WO2017003166A1 - Composition for early diagnosis of diabetes using metabolome analysis - Google Patents

Composition for early diagnosis of diabetes using metabolome analysis Download PDF

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WO2017003166A1
WO2017003166A1 PCT/KR2016/006937 KR2016006937W WO2017003166A1 WO 2017003166 A1 WO2017003166 A1 WO 2017003166A1 KR 2016006937 W KR2016006937 W KR 2016006937W WO 2017003166 A1 WO2017003166 A1 WO 2017003166A1
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diabetes
concentration
group
early diagnosis
early
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French (fr)
Korean (ko)
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박영자
메드리안노칼앤젤로
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고려대학교 산학협력단
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/62Detectors specially adapted therefor
    • G01N30/72Mass spectrometers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/62Detectors specially adapted therefor
    • G01N30/72Mass spectrometers
    • G01N30/7233Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806Determination of free amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848Methods of protein analysis involving mass spectrometry
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/04Endocrine or metabolic disorders
    • G01N2800/042Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention relates to a composition for diagnosing diabetes early, based on changes in metabolites in the blood.
  • the national R & D project supporting the present invention is a disease overcoming technology development project of the Ministry of Health and Welfare, research project number HI14C2686, metabolic research-based metabolic syndrome, new biomarker discovery and clinical significance securing project, supported by Korea University Industry-Academic Cooperation Group. It was.
  • type 2 diabetes occurs when the regulation of insulin secretion is smooth due to a lack of exercise, obesity, or stress, but insulin fails to function and blood glucose control fails.
  • the prevalence of diabetes in people aged 30 and over reaches 9.1%, and the prevalence increases rapidly after age 40, reaching 20% in their 50s. The rapid increase in prevalence is presumably due to changes in environmental factors such as improved nutrition and lack of exercise.
  • insulin-independent type 2 diabetes accounts for 90-95% of all diabetic patients.
  • people in developing countries gradually reduce physical activity and increase obesity. This is increasing at a frightening rate.
  • the proportion of fat in Korean diets has increased from 7.2% in 1969 to 18.5% in 2007 over the last decade, and the mortality rate from diabetes has increased rapidly from 7.4% in 1988 to 22.9% in 2007.
  • the existing diagnosis of diabetes is only diagnosed with blood glucose level or glycated hemoglobin level, but since this is a clinical sign after the progress of diabetes is sufficiently progressed, it is difficult to use it as an early diagnosis tool. Cannot be secured.
  • the present invention has been made to solve the above problems, an object of the present invention is to observe the changes in the metabolites in the blood of patients suspected of diabetes, based on their concentration changes, diabetic early diagnosis composition To provide.
  • the present invention provides a composition / kit for early diagnosis of diabetes, comprising a detection agent for a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and combinations thereof. .
  • the diabetes may be early diabetes or type 2 diabetes.
  • the concentration of the metabolite selected from the group consisting of proline and cholesterol when the concentration of the metabolite selected from the group consisting of proline and cholesterol is increased compared to the normal control, it may indicate an increased risk of developing diabetes.
  • the concentration of the metabolite selected from the group consisting of leucine, lysine, and phenylalanine may be reduced compared to the normal control, it may indicate an increased risk of developing diabetes.
  • the present invention comprises the steps of measuring the concentration of a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof from a biological sample isolated from the subject; And it provides a method for providing information for the early diagnosis of diabetes, comprising the step of comparing the measured concentration of the metabolite with a normal control.
  • a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof from a biological sample isolated from the subject.
  • the diabetes may be type 2 diabetes or early diabetes.
  • the biological sample may be blood, serum, or plasma.
  • the step of measuring the concentration of the metabolite may be measured by chromatography / mass spectrometry.
  • the present invention also provides a method for early diagnosis of diabetes, comprising administering the composition to a subject.
  • the present invention also provides the use of the composition for the early diagnosis of diabetes.
  • the present invention provides a biomarker for diagnosing early diabetes based on the change in the concentration of metabolites in the blood using metabolomics, and these can be applied as a leading material for the development of diabetes therapeutics.
  • 1 is a result of comparing the change in the concentration of leucine in the blood of the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes using Q-TOF MS and QQQ MS.
  • 3 is a result of comparing the change in the concentration of valine in the blood of the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes using Q-TOF MS and QQQ MS.
  • NDM non-diabetic group
  • PDM pre-diabetic group
  • DM type 2 diabetic group
  • NDM non-diabetic group
  • PDM pre-diabetic group
  • DM type 2 diabetic group
  • NDM non-diabetic group
  • PDM pre-diabetic group
  • DM type 2 diabetic group
  • FIG. 8 shows the results of comparing lysine concentration changes in blood of non-diabetic (NDM), pre-diabetic (PDM), and type 2 diabetic (DM) groups using Q-TOF MS.
  • NDM non-diabetic group
  • PDM pre-diabetic group
  • DM type 2 diabetic group
  • the present inventors collected blood before, during, and after the onset of diabetes by liquid chromatography. Through graphimetric / mass spectrometry, we observed changes in metabolite profiles, especially amino acids that are easily observed in the body, and confirmed their concentration changes.Leucine, Lysine, and Proline Obvious changes in concentrations of Phenylalanine, and Cholesterol were observed and used as biomarkers for early diagnosis of diabetes.
  • the present invention provides a composition / kit for early diagnosis of diabetes, comprising a detection agent for a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and combinations thereof, and more specifically, for early diagnosis of diabetes.
  • a detection agent for a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and combinations thereof, and more specifically, for early diagnosis of diabetes.
  • plasma biomarkers for leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof are provided.
  • the term “detection agent” means an agent for quantitatively detecting leucine, lysine, proline, phenylalanine, or cholesterol from a biological sample isolated from a diabetic patient, and the agent is not particularly limited.
  • the component to be detected is an amino acid, it may be a primer, a probe, an aptamer, or an antibody capable of complementary binding, and for other components, it may be a reagent or a chemical that can quantify them.
  • the term “metabolite” refers to a metabolite obtained from a sample of biological origin, and preferably, a sample of biological origin from which the metabolite can be obtained is whole blood, more preferably plasma. .
  • whole blood can be pretreated to detect the metabolite.
  • whole blood can be pretreated to detect the metabolite.
  • it may include filtration, distillation, extraction, separation, concentration, inactivation of interference components, addition of reagents, and the like.
  • the metabolites may include substances produced by metabolic and metabolic processes or substances generated by chemical metabolism by biological enzymes and molecules.
  • the "diabetes" disease to be diagnosed with the composition of the present invention may preferably be early diabetes or type 2 diabetes.
