KR102583808B1 - Method and kit for diagnosing diabetes using tear samples - Google Patents
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Abstract
본 발명은 당뇨병 환자로부터 분리된 눈물 내 대사체를 검출하는 물질을 포함하는 당뇨병 진단용 조성물 또는 당뇨병 진단을 위한 정보제공방법에 관한 것이다. 본 발명은 당뇨병 환자의 눈물을 GC/TOF MS 분석하여 대조군 대비 높거나 낮은 수준을 보이는 대사체를 규명하고, 트레오닌, 펜타데칸산, 만노스, 트레오스, 솔비톨, 세린, 알라닌 또는 스쿠알렌 수준이 대조군 대비 높을 경우 또는 1,5-안하이드로글루시톨 또는 베타-알라닌 수준이 대조군 대비 낮을 경우에는 대조군 대비 당뇨병이 발병했을 가능성이 높다고 진단할 수 있다.The present invention relates to a composition for diagnosing diabetes containing a substance for detecting metabolites in tears isolated from diabetic patients, or a method for providing information for diagnosing diabetes. The present invention analyzes the tears of diabetic patients through GC/TOF MS to identify metabolites at high or low levels compared to the control group, and determines the levels of threonine, pentadecanoic acid, mannose, throse, sorbitol, serine, alanine, or squalene compared to the control group. If the level is high or if the level of 1,5-anhydroglucitol or beta-alanine is low compared to the control group, it can be diagnosed that there is a high possibility of developing diabetes compared to the control group.
Description
본 발명은 눈물의 대사물질 분석을 통해 당뇨병을 진단하거나 치료 후 당뇨병의 경과를 모니터링 하기 위한 것이다.The present invention is intended to diagnose diabetes or monitor the progress of diabetes after treatment through analysis of metabolites in tears.
당뇨병은 인슐린의 작용이나 분비에 문제가 생겨서 만성적인 고혈당을 초래하는 질환으로 전 세계적으로 수백만 명의 환자가 겪는 흔한 질환이다. 당뇨병은 다양한 유전적, 환경적 요인에 의하여 유발될 수 있다. 이러한 당뇨병은 당뇨병성 망막병증, 신증, 신경병증, 뇌졸중, 심근경색, 그리고 족부 괴저 등 다양한 합병증을 유발하여 환자의 건강에 심각한 영향을 미칠 수 있으며 사망에까지 이를 수 있다. Diabetes is a disease that causes chronic hyperglycemia due to problems with the action or secretion of insulin and is a common disease suffered by millions of patients worldwide. Diabetes can be caused by various genetic and environmental factors. This type of diabetes can have a serious impact on the patient's health by causing various complications such as diabetic retinopathy, nephropathy, neuropathy, stroke, myocardial infarction, and foot gangrene, and can even lead to death.
당뇨병 진단방법의 개발은 조기 진단을 통해 빠르고 적절한 치료를 가능하게 하여 환자의 예후를 좋게 할 수 있고, 적절한 모니터링 시스템을 마련하는 것은 고혈당이나 저혈당을 피하고 적절한 혈당 수치를 유지할 수 있도록 하여 합병증 예방에 매우 중요하다. The development of a diabetes diagnosis method can improve the patient's prognosis by enabling quick and appropriate treatment through early diagnosis, and establishing an appropriate monitoring system is very important in preventing complications by avoiding hyperglycemia or hypoglycemia and maintaining appropriate blood sugar levels. It is important.
