KR102596890B1 - A Biomarker for Diagnosing Diabetic Nephropathy Comprising Urinary Glycated Albumin - Google Patents
A Biomarker for Diagnosing Diabetic Nephropathy Comprising Urinary Glycated Albumin Download PDFInfo
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- diabetic nephropathy
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- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
본 발명은 당뇨병성 신병증(diabetic nephropathy)의 진단용 조성물 및 혈당 변화량 측정용 조성물에 관한 것이다. 본 발명은 소변 내 당화 알부민의 비침습적 측정만으로도 당뇨병성 신병증 및 혈당의 변화량에 대한 신뢰도 높은 진단 정보를 제공한다. 본 발명은 당뇨병성 신병증에 있어서 사구체 손상에 선행하는 신세뇨관의 손상은 물론 혈당 변화에 대한 정보까지 제공함으로써, 질환의 조기 진단 뿐 아니라 질환의 진행 정도에 대한 효율적인 바이오마커로 유용하게 이용될 수 있다. The present invention relates to a composition for diagnosing diabetic nephropathy and a composition for measuring change in blood sugar level. The present invention provides highly reliable diagnostic information on diabetic nephropathy and changes in blood sugar levels through non-invasive measurement of glycated albumin in urine. The present invention provides information on blood sugar changes as well as renal tubular damage that precedes glomerular damage in diabetic nephropathy, so it can be useful as an efficient biomarker for the extent of disease progression as well as early diagnosis of the disease. there is.
Description
본 발명은 소변 내 당화 알부민의 농도를 측정함으로써 당뇨병성 신병증을 조기 진단하고 대상체 내 혈당 변동성을 예측하는 방법에 관한 것이다.The present invention relates to a method for early diagnosis of diabetic nephropathy and prediction of blood sugar variability in a subject by measuring the concentration of glycated albumin in urine.
당뇨병은 포도당-비관용(intolerance)을 초래하는 인슐린의 상대적 또는 절대적 부족으로 특징되는 만성 대사질환의 일종으로, 혈중 포도당의 농도가 높아지는 고혈당을 특징으로 하며, 이로 인하여 여러 증상 및 징후를 일으키고 소변에서 포도당을 배출하게 된다. 현대인의 식생활 변화 및 운동 부족으로 인해 인체 고유의 에너지 대사과정에 커다란 변화가 발생하면서, 당뇨병의 유병률이 현저히 증가하고 있다. Diabetes is a type of chronic metabolic disease characterized by a relative or absolute lack of insulin that leads to glucose intolerance. It is characterized by hyperglycemia, which increases the concentration of glucose in the blood, causing various symptoms and signs and excretion in the urine. Glucose is excreted. Due to changes in modern people's eating habits and lack of exercise, significant changes occur in the human body's inherent energy metabolism process, and the prevalence of diabetes is significantly increasing.
당뇨병은 제1형 당뇨, 제2형 당뇨로 구분된다. 소아 당뇨병으로도 불리는 제1형 당뇨는 인슐린 의존성 당뇨병으로서, β-세포의 파괴에 의해 주로 초래된다. 제2형 당뇨는 인슐린 비의존성 당뇨병으로서, 식사 후 불충분한 인슐린 분비에 의해 초래되거나 또는 인슐린 내성에 의해 초래된다. 제2형 당뇨는 식생활의 서구화에 따른 고열량, 고지방, 고단백의 식단, 운동 부족 및 스트레스 등 환경적인 요인이 크게 작용하는 것으로 보이지만, 이 외에 특정 유전자의 결함에 의해서도 당뇨병이 생길 수 있으며, 췌장 수술, 감염, 약제에 의해서도 생길 수 있다. 당뇨병의 95% 이상은 제2형 당뇨병이다.Diabetes is divided into type 1 diabetes and type 2 diabetes. Type 1 diabetes, also called juvenile diabetes, is insulin-dependent diabetes and is mainly caused by destruction of β-cells. Type 2 diabetes is non-insulin dependent diabetes, which is caused by insufficient insulin secretion after a meal or by insulin resistance. Type 2 diabetes appears to be largely caused by environmental factors such as high-calorie, high-fat, and high-protein diets due to the westernization of eating habits, lack of exercise, and stress, but diabetes can also be caused by defects in specific genes, such as pancreatic surgery, It can also be caused by infection or medication. More than 95% of diabetes is type 2 diabetes.