  • the term "early diabetes” includes a condition in which blood sugar is slightly higher than normal but needs additional information before it is confirmed as diabetes. Most people have an early diabetes course before they are confirmed with type 2 diabetes. The rise in blood sugar levels in early diabetes is due to insulin resistance problems and does not automatically progress to diabetes, but is at risk of developing diabetes. Early determination of the progression from early diabetes to diabetes is very important from the perspective of preventive treatment. On the other hand, early diabetes may be a risk factor for the development of heart disease, and people with early diabetes, like those with type 2 diabetes, may be overweight, have high blood pressure, and have abnormal cholesterol concentrations.
  • type 2 diabetes refers to diabetes that occurs when insulin is normally secreted but fails to function properly, and is also referred to as "gender diabetes or insulin-independent diabetes".
  • Type 2 diabetes occurs when cells do not respond effectively to the insulin produced by the pancreas. This condition is called insulin resistance. Insulin-resistant patients initially produce more insulin to maintain normal blood sugar, which in turn causes the pancreas to lose its insulin requirements, resulting in elevated blood sugar. That is, in the pre-diabetic state (early stage), normal glucose tolerance is maintained (normal glucose tolerance), but after a certain time, the pancreatic beta cells to compensate for insulin resistance begins to fail, hyperglycemia (hyperglycemia), which results in type 2 diabetes. Therefore, there is a problem in that early diabetes, which can progress to type 2 diabetes, cannot be diagnosed early by the existing diabetes diagnosis method based on blood sugar, such as a fasting blood sugar test.
  • diagnosis refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition (eg, identifying early diabetes), Determining the prognosis of a subject with a particular disease or condition, or therametrics (eg, monitoring the condition of the subject to provide information about treatment efficacy).
  • the present invention comprises the steps of measuring the concentration of a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof from a biological sample isolated from the subject; And it provides a method for providing information for the early diagnosis of diabetes, comprising the step of comparing the measured concentration of the metabolite with a normal control.
  • a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof from a biological sample isolated from the subject.
  • the biological sample may include solid tissue samples such as blood and other liquid samples of biological origin, biopsy samples, tissue cultures or cells derived therefrom. More specifically, it may be tissue, extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like, more preferably whole blood, and more preferably Preferably plasma, but is not limited thereto.
  • the biological sample may be pretreated prior to use in detection and may include, for example, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like, including animals, preferably mammals, Most preferably it can be obtained from humans.
  • the concentration of the metabolite can be measured by chromatography / mass spectrometry.
  • Chromatography used in the present invention is Gas Chromatography, Liquid-Solid Chromatography (LSC), Paper Chromatography (PC), Thin-Layer Chromatography (TLC) ), Gas-solid chromatography (GSC), liquid-liquid chromatography (Liquid-Liquid Chromatography, LLC), foam chromatography (Foam Chromatography, FC), emulsion chromatography (Emulsion Chromatography, EC), Gas-Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatograhy (GFC) or Gel Permeation Chromatography (GPC)
  • the chromatography used in the present invention may be liquid chromatography
  • the mass spectrometer used in the present invention may be Q-TOF MS.
  • Metabolites of the present invention are separated from each component in the liquid chromatography, using the information obtained through the Q-TOF MS can identify the components through the structural information (elemental composition) as well as accurate molecular weight information.
  • diabetes can be diagnosed early by comparing the measured concentration of the metabolite with the normal control. Specifically, when the concentration of the metabolite selected from the group consisting of proline and cholesterol is increased compared to the normal control group, it indicates an increased risk of developing diabetes, on the contrary, the leucine, lysine, and phenylalanine are selected from the group consisting of If the concentration of the metabolite is reduced compared to the normal control, it indicates an increased risk of developing diabetes.
  • the term "increase in metabolic concentration” means that the metabolite concentration in the sample of a patient group having a high likelihood of developing diabetes is measurably increased as compared with a normal group before the onset of diabetes, preferably 70 It means increased by more than%, more preferably increased by more than 30%.
  • the term "reduction of metabolic concentration” means that the metabolite concentration in the blood of a patient group having a high likelihood of developing diabetes is measurably reduced as compared with a normal group before the onset of diabetes, preferably 40 It means reduced by more than%, more preferably reduced by more than 20%.
  • Example 1 on the basis of the results of Example 1, it was intended to confirm the biomarkers and their tendency to determine whether the progression to diabetes in the pre-diabetic state early.
  • Example 2-1 50 ⁇ l of the serum sample prepared in Example 2-1 was treated with 200 ⁇ l of acetonitrile (1: 4 v / v), and then centrifuged at 14,000 ⁇ g for 5 minutes. The samples were then analyzed by ultrafast liquid chromatography (C18 Synchronis aQ 1.9 ⁇ m 1002.1 mm (Thermo Scientific, MA, USA)) coupled with a Q-TOF 6550 mass spectrometer (Agilent, CA, USA). As a mobile phase, H 2 O and acetonitrile with 0.1% of formic aicd added respectively were used. As a% concentration of distilled water with respect to a solvent, 1 min in 95% distilled water and 55% distilled water by adding a straight tool.

Abstract

The present invention relates to a composition for early diagnosis of diabetes using metabolome analysis and, more specifically, provides biomarkers which can diagnose the early stage of diabetes on the basis of concentration change of metabolome in blood by using metabolomics, and the biomarkers can be applied to leading materials for developing therapeutic agents of diabetes.

Description

대사체 분석을 이용한 당뇨병 조기 진단용 조성물 Early Diabetes Diagnosis Using Metabolite Analysis
본 발명은 혈액 내 대사체의 변화를 기반으로 하는, 당뇨병 조기 진단용 조성물에 관한 것이다. 본 발명을 지원한 국가연구개발사업은 보건복지부 질환극복기술개발사업으로서, 연구 과제번호 HI14C2686, 대사체 연구 기반 대사증후군 신규 바이오마커 발굴 및 임상적 유의성 확보사업이며, 주관기관인 고려대학교 산학협력단이 지원하였다. The present invention relates to a composition for diagnosing diabetes early, based on changes in metabolites in the blood. The national R & D project supporting the present invention is a disease overcoming technology development project of the Ministry of Health and Welfare, research project number HI14C2686, metabolic research-based metabolic syndrome, new biomarker discovery and clinical significance securing project, supported by Korea University Industry-Academic Cooperation Group. It was.
