodiment, processing preceding the determination of biomarkers in the assay sample is performed by a human.

[0545] like Figure 2 As shown, the computer system 30 includes a computer main body 300, an input unit 301, and a display unit 302 for displaying information about the object to be tested, evaluation results, etc.

[0546] The processor of computer system 30 executes a computer program installed on hard disk 313 for evaluating the intestinal barrier function of the subject, based on the measured values ​​of biomarkers.

[0547] like Figure 2 As shown, the computer main body 300 includes a CPU (Central Processing Unit) 310, ROM (Read Only Memory) 311, RAM (Random Access Memory) 312, hard disk 313, input / output interface 314, reading device 315, communication interface 316, and image output interface 317. The CPU 310, ROM 311, RAM 312, hard disk 313, input / output interface 314, reading device 315, communication interface 316, and image output interface 317 are communicatively connected via bus 318. The measuring device 20 is communicatively connected to the computer system 30 via communication interface 316.

[0548] CPU 310 can execute programs stored in ROM 311 or hard disk 313 and programs loaded into RAM 312. By executing programs through CPU 330, device 10 functions as a device for evaluating the intestinal barrier function of a subject.

[0549] ROM311 is composed of mask ROM, PROM, EPROM, EEPROM, etc. ROM311 records the computer program executed by CPU310 and the data used in the execution of the computer program.

[0550] RAM312 is composed of SRAM, DRAM, etc. RAM312 is used to read programs recorded in ROM311 and hard disk 313. In addition, RAM312 is used as the working area of ​​CPU310 when executing programs.

[0551] The hard disk 313 contains computer programs such as the operating system and application programs that are executed by the CPU 310, as well as the data used in the execution of these computer programs.

[0552] The reading device 315 comprises a floppy disk drive, a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a memory stick reader, a solid-state drive, etc. The reading device 315 can read programs or data recorded on the portable recording medium 400.

[0553] The input / output interface 314 comprises serial interfaces such as USB, IEEE 1394, and RS-232C; parallel interfaces such as SCSI, IDE, and IEEE 1284; and analog interfaces such as D / A converters and A / D converters. The input / output interface 314 is connected to input units 301 such as a keyboard and mouse. The operator can input various commands to the computer main unit 300 through the input units 301.

[0554] The communication interface 316 is, for example, an Ethernet (registered trademark) interface. The computer body 300 can use the communication interface 316 to send printing data to a printer, the computer of the object or service provider, the object's smartphone terminal, etc.

[0555] The image output interface 317 is connected to a display unit 302, which is composed of an LCD, CRT, or the like. Thus, the display unit 302 can output an image signal corresponding to the image data provided by the CPU 310. The display unit 302 displays an image (screen) based on the input image signal. The display unit 302 can display the original data itself, or it can convert the original data into illustrations, diagrams, etc., for easier understanding by the viewer.

[0556] The following is for reference Figures 3-5 This explains the processing sequence of device 10.

[0557] like Figure 3 As shown, in step S101, CPU 310 obtains the measurement values ​​of biomarkers from the test sample of the object. The description of the test sample from the object and the measurement values ​​of biomarkers is the same as in Scheme 1.

[0558] In one embodiment, the measuring device 20 performs a measurement of the biomarker from the analyte of the subject, obtains the biomarker measurement value (e.g., absolute concentration, relative concentration, or composite variable), and sends it to the computer system 30. The description of the analyte from the subject, the biomarker measurement, and the biomarker measurement value is the same as in Scheme 1. The CPU 310 stores the biomarker measurement value sent by the measuring device 20 in the hard disk 313. In step S301, the CPU 310 retrieves the biomarker measurement value stored in the hard disk 313.

[0559] In another embodiment, the measuring device 20 performs a measurement of the biomarker from the analyte of the subject, obtains the measurement value required to calculate the biomarker's measured value (e.g., the measurement value required to calculate the relative concentration or composite variable), and sends it to the computer system 30. The description of the analyte from the subject, the biomarker measurement, and the biomarker measurement value is the same as in embodiment 1. Based on the measurement value sent by the measuring device 20, the CPU 310 calculates the biomarker's measured value (e.g., relative concentration or composite variable) and stores it in the hard disk 313. In step S301, the CPU 310 retrieves the biomarker's measured value stored in the hard disk 313.

[0560] In another embodiment, the operator inputs the measured values ​​(e.g., absolute concentration, relative concentration, or composite variable) of a biomarker obtained in advance through the measurement of a biomarker in the test sample from the subject, from the input unit 301. The description of the test sample from the subject, the measurement of the biomarker, and the measured values ​​of the biomarker are the same as in embodiment 1. The CPU 310 stores the input measured values ​​of the biomarker in the hard disk 313. In step S301, the CPU 310 retrieves the measured values ​​of the biomarker stored in the hard disk 313.

[0561] In another embodiment, the operator inputs from the input unit 301 the measurement values ​​needed to calculate the biomarker measurement values ​​obtained in advance through the measurement of biomarkers from the analyte of the subject (e.g., the measurement values ​​needed to calculate relative concentrations or composite variables). The description of the analyte from the subject, the biomarker measurement, and the biomarker measurement values ​​is the same as in Embodiment 1. The CPU 310 calculates the biomarker measurement values ​​(e.g., relative concentrations or composite variables) based on the input measurement values ​​and stores them in the hard disk 313. In step S301, the CPU 310 retrieves the biomarker measurement values ​​stored in the hard disk 313.

