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 dry excrement or wet excrement, the biomarker including one or more substances selected from group A when the subject is dry excrement, and the biomarker including one or more substances selected from group B when the subject is wet excrement, and the biomarker including one or more substances selected from group A when the subject is wet excrement. The biomarker includes one or more substances selected from group B.

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 Aet 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] Solutions for solving problems

[0030] In this invention, group A consists of the substances shown in Table 1.

[0031] Table 1-1

[0032] Table 1 (Group A)

[0033] No. Substance name 1 2-Formylanthranilic acid 2 Diethanolamine 3 Glycerol 4 5,6-Dihydroxyindole 5 N-Imineformyl-L-glutamic acid 6 Leucine 7 Myristoleic acid 8 3-Aminopropane-1,2-diol 9 4-Guanidinobutyric acid 10 Phenlyalanine 11 2′-Deoxyadenosine and 5′-Deoxyadenosine 12 Lipoamide 13 3-Guanidinopropionic acid 14 Acetanilide 15 Histidine 16 γ-glutamyl-2-aminobutyric acid 17 Methionine sulfoxide 18 Phenylhydrazine 19 Tryptophan 20 Tyrosine 21 Hydroxyindole 22 Alanine 23 p-Aminobenzoic acid 24 Penicillamine 25 2-Cyanopyridine 26 Glutamine 27 2,4-Diaminobutyric acid 28 Trigonelline 29 2-Methylserine 30 Glycylglycine (Gly-Gly) 31 N,N-Dimethylhistidine 32 Valine 33 N-Acetylphenylalanine 34 Isoleucine 35 Methionine 36 γ-Glu-Val-Gly 37 N-acetylaspartic acid 38 Threonic acid

[0034] Table 1-2

[0035] Table 1 (continued)

[0036]

[0037] Table 1-3

[0038] Table 1 (continued)

[0039]

[0040] Table 1-4

[0041] Table 1 (continued)

[0042]

[0043] In this invention, group B consists of the substances shown in Table 2.

[0044] Table 2-1

[0045] Table 2 (Group B)

[0046] No. Substance name 1 2-Formylanthranilic acid 2 Glycerol 3 N-Imineformyl-L-glutamic acid 4 5,6-Dihydroxyindole 5 3-Aminopropane-1,2-diol 6 4-Guanidinobutyric acid 7 2′-Deoxyadenosine and 5′-Deoxyadenosine 8 Acetanilide 9 Pyrophosphate 10 Myristoleic acid 11 Diethanolamine 12 Thiaproline 13 Hydroxyindole 14 2,6-Diaminopimelicacid 15 p-Aminobenzoic acid 16 Lipoamide 17 Phenylhydrazine 18 3-Guanidinopropionic acid 19 N-acetylaspartic acid 20 2,4-Diaminobutyric acid 21 γ-glutamyl-2-aminobutyric acid 22 Cysteine 23 Glycerol 3-phosphate 24 Glucose 6-phosphate 25 Mucic acid 26 Trigonelline 27 N-Carbamoylaspartic acid 28 Threonic acid 29 Glycocholic acid 30 Isobutyric acid and butyric acid 31 2-Deoxyribose 1-phosphate 32 Arginine ethyl ester 33 N-Methylglutamic acid 34 Pterin 35 N-Acetylphenylalanine 36 Ribulose 5-phosphate 37 Fructose 6-phosphate 38 Sedoheptulose 7-phosphate

[0047]

Table 2-2

[0048] Table 2 (continued)

[0049]

[0050]

Table 2-3

[0051] Table 2 (continued)

[0052]

[0053] The present invention includes the following technical solutions.

[0054] [1] A method for evaluating the intestinal barrier function of a subject, the method comprising the following steps:

[0055] (1a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0056] (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain a comparison result; and

[0057] (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0058] The sample to be tested is either dry or wet feces.

[0059] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0060] When the test subject is wet feces, the biomarker contains one or more substances selected from group B below.

[0061] [2] A method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps:

[0062] (2a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0063] (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain a comparison result; and

[0064] (2c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 2b.

[0065] The sample to be tested is either dry or wet feces.

[0066] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0067] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0068] [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:

[0069] (3a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0070] (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result;

[0071] (3c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 3b; and

[0072] (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.

[0073] The sample to be tested is either dry or wet feces.

[0074] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0075] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0076] [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:

[0077] (4a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0078] (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain a comparison result; and

[0079] (4c) The step of evaluating the risk or the likelihood based on the comparison results obtained in step 4b.

[0080] The sample to be tested is either dry or wet feces.

[0081] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0082] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0083] [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:

[0084] (5a) The step of measuring biomarkers in a sample from the subject and obtaining the measured values ​​of the biomarkers; and

[0085] (5b) The step of comparing the measured value obtained in step 5a with the reference value and obtaining the comparison result as the data.

[0086] The sample to be tested is either dry or wet feces.

[0087] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0088] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0089] [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:

[0090] (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0091] (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;

[0092] (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain a comparison result; and

[0093] (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.

[0094] The sample to be tested is either dry or wet feces.

[0095] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0096] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0097] [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:

[0098] (7a) The step of administering the candidate substance or the candidate composition to a subject whose intestinal barrier function has been reduced;

[0099] (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;

[0100] (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain a comparison result; and

[0101] (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.

[0102] The sample to be tested is either dry or wet feces.

[0103] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0104] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0105] [8] The method according to any one of [1] to [7], wherein the intestinal barrier function is the barrier function of the small intestine.

[0106] [9] The method according to any one of [1] to [8], wherein, when the test subject is dried feces, the biomarker comprises two, three, or four or more substances selected from group A.

[0107] When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

[0108]

[10] The method according to any one of [1] to [9], wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol and 5,6-dihydroxyindole.

[0109] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

[0110]

[11] According to the method of

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

[0111]

[12] The method according to any one of [1] to [9], wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0112] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

[0113]

[13] According to the method of

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

[0114]

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

[0115] The sample to be tested is either dry or wet feces.

[0116] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0117] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0118]

[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.

[0119] The sample to be tested is either dry or wet feces.

[0120] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0121] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0122]

[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.

[0123] The sample to be tested is either dry or wet feces.

[0124] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0125] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0126]

[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.

[0127] The sample to be tested is either dry or wet feces.

[0128] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0129] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0130]

[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.

[0131] The sample to be tested is either dry or wet feces.

[0132] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0133] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0134]

[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.

[0135] The sample to be tested is either dry or wet feces.

[0136] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0137] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0138]

[20] The biomarker according to any one of

[14] to

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

[0139]

[21] The biomarker according to any one of

[14] to

[20] , wherein, when the test subject is dried feces, the biomarker comprises two, three, or four or more substances selected from group A.

[0140] When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

[0141]

[22] The biomarker according to any one of

[14] to

[21] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol and 5,6-dihydroxyindole.

[0142] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

[0143]

[23] According to the biomarker described in

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

[0144]

[24] The biomarker according to any one of

[14] to

[21] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0145] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

[0146]

[25] According to the biomarker described in

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

[0147]

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

[0148] The sample to be tested is either dry or wet feces.

[0149] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0150] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0151]

[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.

[0152] The sample to be tested is either dry or wet feces.

[0153] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0154] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0155]

[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.

[0156] The sample to be tested is either dry or wet feces.

[0157] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0158] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0159]

[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.

[0160] The sample to be tested is either dry or wet feces.

[0161] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0162] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0163]

[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.

[0164] The sample to be tested is either dry or wet feces.

[0165] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0166] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0167]

[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.

[0168] The sample to be tested is either dry or wet feces.

[0169] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0170] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0171]

[32] The use according to any one of

[26] to

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

[0172]

[33] According to any one of

[26] to

[32] , wherein, when the test subject is dried feces, the biomarker comprises two, three, or four or more substances selected from group A.

[0173] When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

[0174]

[34] According to any one of

[26] to

[33] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol and 5,6-dihydroxyindole.

[0175] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

[0176]

[35] According to the use described in

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

[0177]

[36] According to any one of

[26] to

[33] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0178] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

[0179]

[37] According to the use described in

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

[0180]

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

[0181] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0182] The sample to be tested is either dry or wet feces.

[0183] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0184] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0185]

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

[0186] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0187] The sample to be tested is either dry or wet feces.

[0188] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0189] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0190]

[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.

[0191] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0192] The sample to be tested is either dry or wet feces.

[0193] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0194] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0195]

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

[0196] The kit contains one or more reagents for determining biomarkers in the analyte from the subject.

[0197] The sample to be tested is either dry or wet feces.

[0198] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0199] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0200]

[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.

[0201] The kit contains one or more reagents for determining biomarkers in analytes obtained from subjects with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subjects.

[0202] The sample to be tested is either dry or wet feces.

[0203] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0204] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0205]

[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.

[0206] 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.

[0207] The sample to be tested is either dry or wet feces.

[0208] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0209] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0210]

[44] The kit according to any one of

[38] to

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

[0211]

[45] The kit according to any one of

[38] to

[44] , wherein, when the test subject is dried feces, the biomarker comprises two, three, or four or more substances selected from group A.

[0212] When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

[0213]

[46] The kit according to any one of

[38] to

[45] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole.

[0214] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

[0215]

[47] According to the kit described in

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

[0216]

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

[38] to

[45] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0217] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

[0218]

[49] According to the kit described in

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

[0219]

[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.

[0220]

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

[0221] The method includes the following steps:

[0222] (S101) Step of obtaining the measurement value of biomarkers from the test body of the object;

[0223] (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain a comparison result; and

[0224] (S103) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step S102.

[0225] The sample to be tested is either dry or wet feces.

[0226] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0227] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0228]

[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.

[0229] The method includes the following steps:

[0230] (S201) The step of obtaining the measurement value of the biomarker in the test body of the object;

[0231] (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result;

[0232] (S203) A step of diagnosing the disease or symptom based on the comparison results obtained in step S202.

[0233] The sample to be tested is either dry or wet feces.

[0234] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0235] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0236]

[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.

[0237] The method comprises the following steps:

[0238] (S301) The step of obtaining the measurement value of the biomarker in the test body from the object;

[0239] (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain a comparison result; and

[0240] (S303) The step of evaluating the risk or the likelihood based on the comparison results obtained in step S302.

[0241] The sample to be tested is either dry or wet feces.

[0242] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0243] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0244]

[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.

[0245] The method comprises the following steps:

[0246] (S401) The step of obtaining the measurement value of the biomarker from the test sample of the subject; and

[0247] (S402) The step of comparing the measured value obtained in step S401 with the reference value and obtaining the comparison result as the data.

[0248] The sample to be tested is either dry or wet feces.

[0249] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0250] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0251]

[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.

[0252] The method comprises the following steps:

[0253] (S501) A step of obtaining the determination value of a biomarker in a test body obtained after administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0254] (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain a comparison result; and

[0255] (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.

[0256] The sample to be tested is either dry or wet feces.

[0257] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0258] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0259]

[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.

[0260] The method includes the following steps:

[0261] (S601) A step of obtaining the determination value of a biomarker in a test body obtained after administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0262] (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain a comparison result; and

[0263] (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.

[0264] The sample to be tested is either dry or wet feces.

[0265] When the test subject is dried feces, the biomarker contains one or more substances selected from group A.

[0266] When the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0267]

[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.

[0268]

[58] The computer program according to any one of

[51] to

[57] , wherein, when the test subject is dried feces, the biomarker comprises two, three, or four or more substances selected from group A.

[0269] When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

[0270]

[59] The computer program according to any one of

[51] to

[58] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole.

[0271] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

[0272]

[60] According to the computer program described in

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

[0273]

[61] The computer program according to any one of

[51] to

[58] , wherein, when the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0274] When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

[0275]

[62] According to the computer program described in

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

[0276]

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

[51] to

[62] .

[0277] The effects of the invention

[0278] 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.

[0279] 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.

[0280] In addition, the present invention provides a method for treating diseases or symptoms caused by a decrease in intestinal barrier function in a subject.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] In addition, the present invention provides a method for manufacturing food or pharmaceutical compositions. Attached Figure Description

[0286] Figure 1 This is a schematic diagram of an apparatus according to one embodiment of the present invention.

[0287] Figure 2 This is a block diagram illustrating the hardware structure of an apparatus according to one embodiment of the present invention.

