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WO2020044497A1 - Méthode de prédiction de risque, ou de détection, d'une stéatose hépatique non alcoolique (nafld) ou shna, trousse de réactif de diagnostic pour la détection de nafld ou nash, méthode de détermination du taux de progression d'une fibrose hépatique chez un sujet, et trousse de réactif de diagnostic pour la détermination du taux de progression d'une fibrose hépatique chez un sujet - Google Patents

Méthode de prédiction de risque, ou de détection, d'une stéatose hépatique non alcoolique (nafld) ou shna, trousse de réactif de diagnostic pour la détection de nafld ou nash, méthode de détermination du taux de progression d'une fibrose hépatique chez un sujet, et trousse de réactif de diagnostic pour la détermination du taux de progression d'une fibrose hépatique chez un sujet Download PDF

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Publication number
WO2020044497A1
WO2020044497A1 PCT/JP2018/032138 JP2018032138W WO2020044497A1 WO 2020044497 A1 WO2020044497 A1 WO 2020044497A1 JP 2018032138 W JP2018032138 W JP 2018032138W WO 2020044497 A1 WO2020044497 A1 WO 2020044497A1
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total amount
carnitine
acylcarnitine
nash
nafld
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English (en)
Japanese (ja)
Inventor
勇人 中川
健一郎 榎奥
直人 藤原
小池　和彦
直樹 曲尾
泰仁 青木
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University of Tokyo NUC
Sekisui Medical Co Ltd
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University of Tokyo NUC
Sekisui Medical Co Ltd
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Priority to PCT/JP2018/032138 priority Critical patent/WO2020044497A1/fr
Priority to JP2019513860A priority patent/JP6592637B1/ja
Publication of WO2020044497A1 publication Critical patent/WO2020044497A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a method for detecting NAFLD or NASH or estimating a risk.
  • the present invention also relates to a diagnostic kit for detecting NAFLD or NASH.
  • the present invention also relates to a method for determining the degree of progression of hepatic fibrosis in a subject, and a diagnostic reagent kit for determining the degree of progression of hepatic fibrosis in a subject.
  • Non-alcoholic fatty liver disease is a general term for fatty liver not caused by alcohol.
  • NAFLD is classified into nonalcoholic steatohepatitis (NASH), which also becomes a home of cirrhosis and liver cancer, and nonalcoholic fatty liver (NAFL), whose disease state hardly progresses.
  • NASH nonalcoholic steatohepatitis
  • NAFL nonalcoholic fatty liver
  • biopsy of liver tissue is essential.
  • the biopsy of liver tissue makes it possible to grasp the amount of fat, the degree of inflammation, and the degree of progression of fibrosis, and to diagnose NASH.
  • a biopsy of liver tissue involves puncturing the liver with a needle to collect a portion of the tissue or cells, which places an excessive burden on the patient and healthcare professionals, and also involves complications. It involves risks. Therefore, there is a demand for the development of a new NASH diagnostic method which can be performed more easily and does not burden patients and medical staff.
  • Acylcarnitine is a compound formed by combining fatty acid and carnitine when a fatty acid is transported to the inner mitochondrial membrane in a living body. More specifically, acylcarnitine is produced from acyl-CoA and carnitine by the action of carnitine palmitoyltransferase I present in the outer mitochondrial membrane. Analysis of acylcarnitine in biological samples such as blood and urine is an analysis item in screening for congenital metabolic disorders in neonates.
  • Patent Document 1 discloses a biomarker for diagnosing fatty liver disease and a method for measuring the same.
  • the amount of palmitoylcarnitine or acetylcarnitine is used in combination with the measured value of another biomarker, and the amount of acylcarnitine alone is measured to reduce fatty liver disease. Diagnosis is neither disclosed nor suggested.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, by measuring the total amount of acylcarnitine in a biological sample and comparing it with a reference value, the burden on patients and healthcare professionals is reduced, and NAFLD and NASH The present inventors have found that early diagnosis is possible and that the degree of progression of liver fibrosis can be determined, and have completed the present invention. Specifically, the present invention is as follows.
  • ⁇ 1> a method for detecting NAFLD or NASH or predicting a risk, comprising measuring the total amount of acylcarnitine in a biological sample from a subject and comparing the total amount of acylcarnitine with a reference value, ⁇ 2> The method for detecting NAFLD or NASH or predicting risk according to ⁇ 1>, wherein the biological sample is blood, serum or plasma.
