JP6592628B1 - A method for detecting fatty liver disease or predicting risk, a diagnostic kit and biomarker for detecting fatty liver disease, a method for determining the degree of liver fibrosis in a subject, and the degree of liver fibrosis in a subject Biomarker for judgment - Google Patents

Abstract

It is an object of the present invention to provide a new method for diagnosing fatty liver disease that can be easily implemented and does not place a burden on patients and medical staff. A long chain selected from the group consisting of octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, and octadecadienoylcarnitine in a body fluid sample. The said subject can be solved by measuring the quantity of any one or more of acyl carnitine, and comparing with a reference value. [Selection figure] None

Description

  The present invention relates to a method for detecting fatty liver disease or predicting risk. The present invention also relates to biomarkers and diagnostic kits for fatty liver disease. The present invention also relates to a method for determining the progress of liver fibrosis in a subject and a biomarker for determining the progress of liver fibrosis in a subject.

  Nonalcoholic fatty liver disease (NAFLD) is a general term for fatty liver that does not cause alcohol. NAFLD is classified into non-alcoholic steatohepatitis (NASH), which is also the home of cirrhosis and liver cancer, and non-alcoholic fatty liver (NAFL) in which the disease state hardly progresses. NAFLD and NASH are fatty liver diseases that have attracted a great deal of attention recently in Japan and abroad. In Japan, it is estimated that about 10 million people suffer from NAFLD and about 1 to 2 million people suffer from NASH. In this specification, the term “NASH” includes cirrhosis in which NASH has progressed and hepatocellular carcinoma in which NASH has progressed.

  Biopsy of liver tissue is essential for a definitive diagnosis of NASH. By biopsy of liver tissue, it is possible to grasp the amount of fat, the degree of inflammation, and / or the degree of progression of fibrosis, and diagnose NASH. However, a biopsy of liver tissue involves the collection of a part of tissue or cells by inserting a needle into the liver, which imposes an excessive burden on patients and medical workers, as well as complications, etc. There are risks. Therefore, it is desired to develop a new diagnostic method for fatty liver diseases such as NASH that can be carried out more simply and does not place a burden on patients and medical staff.

  Acylcarnitine is a compound formed by combining fatty acid and carnitine when the fatty acid is transported to the inner mitochondrial membrane in vivo. More specifically, acylcarnitine is produced from acyl CoA and carnitine by the action of carnitine palmitoyltransferase I (CPTI) present in the outer mitochondrial membrane. After the acylcarnitine enters the mitochondrial inner membrane, it is again separated into carnitine and acyl CoA by carnitine palmitoyltransferase I (CPTII), and the acyl CoA undergoes β-oxidation and is used for energy production. Acylcarnitine concentrations in tissues and blood fluctuate due to abnormalities in these metabolic pathways, and acylcarnitine analysis in biological samples such as blood and urine is an analysis item in screening for inborn errors of metabolism in newborns .

  Non-patent document 1 shows that when metabolomic analysis was performed between a hepatocellular carcinoma tissue and a noncancerous tissue, oleoylcarnitine and palmitoylcarnitine were detected more in hepatocellular carcinoma tissue than in noncancerous tissue. Is disclosed. Non-Patent Document 2 discloses that patients suffering from hepatocellular carcinoma had higher serum acylcarnitine levels than healthy subjects. However, the hepatocellular carcinoma tissue used in these reports is not derived from fatty liver disease. More specifically, the hepatocellular carcinoma tissue used in these reports is not a cancer that has progressed to NASH, and therefore the relationship between fatty liver disease and acylcarnitine levels remains unclear. . In addition, the possibility that acylcarnitine is released from other tissues different from the liver cannot be denied due to hepatocellular carcinoma changing the metabolic mechanism of other tissues.

Budhu A et al. Gastroenterology 2013 May; 144 (5): 1066-1075 Yaligar J et al. Scientific Reports volume 6 Article Number 20299 (2016)

  An object of the present invention is to provide a new diagnostic method for fatty liver disease that can be carried out more simply and does not place a burden on patients and medical staff. Another object of the present invention is to provide a new method for determining the degree of progression of liver fibrosis in a subject who may suffer from fatty liver disease without burdening the patient and medical staff. is there.

