Liver disease detection method

Abstract

To provide a novel method for detecting a liver disease in a non-invasive and simple manner, and a novel method of screening a therapeutic agent for a liver disease.SOLUTION: Provided are a method for assisting in detection of a liver disease using a volatile oxidation lipid in exhaled gas as an index; and a method of screening a therapeutic agent for a liver disease.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a method for assisting in the detection of liver diseases. The present invention also relates to a system for assisting in the detection of liver diseases. Furthermore, the present invention relates to a method for screening therapeutic agents for liver diseases.

Background Art

[0002] Currently, blood sampling, urine tests, image analysis, etc. are mainly used for disease diagnosis, severity determination, and drug effect determination, and a determination method using exhaled breath is not generally performed.

[0003] As a determination method using exhaled breath, a method using a medical device that determines the successful eradication of Helicobacter pylori based on the ammonia concentration in exhaled breath is known. Also, for example, Patent Document 1 focuses on n-alkanes having 2 to 20 carbon atoms in exhaled breath and describes a method for determining the presence or absence of a disease or the aging of a mammal including a human. However, regarding the compounds in exhaled breath that are excreted, little information has been reported that can provide clinically useful disease markers, including Patent Document 1.

[0004] By the way, fatty liver classified as a digestive disease includes alcoholic and non-alcoholic types, and the latter is called non-alcoholic fatty liver disease (NAFLD). Among them, there are simple fatty liver that does not progress and non-alcoholic steatohepatitis (NASH) that progresses to liver cirrhosis (LC) or liver cancer, and the differentiation between the two is important when grasping the prognosis and considering treatment strategies. However, in current medicine, liver biopsy is the only diagnostic method, which is invasive and difficult to widely implement, and is not fully evaluated in actual clinical practice. Also, for this reason, there is a problem that undiagnosed NAFLD progresses to hepatitis C or liver cirrhosis, making treatment difficult.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, an object of the present invention is to provide a novel method for non-invasively and simply detecting liver diseases. Another object of the present invention is to provide a novel method for screening therapeutic agents for liver diseases.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that it is possible to determine liver diseases by using volatile oxidized lipids (for example, 1-octen-3-ol, 2-pentanone) in exhaled breath as an index. In addition, the present inventors have found that it is possible to screen therapeutic agents for liver diseases by using volatile oxidized lipids (for example, toluene / benzaldehyde) in exhaled breath as an index. Based on the above findings, the present invention has been completed.

[0008] That is, one aspect of the present invention relates to the following. 〔1〕A method for assisting the detection of liver diseases, comprising the step of detecting volatile oxidized lipids in exhaled breath collected from a subject, and determining that the subject has a liver disease when volatile oxidized lipids are present in the exhaled breath collected from the subject. 〔2〕(i) A step of measuring the amount of volatile oxidized lipids in exhaled breath collected from a subject, and (ii) A step of comparing the amount of volatile oxidized lipids in exhaled breath collected from the subject with the amount of volatile oxidized lipids in exhaled breath collected from a healthy subject, A method for assisting the detection of liver diseases, wherein when the amount of volatile oxidized lipids in exhaled breath collected from the subject is greater than the amount of volatile oxidized lipids in exhaled breath collected from the healthy subject, it is determined that the subject has a liver disease. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the volatile oxidized lipid is at least one selected from the group consisting of 1-octen-3-ol and 2-pentanone. 〔4〕The method according to any one of 〔1〕 to 〔3〕, wherein the liver disease is at least one selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. 〔5〕The method according to 〔1〕, further comprising, before the step of detecting the volatile oxidized lipid, a step of collecting exhaled gas from a subject (i’). 〔6〕The method according to 〔2〕, further comprising, before the step (i), a step of collecting exhaled gas from a subject (i’). 〔7〕A detection unit for detecting a volatile oxidized lipid in exhaled gas collected from a subject, and A determination unit that determines that the subject has a liver disease when a volatile oxidized lipid is present in the exhaled gas collected from the subject based on the detection result. A system for assisting in the detection of liver disease, comprising the above. 〔8〕A measurement unit for measuring the amount of a volatile oxidized lipid in exhaled gas collected from a subject, and A determination unit that compares the amount of the volatile oxidized lipid in the exhaled gas collected from the subject with the amount of the volatile oxidized lipid in the exhaled gas collected from a healthy subject, and determines that the subject has a liver disease when the amount of the volatile oxidized lipid in the exhaled gas collected from the subject is greater than the amount of the volatile oxidized lipid in the exhaled gas collected from the healthy subject. A system for assisting in the detection of liver disease, comprising the above. 〔9〕The system according to 〔7〕 or 〔8〕, wherein the volatile oxidized lipid is at least one selected from the group consisting of 1-octen-3-ol and 2-pentanone. 〔10〕The system according to any one of 〔7〕 to 〔9〕, wherein the liver disease is at least one selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. The system according to any one of [7] to