[0562] like Figure 3As shown, in step S102, the CPU 310 compares the measured value of the biomarker with the reference value stored in the hard disk 313 to obtain a comparison result. The explanations regarding the reference value, comparison, and comparison result are the same as in Scheme 1. The CPU 310 stores the obtained comparison result in the hard disk 313. The CPU 310 may also output the obtained comparison result and display it on the display unit 302, or have it printed by a printer, or transmit it to another computer or application (such as a smartphone). Alternatively, the CPU 310 may display the measured value of the biomarker, the reference value, etc., as reference information on the display unit 302 when outputting the comparison result, or have it printed by a printer, or transmit it to another computer or application (such as a smartphone). Furthermore, the CPU 310 may also display lifestyle suggestions for improving intestinal barrier function, recommended food intake (including functional foods, etc.), etc., along with the comparison result on the display unit 302, or have it printed by a printer, or transmit it to another computer or application (such as a smartphone). This allows for the provision of indicators to doctors, nurses, caregivers, examiners, examination operators, service providers, and the individuals themselves for evaluating the intestinal barrier function.

[0563] like Figure 3 As shown, in step S103, CPU 310 evaluates the intestinal barrier function of the subject based on the comparison results obtained in step S102. The evaluation process is the same as in Scheme 1. CPU 310 stores the evaluation results in hard disk 313. CPU 310 can also output the obtained evaluation results, displaying them on display unit 302, printing them, or transmitting them to other computers or applications (such as smartphones). Furthermore, CPU 310 can also display biomarker measurements, baseline values, etc., as reference information on display unit 302 when outputting the evaluation results, or print them, or transmit them to other computers or applications (such as smartphones). Additionally, CPU 310 can also display lifestyle suggestions for improving intestinal barrier function, recommended food intake (including functional foods), etc., along with the evaluation results on display unit 302, or print them, or transmit them to other computers or applications (such as smartphones). Thus, indicators for evaluating the intestinal barrier function of the subject can be provided to doctors, nurses, caregivers, examiners, examination operators, service providers, and the subject themselves.

[0564] Regarding each substance selected from the group of substances of this invention, the CPU310 can evaluate the intestinal barrier function of the subject based on whether the comparison results obtained in step S102 meet the criteria shown in Table 4. Specifically, regarding each substance whose criterion is "high" as shown in Table 4, such as... Figure 4As shown, the CPU 310 determines whether the measured value obtained in step 101 is higher than the reference value (i.e., whether the comparison result meets the reference values ​​shown in Table 4) (step S103a). If the measured value obtained in step 101 is higher than the reference value (i.e., the comparison result meets the reference values ​​shown in Table 4), it is evaluated as positive (step S103b). If the measured value obtained in step 101 is lower than the reference value (i.e., the comparison result does not meet the reference values ​​shown in Table 4), it is evaluated as negative (step S103c). Furthermore, regarding substances for which the reference value shown in Table 4 is "low", such as... Figure 5 As shown, the CPU310 determines whether the measured value obtained in step 101 is lower than the benchmark value (i.e., whether the comparison result meets the benchmark shown in Table 4) (step S103d). If the measured value obtained in step 101 is lower than the benchmark value (i.e., the comparison result meets the benchmark shown in Table 4), it is evaluated as positive (step S103e). If the measured value obtained in step 101 is higher than the benchmark value (i.e., the comparison result does not meet the benchmark shown in Table 4), it is evaluated as negative (step S103f).

[0565] Plan 28

[0566] Solution 28 of the present invention relates to a computer program for causing a computer to perform a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function. Hereinafter, "disease or symptom caused by a decrease in intestinal barrier function" will sometimes be referred to as "disease or symptom".

[0567] The method for enabling a computer to execute a computer program according to scheme 28 includes the following steps:

[0568] (S201) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0569] (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result;

[0570] (S203) Based on the comparison results obtained in step S202, the step of diagnosing the disease or symptoms of the subject caused by the reduction of intestinal barrier function, wherein the test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0571] The explanation regarding Scheme 2 also applies to Scheme 28, unless otherwise specified. Similarly, the explanation regarding Scheme 27 also applies to Scheme 28, unless otherwise specified. When applying this, replace "Step S101" with "Step S201", "Step S102" with "Step S202", "Step S103" with "Step S203", "evaluate the intestinal barrier function of the subject" with "diagnose the disease or symptoms of the subject", "accuracy of evaluating the intestinal barrier function of the subject" with "accuracy of diagnosing the disease or symptoms of the subject", "positive evaluation" with "positive diagnosis", and "negative evaluation" with "negative diagnosis".

[0572] Plan 29

[0573] Solution 29 of the present invention relates to a computer program for causing a computer to execute a method for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function, or the likelihood that the subject has already developed said disease or symptoms. Hereinafter, the "risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function" is sometimes referred to as "occurrence risk," and the "likelihood that a subject has already developed a disease or symptoms caused by a reduction in intestinal barrier function" is sometimes referred to as "potential likelihood."