소아 당뇨병이라 불리는 제1형 당뇨병과 달리, 제2형 당뇨병은 운동부족, 비만 또는 스트레스 등에 의한 후천적 요인으로 인슐린의 분비 조절은 원활하나 인슐린이 제기능을 하지 못하여 혈당 조절이 실패하는 경우 발생한다. 2008년 질병 관리본부의 통계에 따르면, 30세 이상 국민에서 당뇨병의 유병률은 9.1%에 달하며, 40세가 넘으면 유병률이 급격히 증가하여 50대에는 20%에 이르는 것으로 나타났다. 이러한 급격한 유병률의 증가는 영양 상태의 개선과 운동의 부족 등 환경적 요인의 변화가 가장 주된 원인인 것으로 추정된다. 우리나라의 경우, 인슐린 비의존형의 제2형 당뇨 환자가 전체 당뇨 환자의 90-95%를 차지하고 있으며, 선진국뿐만 아니라 개발 도상국의 사람들도 점차 신체 활동은 줄고 비만은 늘어나면서, 제2형 당뇨병의 발생이 무서운 속도로 증가하고 있다. 최근 10년간 한국인의 식단에서 지방이 차지하는 비율이 1969년 7.2%에서 2007년 18.5%까지 증가하였으며, 당뇨병으로 인한 사망률은 빠르게 증가하여 1988년 7.4%에서 2007년 22.9%에 이르렀다.Unlike type 1 diabetes, which is called pediatric diabetes, type 2 diabetes occurs when the regulation of insulin secretion is smooth due to a lack of exercise, obesity, or stress, but insulin fails to function and blood glucose control fails. According to the statistics of the Centers for Disease Control and Prevention in 2008, the prevalence of diabetes in people aged 30 and over reaches 9.1%, and the prevalence increases rapidly after age 40, reaching 20% in their 50s. The rapid increase in prevalence is presumably due to changes in environmental factors such as improved nutrition and lack of exercise. In Korea, insulin-independent type 2 diabetes accounts for 90-95% of all diabetic patients. In addition to developed countries, people in developing countries gradually reduce physical activity and increase obesity. This is increasing at a frightening rate. The proportion of fat in Korean diets has increased from 7.2% in 1969 to 18.5% in 2007 over the last decade, and the mortality rate from diabetes has increased rapidly from 7.4% in 1988 to 22.9% in 2007.
기존의 당뇨병 진단에는 혈당 수치나 당화 혈색소의 수치로만 진단이 되지만, 이는 당뇨의 진행이 충분히 진행되고 나서의 임상학적 징후이므로 조기 진단의 도구로 쓰기는 힘들며, 하나의 혈액학적 수치로는 진단의 정확성을 담보할 수 없다.The existing diagnosis of diabetes is only diagnosed with blood glucose level or glycated hemoglobin level, but since this is a clinical sign after the progress of diabetes is sufficiently progressed, it is difficult to use it as an early diagnosis tool. Cannot be secured.
최근, 대사체 연구는 인간 혈장 대사체들이 인슐린 저항성, 제2형 당뇨병 및 당뇨병 전증으로 발생한 혈당 부하와 관련이 있음을 보여주고 있다 (한국 공개특허 10-2012-0134272). 하지만, 최근의 혈장 대사체 연구들은 당뇨병 진단 방법 개발하거나 치료제 효과를 높이는데 목표를 두고 있다. 특히, 초기 당뇨병 환자 및 건강한 사람 간의 염증 마커, 산화적 마커 및 동맥 경직 마커의 차이점 및 대사체 변화에 대한 포괄적인 이해는 아직까지 연구되어 있지 않다.Recently, metabolite studies have shown that human plasma metabolites are associated with glycemic load caused by insulin resistance, type 2 diabetes and prediabetes (Korean Patent Publication 10-2012-0134272). However, recent plasma metabolite studies are aimed at developing methods for diagnosing diabetes or enhancing the effectiveness of the treatment. In particular, a comprehensive understanding of the differences between inflammatory markers, oxidative markers and arterial stiffness markers and metabolic changes between early diabetics and healthy people has not been studied yet.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명의 목적은 당뇨병이 의심되는 환자들의 혈액 내 대사체들의 변화를 관찰하고, 이들의 농도 변화를 기반으로 하는, 당뇨병 조기 진단용 조성물을 제공하는데 있다.The present invention has been made to solve the above problems, an object of the present invention is to observe the changes in the metabolites in the blood of patients suspected of diabetes, based on their concentration changes, diabetic early diagnosis composition To provide.
또한, 본 발명의 목적은 당뇨병 진단를 위한 상기 조성물의 의약적 용도를 제공하는데 있다. It is also an object of the present invention to provide a medicinal use of the composition for diagnosing diabetes.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기 과제를 해결하기 위한 수단으로서, 본 발명은 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합으로 이루어지는 군으로부터 선택되는 대사체에 대한 검출 제제를 포함하는, 당뇨병 조기 진단용 조성물/키트를 제공한다.As a means for solving the above problems, the present invention provides a composition / kit for early diagnosis of diabetes, comprising a detection agent for a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and combinations thereof. .
본 발명의 일 구현예로서, 상기 당뇨병은 초기 당뇨병 또는 제2형 당뇨병일 수 있다.In one embodiment of the present invention, the diabetes may be early diabetes or type 2 diabetes.
본 발명의 또 다른 구현 예로서, 상기 프롤린 및 콜레스테롤로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 증가된 경우, 증가된 당뇨병 발병 위험도를 나타내는 것일 수 있다. As another embodiment of the present invention, when the concentration of the metabolite selected from the group consisting of proline and cholesterol is increased compared to the normal control, it may indicate an increased risk of developing diabetes.
본 발명의 또 다른 구현 예로서, 상기 루신, 라이신, 및 페닐알라닌으로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 감소된 경우, 증가된 당뇨병 발병 위험도를 나타내는 것일 수 있다. As another embodiment of the present invention, when the concentration of the metabolite selected from the group consisting of leucine, lysine, and phenylalanine may be reduced compared to the normal control, it may indicate an increased risk of developing diabetes.
또한, 본 발명은 피검체에서 분리된 생체 시료로부터 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합으로 구성된 군으로부터 선택되는 대사체의 농도를 측정하는 단계; 및 상기 측정된 대사체의 농도를 정상 대조군과 비교하는 단계를 포함하는, 당뇨병 조기 진단을 위한 정보제공 방법을 제공한다. In addition, the present invention comprises the steps of measuring the concentration of a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof from a biological sample isolated from the subject; And it provides a method for providing information for the early diagnosis of diabetes, comprising the step of comparing the measured concentration of the metabolite with a normal control.
본 발명의 일 구현예로서, 상기 당뇨병은 제2형 당뇨병이거나 초기 당뇨병 일 수 있다.In one embodiment of the present invention, the diabetes may be type 2 diabetes or early diabetes.
본 발명의 다른 구현예로서, 상기 생체 시료는 혈액, 혈청, 또는 혈장일 수 있다. In another embodiment of the present invention, the biological sample may be blood, serum, or plasma.
본 발명의 또 다른 구현예로서, 상기 대사체의 농도를 측정하는 단계는 크로마토그래피/질량 분석법을 통해 측정하는 것일 수 있다. In another embodiment of the present invention, the step of measuring the concentration of the metabolite may be measured by chromatography / mass spectrometry.