대사체학은 생체의 대사적 변화에 따른 종합적인 대사물질의 변화, 즉 다양한 생리학적, 병리학적 상태를 규명하는 학문이다. 당뇨병은 유전적, 환경적 인자들이 복합적으로 작용하여 발생하는 것이기 때문에 그 병리학을 개별적인 유전자 및 식습관만으로 완전히 이해하기는 매우 어렵다. 그러나 당뇨병의 발생은 필연적으로 대사물질의 변화가 동반되므로, 대사체학적 접근을 통해서 직접적인 생리 변화를 추측하고 병인학을 이해할 수 있다.Metabolomics is a study that investigates comprehensive changes in metabolites due to metabolic changes in the living body, that is, various physiological and pathological conditions. Because diabetes is caused by a combination of genetic and environmental factors, it is very difficult to fully understand its pathology based on individual genes and eating habits. However, since the occurrence of diabetes is inevitably accompanied by changes in metabolites, direct physiological changes can be inferred and etiology understood through a metabolomics approach.
눈물은 각막의 표면을 유지하며 감염을 억제하는 역할을 할 뿐만 아니라, 영양분을 공급하기 위한 수단으로 사용되며 다양한 대사물질을 포함하고 있으므로 눈물은 질병의 원인이 될 수 있는 대사물질을 포함하고 있을 가능성이 매우 높다. 이러한 특성을 바탕으로 눈물 내 대사물질을 감별하는 키트를 개발하여 질병을 진단할 수 있게 되면 개인이 전문인력의 도움 없이 비침습적으로 간편히 질병을 자가 진단할 수 있다. 따라서 눈물을 이용해서 당뇨병을 진단하는 키트를 발명하는 것은 잠재적 환자가 일상에서 자가 진단을 가능하게 하여 조기 진단의 가능성을 높일 뿐 아니라 진단 후 당대사에 대한 적절한 모니터링을 가능하게 하므로, 병리학 이해를 통한 치료제의 개발도 기대될 수 있어서 매우 중요하다. Tears not only maintain the surface of the cornea and play a role in suppressing infection, but are also used as a means of supplying nutrients and contain various metabolites, so there is a possibility that tears contain metabolites that can cause disease. This is very high. Based on these characteristics, if a kit to identify metabolites in tears can be developed to diagnose diseases, individuals can easily self-diagnose diseases non-invasively without the help of professional personnel. Therefore, inventing a kit to diagnose diabetes using tears not only increases the possibility of early diagnosis by enabling potential patients to self-diagnose in their daily lives, but also enables appropriate monitoring of sugar metabolism after diagnosis, thereby enabling an understanding of pathology. This is very important as it can also lead to the development of treatments.
본 발명은 당뇨병 환자에 특이적인 눈물 대사체 바이오마커를 제시하고 대사물질을 통해 정상인과 환자를 감별할 수 있는 모델을 제공하는 것을 목적으로 한다.The purpose of the present invention is to present tear metabolite biomarkers specific to diabetic patients and to provide a model that can distinguish patients from normal people through metabolites.
1. 당뇨병 환자로부터 분리된 눈물 내 1,5-안하이드로글루시톨, 베타-알라닌, 트레오닌, 펜타데칸산, 만노스, 트레오스, 솔비톨, 세린, 알라닌, 스쿠알렌으로 이루어진 군에서 선택되는 적어도 하나의 대사체를 검출하는 물질을 포함하는, 당뇨병 진단용 조성물.1. At least one agent selected from the group consisting of 1,5-anhydroglucitol, beta-alanine, threonine, pentadecanoic acid, mannose, throse, sorbitol, serine, alanine, and squalene in tears isolated from diabetic patients. A composition for diagnosing diabetes, comprising a substance that detects metabolites.
2. 위 1의 조성물을 포함하는 당뇨병 진단용 키트.2. A kit for diagnosing diabetes containing the composition of 1 above.
3. 당뇨병 환자로부터 분리된 눈물 시료에서 확인된 1,5-안하이드로글루시톨, 베타-알라닌, 트레오닌, 펜타데칸산, 만노스, 트레오스, 솔비톨, 세린, 알라닌, 스쿠알렌으로 이루어진 군에서 선택되는 적어도 하나의 대사체의 수준을 대조군과 비교하는 단계를 포함하는 당뇨병 진단을 위한 정보제공방법.3. Selected from the group consisting of 1,5-anhydroglucitol, beta-alanine, threonine, pentadecanoic acid, mannose, throse, sorbitol, serine, alanine, and squalene identified in tear samples isolated from diabetic patients. A method of providing information for diagnosing diabetes, comprising comparing the level of at least one metabolite with a control group.