[0574] The method for enabling a computer to execute a computer program according to scheme 29 includes the following steps:

[0575] (S301) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0576] (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain the comparison result; and

[0577] (S303) The step of evaluating the risk or the likelihood based on the comparison results obtained in step S302.

[0578] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0579] The explanation regarding Scheme 4 also applies to Scheme 29, unless otherwise specified. Similarly, the explanation regarding Scheme 27 also applies to Scheme 29, unless otherwise specified. When applying this, "Step S101" is replaced with "Step S301", "Step S102" with "Step S302", "Step S103" with "Step S303", "evaluating the intestinal barrier function of the subject" with "evaluating the risk or likelihood of developing the disease", and "the accuracy of evaluating the intestinal barrier function of the subject" with "the accuracy of evaluating the risk or likelihood of developing the disease".

[0580] Plan 30

[0581] Solution 30 of the present invention relates to a computer program for causing a computer to execute a method for obtaining data on diseases or symptoms caused by a decrease in intestinal barrier function for a diagnostic subject. Hereinafter, "diseases or symptoms caused by a decrease in intestinal barrier function" will sometimes be referred to as "diseases or symptoms".

[0582] The method for enabling a computer to execute a computer program according to Scheme 30 includes the following steps:

[0583] (S401) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0584] (S402) The step of comparing the measured value obtained in step S401 with the baseline value and obtaining the comparison result as data for diagnosing the disease or symptoms caused by the decrease in intestinal barrier function in the subject.

[0585] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0586] The explanation regarding Scheme 5 also applies to Scheme 30, unless otherwise specified. Similarly, the explanation regarding Scheme 27 also applies to Scheme 30, unless otherwise specified. When applying this, "step S101" is replaced with "step S401", "step S102" is replaced with "step S402", "evaluate the intestinal barrier function of the subject" is replaced with "obtain data for diagnosing the subject's disease or symptoms", and "accuracy of evaluating the intestinal barrier function of the subject" is replaced with "accuracy of diagnosing the subject's disease or symptoms".

[0587] Option 31

[0588] Scheme 31 of the present invention relates to a computer program for causing a computer to execute a method for screening substances or compositions having an effect on improving intestinal barrier function or having a preventive or ameliorative effect on diseases or symptoms caused by a reduction in intestinal barrier function. Hereinafter, "the preventive or ameliorative effect on diseases or symptoms caused by a reduction in intestinal barrier function" is sometimes referred to as "the preventive or ameliorative effect on diseases or symptoms".

[0589] The method for enabling a computer to execute a computer program according to Scheme 31 includes the following steps:

[0590] (S501) A step of obtaining the determination value of the biomarker in the test body obtained after administering the candidate substance or candidate composition to the subject from a subject with reduced intestinal barrier function;

[0591] (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain the comparison result; and

[0592] (S503) Based on the comparison results obtained in step S502, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0593] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0594] The explanation regarding Scheme 6 also applies to Scheme 31, unless otherwise specified. Similarly, the explanation regarding Scheme 27 also applies to Scheme 31, unless otherwise specified. When applying this, "step S101" is replaced with "step S501," "step S102" with "step S502," "step S103" with "step S503," "evaluation of the intestinal barrier function of the subject" is replaced with "evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms," and "accuracy of evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms."

[0595] Option 32

[0596] Scheme 32 of the present invention relates to a computer program for causing a computer to execute a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function. Hereinafter, the "prevention or improvement effect on diseases or symptoms caused by a decrease in intestinal barrier function" is sometimes referred to as the "prevention or improvement effect on diseases or symptoms".

[0597] The method for enabling a computer to execute a computer program according to scheme 32 includes the following steps:

[0598] (S601) A step of obtaining the determination value of the biomarker in the test body obtained after administering the candidate substance or candidate composition to the subject from a subject with reduced intestinal barrier function;

[0599] (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain the comparison result; and

[0600] (S603) Based on the comparison results obtained in step S602, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0601] The test sample is urine, and the biomarker contains one or more substances selected from the group of substances of the present invention.

[0602] The explanation regarding Scheme 7 also applies to Scheme 32, unless otherwise specified. Similarly, the explanation regarding Scheme 27 also applies to Scheme 32, unless otherwise specified. When applying this, "step S101" is replaced with "step S601", "step S102" with "step S602", "step S103" with "step S603", "evaluation of the intestinal barrier function of the subject" is replaced with "evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms", and "accuracy of evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms".

[0603] Option 33

[0604] Scheme 33 of the present invention relates to a computer-readable recording medium having recorded a computer program as described in any one of schemes 27 to 32.

[0605] As a computer-readable recording medium containing a program, examples include non-transitory recording media such as ROM, floppy disk (registered trademark), hard disk, optical disk, optical disc, CD-ROM, magnetic tape, and non-volatile memory cards.

[0606] Example

[0607] The present invention will now be described based on embodiments.