As a result of intensive studies to solve the above problems, the present inventor measured the amount of a specific long-chain acylcarnitine in a body fluid sample, for example, serum, and compared it with a reference value, thereby burdening the patient and medical staff. The inventors have found that it is possible to easily diagnose an early stage of fatty liver disease and determine the degree of progression of liver fibrosis, and have completed the present invention.
Specifically, the present invention is as follows.
<1> selected from the group consisting of octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, and octadecadienoylcarnitine in a body fluid sample from the subject. Measuring the amount of any one or more of the long-chain acylcarnitines and comparing the measured amount of any one or more of the long-chain acylcarnitines to a reference value. Disease detection or risk prediction method,
<2> The method for detecting fatty liver disease or predicting risk according to <1>, wherein the body fluid sample is blood, serum, or plasma.
<3> The method for detecting fatty liver disease or predicting risk according to <1> or <2>, wherein the fatty liver disease is NASH.
<4> A method of detecting fatty liver disease or predicting risk according to any one of <1> to <3>, wherein the degree of fibrosis in the liver is determined.
<5> Measure the amount of any one or more medium-short chain acylcarnitines having 2 to 10 carbon atoms in the acyl group and the measured amount of any one or more medium-short chain acylcarnitines as a reference value And a method for detecting fatty liver disease or predicting risk according to any one of <1> to <4>,
<6> The method for detecting fatty liver disease or predicting risk according to <5>, wherein the medium-short chain acylcarnitine is carnitine isovalerate.
<7> A method for detecting fatty liver disease or predicting risk in a subject, wherein an index score is calculated using the following formula:
[Formula 1]
AC14: 1 + AC18: 1-AC5: 0
(Where AC14: 1 indicates the amount of tetradecenoylcarnitine in the body fluid sample from the subject, AC18: 1 indicates the amount of octadecenoylcarnitine in the body fluid sample from the subject, and AC5: 0 indicates the amount of carnitine isovalerate in the body fluid sample from the subject)
Comparing the calculated index score with a reference value,
<8> A method for detecting fatty liver disease or predicting risk in a subject, and calculating an index score using the following formula:
[Formula 2]
(AC16: 0 + AC18: 1) × 100 / AC2: 0
(Where AC16: 0 indicates the amount of hexadecanoylcarnitine in the body fluid sample from the subject, AC18: 1 indicates the amount of octadecenoylcarnitine in the body fluid sample from the subject, and AC2: 0 indicates the amount of acetylcarnitine in the body fluid sample from the subject)
Comparing the calculated index score with a reference value,
<9> The method for detecting fatty liver disease or predicting risk according to <8>, wherein the body fluid sample is blood, serum, or plasma, and the fatty liver disease is hepatocellular carcinoma with advanced NASH. ,
<10> A biomarker in a body fluid for diagnosing fatty liver disease,
The biomarker, characterized in that it is selected from the group consisting of octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, and octadecadienoylcarnitine,
<11> The biomarker according to <10>, wherein the body fluid sample is blood, serum or plasma.
<12> The biomarker according to <10> or <11>, wherein the fatty liver disease is NASH.
<13> The biomarker according to any one of <10> to <12>, wherein the degree of progression of fibrosis in the liver is determined,
<14> A diagnostic kit for detecting fatty liver disease, comprising octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, and octadecadie A diagnostic kit comprising a reagent for measuring the amount of any one or more long-chain acylcarnitines selected from the group consisting of noylcarnitine,
<15> Octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, in a body fluid sample from a subject who may suffer from fatty liver disease And measuring the amount of any one or more of the long-chain acylcarnitines selected from the group consisting of octadecadienoylcarnitine and comparing the amount of any one or more of the measured long-chain acylcarnitines with a reference value A method for determining the degree of progression of liver fibrosis in the subject,
<16> The method for determining the degree of progression of liver fibrosis in the subject according to <15>, wherein the body fluid sample is blood, serum, or plasma,
<17> Measure the amount of any one or more medium-short chain acylcarnitines having 2 to 10 carbon atoms in the acyl group and the measured amount of one or more medium-short chain acylcarnitines A method for determining the degree of progression of liver fibrosis in the subject according to <15> or <16>, further comprising comparing
<18> The method for determining the degree of progression of liver fibrosis in a subject according to <17>, wherein the medium-short chain acylcarnitine is carnitine isovalerate,
<19> A method for determining the degree of progression of liver fibrosis in a subject who may be suffering from fatty liver disease, wherein an index score is calculated using the following formula: [Formula 1]
AC14: 1 + AC18: 1-AC5: 0
(Where AC14: 1 indicates the amount of tetradecenoylcarnitine in the body fluid sample from the subject, AC18: 1 indicates the amount of octadecenoylcarnitine in the body fluid sample from the subject, and AC5: 0 indicates the amount of carnitine isovalerate in the body fluid sample from the subject)
Comparing the calculated index score with a reference value, and <20> for determining the degree of progression of liver fibrosis in a subject potentially having fatty liver disease A biomarker in body fluid,
The biomarker described above, wherein the biomarker is selected from the group consisting of octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, and octadecadienoylcarnitine.

  According to the present invention, it is possible to easily diagnose fatty liver disease without imposing a burden on patients and medical staff. According to the present invention, fatty liver disease such as NASH can be detected before the subject fatty liver disease progresses to hepatocellular carcinoma derived from fatty liver disease. According to the present invention, it is possible to easily determine the degree of progression of liver fibrosis in a subject without burdening the patient and medical staff.

Various long chains in hepatocellular carcinoma tissue (HFD-HCC) of mice fed a high fat diet after administration of the chemical carcinogen Dietylnitrosamine (DEN) and non-tumor tissues (ND-NT) of DEN-injected mice fed a normal diet It is a figure which shows the comparison of the quantity of acylcarnitine. Non-tumor tissue (ND-NT) of DEN-treated mice fed normal diet, hepatocellular carcinoma tissue (ND-HCC) of DEN-treated mice fed normal diet, non-tumor tissue of DEN-treated mice fed high fat diet It is a figure which shows the quantity of various long-chain acyl carnitines in each of the hepatocellular carcinoma tissue (HFD-HCC) of the DEN administration mouse | mouth which gave the high fat diet (HFD-NT). It is a schematic diagram which shows the metabolic pathway of acylcarnitine in the living body. Non-tumor tissue (ND-NT) of DEN-treated mice fed normal diet, hepatocellular carcinoma tissue (ND-HCC) of DEN-treated mice fed normal diet, non-tumor tissue of DEN-treated mice fed high fat diet The expression levels of ACSL4, CPT1A, CACT, and CPT2 investigated using real-time PCR in each of the hepatocellular carcinoma tissues (HFD-HCC) of DEN-injected mice fed with (HFD-NT) and a high fat diet are shown. FIG. It is a schematic diagram showing a mechanism in which accumulation of long-chain acylcarnitine occurs in hepatocellular carcinoma tissue. It is a figure which shows the analysis result of the amount of long-chain acyl carnitine in the NAFLD patient serum of the progress degree of various fibrosis. It is a figure which shows the analysis result of the amount of short-chain acyl carnitine in the NAFLD patient serum of various fibrosis progressing degree. It is a figure which shows the result of having analyzed the amount of long-chain acyl carnitine in the NASH patient (HCC (+)) which has hepatocellular carcinoma, and the NASH patient (HCC (-)) which does not have hepatocellular carcinoma. It is a figure which shows the result of having analyzed the effectiveness as a marker of the progress of the fibrosis of the formula AC14: 1 + AC18: 1-AC5: 0 in the serum of the NAFLD patient of various fibrosis progress. Hepatoma-related factors of formula (AC16: 0 + AC18: 1) × 100 / AC2: 0 in NASH patients with hepatocellular carcinoma (HCC (+)) and NASH patients without hepatocellular carcinoma (HCC (−)) It is a figure which shows the result of having analyzed the effectiveness as. It is a figure which shows the analysis result of the relationship between the amount of long-chain acyl carnitine and serum inflammatory cytokine in NAFLD patient serum. It is a figure which shows the analysis result of the relationship between the amount of long-chain acyl carnitine and a serum carcinogenic risk marker in a NAFLD patient serum.