4. 위 3에 있어서, 트레오닌, 펜타데칸산, 만노스, 트레오스, 솔비톨, 세린, 알라닌 또는 스쿠알렌 수준이 대조군 대비 높으면 대조군 대비 당뇨병이 발병했을 가능성이 높다고 진단하는 단계를 더 포함하는 당뇨병 진단을 위한 정보제공방법.4. In step 3 above, for diagnosing diabetes, further comprising the step of diagnosing that diabetes is more likely to have occurred compared to the control group if the level of threonine, pentadecanoic acid, mannose, throse, sorbitol, serine, alanine, or squalene is high compared to the control group. How to provide information.
5. 위 3에 있어서, 1,5-안하이드로글루시톨 또는 베타-알라닌 수준이 대조군 대비 낮으면 대조군 대비 당뇨병이 발병했을 가능성이 높다고 진단하는 단계를 더 포함하는 당뇨병 진단을 위한 정보제공방법.5. In 3 above, a method of providing information for diagnosing diabetes, further comprising diagnosing that if the level of 1,5-anhydroglucitol or beta-alanine is lower than that of the control group, the likelihood of developing diabetes is high compared to the control group.
본 발명은 당뇨병 발병여부에 따라서 당뇨병 환자의 눈물에서 특이적으로 증감하는 대사체 바이오마커를 발굴하여, 이를 기반으로 신속하고 정확하게 비침습적으로 당뇨병을 진단할 수 있을 뿐만 아니라 치료 후 모니터링이 가능한 생체표지자를 제공할 수 있다. The present invention discovers metabolite biomarkers that specifically increase or decrease in the tears of diabetic patients depending on the occurrence of diabetes, and based on this, not only can diabetes be diagnosed quickly and accurately non-invasively, but also a biomarker that can be monitored after treatment. can be provided.
또한 본 발명은 대사체 분석을 통한 당뇨병 진단 키트에 적용될 수 있다. 또한 당대사 및 당뇨병 합병증 등의 다양한 병인학 연구에 이용될 수 있다.Additionally, the present invention can be applied to a diabetes diagnosis kit through metabolite analysis. It can also be used for various etiological studies, such as sugar metabolism and diabetes complications.
도 1은 OPLS-DA를 이용한 당뇨병 환자와(Diabetes_Tears) 건강한 대조군 (Control Tears) 간의 비교 분석 결과로, 눈물 대사체 프로파일을 나타낸다 (A: score plot; B: permutation tests).
도 2는 Mann Whitney U test와 MetaMapp에 기반하여 당뇨병 환자의 대사물질 변화를 나타낸 것으로, 각 도형은 각 대사물질을 의미한다 (빨간색: 대조군 대비 당뇨병 환자에서 유의미하게 수준이 높은 대사물질; 파란색: 당뇨병 환자에서 유의미하게 감소한 대사물질; 주황색: 그룹 간 통계적 유의미성이 없는 대사물질을 의미). P-value <0.05은 Mann whitney U test 결과에 기반하여 통계적으로 유의미함을 나타낸다.Figure 1 shows the results of comparative analysis between diabetic patients (Diabetes_Tears) and healthy controls (Control Tears) using OPLS-DA, showing tear metabolite profiles (A: score plot; B: permutation tests).
Figure 2 shows changes in metabolites in diabetic patients based on the Mann Whitney U test and MetaMapp, where each figure represents each metabolite (red: metabolites with significantly higher levels in diabetic patients compared to the control group; blue: diabetes Metabolites significantly decreased in patients; orange: indicates metabolites without statistical significance between groups). P-value <0.05 indicates statistical significance based on Mann whitney U test results.