[10] , further comprising a collection unit that collects exhaled gas from a subject. 〔12〕A step of collecting a first exhaled gas from a non-human animal suffering from a liver disease and measuring the amount of volatile oxidized lipids in the first exhaled gas, A step of administering a test substance to the non-human animal, A step of collecting a second exhaled gas from the non-human animal administered with the test substance and measuring the amount of volatile oxidized lipids in the second exhaled gas, and A step of comparing the amount of volatile oxidized lipids in the first exhaled gas with the amount of volatile oxidized lipids in the second exhaled gas, A method for screening a therapeutic agent for liver disease, wherein when the amount of volatile oxidized lipids in the second exhaled gas is reduced compared to the amount of volatile oxidized lipids in the first exhaled gas, the test substance is a candidate for a therapeutic agent for liver disease. 〔13〕The method according to

[12] , wherein the volatile oxidized lipid is at least one selected from the group consisting of Toluene / benzaldehyde, 1-octen-3-ol, and 2-pentanone. 〔14〕The method according to

[12] or

[13] , wherein the liver disease is at least one selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver cirrhosis. 〔15〕(i) A step of measuring the amount of volatile oxidized lipids in exhaled gas collected from a subject, and (iii) A method for assisting in the determination of the severity of liver disease, comprising a step of classifying the subject according to the stage of the severity of liver disease based on the amount of volatile oxidized lipids in the exhaled gas collected from the subject. 〔16〕The method according to

[15] , wherein in the step (iii), the subject is classified into any one of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and liver cirrhosis. 〔17〕(i) A step of measuring the amount of volatile oxidized lipids in exhaled gas collected from a subject, and (iv) including a step of comparing the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a subject suffering from a liver disease, A method for assisting in the determination of the severity of a liver disease, wherein when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is relatively large, it is determined that it is an early stage of the disease state, and when the amount of volatile oxidized lipids is relatively small, it is determined that it is a late stage of the disease state.

Advantages of the Invention

[0009] According to the present invention, based on the volatile oxidized lipids in exhaled gas, liver diseases can be detected non-invasively and simply. Further, according to the present invention, a method for screening therapeutic drugs for liver diseases can be provided. Therefore, the present invention is extremely useful in the detection and treatment of liver diseases.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

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Figure 5

Figure 6

Mode for Carrying Out the Invention

[0011] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited thereto.

[0012] (1. Definitions) In the present specification, "liver disease" means a disease caused by an abnormality of the liver. The liver disease is not particularly limited, and examples thereof include non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis (LC), hepatocellular carcinoma, liver failure, and the like. Preferably, it can be NAFLD, NASH, or cirrhosis.

[0013] In the present specification, "exhaled breath" means a gas discharged from the body along with breathing. The exhaled breath contains, for example, nitrogen, oxygen, carbon dioxide, and other trace components (for example, hydrogen, carbon monoxide, nitrogen oxides, hydrogen sulfide, volatile organic compounds (VOCs), etc.).

[0014] In the present specification, "volatile oxidized lipid" means a general term for lipid species showing volatility under atmospheric pressure.

[0015] In the present specification, "subject" means a person to be determined whether or not suffering from liver disease. The subject may be not only a human but also a non-human mammal, a non-mammal, or the like. Preferably, the subject is a human.

[0016] As used herein, the term "healthy subject" refers to a person who does not suffer from liver disease. Whether a subject is a "healthy subject" can be determined by performing general tests related to liver disease. Such tests include, for example, tests using serum aminotransferase, bilirubin, and alkaline phosphatase as indicators.

[0017] (2. Method for assisting in the detection of liver disease) <Aspect 1> The method for assisting in the detection of liver disease in the present invention (hereinafter referred to as "this detection assistance method") includes a step of detecting volatile oxidized lipids in the exhaled gas collected from a subject, and when volatile oxidized lipids are present in the exhaled gas collected from the subject, it is determined that the subject suffers from liver disease.

[0018] The present inventors focused on volatile organic compounds in exhaled gas regarding the detection of liver disease. Among the volatile organic compounds in exhaled gas, particular attention was paid to volatile oxidized lipids. When analysis was performed using a unique volatile oxidized lipid library equipped with 500 types of oxidized lipids developed by the present inventors (previously, about 50 types of oxidized lipids were equipped), it was found that volatile oxidized lipids in exhaled gas are useful for the detection of liver disease. In addition, among the volatile oxidized lipids, it was found that 1-octen-3-ol and 2-pentanone can detect liver disease with high accuracy. There has been no report on the technique of detecting liver disease using volatile oxidized lipids in exhaled gas as an indicator, and the present invention is extremely useful in the diagnosis of liver disease and the like.