[1] Fatty liver disease detection or risk prediction method (body fluid sample from the subject)
Examples of body fluid samples that can be analyzed in the present invention include body fluids derived from living organisms. The body fluid sample that can be analyzed in the present invention is more preferably blood, serum, plasma, urine, saliva, sputum, tears, otorrhea, or prostate fluid, more preferably blood, serum, or plasma, still more preferably. Is the blood, serum or plasma of a subject suspected of having fatty liver disease (eg, NAFLD or NASH). Living organisms or subjects include humans or animals (eg, mice, guinea pigs, rats, monkeys, dogs, cats, hamsters, horses, cows, and pigs), preferably humans.

(Acylcarnitine)
In the present invention, acylcarnitine refers to a group of compounds represented by Formula 1.
Formula _{1: (CH 3) 3 N} + CH 2 CH (OR 1) CH 2 COO -
(In Formula 1, R ¹ represents a saturated or unsaturated fatty acid residue having 2 to 30 carbon atoms, and the hydrogen atom bonded to the fatty acid residue may be substituted with an oxygen atom or a hydroxy group.)

  Acylcarnitine is composed of acetylcarnitine, propionylcarnitine, stearoylcarnitine, oleylcarnitine, depending on the carbon chain length of the fatty acid moiety, the presence or absence and number of unsaturated bonds, substitution of oxygen atoms or hydroxy groups of hydrogen atoms bound to the carbon chain , Linoleyl carnitine, malonyl carnitine, 3-hydroxy carnitine, etc., but in the present specification they are generically described as acyl carnitine. In the present specification, free carnitine to which no fatty acid is bound is not included in acylcarnitine.

  Examples of saturated or unsaturated fatty acid residues include those listed in Table 1. Acylcarnitine is also used as an evaluation item in screening for inborn errors of metabolism such as fatty acid metabolism abnormalities. Table 2 shows the main acylcarnitines used in clinical evaluation items such as screening for inborn errors of metabolism.

(Long-chain acylcarnitine)
In this specification, “long-chain acylcarnitine” means acylcarnitine in which the fatty acid residue (acyl group) has 12 to 30 carbon atoms. In the method for detecting fatty liver disease or predicting risk according to the present invention, octadecenoylcarnitine, tetradecenoylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, and octadecadienoylcarnitine The amount of any one or more of long-chain acylcarnitines selected from the group consisting of the following groups (hereinafter sometimes referred to as 6 types of long-chain acylcarnitines) is measured. More preferably, the long chain acylcarnitine to be measured is one or more selected from tetradecenoylcarnitine and octadecenoylcarnitine, and most preferably octadecenoylcarnitine.

  Intracellular long chain fatty acids are esterified to acyl CoA by the acyl CoA (acyl-CoA) synthase (ACSL) family. This acyl CoA is then converted to acylcarnitine by binding to carnitine by carnitine palmitoyltransferase 1 (CPT1). After entering the mitochondrial inner membrane via carnitine acylcarnitine translocase (CACT), acylcarnitine is converted again to carnitine and acylCoA by carnitine palmitoyltransferase 2 (CPT2), and acyl CoA undergoes β-oxidation to produce energy. Used for (See FIG. 3).

  The present inventors sampled liver cancer tissues of liver cancer model mice related to obesity and investigated changes in gene expression levels related to the above pathway. As a result, the expression levels of ACSL4 and CPT1A, one of the ACSL families, were found. It was found that the expression level of CPT2 increased and decreased. Due to these changes in the expression level, reconversion of long-chain acylcarnitine to acyl-CoA by CPT2 does not function in liver cancer tissues of liver cancer model mice related to obesity. Therefore, it is considered that accumulation of long-chain acylcarnitine occurs in the liver cancer tissue of liver cancer model mice related to obesity (see FIG. 5).

(Method for measuring the amount of long-chain acylcarnitine)
The amount of long-chain acylcarnitine can be measured by, for example, a capillary electrophoresis-mass spectrometer, a liquid chromatograph-tandem mass spectrometer (LC-MS / MS), or the like. A combination of these methods can also be used. In the present specification, “liquid chromatograph-tandem mass spectrometer” or “LC-MS / MS” is an ion generated by ionizing an analysis target component separated by a liquid chromatograph (LC) through a dedicated interface. Is separated by a mass spectrometer (MS) to dissociate and / or fragment ions having a specific mass, and the ions are detected by the mass spectrometer.