본 발명은 당뇨병 환자로부터 분리된 눈물 내 1,5-안하이드로글루시톨, 베타-알라닌, 트레오닌, 펜타데칸산, 만노스, 트레오스, 솔비톨, 세린, 알라닌, 스쿠알렌으로 이루어진 군에서 선택되는 적어도 하나의 대사체를 검출하는 물질을 포함하는, 당뇨병 진단용 조성물을 제공한다:The present invention provides at least one selected from the group consisting of 1,5-anhydroglucitol, beta-alanine, threonine, pentadecanoic acid, mannose, throse, sorbitol, serine, alanine, and squalene in tears isolated from diabetic patients. Provided is a composition for diagnosing diabetes, comprising a substance that detects metabolites of:
상기 당뇨병은 인슐린 작용의 절대적 혹은 상대적 결핍으로 고혈당과 이에 수반되는 대사 장애일 수 있다.The diabetes may be hyperglycemia and accompanying metabolic disorders due to absolute or relative deficiency of insulin action.
상기 1,5-안하이드로글루시톨은 1,5-AG로도 알려진 물질로, 거의 모든 음식에서 발견되는 자연 발생 단당류일 수 있으며, 건강한 개체의 혈중에 글루코스에 뒤이어 많이 존재하는 글루코스 유도체의 일종으로 화학적 및 생화학적으로 생체 내에서 매우 안정한 물질일 수 있고, 거의 대사되지 않고 요중에 배설될 수 있다.The 1,5-anhydroglucitol, also known as 1,5-AG, may be a naturally occurring monosaccharide found in almost all foods, and is a type of glucose derivative that is present in large quantities after glucose in the blood of healthy individuals. Chemically and biochemically, it can be a very stable substance in vivo, and can be excreted in urine without being metabolized.
상기 베타-알라닌은 β 탄소에 아미노기가 결합하고 있는 자연적으로 생성되는 β-아미노산일 수 있다.The beta-alanine may be a naturally occurring β-amino acid in which an amino group is bonded to the β carbon.
상기 트레오닌은 알파-아미노산의 하나로 필수 아미노산일 수 있다.The threonine is one of the alpha-amino acids and may be an essential amino acid.
상기 펜타데칸산은 펜타데실산으로도 불리며 자연계에 드물게 존재하는 포화 지방산일 수 있다The pentadecanoic acid is also called pentadecylic acid and may be a saturated fatty acid that rarely exists in nature.
상기 만노스는 6개의 탄소원자가 포함된 단당류이고, 알데하이드기를 가지고 있는 알도스로 물질대사, 특히 특정 단백질의 글리코실화에 중요할 수 있다.The mannose is a monosaccharide containing 6 carbon atoms, and as an aldose with an aldehyde group, it may be important in metabolism, especially glycosylation of specific proteins.
상기 트레오스는 탄소 원자가 포함된 단당류이고, 알데하이드기를 가지고 있는 알도스일 수 있다.The throse is a monosaccharide containing a carbon atom and may be an aldose having an aldehyde group.
상기 솔비톨은 포도당과 같은 육탄당을 환원하여 얻는 6가 알코올의 일종일 수 있다.The sorbitol may be a type of hexahydric alcohol obtained by reducing hexose sugars such as glucose.
상기 세린은 일반적으로 동물성 단백질에서 발견되는 20개의 아미노산 중 하나로 유기 화합물일 수 있다.The serine is one of 20 amino acids generally found in animal proteins and may be an organic compound.
상기 알라닌은 20개 아미노산 중 하나로, α-아미노산일 수 있다.The alanine is one of 20 amino acids and may be an α-amino acid.
상기 스쿠알렌은 상어간유, 올리브, 아마란스 씨, 쌀겨, 맥아 등에 많이 함유되어 있는 불포화 탄화수소로서 인체의 여러 조직에도 존재하며, 체내에서 스테로이드 호르몬과 비타민D, 담즙산, 콜레스테롤의 생합성에도 이용될 수 있다.The squalene is an unsaturated hydrocarbon contained in large quantities in shark liver oil, olives, amaranth seeds, rice bran, malt, etc., and is also present in various tissues of the human body, and can also be used in the biosynthesis of steroid hormones, vitamin D, bile acid, and cholesterol in the body.
