KR102122988B1 - Biomarker for prognostic analysis of intermittent fasting - Google Patents

Abstract

The present specification describes a metabolite whose expression level is changed by an intermittent fasting diet, a composition, kit for prognostic analysis of intermittent fasting including the metabolite, and a method for prognostic analysis of intermittent fasting. According to the contents described in the present invention, it is possible to analyze the effect of intermittent fasting in vivo through changes in expression levels of metabolites and their metabolic pathway analysis in vivo.

Description

Biomarker for prognostic analysis of intermittent fasting

This specification describes biomarkers that change by intermittent fasting and how to use them.

As interest in health and diet increases, interest in intermittent fasting (IF) is also increasing as one of the methods. The size of the domestic diet market is 10 trillion won, which is growing rapidly compared to 7 trillion won 5 years ago. According to data released by the Korea Centers for Disease Control and Prevention, the prevalence of obesity (body mass index over 25) in 2005 was 34.8%. In 2016, it was 37%. In particular, in male adults (over 30 years old), 1 in 2 (43.3%) was obese, and in female adults (over 30 years old), 1 in 3 (30.0%) was obese. However, intermittent fasting methods and weight control through the same, there is a lack of information other than the above-mentioned efficacy, and the need for research on the fundamental action and efficacy of intermittent fasting has emerged.

Previous studies have reported that caloric restriction or intermittent fasting has a neuroprotective effect and improved functional results in animal models of stroke, Parkinson's disease and Huntington's disease (Mark P.Mattson, Beneficial effects of intermittent fasting and caloric). restriction on the cardiovascular and cerebrovascular systems. J Nutr Biochem. 2005, 16, 3, 129-137) , according to animal studies, intermittent fasting is to help the brain by increasing the stress resistance mechanism of the cells to lower oxidative stress levels. In addition, large-scale human cohort studies have shown that people with fewer calories have a lower risk of developing dementia, and that diabetes and cardiovascular disease caused by excessive consumption is associated with an increased risk of brain disease. (Bronwen Martin, Caloric restriction and intermittent fasting: Two potential diets for successful brain aging, Ageing Res Rev. 2006 Aug; 5(3), 332-353)

Republic of Korea Patent Publication No. 10-2011-0069610

Mark P.Mattson, Beneficial effects of intermittent fasting and caloric restriction on the cardiovascular and cerebrovascular systems. J Nutr Biochem. 2005, 16, 3, 129-137 Bronwen Martin, Caloric restriction and intermittent fasting: Two potential diets for successful brain aging, Ageing Res Rev. 2006 Aug; 5(3), 332-353

In one aspect, the problem to be solved by the present invention is to provide a biomarker whose expression level is changed by an intermittent fasting diet.

In another aspect, the problem to be solved by the present invention is to provide a composition, kit for intermittent fasting prognostic analysis using the biomarker, and a method for prognostic analysis of intermittent fasting.

In order to solve the above problems, one embodiment of the present invention is glutamine (Glutamine), methionine (Methionine), tryptophan (Hopcysteine), homoserine (Homoserine), pyroglutamic acid (Pyroglutamic acid), N-6 -Acetyl-lysine (N6-Acetyl-lysine), 2-keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide, 2-Keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl-butrescine ( Gamma-glutamyl-putrescine) and at least one selected from the group consisting of oxalic acid (Oxalacetic acid), phenylalanine (Phenylalanine) and phenylpyruvic acid (Phenylpyruvic acid) further comprises at least one selected from the group consisting of Provided is a composition for intermittent fasting prognosis analysis comprising a reagent for detecting metabolites.

One embodiment of the present invention, glutamine (Glutamine), methionine (Methionine), tryptophan (Tryptophan), homocysteine (Homocysteine), homoserine (Homoserine), pyroglutamic acid (Pyroglutamic acid), N6-acetyl-lysine (N6-Acetyl- lysine, 2-Keto-6-aminocaproate, 5-Glutamyl-glutamyl-peptide, 2-keto-6-acetamido Group consisting of 2-Keto-6-acetamidocaproate, 2-Amino-5-oxohexanoate and Gamma-glutamyl-putrescine Measurement to measure the expression level of a metabolite containing at least one selected from, and optionally further comprising at least one selected from the group consisting of oxalacetic acid, phenylalanine and phenylpyruvic acid Provided is a kit for intermittent fasting prognosis analysis comprising a part.