(Standard value)
The method for detecting fatty liver disease or predicting risk according to the present invention includes comparing the amount of any one or more long-chain acylcarnitines out of six long-chain acylcarnitines with a reference value. In the method for detecting fatty liver disease or predicting risk according to the present invention, the index indicates that the amount of any of the six long-chain acylcarnitines in the subject is higher than the amount of the long-chain acylcarnitine in the healthy subject group Detection of fatty liver disease and prediction of risk. Specifically, for example, whether fatty liver disease is detected when the amount of any of the six types of long-chain acylcarnitines in the subject is equal to or greater than a threshold value (reference value) for determination with a healthy person group Or, it can be determined that there is a high possibility of developing.

  A numerical range can also be used as a reference value. When diagnosing whether or not a patient has a fatty liver disease, the body fluid of a subject previously diagnosed as having a fatty liver disease and a subject diagnosed as not having a fatty liver disease The range of the amount of any one of the six types of long-chain acylcarnitines in the sample is measured, and the amount of the long-chain acylcarnitine in the subject body fluid sample is the length in the body fluid sample of the healthy subject. If it falls within the range of chain acylcarnitine levels, the subject is likely not suffering from fatty liver disease, and the amount of long chain acylcarnitine in the body fluid sample of the subject suffering from fatty liver disease If it falls in range, it is likely that you have fatty liver disease.

  The judgment threshold (reference value) is expected to change depending on various conditions such as the underlying disease, sex, age, etc., but those skilled in the art can appropriately select an appropriate population corresponding to the subject. The normal value range or the threshold for determination can be determined by performing statistical processing on the data obtained from the population.

  In the method for detecting fatty liver disease or predicting risk according to the present invention, the amount of one specific long-chain acylcarnitine among the six long-chain acylcarnitines may be compared with a reference value. The amount of long chain acylcarnitine may be compared to a reference value for each long chain acylcarnitine.

  In the method for detecting fatty liver disease or predicting risk according to the present invention, the amount of medium-short chain acylcarnitine whose acyl group has 2 to 10 carbon atoms is measured, and the amount of medium-short chain acylcarnitine is the reference value. It is preferable that it further includes comparing with. More preferably, the medium and short chain acyl carnitine is carnitine isovalerate. Hereinafter, the medium-short chain acylcarnitine whose acyl group has 2 to 10 carbon atoms may be simply referred to as medium-short chain acylcarnitine.

  Specifically, in the method for detecting fatty liver disease or predicting risk according to the present invention, the amount of any medium-short chain acylcarnitine in the subject is lower than the amount of the medium-short chain acylcarnitine in the healthy group. Can be used to detect fatty liver disease and predict risk more precisely. Specifically, for example, when the amount of any medium- and short-chain acylcarnitine in a subject is equal to or less than a threshold value (reference value) for determination with a healthy person group, fatty liver disease is detected or developed It can be determined that the possibility is higher.

  A numerical range can also be used as a reference value. When diagnosing whether or not a patient has a fatty liver disease, the body fluid of a subject previously diagnosed as having a fatty liver disease and a subject diagnosed as not having a fatty liver disease Measure the range of any medium-short chain acylcarnitine in the sample, and the medium-short chain acylcarnitine in the subject body fluid sample is the range of the amount of the medium-short chain acylcarnitine in the body fluid sample of the healthy subject If the subject is more likely not to have fatty liver disease and falls within the range of medium and short chain acylcarnitine levels in the body fluid sample of the subject suffering from fatty liver disease Are more likely to have fatty liver disease.

By calculating the index score using the following calculation formula and comparing the calculated index score with a reference value, it is possible to more accurately detect fatty liver disease or predict risk.
[Formula 1]
AC14: 1 + AC18: 1-AC5: 0
(Where AC14: 1 indicates the amount of tetradecenoylcarnitine in the body fluid sample from the subject, AC18: 1 indicates the amount of octadecenoylcarnitine in the body fluid sample from the subject, and AC5: 0 indicates the amount of carnitine isovalerate in the body fluid sample from the subject)

By calculating the index score using the following calculation formula and comparing the calculated index score with a reference value, it is possible to more accurately detect fatty liver disease or predict risk.
[Formula 2]
(AC16: 0 + AC18: 1) × 100 / AC2: 0
(Where AC16: 0 indicates the amount of hexadecanoylcarnitine in the body fluid sample from the subject, AC18: 1 indicates the amount of octadecenoylcarnitine in the body fluid sample from the subject, and AC2: 0 indicates the amount of acetylcarnitine in the body fluid sample from the subject)
When using the above formula, the fatty liver disease is preferably hepatocellular carcinoma with advanced NASH.

(Fatty liver disease)
As used herein, “fatty liver disease” means a liver disease caused by fibrosis and / or fat deposition of hepatocytes. The fatty liver disease is preferably NAFLD. NAFLD includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), cirrhosis with advanced NASH, and hepatocellular carcinoma with advanced NASH. The fatty liver disease is more preferably NASH. In the present specification, the terms “NAFLD” and “NASH” can include hepatocellular carcinoma in which NASH has progressed, but preferably do not include it.

In the method for detecting fatty liver disease or predicting risk according to the present invention, NASH can detect or predict risk before NASH progresses to hepatocellular carcinoma. Therefore, it is more preferable from the viewpoint of early diagnosis that the fatty liver disease is NASH without hepatocellular carcinoma.
In addition, as shown in Examples described later, the amount of long-chain acylcarnitine and liver fibrosis have a significant positive correlation. Therefore, from the viewpoint of detection sensitivity, the fatty liver disease is preferably cirrhosis in which NASH has progressed or hepatocellular carcinoma in which NASH has progressed, and most preferably hepatocellular carcinoma in which NASH has progressed.

