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WO2020066162A1 - Procédé de détection de la stéatose hépatique non alcoolique, kit de détection de la stéatose hépatique non alcoolique, et biomarqueur destiné à être utilisé dans la détection de la stéatose hépatique non alcoolique - Google Patents

Procédé de détection de la stéatose hépatique non alcoolique, kit de détection de la stéatose hépatique non alcoolique, et biomarqueur destiné à être utilisé dans la détection de la stéatose hépatique non alcoolique Download PDF

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Publication number
WO2020066162A1
WO2020066162A1 PCT/JP2019/024293 JP2019024293W WO2020066162A1 WO 2020066162 A1 WO2020066162 A1 WO 2020066162A1 JP 2019024293 W JP2019024293 W JP 2019024293W WO 2020066162 A1 WO2020066162 A1 WO 2020066162A1
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WO
WIPO (PCT)
Prior art keywords
liver disease
fatty liver
alcoholic fatty
acid
detecting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2019/024293
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English (en)
Japanese (ja)
Inventor
宏隆 藤本
青木　豊
佐藤　孝明
木村　武志
勝紀 増田
勇司 平家
静香 宇山
鈴木　一彦
まり子 浅見
ケビン 浦山
邦好 林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
StLuke's International University
Shimadzu Corp
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StLuke's International University
Shimadzu Corp
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Priority to JP2020547977A priority Critical patent/JP6998023B2/ja
Priority to TW108121787A priority patent/TWI721462B/zh
Publication of WO2020066162A1 publication Critical patent/WO2020066162A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for detecting non-alcoholic fatty liver disease, a kit for detecting non-alcoholic fatty liver disease, and a biomarker for detecting non-alcoholic fatty liver disease.
  • a method for detecting a non-alcoholic fatty liver disease comprises obtaining a sample derived from a body fluid of a mammal, and obtaining 2-aminoadipic acid, 2-oxocaproic acid, Detecting at least one molecule selected from the group consisting of 2-oxoglutarate, alanine, glutamic acid, tyrosine, lactic acid, uric acid, valine, pyruvate, phenylalanine, and proline.
  • the mammal obtained based on a magnitude of a detection signal corresponding to the molecule in the detection.
  • the concentration of the molecule in the sample is calculated based on the magnitude of the detection signal, and the calculated concentration is calculated. It is preferable to output information on whether or not the mammal has a non-alcoholic fatty liver disease obtained based on whether or not the concentration satisfies a condition based on a predetermined threshold.
  • the detection is performed by gas chromatography, liquid chromatography, mass spectrometry, gas chromatography / mass. It is preferably performed by at least one of analysis, liquid chromatography / mass spectrometry, nuclear magnetic resonance spectroscopy, and immunological detection.
  • the mammal is preferably a human.
  • FIG. 1 is a flowchart illustrating a flow of a detection method according to an embodiment.
  • the method for detecting non-alcoholic fatty liver disease in the following embodiment detects a predetermined molecule in a sample derived from a target individual, and determines whether or not this individual has NAFLD based on the detection. Is to obtain information about.
  • NAFLD non-alcoholic fatty liver disease
  • the NAFLD marker in the present embodiment is selected from the group consisting of 2-aminoadipic acid, 2-oxocaproic acid, 2-oxoglutarate, alanine, glutamic acid, tyrosine, lactic acid, uric acid, valine, pyruvate, phenylalanine, and proline. Is done.
  • at least one marker selected from this group is detected, and the individual is determined to be NAFLD based on the magnitude of a detection signal corresponding to the detected marker. It is possible to acquire information as to whether or not the patient suffers from.
  • the detection reagent can include an indicator that develops a color by a chemical reaction with the marker, and the concentration of the marker can be detected from the color tone of the sample solution to which the indicator has been added.
  • a kit for detecting non-alcoholic fatty liver disease comprising a consumable used for the above-described gas chromatography, liquid chromatography, mass spectrometry or nuclear magnetic resonance spectroscopy, or a detection kit comprising the above-mentioned detection reagent is provided.
  • the method of pretreatment before subjecting the sample to the detection device to detect the marker is not particularly limited.
  • an internal standard is added to the sample, and after the solvent extraction, centrifugal concentration, lyophilization, and derivatization can be appropriately performed.
  • the internal standard can be appropriately selected according to the m / z of the marker to be detected, the type of sample (serum, plasma, etc.), and the like. For example, when a region of m / z 500 or less is measured in a serum sample, 2-isopropylmalic acid or the like, which is a synthetic compound not contained in serum, can be appropriately used.