One embodiment of the present invention comprises the steps of measuring the expression level of a metabolite from a biological sample isolated from an individual prior to intermittent fasting; Measuring the expression level of metabolites from biological samples isolated from the individual after intermittent fasting; And comparing the metabolite expression levels before and after the intermittent fasting, wherein the metabolites are glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid. (Pyroglutamic acid), N6-acetyl-lysine, 2-Keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide (5-Glutamyl -glutamyl-peptide, 2-keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glu 1 or more selected from the group consisting of Tamil-Futresin (Gamma-glutamyl-putrescine), and optionally selected from the group consisting of Oxalacetic acid, Phenylalanine and Phenylpyruvic acid Provided is an intermittent fasting prognostic analysis method that further includes a species or more.

Intermittent fasting causes a change in lifestyle and affects the regulation of a person's biometabolism. Accordingly, one embodiment of the present invention provides metabolites in which the expression level in vivo is changed by intermittent fasting as a biomarker, and the effect of intermittent fasting in vivo through analysis of the expression levels of the biomarkers and in vivo metabolic pathway analysis. Can be analyzed. In addition, through analysis of the biomarkers, it is possible to determine which biopathy intermittent fasting affects various diseases related to metabolic syndrome, such as stroke and cardiovascular disease, and develop alternative medicines and health functional foods through clinical diagnosis and metabolomics It can also be useful.

1 is a diagram schematically showing steps as an experiment conducted to identify a biomarker according to an embodiment of the present invention.
2 is a diagram showing an OPLS-DA score plot analyzed by UPLC-Q-TOF-MS for metabolites changing by intermittent fasting.
Figure 3 is a diagram showing the s-plot analyzed by UPLC-Q-TOF-MS metabolites changing by intermittent fasting.
4A to 4F and 5A to 5I are box-whisker plots showing fluctuations according to intermittent fasting by metabolites according to an embodiment of the present invention.
FIG. 6 is a view showing associations and associated biometabolism pathways by structural similarities of metabolites according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The inventors have identified biomarkers that are overexpressed or underexpressed by an intermittent fasting diet.

In the present specification, "intermittent fasting (IF)" refers to repeating a regular or irregular period for a certain period of fasting, which does not eat food for a period of time and maintains an empty stomach for a period of time. In one embodiment, the intermittent fasting is the "time-restricted feeding (TRF)" in which the intermittent fasting fasts for a certain period of time or more every day for a period of time and a normal or complementary diet for several days after fasting for several days for a period of time ( It may include "whole-day fasting" that repeats the act of ingesting 補食. In one embodiment, the "time-limited diet" may be to maintain an fasting time of 12 hours or more and less than 24 hours per day, specifically, 12 hours or more, 13 hours or more, 14 hours or more, and 15 hours of 24 hours per day. Above, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more or 23 hours or more may be to maintain the fasting time. As an example, the "hole-day fasting" may be ingesting a normal diet or a diet for 1 to 6 days after fasting for 1 to 6 days of the week, specifically, 1 day or more, 2 days or more of the week It may include fasting for 3 days or more, 4 days or more, 5 days or more, or 6 days.

As used herein, the term "fasting" of intermittent fasting means not only eating no food or drink, but also taking only water, eating tea or coffee excluding sugar or dairy products, or having fewer calories than normal diet. It is the meaning of Choi Kwang-Woo, which includes all of eating a diet. The "less than normal diet" is, for example, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% of normal dietary calories Or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less may be taken.

The intermittent fasting is known to affect the prevention or improvement of various diseases such as brain disease and cardiovascular disease, and is also known to be effective in controlling weight through calorie restriction.

In the present specification, the term "biomarker" refers to an index capable of determining a change induced inside an organism due to an external influence, and protein, DNA, RNA, and metabolites may be used as biomarkers. Biomarkers can be used to objectively measure, predict, or monitor the reaction or action of a subject's normal or pathological condition, diet, and drug intake. Biomarkers are used to diagnose or predict various diseases related to metabolic syndrome, such as stroke and cardiovascular disease, and biomarkers are also used in food or new drug development.