[0019] The volatile oxidized lipids are not particularly limited as long as they can be contained in the exhaled gas of a subject suffering from liver disease. Examples include 1-octen-3-ol, 2-pentanone, 2-methylfuran, 2-ethylfuran, 2-pentylfuran, hexanal, heptanal, octanal, 1-octen-3-one, 2-octenal, etc. Among them, from the viewpoint of detecting liver disease with high accuracy, 1-octen-3-ol and 2-pentanone are preferred.

[0020] Volatile oxidized lipids may be used alone or in combination of multiple ones. From the viewpoints of discrimination ability and sensitivity, it is preferable to use a combination of multiple volatile oxidized lipids. The combination of volatile oxidized lipids is not particularly limited as long as it exhibits the effects of the present invention. For example, a combination of 1-octen-3-ol and other volatile oxidized lipids (e.g., the volatile oxidized lipids exemplified above), a combination of 2-pentanone and other volatile oxidized lipids (e.g., the volatile oxidized lipids exemplified above), etc. can be mentioned. A combination of 1-octen-3-ol and 2-pentanone is preferable.

[0021] The method for detecting volatile oxidized lipids in exhaled gas is not particularly limited. For example, liquid chromatography (LC), gas chromatography (GC), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc. can be mentioned.

[0022] When volatile oxidized lipids are present in the exhaled gas collected from the subject based on the detection result, it is determined that the subject has a liver disease.

[0023] <Aspect 2> Further, in one embodiment of the present invention, this detection assistance method is a method including the following steps. (i) A step of measuring the amount of volatile oxidized lipids in the exhaled gas collected from the subject, (ii) A step of comparing the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a healthy subject.

[0024] As a result of the comparison in step (ii), when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is larger than the amount of volatile oxidized lipids in the exhaled gas collected from the healthy subject, it is determined that the subject has a liver disease. By having the above configuration, this detection assistance method can assist in the detection of liver diseases in subjects.

[0025] (Step (i)) In Step (i), the amount of volatile oxidized lipids in the exhaled gas collected from the subject is measured.

[0026] Regarding the volatile oxidized lipids, the description of the volatile oxidized lipids in the above <Aspect 1> is incorporated herein by reference.

[0027] As the volatile oxidized lipids, the above ones may be used alone or in combination of a plurality. From the viewpoints of discrimination ability and sensitivity, it is preferable to use a combination of a plurality of volatile oxidized lipids. The combination of volatile oxidized lipids is not particularly limited as long as it exhibits the effects of the present invention. For example, a combination of 1-octen-3-ol and other volatile oxidized lipids (e.g., the volatile oxidized lipids exemplified above), a combination of 2-pentanone and other volatile oxidized lipids (e.g., the volatile oxidized lipids exemplified above), etc. may be mentioned. A combination of 1-octen-3-ol and 2-pentanone is preferable.

[0028] The method for measuring the amount of volatile oxidized lipids in the exhaled gas is not particularly limited, and examples thereof include liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc.

[0029] (Step (ii)) In Step (ii), the amount of volatile oxidized lipids in the exhaled gas collected from the subject is compared with the amount of volatile oxidized lipids in the exhaled gas collected from a healthy subject.

[0030] The comparison of the amounts of volatile oxidized lipids can be performed, for example, using liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc.

[0031] When, as a result of the comparison, the amount of volatile oxidized lipids in the exhaled gas collected from the subject is greater than the amount of volatile oxidized lipids in the exhaled gas collected from the healthy subject, it is determined that the subject has a liver disease.

[0032] <Others> In one embodiment of the present invention, the detection assistance method may further include a step of collecting exhaled gas from a subject (i') before the detection step of Embodiment 1 or step (i) of Embodiment 2.

[0033] For the collection of exhaled gas from a subject, any method known in the art can be used. Examples include ReCIVA (registered trademark) Breath Sampler (Owlstone Medical, Cambridge, UK), RTube (trademark) Breath Condensate Collection Device (Respiratory Research, Austin, Texas, USA), etc. From the perspective of preventing contamination by ambient pollution, ReCIVA (registered trademark) Breath Sampler (Owlstone Medical, Cambridge, UK) is preferred.

[0034] In this detection assistance method, the liver disease can be the liver disease described in the above (1. Definition).

[0035] (3. System for assisting the detection of liver disease) <Embodiment 1> The system for assisting the detection of liver disease in the present invention (hereinafter referred to as "this system") includes a detection unit that detects volatile oxidized lipids in exhaled gas collected from a subject, and based on the detection result, when volatile oxidized lipids are present in the exhaled gas collected from the subject, a determination unit that determines that the subject has a liver disease.