또한, 본 발명은 상기 조성물을 개체에 투여하는 단계를 포함하는, 당뇨병 조기 진단 방법을 제공한다. The present invention also provides a method for early diagnosis of diabetes, comprising administering the composition to a subject.
또한, 본 발명은 상기 조성물의 당뇨병 조기 진단 용도를 제공한다.The present invention also provides the use of the composition for the early diagnosis of diabetes.
본 발명은 대사체학을 이용하여 혈액 내 대사체들의 농도 변화를 기반으로 초기 당뇨병을 진단할 수 있는 바이오마커를 제공하며, 이들은 당뇨병 치료제 개발을 위한 선도물질로 적용할 수 있다.The present invention provides a biomarker for diagnosing early diabetes based on the change in the concentration of metabolites in the blood using metabolomics, and these can be applied as a leading material for the development of diabetes therapeutics.
도 1은 Q-TOF MS 및 QQQ MS를 이용하여 당뇨 발병 전의 정상군 (Pre-DM)과 당뇨 발병군 (DM)의 혈액 내 루신의 농도 변화를 비교한 결과이다. 1 is a result of comparing the change in the concentration of leucine in the blood of the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes using Q-TOF MS and QQQ MS.
도 2는 Q-TOF MS 및 QQQ MS를 이용하여 당뇨 발병 전의 정상군 (Pre-DM)과 당뇨 발병군 (DM)의 혈액 내 타이로신의 농도 변화를 비교한 결과이다. 2 is a result of comparing the concentration changes of tyrosine in blood of the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes using Q-TOF MS and QQQ MS.
도 3은 Q-TOF MS 및 QQQ MS를 이용하여 당뇨 발병 전의 정상군 (Pre-DM)과 당뇨 발병군 (DM)의 혈액 내 발린의 농도 변화를 비교한 결과이다.3 is a result of comparing the change in the concentration of valine in the blood of the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes using Q-TOF MS and QQQ MS.
도 4는 Q-TOF MS 및 QQQ MS를 이용하여 당뇨 발병 전의 정상군 (Pre-DM)과 당뇨 발병군 (DM)의 혈액 내 페닐알라닌의 농도 변화를 비교한 결과이다.4 is a result of comparing the change in the concentration of phenylalanine in the blood of the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes using Q-TOF MS and QQQ MS.
도 5는 Q-TOF MS를 이용하여, 당뇨 비발병군 (NDM), 전당뇨 발병군 (PDM), 및 제2형 당뇨 발병군 (DM)의 혈액 내 콜레스테롤의 농도 변화를 비교한 결과이다.  5 is a result of comparing the concentration of cholesterol in the blood of the non-diabetic group (NDM), pre-diabetic group (PDM), and type 2 diabetic group (DM) using Q-TOF MS.
도 6은 Q-TOF MS를 이용하여, 당뇨 비발병군 (NDM), 전당뇨 발병군 (PDM), 및 제2형 당뇨 발병군 (DM)의 혈액 내 프롤린의 농도 변화를 비교한 결과이다. 6 is a result of comparing the concentration of proline in the blood of the non-diabetic group (NDM), pre-diabetic group (PDM), and type 2 diabetic group (DM) using Q-TOF MS.
도 7은 Q-TOF MS를 이용하여, 당뇨 비발병군 (NDM), 전당뇨 발병군 (PDM), 및 제2형 당뇨 발병군 (DM)의 혈액 내 루신의 농도 변화를 비교한 결과이다. 7 is a result of comparing the concentration of leucine in the blood of non-diabetic group (NDM), pre-diabetic group (PDM), and type 2 diabetic group (DM) using Q-TOF MS.
도 8은 Q-TOF MS를 이용하여, 당뇨 비발병군 (NDM), 전당뇨 발병군 (PDM), 및 제2형 당뇨 발병군 (DM)의 혈액 내 라이신의 농도 변화를 비교한 결과이다. FIG. 8 shows the results of comparing lysine concentration changes in blood of non-diabetic (NDM), pre-diabetic (PDM), and type 2 diabetic (DM) groups using Q-TOF MS.
도 9는 Q-TOF MS를 이용하여, 당뇨 비발병군 (NDM), 전당뇨 발병군 (PDM), 및 제2형 당뇨 발병군 (DM)의 혈액 내 페닐알라닌의 농도 변화를 비교한 결과이다. 9 is a result of comparing the concentration change of phenylalanine in the blood of the non-diabetic group (NDM), pre-diabetic group (PDM), and type 2 diabetic group (DM) using Q-TOF MS.
이하, 본 발명을 상세히 설명하기로 한다. Hereinafter, the present invention will be described in detail.
당뇨병은 대사성 질환이므로 대사회로의 차이에서 나오는 대사체 농도의 변화가 조기 진단의 중요한 요소가 될 수 있을 것으로 판단하였으며, 이에, 본 발명자들은, 당뇨 발병 전, 초기, 및 후의 혈액을 채취하여 액체크로마토그래피/질량분석을 통해 대사체 프로파일, 특히 체내에서 관찰이 잘 되는 아미노산 등을 위주로 변화를 관찰함과 동시에 이들의 농도 변화를 함께 확인한 결과, 루신 (Leucine), 라이신 (Lysine), 프롤린 (Proline), 페닐알라닌 (Phenylalanine), 및 콜레스테롤 (Cholesterol)의 뚜렷한 농도 변화를 관찰하고, 이들을 당뇨병 조기 진단을 위한 바이오마커로 사용하였다.Since diabetes is a metabolic disease, it was determined that the change in metabolite concentration from the metabolic circuit could be an important factor for early diagnosis. Therefore, the present inventors collected blood before, during, and after the onset of diabetes by liquid chromatography. Through graphimetric / mass spectrometry, we observed changes in metabolite profiles, especially amino acids that are easily observed in the body, and confirmed their concentration changes.Leucine, Lysine, and Proline Obvious changes in concentrations of Phenylalanine, and Cholesterol were observed and used as biomarkers for early diagnosis of diabetes.
본 발명은 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합으로 이루어지는 군으로부터 선택되는 대사체에 대한 검출 제제를 포함하는, 당뇨병 조기 진단용 조성물/키트를 제공하며, 보다 구체적으로, 당뇨병 조기 진단을 위한 혈장 내 바이오마커로서, 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합을 제공한다.The present invention provides a composition / kit for early diagnosis of diabetes, comprising a detection agent for a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and combinations thereof, and more specifically, for early diagnosis of diabetes. As plasma biomarkers for leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof are provided.