  • ⁇ 3> the method for detecting NAFLD or NASH or predicting risk according to ⁇ 1> or ⁇ 2>, wherein the degree of progression of fibrosis in the liver is determined; ⁇ 4> including measuring the total amount of carnitine, which is the total amount of free carnitine and the total amount of acyl carnitine, and measuring the total amount of acyl carnitine by subtracting the amount of free carnitine from the total amount of carnitine, from ⁇ 1> to ⁇ 3>
  • a diagnostic reagent kit for detecting NAFLD or NASH comprising an acylcarnitine composition as an internal standard solution of a mass spectrometer,
  • the acylcarnitine composition comprises acetylcarnitine, propionylcarnitine, butyrylcarnitine, carnitine isovalerate, carnitine 3-hydroxy-isovalerate, glutarylcarnitine, octanoylcarnitine, decanoylcarnitine, dodecanoylcarnitine, tetradecanoyl
  • the diagnostic kit comprising at least one of carnitine, tetradecenoylcarnitine, palmitoylcarnitine, and stearoylcarnitine, ⁇ 8>
  • a subject comprising: measuring the total amount of acylcarnitine in a biological sample from a subject possibly having NAFLD or NASH, and comparing the measured total amount of acylcarnitine with a reference value.
  • Method for determining the degree of progression of liver fibrosis in ⁇ 9> including: measuring the total amount of carnitine, which is the total amount of free carnitine and the total amount of acyl carnitine, and measuring the total amount of acyl carnitine by subtracting the amount of free carnitine from the total amount of carnitine.
  • a diagnostic kit for determining the degree of progression of hepatic fibrosis in a subject possibly having NAFLD or NASH the diagnostic kit including a reagent for measuring the total amount of acylcarnitine, and ⁇ 11> a diagnostic reagent kit comprising an acylcarnitine composition as an internal standard solution of a mass spectrometer for determining the degree of liver fibrosis in a subject possibly having NAFLD or NASH.
  • the acylcarnitine composition comprises acetylcarnitine, propionylcarnitine, butyrylcarnitine, carnitine isovalerate, carnitine 3-hydroxy-isovalerate, glutarylcarnitine, octanoylcarnitine, decanoylcarnitine, dodecanoylcarnitine, tetradecanoyl
  • the above-mentioned diagnostic kit comprising at least one of carnitine, tetradecenoylcarnitine, palmitoylcarnitine, and stearoylcarnitine.
  • diagnosis of NAFLD and NASH can be easily performed without imposing a burden on patients and medical staff.
  • NAFLD and NASH can be detected before the subject progresses to hepatocellular carcinoma.
  • Advantageous Effects of Invention According to the present invention, it is possible to easily determine the degree of progression of liver fibrosis without placing a burden on patients and medical staff.
  • FIG. 4 is a violin diagram showing serum acylcarnitine levels and free carnitine levels in NAFLD patients with hepatocellular carcinoma and NAFLD patients without hepatocellular carcinoma. It is a graph which shows ROC curve in each of serum acyl carnitine, alpha-fetoprotein (AFP), and des-gamma-carboxyprothrombin (DCP). It is a graph which shows the serum acylcarnitine density
  • Figure 4 is a graph showing serum acylcarnitine concentrations in NAFLD patients with various degrees of inflammation, balloon-like hypertrophy, and fatty liver.
  • FIG. 3 is a plot showing a correlation between the total amount of acylcarnitine measured using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS) and the total amount of acylcarnitine measured using an enzyme cycling method.
  • LC-MS / MS liquid chromatograph-
  • the biological sample that can be analyzed in the present invention examples include a solid tissue and a body fluid mainly derived from a living body (organism), and a body fluid is preferably used.
  • the biological sample that can be analyzed in the present invention is more preferably a body fluid such as blood, serum, plasma, urine, saliva, sputum, tear fluid, otorrhea, or prostate fluid, and still more preferably blood, serum or plasma. And more preferably blood, serum or plasma of a subject possibly having NAFLD and / or NASH.
  • the living body or subject includes a human or an animal (eg, mouse, guinea pig, rat, monkey, dog, cat, hamster, horse, cow, and pig), and is preferably a human.
  • 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 ⁇
  • R 1 represents a saturated or unsaturated fatty acid residue having 2 to 30 carbon atoms, and a hydrogen atom bonded to the fatty acid residue may be substituted with an oxygen atom or a hydroxy group.