[0036] In the detection unit of this system, volatile oxidized lipids in the exhaled gas collected from the subject are detected. The detection unit is not particularly limited as long as it can detect volatile oxidized lipids in the exhaled gas. For example, liquid chromatography (LC), gas chromatography (GC), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc. can be mentioned. The information obtained by the detection unit (information regarding the presence or absence of volatile oxidized lipids in the exhaled gas collected from the subject) is transmitted to the determination unit in this system.

[0037] In the determination unit, based on the detection result, when volatile oxidized lipids are present in the exhaled gas collected from the subject, it is determined that the subject has a liver disease.

[0038] The determination unit is not particularly limited, but examples include liquid chromatography (LC), gas chromatography (GC), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc.

[0039] In one embodiment of the present invention, the detection unit and the determination unit may coexist in one device. That is, it may be a device that can perform detection and determination with one device.

[0040] <Aspect 2> Also, in one embodiment of the present invention, this system includes a measurement unit that measures the amount of volatile oxidized lipids in the exhaled gas collected from the subject, and compares the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a healthy subject. When the amount of volatile oxidized lipids in the exhaled gas collected from the subject is greater than the amount of volatile oxidized lipids in the exhaled gas collected from the healthy subject, it is a system comprising a determination unit that determines that the subject has a liver disease.

[0041] In the measurement unit of this system, the amount of volatile oxidized lipids in the exhaled gas collected from the subject is measured. The measurement unit is not particularly limited as long as it can measure the amount of volatile oxidized lipids in the exhaled gas. For example, liquid chromatography (LC), gas chromatography (GC), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc. can be mentioned. The information obtained by the measurement unit (information regarding the amount of volatile oxidized lipids in the exhaled gas collected from the subject and the amount of volatile oxidized lipids in the exhaled gas collected from healthy subjects) is transmitted to the determination unit in this system.

[0042] In the determination unit, the amount of volatile oxidized lipids in the exhaled gas collected from the subject is compared with the amount of volatile oxidized lipids in the exhaled gas collected from healthy subjects. As a result of the comparison, when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is greater than the amount of volatile oxidized lipids in the exhaled gas collected from the healthy subjects, it is determined that the subject has a liver disease.

[0043] The determination unit is not particularly limited, but examples include liquid chromatography (LC), gas chromatography (GC), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), supercritical chromatography (SFC), LC-MS / MS, GC-MS, GC-FID, TD GC / MS, etc.

[0044] In one embodiment of the present invention, the detection unit and the determination unit may coexist in one device. That is, it may be a device capable of performing detection and determination with one device.

[0045] <Others> In one embodiment of the present invention, this system may further include a collection unit that collects exhaled gas from the subject. By providing the system with the collection unit, a series of processes from the collection of exhaled gas from the subject to the determination of the subject's liver disease can be performed in one stop.

[0046] The collection unit in this system is not particularly limited as long as it can collect exhaled gas from the subject. Examples include ReCIVA (registered trademark) Breath Sampler (Owlstone Medical, Cambridge, UK), RTube (trademark) Breath Condensate Collection Device (Respiratory Research, Austin, Texas, USA), and the like.

[0047] In this system, the volatile oxidized lipids to be measured can be the volatile oxidized lipids described in the above (2. Method for assisting in the detection of liver diseases).

[0048] In this system, the liver disease can be the liver disease described in the above (1. Definition).

[0049] (4. Method for screening therapeutic agents for liver diseases) The method for screening a therapeutic agent for liver diseases in the present invention (hereinafter referred to as "this screening method") includes the steps of collecting a first exhaled gas from a non-human animal suffering from a liver disease and measuring the amount of volatile oxidized lipids in the first exhaled gas, administering a test substance to the non-human animal, collecting a second exhaled gas from the non-human animal to which the test substance has been administered and measuring the amount of volatile oxidized lipids in the second exhaled gas, and comparing the amount of volatile oxidized lipids in the first exhaled gas with the amount of volatile oxidized lipids in the second exhaled gas. When the amount of volatile oxidized lipids in the second exhaled gas is reduced compared to the amount of volatile oxidized lipids in the first exhaled gas, the test substance is considered a candidate for a therapeutic agent for liver diseases.

[0050] In this screening method, a therapeutic agent for liver diseases can be screened by using as an index the amount of volatile oxidized lipids in the exhaled gas collected from the subject before and after the administration of the test substance. In this screening method, the terms "first" and "second" for exhaled gas are added for convenience to distinguish the exhaled gas collected from the subject before and after the administration of the test substance.

[0051] The type of non-human animals suffering from liver diseases is not particularly limited and can be, for example, mammals other than humans, such as mice, rats, monkeys, dogs, etc. Non-human animals suffering from liver diseases can be liver disease model animals. Liver disease model animals can be obtained commercially or can also be constructed by ordinary methods in the art.

[0052] In this screening method, the liver disease can be the liver disease described in the above (1. Definition).