[0288] Figure 3 This is a flowchart illustrating the processing sequence of an apparatus according to one embodiment of the present invention.

[0289] Figure 4 This is a flowchart illustrating the processing sequence of an apparatus according to one embodiment of the present invention.

[0290] Figure 5 This is a flowchart illustrating the processing sequence of an apparatus according to one embodiment of the present invention. Detailed Implementation

[0291] 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 .

[0292] <<Option 1>>

[0293] Scheme 1 of the present invention relates to a method for evaluating the intestinal barrier function of a subject.

[0294] In the method of scheme 1, biomarkers from the test subjects are used as indicators to evaluate the intestinal barrier function of the subjects.

[0295] The subject can be any animal that excretes feces; there are no special 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 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 domestic fowl (e.g., chickens, wild ducks, domestic ducks).

[0296] Feces are excretions from the anus. Feces from the subject can be obtained from the subject's excretions using conventional methods and can be used as the test sample.

[0297] Based on the mass of the dried feces, the moisture content in the dried feces is preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less. The lower limit for the moisture content in the dried feces is zero. Dried feces can be obtained by drying feces that have just been expelled from the anus. The drying process can be carried out, for example, using a commercially available freeze dryer. In the freeze drying process, the processing temperature is, for example, above -80°C and below 0°C, and the processing time is, for example, above 24 hours and below 72 hours.

[0298] Based on the mass of the wet stool, the water content in the wet stool is preferably 40% by mass or more, more preferably 50% by mass or more, and even more preferably 60% by mass or more. There is no particular upper limit, but it can be, for example, 95% by mass or less, 90% by mass or less, 80% by mass or less, or 70% by mass or less. These upper limits can be combined with any of the lower limits. The wet stool can be stool just expelled from the anus, or stool obtained by freezing and thawing stool just expelled from the anus. The freezing temperature is preferably -100°C or higher and -10°C or lower, more preferably -80°C or higher and -15°C or lower, and even more preferably -80°C or higher and -60°C or lower. Freezing can be performed, for example, by collecting stool just expelled from the anus in a special container for stool collection (e.g., a stool collection tube with a screw cap and a sampling spoon (manufactured by Sarstedt)). The freezing time is preferably within 180 days from the date of stool collection, more preferably within 60 days from the date of stool collection, and even more preferably within 30 days from the date of stool collection. That is, it is preferable to use wet feces within 180 days from the date of collection for the determination of biomarkers, more preferably to use wet feces within 60 days from the date of collection for the determination of biomarkers, and even more preferably to use wet feces within 30 days from the date of collection for the determination of biomarkers.

[0299] 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.

[0300] The test sample from the subject can be either dry or wet stool. Using dry or wet stool as the test sample allows for a simple assessment of the subject's intestinal barrier function.

[0301] When the test sample from the subject is dried feces, one or more substances selected from group A are used as biomarkers to evaluate the subject's intestinal barrier function. That is, when the test sample from the subject is dried feces, the biomarker in the test sample from the subject contains one or more substances selected from group A.

[0302] Biomarkers from the test samples of the subjects may contain one substance selected from group A, or two or more substances selected from group A. From the viewpoint of improving the accuracy of evaluating the intestinal barrier function of the subjects, biomarkers from the test samples of the subjects preferably contain two, three, or four or more substances selected from group A.

[0303] When the test sample from the subject is wet feces, one or more substances selected from group B are used as biomarkers for evaluating the subject's intestinal barrier function. That is, when the test sample from the subject is wet feces, the biomarker in the test sample from the subject contains one or more substances selected from group B.

[0304] Biomarkers from the test samples of the subjects may contain one substance selected from group B, or two or more substances selected from group B. From the viewpoint of improving the accuracy of evaluating the intestinal barrier function of the subjects, biomarkers from the test samples of the subjects preferably contain two, three, or four or more substances selected from group B.

[0305] When the test sample from the subject is dried feces, the intestinal barrier function of the subject can be evaluated with high accuracy by using one or more substances selected from group A as biomarkers. Substances No. 1–15 and No. 63–86 in group A have higher accuracy in evaluating intestinal barrier function compared with other substances in group A, and substances No. 1–4 and No. 63–66 in group A have particularly high accuracy in evaluating intestinal barrier function compared with other substances in group A.

[0306] When the test sample is wet feces, the intestinal barrier function of the subject can be evaluated with high accuracy by using one or more substances selected from group B as biomarkers. Substances No. 1–11 and No. 46–67 in group B have higher accuracy in evaluating intestinal barrier function compared with other substances in group B, and substances No. 1–4 and No. 46–49 in group B have particularly high accuracy in evaluating intestinal barrier function compared with other substances in group B.

[0307] In one embodiment, the test sample from the subject is dried feces, and the biomarker in the test sample contains one or more substances selected from No. 1 to No. 4 of Group A (2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole). 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 may also contain one or more other substances selected from Group A (e.g., one or more substances selected from No. 5 to No. 62 of Group A).

[0308] In another embodiment, the test sample from the subject is wet feces, and the biomarker in the test sample contains one or more substances selected from No. 1 to No. 4 of Group B (2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid, and 5,6-dihydroxyindole). 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 may also contain one or more other substances selected from Group B (e.g., one or more substances selected from No. 5 to No. 45 of Group B).

[0309] In another embodiment, the test sample from the subject is dried feces, and the biomarker in the test sample from the subject includes one or more substances selected from No. 63 to No. 66 of Group A (3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate). 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 Group A (e.g., one or more substances selected from No. 67 to No. 128 of Group A).

[0310] In another embodiment, the test sample from the subject is wet feces, and the biomarker in the test sample from the subject includes one or more substances selected from No. 46 to No. 49 of Group B (6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline, and 3-methyladipic acid). 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 Group B (e.g., one or more substances selected from No. 50 to No. 99 of Group B).

[0311] In another embodiment, the test sample from the subject is dried feces, and the biomarker in the test sample from the subject includes substance No. 1 of Group A (2-formamidobenzoic acid). 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 Group A (e.g., one or more substances selected from substances No. 2 to No. 62 of Group A).

[0312] In another embodiment, the test sample from the subject is dried feces, and the biomarker in the test sample from the subject includes substance No. 2 (diethanolamine) from group A. 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 group A (e.g., one or more substances selected from substances No. 1 and No. 3 to No. 62 in group A).

[0313] In another embodiment, the test sample from the subject is dried feces, and the biomarker in the test sample from the subject includes substance No. 4 of Group A (5,6-dihydroxyindole). This can improve the accuracy of evaluating the subject's intestinal barrier function. In addition to this one substance, the biomarker in the test sample from the subject may also include one or more other substances selected from Group A (e.g., one or more substances selected from substances No. 1 to No. 3 and No. 5 to No. 62 of Group A).

[0314] In another embodiment, the test sample from the subject is dried feces, and the biomarker in the test sample from the subject includes substance No. 6 (leucine) from group A. This can improve the accuracy of evaluating the subject's intestinal barrier function. In addition to this one substance, the biomarker in the test sample from the subject may also include one or more other substances selected from group A (e.g., one or more substances selected from substances No. 1 to No. 5 and No. 7 to No. 62 in group A).

[0315] In another embodiment, the test sample from the subject is wet feces, and the biomarker in the test sample from the subject includes substance No. 1 in group B (2-formamidobenzoic acid). 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 group B (e.g., one or more substances selected from substances No. 2 to No. 45 in group B).

[0316] In another embodiment, the test sample from the subject is wet feces, and the biomarker in the test sample from the subject includes substance No. 4 from group B (5,6-dihydroxyindole). This can improve the accuracy of evaluating the subject's intestinal barrier function. In addition to this one substance, the biomarker in the test sample from the subject may also include one or more other substances selected from group B (e.g., one or more substances selected from substances No. 1 to No. 3 and No. 5 to No. 45 from group B).

[0317] In another embodiment, the test sample from the subject is wet feces, and the biomarker in the test sample from the subject includes substance No. 6 (4-guanidinobutyric acid) from group B. This can improve the accuracy of evaluating the subject's intestinal barrier function. In addition to this one substance, the biomarker in the test sample from the subject may also include one or more other substances selected from group B (e.g., one or more substances selected from substances No. 1 to No. 5 and No. 7 to No. 45 in group B).

[0318] In another embodiment, the test sample from the subject is wet feces, and the biomarker in the test sample from the subject includes substance No. 7 from group B (2'-deoxyadenosine and 5'-deoxyadenosine). This can improve the accuracy of evaluating the subject's intestinal barrier function. In addition to this one substance, the biomarker in the test sample from the subject may also include one or more other substances selected from group B (e.g., one or more substances selected from substances No. 1 to No. 6 and No. 8 to No. 45 from group B).

[0319] Option 1 includes the following steps:

[0320] (1a) The step of measuring biomarkers from the test sample of the subject and obtaining the measured values ​​of the biomarkers; and

[0321] (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain the comparison result; and

[0322] (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0323] <Step 1a>

[0324] Step 1a is the step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers.

[0325] The test sample from the subject is either dry or wet feces.

[0326] When the test sample from the subject is dried feces, one or more substances selected from group A are used as biomarkers for evaluating the subject's intestinal barrier function. That is, when the test sample from the subject is dried feces, the biomarkers measured in step 1a contain one or more substances selected from group A.

[0327] If the biomarker measured in step 1a contains one substance selected from group A, then that one substance is measured in step 1a. If the biomarker measured in step 1a contains two or more substances selected from group A, 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 group A (i.e., in step 1a, two, three, or four or more substances selected from group A are measured).

[0328] When the test sample from the subject is wet feces, one or more substances selected from group B are used as biomarkers for evaluating the subject's intestinal barrier function. That is, when the test sample from the subject is wet feces, the biomarkers measured in step 1a contain one or more substances selected from group B.

[0329] If the biomarker measured in step 1a contains one substance selected from group B, then that one substance is measured in step 1a. If the biomarker measured in step 1a contains two or more substances selected from group B, 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 group B (i.e., in step 1a, two, three, or four or more substances selected from group B are measured).

[0330] "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".

[0331] Concentration can be relative or absolute, but is preferably absolute.

[0332] 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.

[0333] Substance No. 11 in Group A consists of two substances (2'-deoxyadenosine and 5'-deoxyadenosine). Regarding substance No. 11 in Group A, these two substances are treated as a single entity. Therefore, the concentration of substance No. 11 in Group A refers to the combined concentration of these two substances.

[0334] Substance No. 58 in Group A consists of two substances (3'-cytidine and 2'-cytidine).

[0335] Regarding substance No. 58 in Group A, these two substances are treated as a whole and treated as one substance. Therefore, the concentration of substance No. 58 in Group A refers to the combined concentration of 3'-cytidine and 2'-cytidine.

[0336] Substance No. 7 in Group B consists of two substances (2'-deoxyadenosine and 5'-deoxyadenosine). Regarding substance No. 7 in Group B, these two substances are treated as a single entity. Therefore, the concentration of substance No. 7 in Group B refers to the combined concentration of these two substances.

[0337] Substance No. 30 in Group B consists of two substances (isobutyric acid and butyric acid). Regarding substance No. 30 in Group B, these two substances are treated as a single entity. Therefore, the concentration of substance No. 30 in Group B refers to the combined concentration of these two substances.

[0338] Substance No. 41 in Group B consists of two substances (3'-cytidine and 2'-cytidine). Regarding substance No. 41 in Group B, these two substances are treated as a single entity. Therefore, the concentration of substance No. 41 in Group B refers to the combined concentration of these two substances.

[0339] In this invention, "DL" (refer to No. 118 in Group A and No. 94 in Group B) refers to a mixture of D-body and L-body.

[0340] 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.

[0341] 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.

[0342] 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).

[0343] 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.

[0344] 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).

[0345] 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.

[0346] The determination of substances No. 1 to No. 62 in Group A is preferably performed by CE-MS (especially CE-FTMS), and the determination of substances No. 63 to No. 128 in Group A is preferably performed by LC-MS (especially LC-MS / MS). The m / z values ​​of each substance in Group A are shown in Table 18.

[0347] The determination of substances No. 1 to No. 45 in Group B is preferably performed by CE-MS (especially CE-FTMS), and the determination of substances No. 46 to No. 99 in Group B is preferably performed by LC-MS (especially LC-MS / MS). The m / z values ​​of each substance in Group B are shown in Table 16.