당뇨병성 신병증(diabetic nephropathy)은 당뇨병 환자에서 신장으로 공급되는 동맥에 고혈당으로 인한 동맥경화가 발생하여 신장 내 작은 혈관들이 손상됨으로써 신장 기능이 저하되는 질환이다. 신장 기능의 이상으로 인해 단백뇨가 발생하고 체내 물과 염분의 정체로 체중증가와 발목 부종 등의 증상과 함께 혈압 상승, 구토, 식욕상실, 피로감, 근육경련 등의 증상을 수반한다. 특히, 당뇨병으로 인한 말기신질환(End-stage renal disease) 환자는 ESRD를 수반하지 않는 당뇨병 환자에 비해 사망률이 2배 정도 상승하므로, ESRD 및 만성신부전(Chronic renal disease, CKD)을 포함하는 당뇨병성 신병증에 대한 신뢰도 높은 바이오마커의 발굴이 매우 중요하다.Diabetic nephropathy is a disease in which arteriosclerosis occurs in the arteries supplying the kidneys in diabetic patients due to high blood sugar, damaging small blood vessels within the kidneys, thereby reducing kidney function. Proteinuria occurs due to abnormal kidney function, and water and salt retention in the body causes symptoms such as weight gain and ankle swelling, as well as increased blood pressure, vomiting, loss of appetite, fatigue, and muscle cramps. In particular, patients with end-stage renal disease due to diabetes have a mortality rate that is approximately twice that of patients with diabetes without ESRD, so diabetic renal disease including ESRD and chronic renal disease (CKD) It is very important to discover highly reliable biomarkers for the disease.
현재까지 2형 당뇨병과 관련된 당뇨병성 신병증의 진단을 위해 알부민뇨 (albuminuria)와 신사구체여과율(estimated glomerular filtration rate, eGFR)이 적용되어 왔으나, 이들은 당뇨병성 신병증으로 인한 사구체의 손상을 대변할 수는 있으나 사구체 손상에 선행하는 신세뇨관의 손상을 반영하지 못할 뿐 아니라 환자 내에서의 혈당 변화에 대한 정보도 제공하지 못한다. 이에. 이러한 기존의 당뇨병성 신병증 진단 방법의 단점을 보완하면서 보다 간편하고도 정확하게 조기 진단할 수 있는 새로운 바이오마커의 개발이 요구되고 있다.To date, albuminuria and estimated glomerular filtration rate (eGFR) have been used to diagnose diabetic nephropathy related to type 2 diabetes, but these cannot represent glomerular damage due to diabetic nephropathy. However, it not only does not reflect renal tubular damage that precedes glomerular damage, but also does not provide information on changes in blood sugar levels within the patient. Therefore. There is a need for the development of new biomarkers that can make early diagnosis more easily and accurately while compensating for the shortcomings of existing diabetic nephropathy diagnostic methods.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.
본 발명자들은 당뇨병 환자의 삶의 질과 사망률에 중대한 영향을 미치는 주요 합병증인 당뇨병성 신병증에 대한 정확하고도 신속한 진단 방법을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 소변 내 당화 알부민의 농도가 공복 혈당, 혈청 HbA1c, 혈청 당화 알부민, 혈청 크레아티닌 수준과 같은 이미 검증된 혈청 파라미터들과 유의한 양의 상관관계를 가지고, 추정 사구체 여과율(eGFR)과 같은 신장 기능 표지자와 유의한 음의 상관관계를 가져, 소변 내 당화 알부민의 비침습적 측정만으로도 이들 혈청 파라미터들을 간접적으로 예측함으로써 당뇨병성 신병증, 이로 인한 신세뇨관 손상 여부 및 혈당의 변화량 등에 대한 신뢰도 높은 진단 정보를 수득할 수 있음을 발견함으로써, 본 발명을 완성하게 되었다.The present inventors have made extensive research efforts to develop an accurate and rapid diagnostic method for diabetic nephropathy, a major complication that significantly affects the quality of life and mortality of diabetic patients. As a result, the concentration of urinary glycated albumin had a significant positive correlation with already validated serum parameters such as fasting blood sugar, serum HbA1c, serum glycated albumin, and serum creatinine levels, and kidney function such as estimated glomerular filtration rate (eGFR). It has a significant negative correlation with functional markers and indirectly predicts these serum parameters only through non-invasive measurement of glycated albumin in urine, providing highly reliable diagnostic information on diabetic nephropathy, renal tubular damage resulting from it, and changes in blood sugar levels. By discovering that it is possible to obtain, the present invention was completed.