  • the method of derivatization is not particularly limited.
  • a methoxyamine solution can be added to a lyophilized sample to form an oxime, and then N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) can be added for silylation.
  • MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide
  • the pretreated sample is introduced into the detection device.
  • FIG. 1 is a flowchart showing the flow of the method for detecting non-alcoholic fatty liver disease of the present embodiment.
  • the detection method of the present embodiment is a method of in vitro detecting a marker from a sample derived from the blood of a subject in order to diagnose NAFLD.
  • the flowchart of FIG. 1 shows an example in which a marker is detected by GC / MS, the present invention is not limited to this.
  • step S1001 blood is collected by a medical worker or the like, and blood is obtained from the subject.
  • step S1003 starts.
  • step S1003 the examiner, the user of the detection device, and the like perform pretreatment on the blood to prepare a sample derived from the blood.
  • step S1005 starts.
  • step S1009 the marker concentration in the sample is calculated from the magnitude of the detection signal corresponding to the marker.
  • step S1011 starts.
  • step S1011 it is determined whether the calculated marker concentration satisfies a condition based on a predetermined threshold (marker threshold), and data on whether the subject has NAFLD is created based on the determination result. Is done.
  • step S1013 starts.
  • step S1013 information on whether the subject has NAFLD is output to a display device or the like based on the data created in step S1011.
  • step S1013 ends, the process ends.
  • the method for detecting non-alcoholic fatty liver disease of the present embodiment comprises obtaining a sample derived from human blood, and obtaining 2-aminoadipate, 2-oxocaproate, 2-oxoglutarate, Detecting at least one molecule selected from the group consisting of alanine, glutamic acid, tyrosine, lactic acid, uric acid, valine, pyruvate, phenylalanine, and proline.
  • alanine glutamic acid, tyrosine, lactic acid, uric acid, valine, pyruvate, phenylalanine, and proline.
  • the marker is detected by gas chromatography, liquid chromatography, mass spectrometry, gas chromatography / mass spectrometry, liquid chromatography / mass spectrometry, nuclear magnetic resonance spectroscopy or It can be performed by an immunological detection method. Thereby, each component contained in the sample can be separated, and screening can be performed more precisely.
  • the biomarker for detecting non-alcoholic fatty liver disease is 2-aminoadipate, 2-oxocaproate, 2-oxoglutarate, alanine, glutamic acid, tyrosine, lactic acid, uric acid, valine in a sample. , Pyruvate, phenylalanine, and proline.
  • these molecules are markers obtained by analysis based on large-scale cases, as described in Examples described later, accurate screening can be performed.
  • the serum samples were shaken at 37 ° C. for 30 minutes at 1200 rpm.
  • the sample after shaking was centrifuged at 16,000 ⁇ g and 25 ° C. for 5 minutes.
  • 150 ⁇ L of the supernatant after centrifugation was previously added to a tube to which 140 ⁇ L of water had been added, and the mixture was stirred using a vortex mixer.
  • the solution after stirring was centrifuged at 16,000 ⁇ g and 25 ° C. for 5 minutes.
  • 180 ⁇ L of the supernatant after centrifugation was collected in a new tube. Concentration was performed by a centrifugal evaporator at room temperature for 60 minutes.
  • the sample after concentration was left at ⁇ 80 ° C. for 30 minutes to freeze.
  • the diagnostic ability of NAFLD was evaluated for each metabolite by using an ROC (Receiver Operating Characteristic) curve and using the area (AUC) of the portion below the ROC curve.
  • ROC Receiveiver Operating Characteristic
  • AUC area of the portion below the ROC curve.
  • the study with multiple metabolites used the LASSO method.
  • evaluation was made using c-statistics.
  • the dependent variables were the NAFLD group / normal group, ROC analysis was performed for each metabolite, and AUC was examined.
  • the following table shows the AUC for each metabolite.
  • the diagnosis ability was examined using a single metabolite. However, the same examination was conducted using a plurality of metabolites. As a result of the examination by the LASSO method, 70 metabolites were selected, and in that case, the AUC was 0.870.
  • the 70 metabolites are as follows: Boric acid, phenol, 2-hydroxyisobutyric acid, glycolic acid, alanine, glycine, 2-hydroxybutyric acid, p-cresol, 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 2-hydroxyisovaleric acid, 2-aminobutyric acid, 3-hydroxyisovaleric acid, valine, urea, caprylic acid, glycerol, aceturic acid, phosphoric acid, isoleucine, proline, succinic acid, fumaric acid, serine, glutaric acid, 2-deoxytetronic acid, decanoic acid, aspartic acid, methionine , Pyroglutamic acid, hydroxyproline, glutamic acid, phenylalanine, lauric acid, asparagine, 2-aminoadipic acid, aconitic acid, glutamine, histidine, tyrosine, palmitoleic acid, uric acid, stearic acid,