[0348] 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).

[0349] 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)

[0350] 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.

[0351] There are no special restrictions on n as long as it is an integer greater than or equal to 2, such as integers greater than or equal to 2 and less than or equal to 128 (e.g., 3, 4, 6, 10, 50, 90, 99, 128).

[0352] 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.

[0353] The test sample obtained from the subject is dried feces, and the biomarker measured in step 1a contains two or more substances selected from No. 1 to No. 4 of group A (2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole). 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 3 can be used to calculate the composite variable from the concentration of the two or more substances based on the formula (1). In Table 3, "CFD_A" represents 2-formamidobenzoic acid, "CFD_B" represents diethanolamine, "CFD_C" represents glycerol, and "CFD_D" represents 5,6-dihydroxyindole.

[0354]

[0355] The test sample from the subject is dried feces, and the biomarkers measured in step 1a include two or more substances selected from No. 63 to No. 66 of group A (3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate). 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 4 can be used to determine the composite variable based on the concentration of the two or more substances according to the formula (1). In Table 4, “LFD_A” represents 3-methyl adipic acid, “LFD_B” represents 2-hydroxyquinoline, “LFD_C” represents 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione, and “LFD_D” represents 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0356]

[0357] When the test sample from the subject is wet feces, and the biomarker measured in step 1a contains two or more substances selected from No. 1 to No. 4 of group B (2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid, and 5,6-dihydroxyindole), and the relative area value (relative concentration) of each of the two or more substances is determined by CE-FTMS under the conditions described in the examples, the composite variable can be obtained from the concentration of the two or more substances based on the concentration of the two or more substances using, for example, the coefficients and constants shown in Table 5. In Table 5, “CFW_A” represents 2-formamidobenzoic acid, “CFW_B” represents glycerol, “CFW_C” represents N-iminoformyl-L-glutamic acid, and “CFW_D” represents 5,6-dihydroxyindole.

[0358]

[0359] When the test sample from the subject is wet feces, and the biomarker measured in step 1a contains two or more substances selected from No. 46 to No. 49 of group B (6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline, and 3-methyl adipic acid), and the relative area values ​​(relative concentrations) of the two or more substances are determined by LC-MS / MS under the conditions described in the examples, for example, the coefficients and constants shown in Table 6 can be used to determine the composite variable from the concentrations of the two or more substances based on Equation (1). In Table 6, “LFW_A” represents 6-methylquinoline, “LFW_B” represents N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, “LFW_C” represents 2-hydroxyquinoline, and “LFW_D” represents 3-methyl adipic acid.

[0360]

[0361] <Step 1b>

[0362] Step 1b is to compare the measured value obtained in step 1a with the benchmark value to obtain the comparison result.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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).

[0368] 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.

[0369] If two or more substances from the analyte are measured in step 1a, then 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, if substances No. 1 and No. 2 in group A are measured in step 1a, then 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.

[0370] 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).

[0371] 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.

[0372] 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.

[0373] <Step 1c>

[0374] Step 1c is a step to evaluate the intestinal barrier function of the subjects based on the comparison results obtained in step 1b.

[0375] 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.

[0376] 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.

[0377] The intestinal barrier function, particularly the physical barrier function such as tight junction structures, is the subject of evaluation.

[0378] Regarding the substances selected from Group A, the intestinal barrier function of the subjects can be evaluated based on whether the comparison results obtained in step 1b meet the criteria shown in Table 7. Specifically, for each substance with a "high" criterion in Table 7, 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 7), 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 7), it can be evaluated as negative. Conversely, for substances with a "low" criterion in Table 7, 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 7), 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 7), it can be evaluated as negative. It should be noted that the criteria shown in Table 7 can also be applied to schemes other than Scheme 1.

[0379] Regarding the substances selected from Group B, the intestinal barrier function of the subjects can be evaluated based on whether the comparison results obtained in step 1b meet the criteria shown in Table 8. Specifically, for substances with a "high" criterion in Table 8, a positive result can be evaluated 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 8), and a negative result can be evaluated 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 8). Similarly, for substances with a "low" criterion in Table 8, a positive result can be evaluated 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 8), and a negative result can be evaluated 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 8). It should be noted that the criteria shown in Table 8 can also be applied to schemes other than Scheme 1.

[0380] In step 1a, if the comparison result of one substance selected from group A meets the criteria shown in Table 7, it can be evaluated as positive; if the comparison result of one substance does not meet the criteria shown in Table 7, it can be evaluated as negative.

[0381] In step 1a, if two or more substances selected from group A 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 7; 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 7. The more substances whose comparison results meet the criteria shown in Table 7, the higher the probability of a positive result. Therefore, in step 1a, measuring two or more substances selected from group A (e.g., two, three, or four or more) can improve the accuracy of evaluating the intestinal barrier function of the subject.

[0382] In step 1a, if the comparison result of one substance selected from group B meets the criteria shown in Table 8, it can be evaluated as positive; if the comparison result of one substance does not meet the criteria shown in Table 8, it can be evaluated as negative.

[0383] In step 1a, if two or more substances selected from group B 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 8; 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 8. The more substances whose comparison results meet the criteria shown in Table 8, the higher the probability of a positive result. Therefore, in step 1a, measuring two or more substances selected from group B (e.g., two, three, or four or more) can improve the accuracy of evaluating the intestinal barrier function of the subject.

[0384] Table 71

[0385] Table 7 (Group A)

[0386] No. Substance name benchmark 1 2-Formamidobenzoic acid high 2 diethanolamine Low 3 glycerin Low 4 5,6-Dihydroxyindole high 5 N-Imineformyl-L-glutamic acid high 6 Leucine Low 7 Myristoleic acid Low 8 3-Aminopropane-1,2-diol Low 9 4-Guidinobutyric acid Low 10 Phenylalanine Low 11 2′-Deoxyadenosine and 5′-Deoxyadenosine high 12 Thioctamide Low 13 3-Guidinopropionic acid Low 14 Acetaniline high 15 Histidine Low 16 γ-Glutamyl-2-aminobutyric acid high 17 Methionine sulfoxide Low 18 phenylhydrazine Low 19 Tryptophan Low 20 Tyrosine Low 21 Hydroxyindole high 22 Alanine Low 23 p-Aminobenzoic acid high 24 Penicillamine Low 25 2-Cyanopyridine Low 26 glutamine Low 27 2,4-Diaminobutyric acid high 28 Trigonelline Low 29 2-Methylserine Low 30 glycyl glycine Low 31 N,N-Dimethylhistidine Low 32 Valine Low 33 N-acetylphenylalanine Low 34 Isoleucine Low 35 Methionine Low 36 γ-Glutamylvaline glycine Low 37 N-acetylaspartic acid high 38 Threonic acid Low 39 Alloleucine Low 40 Arginine ethyl ester Low

[0387] Table 72

[0388] Table 7 (continued)

[0389] No. Substance name benchmark 41 2,6-Diaminopimelic acid high 42 Glucose 6-phosphate high 43 adenosine Low 44 mucic acid Low 45 thymidine Low 46 Glycinecholic acid Low 47 2-Deoxyribose-1-phosphate high 48 Isoleucylprolylproline Low 49 Sedum heptanose-7-phosphate high 50 fumaric acid Low 51 N-acetylmethionine Low 52 Fructose-6-phosphate high 53 Glutamine glutamic acid Low 54 Cysteine Low 55 N-acetylglucosamine 1-phosphate high 56 Ribose 5-phosphate high 57 Glucose 1-phosphate high 58 3′-cytidine and 2′-cytidine high 59 dihydroxyacetone phosphate high 60 4-Oxovaleric acid Low 61 2-Deoxyglucose-6-phosphate high 62 Glucosamine 6-phosphate high 63 3-Methyladipic acid Low 64 2-Hydroxyquinoline Low 65 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione Low 66 2,4-Dihydroxyheptadec-16-en-1-yl acetate Low 67 α-Aspartylphenylalanine Low 68 N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide high 69 1-(2,4-Dihydroxyquinolin-3-yl)ethane-1-one Low 70 4-(Diethylamino)salicylaldehyde high 71 Atenol Low 72 5-Hydroxyindole high 73 6-Methylquinoline high 74 Guanine Low 75 Carboxy-ibuprofen Low 76 3-Isopropylmalic acid Low 77 bis(methylbenzylidene)sorbitol Low 78 2-Furfuric acid Low 79 whey acid high

[0390] Table 7-3

[0391] Table 7 (continued)

[0392] No. Substance name benchmark 80 2-Acetamino-N-β-Aspartic-2-deoxypyranohexosamine Low 81 flavonoids high 82 1-Propyl-1H-benzo[d]imidazolium hydrobromide high 83 4-Hydroxybenzenesulfonic acid Low 84 Xanthohumol Low 85 laurolactam Low 86 adenine Low 87 2′-Deoxyguanosine Low 88 6-Hydroxypyridinecarboxylic acid Low 89 D-(-)-quinic acid Low 90 4-{[(4,6-dimethoxypyrimidin-2-yl)amino]methylene}-2-phenyl-4,5-dihydro-1,3-oxazol-5-one Low 91 4-[(6E)-3-hydroxy-8,10-dimethyl-2-(methylamino)-6-dodecen-1-yl]phenol Low 92 2-Cyano-3-(Tetrahydro-3-thiophenylamino)ethyl acrylate Low 93 3-Succinopyridine Low 94 proline Low 95 L-Threo-3-phenylserine high 96 (24-Diamino-5-pyrimidinyl)(3,45-trimethoxyphenyl, methyl ketone) Low 97 3-Morpholin-4-tetrahydro-1H-pyrrole-1-ylcyclobut-3-ene-1,2-dione Low 98 16-Hydroxyhexadecanoic acid Low 99 clove acid Low 100 Tranexamic acid Low 101 3,5-Dimethylisoxazole-4-carboxylic acid 4-(tert-butyl)phenyl ester Low 102 3-Methylhistidine Low 103 5-Methoxyindole high 104 Perilla acid Low 105 6-Methyl[1,2,4]triazolo[4,3-b]pyridazine-8-ol high 106 Piperonitrile high 107 N1-(5-chloro-2-hydroxyphenyl)-2-(morpholinylmethyl)benzamide Low 108 N-methylhistamine Low 109 1-(1,3-benzodioxono-5-yl)-2-(dimethylamino)-1-butanone Low 110 (-)-Gallic acid Low 111 Polysaccharin Low 112 4-Indolecarboxaldehyde high 113 Uracil high 114 dihydrothymidine Low 115 3-Methylglutaric acid Low 116 1,5-Dehydro-D-glucol Low

[0393]

Table 7-4

[0394] Table 7 (continued)

[0395] No. Substance name benchmark 117 Pentamethrin Low 118 DL-arginine Low 119 (R)-Equivalerate high 120 Theobromine Low 121 1,5-Isoquinolinediol Low 122 12-Hydroxydodecanoic acid Low 123 1,3,5-Trihydroxy-4-{[(2E)-3-(3-hydroxy-4-methoxyphenyl)prop-2-enoyl]oxy}cyclohexane-1-carboxylic acid Low 124 3-Hydroxypyridinecarboxylic acid Low 125 Theophylline Low 126 3,4-Dihydroxybenzaldehyde Low 127 Acetylcholine high 128 Tridecanoic acid high

[0396] Table 8-1

[0397] Table 8 (Group B)

[0398] NO. Substance name benchmark 1 2-Formamidobenzoic acid high 2 glycerin Low 3 N-Imineformyl-L-glutamic acid high 4 5,6-Dihydroxyindole high 5 3-Aminopropane-1,2-diol Low 6 4-Guidinobutyric acid Low 7 2′-Deoxyadenosine and 5′-Deoxyadenosine high 8 Acetaniline high 9 Pyrophosphate high 10 Myristoleic acid Low 11 diethanolamine Low 12 Thioproline high 13 Hydroxyindole high 14 2,6-Diaminopimelic acid high 15 p-Aminobenzoic acid high 16 Thioctamide Low 17 phenylhydrazine Low 18 3-Guidinopropionic acid Low 19 N-acetylaspartic acid high 20 2,4-Diaminobutyric acid high 21 γ-Glutamyl-2-aminobutyric acid high 22 Cysteine Low 23 Glycerol 3-phosphate high 24 Glucose 6-phosphate high 25 mucic acid Low 26 Trigonelline Low 27 N-Carbamoyl Aspartic Acid high 28 Threonic acid Low 29 Glycinecholic acid Low 30 Isobutyric acid and butyric acid high 31 2-Deoxyribose-1-phosphate high 32 Arginine ethyl ester Low 33 N-methylglutamic acid high 34 Butterfly high 35 N-acetylphenylalanine Low 36 Ribulose 5-phosphate high 37 Fructose-6-phosphate high 38 Sedum heptanose-7-phosphate high 39 N-acetylglucosamine 1-phosphate high 40 Ribose 5-phosphate high