  • Acylcarnitine can be acetylcarnitine, propionylcarnitine, stearoylcarnitine, oleylcarnitine, or the like, depending on the carbon chain length of the fatty acid portion, the presence and number of unsaturated bonds, the number of unsaturated bonds, the replacement of hydrogen atoms bonded to carbon chains with oxygen atoms or hydroxy groups, and the like.
  • Linoleylcarnitine, malonylcarnitine, 3-hydroxycarnitine, etc. and they are generically referred to as acylcarnitine in this specification.
  • free carnitine to which no fatty acid is bound is not included in acylcarnitine. That is, the “total amount of acylcarnitine” indicates the total amount of acylcarnitine excluding the amount of free carnitine to which no fatty acid is bound.
  • saturated or unsaturated fatty acid residue examples include those shown in Table 1.
  • Acylcarnitine is also used as an evaluation item in screening for inborn errors of metabolism such as fatty acid metabolism abnormality.
  • Table 2 shows the main acylcarnitines used in clinical evaluation items such as screening for inborn errors of metabolism.
  • the method for detecting NAFLD or NASH or estimating risk according to the present invention includes measuring the total amount of acylcarnitine in a biological sample from a subject.
  • the total amount of acylcarnitine may be measured in a lump by measuring the amount of acylcarnitine present in the biological sample using an enzyme cycling method or the like, or may be individually determined using a liquid chromatograph-tandem mass spectrometer or the like. May be measured and the amounts thereof may be summed.
  • the total amount of acylcarnitine is preferably at least acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine (C4), carnitine isovalerate (C5).
  • Carnitine 3-hydroxy-isovalerate C5OH
  • glutarylcarnitine C5DC
  • hexanoylcarnitine C6
  • octanoylcarnitine C8
  • decanoylcarnitine C10)
  • dodecanoylcarnitine C12
  • tetradeca Noylcarnitine C14
  • tetradecenoylcarnitine C14: 1
  • palmitoylcarnitine C16
  • 3-hydroxy-hexadecanoylcarnitine C16OH
  • stearoylcarnitine C18
  • octadecenoylka Nichin C18: 1
  • 3-hydroxy - octadecenyl octanoyl carnitine containing an amount of 17 kinds of acylcarnitine (C18 1 OH).
  • acylcarnitines are acylcarnitines used as evaluation items for inborn errors of metabolism, and are particularly susceptible to diseases when fatty acid metabolism and organic acid metabolism are caused by a disease, and have a disease.
  • Acylcarnitine is likely to cause a quantitative difference between a subject and a healthy person.
  • a diagnostic method using a tandem mass spectrometer is essential for measuring the amount of acylcarnitine.
  • the “total amount of acylcarnitine” refers to the total amount of acylcarnitine, that is, the total amount of all types of acylcarnitines present in a biological sample. The total amount of a particular acylcarnitine.
  • the total amount of acylcarnitine can be measured, for example, by a capillary electrophoresis-mass spectrometer, a liquid chromatograph-tandem mass spectrometer (LC-MS / MS), an enzyme cycling method, or the like. These methods can be used in combination.
  • the enzyme cycling method the measurement can be performed by a general-purpose automatic analyzer, for example, LABOSPECT 008 (manufactured by Hitachi High-Technologies Corporation), so that the complicated operation can be omitted and the measurement can be performed quickly at low cost. is there.
  • a 2-fraction test ie, measuring the total amount of carnitine, which is the total amount of free carnitine and the total amount of acyl carnitine, and subtracting the amount of free carnitine, ie, the amount of carnitine to which no fatty acid is bound, from the total amount of carnitine By doing so, the total amount of acylcarnitine can also be measured.
  • the two-fraction test can be performed using the enzyme cycling method.
  • liquid chromatograph-tandem mass spectrometer or “LC-MS / MS” refers to an ion generated by ionizing a component to be analyzed separated by liquid chromatography (LC) through a dedicated interface.
  • LC liquid chromatography
  • MS mass spectrometer
  • the term “enzyme cycling method” refers to a technique for measuring the concentration of a trace substance by utilizing the action of an enzyme that promotes a specific chemical reaction.