상기 “진단”은 특정 질병 또는 질환에 대한 한 객체의 감수성을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후를 판정하는 것, 또는 테라메트릭스(therametrics)를 포함할 수 있다.The “diagnosis” refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or condition, and determining the prognosis of an object suffering from a specific disease or condition. It may include things, or therametrics.
본 발명은 상기 당뇨병 진단용 조성물을 포함하는 당뇨병 진단용 키트를 제공한다:The present invention provides a kit for diagnosing diabetes including the composition for diagnosing diabetes:
“당뇨병 진단용 키트”는 당뇨병 진단용 조성물이 포함된 키트를 의미하며, 상기 키트는 키트를 사용하기 위한 방법이 기재된 설명서를 더 포함할 수 있다.“Diagnosis kit for diabetes” refers to a kit containing a composition for diagnosing diabetes, and the kit may further include instructions describing how to use the kit.
또한 본 발명은 당뇨병 환자로부터 분리된 눈물 시료에서 확인된 1,5-안하이드로글루시톨, 베타-알라닌, 트레오닌, 펜타데칸산, 만노스, 트레오스, 솔비톨, 세린, 알라닌, 스쿠알렌으로 이루어진 군에서 선택되는 적어도 하나의 대사체의 수준을 대조군과 비교하는 단계를 포함하는 당뇨병 진단을 위한 정보제공방법을 제공한다:In addition, the present invention is a group consisting of 1,5-anhydroglucitol, beta-alanine, threonine, pentadecanoic acid, mannose, throse, sorbitol, serine, alanine, and squalene identified in tear samples isolated from diabetic patients. An informational method for diagnosing diabetes is provided, comprising comparing the level of at least one selected metabolite to a control group:
상기 대조군은 당뇨병이 없는 정상인 또는 정상인으로부터 분리된 눈물 시료를 의미하는 것일 수 있다.The control group may refer to a normal person without diabetes or a tear sample separated from a normal person.
상기 당뇨병 환자로부터 분리된 눈물 시료에서 확인된 대사체의 수준을 대조군과 비교하는 단계는 우선적으로 당뇨병 환자 및 정상 대조군에서의 대사체 농도를 측정하는 단계를 포함하고, 상기 대사체의 농도를 비교하여 건강한 대조군 대비 당뇨병 환자군에서 대사체 농도가 유의하게 높거나 낮은 것을 판단하는 것을 의미한다.The step of comparing the level of metabolites identified in the tear sample isolated from the diabetic patient with the control group includes first measuring the concentration of the metabolite in the diabetic patient and the normal control group, and comparing the concentrations of the metabolites. This means determining whether the metabolite concentration is significantly higher or lower in the diabetic patient group compared to the healthy control group.