An embodiment of the present invention provides a biomarker whose expression level in the body changes by intermittent fasting. In one embodiment, the biomarker is glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, N-6-acetyl-lysine N6-Acetyl-lysine, 2-Keto-6-aminocaproate, 5-Glutamyl-glutamyl-peptide, 2-Keto-6 -Acetamidocaproate (2-Keto-6-acetamidocaproate), 2-amino-5-oxohexanoate, gamma-glutamyl-putrescine (Gamma-glutamyl-putrescine) ), oxalic acid (Oxalacetic acid), phenylalanine (Phenylalanine) and phenylpyruvic acid (Phenylpyruvic acid) may include one or more metabolites selected from the group consisting of.

The term "comprising" in the present specification means that other components may be further included instead of excluding other components, unless otherwise specified.

The present invention may provide a composition or kit for analyzing the prognosis of intermittent fasting and a method capable of analyzing the prognosis of intermittent fasting as another embodiment using the biomarker.

The term "prognosis" as used herein refers to results such as expected or predicted in vivo action, progression or symptoms following intermittent fasting.

One embodiment of the invention is glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, N6-acetyl-lysine, 2-keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide, 2-keto Intermittent fasting with a detection reagent for a metabolite comprising at least one member selected from the group consisting of -6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl-butresin Compositions for prognostic analysis can be provided. As an embodiment, the metabolite may further include at least one selected from the group consisting of oxalic acid, phenylalanine and phenylpyruvic acid.

The detection reagent is not limited as long as it is capable of detecting the metabolite from an individual or a subject, or a biological sample obtained from the subject or subject, and may include all. As an embodiment, the detection reagent may include one or more of mass spectrometry, multiple reaction monitoring (MRM) analysis, and selected reaction monitoring (SRM) analysis reagent.

As used herein, the term “individual” or “subject” refers to an individual or subject performing intermittent fasting, such as a primate such as a human or a monkey, a rodent such as a mouse or a rat, or a cow, horse, pig, dog, etc. It can include any mammal.

The term "biological sample" as used herein refers to a sample obtained from an organism. The biological sample includes the broadest meaning including cells, tissues, and body fluids obtained from a subject or an individual.

Another embodiment of the present invention is glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, N6-acetyl-lysine, 2-keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide, 2- Keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl-butresin measurement unit for measuring the expression level of metabolites containing at least one selected from the group consisting of An intermittent fasting prognostic assay kit can be provided. As an embodiment, the metabolite may further include at least one selected from the group consisting of oxalic acid, phenylalanine and phenylpyruvic acid.

As an example, the kit may include a substance, a composition capable of detecting the metabolite, or a detection and/or quantitation device, and the composition may include a composition for intermittent fasting prognosis analysis as described herein. have. As an embodiment, the device may include one or more of devices selected from mass spectrometry, multiple reaction monitoring (MRM) analysis and selected reaction monitoring (SRM) analysis devices. As an example, the quantitative device is for measuring the expression level of each metabolite, that is, concentration, and examples thereof include chromatography and mass spectrometry. As an example, the chromatography may include liquid chromatography (LC), liquid-solid chromatography (LSC), liquid-liquid chromatography (LLC), gas-liquid chromatography (GLC), and the like. Liquid chromatography, and more specifically, may be ultra performance liquid chromatography (UPLC), but is not limited thereto. As an example, the mass spectrometer is a chemical electrospray ionization (ESI), a matrix-assisted laser desorption/ionization (MALDI) and an atmospheric pressure chemical ionization (APCI), orbitrap, or quadrupole time-of-flight (Q-TOF). ) And the like. Specifically, the mass spectrometer may use a Waters Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer, but is not limited thereto. The chromatography can separate each metabolite using different mobility for each molecule, and the mass spectrometer can identify the metabolite through structural information as well as molecular weight information of the analysis object.

One embodiment of the present invention may provide an intermittent fasting prognostic analysis method, the method comprising: measuring the expression level of a metabolite from a biological sample isolated from an individual before intermittent fasting; Measuring the expression level of metabolites from biological samples isolated from the individual after intermittent fasting; And comparing the metabolite expression levels before and after the intermittent fasting, wherein the metabolites are glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, N6-acetyl-lysine, 2-keto-6-aminocapro. Eight, 5-glutamyl-glutamyl-peptide, 2-keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl-putrescine. It may include the above. As an embodiment, the metabolite may further include at least one selected from the group consisting of oxalic acid, phenylalanine and phenylpyruvic acid.