  • Acyl carnitine esterase and carnitine dehydrogenase can be used as enzymes for the measurement of acyl carnitine by the enzyme cycling method.More specifically, carnitine dehydrogenase can be used for measuring the amount of free carnitine, and the total amount of carnitine can be measured. Acylcarnitine esterase and carnitine dehydrogenase can be used for the measurement. Then, ⁇ -thionicotinamide adenine dinucleotide oxidized form (Thio-NAD + ) and ⁇ -nicotinamide adenine dinucleotide reduced form (NADH) can be used as coenzymes.
  • Thio-NAD + ⁇ -thionicotinamide adenine dinucleotide oxidized form
  • NADH ⁇ -nicotinamide adenine dinucleotide reduced form
  • the method for detecting NAFLD or NASH or predicting a risk according to the present invention includes comparing the total amount of measured acylcarnitine with a reference value.
  • a threshold for determining a healthy person group
  • NAFLD or NASH is detected or the possibility of onset is high. Can be determined.
  • a range of numerical values can be used as a reference value.
  • a biological sample of a subject previously diagnosed as having NAFLD or NASH and a subject diagnosed as not having NAFLD or NASH may be used.
  • the range of the total amount of acylcarnitine is measured, and if the total amount of acylcarnitine in the biological sample of the subject falls within the range of the total amount of acylcarnitine in the biological sample of a healthy subject, the subject is suffering from NAFLD or NASH. If the subject is likely to be in the range of the total amount of acylcarnitine in the biological sample of the subject suffering from NAFLD or NASH, then the subject is likely to have NAFLD or NASH.
  • the judgment threshold value (reference value) is expected to change depending on various conditions, for example, the underlying disease, gender, age, etc. However, those skilled in the art can appropriately select an appropriate population corresponding to the subject. By performing statistical processing on data obtained from the population, a normal value range or a threshold for determination can be determined.
  • the total amount of acylcarnitine may be compared with a reference value, and the total amount of the 17 types of acylcarnitines considered to be equivalent to the total amount of acylcarnitine may be compared with the reference value.
  • the total amount obtained by adding any one or more acylcarnitines to the 17 types of acylcarnitines may be compared with a reference value. You may compare.
  • the method for detecting NAFLD or NASH or predicting risk according to the present invention can also monitor the progress of NAFLD or NASH in a subject.
  • the target total amount of acylcarnitine at a specific time point is used as a determination threshold (reference value).
  • the subject's total amount of acylcarnitine is measured again, and if the total amount of acylcarnitine is higher than the previous measurement, NAFLD or NASH progresses. Can be determined to be.
  • the total amount of acylcarnitine is smaller than the previously measured value, it can be determined that NAFLD or NASH has not progressed.
  • NAFLD includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), cirrhosis with advanced NASH, and hepatocellular carcinoma with advanced NASH.
  • NAFLD non-alcoholic fatty liver
  • NASH non-alcoholic steatohepatitis
  • cirrhosis with advanced NASH hepatocellular carcinoma with advanced NASH.
  • the terms “NAFLD” and “NASH” can include hepatocellular carcinoma with advanced NASH, but preferably do not. That is, NAFLD is preferably non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and / or cirrhosis with advanced NASH.
  • the diagnostic agent kit for detecting NAFLD or NASH of the present invention can include a reagent for measuring the total amount of acylcarnitine.
  • the reagent may be a reagent for measuring the amount of individual acylcarnitines and summing up the amounts, or a reagent for measuring the amount of acylcarnitine present in the biological sample at once. There may be.
  • the reagent for measuring the total amount of acylcarnitine is preferably a combination of a reagent for measuring the amount of free carnitine and a reagent for measuring the total amount of carnitine.
  • the combination of the reagent for measuring the amount of free carnitine and the reagent for measuring the total amount of carnitine is preferably a reagent for using an enzyme cycling method.
  • the diagnostic reagent kit for detecting NAFLD or NASH of the present invention comprises, as an internal standard solution of a mass spectrometer, acetylcarnitine, propionylcarnitine, butyrylcarnitine, carnitine isovalerate, carnitine 3-hydroxy-isovalerate, gluta
  • An acylcarnitine composition comprising at least one of rilcarnitine, octanoylcarnitine, decanoylcarnitine, dodecanoylcarnitine, tetradecanoylcarnitine, tetradecenoylcarnitine, palmitoylcarnitine, and stearoylcarnitine can be included.
  • the acylcarnitine contained in the acylcarnitine composition is preferably isotopically labeled.
  • the isotope label include a label with a radioisotope and a label with an element such as carbon, nitrogen, oxygen, and deuterium, and a label with deuterium is preferable.