상기 당뇨병 환자 및 정상 대조군에서의 대사체 농도를 측정하는 단계는 정량기기, 즉 크로마토그래피/질량분석기에 의해 정량화될 수 있다. 상기 정량기기는 해당 분야에서 통상적으로 사용되는 모든 정량 목적의 크로마토그래피일 수 있고, 정량기기의 종류는 예를 들면 가스 크로마토그래피, 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)일 수 있고, 바람직하게는 GC/TOF MS(gas chromatography/time-of-flight mass spectrometry) 분석기기일 수 있으나, 이에 제한되는 것은 아니다.The step of measuring metabolite concentrations in diabetic patients and normal controls can be quantified using a quantitative device, that is, chromatography/mass spectrometry. The quantitative equipment may be any chromatography for quantitative purposes commonly used in the relevant field, and types of quantitative equipment include, for example, gas chromatography, liquid-solid chromatography (LSC), and paper chromatography. (Paper Chromatography, PC), Thin-Layer Chromatography (TLC), Gas-Solid Chromatography (GSC), Liquid-Liquid Chromatography (LLC), Foam Chromatography (Foam Chromatography, FC), Emulsion Chromatography (EC), Gas-Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatography (Gel Filtration Chromatography, It may be GFC) or Gel Permeation Chromatography (GPC), preferably a GC/TOF MS (gas chromatography/time-of-flight mass spectrometry) analysis device, but is not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것으로, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. The following examples illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예Example
1. 실험방법1. Experimental method
(1) 당뇨병 환자 또는 정상인의 눈물 채취(1) Collection of tears from diabetic patients or normal people
본 발명은 임상연구를 위해 경희의대 강동경희대학교병원 IRB (임상연구 기관윤리위원회)의 승인을 거쳐 강동경희대학교병원의 내분비내과에 진료중인 당뇨병 환자와 당뇨병이 없는 정상인을 대상으로 연구에 동의한 대상자에 한하여 연구를 진행하였다.For clinical research, the present invention was approved by the IRB (Clinical Research Institutional Ethics Committee) of Kyung Hee University Hospital at Kangdong Kyung Hee University Hospital, and subjects who consented to the study were diabetic patients receiving treatment at the Department of Endocrinology at Kang Dong Kyung Hee University Hospital and normal people without diabetes. The study was conducted limited to .
당뇨병 환자 또는 정상인의 눈물 내 대사체를 동정하기 위해, 총 44명의 당뇨병 환자와 당뇨병이 없는 건강한 성인을 대조군으로 하여 양안을 포함하여 54개의 눈물 샘플을 채취하였다. To identify metabolites in the tears of diabetic patients or normal people, 54 tear samples, including both eyes, were collected from a total of 44 diabetic patients and healthy adults without diabetes as controls.
(2) GC/TOF MS 및 OPLS-DA 분석(2) GC/TOF MS and OPLS-DA analysis
각 눈물 샘플 10 μl에 순수 메탄올 990 μl을 섞고 강하게 혼합(vortexing)한 후에 원심분리하여 대사체를 추출하였다. 이후 추출한 대사체를 유도체화 시킨 후 GC/TOF MS 를 이용하여 분석하는데, 그 과정은 다음과 같다. 추출한 검체를 스피드 백으로 건조시킨 후에 5 μl의 40%(w/v)농도의 O-methylhydroxylamine hydrochloride in pyridine을 넣고 30°C, 200 rpm에서 90분간 반응을 시킨다. 그리고 45 μl의 N-methyl-N-(trimethylsilyl)trifluoroacetamide를 넣고 37°C, 200 rpm에서 30분간 반응을 실시한다. Metabolites were extracted by mixing 10 μl of each tear sample with 990 μl of pure methanol, mixing vigorously (vortexing), and centrifuging. Afterwards, the extracted metabolites are derivatized and analyzed using GC/TOF MS. The process is as follows. After drying the extracted sample with a speed bag, add 5 μl of 40% (w/v) O-methylhydroxylamine hydrochloride in pyridine and react at 30°C and 200 rpm for 90 minutes. Then, add 45 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide and conduct the reaction at 37°C and 200 rpm for 30 minutes.
GC/TOF MS 분석을 위한 기기 조건은 다음과 같다. 분석할 때 사용한 컬럼은 RTX-5Sil MS capillary column (30 m length, 0.25 mm film thickness, and 25 mm inner diameter)이며, GC 컬럼 온도 조건은 먼저 50°C에서 5분간 유지시킨 후 330°C까지 승온 시킨 후 1분간 유지하였다. 1μl의 검체를 splitless로 injection 하였다. Transfer line 온도와 Ion source 온도는 각각 280°C, 250°C로 유지시켰다. Instrument conditions for GC/TOF MS analysis are as follows. The column used for analysis was RTX-5Sil MS capillary column (30 m length, 0.25 mm film thickness, and 25 mm inner diameter), and the GC column temperature conditions were first maintained at 50°C for 5 minutes and then raised to 330°C. After doing so, it was maintained for 1 minute. 1μl of sample was injected splitless. Transfer line temperature and ion source temperature were maintained at 280°C and 250°C, respectively.