본 발명에서 사용되는 용어, "검출 제제"는 당뇨병 환자로부터 분리된 생체 시료로부터 루신, 라이신, 프롤린, 페닐알라닌, 또는 콜레스테롤을 정량적으로 검출하기 위한 제제를 의미하며, 상기 제제는 특별히 제한되는 것은 아니나, 검출하고자 하는 성분이 아미노산의 경우, 상보적 결합이 가능한 프라이머, 프로브, 압타머, 또는 항체일 수 있으며, 이 외의 성분들에 대해서는 이들을 정량화할 수 있는 시약 또는 화학 물질일 수 있다.As used herein, the term “detection agent” means an agent for quantitatively detecting leucine, lysine, proline, phenylalanine, or cholesterol from a biological sample isolated from a diabetic patient, and the agent is not particularly limited. When the component to be detected is an amino acid, it may be a primer, a probe, an aptamer, or an antibody capable of complementary binding, and for other components, it may be a reagent or a chemical that can quantify them.
본 발명에서 사용되는 용어, “대사체”는 생체 기원의 시료로부터 수득한 대사물질을 말하며 바람직하게, 상기 대사체를 수득할 수 있는 생체 기원의 시료는 전혈이고, 더욱 바람직하게는 혈장일 수 있다. 한 구체예로서, 상기 대사체를 검출하기 위해 전혈을 전처리할 수 있다. 예를 들어, 여과, 증류, 추출, 분리, 농축, 방해 성분의 불활성화, 시약의 첨가 등을 포함할 수 있다. 또한, 상기 대사체는 대사 및 대사 과정에 의해 생산된 물질 또는 생물학적 효소 및 분자에 의한 화학적 대사작용으로 발생한 물질 등을 포함할 수 있다.As used herein, the term “metabolite” refers to a metabolite obtained from a sample of biological origin, and preferably, a sample of biological origin from which the metabolite can be obtained is whole blood, more preferably plasma. . In one embodiment, whole blood can be pretreated to detect the metabolite. For example, it may include filtration, distillation, extraction, separation, concentration, inactivation of interference components, addition of reagents, and the like. In addition, the metabolites may include substances produced by metabolic and metabolic processes or substances generated by chemical metabolism by biological enzymes and molecules.
본 발명의 조성물에 의한 진단 대상 질병인 "당뇨병"은 바람직하게 초기 당뇨병 또는 제2형 당뇨병일 수 있다.The "diabetes" disease to be diagnosed with the composition of the present invention may preferably be early diabetes or type 2 diabetes.
본 발명에서 사용되는 용어, “초기 당뇨병”은 혈당이 정상보다 다소 높지만 당뇨병으로 확진되기까지 추가적인 정보가 필요한 상태를 포함한다. 대부분의 사람은 제2형 당뇨병으로 확진되기 전에 초기 당뇨병 과정을 거친다. 초기 당뇨병에서 나타나는 혈당의 상승은 인슐린 저항성 문제로 인해 시작되며, 초기 당뇨병에 있다고 해서 자동적으로 당뇨병으로 진행하지는 않으나, 당뇨병으로 진행될 위험성이 있는 상태에 놓인다. 이러한 초기 당뇨병에서 당뇨병으로의 진행 여부를 조기에 판단하는 것은 예방적 치료의 관점에서 매우 중요하다. 한편, 초기 당뇨병은 심장 질환 발생의 위험 요인이 될 수 있고, 제2형 당뇨병 환자와 마찬가지로 초기 당뇨병의 사람들도 과체중인 경향이 있으며, 혈압이 높고 비정상적인 콜레스테롤 농도를 보일 수 있다. As used herein, the term "early diabetes" includes a condition in which blood sugar is slightly higher than normal but needs additional information before it is confirmed as diabetes. Most people have an early diabetes course before they are confirmed with type 2 diabetes. The rise in blood sugar levels in early diabetes is due to insulin resistance problems and does not automatically progress to diabetes, but is at risk of developing diabetes. Early determination of the progression from early diabetes to diabetes is very important from the perspective of preventive treatment. On the other hand, early diabetes may be a risk factor for the development of heart disease, and people with early diabetes, like those with type 2 diabetes, may be overweight, have high blood pressure, and have abnormal cholesterol concentrations.
본 발명에서 사용되는 용어, “제2형 당뇨병”은 인슐린은 정상적으로 분비되나 인슐린이 제 기능을 못하는 경우에 발병하는 당뇨병을 말하며, “성인형 당뇨병 또는 인슐린 비의존형 당뇨병”으로도 불린다. 제2형 당뇨병은 세포가 췌장에서 생성된 인슐린에 효과적으로 반응하지 않는 경우에 발생하는데 이러한 상태를 인슐린 저항성이라고 한다. 인슐린 저항성이 있는 환자들은 처음에 정상적인 혈당을 유지하기 위해 추가로 더 많은 인슐린을 생산하는데, 결국에는 인슐린 저항성에 의해 췌장이 인슐린 요구량을 감당할 수 없게 되어 혈당이 상승하게 된다. 즉, 전당뇨 상태 (초기 단계)에서는, 정상적인 혈당이 유지되나 (normal glucose tolerance), 일정 시간이 경과 후, 인슐린 저항성에 대한 췌장 베타 세포 (pancreatic beta cell)의 보상 작용이 실패하기 시작하면서, 고혈당증 (hyperglycemia)로 진행되고, 결과적으로, 제2형 당뇨 상태에 이르게 된다. 따라서, 공복혈당검사 (Fasting blood sugar test)와 같은, 혈당에 기반하는 기존의 당뇨병 진단 방법만으로는, 제2형 당뇨병으로 진행될 수 있는, 초기 당뇨병을 조기에 진단할 수 없다는 문제점이 있다.As used herein, the term "type 2 diabetes" refers to diabetes that occurs when insulin is normally secreted but fails to function properly, and is also referred to as "gender diabetes or insulin-independent diabetes". Type 2 diabetes occurs when cells do not respond effectively to the insulin produced by the pancreas. This condition is called insulin resistance. Insulin-resistant patients initially produce more insulin to maintain normal blood sugar, which in turn causes the pancreas to lose its insulin requirements, resulting in elevated blood sugar. That is, in the pre-diabetic state (early stage), normal glucose tolerance is maintained (normal glucose tolerance), but after a certain time, the pancreatic beta cells to compensate for insulin resistance begins to fail, hyperglycemia (hyperglycemia), which results in type 2 diabetes. Therefore, there is a problem in that early diabetes, which can progress to type 2 diabetes, cannot be diagnosed early by the existing diabetes diagnosis method based on blood sugar, such as a fasting blood sugar test.
본 명세서에서 용어 “진단”은 특정 질병 또는 질환에 대한 객체의 감수성 (susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것 (예컨대, 초기 당뇨병의 동정), 특정 질병 또는 질환에 걸린 한 객체의 예후 (prognosis)를 판정하는 것, 또는 테라메트릭스 (therametrics) (예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 포함한다.As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition (eg, identifying early diabetes), Determining the prognosis of a subject with a particular disease or condition, or therametrics (eg, monitoring the condition of the subject to provide information about treatment efficacy).