In one embodiment of the present invention, the gamma-glutamyl-putresin may increase the expression level after intermittent fasting. In one embodiment, the glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, N6-acetyl-lysine, 2-keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide, 2-keto-6 -One or more metabolites selected from the group consisting of acetamidocaproate, 2-amino-5-oxohexanoate, oxalic acid, phenylalanine and phenylpyruvic acid may have reduced expression levels after intermittent fasting.

The biological sample according to an embodiment of the present invention may include cells, tissues, body fluids, etc. obtained by being separated from an individual. Specifically, the biological sample may include one or more selected from the group consisting of blood, serum, plasma, saliva, urine, tissue, intracellular fluid, and extracellular fluid, but if the metabolites in a bird can be detected, It is not limited. More specifically, the biological sample may be blood, plasma, platelets, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucous fluid, amniotic fluid, or a combination thereof, and more specifically, serum separated from blood. Can be

In the present specification, the term "expression level" means detection of the target metabolite expression level, and more specifically, quantitative detection of the target metabolite expression level, for example, content, concentration, etc. in a biological sample. It may mean.

As an example, the step of measuring the expression level of a metabolite from a biological sample isolated from an individual prior to the intermittent fasting is to collect and separate a biological sample from the individual before providing an intermittent fasting diet, and centrifuge it with an organic solvent. Separation may include precipitating proteins and extracting metabolites.

In another embodiment, the step of measuring the expression level of a metabolite from a biological sample isolated from an individual after the intermittent fasting is to collect a biological sample prior to the intermittent fasting and provide the isolated individual with an intermittent fasting diet for a period of time. It may include steps. The intermittent fasting is, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, 10 weeks or more, 11 weeks or more , 12 weeks or more, 13 weeks or more, 14 weeks or more, 15 weeks or more, 16 weeks or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, and the like.

As an embodiment, the step of measuring the expression level of the metabolite from the biological sample isolated from the individual after the intermittent fasting, after providing the intermittent fasting for a certain period of time, collecting and separating the biological sample from the individual, and It may include the step of precipitating the protein using centrifugation and extracting the metabolite.

As an embodiment, the step of measuring the expression level of the metabolite from the biological sample isolated from the individual before and after the intermittent fasting is selected from the group consisting of mass spectrometry, multiple reaction monitoring (MRM) analysis, and selected reaction monitoring (SRM) analysis. It may be performed by a method comprising one or more. The step may include measuring the expression level of the metabolite to obtain an overall metabolite spectrum. Specifically, the step of measuring the expression level of the metabolite from the biological sample isolated from the individual before and after the intermittent fasting may include liquid chromatography-mass spectrometry (LC-MS). As an example, the liquid chromatography-mass spectrometry (LC-MS) may include UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectroscopy). The UPLC-MS is used to enhance peak dose and enhance the separation of complex metabolites, and UPLC-Q-TOF-MS (Ultra Performance Liquid Chromatography-Quadrupole) for full-length profiling (non-targeting metabolite analysis) of metabolites. -Time of flight--Mass Spectroscopy) can be used.

As an example, the step of comparing the metabolite expression level before and after the intermittent fasting may include a multivariate analysis step. In addition, as an example, the step of comparing the metabolic expression levels before and after the intermittent fasting may include a metabololomics technique. The metabolomic technique is a research technique that analyzes metabolites in vivo that change according to changes in disease or environment along with multivariate data analysis, and can be effectively used for qualitative evaluation of biologically derived substances such as metabolites. One embodiment of the present invention can observe and analyze the overall change in the metabolite profile in the biological sample by the intermittent fasting diet using metabolomic technique, thereby enabling the expression level by the intermittent fasting diet of the metabolites. It can be analyzed and used in the study of related metabolic pathways. Another embodiment of the present invention in view of the above may further include performing additional statistical analysis on the significant candidate metabolite group after the step of comparing the metabolite expression level before and after the intermittent fasting.