[0399] Table 8-2

[0400] Table 8 (continued)

[0401] NO. Substance name benchmark 41 3′-cytidine and 2′-cytidine high 42 dihydroxyacetone phosphate high 43 4-Oxovaleric acid Low 44 Propranolol high 45 2-Deoxyglucose-6-phosphate high 46 6-Methylquinoline high 47 N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide high 48 2-Hydroxyquinoline Low 49 3-Methyladipic acid Low 50 L-Threo-3-phenylserine high 51 4-(Diethylamino)salicylaldehyde high 52 5-Methoxyindole high 53 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione Low 54 4-Indolecarboxaldehyde high 55 Carboxy-ibuprofen Low 56 Piperonitrile high 57 5-Hydroxyindole high 58 whey acid high 59 Glucose 1-phosphate high 60 L-Alanyl-L-proline high 61 2-Acetamino-N-β-Aspartic-2-deoxypyranohexosamine Low 62 α-Aspartylphenylalanine Low 63 6-Hydroxymelatonin high 64 flavonoids high 65 Atenol Low 66 (R)-Equivalerate high 67 2,4-Dihydroxyheptadec-16-en-1-yl acetate Low 68 Guanine Low 69 1-(4-methyl-2-pyridyl)pyrrolidine-2,5-dione high 70 laurolactam Low 71 Omesartan high 72 adenine Low 73 Uracil high 74 6-Methyl[1,2,4]triazolo[4,3-b]pyridazine-8-ol high 75 1-Propyl-1H-benzo[d]imidazolium hydrobromide high 76 pseudouridine high 77 D-(-)-quinic acid Low 78 Acetylcholine high

[0402] Table 8-3

[0403] Table 8 (continued)

[0404] NO. Substance name benchmark 79 3-Isopropylmalic acid Low 80 2-Methoxy-5-(1H-1,2,4-triazol-5-yl)-4-(trifluoromethyl)pyridine high 81 3-Amino-2-naphthoic acid high 82 ornithine high 83 2′-Deoxyguanosine Low 84 bis(methylbenzylidene)sorbitol Low 85 2-Furfuric acid Low 86 3,5-Dimethylisoxazole-4-carboxylic acid 4-(tert-butyl)phenyl ester Low 87 Pyridoxine high 88 Tridecanoic acid high 89 3-Methylhistidine Low 90 hexanoic acid high 91 Azobenzene high 92 (-)-Gallic acid Low 93 3-(2-thienyl)-1,2,4-oxadiazole-5-carbonylhydrazine high 94 DL-lactic acid high 95 6-Hydroxypyridinecarboxylic acid Low 96 proline Low 97 1,3,5-Trihydroxy-4-{[(2E)-3-(3-hydroxy-4-methoxyphenyl)prop-2-enoyl]oxy}cyclohexane-1-carboxylic acid Low 98 Prolyl glycine high 99 4-[(6E)-3-hydroxy-8,10-dimethyl-2-(methylamino)-6-dodecen-1-yl]phenol Low

[0405] The baseline values ​​for each substance selected from Group A or Group B can be set using conventional methods.

[0406] In one embodiment, for each substance selected from group A or group B, the average value of the measured values ​​of each substance in the test sample obtained from the 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 it is a human) can be used as the baseline value for each substance. Hereinafter, this embodiment (hereinafter referred to as "Embodiment 1-1") will be described.

[0407] 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).

[0408] If the test sample used in step 1a is dried feces, the test sample obtained from the negative group is also dried feces.

[0409] If the test sample used in step 1a is wet feces, the test sample obtained from the negative group is also wet feces.

[0410] 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.

[0411] If the measured value obtained in step 1a is the relative concentration of one substance selected from group A or group B, the reference value for that substance used in step 1b is the average relative concentration of that 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. If 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. If 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.

[0412] If the measured value obtained in step 1a is the absolute concentration of one substance selected from group A or group B, the reference value for that substance used in step 1b is the average absolute concentration of that 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.

[0413] If the measured values ​​obtained in step 1a are relative concentrations of two or more substances selected from group A or group B, the reference values ​​for each substance used in step 1b are the average relative concentrations of each substance in the analyte obtained from the negative control group. The relative concentration of the latter is synonymous with the relative concentration of the former and is calculated in the same way. If 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. If 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.

[0414] If the measured values ​​obtained in step 1a are the absolute concentrations of two or more substances selected from group A or group B, the reference values ​​for each substance used in step 1b are the average values ​​of the absolute concentrations of each substance in the test sample obtained from the negative group.

[0415] Regarding the substances selected from group A, the intestinal barrier function of the subject is preferably evaluated based on whether the comparison results obtained in step 1b meet the criteria shown in Table 9. Regarding the substances selected from group B, the intestinal barrier function of the subject is preferably evaluated based on whether the comparison results obtained in step 1b meet the criteria shown in Table 10.

[0416] Specifically, in Table 9 or 10, 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 9 or 10, 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 even 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.

[0417] Taking substance No. 1 in group A as an example, if the measured value obtained in step 1a is more than twice the average value, it is preferably evaluated as positive; if the measured value obtained in step 1a is more than three times the average value, it is more preferably evaluated as positive; and if the measured value obtained in step 1a is more than five times the average value, it is even more preferably evaluated as positive.

[0418] Taking substance No. 2 in group A as an example, if the measured value obtained in step 1a is less than 0.8 times the average value, it is preferably evaluated as positive; if the measured value obtained in step 1a is less than 0.7 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.

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431] In another embodiment, for each substance selected from group A or group B, the median value of the measured values ​​of each substance in the test sample obtained from the negative group (the 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 it is a human) can be used as the reference value for each substance. Hereinafter, this embodiment (hereinafter referred to as "Embodiment 1-2") will be described.

[0432] The description of Implementation 1-1 also applies to Implementation 1-2, unless otherwise specified. In application, "average value" is replaced with "median value".

[0433] In yet another embodiment, for each substance selected from group A or group B, the cutoff value obtained from the ROC (Receiver Operating Characteristic) curve of each substance can be used as the baseline value for each substance. This embodiment (hereinafter referred to as "Embodiments 1-3") will be described below.

[0434] 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).

[0435] When the test sample used in step 1a is dried feces, the test samples obtained from the negative group and the positive group are also dried feces.

[0436] If the test sample used in step 1a is wet feces, the test samples obtained from the negative group and the positive group are also wet feces.

[0437] When the measured value obtained in step 1a is the relative concentration of one substance selected from group A or group B, the concentration of that substance used in the preparation of its ROC curve is also a relative concentration. The latter relative concentration is synonymous with the former relative concentration and is calculated in the same way. If the former relative concentration is a relative area value CE, the latter relative concentration is also a relative area value CE. If the former relative concentration is a relative area value LC, the latter relative concentration is also a relative area value LC. When the concentration used in the preparation of the ROC curve for that substance is a relative concentration, the cutoff value obtained from the ROC curve for that substance is also a relative concentration.

[0438] If the measured value obtained in step 1a is the absolute concentration of one substance selected from group A or group B, the concentration of that substance used in the preparation of its ROC curve 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.

[0439] When the measured values ​​obtained in step 1a are relative concentrations of two or more substances selected from group A or group B, 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 way. If 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. If 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 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.

[0440] If the measured values ​​obtained in step 1a are absolute concentrations of two or more substances selected from group A or group B, 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.

[0441] 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.

[0442] The specific examples of the cutoff values ​​for each substance selected from Group A are shown in Table 18, and the specific examples of the cutoff values ​​for each substance selected from Group B are shown in Table 16.

[0443] The cutoff values ​​for each substance selected from Group A are preferably values ​​within the range 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 Group A can be values ​​within the range above the value shown in P1 of Table A and below the value shown in P2 of Table A, or values ​​within the range 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 Group A can be values ​​shown in P1 of Table A, values ​​shown in P2 of Table A, or values ​​shown in P3 of Table A.

[0444] The cutoff values ​​for each substance selected from Group B 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 each substance selected from Group B 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 each substance selected from Group B can be values ​​shown in Q1 of Table B, values ​​shown in Q2 of Table B, or values ​​shown in Q3 of Table B.

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456] In another embodiment, regarding two or more substances selected from group A or group B, the concentrations of these two or more substances in the test sample obtained from a negative group (a group of individuals with no decreased intestinal barrier function) and a positive group (a group of individuals with decreased intestinal barrier function) composed 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 these two or more substances. An ROC curve is generated based on the obtained composite variable, and the cutoff value obtained from the generated ROC curve is used as the reference value for these two or more substances. Hereinafter, this embodiment (hereinafter referred to as "Embodiments 1-4") will be described.

[0457] Implementation methods 1-4 are applicable to cases where the measured values ​​obtained in step 1a are composite variables derived from the concentrations of two or more substances selected from group A or group B based on the formula (1).

[0458] When the measured value obtained in step 1a is a composite variable derived from the relative concentrations of two or more substances selected from group A or group B using equation (1), the concentration used when determining the cutoff value is also a relative concentration. The latter relative concentration is synonymous with the former relative concentration and is calculated in the same way. If 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. If 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.

[0459] When the measured value obtained in step 1a is a composite variable derived from the absolute concentrations of two or more substances selected from group A or group B using equation (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 calculated in the same way.

[0460] 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.

[0461] The test sample is dried feces, and the biomarkers measured in step 1a include two or more substances selected from No. 1 to No. 4 of group A (2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole). When the relative area values ​​(relative concentrations) of these 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 3, and the specific examples of the cutoff values ​​are shown in Table 11. In Table 11, "CFD_A", "CFD_B", "CFD_C", and "CFD_D" are synonymous with those in Table 3.

[0462] Table 11)

[0463] Table 11 (Dried feces, Group A, No. 1 to No. 4)

[0464]

[0465] The cutoff values ​​for No. 1 to No. 11 in Table 11 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 11 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 11 can be values ​​shown in R1 of Table C, values ​​shown in R2 of Table C, or values ​​shown in R3 of Table C.

[0466] Table C

[0467] Table C

[0468] No. R1 R2 R3 1 -0.3903 -0.3002 -0.2101 2 0.0558 0.0797 0.1036 3 0.1781 0.2544 0.3307 4 -0.2123 -0.1633 -0.1143 5 -0.0568 -0.0437 -0.0306 6 -0.4684 -0.3603 -0.2522 7 0.1085 0.1550 0.2015 8 0.1633 0.2333 0.3033 9 -0.0430 -0.0331 -0.0232 10 0.1985 0.2835 0.3686 11 0.0854 0.1220 0.1586

[0469] The test sample was dried feces, and the biomarkers measured in step 1a included two or more substances selected from No. 63 to No. 66 of Group A (3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate). When the relative area values ​​(relative concentrations) of these two or more substances were determined by LC-MS / MS under the conditions described in the examples, the specific examples of coefficients and constants are shown in Table 4, and the specific examples of cutoff values ​​are shown in Table 12. In Table 12, “LFD A”, “LFD_B”, “LFD_C”, and “LFD_D” are synonymous with those in Table 4.

[0470] Table 12)

[0471] Table 12 (Dried feces, Group A, No. 63–No. 66)

[0472]

[0473] The cutoff values ​​for No. 1 to No. 11 in Table 12 are preferably values ​​within the range of above the value shown in S1 of Table D and below the value shown in S3 of Table D. For example, the cutoff values ​​for No. 1 to No. 11 in Table 12 can be values ​​within the range of above the value shown in S1 of Table D and below the value shown in S2 of Table D, or values ​​within the range of above the value shown in S2 of Table D and below the value shown in S3 of Table D. The cutoff values ​​for No. 1 to No. 11 in Table 12 can be values ​​shown in S1 of Table D, values ​​shown in S2 of Table D, or values ​​shown in S3 of Table D.