[0053] The method for administering a test substance to a non-human animal is not particularly limited as long as the screening can be appropriately performed, and it may be systemic or local administration, or oral or parenteral administration. The dosage is also not particularly limited and can be appropriately set by those skilled in the art according to the type of test substance, the type and severity of liver disease, the non-human animal to be administered, etc.

[0054] As the test substance, any substance can be used. The type of test substance is not particularly limited, and for example, compounds present in extracts of natural products, low molecular weight synthetic compounds, synthetic peptides, antibodies, antisense RNAs, siRNAs, miRNAs, etc. are used. Also, the test substance may be a compound contained in a compound library (for example, a compound library of low molecular weight compounds), a phage display library, a combinatorial library, etc.

[0055] Also, as the test substance, those commercially available can be used, or those constructed by ordinary methods in the art can be used. Those skilled in the art can prepare the test substance based on ordinary methods in the art.

[0056] The method for collecting exhaled gas and the method for measuring the amount of volatile oxidized lipids in exhaled gas can be the methods described in the above (2. Method for assisting the detection of liver diseases).

[0057] In this screening method, the volatile oxidized lipid is not particularly limited as long as it is a volatile oxidized lipid contained in the exhaled gas of a non-human animal suffering from liver disease. Examples thereof include toluene / benzaldehyde, 1-octen-3-ol, 2-pentanone, 2-methylfuran, 2-ethylfuran, 2-pentylfuran, hexanal, heptanal, octanal, 1-octen-3-one, 2-octenal, and the like. The volatile oxidized lipid is preferably toluene / benzaldehyde.

[0058] The comparison between the amount of the volatile oxidized lipid in the first exhaled gas and the amount of the volatile oxidized lipid in the second exhaled gas can be carried out according to the method described in the above (2. Method for assisting the detection of liver disease).

[0059] Further, in one embodiment of the present invention, a step of measuring the amount of a volatile oxidized lipid in a first exhaled gas collected from a non-human animal suffering from liver disease, a step of administering a test substance to the non-human animal, collecting a second exhaled gas from the non-human animal to which the test substance has been administered, and measuring the amount of the volatile oxidized lipid in the second exhaled gas, and a step of comparing the amount of the volatile oxidized lipid in the first exhaled gas with the amount of the volatile oxidized lipid in the second exhaled gas, wherein when the amount of the volatile oxidized lipid in the second exhaled gas is reduced compared to the amount of the volatile oxidized lipid in the first exhaled gas, the test substance is considered to be a candidate for a therapeutic agent for liver disease, and a screening method for a therapeutic agent for liver disease is provided.

[0060] (5. Method for assisting in determining the severity of liver disease) In one embodiment of the present invention, there is provided a method for assisting in determining the severity of liver disease, including (i) a step of measuring the amount of a volatile oxidized lipid in exhaled gas collected from a subject, and (iii) a step of classifying the subject according to the stage of the severity of liver disease based on the amount of the volatile oxidized lipid in the exhaled gas collected from the subject.

[0061] For step (i), the description of (step (i)) in the above (2. Method for assisting the detection of liver disease) is incorporated by reference.

[0062] Step (iii) classifies the subject according to the stage of the severity of the liver disease based on the amount of volatile oxidized lipids in the exhaled gas collected from the subject measured in step (i). Volatile oxidized lipids are generally produced through a lipid peroxidation reaction involving strong oxidative stress and iron. Furthermore, the lipid peroxidation reaction induces cell death called ferroptosis and progresses to tissue damage. Therefore, it is considered that the production of volatile oxidized lipids increases in the early stage of the disease, and decreases as fibrosis progresses with the progression of the disease state. Thus, by using the volatile oxidized lipids in the exhaled gas as an index, the subject can be classified according to the stage of the severity of the liver disease. Also, in one aspect, for example, when the amount of volatile oxidized lipids is relatively large, it can be determined that it is the initial stage of the disease state, and when the amount of volatile oxidized lipids is relatively small, it can be determined that it is the late stage of the disease state.

[0063] In one embodiment of the present invention, in step (iii), preferably, the subject is classified into any one of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and liver cirrhosis.

[0064] Also, in another embodiment of the present invention, there is provided a method for assisting in the determination of the severity of a liver disease, including: (i) a step of measuring the amount of volatile oxidized lipids in the exhaled gas collected from the subject, and (iv) a step of comparing the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a subject suffering from a liver disease. When the amount of volatile oxidized lipids in the exhaled gas collected from the subject is relatively large, it is determined that it is the initial stage of the disease state, and when the amount of volatile oxidized lipids is relatively small, it is determined that it is the late stage of the disease state.

[0065] Step (i) incorporates the description of (Step (i)) in the above (2. Method for assisting in the detection of liver disease).