(Fatty liver disease detection or risk prediction method)
The method for detecting fatty liver disease or predicting risk according to the present invention can monitor the degree of progression of fatty liver disease. In this case, the amount of any of the six types of long-chain acylcarnitines in the subject at a specific time point is used as a determination threshold value (reference value). After a period of time (eg after 1, 3, 6, or 12 months), the amount of this long chain acylcarnitine in this subject is measured again and the amount of this long chain acylcarnitine is compared to the previous measurement. If so, it can be determined that fatty liver disease is progressing. Conversely, if the amount of this long-chain acylcarnitine is the same or small compared to the previous measurement value, it can be determined that fatty liver disease has not progressed. As described above, in addition to the amount of any one of the six types of long-chain acylcarnitines, the amount of any one or more medium-short chain acylcarnitines in which the carbon number of the acyl group is C2-10 (preferably , The amount of carnitine isovalerate) can also be measured after a certain time and after a certain period of time.

  After performing the method for detecting fatty liver disease or predicting risk according to the present invention, if necessary, the method for detecting another fatty liver disease (for example, NASH) in patients and / or fatty liver disease Administration of a therapeutic agent (eg, NASH) to the patient may be performed.

[2] Biomarker for diagnosing fatty liver disease In the present specification, a biomarker is a peptide, protein, nucleic acid, lipid or other low-molecular compound related to a predetermined disease, The increase or decrease in the concentration reflects the presence or progression of the disease. In the biomarker for diagnosing fatty liver disease of the present invention, it is an indicator that the amount of any one of the six types of long-chain acylcarnitines in the subject is higher than the amount of the long-chain acylcarnitines in the healthy subject group As a fatty liver disease can be diagnosed. Specifically, for example, when the amount of any long-chain acylcarnitine in the subject is equal to or greater than the threshold for determination (reference value) with the healthy human group, fatty liver disease can be detected or developed It can be determined that the property is high.

  In the biomarker for diagnosing fatty liver disease of the present invention, a numerical value range can be used as a reference value. When diagnosing whether or not a patient has a fatty liver disease, the body fluid of a subject previously diagnosed as having a fatty liver disease and a subject diagnosed as not having a fatty liver disease The range of the amount of any of the six long-chain acylcarnitines in the sample is measured, and the long-chain acylcarnitine in the subject body fluid sample is converted into the long-chain acylcarnitine in the healthy subject body fluid sample. If within the range, the subject is likely not suffering from fatty liver disease and falls within the long chain acylcarnitine level in the body fluid sample of the subject suffering from fatty liver disease If so, you are likely to have fatty liver disease.

[3] Diagnostic kit The diagnostic kit for detecting fatty liver disease of the present invention contains a reagent for measuring the amount of any one or more of the six types of long-chain acylcarnitines. The reagent may contain a reagent for measuring the amount of one specific long-chain acylcarnitine, or may contain a reagent for measuring the amount of two or more long-chain acylcarnitines. The diagnostic kit for detecting fatty liver disease of the present invention is preferably one of six long-chain acylcarnitines using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS). A reagent for measuring one or more quantities.

[4] Method for Determining the Progression Level of Liver Fibrosis in a Subject In the method for determining the progression degree of liver fibrosis in a subject according to the present invention, depending on the amount of any of the six types of long-chain acylcarnitines in the subject body fluid sample. Thus, the progress of fibrosis in the liver can be determined. In this specification, “liver fibrosis” means that connective tissue containing fibrous components such as collagen fibers and / or extracellular matrix is increased in the liver in large quantities due to chronic liver inflammation, and the tissue is hardened. Means. The method for determining the degree of progression of liver fibrosis of the subject of the present invention can determine the degree of progression of liver fibrosis caused by NAFLD or NASH in a subject who may be affected by NAFLD or NASH. It is also possible to determine the degree of progression of liver fibrosis caused by NAFLD or NASH in a subject already suffering from NAFLD or NASH. The method for measuring long-chain acylcarnitine and the body fluid sample are as described above.

(Standard value)
The method for determining the degree of progression of liver fibrosis of the subject of the present invention comprises comparing the measured amount of long-chain acylcarnitine with a reference value. In the method for determining the degree of progression of liver fibrosis according to the present invention of the present invention, it is preferable to use a numerical range as a reference value. When determining the degree of progression of liver fibrosis in a subject, the range of the amount of any of the six types of long-chain acylcarnitines in the body fluid samples of subjects at various liver fibrosis stages is measured in advance. If the amount of this long chain acylcarnitine falls within the range of long chain acylcarnitines in a body fluid sample at a particular liver fibrosis stage, the subject is likely to be at that particular liver fibrosis stage.
The classification of liver fibrosis stages is, for example, F0: no fibrosis, F1: fibrosis in the central part of the leaflet, F2: fibrosis in the central part of the leaflet and portal vein area, F3: formation of fibrous bridge, F4: cirrhosis, HCC : Can follow the distinction of hepatocellular carcinoma. The method for determining the degree of progression of liver fibrosis of the subject of the present invention can determine at which stage F0 to F4 the subject liver is. However, the classification is not limited to the above F0 to F4, and different classifications may be used.

  The judgment threshold (reference value) is expected to change depending on various conditions such as the underlying disease, sex, age, etc., but those skilled in the art can appropriately select an appropriate population corresponding to the subject. The normal value range or the threshold for determination can be determined by performing statistical processing on the data obtained from the population.