  • the solvent for dissolving the acylcarnitine include an organic solvent. Among the organic solvents, organic solvents having 1 to 5 carbon atoms are preferable, alcohols having 1 to 5 carbon atoms and acetonitrile are more preferable, and methanol, ethanol and isopropanol are further preferable.
  • the diagnostic reagent kit of the present invention can also include other test reagents, sample diluents, instructions for use, and the like.
  • Other test reagents include test reagents for amino acids and succinylacetone that are to be measured at the same time, and specifically include these internal standard solutions.
  • liver fibrosis refers to a massive increase in connective tissue containing fibrous components such as collagen fibers and / or extracellular matrix in the liver due to chronic inflammation of the liver and hardening of the tissue.
  • the method for determining the degree of progression of liver fibrosis in 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 suffering from NAFLD or NASH, Alternatively, in a subject already suffering from NAFLD or NASH, the degree of progression of liver fibrosis caused by NAFLD or NASH can be determined.
  • Acylcarnitine, the total amount of acylcarnitine, and the method for measuring the total amount of acylcarnitine are as described above.
  • the method for determining the degree of progression of hepatic fibrosis in the subject of the present invention includes comparing the total amount of measured acylcarnitine with a reference value.
  • a range of numerical values can be used as a reference value.
  • the classification of the liver fibrosis stage is, for example, F0: no fibrosis, F1: fibrosis in the central lobule, F2: fibrosis in the central lobule + portal vein area, F3: fibrous cross-link formation, F4: liver cirrhosis, HCC A: The hepatocellular carcinoma can be distinguished. According to the method for determining the degree of progression of liver fibrosis in a subject of the present invention, it is possible to determine the stage of the subject liver in F0 to F4. However, the classification is not limited to F0 to F4, and a different classification may be used.
  • the judgment threshold value (reference value) is expected to change depending on various conditions, for example, the underlying disease, gender, age, etc. However, those skilled in the art can appropriately select an appropriate population corresponding to the subject. By performing statistical processing on data obtained from the population, a normal value range or a threshold for determination can be determined.
  • the method of determining the degree of liver fibrosis in a subject can monitor the degree of liver fibrosis in a subject.
  • the target total amount of acylcarnitine at a specific time point is used as a determination threshold (reference value).
  • the subject's total amount of acylcarnitine is measured again. If the total amount of acylcarnitine is higher than the previous measurement, liver fibrosis progresses Can be determined to be. Conversely, if the total amount of acylcarnitine is equal to or less than the previous measurement value, it can be determined that liver fibrosis has not progressed.
  • LC-MS / MS method liquid chromatography-tandem mass spectrometry
  • NeoSMAAT registered trademark
  • NeoSMAAT registered trademark
  • C0 carnitine
  • C2 acetylcarnitine
  • C3 propionylcarnitine
  • C4 butyrylcarnitine
  • C5 carnitine isovalerate
  • Carnitine hydroxy-isovalerate C5OH
  • glutarylcarnitine C5DH
  • octanoylcarnitine C8
  • decanoylcarnitine C10
  • dodecanoylcarnitine C12
  • tetradecanoylcarnitine C14
  • tetradecenoyl Carnitine C14: 1
  • palmitoylcarnitine C16
  • stearoylcarnitine C18
  • NeoSMAAT registered trademark
  • 10 ⁇ L of an internal standard stock solution attached to NeoSMAAT was added to 10 ⁇ L of a human serum specimen.
  • 200 ⁇ L of ethanol was further 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.
  • the mixture was concentrated to dryness under a stream of nitrogen (40 ° C.).
  • 100 ⁇ L of formic acid / acetonitrile (0.05: 10) was added and stirred with a vortex mixer for 10 seconds.
  • 100 ⁇ L of acetonitrile was added, and the mixture was stirred with a vortex mixer for 10 seconds.
  • LC-MS / MS The conditions of LC-MS / MS are as follows.
  • the peak area value, the peak area ratio, the regression value, and the measured value were calculated using Analyst ver. 1.4.2 (manufactured by SCIEX), which is software for LC-MS / MS.
  • C16OH peak area value / deuterium-substituted C16 peak area value was calculated, and the concentration value calculated from the C16OH peak area value and the internal standard C16 peak area value was divided by the conversion coefficient to obtain a conversion concentration value.
  • the conversion concentration value was calculated.