[0066] In step (iv), the amount of volatile oxidized lipids in the exhaled gas collected from the subject is compared with the amount of volatile oxidized lipids in the exhaled gas collected from a subject suffering from liver disease. Step (iv) is performed according to the method described in step (ii) of the above (2. Method for assisting in the detection of liver disease).

[0067] As a result of the comparison, when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is relatively large, it is determined that it is the initial stage of the disease state, and when the amount of volatile oxidized lipids is relatively small, it is determined that it is the late stage of the disease state.

[0068] In one embodiment of the present invention, in step (iv), it is determined that the subject has any one of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and liver cirrhosis.

Example

[0069] The present invention will be described in more detail below using examples, but this does not limit the scope of the present invention. The documents cited throughout the present specification are incorporated herein by reference in their entirety.

[0070] (1. Test tube experiment for constructing a volatile oxidized lipid database) In a vial for solid-phase microextraction (SPME), polyunsaturated fatty acids (500 μM), 2,2’-Azobis(2-Methylpropionamidine) Dihydrochloride (50 mM), and Hemin (10 μM) were mixed in PBS (pH 7.4, containing 0.1% ethanol). As polyunsaturated fatty acids, linoleic acid (FA18:2), arachidonic acid (FA20:4), eicosapentaenoic acid (FA20:5), and docosahexaenoic acid (FA22:6) were used. After covering and sealing the vial, the reaction was carried out at 37 °C for 2 hours. Then, the analysis was performed by SPME-GC / MS shown below.

[0071] (SPME-GC / MS analysis conditions) The GC / MS device used was an Agilent 5977B GC / MSD. The SPME-autosampler device used was a PAL-RTC gas chromatograph multifunction sampler. The GC / MS separation column used was an HP-5ms (60m x 0.25mm, ID x 0.25μM). Helium gas was used as the carrier gas, and the flow rate was set to 1.5mL / min. The inlet temperature was set to 250°C. The column oven was heated at 35°C for 4 minutes, then at 5°C / min to 150°C, and then at 10°C / min to 280°C, and held for 10 minutes. The EI method was used for ionization, and Scan analysis (test tube experiment) and SIM analysis (cultured cell experiment) were performed. The GC / MS analysis was controlled by MassHunter, and the autosampler was controlled by PAL Sample Control.

[0072] The SPME-Arrow fiber used for extraction was a SARR15-DVB / CWR120 / 20-P1 SPME Arrow. The vial was stirred at 40°C and 500 rpm for 900 seconds, and then volatile molecules in the gas phase of the vial were extracted under the same conditions for 600 seconds. The SPME-Arrow fiber was then connected to the GC / MS injection port, and the volatile molecules were desorbed at 250°C for 60 seconds.

[0073] The detected volatile molecules were analyzed using MassHunter Workstation, Qualitative Analysis. Compound searches were performed using the NIST mass spectral library included in NIST MS Search ver.2.4. Candidate compounds were selected by comparing the mass spectra obtained in the experiment. Furthermore, structures were identified by comparison with the GC / MS analysis results of standard samples of the candidate compounds. Peaks that were not annotated in the library search were designated as Unknown. In addition, peak area values ​​were calculated from the mass chromatogram of the fragment peak that showed the highest intensity for each peak.

[0074] The results are shown in Figure 1. As shown in Figure 1, it was found that various volatile oxidized lipids were produced from each polyunsaturated fatty acid.

[0075] (2. Ferroptosis cell headspace collection and analysis experiment) HepG2 cells were seeded in cell culture dishes (100 mM) at 2,000,000 cells / dish and incubated for 24 hours to allow adhesion. A ferroptosis inducer ([RSL-3]; 5.0 μM), an apoptosis inducer ([STS]; 1 μM), and a ferroptosis inhibitor ([Lip-1]; 5.0 μM) were added and incubated for 4 hours (RSL-3) and 24 hours (STS), respectively. Then, the cells and culture medium were transferred to an SPME vial and analyzed by SPME-GC / MS. The results are shown in Figure 2.

[0076] As shown in Figure 2(A), it was found that when HepG2 cells were treated with RSL-3, particularly, a large amount of 1-octen-3-ol was released into the gas phase. Also, as shown in Figure 2(B), it was found that 1-octen-3-ol did not increase during cell death induction other than ferroptosis.

[0077] (3. Collection and analysis experiment of volatile oxidized lipids in the exhaled gas of NASH model mice) Five-week-old male C57BL / 6J mice were purchased from CLEA Japan, Inc. All mice were housed in an environment with a temperature of 24 ± 1°C, humidity of 60 ± 10%, and a 12-hour light-dark cycle, and were intermittently provided with drinking water and food in an environment where they could freely ingest them. The mice that had been acclimated for one week were randomly divided into groups of 6 each and fed a normal diet (SD) or an L-amino acid diet (CDAHFD); Research Diets A06071302 containing 60 kcal% fat and 0.1% methionine and no added choline for 3 and 6 weeks to create NASH model mice.