  The method for determining the progress of liver fibrosis of the subject of the present invention can monitor the progress of liver fibrosis of the subject. In this case, the amount of any of the six types of long-chain acylcarnitines in the target body fluid sample at a specific time is used as a determination threshold value (reference value). This long chain acylcarnitine in this subject is measured again after a certain period of time (eg after 1, 3, 6, or 12 months) and if the amount is large compared to previous measurements, liver fibrosis will progress It can be determined that Conversely, if the amount is the same or less than the previous measurement, it can be determined that liver fibrosis has not progressed.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

[Example 1] Comparison of the amount of various long-chain acylcarnitines in HFD-HCC and ND-NT In order to examine the metabolic changes characteristic of hepatocellular carcinoma associated with obesity, normal diet (ND) or high fat Liquid chromatography-mass spectrometry (LC-MS) was performed on non-tumor tissue (NT) and hepatocellular carcinoma tissue (HCC) obtained from 8 month old diethylnitrosamine (DEN) -administered mice fed diet (HFD). The non-target metabolomic profiling used was performed. DEN-administered mice fed with a high fat diet are widely recognized as hepatoma model mice associated with obesity.

  C57BL / 6 mice were purchased from Nippon Claire Co., Ltd. Diethylnitrosamine (Sigma Co.) was dissolved in phosphate buffered saline and injected intraperitoneally into mice on day 14 after birth (25 mg / kg). When the mice were 6 weeks old, administration of a high fat diet (HFD) (D12492; Research Diets Inc) or a normal diet (ND) was started. Administration of a normal diet (ND) or a high fat diet (HFD) was continued until the age of 8 months, and DEN-administered mice fed with a normal diet or a high fat diet were obtained. Thereafter, a non-tumor tissue (NT) and a hepatocellular carcinoma tissue (HCC) were sampled from a mouse liver administered with a normal diet (ND) or a high fat diet (HFD), and ND-NT (normal diet / non-tumor tissue). ), ND-HCC (normal diet / tumor tissue), HFD-NT (high fat diet / non-tumor tissue), and HFD-HCC (high fat diet / tumor tissue), liquid chromatography-mass spectrometry (LC-MS) ) Was used in the following procedure.

(Extraction of metabolites)
Metabolite extraction and metabolite analysis were performed at Human Metabolome Technologies (HMT; Tsuruoka City). Briefly, for CE-TOFMS analysis, approximately 50 mg of frozen tissue was sedimented at 0 ° C. in 1,500 μL of 50% acetonitrile / milli Q water containing an internal standard (H3304-1002; HMT). The tissue was homogenized three times for 120 seconds at 1,500 rpm using a tissue homogenizer (Micro Smash MS100R; Fuji Digital Biology). The homogenate was centrifuged at 2,300 × g and 4 ° C. for 5 minutes. Subsequently, 800 μL of the upper aqueous layer was centrifuged using a Millipore 5-kDa cut-off filter (UltrafreeMC-PLHCC; HMT) to remove the polymer (9,100 × g, 4 ° C., 120 minutes). The filtrate was concentrated by centrifugation and resuspended in 50 μL MilliQ water for CE-TOFMS analysis with HMT.

  For liquid chromatography TOFMS (LC-TOFMS) analysis, approximately 50 mg of frozen tissue was precipitated at 0 ° C. in 500 μL of 1% formic acid / acetonitrile containing an internal standard solution (H3304-1002; HMT). The tissue was homogenized as above, and after addition of 167 μL of MilliQ water, the mixture was homogenized again and the homogenate was centrifuged (2,300 × g, 4 ° C., 5 minutes). Next, 500 μL of 1% formic acid / acetonitrile and 167 μL of milli-Q water were mixed with the supernatant, and the mixed solution was filtered through a 3-kDa cut-off filter (NANOCEP 3K OMEGA; PALL) to remove the polymer. Further filtration was performed using a hybrid SPE phospholipid cartridge (55261-U; Supelco) to remove phospholipids. The filtrate was dried and resuspended in 100 μL of isopropanol / milli Q water for LC-TOFMS analysis.

(Analysis of metabolites)
Metabolomic analysis was performed on CE-TOFMS and ionic and nonionic metabolites, respectively, based on the methods described in Ooga et al. Mol Biosystem 2011; 7: 1217-1223 and Sugimoto et al. Metabolics 2010; 6: 78-95, respectively. LC-TOFMS was used with the HMT DualScan package. Briefly, an Agilent CE-F system using an Agilent CE-MS system equipped with an Agilent 6210 TOFMS, an Agilent 1100 isocratic HPLC pump, an Agilent G1603A CE-MS adapter kit, and an Agilent G1607A CE-ESI-MS spray kit (Agilent Technologies) is used. Went. This system is based on Agilent G2201A ChemStation software version B. 03.01 for CE (Agilent Technologies) controlled by fused silica capillaries (50 μm id × 80 cm full length) as commercial electrolyte buffers as electrolytes (for cation and anion analysis, H3301-1001 and H3302-, respectively) 1021; HMT). The spectrophotometer was scanned from 50 to 1,000 m / z. LC-TOFMS analysis was performed using an Agilent LC System (Agilent 1200 series RRLC system SL) equipped with Agilent 6230 TOFMS (Agilent Technologies). This system was installed on an Agilent G2201A ChemStation software version B.O. equipped with an ODS column (2 × 50 mm, 2 μm). Controlled by 03.01 (Agilent Technologies).

  Peaks were extracted using MasterHands automatic integration software (Keio University, Tsuruoka City) to obtain peak information including m / z, peak area, and migration time (MT). Signal peaks corresponding to known metabolite isotopomers, adduct ions, and other product ions are excluded, and the remaining peaks are based on MT and RT determined using TOFMS and their m / z values. Annotated by the HMT metabolite database. The area of the annotated peak was then normalized based on the internal standard level and sample volume to obtain the relative level of each metabolite. Based on a one-point calibration with each standard compound, 110 major metabolites were quantified absolutely. Hierarchical cluster analysis and principal component analysis (PCA) were performed using PeakStat and SampleStat, which are HMT proprietary software, respectively. The detected metabolites were displayed on the metabolic pathway map using VANTED software.