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Un procédé de détection de la stéatose hépatique non alcoolique comprend les étapes suivantes consistant à : obtenir un échantillon dérivé d'un fluide corporel d'un mammifère; et détecter au moins une molécule choisie dans le groupe constitué par l'acide 2-aminoadipique, l'acide 2-oxocaproïque, l'acide 2-oxoglutarique, l'alanine, l'acide glutamique, la tyrosine, l'acide lactique, l'acide urique, la valine, l'acide pyruvique, la phénylalanine et la proline dans l'échantillon.
PCT/JP2019/024293 2018-09-26 2019-06-19 Procédé de détection de la stéatose hépatique non alcoolique, kit de détection de la stéatose hépatique non alcoolique, et biomarqueur destiné à être utilisé dans la détection de la stéatose hépatique non alcoolique Ceased WO2020066162A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2020547977A JP6998023B2 (ja) 2018-09-26 2019-06-19 非アルコール性脂肪肝疾患の検出方法、非アルコール性脂肪肝疾患検出用キットおよび非アルコール性脂肪肝疾患検出用バイオマーカー
TW108121787A TWI721462B (zh) 2018-09-26 2019-06-21 非酒精性脂肪肝疾病的檢測方法、非酒精性脂肪肝疾病檢測用試劑盒和非酒精性脂肪肝疾病檢測用生物標記

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JP2018180886 2018-09-26
JP2018-180886 2018-09-26

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TW (1) TWI721462B (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113176372A (zh) * 2021-05-20 2021-07-27 北京化工大学 一种检测发酵液中己二酸含量的气相色谱方法
WO2024237258A1 (fr) 2023-05-17 2024-11-21 株式会社島津製作所 Procédé d'identification d'une stéatose hépatique non alcoolique, et biomarqueur
WO2024237259A1 (fr) * 2023-05-17 2024-11-21 株式会社島津製作所 Procédé d'évaluation du risque d'apparition d'une stéatose hépatique non alcoolique, et biomarqueurs

Citations (5)

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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
JP2011503547A (ja) * 2007-11-02 2011-01-27 メタボロン、インコーポレイテッド 脂肪肝疾患用のバイオマーカー及びその使用方法
WO2013002381A1 (fr) * 2011-06-30 2013-01-03 味の素株式会社 Procédé d'évaluation de maladie du foie gras, dispositif d'évaluation de maladie du foie gras, procédé d'évaluation de maladie du foie gras, programme d'évaluation de maladie du foie gras, système d'évaluation de maladie du foie gras, dispositif terminal de communication d'informations et procédé de recherche de substance utilisée pour empêcher ou soigner une maladie du foie gras
JP2016065873A (ja) * 2010-06-10 2016-04-28 メタノミクス ヘルス ゲーエムベーハー 肝疾患を診断する方法
JP2018502286A (ja) * 2014-11-19 2018-01-25 メタボロン，インコーポレイテッド 脂肪肝疾患のバイオマーカーおよびその使用方法

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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
JP2011503547A (ja) * 2007-11-02 2011-01-27 メタボロン、インコーポレイテッド 脂肪肝疾患用のバイオマーカー及びその使用方法
JP2016065873A (ja) * 2010-06-10 2016-04-28 メタノミクス ヘルス ゲーエムベーハー 肝疾患を診断する方法
WO2013002381A1 (fr) * 2011-06-30 2013-01-03 味の素株式会社 Procédé d'évaluation de maladie du foie gras, dispositif d'évaluation de maladie du foie gras, procédé d'évaluation de maladie du foie gras, programme d'évaluation de maladie du foie gras, système d'évaluation de maladie du foie gras, dispositif terminal de communication d'informations et procédé de recherche de substance utilisée pour empêcher ou soigner une maladie du foie gras
JP2018502286A (ja) * 2014-11-19 2018-01-25 メタボロン，インコーポレイテッド 脂肪肝疾患のバイオマーカーおよびその使用方法

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LAI, YI-SYUAN ET AL.: "Mass-spectrometry-based serum metabolomics of a C57BL/6J mouse model of high-fat-diet-induced non-alcoholic fatty liver disease development", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 63, no. 35, 9 September 2015 (2015-09-09), pages 7873 - 7884, XP055664718, DOI: 10.1021/acs.jafc.5b02830 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113176372A (zh) * 2021-05-20 2021-07-27 北京化工大学 一种检测发酵液中己二酸含量的气相色谱方法
WO2024237258A1 (fr) 2023-05-17 2024-11-21 株式会社島津製作所 Procédé d'identification d'une stéatose hépatique non alcoolique, et biomarqueur
WO2024237259A1 (fr) * 2023-05-17 2024-11-21 株式会社島津製作所 Procédé d'évaluation du risque d'apparition d'une stéatose hépatique non alcoolique, et biomarqueurs

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JPWO2020066162A1 (ja) 2021-08-30
TWI721462B (zh) 2021-03-11
TW202024636A (zh) 2020-07-01
JP6998023B2 (ja) 2022-02-10

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