한편, 본 발명의 다른 양태로서, 본 발명은 피검체에서 분리된 생체 시료로부터 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합으로 구성된 군으로부터 선택되는 대사체의 농도를 측정하는 단계; 및 상기 측정된 대사체의 농도를 정상 대조군과 비교하는 단계를 포함하는, 당뇨병 조기 진단을 위한 정보제공 방법을 제공한다. On the other hand, as another aspect of the present invention, the present invention comprises the steps of measuring the concentration of a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol and combinations thereof from a biological sample isolated from the subject; And it provides a method for providing information for the early diagnosis of diabetes, comprising the step of comparing the measured concentration of the metabolite with a normal control.
본 발명에서, 상기 생체 시료는 혈액 및 생물학적 기원의 기타 액상 시료, 생검 표본, 조직 배양과 같은 고형 조직 시료 또는 이로부터 유래된 세포가 포함할 수 있다. 보다 구체적으로, 조직, 추출물, 세포 용해물, 전혈, 혈장, 혈청, 침, 안구액, 뇌척수액, 땀, 뇨, 젖, 복수액, 활액, 복막액 등일 수 있으며, 보다 바람직하게 전혈이고, 더욱 바람직하게는 혈장일 수 있으나, 이에 제한되는 것은 아니다. 상기 생체 시료는 검출에 사용하기 전에 전처리할 수 있으며, 예를 들어, 여과, 증류, 추출, 농축, 방해 성분의 불활성화, 시약의 첨가 등을 포함할 수 있고, 동물, 바람직하게는 포유동물, 가장 바람직하게는 인간으로부터 수득될 수 있다. In the present invention, the biological sample may include solid tissue samples such as blood and other liquid samples of biological origin, biopsy samples, tissue cultures or cells derived therefrom. More specifically, it may be tissue, extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like, more preferably whole blood, and more preferably Preferably plasma, but is not limited thereto. The biological sample may be pretreated prior to use in detection and may include, for example, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like, including animals, preferably mammals, Most preferably it can be obtained from humans.
본 발명에서, 상기 대사체의 농도는 크로마토그래피/질량 분석법에 의해 측정 될 수 있다. 본 발명에서 이용되는 크로마토그래피는 가스 크로마토그래피 (Gas Chromatography), 액체-고체 크로마토그래피 (Liquid-Solid Chromatography, LSC), 종이 크로마토그래피 (Paper Chromatography, PC), 박층 크로마토그래피 (Thin-Layer Chromatography, TLC), 기체-고체 크로마토그래피 (Gas-Solid Chromatography, GSC), 액체-액체 크로마토그래피 (Liquid-Liquid Chromatography, LLC), 포말 크로마토그래피 (Foam Chromatography, FC), 유화 크로마토그래피 (Emulsion Chromatography, EC), 기체-액체 크로마토그래피 (Gas-Liquid Chromatography, GLC), 이온 크로마토그래피 (Ion Chromatography, IC), 겔 여과 크로마토그래피 (Gel Filtration Chromatograhy, GFC) 또는 겔 투과 크로마토그래피 (Gel Permeation Chromatography, GPC)를 포함하나, 이에 제한되지 않고 당업계에서 통상적으로 사용되는 모든 정량용 크로마토그래피를 사용할 수 있다. 바람직하게는, 본 발명에서 이용되는 크로마토그래피는 액체 크로마토그래피일 수 있고, 본 발명에서 이용되는 질량분석기는 Q-TOF MS일 수 있다. In the present invention, the concentration of the metabolite can be measured by chromatography / mass spectrometry. Chromatography used in the present invention is Gas Chromatography, Liquid-Solid Chromatography (LSC), Paper Chromatography (PC), Thin-Layer Chromatography (TLC) ), Gas-solid chromatography (GSC), liquid-liquid chromatography (Liquid-Liquid Chromatography, LLC), foam chromatography (Foam Chromatography, FC), emulsion chromatography (Emulsion Chromatography, EC), Gas-Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatograhy (GFC) or Gel Permeation Chromatography (GPC) However, the present invention is not limited thereto, and any quantitative chromatography commonly used in the art may be used. Preferably, the chromatography used in the present invention may be liquid chromatography, and the mass spectrometer used in the present invention may be Q-TOF MS.
본 발명의 대사체는 액체 크로마토그래피에서 각 성분들이 분리되며, Q-TOF MS를 거쳐 얻어진 정보를 이용하여 정확한 분자량 정보뿐만 아니라 구조 정보 (elemental composition)를 통해 구성 성분을 확인할 수 있다.Metabolites of the present invention are separated from each component in the liquid chromatography, using the information obtained through the Q-TOF MS can identify the components through the structural information (elemental composition) as well as accurate molecular weight information.
본 발명에서, 상기 측정된 대사체의 농도를 정상 대조군과 비교함으로써, 당뇨병을 조기 진단할 수 있다. 구체적으로, 상기 프롤린 및 콜레스테롤로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 증가된 경우, 증가된 당뇨병 발병 위험도를 나타내며, 이와 반대로, 상기 루신, 라이신, 및 페닐알라닌으로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 감소된 경우, 증가된 당뇨병 발병 위험도를 나타낸다. In the present invention, diabetes can be diagnosed early by comparing the measured concentration of the metabolite with the normal control. Specifically, when the concentration of the metabolite selected from the group consisting of proline and cholesterol is increased compared to the normal control group, it indicates an increased risk of developing diabetes, on the contrary, the leucine, lysine, and phenylalanine are selected from the group consisting of If the concentration of the metabolite is reduced compared to the normal control, it indicates an increased risk of developing diabetes.
본 발명에서 사용되는 용어, "대사체 농도의 증가"는 당뇨 발병 전의 정상군에 비해 당뇨 발병 가능성이 높은 환자군의 시료 내 대사체 농도가 측정 가능할 정도로 유의하게 증가된 것을 의미하며, 바람직하게는 70% 이상 증가된 것을 의미하고, 보다 바람직하게는 30% 이상 증가된 것을 의미한다. As used herein, the term "increase in metabolic concentration" means that the metabolite concentration in the sample of a patient group having a high likelihood of developing diabetes is measurably increased as compared with a normal group before the onset of diabetes, preferably 70 It means increased by more than%, more preferably increased by more than 30%.