  • acetyl carnitine (C2), propionyl carnitine (C3), butyryl carnitine (C4), carnitine isovalerate (C5), carnitine 3-hydroxy-isovalerate (C5OH), glutaryl carnitine (C5DC), hexa Noylcarnitine (C6), octanoylcarnitine (C8), decanoylcarnitine (C10), dodecanoylcarnitine (C12), tetradecanoylcarnitine (C14), tetradecenoylcarnitine (C14: 1), palmitoylcarnitine (C16) ), 3-hydroxy-hexadecanoylcarnitine (C16OH), stearoylcarnitine (C18), octadecenoylcarnitine (C18: 1), and 3-hydroxy-octadecenoylcarnitine (C18: 1OH).
  • Example 2 Verification of usefulness of total amount of acylcarnitine compared to other biomarkers Using the measurement results of total amount of acylcarnitine in Example 1, the usefulness of total amount of acylcarnitine in detecting NASH-related hepatocellular carcinoma was evaluated. Compared to biomarkers. As a control, tumor markers ⁇ -fetoprotein (AFP) and des- ⁇ -carboxyprothrombin (DCP) were used.
  • AFP ⁇ -fetoprotein
  • DCP des- ⁇ -carboxyprothrombin
  • Statistical analysis was performed using Wilcoxon rank sum test, Fisher's exact test, and Jonckheree-Terpstra test to test differences between groups corresponding to changes in continuity and classification. Serum acylcarnitine levels and the predictive performance of other tumor markers, ⁇ -fetoprotein (AFP) and des- ⁇ -carboxyprothrombin (DCP), were evaluated by the area under the ROC curve (AUROC). The results are shown in FIG.
  • AUROC of AFP was 0.90
  • AUROC of total acylcarnitine was 0.72
  • AUROC of DCP was 0.62.
  • AUROC of the total amount of acylcarnitine showed a higher value than DCP used as a tumor marker. Therefore, it was found that the total amount of acylcarnitine can be used as a biomarker in detecting NASH-related hepatocellular carcinoma.
  • Example 3 Comparison of total amount of acylcarnitine in NAFLD patients with various degrees of fibrosis progression The serum sample measured in Example 1 was further subdivided according to the degree of fibrosis progression of the patient as follows. The total amount of acylcarnitine and the amount of free carnitine contained in serum samples categorized according to the degree of progress were compared. The results are shown in FIG.
  • the total amount of acylcarnitine tended to increase as fibrosis progressed to F0, F1-2, F3-4, and HCC.
  • the p value between the F0 group and the F1-2 group was 0.041
  • the p value between the F0 group and the F3-4 group was 0.0002. there were.
  • free carnitine no quantitative change could be confirmed even if fibrosis progressed. Therefore, it was found that the degree of progression of fibrosis can be determined by measuring the total amount of acylcarnitine.
  • Example 1 Comparison of total amount of acylcarnitine based on diagnostic criteria other than fibrosis
  • the serum sample measured in Example 1 was used for NASF (NAFLD Activity) on page 82 of the Japanese Society of Gastroenterology NAFLD / NASH Clinical Practice Guidelines (2014 edition). Score) are classified according to the degree of progression of inflammation (Lobular inflammation), balloon-like hypertrophy (Ballooning), and fatty liver (Steatosis) of the liver of the patient described in (Score), and the total amount of acylcarnitine between patients at each degree of progression is calculated. Compared.
  • the classification criteria are as follows.
  • stage 0 no lesion
  • stage 1 2 or less lesions in 200-fold field of view
  • stage 2 2 to 4 lesions in 200-fold field of view
  • stage 3 4 or more lesions in 200-fold field of view
  • Balloon-like hypertrophy stage 0: no hypertrophy
  • stage 1 small number of balloon-like degenerated cells
  • stage 2 large number of balloon-like degenerated cells
  • Fatty liver Stage 1: 5-33%
  • Stage 3 66% or more.
  • FIG. 4 shows the results.
  • any of the degree of inflammation, balloon-like hypertrophy, and fatty liver no significant difference was found in the total amount of acylcarnitine between the patient groups at each stage.
  • the total amount of acylcarnitine by the enzyme cycling method was measured at the request of SRL.
  • SRL measures the total amount of acylcarnitine using a measurement kit (T-Carnitine reagent Kainos and F-Carnitine reagent Kainos) from Kainos.