[0608] The lactulose-mannitol test, a well-known method for assessing intestinal barrier function, was performed on 108 subjects. The lactulose-mannitol test was conducted as follows: Subjects were instructed to finish their meal before 9 PM the day before the test and were allowed to drink water before bedtime. On the day of the test, subjects were not allowed to eat or drink, and a glucose tolerance test was performed. Subjects ingested a glucose solution (a solution containing 10 g lactulose, 5 g mannitol, and 20 g sucrose dissolved in 100 mL of water). After ingesting the glucose solution, subjects ingested at least 150 mL of water at 2–3 hours, 3–4 hours, 4–5 hours, and 5–6 hours later. After water ingestion, urine was collected from 0–6 hours later, and the amounts of lactulose and mannitol in the urine were determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The LC-MS / MS was performed as follows.

[0609] The assay sample used in LC-MS / MS was prepared from urine as follows: Add 400 μL of cooled acetonitrile to 50 μL of urine and stir thoroughly. Incubate on ice for 1 hour. Further stir the resulting mixture, add 800 μL of 75% acetonitrile and stir thoroughly. Filter the mixture through a Millex FH 0.45 μm filter before use as the assay sample. No internal standard was used.

[0610] Measurements based on LC-MS / MS and subsequent data analysis were performed using a QTRAP 4500 LC-MS / MS system (Thermo Fisher Scientific). As columns, Agilent Advance Bio MS SpentMedia was used. 2.1 × 100 mm (Agilent Technologies). The determination conditions for LC-MS / MS are as follows.

[0611] Mobile phase A: 10 mM ammonium formate (pH 10.5)

[0612] Mobile phase B: Acetonitrile (90%) / 100mM ammonium formate (pH 10.5) (10%)

[0613] Flow conditions: 0-15 min B 97%-89%, 15-18 min B 89%

[0614] Flow rate: 0.4 mL / min

[0615] Injection volume: 5μL

[0616] Temperature: 35℃

[0617] Ion spray voltage: -4500V

[0618] After determining the amount of lactulose and mannitol in urine by LC-MS / MS (hereinafter referred to as "excretion"), the excretion rate (%) of each is calculated based on the following formula.

[0619] Excretion rate = Excretion volume (μmol / mL) × Molecular weight (μg / μmol) × Urine volume (mL) / Sugar intake (μg) × 100

[0620] After calculating the excretion rate (%), the lactulose / mannitol ratio is calculated based on the following formula.

[0621] Lactulose / mannitol ratio = Lactulose excretion rate / Mannitol excretion rate

[0622] Twenty-seven subjects with a lactulose / mannitol ratio of 0.025 or higher were designated as the positive group, and 27 subjects with a lactulose / mannitol ratio of 0.015 or lower were designated as the negative group.

[0623] Urine and serum samples were obtained from the negative and positive groups and used in the following comparative examples and embodiments.

[0624] The serum was obtained as follows: On the day of the lactulose-mannitol test, 20 mL of blood was collected from the subject, a portion of which was separated into serum and stored in a deep freezer (-80°C) until use.

[0625] The urine was obtained as follows. On the day of the examination, 10 mL of midstream urine was collected from the first urine excreted in the morning (urination at bedtime, collected immediately after waking up) and stored in a deep freezer (-80°C) until use.

[0626] Comparative Example 1

[0627] For each serum sample obtained from the negative and positive groups, the concentration of clathrin in the serum was determined according to the ELISA method. The ELISA method was performed using a clathrin ELISA kit (Immundiagnostik AG, Germany) according to the manufacturer's protocol.

[0628] Using the serum ligin concentrations in the negative and positive groups, ROC (Receiving Controller Operational Characteristic) curves were constructed. The area under the ROC curve (AUC) and cutoff value were then calculated from the constructed ROC curves. The ROC curves, AUC, and cutoff value were obtained using the statistical analysis software "EXCEL Statistics" (developed by Social Survey Research Information Co., Ltd.). The ROC curve is a curve plotted with sensitivity on the vertical axis and (1-specificity) on the horizontal axis, showing the serum ligin concentrations in the negative and positive groups. The AUC is the area under the ROC curve. The point on the ROC curve with the smallest distance from the upper left corner of the ROC curve was used as the cutoff value. The results are shown in Table 8.

[0629] [Table 8]

[0630] Table 8

[0631] substance name Determination methods AUC Cutoff value ligin ELISA 0.638 39.70

[0632] Example 1

[0633] (1) Metabolomics analysis of urine

[0634] For each urine sample obtained from both the negative and positive groups, a comprehensive metabolomics analysis was performed to determine the metabolites in each urine sample. Metabolomics analysis was conducted using capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0635] The sample for CE-FTMS determination was prepared from urine as follows: 20 μL of an aqueous solution containing an internal standard (100 μM) and 60 μL of Milli-Q water were added to 20 μL of urine, and the mixture was vortexed. The mixture was then transferred to an ultrafiltration tube, centrifuged (4 °C, 9100 × g, 60 min), and ultrafiltered for use as the determination sample. It should be noted that the internal standard used was Human Metabolome Technologies (HMT) internal standard (H3304-1002; HMT).

[0636] CE-FTMS measurements and subsequent data analysis were performed using an Agilent CE system (Agilent Technologies). Capillary electrophoresis was performed using a fused silica capillary. The CE-FTMS measurement conditions are described below.

[0637] [Catonic metabolites (cationic model)]

[0638] Running buffer: Cation buffer (p / n: H3301-1001)

[0639] Rinse buffer: Cation buffer (p / n: H3301-1001)

[0640] Sample injection: Inject at 50 mbar for 10 seconds.