In addition, the present invention may include a method for determining whether or not intermittent fasting progresses, including monitoring the expression level of the metabolites of a subject as an embodiment. According to the above method, it is possible to determine whether the intermittent fasting is progressing effectively by monitoring the increase or decrease in the expression level of the metabolites, and based on this, it is possible to determine whether to change or maintain the intermittent fasting method or to stop the progression. Can be. Furthermore, an embodiment may also provide a method for controlling or improving a specific disease, such as a metabolic syndrome disease, such as a weight control method or a cardiovascular disease, including the method for determining whether the intermittent fasting progresses.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples and drawings. However, the following examples and drawings are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example]

As an example of the present invention, it was confirmed that the metabolites of the present invention functioned as biomarkers by comparing the in vivo expression levels of metabolites before and after intermittent fasting.

Preparation of biological samples

Nine healthy men and women who met the following conditions were selected: age 20-65 years, body mass index (BMI) of 23.0 kg/m ² or higher, stable body weight, thyroid, diabetes, aspartate amino for 3 months before study Levels of transferase (AST), alanine amino transferase (ALT), serum creatinine, etc. are normal and free of related diseases.

From these, blood was collected as a biological sample before and after providing an intermittent fasting diet as shown in FIG. 1. At this time, the intermittent fasting diet consumed 25% (approximately 500 kcal) of the recommended daily energy intake 3 times a week for 8 weeks between 12 PM and 2 PM, and the diet was freely consumed on days other than the fasting day. The collected blood samples were separated into serum and stored at -80°C until analysis. The separated serum sample was subjected to protein precipitation and metabolite extraction using cold acetonitrile as an extraction solvent. Specifically, the separated serum was maintained at -20°C for 20 minutes, then centrifuged at 10,000 rpm for 7 minutes and the supernatant was taken. The separated supernatant was completely dried, and the dried samples were redissolved in 5% acetonitrile to prepare a sample for measuring the expression level of metabolites in the sample.

Metabolites Expression level measurement

The expression level of metabolites in the serum samples of each of the prepared subjects was measured using UPLC-Q-TOF-MS (ultra performance liquid chromatography quadrupole-time of flight-mass spectrometry). Chromatographic separation is performed on a Waters Acquity High Performance Liquid Chromatography (HPLC) BEH C18 column (100 mm x 2.1 mm id x 1.7 μm particle size) utilizing a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA). Did. The solvent for chromatographic separation was carried out with a gradient of 95% acetonitrile with 0.1% formic acid as mobile phase A and 5% acetonitrile with 0.1% formic acid as mobile phase B. The gradient was maintained at 0% B solvent for the first 3 minutes, then increased linearly from 0°C to 50% for 7 minutes, then increased linearly from 50°C to 90% for 2 minutes, and then decreased to 0% for 2 minutes. The overall analysis time, including re-equilibration of the column as initial conditions, was 16 minutes. The injection volume was 5 μL and the flow rate was 0.35 ml/min.

For mass spectrometry, Waters Q-TOF Premier (Micromass MS Technologies, Manchester, UK) was used. The time of flight (TOF) data was collected between m/z 50 and 1,200 in negative (-) and positive (+) ions. The desolvation gas (nitrogen) was set at 600 L/h at a temperature of 200? The cone gas (nitrogen) was set at 60 L/h and the source temperature was 120°C. The capillary and cone voltages were set at 3.0 kV and 36 V, respectively. Data were collected with a scan accumulation time of 0.2 s in centroid mode. All analyzes were obtained using independent reference sprays through lock spray interference to ensure accuracy and reproducibility; Leucine enkephalin ions were used as lock mass (m/z 554.2615 (-) and 556.2771 (+)) at a flow rate of 5 μL/min.

FIG. 2 is a diagram showing the OPLS-DA score plot of the metabolites analyzed by UPLC-Q-TOF-MS. The blue dots mean before intermittent fasting (Pre_IF), and the red dots mean after intermittent fasting (P0_IF), where individual dots represent each individual sample (experimental group). It was confirmed through the OPLS-DA score plot of FIG. 2 that the division between the two groups was made, and the S-plot of FIG. 3 was shown to confirm the factors influencing the division between these groups among variables of individual data. In Figure 3, the metabolites at both ends represent metabolites with significant differences in the two groups. FIG. 3 is an S-plot showing the contribution of each individual bin to the separation of FIG. 2. The p(corr)[1] axis represents the correlation of each bin to the predicted change in FIG. 2, and the p[1] axis represents the magnitude of the spectral bin. In Figure 3, the metabolites at both ends represent metabolites with significant differences in the two groups.