이후, GC/TOF MS (gas chromatography/time-of-flight mass spectrometry) 분석을 수행하였다. 우선 결과 값을 통계처리 가능한 수치로 변환하고, 이를 이용하여 통계학적으로 두 생체시료군 (당뇨병 환자군 및 정상 대조군)의 차별성을 검증하기 위해 교부분 최소제곱회귀법(Orthogonal partial least squares discriminant analysis: OPLS-DA)을 수행하여, 두 생체시료군 간의 유의적인 차이를 나타내는 대사체 바이오마커를 선정하고, 해당 바이오마커들에 대해 추가적으로 분석하고 검증하였다.Afterwards, GC/TOF MS (gas chromatography/time-of-flight mass spectrometry) analysis was performed. First, the result is converted into a numerical value that can be statistically processed, and then the result is used to statistically verify the discrimination between the two biological sample groups (diabetic patient group and normal control group) using orthogonal partial least squares discriminant analysis (OPLS-). DA) was performed to select metabolite biomarkers that showed significant differences between the two biological sample groups, and the corresponding biomarkers were additionally analyzed and verified.
2. 실험결과2. Experiment results
(1) GC/TOF MS 분석 결과(1) GC/TOF MS analysis results
GC/TOF MS 결과를 보유하고 있는 library에서 찾아 동정하여, 결과적으로 눈물에서 48개의 대사체를 동정하였다 (표 1). 구체적으로는 아민류, 아미노산류, 당 및 당 알코올류, 지방산류, 인산류, 유기산류 등을 포함한 총 48개의 대사체를 동정 및 상대정량하였다. 이를 기반으로 당뇨병 환자와 건강한 대조군의 대사체 차이가 있는지 살펴보기 위하여 OPLS-DA를 이용하여 구분하였다.By searching and identifying GC/TOF MS results from a library that holds them, 48 metabolites were identified in tears (Table 1). Specifically, a total of 48 metabolites including amines, amino acids, sugars and sugar alcohols, fatty acids, phosphoric acids, and organic acids were identified and relative quantified. Based on this, OPLS-DA was used to determine whether there were differences in metabolites between diabetic patients and healthy controls.
(2) OPLS-DA를 이용한 당뇨병 환자군 및 건강한 대조군간 대사체 발현 차이 확인(2) Confirmation of differences in metabolite expression between diabetic patient group and healthy control group using OPLS-DA
실시예 1로부터 동정한 48개 대사체의 발현 수준을 확인하고 당뇨병 환자와 건강한 대조군 간의 대사체 프로파일링 차이를 비교하기 위하여 OPLS-DA를 실시하였다.OPLS-DA was performed to confirm the expression levels of 48 metabolites identified in Example 1 and to compare differences in metabolite profiling between diabetic patients and healthy controls.
그 결과, 당뇨병 환자와 건강한 대조군의 눈물 내 대사체 프로파일이 명확하게 차이가 나는 것을 확인하였다(도 1A). OPLS-DA 모델의 t[1] 축을 기준으로 건강한 대조군이 양수값을 띠며 당뇨병 환자가 음수값을 띠어서 서로 대사체 프로파일이 완전히 구분되는 것을 확인하였다(표 2). 당뇨병 환자와 건강한 대조군을 구분하는 OPLS-DA 모델이 통계적으로 유의미하게 외부 샘플에도 적용이 가능한지 살펴보기 위하여 permutation test를 진행하였고, 그 R2 값과 Q2 값이 original points보다 낮아서 모델이 통계적으로 유효함을 살펴볼 수 있었다 (도 1B). As a result, it was confirmed that the metabolite profiles in the tears of diabetic patients and healthy controls were clearly different (Figure 1A). Based on the t[1] axis of the OPLS-DA model, healthy controls had positive values and diabetic patients had negative values, confirming that the metabolite profiles were completely distinct from each other (Table 2). A permutation test was performed to see whether the OPLS-DA model, which distinguishes diabetic patients from healthy controls, can be applied to external samples in a statistically significant way. The R2 and Q2 values were lower than the original points, showing that the model was statistically valid. could be observed (Figure 1B).