[0641] CE voltage: positive, 30kV

[0642] MS ionization: ESI cathode

[0643] MS capillary voltage: 4,000V

[0644] MS scan range: m / z 60-900

[0645] Sheath fluid: HMT sheath fluid (p / n: I3301-1040)

[0646] [Anionic Metabolites (Anionic Pattern)]

[0647] Running buffer: Anion exchange buffer (p / n: H3302-1023)

[0648] Rinse buffer: Anion exchange buffer (p / n: H3302-1023)

[0649] Sample injection: 50 mbar pressurized injection, 22 seconds

[0650] CE voltage: positive, 30kV

[0651] MS ionization: ESI negative electrode

[0652] MS capillary voltage: 3,500V

[0653] MS scan range: m / z 70-1,050

[0654] Sheath fluid: HMT sheath fluid (p / n: I3301-1040)

[0655] The analyte sample used in LC-MS / MS was prepared from urine as follows: Add 200 μL of cooled methanol containing 0.15% formic acid to every 50 μL of urine and stir thoroughly. Incubate on ice for 1 hour. Centrifuge (12000 rpm, 10 min, 4 °C), and dry 200 μL of the supernatant under nitrogen. For analysis using an octadecylsilane (ODS) column, dissolve in 200 μL of 10% methanol (containing 0.1% formic acid); for analysis using a hydrophilic interaction liquid chromatography (HILIC) column, dissolve in 200 μL of 50% acetonitrile. When using an ODS column, the sample was filtered through a 0.45 μm aqueous filter (GL disc 4A). When using a HILIC column, the sample was filtered through a 0.45 μm Millex FH filter. No internal standard was used. It should be noted that, for comprehensive analysis of metabolites, two types of columns (ODS column and HILIC column) are used to detect a variety of substances. Compared with the HILIC column, the ODS column is more suitable for determining hydrophobic substances, while the HILIC column is more suitable for determining hydrophilic substances.

[0656] The determinations and subsequent data analysis based on LC-MS / MS were performed using the Q Exactive Plus Orbitrab LC-MS / MS system (Thermo Fisher Scientific). Thermo Scientific Hypersil Gold C18 (150 mm L., 2.1 mm ID, 1.9 μm) (Thermo Fisher Scientific) was used as the ODS column, and Merck SeQuant ZIC pHILIC (150 mm L., 2.1 mm ID, 5 μm) (Merck Millipore) was used as the HILIC column. The LC-MS / MS determination conditions are described below.

[0657] [Determination using an ODS column]

[0658] Mobile phase A: An aqueous solution containing 0.1% formic acid by mass

[0659] Mobile phase B: Methanol containing 0.1% formic acid by mass

[0660] Flow conditions: 0-3 min B 0.5%, 3-10 min B 0.5-50%, 10-12 min B 50-98%, 12-32 min B 98%, 32-33 min B 98-0.5%, 33-40 min B 0.5%.

[0661] Flow rate: 0.1 mL / min for 0-3 min, 0.25 mL / min for 3-40 min

[0662] Injection volume: 2μL

[0663] Temperature: 55℃

[0664] MS ionization: Switching modes (ESI positive electrode, ESI negative electrode)

[0665] MS capillary voltage: 2.5kV

[0666] MS scan range: m / z 70-1050

[0667] [Measurements performed using a HILIC column]

[0668] Mobile phase A: 10 mM ammonium acetate aqueous solution (pH 9.6)

[0669] Mobile phase B: Acetonitrile

[0670] Flow conditions: 0-3.5 min B 90%, 3.5-18 min B 90-30%, 18-21 min B 30%, 21-22 min B 30-90%, 22-30 min B 90%

[0671] Flow rate: 0.25 mL / min

[0672] Injection volume: 2μL

[0673] Temperature: 20℃

[0674] MS ionization: Switching modes (ESI positive electrode, ESI negative electrode)

[0675] MS capillary voltage: 2.5kV

[0676] MS scan range: m / z 70-1050

[0677] For each urine sample obtained from the negative and positive groups, the peaks from each substance were determined based on their m / z values ​​from the peaks detected by CE-FTMS, and the peak areas of each substance were calculated. The m / z values ​​of each substance are shown in Table 9. When calculating the peak areas, the automatic integration software MasterHands ver. 2.17.5.19 (developed by Keio University) was used to extract only peaks with a signal-to-noise (S / N) ratio of 3 or higher from the peaks detected by CE-FTMS. The relative area values ​​(relative concentrations) of each substance in each urine sample were calculated based on the following formula. In the following formula, "internal standard for anions" refers to the value of the internal standard (H3304-1002; HMT) measured in anion mode; "internal standard for cations" refers to the value of the internal standard (H3304-1002; HMT) measured in cation mode.