[0078] For the collection of mouse exhaled gas, Bio-monitoring-stainless steel tubes were used as heated desorption gas collection tubes. Control NASH model mice were transferred into a 500 mL flask and sealed. A mixed gas (nitrogen: 78%, oxygen 21%) was introduced into the flask at a flow rate of 20 mL / min from one side through a line. Further, the other line was connected to the heated desorption gas collection tube to collect the volatile molecules released from the mice for 1 hour. After collection, the collection tube was disconnected from the line and analyzed by TD-GC / MS as shown below.

[0079] (Heated Desorption TD-GC / MS Analysis Conditions) The GC / MS instrument used was Shimadzu GC / MS-QP2020NX. The heated desorption system used was Shimadzu TD-30R. The GC / MS separation column used was HP-5ms (60 m × 0.25 mM, I.D. × 0.25 μM). Helium gas was used as the carrier gas, and the flow rate was set at 1.5 mL / min. The inlet temperature was set at 250 °C. The temperature increase conditions of the column oven were to hold at 35 °C for 4 minutes, then increase the temperature to 150 °C at 5 °C / min, and then increase the temperature to 280 °C at 10 °C / min and hold for 10 minutes. The ionization method used was the EI method, and SIM analysis was performed. The analysis and detection of the volatile oxidized lipids by GC / MS were carried out using Shimadzu LabSolutions.

[0080] The results are shown in Figure 3. As shown in Figure 3, it was found that the amount of 1-octen-3-ol in the exhaled gas increased with the progression of the disease in the NASH model mouse group compared with the normal diet group.

[0081] (4. Analysis Experiment of Volatile Oxidized Lipids in NASH Model Mouse Tissues) The prepared model mice were dissected, and each organ was collected and extracted by the improved Cold MeOH method. Specifically, 1 mL of extraction solution (methanol containing 100 μM dibutyl hydroxytoluene (BHT) and 100 μM ethylenediaminetetraacetic acid (EDTA)) was added to 50 mg of frozen tissue, and homogenized using a Macro Smash Homogeniser. Sonication was performed for 5 minutes in ice, and after standing for 5 minutes, 700 μL of the supernatant obtained by centrifugation (6000 g, 10 / min, 4 °C) was collected. The extraction samples were stored at -80 °C until immediately before measurement.

[0082] The results are shown in Figure 4. As shown in Figure 4, it was found that 1-octen-3-ol increased with the progression of the disease state only in the liver with lesions.

[0083] (5. Changes in the amount of volatile oxidized lipids at each stage of liver disease) The following experiment was conducted to examine the relationship between each stage from non-alcoholic fatty liver disease to liver cirrhosis and the amounts of 1-octen-3-ol and 2-pentanone in exhaled gas.

[0084] (Subjects) The subjects were 17 patients with liver diseases (6 with NAFLD, 9 with NASH, and 2 with LC) and 19 healthy individuals.

[0085] (Exhaled gas sampling) The collection of breath biopsy samples was performed for all subjects in a single room at Hospital (Tokyo) between February 2023 and July 2023. Exhaled samples were collected by adsorption onto four 1 / 4-inch × 3 1 / 2-inch inert-coated stainless-steel tubes (Tenax TA / carbograph 5TD adsorbent, Markes International Ltd, Llantrisant, UK) through a ReCIVA® Breath Sampler (Owlstone Medical, Cambridge, UK). Prior to use for each sampling, the tubes were conditioned for 4 hours with N2 flow at 20 psi and 320 °C using a TC-20 (Markes International Ltd). 1.5 L of exhaled breath per tube was sampled at 225 mL / m. A CASPER Portable Air Supply (Owlstone Medical, Cambridge, UK) was used to minimize ambient contamination. Subjects were fitted with a Breath Sample Collection Mouthpiece (Owlstone Medical, Cambridge, UK) and sampled by breathing for approximately 10 minutes. The tubes were stored at a temperature of 4 - 8 °C for no more than 4 weeks prior to analysis. The ReCIVA breath sampler was attached to a glass shot bottle instead of the subject, and blank sampling performed in the same room was also carried out using the same procedure.

[0086] Table 1 shows information on the subjects who participated in this study. Results were calculated as the median (25% - 75%). BMI was significantly higher in the liver disease group (mean difference, 4.3, 95% CI, 1.7 - 6.8, Cohen’d, 1.2, p < 0.01).

Table 1

[0087] (Thermal Desorption TD-GC / MS Analysis Conditions) The sampled exhaled gas sample was analyzed in the same manner as described in the above (3. Collection and analysis experiment of volatile oxidized lipids in the exhaled gas of NASH model mice).