Data analysis

Preprocessing for UPLC-Q-TOF-MS analysis was performed using MassLynx ^TM (Mass Spectrometry Software, Waters) software. Send the result data to Excel (Microsoft, Redmond, WA, USA) to perform statistical analysis using SIMCA-P+ 14.0 software (Umetrics, Umea, Sweden) and SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA). Was performed. Multivariate analysis techniques including Unsupervised principal component analysis (PCA) and supervised orthogonal projection to latent structures discriminate analysis (OPLSDA) were used to compare samples before and after intermittent fasting and to identify major metabolites mutated by intermittent fasting. The dataset was pareto scaled before PCA and OPLS-DA modeling. Metabolites with the largest variable importance in the projection (VIP) values in the model were considered as potential biomarkers. Metabolites appearing at both ends of the s-plot in FIG. 3 and satisfying both VIP values> 1 and p-values <0.05 were selected as significant biomarkers.

Table 1 below shows the individual values of metabolites that satisfy the conditions.

RT m/z Name VIP p-value Folding change Regulation One 6.26 188.0771 2-Keto-6-acetamidocaproate 6.78 2.53E-03 0.4 Down 2 4.52 166.0907 Phenylalanine 5.33 3.66E-03 0.31 Down 3 4.52 120.0839 Homoserine 5.05 1.88E-03 0.31 Down 4 6.27 205.1013 Tryptophan 4.02 5.37E-03 0.42 Down 5 1.75 165.0616 Phenylpyruvic acid 2.82 3.41E-03 0.42 Down 6 6.3 189.0741 N6-Acetyl-lysine 2.71 9.26E-04 0.3 Down 7 6.4 146.0447 2-Keto-6-aminocaproate 2.15 1.14E-06 0.07 Down 8 1.75 136.081 Homocysteine 1.88 1.66E-02 0.5 Down 9 1.27 150.0649 Methionine 1.73 2.31E-02 0.46 Down 10 6.25 146.0655 2-Amino-5-oxohexanoate 1.56 5.83E-03 0.42 Down 11 1.46 130.0604 Pyroglutamic acid 1.48 8.47E-03 0.39 Down 12 2.69 276.1534 5-Glutamyl-glutamyl-peptide 1.4 1.49E-03 0.13 Down 13 1.26 133.0362 Oxalacetic acid 1.26 2.16E-02 0.53 Down 14 1.74 147.0489 Glutamine 1.06 9.47E-03 0.46 Down 15 2.63 218.1539 Gamma-glutamyl-putrescine 1.03 1.26E-02 - Up

Table 2 below shows individual values of metabolites satisfying the corresponding conditions.

Name Pre-IF Post-IF Increase / decrease rate% (average) min max min max One 2-Keto-6-acetamidocaproate 228.91 1307.98 0 1012.56 -40.16 2 Phenylalanine 45.16 824.00 0 688.81 -30.58 3 Homoserine 117.91 657.90 0 638.65 -30.59 4 Tryptophan 139.92 566.43 0 517.92 -42.08 5 Phenylpyruvic acid 70.27 242.23 0 236.20 -41.97 6 N6-Acetyl-lysine 23.64 218.90 0 144.85 -30.16 7 2-Keto-6-aminocaproate 30.33 98.92 0 39.44 -6.75 8 Homocysteine 17.31 156.98 0 126.76 -49.68 9 Methionine 24.36 147.83 0 97.19 -45.58 10 2-Amino-5-oxohexanoate 30.86 96.94 0 81.02 -42.12 11 Pyroglutamic acid 25.79 102.84 0 67.51 -39.17 12 5-Glutamyl-glutamyl-peptide 2.49 61.80 0 30.99 -12.57 13 Oxalacetic acid 26.21 90.89 0 64.15 -52.62 14 Glutamine 14.32 55.51 0 40.84 -45.83 15 Gamma-glutamyl-putrescine 0 0 0 41.09 N/A

4A-4F and FIGS. 5A-5I are box-whisker plots showing fluctuations due to intermittent fasting of the selected metabolites. Among the metabolites, glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, N6-acetyl-lysine, 2-keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide, 2-keto-6-acet Amidocaproate, 2-amino-5-oxohexanoate, oxalic acid, phenylalanine and phenylpyruvic acid had decreased expression levels after intermittent fasting, and gamma-glutamyl-putrescine increased expression levels after intermittent fasting, The overall expression level was -6.75% to -52.63%.