[0678] [In cationic mode]

[0679] The relative surface area of ​​each substance in urine = (peak surface area of ​​each substance) / (surface area of ​​creatinine) × 1000

[0680] [In anion mode]

[0681] The relative surface area of ​​each substance in urine is calculated as follows: (peak area of ​​each substance / surface area of ​​the internal standard for anions) / (surface area of ​​creatinine / surface area of ​​the internal standard for cations) × 1000

[0682] Urine samples (samples No. 1–No. 27) from the negative control group (27 individuals) and urine samples (samples No. 28–No. 54) from the positive control group (27 individuals) were sequentially measured by LC-MS / MS. Then, using the NormalizeArea node of the small molecule structure identification software Compound Discoverer 3.1 (Thermo Fisher Scientific), regression curves were constructed based on the peak area values ​​of each substance in the mixture (mixed sample) obtained by mixing the urine samples (samples No. 1–No. 27) from the negative control group (27 individuals) and the urine samples (samples No. 28–No. 54) from the positive control group (27 individuals). The peak area values ​​of each substance in each sample were corrected, and the relative area values ​​of each substance in each urine sample were calculated.

[0683] Specifically, along with the measurements of each sample No. 1 to No. 54, after measuring every 15 samples in a mixed sample, subsequent data analysis was performed, and the peak area of ​​each substance in the mixed sample was calculated in the same manner as described above (i.e., the measurements of each sample No. 1 to No. 15 → mixed sample → samples No. 16 to No. 30 → mixed sample were repeated until the measurement of sample No. 54, and subsequent data analysis was performed, and the peak area of ​​each substance in the mixed sample was calculated in the same manner as described above). The peak area values ​​of each substance in each sample were corrected in the same way as described above, thereby calculating the relative area values ​​of each substance in each urine sample.

[0684] It should be noted that the correction using mixed samples is performed to correct for errors caused by the effects of time-dependent decay of the peak area values ​​of each substance.

[0685] ROC (Responder Operating Characteristic) curves were constructed using the relative area values ​​of each substance in each urine sample. The area under the ROC curve (AUC) and cutoff value were then calculated from the ROC curves. The ROC curves, AUC, and cutoff values ​​were obtained using the statistical analysis software "EXCEL Statistics" (developed by Social Survey Research Information Co., Ltd.). The ROC curve plots the relative area values ​​of each substance in each urine sample, with sensitivity on the vertical axis and (1-specificity) on the horizontal axis. The AUC is the area under the ROC curve. The point on the ROC curve with the smallest distance from the upper left corner of the ROC curve was used as the cutoff value. The results are shown in Table 9. The closer the AUC value is to 1, the higher the accuracy of evaluating intestinal barrier function. The accuracy can be categorized as follows: when the AUC value is below 0.6, the accuracy of the evaluation is low; when the AUC value is above 0.6 but below 0.7, the accuracy of the evaluation is slightly higher; when the AUC value is above 0.7 but below 0.8, the accuracy of the evaluation is high; and when the AUC value is above 0.8, the accuracy of the evaluation is very high.

[0686] Table 9 shows only the results for substances with an AUC of 0.65 or higher (substances No.1 to No.61 in the substance group of the present invention) that were comprehensively measured.

[0687] Regarding substances No. 1 to No. 61 in the material group of this invention, the average value of the positive group, the average value of the negative group, and the ratio of the average value of the positive group to the average value of the negative group (average value of the positive group / average value of the negative group) were calculated using the relative area values ​​of each substance in each urine sample. The results are shown in Table 10. Table 10 also shows the trends in the positive group. "Increase" indicates that there is a tendency to increase in the positive group, and "decrease" indicates that there is a tendency to decrease in the positive group.

[0688] As shown in Tables 9 and 10, when the test sample is urine, the intestinal barrier function of the subject can be evaluated with high accuracy by using one or more substances selected from the material group of the present invention as biomarkers. The closer the AUC is to 1, the higher the accuracy of evaluating intestinal barrier function. Therefore, the substances No. 1 to No. 4 in the material group of the present invention (4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine) and the substances No. 39 to No. 42 in the material group of the present invention (cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzyl)sorbitol) have particularly high accuracy in evaluating intestinal barrier function.

[0689] [Table 9-1]

[0690]

[0691] [Table 9-2]

[0692]

[0693] [Table 9-3]

[0694]

[0695] [Table 10-1]

[0696]

[0697] [Table 10-2]

[0698]

[0699] [Table 10-3]

[0700]

[0701] Composite variables (statistical analysis values)

[0702] (2-1) Substances No. 1 to No. 4 in the material group of the present invention

[0703] For two or more substances selected from No. 1 to No. 4 of the material group of this invention (4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine), the relative area values ​​obtained in (1) are used to calculate the composite variable based on the following formula. An ROC curve is then constructed based on the calculated composite variable, and the AUC and cutoff value are obtained from the constructed ROC curve. The results are shown in Table 11. The coefficients and constants are obtained through multinomial logistic regression analysis. The coefficients and constants are shown in Table 2. In Table 11, “CU_A” represents 4-acetaminophen, “CU_B” represents N-acetylputrescine, “CU_C” represents 3-amino-2-piperidinone, and “CU_D” represents O-succinylhomoserine.

[0704] Statistical analysis value = (coefficient 1 × (concentration of the first substance)) + (coefficient 2 × (concentration of the second substance)) + ... + (coefficient n × (concentration of the nth substance)) + constant term

[0705] As shown in Table 11, it is clear that by combining two or more substances selected from No.1 to No.4 of the substance group of the present invention, the AUC of the ROC curve can be increased, that is, the accuracy of evaluating intestinal barrier function can be improved.