The amount of various long chain acylcarnitines in four tissues was analyzed. The results are shown in FIGS. In FIG. 1, “20” in AC (20: 1) represents the carbon number of the acyl group, and “1” represents the number of unsaturated bonds. In FIG. 1, the number “9.8” and the size of the circle represent the relative amount of AC (20: 1) in HFD-HCC, where AC (20: 1) in ND-NT is 1. Show.
HFD-HCC was found to have increased amounts of various types of long-chain acylcarnitine (AC) compared to ND-NT (FIG. 1).

In FIG. 2, the data is displayed as a relative amount to the average amount in ND-NT tissue. * Indicates P <0.05 for ND-NT tissue, † indicates P <0.05 for ND-HCC tissue, ‡ indicates HFD-NT tissue In contrast, P <0.05.
HFD-HCC tended to have a longer amount of long-chain acylcarnitine than each of ND-HCC and HFD-NT. In particular, three types of AC12: 1, AC16: 0, and AC18: 1 were significantly higher in HFD-HCC tissue than all the other three types of tissue.

[Example 2] Verification of acylcarnitine metabolism-related gene expression in HCC related to obesity The DEN-injected mice obtained in Example 1 and fed with a normal diet or a high-fat diet were used in the experiment. In each tissue of ND-NT, ND-HCC, HFD-NT, and HFD-HCC, the gene expression levels of ACSL4, CPT1A, CACT, and CPT2 related to acylcarnitine metabolism were investigated using real-time PCR. The results are shown in FIG.

  In FIG. 4, the data is displayed as a relative amount with respect to the gene expression level in the ND-NT tissue. * Indicates P <0.05 for ND-NT tissue, † indicates P <0.05 for ND-HCC tissue, ‡ indicates HFD-NT tissue In contrast, P <0.05. Strong expression of CPT1A and ACSL4 was detected in ND-HCC and HFD-HCC tissues. On the other hand, the expression level of CPT2 is decreased in ND-HCC and HFD-HCC tissues. In particular, the decrease in the expression level in HFD-HCC tissues is ND-NT, ND-HCC, and HFD-NT. There was a significant difference. In HFD-HCC, the expression level of CACT was also significantly lower than that of ND-NT. This result shows that in hepatocellular carcinoma tissue, conversion of fatty acids to long chain acylcarnitine is increased in HFD-HCC tissue, while reconversion of long chain acylcarnitine to acyl CoA is suppressed. Show. That is, this result explains why long-chain acylcarnitine species accumulate in hepatocellular carcinoma tissue (see FIG. 5). For CPT1A and CPT2, a test was conducted to confirm the expression level in each tissue using Western blot analysis, but the same tendency was observed.

[Example 3] Verification using human serum sample Using 241 samples of NAFLD patient-preserved serum, liquid chromatography-tandem mass spectrometry (LC-MS / MS method) and NeoSMAAT (registered trademark) (manufactured by Sekisui Medical) ) Was used to measure the concentration of various acylcarnitines, and the relationship between NAFLD and the pathological condition was examined. The relationship between serum acylcarnitine concentration and serum inflammatory cytokine concentration or serum carcinogenic risk marker concentration was also examined.

1. Patient serum samples NAFLD patient sera were classified into F0-4 and hepatocellular carcinoma as follows according to the degree of fibrosis.
F0: No fibrosis F1: Fibrosis in the central part of the leaflet F2: Fibrosis in the central part of the leaflet + portal vein region F3: Fibrous bridge formation F4: Cirrhosis HCC: Hepatoma 241 There are 38 specimens, F1 74 specimens, F2 63 specimens, F4 25 specimens, and hepatocellular carcinoma 23 specimens.

2. Measurement of serum long-chain acylcarnitine The collected serum samples were measured using liquid chromatograph-tandem mass spectrometry (LC-MS / MS method) and NeoSMAAT (registered trademark). NeoSMAAT® includes carnitine (C0), acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine (C4), carnitine isovalerate (C5), 3- Hydroxy-isovaleric acid carnitine (C5OH), glutarylcarnitine (C5DH), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecanoylcarnitine (C14), tetradecenoyl Carnitine (C14: 1), palmitoylcarnitine (C16), and stearoylcarnitine (C18) are included as internal standards. By calculating the ratio of the peak area of each acylcarnitine or free carnitine that is the measurement target substance to the peak area of the internal standard substance, the amount and concentration of the measurement target substance present in the serum sample can be obtained.

  10 μL of an internal standard stock solution attached to NeoSMAAT (registered trademark) was added to 10 μL of human serum specimen. An additional 200 μL of ethanol was added and stirred for 10 seconds. Centrifugation (10000 rpm, 4 ° C., 5 minutes) was performed, and the supernatant was transferred to a glass test tube. It was concentrated to dryness under a nitrogen stream (40 ° C.). 100 μL of formic acid / acetonitrile (0.05: 10) was added and stirred for 10 seconds with a vortex mixer. 100 μL of acetonitrile was added and stirred with a vortex mixer for 10 seconds. Centrifugation (2000 rpm, 4 ° C., 3 minutes) was performed, the supernatant was transferred to an HPLC vial, and the amount of acylcarnitine was measured by LC-MS / MS. The conditions of LC-MS / MS are as follows.