[0474] Table D

[0475] Table D

[0476] No. S1 S2 S3 1 -0.7329 -0.5638 -0.3947 2 -0.3896 -0.2997 -0.2098 3 -0.8272 -0.6363 -0.4454 4 0.0942 0.1345 0.1749 5 -0.2565 -0.1973 -0.1381 6 -0.0884 -0.0680 -0.0476 7 -0.4958 -0.3814 -0.2670 8 -0.9893 -0.7610 -0.5327 9 -0.7192 -0.5532 -0.3872 10 -0.1622 -0.1248 -0.0874 11 -0.6986 -0.5374 -0.3762

[0477] The test sample is wet feces, and the biomarkers measured in step 1a include two or more substances selected from No. 1 to No. 4 of group B (2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid, and 5,6-dihydroxyindole). When the relative area values ​​(relative concentrations) of these two or more substances are determined by CE-FTMS under the conditions described in the examples, the specific examples of coefficients and constants are shown in Table 5, and the specific examples of cutoff values ​​are shown in Table 13. In Table 13, "CFWA", "CFW B", "CFWC", and "CFWD" are synonymous with those in Table 5.

[0478] Table 13)

[0479] Table 13 (Wet feces, Group B, No. 1 to No. 4)

[0480]

[0481] The cutoff values ​​for No. 1 to No. 11 in Table 13 are preferably values ​​within the range of above the value shown in T1 of Table E and below the value shown in T3 of Table E. For example, the cutoff values ​​for No. 1 to No. 11 in Table 13 can be values ​​within the range of above the value shown in T1 of Table E and below the value shown in T2 of Table E, or values ​​within the range of above the value shown in T2 of Table E and below the value shown in T3 of Table E. The cutoff values ​​for No. 1 to No. 11 in Table 13 can be values ​​shown in T1 of Table E, values ​​shown in T2 of Table E, or values ​​shown in T3 of Table E.

[0482] Table E

[0483] Table E

[0484] No. T1 T2 T3 1 0.0286 0.0409 0.0532 2 0.2062 0.2945 0.3829 3 0.4859 0.6941 0.9023 4 0.2028 0.2897 0.3766 5 0.1742 0.2488 0.3234 6 0.1919 0.2742 0.3565 7 0.1460 0.2086 0.2712 8 0.1992 0.2846 0.3700 9 0.1449 0.2070 0.2691 10 0.1050 0.1500 0.1950 11 0.0250 0.0357 0.0464

[0485] When the test sample is wet feces, and the biomarker measured in step 1a contains two or more substances selected from No. 46 to No. 49 of group B (6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline, and 3-methyladipic acid), and 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 the coefficients and constants are shown in Table 6, and the specific examples of the cutoff values ​​are shown in Table 14. In Table 14, "LFW A", "LFW B", "LFWC", and "LFWD" are synonymous with those in Table 6.

[0486]

Table 14

[0487] Table 14 (Wet feces, Group B, No. 46–No. 49)

[0488]

[0489] The cutoff values ​​for No. 1 to No. 11 in Table 14 are preferably values ​​within the range of above the value shown in U1 of Table F and below the value shown in U3 of Table F. For example, the cutoff values ​​for No. 1 to No. 11 in Table 14 can be values ​​within the range of above the value shown in U1 of Table F and below the value shown in U2 of Table F, or values ​​within the range of above the value shown in U2 of Table F and below the value shown in U3 of Table F. The cutoff values ​​for No. 1 to No. 11 in Table 14 can be values ​​shown in U1 of Table F, values ​​shown in U2 of Table F, or values ​​shown in U3 of Table F.

[0490] Table F

[0491] Table F

[0492] No. U1 U2 U3 1 0.1982 0.2831 0.3680 2 0.0902 0.1288 0.1674 3 0.3073 0.4390 0.5707 4 0.0216 0.0309 0.0402 5 -0.8317 -0.6398 -0.4479 6 -0.5612 -0.4317 -0.3022 7 0.0988 0.1412 0.1836 8 -0.0632 -0.0486 -0.0340 9 0.2895 0.4135 0.5376 10 -0.4060 -0.3123 -0.2186 11 0.0764 0.1091 0.1418

[0493] <<Option 2>>

[0494] 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".

[0495] The method of Scheme 2 of the present invention includes the following steps:

[0496] (2a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0497] (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain the comparison result; and

[0498] (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.

[0499] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0500] 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".

[0501] In Option 2, "positive" means that the subject has contracted a disease or symptoms, and "negative" means that the subject has not contracted a disease or symptoms.

[0502] 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.

[0503] 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.

[0504] 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.

[0505] 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.

[0506] 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.

[0507] <<Option 3>>

[0508] 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".

[0509] The method of Scheme 3 of the present invention includes the following steps:

[0510] (3a) The step of measuring biomarkers in the test sample from the subject and obtaining the measured values ​​of the biomarkers;

[0511] (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result;

[0512] (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

[0513] (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.

[0514] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0515] 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".

[0516] 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.

[0517] 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.

[0518] 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.

[0519] 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).

[0520] <<Option 4>>

[0521] 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."

[0522] Option 4 includes the following steps:

[0523] (4a) The step of measuring biomarkers from the test sample of the subject and obtaining the measured values ​​of the biomarkers; and

[0524] (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain the comparison result; and

[0525] (4c) Based on the comparison results obtained in step 4b, the step of evaluating the risk or likelihood of occurrence,

[0526] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0527] 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".

[0528] 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.

[0529] 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.

[0530] 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.

[0531] The description of diseases or symptoms caused by decreased intestinal barrier function is the same as in Scheme 2.

[0532] <<Option 5>>

[0533] 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".

[0534] Option 5 includes the following steps:

[0535] (5a) The step of measuring biomarkers from the test sample of the subject and obtaining the measured values ​​of the biomarkers; and

[0536] (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.

[0537] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0538] 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".

[0539] 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.

[0540] 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.

[0541] The description of diseases or symptoms caused by decreased intestinal barrier function is the same as in Scheme 2.

[0542] For each substance selected from Group A, comparison results that meet the criteria shown in Table 7 can be used as data for a positive diagnosis, and comparison results that do not meet the criteria shown in Table 7 can be used as data for a negative diagnosis. Specifically, for each substance with a "high" criterion in Table 7, a comparison result where the measured value obtained in step 5a is higher than the criterion value can be used as data for a positive diagnosis, and a comparison result where the measured value obtained in step 5a is lower than the criterion value can be used as data for a negative diagnosis. Similarly, for substances with a "low" criterion in Table 7, a comparison result where the measured value obtained in step 5a is lower than the criterion value can be used as data for a positive diagnosis, and a comparison result where the measured value obtained in step 5a is higher than the criterion value can be used as data for a negative diagnosis.

[0543] For each substance selected from Group B, comparison results that meet the criteria shown in Table 8 can be used as data for a positive diagnosis, and comparison results that do not meet the criteria shown in Table 8 can be used as data for a negative diagnosis. Specifically, for substances with a "high" criterion in Table 8, a comparison result where the measured value obtained in step 5a is higher than the criterion value can be used as data for a positive diagnosis, and a comparison result where the measured value obtained in step 5a is lower than the criterion value can be used as data for a negative diagnosis. Similarly, for substances with a "low" criterion in Table 8, a comparison result where the measured value obtained in step 5a is lower than the criterion value can be used as data for a positive diagnosis, and a comparison result where the measured value obtained in step 5a is higher than the criterion value can be used as data for a negative diagnosis.

[0544] In step 5a, if the comparison result of one substance selected from group A meets the criteria shown in Table 7, the comparison result can be used as data for a negative diagnosis. If the comparison result of one substance does not meet the criteria shown in Table 7, the comparison result can be used as data for a negative diagnosis.

[0545] In step 5a, when measuring two or more substances selected from group A, if the comparison result of at least one of the two or more substances meets the criteria shown in Table 7, the comparison result can be used 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 7, the comparison result can be used as data for a negative diagnosis. The more substances whose comparison results meet the criteria shown in Table 7, the higher the probability of a positive result. Therefore, in step 5a, by measuring two or more substances selected from group A (e.g., two, three, or four or more), the accuracy of diagnosing the subject's disease or symptoms can be improved.

[0546] In step 5a, if the comparison result of one substance selected from group B meets the criteria shown in Table 8, the comparison result can be used as data for diagnosis of positive. If the comparison result of one substance does not meet the criteria shown in Table 8, the comparison result can be used as data for diagnosis of negative.

[0547] In step 5a, when measuring two or more substances selected from group B, if the comparison result of at least one of the two or more substances meets the criteria shown in Table 8, the comparison result can be used 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 8, the comparison result can be used as data for a negative diagnosis. The more substances whose comparison results meet the criteria shown in Table 8, the higher the probability of a positive result. Therefore, in step 5a, by measuring two or more substances selected from group B (e.g., two, three, or four or more), the accuracy of diagnosing the subject's disease or symptoms can be improved.

[0548] When Scheme 1, embodiment 1-1 is applied to Scheme 5, for each substance selected from group A, it is preferable to obtain comparison results based on the benchmarks shown in Table 9 as data for diagnosis, and for each substance selected from group B, it is preferable to obtain comparison results based on the benchmarks shown in Table 10 as data for diagnosis.

[0549] Specifically, in Table 9 or 10, 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 9 or 10, 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.

[0550] Taking substance No. 1 in group A as an example, it is preferable to obtain a comparison result that is more than twice 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 that is more than three 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 that is more than five times the average value of the measurement obtained in step 5a as the data for diagnosing a positive result.

[0551] Taking substance No. 2 in group A as an example, it is preferable to obtain a comparison result that is less than 0.8 times the average value of the measured value obtained in step 5a as the data for diagnosing a positive result, more preferably to obtain a comparison result that is less than 0.7 times the average value of the measured value obtained in step 5a as the data for diagnosing a positive result, and even more preferably to obtain a comparison result that is less than 0.5 times the average value of the measured value obtained in step 5a as the data for diagnosing a positive result.

[0552] 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.

[0553] 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.

[0554] 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.

[0555] <<Option 6>>

[0556] 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".

[0557] Option 6 includes the following steps:

[0558] (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0559] (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

[0560] (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain the comparison result; and

[0561] (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.

[0562] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0563] 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".

[0564] 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.

[0565] 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.

[0566] 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.

[0567] The description of diseases or symptoms caused by decreased intestinal barrier function is the same as in Scheme 2.

[0568] 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.

[0569] Step 6a is the step of administering the candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced.

[0570] 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.

[0571] 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.

[0572] 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.

[0573] 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).

[0574] 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.

[0575] 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).

[0576] 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.

[0577] 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.

[0578] 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.

[0579] Step 6c is to compare the measured value obtained in step 6b with the benchmark value to obtain the comparison result.

[0580] 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.

[0581] 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.

[0582] 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.

[0583] Regarding the substances selected from Group A, the improvement effect on intestinal barrier function or the prevention or improvement effect on disease or symptoms can be evaluated based on whether the comparison results obtained in step 6c meet the criteria shown in Table 7. Specifically, for each substance with a "high" criterion shown in Table 7, 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 7), 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 7), it can be evaluated as positive. Conversely, for substances with a "low" criterion shown in Table 7, 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 7), 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 7), it can be evaluated as positive.

[0584] Regarding the substances selected from Group B, the improvement effect on intestinal barrier function or the prevention or improvement effect on disease or symptoms can be evaluated based on whether the comparison results obtained in step 6c meet the criteria shown in Table 8. Specifically, for substances with a "high" criterion shown in Table 8, 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 8), 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 8), it can be evaluated as positive. Conversely, for substances with a "low" criterion shown in Table 8, 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 8), 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 8), it can be evaluated as positive.

[0585] In step 6b, if the comparison result of one substance selected from group A meets the criteria shown in Table 7, it can be evaluated as negative; if the comparison result of one substance does not meet the criteria shown in Table 7, it can be evaluated as positive.

[0586] In step 6b, if two or more substances selected from group A 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 7, 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 7. The more substances whose comparison results do not meet the criteria shown in Table 7, the higher the probability of a positive result. Therefore, in step 6b, by measuring two or more substances selected from group A (e.g., two, three, or four or more), the accuracy of evaluating the improvement effect of candidate substances or candidate compositions on intestinal barrier function or the prevention or improvement effect on diseases or symptoms can be improved.