본 발명에서 사용되는 용어, "대사체 농도의 감소"는 당뇨 발병 전의 정상군에 비해 당뇨 발병 가능성이 높은 환자군의 혈액 내 대사체 농도가 측정 가능할 정도로 유의하게 감소된 것을 의미하며, 바람직하게는 40% 이상 감소된 것을 의미하고, 보다 바람직하게는 20% 이상 감소된 것을 의미한다. As used herein, the term "reduction of metabolic concentration" means that the metabolite concentration in the blood of a patient group having a high likelihood of developing diabetes is measurably reduced as compared with a normal group before the onset of diabetes, preferably 40 It means reduced by more than%, more preferably reduced by more than 20%.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예Example 1. LC/ LC / TOFTOF MS 등을 이용한 당뇨 발병  Diabetes outbreak using MS 전 후의Before 대사체Metabolite 변화 확인 Confirm change
본 실시예에서는, 당뇨 발병에 따른 대사체의 혈중 농도 변화를 관찰하여 당뇨와 혈중 대사체간 관련성을 확인하고자 하였다. 구체적으로, 당뇨 발병 전의 정상군 (Pre-DM)과 당뇨 발병군 (DM)으로부터 혈액을 채취한 뒤, 상기 혈액 내 존재하는 4개의 아미노산 (루신, 타이로신, 발린, 페닐알라닌)을 Q-TOF MS 및 QQQ MS를 통해 정량화한 후, paired t test로 유의성을 검증하였다 (p<=0.05). In this example, the relationship between diabetes and blood metabolites was examined by observing changes in blood concentrations of metabolites following diabetes. Specifically, after collecting blood from the normal group (Pre-DM) and the diabetic group (DM) before the onset of diabetes, the four amino acids (leucine, tyrosine, valine, phenylalanine) present in the blood were Q-TOF MS and After quantification through QQQ MS, the significance was verified by paired t test (p <= 0.05).
그 결과, 도 1 내지 4에 나타낸 바와 같이, 혈중 루신, 타이로신, 발린, 및 페닐알라닌의 농도는, Q-TOF MS 및 QQQ MS 모두 일관성 있게 당뇨 발병군 (DM)에서 유의적으로 높게 검출되었다. 상기 결과로부터, 루신, 타이로신, 발린, 및 페닐알라닌 등과 같은 대사체들은 당뇨의 진단을 위한 바이오마커로 활용될 수 있음을 알 수 있었다. As a result, as shown in Figs. 1 to 4, the concentrations of leucine, tyrosine, valine, and phenylalanine in blood were significantly detected in the diabetic group (DM) consistently in both Q-TOF MS and QQQ MS. From the above results, it can be seen that metabolites such as leucine, tyrosine, valine, and phenylalanine can be used as biomarkers for diagnosing diabetes.
실시예Example 2. LC/ 2. LC / TOFTOF MS 등을 이용한  Using MS 전당뇨Prediabetes 상태에서의  In state 대사체Metabolite 변화 확인 Confirm change
본 실시예에서는, 상기 실시예 1의 결과에 근거하여, 전당뇨 상태에서 당뇨로의 진행 여부를 조기에 판단할 수 있는 바이오마커 및 이들의 경향성을 확인하고자 하였다. In the present embodiment, on the basis of the results of Example 1, it was intended to confirm the biomarkers and their tendency to determine whether the progression to diabetes in the pre-diabetic state early.
2-1. 실험 대상 2-1. Test subject
고려대학교 생명 윤리 위원회 (Korea university institutional review board, IRB)의 승인 하에서, 윤리 가이드라인 (KU-IRB-15-19-A-1)에 따라 진행되었다. 실험 대상은 한국인 암 예방 연구 (Korean Cancer Prevention Study (KCPS-))로부터 선정되었으며, 상기 실험 대상자들을 당뇨 비발병군 (NDM), 전당뇨 발병군 (PDM), 제2형 당뇨 발병군 (DM)으로 분류한 뒤, 이들의 혈청 샘플을 3년에 거쳐 채취하였다. 구체적으로, 하기 표 1에 나타내 바와 같이, 총 72명의 실험 대상자 중에서 21명은 실험 기간 내내 당뇨 발병군, 17명은 당뇨 비발병군이었으며, 15명은 2년째 (Follow-up 1), 전당뇨 발병군에서 당뇨 발병군으로 진행되었으며, 19명은 3년째 (Follow-up 2), 전당뇨 발병군에서 당뇨 발병군으로 진행되었다. Under the approval of Korea University institutional review board (IRB), it was conducted in accordance with the ethical guidelines (KU-IRB-15-19-A-1). The subjects were selected from the Korean Cancer Prevention Study (KCPS-) and the subjects were non-diabetic (NDM), pre-diabetic (PDM), and type 2 diabetic (DM) patients. After categorizing them, their serum samples were taken over three years. Specifically, as shown in Table 1, out of a total of 72 test subjects, 21 were diabetic group, 17 were non-diabetic group, and 15 were at 2 years (Follow-up 1), pre-diabetic group. Diabetic progression was progressed, and 19 patients progressed from pre-diabetic to diabetic at 3 years (Follow-up 2).
Figure PCTKR2016006937-appb-T000001
Figure PCTKR2016006937-appb-T000001
또한, 상기 선정된 실험 대상자들에 대한 특성을 하기 표 2에 나타내었다. In addition, the characteristics of the selected test subjects are shown in Table 2 below.
Figure PCTKR2016006937-appb-T000002
Figure PCTKR2016006937-appb-T000002
2-2. LC/2-2. LC / TOFTOF MS 분석에 의한  By MS analysis 대사체Metabolite 분석 analysis
상기 실시예 2-1에서 준비한 50μl의 혈청 샘플에 200μl의 아세토나이트릴 (1:4 v/v)을 처리한 뒤, 14,000xg에서 5분간 원심분리를 실시하였다. 이후, 상기 샘플들은 Q-TOF 6550 질량분석기 (Agilent, CA, USA)가 커플링된 초고속 액체크로마토그래피 (C18 Synchronis aQ 1.9μm 1002.1mm (Thermo Scientific, MA, USA))로 분석하였다. 이동상 (mobile phase)으로 각각 0.1%의 formic aicd가 첨가된 H2O 및 acetonitrile를 이용하였고, 용매에 대한 증류수의 %농도로서, 95%의 증류수에서 1분, 직선농도구배를 가하여 55% 증류수까지 8분, 10% 증류수까지 3분, 95% 증류수로 0.1분 처리하여 액체크로마토그래피의 농도 구배를 형성하였다. 이후, Q-TOF 6550 질량분석기를 이용하여 m/z (mass/charge) 값을 검출한 뒤 (20,000 분리능 (resolution), 50-1,000 ions set), 단변량분석 (univariate analysis), FDR 등의 통계적 분석을 실시하였다.50 μl of the serum sample prepared in Example 2-1 was treated with 200 μl of acetonitrile (1: 4 v / v), and then centrifuged at 14,000 × g for 5 minutes. The samples were then analyzed by ultrafast liquid chromatography (C18 Synchronis aQ 1.9 μm 1002.1 mm (Thermo Scientific, MA, USA)) coupled with a Q-TOF 6550 mass spectrometer (Agilent, CA, USA). As a mobile phase, H 2 O and acetonitrile with 0.1% of formic aicd added respectively were used. As a% concentration of distilled water with respect to a solvent, 1 min in 95% distilled water and 55% distilled water by adding a straight tool. 8 minutes, 3 minutes to 10% distilled water, 0.1 minutes treatment with 95% distilled water to form a concentration gradient of the liquid chromatography. Then, using the Q-TOF 6550 mass spectrometer, m / z (mass / charge) values were detected (20,000 resolution, 50-1,000 ions set), univariate analysis, FDR, etc. Analysis was performed.