  • the measurement kit of Kainos uses acylcarnitine esterase and carnitine dehydrogenase as enzymes, and ⁇ -thionicotinamide adenine dinucleotide oxidized form (Thio-NAD + ) and ⁇ -nicotinamide adenine dinucleotide as coenzymes. Two types of reduced form (NADH) are used.
  • FIG. 5 shows the results.
  • diagnosis of NAFLD and NASH can be easily performed without imposing a burden on patients and medical staff.
  • NAFLD and NASH can be detected before the subject progresses to hepatocellular carcinoma.
  • Advantageous Effects of Invention According to the present invention, it is possible to easily determine the degree of progression of liver fibrosis in a subject without burdening a patient and a medical worker.

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention aborde le problème de la fourniture d'une nouvelle méthode de diagnostic de la stéatose hépatique non alcoolique (NAFLD) ou SHNA, qui peut être réalisée de manière plus facile et ne constitue pas une charge pour les patients ou les prestataires de soins de santé. Il est possible de résoudre ce problème en mesurant la quantité totale d'acylcarnitines dans un échantillon biologiqe et en comparant le résultat avec une valeur seuil.
PCT/JP2018/032138 2018-08-30 2018-08-30 Méthode de prédiction de risque, ou de détection, d'une stéatose hépatique non alcoolique (nafld) ou shna, trousse de réactif de diagnostic pour la détection de nafld ou nash, méthode de détermination du taux de progression d'une fibrose hépatique chez un sujet, et trousse de réactif de diagnostic pour la détermination du taux de progression d'une fibrose hépatique chez un sujet Ceased WO2020044497A1 (fr)

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PCT/JP2018/032138 WO2020044497A1 (fr) 2018-08-30 2018-08-30 Méthode de prédiction de risque, ou de détection, d'une stéatose hépatique non alcoolique (nafld) ou shna, trousse de réactif de diagnostic pour la détection de nafld ou nash, méthode de détermination du taux de progression d'une fibrose hépatique chez un sujet, et trousse de réactif de diagnostic pour la détermination du taux de progression d'une fibrose hépatique chez un sujet
JP2019513860A JP6592637B1 (ja) 2018-08-30 2018-08-30 Ｎａｆｌｄ又はｎａｓｈの検出又はリスクの予測方法、ｎａｆｌｄ又はｎａｓｈを検出するための診断薬キット、対象における肝線維化の進行度の判定方法、及び対象における肝線維化の進行度を判定するための診断薬キット

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PCT/JP2018/032138 WO2020044497A1 (fr) 2018-08-30 2018-08-30 Méthode de prédiction de risque, ou de détection, d'une stéatose hépatique non alcoolique (nafld) ou shna, trousse de réactif de diagnostic pour la détection de nafld ou nash, méthode de détermination du taux de progression d'une fibrose hépatique chez un sujet, et trousse de réactif de diagnostic pour la détermination du taux de progression d'une fibrose hépatique chez un sujet

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PCT/JP2018/032138 Ceased WO2020044497A1 (fr) 2018-08-30 2018-08-30 Méthode de prédiction de risque, ou de détection, d'une stéatose hépatique non alcoolique (nafld) ou shna, trousse de réactif de diagnostic pour la détection de nafld ou nash, méthode de détermination du taux de progression d'une fibrose hépatique chez un sujet, et trousse de réactif de diagnostic pour la détermination du taux de progression d'une fibrose hépatique chez un sujet

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WO2007091623A1 (fr) * 2006-02-08 2007-08-16 Kurume University Composition pharmaceutique comprenant de la méglitinide pour prévenir une fibrose hépatique
JP2010500566A (ja) * 2006-08-08 2010-01-07 テシス バイオサイエンス， インコーポレイテッド 非アルコール性脂肪肝疾患（ｎａｆｌｄ）および非アルコール性脂肪性肝炎（ｎａｓｈ）のマーカーおよびその使用方法
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WO2007091623A1 (fr) * 2006-02-08 2007-08-16 Kurume University Composition pharmaceutique comprenant de la méglitinide pour prévenir une fibrose hépatique
JP2010500566A (ja) * 2006-08-08 2010-01-07 テシス バイオサイエンス， インコーポレイテッド 非アルコール性脂肪肝疾患（ｎａｆｌｄ）および非アルコール性脂肪性肝炎（ｎａｓｈ）のマーカーおよびその使用方法
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2023543763A (ja) * 2020-09-23 2023-10-18 ケンブリッジ エンタープライズ リミティッド バイオマーカー

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