[0587] In step 6b, if one substance selected from group B is measured, the result of the comparison of that substance meets the criteria shown in Table 8, it can be evaluated as negative; if the result of the comparison of that substance does not meet the criteria shown in Table 8, it can be evaluated as positive.

[0588] In step 6b, if two or more substances selected from group B 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 8, 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 8. The more substances whose comparison results do not meet the criteria shown in Table 8, the higher the probability of a positive result. Therefore, in step 6b, by measuring two or more substances selected from group B (e.g., two, three, or four or more), the accuracy of evaluating the improvement effect of candidate substances or candidate compositions on intestinal barrier function or the prevention or improvement effect on diseases or symptoms can be improved.

[0589] <<Option 7>>

[0590] 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".

[0591] The method in Scheme 7 includes the following steps:

[0592] (7a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0593] (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

[0594] (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain the comparison result; and

[0595] (7d) A step of evaluating the effect of candidate substances or candidate compositions on improving intestinal barrier function or preventing or improving disease or symptoms based on the comparison results obtained in step 7c, wherein the test subject is dry feces or wet feces, wherein the biomarker contains one or more substances selected from group A when the test subject is dry feces, and wherein the biomarker contains one or more substances selected from group B when the test subject is wet feces.

[0596] 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".

[0597] <<Option 8>>

[0598] 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 dry or wet stool, wherein, in the case of dry stool, the biomarker contains one or more substances selected from group A, and in the case of wet stool, the biomarker contains one or more substances selected from group B.

[0599] The explanation of Option 1 also applies to Option 8, unless otherwise specified.

[0600] <<Option 9>>

[0601] 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 dry or wet stool. In the case of dry stool, the biomarker contains one or more substances selected from group A, and in the case of wet stool, the biomarker contains one or more substances selected from group B.

[0602] The explanation of Option 2 also applies to Option 9, unless otherwise specified.

[0603] <<Option 10>>

[0604] 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 dry or wet stool. In the case of dry stool, the biomarker contains one or more substances selected from group A, and in the case of wet stool, the biomarker contains one or more substances selected from group B.

[0605] The explanation of Option 4 also applies to Option 10, unless otherwise specified.

[0606] <<Option 11>>

[0607] 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 dry or wet stool, wherein, in the case of dry stool, the biomarker contains one or more substances selected from group A, and in the case of wet stool, the biomarker contains one or more substances selected from group B.

[0608] The explanation of Option 5 also applies to Option 11, unless otherwise specified.

[0609] <<Option 12>>

[0610] 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 from a subject whose intestinal barrier function has been reduced, after the subject has been given a candidate substance or candidate composition, the test sample being dry stool or wet stool, wherein if the test sample is dry stool, the biomarker contains one or more substances selected from group A, and if the test sample is wet stool, the biomarker contains one or more substances selected from group B.

[0611] The explanation of Option 6 also applies to Option 12, unless otherwise specified.

[0612] <<Option 13>>

[0613] 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. The biomarker is a test sample obtained from a subject with decreased intestinal barrier function after administration of the candidate substance or candidate composition to the subject. The test sample is dry or wet stool. In the case of dry stool, the biomarker contains one or more substances selected from group A. In the case of wet stool, the biomarker contains one or more substances selected from group B.

[0614] The explanation of Scheme 7 also applies to Scheme 13, unless otherwise specified.

[0615] <<Option 14>>

[0616] 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 dry or wet stool, wherein, in the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0617] The explanation of Option 1 also applies to Option 14, unless otherwise specified.

[0618] <<Option 15>>

[0619] Scheme 15 of the present invention relates to the use of a biomarker from a subject in a test sample for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function in the subject, the test sample being dry or wet stool, wherein the biomarker comprises one or more substances selected from group A in the case of dry stool, and comprises one or more substances selected from group B in the case of wet stool.

[0620] The explanation of Option 2 also applies to Option 15, unless otherwise specified.

[0621] <<Option 16>>

[0622] Scheme 16 of the present invention relates to the use of a biomarker from a subject in a test sample for evaluating the risk of the subject developing a disease or symptom caused by a reduction in intestinal barrier function or the likelihood that the subject has already developed the disease or symptom, the test sample being dry or wet stool, wherein, in the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0623] The explanation of Option 4 also applies to Option 16, unless otherwise specified.

[0624] <<Option 17>>

[0625] Scheme 17 of the present invention relates to the use of a biomarker from a subject in a test sample for obtaining data for diagnosing diseases or symptoms caused by a decrease in intestinal barrier function in the subject, the test sample being dry or wet stool, wherein, in the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0626] The explanation of Option 5 also applies to Option 17, unless otherwise specified.

[0627] <<Option 18>>

[0628] 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 dry stool or wet stool, wherein in the case of dry stool, the biomarker contains one or more substances selected from group A, and in the case of wet stool, the biomarker contains one or more substances selected from group B.

[0629] The explanation of Option 6 also applies to Option 18, unless otherwise specified.

[0630] <<Option 19>>

[0631] Scheme 19 of the present invention relates to the use of a biomarker, wherein the biomarker 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 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, wherein the test sample is dry stool or wet stool, wherein in the case of dry stool, the biomarker contains one or more substances selected from group A, and in the case of wet stool, the biomarker contains one or more substances selected from group B.

[0632] The explanation of Scheme 7 also applies to Scheme 19, unless otherwise specified.

[0633] <<Option 20>>

[0634] 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 determining a biomarker in a test sample from the subject, the test sample being dry or wet stool, wherein, in the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0635] The explanation of Option 1 also applies to Option 20, unless otherwise specified.

[0636] The kit may contain one reagent or two or more reagents.

[0637] 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.

[0638] In one embodiment, the kit contains one or more reagents for determining the concentration of a 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). Reagents for determining the concentration of the target substance by CE-MS may include, for example, standards selected from group A or group B.

[0639] 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). Reagents for determining the concentration of the target substance by LC-MS may include, for example, standards selected from group A or group B.

[0640] <<Option 21>>

[0641] 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 biomarkers in a test sample from the subject, the test sample being dry or wet stool, wherein, in the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0642] The explanation of Option 2 also applies to Option 21, unless otherwise specified.

[0643] The instructions regarding the reagents are the same as in Scheme 20.

[0644] <<Option 22>>

[0645] 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 a subject has already developed said disease or symptoms. The kit comprises one or more reagents for measuring a biomarker in a test sample from the subject, the test sample being dry or wet stool. In the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0646] The explanation of Option 4 also applies to Option 22, unless otherwise specified.

[0647] The instructions regarding the reagents are the same as in Scheme 20.

[0648] <<Option 23>>

[0649] 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 test sample from the subject, the test sample being dry or wet stool, wherein, in the case of dry stool, the biomarker comprises one or more substances selected from group A, and in the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0650] The explanation of Option 5 also applies to Option 23, unless otherwise specified.

[0651] The instructions regarding the reagents are the same as in Scheme 20.

[0652] <<Option 24>>

[0653] 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 comprises reagents for determining one or more biomarkers 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. The test sample is dry or wet stool. In the case of dry stool, the biomarker comprises one or more substances selected from group A. In the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0654] The explanation of Option 6 also applies to Option 24, unless otherwise specified.

[0655] The instructions regarding the reagents are the same as in Scheme 20.

[0656] <<Option 25>>

[0657] 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 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. The test sample is dry or wet stool. In the case of dry stool, the biomarker comprises one or more substances selected from group A. In the case of wet stool, the biomarker comprises one or more substances selected from group B.

[0658] The explanation of Scheme 7 also applies to Scheme 25, unless otherwise specified.

[0659] The instructions regarding the reagents are the same as in Scheme 20.

[0660] <<Option 26>>

[0661] 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.

[0662] 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.

[0663] The method of Scheme 26 may include the step of screening substances or compositions by the method of Scheme 6.

[0664] <<Option 27>>

[0665] 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.

[0666] The method for enabling a computer to execute a computer program according to scheme 27 includes the following steps:

[0667] (S101) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0668] (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain the comparison result; and

[0669] (S103) Based on the comparison results obtained in step S102, the step of evaluating the intestinal barrier function of the subjects.

[0670] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0671] The explanation of Option 1 also applies to Option 27, unless otherwise specified.

[0672] 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.

[0673] 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.

[0674] 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.

[0675] 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.

[0676] 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).

[0677] The measuring device 20 only needs to be capable of performing the determination of biomarkers in a sample (e.g., a 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 sample, such as the collection of feces from the subject (i.e., collecting feces from the subject's excrement for use as the analyte), the preparation of the sample from the subject's feces, and the setting of the sample in the measuring device 20, can be performed by a human or by the measuring device 20. In one embodiment, processing preceding the determination of biomarkers in the sample is performed by a human.

[0678] 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.

[0679] 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.

[0680] 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.

[0681] 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.

[0682] 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.

[0683] 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.

[0684] 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.

[0685] 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.

[0686] 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.

[0687] 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.

[0688] 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.

[0689] The following is for reference Figures 3-5 This explains the processing sequence of device 10.

[0690] 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.

[0691] 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.

[0692] 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.

[0693] 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.

[0694] 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.

[0695] 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.

[0696] 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.

[0697] Regarding the substances selected from Group A, CPU310 can evaluate the subject's intestinal barrier function based on whether the comparison results obtained in step S102 meet the criteria shown in Table 7. Specifically, for the substances whose criteria are "high" as shown in Table 7, 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 references shown in Table 7) (step S103a). If the measured value obtained in step 101 is higher than the reference value (i.e., the comparison result meets the references shown in Table 7), 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 references shown in Table 7), it is evaluated as negative (step S103c). Furthermore, regarding substances for which the references shown in Table 7 are "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 7) (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 7), 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 7), it is evaluated as negative (step S103f).

[0698] Regarding the substances selected from Group B, CPU310 can evaluate the subject's intestinal barrier function based on whether the comparison results obtained in step S102 meet the criteria shown in Table 8. Specifically, for the substances whose criteria are "high" as shown in Table 8, such as... Figure 4 As 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 references shown in Table 8) (step S103a). If the measured value obtained in step 101 is higher than the reference value (i.e., the comparison result meets the references shown in Table 8), 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 references shown in Table 8), it is evaluated as negative (step S103c). Furthermore, regarding substances for which the references shown in Table 8 are "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 8) (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 8), 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 8), it is evaluated as negative (step S103f).

[0699] <<Option 28>>

[0700] 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".

[0701] The method for enabling a computer to execute a computer program according to scheme 28 includes the following steps:

[0702] (S201) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0703] (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result;

[0704] (S203) Based on the comparison results obtained in step S202, the step of diagnosing the disease or symptoms caused by the reduction of intestinal barrier function of the subject, wherein the test subject is dry stool or wet stool, wherein if the test subject is dry stool, the biomarker contains one or more substances selected from group A, and if the test subject is wet stool, the biomarker contains one or more substances selected from group B.

[0705] 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".

[0706] <<Option 29>>

[0707] 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."

[0708] The method for enabling a computer to execute a computer program according to scheme 29 includes the following steps:

[0709] (S301) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0710] (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain the comparison result; and

[0711] (S303) The step of evaluating the risk or the likelihood based on the comparison results obtained in step S302.

[0712] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0713] 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".

[0714] <<Option 30>>

[0715] 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".

[0716] The method for enabling a computer to execute a computer program according to Scheme 30 includes the following steps:

[0717] (S401) Step of obtaining the measurement value of biomarkers from the test body of the subject;

[0718] (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.

[0719] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0720] 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".

[0721] <<Option 31>>

[0722] 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".

[0723] The method for enabling a computer to execute a computer program according to Scheme 31 includes the following steps:

[0724] (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;

[0725] (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain the comparison result; and

[0726] (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.

[0727] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0728] 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."

[0729] <<Option 32>>

[0730] 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".

[0731] The method for enabling a computer to execute a computer program according to scheme 32 includes the following steps:

[0732] (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;

[0733] (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain the comparison result; and

[0734] (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.

[0735] The test subject is dry or wet feces. If the test subject is dry feces, the biomarker contains one or more substances selected from group A. If the test subject is wet feces, the biomarker contains one or more substances selected from group B.