[0088] The results are shown in Fig. 5. As shown in Fig. 5, it was found that the amount of volatile oxidized lipids varied according to the severity of liver disease. From this, it was shown that the type of liver disease can be determined using the amount of volatile oxidized lipids as an index.

[0089] (6. Judgment of the effect of liver disease therapeutic agents using volatile oxidized lipids as an index) Regarding the effect judgment of ursodeoxycholic acid, which is prescribed to patients with liver damage, attention was paid to the toluene-benzaldehyde ratio. The results are shown in Fig. 6. When taking ursodeoxycholic acid orally, the toluene-benzaldehyde ratio was significantly lower and improved to the level of healthy subjects. By combining VOCs with characteristics according to pharmacokinetics, it was possible to judge the effect of the drug from exhaled gas.

Industrial Applicability

[0090] The present invention is extremely useful particularly in the detection of liver diseases because it can easily detect liver diseases based on volatile oxidized lipids in exhaled gas.

Claims

1. A method for assisting the detection of liver disease, comprising the step of detecting volatile oxidized lipids in exhaled gas collected from a subject, and when volatile oxidized lipids are present in the exhaled gas collected from the subject, determining that the subject has liver disease.

2. (i) a step of measuring the amount of volatile oxidized lipids in exhaled gas collected from a subject, and (ii) a step of comparing the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a healthy subject, and when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is greater than the amount of volatile oxidized lipids in the exhaled gas collected from the healthy subject, determining that the subject has liver disease. A method for assisting the detection of liver disease.

3. The method according to claim 1 or 2, wherein the volatile oxidized lipid is at least one selected from the group consisting of 1-octen-3-ol and 2-pentanone.

4. The method according to claim 1 or 2, wherein the liver disease is at least one selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis.

5. The method according to claim 1, further comprising, before the step of detecting the volatile oxidized lipid, (i') a step of collecting exhaled gas from the subject.

6. The method according to claim 2, further comprising, before the step (i), (i') a step of collecting exhaled gas from the subject.

7. A detection unit for detecting volatile oxidized lipids in exhaled gas collected from a subject, and a determination unit for determining that the subject has liver disease when volatile oxidized lipids are present in the exhaled gas collected from the subject based on the detection result, A system for assisting the detection of liver disease, comprising:

8. A measurement unit for measuring the amount of volatile oxidized lipids in exhaled gas collected from a subject, and a determination unit for comparing the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a healthy subject, and determining that the subject has liver disease when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is greater than the amount of volatile oxidized lipids in the exhaled gas collected from the healthy subject. A system for assisting the detection of liver disease, comprising:

9. The system according to claim 7 or 8, wherein the volatile oxidized lipid is at least one selected from the group consisting of 1-octen-3-ol and 2-pentanone.

10. The system according to claim 7 or 8, wherein the liver disease is at least one selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis.

11. The system according to claim 7 or 8, further comprising a collection unit that collects exhaled gas from a subject.

12. A step of collecting a first exhaled gas from a non-human animal suffering from a liver disease and measuring the amount of a volatile oxidized lipid in the first exhaled gas, A step of administering a test substance to the non-human animal, A step of collecting a second exhaled gas from the non-human animal administered with the test substance and measuring the amount of a volatile oxidized lipid in the second exhaled gas, and A step of comparing the amount of the volatile oxidized lipid in the first exhaled gas with the amount of the volatile oxidized lipid in the second exhaled gas, wherein when the amount of the volatile oxidized lipid in the second exhaled gas is less than the amount of the volatile oxidized lipid in the first exhaled gas, the test substance is considered a candidate for a therapeutic agent for liver disease. A method for screening a therapeutic agent for liver disease.

13. The method according to claim 12, wherein the volatile oxidized lipid is at least one selected from the group consisting of toluene / benzaldehyde, 1-octen-3-ol, and 2-pentanone.

14. The method according to claim 12 or 13, wherein the liver disease is at least one selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis.

15. (i)A step of measuring the amount of a volatile oxidized lipid in exhaled gas collected from a subject, and (iii)A method for assisting in the determination of the severity of a liver disease, comprising a step of classifying the subject according to the stage of the severity of the liver disease based on the amount of the volatile oxidized lipid in the exhaled gas collected from the subject.

16. The method according to claim 15, wherein in the step (iii), the subject is classified into any one of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis.

17. (i)A step of measuring the amount of a volatile oxidized lipid in exhaled gas collected from a subject, and ​ (iv) a step of comparing the amount of volatile oxidized lipids in the exhaled gas collected from the subject with the amount of volatile oxidized lipids in the exhaled gas collected from a subject suffering from a liver disease, A method for assisting in the determination of the severity of a liver disease, wherein when the amount of volatile oxidized lipids in the exhaled gas collected from the subject is relatively large, it is determined that it is an early stage of the disease state, and when the amount of volatile oxidized lipids is relatively small, it is determined that it is a late stage of the disease state.

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