2-3. 실험 결과2-3. Experiment result
도 5 및 도 6에 나타낸 바와 같이, 혈중 콜레스테롤 및 프롤린의 농도는 정상군 (NDM)에 비해 전당뇨 발병군 (PDM)에서 유의적으로 높게 관찰되었으며, 이러한 경향성은 제2형 당뇨 발병군 (DM)에서도 유지되었다. 한편, 도 7 내지 도 9에 나타낸 바와 같이, 혈중 루신, 라이신, 페닐알라닌의 농도는 전당뇨 발병군 (PDM)에서 유의적으로 낮게 관찰되었다. 특히, 루신의 경우, 제2형 당뇨 발병군 (DM)에서는 오히려 정상군에 비해 높게 관찰되었는바, 전당뇨에 보다 특이적 바이오마커로 활용될 수 있음을 알 수 있었다. As shown in Figures 5 and 6, the concentrations of cholesterol and proline in the blood were significantly higher in the pre-diabetic group (PDM) than in the normal group (NDM), and this tendency was observed in the type 2 diabetic group (DM). Was also maintained. On the other hand, as shown in Figures 7 to 9, the concentration of leucine, lysine, phenylalanine in the blood was significantly lower in the pre-diabetic group (PDM). In particular, in the case of Leucine, type 2 diabetes onset (DM) was observed higher than the normal group, it can be seen that it can be used as a more specific biomarker for prediabetes.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (13)

  1. 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합으로 이루어지는 군으로부터 선택되는 대사체에 대한 검출 제제를 포함하는, 당뇨병 조기 진단용 조성물.A composition for early diagnosis of diabetes, comprising a detection agent for a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and a combination thereof.
  2. 제1항에 있어서, The method of claim 1,
    상기 당뇨병은 초기 당뇨병인 것을 특징으로 하는, 당뇨병 조기 진단용 조성물.The diabetes is characterized in that the initial diabetes, diabetic early diagnosis composition.
  3. 제1항에 있어서, The method of claim 1,
    상기 당뇨병은 제2형 당뇨병인 것을 특징으로 하는, 당뇨병 조기 진단용 조성물.The diabetes is characterized in that the type 2 diabetes, diabetes early diagnosis composition.
  4. 제1항 내지 제3항 중 어느 한 항의 조성물을 포함하는, 당뇨병 조기 진단용 키트.A kit for diagnosing diabetes, comprising the composition of any one of claims 1 to 3.
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 프롤린 및 콜레스테롤로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 증가된 경우, 증가된 당뇨병 발병 위험도를 나타내는 것을 특징으로 하는, 당뇨병 조기 진단용 키트.When the concentration of the metabolite selected from the group consisting of proline and cholesterol is increased compared to the normal control, characterized in that the increased risk of developing diabetes, kit for diagnosing diabetes early.
  6. 제4항에 있어서,The method of claim 4, wherein
    상기 루신, 라이신, 및 페닐알라닌으로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 감소된 경우, 증가된 당뇨병 발병 위험도를 나타내는 것을 특징으로 하는, 당뇨병 조기 진단용 키트.When the concentration of the metabolite selected from the group consisting of leucine, lysine, and phenylalanine is reduced compared to the normal control, characterized in that the increased risk of developing diabetes, kit for diagnosing diabetes early.
  7. 피검체에서 분리된 생체 시료로부터 루신, 라이신, 프롤린, 페닐알라닌, 콜레스테롤 및 이들의 조합으로 구성된 군으로부터 선택되는 대사체의 농도를 측정하는 단계; 및Measuring a concentration of a metabolite selected from the group consisting of leucine, lysine, proline, phenylalanine, cholesterol, and combinations thereof from a biological sample isolated from the subject; And
    상기 측정된 대사체의 농도를 정상 대조군과 비교하는 단계를 포함하는, 당뇨병 조기 진단을 위한 정보제공 방법.Comparing the measured concentration of the metabolite with a normal control, Information providing method for the early diagnosis of diabetes.
  8. 제7항에 있어서, The method of claim 7, wherein
    상기 당뇨병는 초기 당뇨병인 것을 특징으로 하는, 당뇨병 조기 진단용 조성물. The diabetes mellitus, characterized in that early diabetes, diabetic early diagnosis composition.
  9. 제7항에 있어서, The method of claim 7, wherein
    상기 당뇨병는 제2형 당뇨병인 것을 특징으로 하는, 당뇨병 조기 진단을 위한 정보제공 방법.The diabetes is characterized in that the type 2 diabetes, information providing method for early diagnosis of diabetes.
  10. 제7항에 있어서, The method of claim 7, wherein
    상기 생체 시료는 혈액, 혈청, 또는 혈장인 것을 특징으로 하는, 당뇨병 조기 진단을 위한 정보제공 방법.The biological sample is blood, serum or plasma, characterized in that the information providing method for the early diagnosis of diabetes.
  11. 제7항에 있어서, The method of claim 7, wherein
    상기 대사체의 농도를 측정하는 단계는 크로마토그래피/질량 분석법을 통해 측정하는 것을 특징으로 하는, 당뇨병 조기 진단을 위한 정보제공 방법.Measuring the concentration of the metabolite is characterized by measuring by chromatography / mass spectrometry, information providing method for the early diagnosis of diabetes.
  12. 제7항에 있어서, The method of claim 7, wherein
    상기 프롤린 및 콜레스테롤로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 증가된 경우, 증가된 당뇨병 발병 위험도를 나타내는 것을 특징으로 하는, 당뇨병 조기 진단을 위한 정보제공 방법. When the concentration of the metabolite selected from the group consisting of proline and cholesterol is increased compared to the normal control, characterized in that the increased risk of developing diabetes, information providing method for early diagnosis of diabetes.
  13. 제7항에 있어서,The method of claim 7, wherein
    상기 루신, 라이신, 및 페닐알라닌으로 이루어진 군으로부터 선택되는 대사체의 농도가 정상 대조군에 비해 감소된 경우, 증가된 당뇨병 발병 위험도를 나타내는 것을 특징으로 하는, 당뇨병 조기 진단을 위한 정보제공 방법.When the concentration of the metabolite selected from the group consisting of leucine, lysine, and phenylalanine is reduced compared to the normal control, characterized in that the increased risk of developing diabetes, information providing method for early diagnosis of diabetes.
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