[0736] 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".

[0737] <<Option 33>>

[0738] 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.

[0739] 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.

[0740] Example

[0741] The present invention will now be described based on embodiments.

[0742] 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.

[0743] 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.

[0744] 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.

[0745] Mobile phase A: 10 mM ammonium formate (pH 10.5)

[0746] Mobile phase B: Acetonitrile (90%) / 100mM ammonium formate (pH 10.5) (10%)

[0747] Flow conditions: 0-15 min B 97%-89%, 15-18 min B 89%

[0748] Flow rate: 0.4 mL / min

[0749] Injection volume: 5μL

[0750] Temperature: 35℃

[0751] Ion spray voltage: -4500V

[0752] 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.

[0753] Excretion rate = Excretion volume (μmol / mL) × Molecular weight (μg / μmol) × Urine volume (mL) / Sugar intake (μg) × 100

[0754] After calculating the excretion rate (%), the lactulose / mannitol ratio is calculated based on the following formula.

[0755] Lactulose / mannitol ratio = Lactulose excretion rate / Mannitol excretion rate

[0756] 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.

[0757] Wet stool and serum were obtained from the negative and positive groups and used in the following comparative examples and embodiments.

[0758] 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.

[0759] The following procedure is used to obtain wet stool samples. Two days prior to the lactulose-mannitol test, or at any time on the day of the test, have the subject collect approximately 10g of freshly excreted stool into a collection container (a screw-capped collection tube with a sampling spoon (Sarstedt)) and store it in a refrigerator until submission. To keep the stool frozen, on the day of the test, place the collection container containing the stool in an insulated container and carry it with you for submission. After submission, store it in a deep freezer (-80°C) until use.

[0760] <<Comparative Example 1>>

[0761] 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.

[0762] Using the serum ligin concentrations in the negative and positive groups, ROC (Receiving Controller Operational Characteristic) curves were constructed. The area under the curve (AUC) and cutoff value were then calculated from the ROC curves. The ROC curves, AUC, and cutoff value were obtained using the statistical analysis software "EXCEL Statistics" (manufactured by Social Survey Research Information Co., Ltd.). The ROC curve is a plot showing the serum ligin concentrations in the negative and positive groups, with sensitivity on the vertical axis and (1-specificity) on the horizontal axis. The AUC is the area under the ROC curve. The cutoff value was taken as the point on the ROC curve that is closest to the upper left corner of the ROC curve. The results are shown in Table 15.

[0763] Table 15

[0764] Table 15

[0765] Substance name Determination methods AUC Cutoff value ligin ELISA 0.638 39.70

[0766] <<Example 1>>

[0767] (1) Metabolomics analysis of wet stool

[0768] A comprehensive metabolomics analysis was performed on all wet stool samples obtained from both the negative and positive groups, determining the metabolites present in each sample. The metabolomics analysis was conducted using capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0769] The sample for CE-FTMS analysis was prepared from wet feces as follows: 500 μL of Milli-Q water containing an internal standard (20 μM) was added to 30–50 mg of wet feces. The mixture was centrifuged under vortex stirring conditions to ensure complete precipitation of the feces without disintegration of the precipitate. 350 μL of the supernatant was transferred to an ultrafiltration unit and centrifuged (4 °C, 9100 × g, 1–2 h) until the solution in the filter cup was almost completely eliminated, thus removing protein. The resulting wet fecal sample was stored at -80 °C. For analysis, after thawing, 20 μL of Milli-Q water was added to 80 μL and stirred; this was used as the analysis sample. It should be noted that the internal standard used was Human Metabolome Technologies (HMT) internal standard (H3304-1002; HMT).

[0770] 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.

[0771] [Catonic metabolites (cationic model)]

[0772] Running buffer: Cation buffer (p / n: H3301-1001)

[0773] Rinse buffer: Cation buffer (p / n: H3301-1001)

[0774] Sample injection: Inject at 50 mbar for 10 seconds.

[0775] CE voltage: positive, 30kV

[0776] MS ionization: ESI cathode

[0777] MS capillary voltage: 4,000V

[0778] MS scan range: m / z 60-900

[0779] Sheath fluid: HMT sheath fluid (p / n: I3301-1040)

[0780] [Anionic Metabolites (Anionic Pattern)]

[0781] Running buffer: Anion exchange buffer (p / n: H3302-1023)

[0782] Rinse buffer: Anion exchange buffer (p / n: H3302-1023)

[0783] Sample injection: 50 mbar pressurized injection, 22 seconds

[0784] CE voltage: positive, 30kV

[0785] MS ionization: ESI negative electrode

[0786] MS capillary voltage: 3,500V

[0787] MS scan range: m / z 70-1,050

[0788] Sheath fluid: HMT sheath fluid (p / n: I3301-1040)

[0789] The analyte samples used in LC-MS / MS were prepared from wet feces as follows: 1600 μL of cooled methanol containing 0.15% formic acid and 300 μL of Milli Q water were added to every 100 mg of wet feces and stirred thoroughly. The mixture was then incubated on ice for 1 hour. The resulting mixture was centrifuged (13000 rpm, 15 min, 4 °C), and 200 μL of the supernatant was dried under nitrogen. For analysis using an octadecylsilane (ODS) column, the supernatant was dissolved in 200 μL of 10% methanol (containing 0.1% formic acid). For analysis using a hydrophilic interaction liquid chromatography (HILIC) column, the supernatant was dissolved 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 Milli Q filter (Millex FH). Internal standards are not 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 to HILIC column, ODS column is more suitable for determining hydrophobic substances, while HILIC column is more suitable for determining hydrophilic substances.

[0790] 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.

[0791] [Determination using an ODS column]

[0792] Mobile phase A: An aqueous solution containing 0.1% formic acid by mass

[0793] Mobile phase B: Methanol containing 0.1% formic acid by mass

[0794] 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%.

[0795] Flow rate: 0.1 mL / min for 0-3 min, 0.25 mL / min for 3-40 min

[0796] Injection volume: 2μL

[0797] Temperature: 55℃

[0798] MS ionization: Switching modes (ESI positive electrode, ESI negative electrode)

[0799] MS capillary voltage: 2.5kV

[0800] MS scan range: m / z 70-1050

[0801] [Measurements performed using a HILIC column]

[0802] Mobile phase A: 10 mM ammonium acetate aqueous solution (pH 9.6)

[0803] Mobile phase B: Acetonitrile

[0804] 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%

[0805] Flow rate: 0.25 mL / min

[0806] Injection volume: 2μL

[0807] Temperature: 20℃

[0808] MS ionization: Switching modes (ESI positive electrode, ESI negative electrode)

[0809] MS capillary voltage: 2.5kV

[0810] MS scan range: m / z 70-1050

[0811] For each wet stool 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 16. 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 value (relative concentration) of each substance in each wet stool sample was calculated based on the following formula.

[0812] The relative area value of each substance in each wet feces = (peak area value of each substance) / (peak area value of the internal standard substance) × (mass of each wet feces))

[0813] The wet stool samples (samples No. 1–No. 27) obtained from the negative control group (27 individuals) and the positive control group (27 individuals) were sequentially measured by LC-MS / MS. Then, using the Normalize Area 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 wet stool samples (samples No. 1–No. 27) obtained from the negative control group (27 individuals) and the wet stool samples (samples No. 28–No. 54) obtained 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 wet stool sample were calculated.

[0814] Specifically, along with the measurements of each sample No. 1 to No. 54, after measuring a mixed sample of 15 samples, 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 wet feces.

[0815] It should be noted that the correction using mixed samples is performed to correct for errors caused by the time-dependent decay of the peak area values ​​of each substance.

[0816] ROC (Responder Operating Characteristic) curves were constructed using the relative area values ​​of each substance in each wet stool 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 wet stool 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 16. 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.

[0817] Table 16 shows only the results for substances with an AUC of 0.65 or higher (substances No.1 to No.99 in Group B) that were fully measured.

[0818] For each substance in Group B, from No. 1 to No. 99, 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 wet stool sample. The results are shown in Table 17. Table 17 also shows the trends in the positive group. "Increase" indicates a trend of increase in the positive group, and "Decrease" indicates a trend of decrease in the positive group.

[0819] The results shown in Tables 16 and 17 indicate that, when the test subjects had wet feces, the intestinal barrier function could be evaluated with high accuracy by using one or more substances selected from Group B as biomarkers. The closer the AUC was to 1, the higher the accuracy in evaluating intestinal barrier function. Therefore, the substances No. 1 to No. 4 in Group B (2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid, and 5,6-dihydroxyindole) and the substances No. 46 to No. 49 in Group B (6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline, and 3-methyladipic acid) showed particularly high accuracy in evaluating intestinal barrier function.

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830] (2) Metabolomics analysis of dried feces

[0831] Approximately 1 g of each wet stool sample obtained from the negative and positive groups was aliquoted into 25 mL centrifuge tubes, and the mass before drying was measured. Each wet stool sample was dried for 2 days using a freeze dryer (RFS2000, Asahi Scientific Corporation), and the mass after drying was measured. The moisture content of each wet stool sample was calculated from the mass before and after drying, and the solid content ratio of each wet stool sample was calculated from the moisture content. The relative area value of each substance in each wet stool sample obtained using the wet stool sample described in (1) was divided by the solid content ratio of each wet stool sample, and the value obtained was used as the relative area value of each substance in each dried stool sample. It should be noted that the values ​​of each biomarker in the dried stool sample did not change significantly when converted from the values ​​of each biomarker in the wet stool sample, or when the dried stool sample was used for analysis.

[0832] ROC (Responsible Response Characteristic) curves were constructed using the relative area values ​​of each substance in each dried stool 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 dried stool 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 18. 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.

[0833] Table 18 shows only the results for substances with an AUC of 0.65 or higher in the metabolites that were fully measured (substances No.1 to No.128 in Group A).

[0834] For each substance in Group A, from No. 1 to No. 128, 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 dried feces. The results are shown in Table 19. Table 19 also shows the trends in the positive group. "Increase" indicates a trend of increase in the positive group, and "Decrease" indicates a trend of decrease in the positive group.

[0835] The results shown in Tables 18 and 19 indicate that, when the test subjects were dry feces, the intestinal barrier function could be evaluated with high accuracy by using one or more substances selected from Group A as biomarkers. Since the accuracy of evaluating intestinal barrier function is higher the closer the AUC is to 1, the substances No. 1–No. 4 in Group A (2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole), and substances No. 63–No. 66 in Group A (3-methyladipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione, and 2,4-dihydroxyheptadec-16-en-1-yl acetate) showed particularly high accuracy in evaluating intestinal barrier function.

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849] (3) Composite variables (statistical analysis values)

[0850] (3-1) Substances No. 1 to No. 4 in Group A

[0851] For two or more substances selected from No. 1 to No. 4 of Group A (2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole), the relative area values ​​obtained in (2) 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 20. The coefficients and constants are obtained through multinomial logistic regression analysis. The coefficients and constants are shown in Table 3. In Table 20, “CFD_A” represents 2-formamidobenzoic acid, “CFD_B” represents diethanolamine, “CFD_C” represents glycerol, and “CFD_D” represents 5,6-dihydroxyindole.

[0852] 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

[0853] As shown in Table 20, it is clear that combining two or more substances selected from No.1 to No.4 in Group A can increase the AUC of the ROC curve, that is, improve the accuracy of evaluating intestinal barrier function.

[0854]

[0855] (3-2) Substances No. 63 to No. 66 in Group A

[0856] For two or more substances selected from No. 63 to No. 66 in Group A (3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate), the composite variable was calculated using the relative area values ​​obtained in (2) in the same manner as in (3-1). An ROC curve was then constructed based on the calculated composite variable, and the AUC and cutoff value were determined from the constructed ROC curve. The results are shown in Table 21. The coefficients and constants were obtained through multinomial logistic regression analysis. The coefficients and constants are shown in Table 4. In Table 21, “LFD_A” represents 3-methyl adipic acid, “LFD_B” represents 2-hydroxyquinoline, “LFD_C” represents 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione, and “LFD_D” represents 2,4-dihydroxyheptadec-16-en-1-yl acetate.

[0857] As shown in Table 21, it is clear that combining two or more substances selected from No. 63 to No. 66 in Group A can increase the AUC of the ROC curve, that is, improve the accuracy of evaluating intestinal barrier function.