The specificity of the biometabolism pathway for the selected biomarker and the correlation between individual biomarkers were evaluated and analyzed, and the results are shown in FIG. 6. FIG. 6 is a diagram showing arrangement of individual candidate metabolites according to the similarity of the structure of individual chemical species and the association of functional groups using the PubChem database. In the schematic, the red color represents the overexpressed metabolite as a result of the intermittent fasting, and the blue color represents the underexpressed metabolite, while the size of the circle represents the increase or decrease. The analysis was made with reference to Chemical similarity network analysis, the documents of which are incorporated herein by reference in their entirety.

As described above, metabolites specifically over-expressed or under-expressed by intermittent fasting were confirmed through the examples. Therefore, metabolites that are mutated by the intermittent fasting of the present invention can be usefully used to analyze the effects of intermittent fasting on the biopathic path as a prognosis for intermittent fasting by analyzing their biopaths.

Claims (11)

N-6-acetyl-lysine, 2-Keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide (5-Glutamyl-glutamyl- peptide), 2-Keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl-fu A composition for determining whether intermittent fasting includes a detection reagent for a metabolite containing at least one selected from the group consisting of trescin (Gamma-glutamyl-putrescine). The method according to claim 1, wherein the metabolites are glutamine, methionine, tryptophan, homocysteine, homoserine, pyroglutamic acid, oxalacetic acid, A composition further comprising at least one member selected from the group consisting of Phenylalanine and Phenylpyruvic acid. The composition of claim 1 or 2, wherein the detection reagent comprises at least one of mass spectrometry, multiple reaction monitoring (MRM) analysis, and selected reaction monitoring (SRM) analysis reagent. N6-Acetyl-lysine, 2-Keto-6-aminocaproate, 5-Glutamyl-glutamyl-peptide , 2-keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl-butrescin (Gamma-glutamyl-putrescine) kit for determining whether intermittent fasting (Intermittent Fasting) comprising a measuring unit for measuring the expression level of the metabolite containing at least one selected from the group consisting of (Gamma-glutamyl-putrescine). The method of claim 4, wherein the metabolite is glutamine (Glutamine), methionine (Methionine), tryptophan (Homocysteine), homoserine (Homoserine), pyroglutamic acid (Oxlacetic acid), Kit further comprising at least one selected from the group consisting of Phenylalanine and Phenylpyruvic acid. Measuring the expression level of metabolites from biological samples isolated from the individual prior to intermittent fasting;
Measuring the expression level of metabolites from biological samples isolated from the individual after intermittent fasting; And
Comparing the metabolic expression level before and after the intermittent fasting,
The metabolite is N6-acetyl-lysine, 2-keto-6-aminocaproate, 5-glutamyl-glutamyl-peptide (5-Glutamyl- glutamyl-peptide, 2-keto-6-acetamidocaproate, 2-amino-5-oxohexanoate and gamma-glutamyl -Method for providing information for determining whether intermittent fasting, including at least one selected from the group consisting of putrescine (Gamma-glutamyl-putrescine). The method of claim 6, wherein the metabolite is glutamine (Glutamine), methionine (Methionine), tryptophan (Homocysteine), homoserine (Homoserine), pyroglutamic acid (Oxlacetic acid), Method further comprising at least one selected from the group consisting of phenylalanine (Phenylalanine) and phenylpyruvic acid (Phenylpyruvic acid). The method according to claim 6 or 7, wherein the biological sample comprises one or more selected from the group consisting of blood, serum, plasma, saliva, urine, tissue, intracellular fluid, and extracellular fluid. The method of claim 6 or 7, wherein the step of measuring the expression level of the metabolite from the biological sample isolated from the individual before and after the intermittent fasting is mass spectrometry, multiple reaction monitoring (MRM) analysis, and selected reaction monitoring (SRM). Method performed by one or more methods selected from the group consisting of assays. The method of claim 6 or 7, wherein measuring the expression level of metabolites from biological samples isolated from the individual before and after the intermittent fasting comprises liquid chromatography-mass spectrometry. The method of claim 6 or 7, wherein comparing metabolic expression levels before and after the intermittent fasting comprises a multivariate analysis step.

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