[0706] [Table 11]

[0707]

[0708] (2-2) Substances No. 39 to No. 42 in the material group of the present invention

[0709] For two or more substances selected from No. 39 to No. 42 of the substance group of this invention (cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzyl)sorbitol), the relative area values ​​obtained in (1) are used to calculate the composite variable in the same way as in (2-1). Based on the calculated composite variable, an ROC curve is constructed, and the AUC and cutoff value are calculated from the constructed ROC curve. The results are shown in Table 12. The coefficients and constants are obtained by multinomial logistic regression analysis. The coefficients and constants are shown in Table 3. In Table 12, “LU_A” represents cyclopentaacetic acid, “LU_B” represents DK cypermethrin, “LU_C” represents paraxanthine, and “LU_D” represents bis(methylbenzyl)sorbitol.

[0710] As shown in Table 12, it is clear that by combining two or more substances selected from No. 39 to No. 42 of the substance group of the present invention, the AUC of the ROC curve can be increased, that is, the accuracy of evaluating intestinal barrier function can be improved.

[0711] [Table 12]

[0712]

Claims

1. A method for evaluating intestinal barrier function in subjects, The method includes the following steps: (1a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers; (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain a comparison result; and (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group consisting of: 4-Acetaminobutyric acid; N-acetyl-corrosive amine; 3-Amino-2-piperidinone; O-succinylhomoserine; sulfotyrosine; Malic acid; Tyrosine; N-Carbamoyl Aspartic Acid; Muscidol; mucic acid; N-acetylaspartic acid; Arginine succinic acid; adipic acid; 2-Oxadipic acid; Phenylalanine; Succinic acid; N-acetylglutamic acid; Glycyl aspartic acid; N-acetylornithine; Ascorbic acid-2-sulfate; Asymmetric dimethylarginine; N-acetylneuraminic acid; 2-Hydroxyglutaric acid; Cyclohexane carboxylic acid; 5-Methylcytosine; cis-aconitic acid; glycine; Tryptophan; Tyramine; Glycerol-2-phosphate; Citric acid; Octylamine and dopamine; Syringic acid; 2'-Deoxyadenosine and 5'-Deoxyadenosine; adenosine diphosphate; Glutathione (GSH); 2-Aminoisobutyric acid and 2-aminobutyric acid; sinapic acid; Cyclopentaacetic acid; DK methamidophos; Paraxanthine; Bis(methylbenzylidene)sorbitol; caffeine; 2-Aminooctanoic acid; (R)-Estrol; Theophylline; Guanidinosuccinic acid; Uracil; 1-Phenylacetyl-3-methyl-5-pyrazolone; 5α-Androstane-3,6,17-trione; Methylmalonic acid; N-acetyl-L-cysteine; Menaquinone; DL-stachyine; adenosine; Arecoline; 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione; Pyrazinamide; Prolylleucine; Norethindrone; and 2-Methylbenzoic acid.

2. A method for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject. The method includes the following steps: (2a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers; (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain a comparison result; and (2c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 2b. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

3. A method for treating a disease or symptoms caused by a decrease in intestinal barrier function in a subject. The method includes the following steps: (3a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers; (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result; (3c) The step of diagnosing the disease or symptoms based on the comparison results obtained in step 3b; as well as (3d) In the event that the subject is diagnosed with the disease or symptoms, the step of administering to the subject a substance or composition for treating the disease or symptoms. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

4. A method for evaluating the risk of a subject developing a disease or symptoms caused by a decrease in intestinal barrier function, or the likelihood that the subject already has said disease or symptoms. The method includes the following steps: (4a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers; (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain a comparison result; and (4c) The step of evaluating the risk or the likelihood based on the comparison results obtained in step 4b. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

5. A method for obtaining data on disease or symptoms caused by decreased intestinal barrier function in a diagnostic subject. The method includes the following steps: (5a) The step of measuring biomarkers in a sample from the subject and obtaining the measured values ​​of the biomarkers; and (5b) The step of comparing the measured value obtained in step 5a with the reference value and obtaining the comparison result as the data. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

6. A method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function. The method includes the following steps: (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced; (6b) The step of determining a biomarker in a test sample obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the biomarker; (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain a comparison result; and (6d) Based on the comparison results obtained in step 6c, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

7. A method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function. The method includes the following steps: (7a) The step of administering the candidate substance or the candidate composition to a subject whose intestinal barrier function has been reduced; (7b) The step of determining a biomarker in a test sample obtained from the subject after administration of the candidate substance or the candidate composition, and obtaining a measured value of the biomarker; (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain a comparison result; and (7d) The step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms, based on the comparison results obtained in step 7c. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

8. The method according to any one of claims 1 to 7, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

9. The method according to any one of claims 1 to 7, wherein, The biomarkers comprise two, three, or four or more substances selected from the group.

10. The method according to any one of claims 1 to 7, wherein, The biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine.

11. The method according to claim 10, wherein, The biomarkers in the test sample were determined by capillary electrophoresis-mass spectrometry.

12. The method according to any one of claims 1 to 7, wherein, The biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzylidene)sorbitol.

13. The method according to claim 12, wherein, The biomarkers in the analyte were determined by liquid chromatography-mass spectrometry.