[0858]

[0859] (3-3) Substances No. 1 to No. 4 in Group B

[0860] For two or more substances selected from No.1 to No.4 in Group B (2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid, and 5,6-dihydroxyindole), the relative area values ​​obtained in (1) were used to calculate the composite variable in the same way as in (3-1). Based on the calculated composite variable, an ROC curve was constructed, and the AUC and cutoff value were determined from the constructed ROC curve. The results are shown in Table 22. The coefficients and constants were obtained through multinomial logistic regression analysis. The coefficients and constants are shown in Table 5. In Table 22, “CFW_A” represents 2-formamidobenzoic acid, “CFW_B” represents glycerol, “CFW_C” represents N-iminoformyl-L-glutamic acid, and “CFW_D” represents 5,6-dihydroxyindole.

[0861] As shown in Table 22, it is clear that combining two or more substances selected from No.1 to No.4 in Group B can increase the AUC of the ROC curve, that is, improve the accuracy of evaluating intestinal barrier function.

[0862]

[0863] (3-4) Substances No. 46 to No. 49 in Group B

[0864] For two or more substances selected from No. 46 to No. 49 in Group B (6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline, and 3-methyl adipic acid), the relative area values ​​obtained in (1) were used to calculate the composite variable in the same way as in (3-1). Based on the calculated composite variable, an ROC curve was constructed, and the AUC and cutoff value were calculated from the constructed ROC curve. The results are shown in Table 23. The coefficients and constants were obtained through multinomial logistic regression analysis. The coefficients and constants are shown in Table 6. In Table 23, “LFW_A” represents 6-methylquinoline, “LFW_B” represents N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, “LFW_C” represents 2-hydroxyquinoline, and “LFW_D” represents 3-methyl adipic acid.

[0865] As shown in Table 23, it is clear that combining two or more substances selected from No.46 to No.49 in Group B can increase the AUC of the ROC curve, that is, improve the accuracy of evaluating intestinal barrier function.

[0866]

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 is either dry or wet feces. When the test subject is dried feces, the biomarker contains one or more substances selected from group A below. Group A: 2-Formamidobenzoic acid; Diethanolamine; glycerin; 5,6-Dihydroxyindole; N-Imineformyl-L-glutamic acid; Leucine; Myristoleic acid; 3-Aminopropane-1,2-diol; 4-Guidinobutyric acid; Phenylalanine; 2'-Deoxyadenosine and 5'-Deoxyadenosine; Thioctyl amide; 3-Guidinopropionic acid; Acetanilide; Histidine; γ-Glutamyl-2-aminobutyric acid; methionine sulfoxide; phenylhydrazine; Tryptophan; Tyrosine; Hydroxyindole; alanine; p-Aminobenzoic acid; Penicillamine; 2-Cyanopyridine; Glutamine; 2,4-Diaminobutyric acid; Trigonelline; 2-Methylserine; Glycylglycine; N,N-dimethylhistidine; Valine; N-acetylphenylalanine; Isoleucine; Methionine; γ-Glutamylvaline glycine; N-acetylaspartic acid; Threonic acid; Isoleucine; Arginine ethyl ester; 2,6-Diaminopimelic acid; Glucose-6-phosphate; adenosine; mucic acid; thymidine; Glycinecholic acid; 2-Deoxyribose-1-phosphate; Isoleucylprolylproline; Crassulacean heptanose 7-phosphate; Fumaric acid; N-acetylmethionine; Fructose-6-phosphate; Glutamine glutamic acid; Cysteine; N-acetylglucosamine 1-phosphate; Ribose 5-phosphate; Glucose-1-phosphate; 3'-cytidine and 2'-cytidine; Dihydroxyacetone phosphate; 4-Oxovaleric acid; 2-Deoxyglucose-6-phosphate; Glucosamine 6-phosphate; 3-Methyladipic acid; 2-Hydroxyquinoline; 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione; 2,4-dihydroxyheptadec-16-en-1-yl acetate; α-Aspartic phenylalanine; N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide; 1-(2,4-Dihydroxyquinolin-3-yl)ethane-1-one; 4-(diethylamino)salicylaldehyde; Atenol; 5-Hydroxyindole; 6-Methylquinoline; Guanine; Carboxy-ibuprofen; 3-Isopropylmalic acid; Bis(methylbenzylidene)sorbitol; 2-Furfuric acid; whey acid; 2-Acetamino-N-β-aspartic-2-deoxypyranohexosamine; Flavosides; 1-Propyl-1H-benzo[d]imidazolium hydrobromide; 4-Hydroxybenzenesulfonic acid; Xanthohumol; laurolactam; Adenine; 2'-Deoxyguanosine; 6-Hydroxypyridinecarboxylic acid; D-(-)-quinic acid; 4-{[(4,6-dimethoxypyrimidin-2-yl)amino]methylene}-2-phenyl-4,5-dihydro-1,3-oxazol-5-one; 4-[(6E)-3-hydroxy-8,10-dimethyl-2-(methylamino)-6-dodecen-1-yl]phenol; Ethyl 2-cyano-3-(tetrahydro-3-thiophenylamino)acrylate; 3-Succinopyridine; Proline; L-Threo-3-phenylserine; (2,4-Diamino-5-pyrimidinyl)(3,4,5-trimethoxyphenyl) methyl ketone; 3-morpholino-4-tetrahydro-1H-pyrrole-1-ylcyclobut-3-ene-1,2-dione; 16-hydroxyhexadecanoic acid; Syringic acid; Tranexamic acid; 3,5-Dimethylisoxazole-4-carboxylic acid 4-(tert-butyl)phenyl ester; 3-Methylhistidine; 5-Methoxyindole; Perilla acid; 6-Methyl[1,2,4]triazolo[4,3-b]pyridazine-8-ol; Piperonitrile; N1-(5-chloro-2-hydroxyphenyl)-2-(morpholinylmethyl)benzamide; N-methylhistamine; 1-(1,3-benzodioxano-5-yl)-2-(dimethylamino)-1-butanone; (-)-epigallocatechin; Polysaccharin; 4-Indolecarboxaldehyde; Uracil; Dihydrothymidine; 3-Methylglutaric acid; 1,5-Dehydro-D-glucol; Pentoxabromide; DL-arginine; (R)-Estrol; Theobromine; 1,5-Isoquinolinediol; 12-Hydroxydodecanoic acid; 1,3,5-Trihydroxy-4-{[(2E)-3-(3-hydroxy-4-methoxyphenyl)prop-2-enoyl]oxy}cyclohexane-1-carboxylic acid; 3-Hydroxypyridinecarboxylic acid; Theophylline; 3,4-Dihydroxybenzaldehyde; Acetylcholine; as well as Tridecanoic acid, When the test subject is wet feces, the biomarker contains one or more substances selected from group B below. Group B: 2-Formamidobenzoic acid; glycerin; N-Imineformyl-L-glutamic acid; 5,6-Dihydroxyindole; 3-Aminopropane-1,2-diol; 4-Guidinobutyric acid; 2'-Deoxyadenosine and 5'-Deoxyadenosine; Acetanilide; Pyrophosphate; Myristoleic acid; Diethanolamine; Thioproline; Hydroxyindole; 2,6-Diaminopimelic acid; p-Aminobenzoic acid; Thioctyl amide; phenylhydrazine; 3-Guidinopropionic acid; N-acetylaspartic acid; 2,4-Diaminobutyric acid; γ-Glutamyl-2-aminobutyric acid; Cysteine; Glycerol 3-phosphate; Glucose 6-phosphate; mucic acid; Trigonelline; N-Carbamoyl Aspartic Acid; Threonic Acid; Glycinecholic acid; Isobutyric acid and butyric acid; 2-Deoxyribose-1-phosphate; Arginine ethyl ester; N-methylglutamic acid; Butterfly's song; N-acetylphenylalanine; Ribulose 5-phosphate; Fructose-6-phosphate; Crassulacean heptanose 7-phosphate; N-acetylglucosamine 1-phosphate; ribose 5-phosphate; 3'-cytidine and 2'-cytidine; Dihydroxyacetone phosphate; 4-Oxovaleric acid; Propranolol; 2-Deoxyglucose-6-phosphate; 6-Methylquinoline; N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide; 2-Hydroxyquinoline; 3-Methyladipic acid; L-Threo-3-phenylserine; 4-(Diethylamino)salicylaldehyde; 5-Methoxyindole; 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione; 4-indolecarboxaldehyde; Carboxy-ibuprofen; Piperonitrile; 5-Hydroxyindole; whey acid; Glucose-1-phosphate; L-alanyl-L-proline; 2-Acetamino-N-β-aspartic-2-deoxypyranohexosamine; α-Aspartic phenylalanine; 6-Hydroxymelatonin; Flavosides; Atenol; (R)-Estrol; 2,4-Dihydroxyheptadec-16-en-1-yl acetate; Guanine; 1-(4-methyl-2-pyridyl)pyrrolidine-2,5-dione; laurolactam; Omeresartan; Adenine; Uracil; 6-Methyl[1,2,4]triazolo[4,3-b]pyridazine-8-ol; 1-Propyl-1H-benzo[d]imidazolium hydrobromide; Pseudouridine; D-(-)-quinic acid; Acetylcholine; 3-Isopropylmalic acid; 2-Methoxy-5-(1H-1,2,4-triazol-5-yl)-4-(trifluoromethyl)pyridine; 3-Amino-2-naphthoic acid; Ornithine; 2'-Deoxyguanosine; Bis(methylbenzylidene)sorbitol; 2-Furfuric acid; 3,5-Dimethylisoxazole-4-carboxylic acid 4-(tert-butyl)phenyl ester; Pyridoxine; Tridecanoic acid; 3-Methylhistidine; Hexanoic acid; Azobenzene; (-)- indicates gallocatechin; 3-(2-thienyl)-1,2,4-oxadiazole-5-carbonylhydrazine; DL-lactic acid; 6-Hydroxypyridinecarboxylic acid; Proline; 1,3,5-Trihydroxy-4-{[(2E)-3-(3-hydroxy-4-methoxyphenyl)prop-2-enoyl]oxy}cyclohexane-1-carboxylic acid; Prolyl glycine; and 4-[(6E)-3-hydroxy-8,10-dimethyl-2-(methylamino)-6-dodecene-1-yl]phenol.

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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 the test sample from the subject and obtaining the measured values ​​of the biomarkers; as well as (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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 comprises 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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, When the test subject is dried feces, the biomarker contains two, three, or four or more substances selected from group A. When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

10. The method according to any one of claims 1 to 7, wherein, When the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

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, When the test sample is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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, When the test subject is dried feces, the biomarker contains two, three, or four or more substances selected from group A. When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

22. The biomarker according to any one of claims 14 to 19, wherein, When the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

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, When the test sample is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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, When the test subject is dried feces, the biomarker contains two, three, or four or more substances selected from group A. When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

34. The use according to any one of claims 26 to 31, wherein, When the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

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, When the test sample is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 from subjects with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subjects. The sample to be tested is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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 is either dry or wet feces. When the test subject is dried feces, the biomarker comprises one or more substances selected from group A of claim 1. When the test subject is wet feces, the biomarker contains one or more substances selected from group B as 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, When the test subject is dried feces, the biomarker contains two, three, or four or more substances selected from group A. When the test subject is wet feces, the biomarker contains two, three, or four or more substances selected from group B.

46. ​​The kit according to any one of claims 38 to 43, wherein, When the test subject is dried feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, diethanolamine, glycerol, and 5,6-dihydroxyindole. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 2-formamidobenzoic acid, glycerol, N-iminoformyl-L-glutamic acid and 5,6-dihydroxyindole.

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, When the test sample is dried feces, the biomarker comprises one or more substances selected from the group consisting of 3-methyl adipic acid, 2-hydroxyquinoline, 7-(tert-butyl)-4-imino-1,2,3,4,5,6-hexahydropyrido[3,4-d]pyridazine-1,5-dione and 2,4-dihydroxyheptadec-16-en-1-yl acetate. When the test subject is wet feces, the biomarker comprises one or more substances selected from the group consisting of 6-methylquinoline, N1-[4-(1,3-oxazol-5-yl)phenyl]cyclopropane-1-carboxamide, 2-hydroxyquinoline and 3-methyladipic acid.

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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