(3) Mann Whitney U test를 이용한 당뇨병 환자-특이적으로 증감한 대사물질의 규명(3) Identification of diabetic patient-specific increased or decreased metabolites using the Mann Whitney U test
당뇨병의 유무에 따라 눈물에서 유의미하게 증감하는 대사물질을 확인하기 위해, 상기 48개의 대사물질에 Mann Whitney U test를 통해서 신뢰구간을 분석하였다 (표 3). 그 결과, 10 개의 대사물질이 당뇨병 환자의 눈물에서 유의미하게 증감하는 것을 확인하였다 (표 4). 구체적으로는 1,5-anhydroglucitol, β-alanine, threonine, pentadecanoic acid, mannose, threose, sorbitol, serine, alanine, squalene 10개의 대사물질이 건강한 대조군보다 당뇨병 환자의 눈물에서 유의적으로 높거나 낮은 것을 확인할 수 있었다. 10개의 대사물질 중 threonine, pentadecanoic acid, mannose, threose, sorbitol, serine, alanine, squalene은 대조군 대비 당뇨병 환자에서 발현 수준이 높았고, 반면 1,5-anhydroglucitol, β-alanine는 대조군 대비 당뇨병 환자에서 발현수준이 낮았다. 이러한 결과는 MetaMapp로 형상화하여 나타내었다 (도 2).To identify metabolites that significantly increase or decrease in tears depending on the presence or absence of diabetes, confidence intervals were analyzed for the 48 metabolites using the Mann Whitney U test (Table 3). As a result, it was confirmed that 10 metabolites significantly increased or decreased in the tears of diabetic patients (Table 4). Specifically, 10 metabolites, 1,5-anhydroglucitol, β-alanine, threonine, pentadecanoic acid, mannose, threose, sorbitol, serine, alanine, and squalene, were found to be significantly higher or lower in the tears of diabetic patients than in healthy controls. I was able to. Among the 10 metabolites, threonine, pentadecanoic acid, mannose, threose, sorbitol, serine, alanine, and squalene had higher expression levels in diabetic patients compared to the control group, while 1,5-anhydroglucitol and β-alanine had higher expression levels in diabetic patients compared to the control group. This was low. These results were visualized in MetaMapp (Figure 2).
당 알코올party and
sugar alcohol
Claims (5)
A composition for diagnosing diabetes, comprising a substance for detecting pentadecanoic acid in tears isolated from diabetic patients.
A kit for diagnosing diabetes comprising the composition of claim 1.
A method of providing information for diagnosing diabetes, comprising comparing the level of pentadecanoic acid identified in tear samples isolated from diabetic patients with a control group.
The method of claim 3, further comprising the step of diagnosing that if the level of pentadecanoic acid is higher than that of the control group, the likelihood of developing diabetes is high compared to the control group.
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KR1020230031286A KR20230038174A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
KR1020230031284A KR20230038172A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
KR1020230031285A KR20230038173A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
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KR1020230031284A Division KR20230038172A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
KR1020230031286A Division KR20230038174A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
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KR1020230031285A KR20230038173A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
KR1020230031287A KR20230038681A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
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KR1020230031287A KR20230038681A (en) | 2021-03-05 | 2023-03-09 | Method and kit for diagnosing diabetes using tear samples |
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Title |
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PITKANEN, ELJA, "Mannose, mannitol, fructose and 1,5-anhydroglucitol concentrations measured by gas chromatography/mass spectrometry in blood plasma of diabetic patients", Clinica Chimica Acta, 1996, 251권, 페이지 91-103 1부.* |
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