14. A biomarker, which is a biomarker derived from a test sample of a subject for evaluating intestinal barrier function in a subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

15. A biomarker, which is a biomarker derived from a test sample of a subject for diagnosing a disease or symptom caused by a decrease in intestinal barrier function. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

16. A biomarker, which is a biomarker from a test sample of a subject for evaluating the risk of a subject developing a disease or symptoms caused by a reduction in intestinal barrier function, or the likelihood that a subject has already developed said disease or symptoms. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

17. A biomarker, which is a biomarker from a test sample of said subject used to obtain data for diagnosing disease or symptoms caused by reduced intestinal barrier function in a subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

18. A biomarker for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, and which is a biomarker in a test sample obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to said subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

19. A biomarker for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, and wherein the biomarker is a biomarker in a analyte obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

20. The biomarker according to any one of claims 14 to 19, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

21. The biomarker according to any one of claims 14 to 19, wherein, The biomarkers comprise two, three, or four or more substances selected from the group.

22. The biomarker according to any one of claims 14 to 19, wherein, The biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine.

23. The biomarker according to claim 22, wherein, The biomarkers in the test sample were determined by capillary electrophoresis-mass spectrometry.

24. The biomarker according to any one of claims 14 to 19, wherein, The biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzylidene)sorbitol.

25. The biomarker according to claim 24, wherein, The biomarkers in the test sample were determined by liquid chromatography-mass spectrometry.

26. Use of a biomarker derived from a subject in the test sample for evaluating the intestinal barrier function of said subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

27. Use of a biomarker derived from a subject in the test sample for the diagnosis of diseases or symptoms in said subject caused by a decrease in intestinal barrier function. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

28. Use of a biomarker from a subject in an assay for evaluating the risk of said subject developing a disease or symptoms caused by a reduction in intestinal barrier function, or the likelihood that said subject already has said disease or symptoms. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

29. Use of a biomarker from a subject for obtaining data for diagnosing diseases or symptoms caused by reduced intestinal barrier function in said subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

30. Use of a biomarker, said biomarker being a biomarker in an analyte obtained from a subject with reduced intestinal barrier function after administration of a candidate substance or candidate composition to said subject, said use being for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

31. Use of a biomarker, said biomarker being a biomarker in an analyte obtained from a subject with reduced intestinal barrier function after administration of a candidate substance or candidate composition to said subject, said use being for evaluating the effect of said candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

32. The use according to any one of claims 26 to 31, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

33. The use according to any one of claims 26 to 31, wherein, The biomarkers comprise two, three, or four or more substances selected from the group.

34. The use according to any one of claims 26 to 31, wherein, The biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine.

35. The use according to claim 34, wherein, The biomarkers in the test sample were determined by capillary electrophoresis-mass spectrometry.

36. The use according to any one of claims 26 to 31, wherein, The biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzylidene)sorbitol.

37. The use according to claim 36, wherein, The biomarkers in the test sample were determined by liquid chromatography-mass spectrometry.

38. A kit for evaluating intestinal barrier function in subjects. The kit contains one or more reagents for determining biomarkers in the analyte from the subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

39. A kit for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject. The kit contains one or more reagents for determining biomarkers in the analyte from the subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

40. A kit for evaluating the risk of a subject developing a disease or symptoms caused by a decrease in intestinal barrier function, or the likelihood that the subject already has said disease or symptoms. The kit contains one or more reagents for determining biomarkers in the analyte from the subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

41. A kit for obtaining data on disease or symptoms caused by decreased intestinal barrier function in a diagnostic subject. The kit contains one or more reagents for determining biomarkers in the analyte from the subject. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

42. A kit for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function. The kit contains one or more reagents for determining biomarkers in analytes obtained after administration of candidate substances or candidate compositions to subjects with impaired intestinal barrier function. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

43. A kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function. The kit contains one or more reagents for determining biomarkers in analytes obtained from subjects with impaired intestinal barrier function after administration of the candidate substance or the candidate composition to the subjects. The sample to be tested was urine. The biomarker comprises one or more substances selected from the group described in claim 1.

44. The kit according to any one of claims 38 to 43, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

45. The kit according to any one of claims 38 to 43, wherein, The biomarkers comprise two, three, or four or more substances selected from the group.

46. ​​The kit according to any one of claims 38 to 43, wherein, The biomarker comprises one or more substances selected from the group consisting of 4-acetaminophen, N-acetylputrescine, 3-amino-2-piperidinone, and O-succinylhomoserine.

47. The kit according to claim 46, wherein, The biomarkers in the test sample were determined by capillary electrophoresis-mass spectrometry.

48. The kit according to any one of claims 38 to 43, wherein, The biomarker comprises one or more substances selected from the group consisting of cyclopentaacetic acid, DK cypermethrin, paraxanthine, and bis(methylbenzylidene)sorbitol.

49. The kit according to claim 48, wherein, The biomarkers in the test sample were determined by liquid chromatography-mass spectrometry.

50. A method for manufacturing a food or pharmaceutical composition, comprising the step of mixing a substance or composition selected by the method of claim 6 with one or more components constituting the food or pharmaceutical composition.

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