System: LC-20A series (manufactured by Shimadzu Corporation) and 4000QTRAP (manufactured by AB Sciex)
<LC conditions>
HPLC column: Inertsil Amide (2.1 × 250 mm, 3 μm, GL Sciences)
Mobile Phase A: 200 mmol / L Ammonium format
Mobile Phase B: Acetonitrile
Gradient:

  As for the amount of 3-hydroxy-hexadecanoylcarnitine (C16OH), a deuterium-substituted C16OH peak area value and a deuterium-substituted C16 peak area value with known concentrations were measured in advance, and the conversion number = deuterium substitution as a conversion factor. C16OH peak area value / deuterium substitution C16 peak area value is calculated, and based on the converted concentration value obtained by dividing the concentration value calculated from the C16OH peak area value and the internal standard C16 peak area value by the conversion factor. Calculated. The amount of octadecenoylcarnitine (C18: 1) is determined in advance by measuring the deuterium substitution C18: 1 area value and deuterium substitution C18 peak area value of known concentrations, and the conversion number = deuterium substitution C18 as a conversion factor. : 1 peak area value / deuterium substitution C18 area value is calculated, and the converted concentration value obtained by dividing the concentration value calculated from the C18: 1 peak area value and the internal standard C18 peak area value by the conversion factor is Based on the calculation. The amount of 3-hydroxy-octadecenoylcarnitine (C18: 1OH) is determined in advance by measuring a deuterium-substituted C18: 1OH area value and a deuterium-substituted C18 peak area value with known concentrations, and the conversion factor = Conversion obtained by calculating deuterium substitution C18: 1OH peak area value / deuterium substitution C18 area value and dividing the concentration value calculated from C18: 1OH area value and internal standard C18 peak area value by the conversion factor Calculation was based on the concentration value.

  The measurement results of AC14: 1 (tetradecenoylcarnitine) and AC18: 1 (octadecenoylcarnitine) are shown in FIG. AC14: 1 and AC18: 1 showed a significant positive correlation with liver fibrosis. (AC14: 1 has a trend test p-value of 0.0087 and AC18: 1 has a trend test p-value of 0.0111). Therefore, it was found that by measuring these long-chain acylcarnitine concentrations in the living body, fatty liver disease can be detected, and the degree of fibrosis in fatty liver disease can be determined. Moreover, the calculation formula AC14: 1 + AC18: 1-AC5: 0 combining long-chain acylcarnitine and short-chain acylcarnitine showed a stronger positive correlation with liver fibrosis (p value 0.000721) (FIG. 9).

  The measurement result of AC5: 0 (carnitine isovalerate) is shown in FIG. For carnitine isovalerate, the p value of the trend test was 0.00249, indicating a significant negative correlation with liver fibrosis. Therefore, it was found that by measuring the concentration of these short-chain acylcarnitines in the living body, fatty liver disease can be detected, and the degree of fibrosis in fatty liver disease can be determined.

  FIG. 8 shows the measurement results of AC18: 1 in NAFLD patients without HCC (total of F0 to 4) and NAFLD patients with HCC. AC18: 1 was significantly higher in NAFLD patients with HCC. Therefore, it was found that hepatocellular carcinoma with advanced NASH can be detected by measuring the amount of AC18: 1 in the living body.

In NAFLD patients without HCC (total of F0-4) and NAFLD patients with HCC, the formula (AC16: 0 + AC18: 1) × 100 / AC2: 0 (where AC16: 0 is a body fluid sample from the subject) Indicates the amount of hexadecanoylcarnitine, AC18: 1 indicates the amount of octadecenoylcarnitine in the body fluid sample from the subject, and AC2: 0 indicates the amount of acetylcarnitine in the body fluid sample from the subject. FIG. 10 shows the result of comparison of the calculated values.
The calculated value of the formula (AC16: 0 + AC18: 1) × 100 / AC2: 0 showed a high value in NAFLD patients with HCC. Therefore, it was found that hepatocellular carcinoma with advanced NASH can be detected by calculating the calculated value of the above formula.

  A diagram showing the relationship between AC18: 1 and serum inflammatory cytokine concentrations is shown in FIG. FIG. 12 shows the relationship between AC18: 1 and serum carcinogenic risk marker concentration. AC18: 1 showed a positive correlation with each of serum inflammatory cytokine levels and serum carcinogenic risk marker levels. Thus, it was found that AC18: 1 can be used as a marker for inflammation and carcinogenesis associated with fatty liver disease.

  According to the present invention, it is possible to easily diagnose fatty liver disease without imposing a burden on patients and medical staff. According to the present invention, NASH can be detected before the subject progresses to hepatocellular carcinoma. According to the present invention, it is possible to easily determine the degree of progression of liver fibrosis in a subject without burdening the patient and medical staff.

Claims (6)

Blood from a subject, in serum or plasma, and comparing with a reference value the amount of tetradecen noil carnitine was measured by measuring the amount of tetradecane Seno dolphins Le Nitinol emissions, fatty in said subject a method for detecting liver disease,
A method for determining the degree of progression of fibrosis in the liver . The method for detecting fatty liver disease according to claim 1 , wherein the fatty liver disease is NASH. Measure the amount of any one or more medium-short chain acylcarnitines whose acyl group has 2 to 10 carbon atoms and compare the measured amount of any one or more medium-short chain acylcarnitines with a reference value The method for detecting the fatty liver disease of Claim 1 or 2 further including this. A biomarker in blood, serum or plasma for diagnosing fatty liver disease,
It is tetradecen noil carnitine, and
The biomarker described above, wherein the biomarker is for determining the degree of fibrosis in the liver . The biomarker according to claim 4 , wherein the fatty liver disease is NASH. A diagnostic kit for detecting fatty liver disease, comprising a reagent for measuring the amount of tetradecane Seno dolphins Le Nitinol down, a diagnostic kit for detecting fatty liver disease,
The diagnostic kit, wherein the reagent determines the degree of fibrosis in the liver .

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