따라서 본 발명의 목적은 당뇨병성 신병증(diabetic nephropathy)의 진단용 조성물을 제공하는 데 있다.Therefore, the purpose of the present invention is to provide a composition for diagnosing diabetic nephropathy.
본 발명의 또 다른 목적은 대상체의 혈당 변화량 측정용 조성물을 제공하는 데 있다.Another object of the present invention is to provide a composition for measuring the change in blood sugar level of a subject.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.
본 발명의 일 양태에 따르면, 본 발명은 소변 내의 당화 알부민 측정용 제제를 유효성분으로 포함하는 당뇨병성 신병증(diabetic nephropathy)의 진단용 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a composition for diagnosing diabetic nephropathy, comprising as an active ingredient an agent for measuring glycated albumin in urine.
본 발명자들은 당뇨병 환자의 삶의 질과 사망률에 중대한 영향을 미치는 주요 합병증인 당뇨병성 신병증에 대한 정확하고도 신속한 진단 방법을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 소변 내 당화 알부민의 농도가 공복 혈당, 혈청 HbA1c, 혈청 당화 알부민, 혈청 크레아티닌 수준과 같은 이미 검증된 혈청 파라미터들과 유의한 양의 상관관계를 가지고, 추정 사구체 여과율(eGFR)과 같은 신장 기능 표지자와 유의한 음의 상관관계를 가져, 소변 내 당화 알부민의 비침습적 측정만으로도 이들 혈청 파라미터들을 간접적으로 예측함으로써 당뇨병성 신병증, 이로 인한 신세뇨관 손상 여부 및 혈당의 변화량 등에 대한 신뢰도 높은 진단 정보를 수득할 수 있음을 발견하였다. The present inventors have made extensive research efforts to develop an accurate and rapid diagnostic method for diabetic nephropathy, a major complication that significantly affects the quality of life and mortality of diabetic patients. As a result, the concentration of urinary glycated albumin had a significant positive correlation with already validated serum parameters such as fasting blood sugar, serum HbA1c, serum glycated albumin, and serum creatinine levels, and kidney function such as estimated glomerular filtration rate (eGFR). It has a significant negative correlation with functional markers and indirectly predicts these serum parameters only through non-invasive measurement of glycated albumin in urine, providing highly reliable diagnostic information on diabetic nephropathy, renal tubular damage resulting from it, and changes in blood sugar levels. It was found that it was possible to obtain.
본 명세서에서 용어“당뇨”는 포도당-비관용(intolerance)을 초래하는 인슐린의 상대적 또는 절대적 부족으로 특징되는 만성질환을 의미한다. 본 발명의 조성물로 진단할 수 있는 신병증의 원인 질환이 되는 당뇨는 모든 종류의 당뇨병을 포함하며, 예를 들어, 제1형 당뇨, 제2형 당뇨 및 유전성 당뇨를 포함한다. 제1형 당뇨는 인슐린 의존성 당뇨병으로서, β-세포의 파괴에 의해 주로 초래된다. 제2형 당뇨는 인슐린 비의존성 당뇨병으로서, 식사 후 불충분한 인슐린 분비에 의해 초래되거나 또는 인슐린 저항성에 의해 초래된다.As used herein, the term “diabetes” refers to a chronic disease characterized by a relative or absolute deficiency of insulin resulting in glucose-intolerance. Diabetes, which is a disease causing nephropathy that can be diagnosed with the composition of the present invention, includes all types of diabetes, for example, type 1 diabetes, type 2 diabetes, and hereditary diabetes. Type 1 diabetes is insulin-dependent diabetes, mainly caused by destruction of β-cells. Type 2 diabetes is non-insulin dependent diabetes, which is caused by insufficient insulin secretion after a meal or by insulin resistance.
본 명세서에서 용어“인슐린 저항성”은 혈당을 낮추는 인슐린의 기능이 떨어져 세포가 포도당을 효과적으로 연소하지 못하는 상태를 의미한다. 인슐린 저항성이 높을 경우, 인체는 너무 많은 인슐린을 만들어 내고 이로 인해 고혈압이나 이상지방혈증은 물론 심장병, 당뇨병 등까지 초래할 수 있다. 특히 제2형 당뇨병에서는 근육과 지방조직에서 인슐린의 증가를 알아채지 못하여, 인슐린의 작용이 일어나지 않는다. As used herein, the term “insulin resistance” refers to a condition in which cells cannot effectively burn glucose due to a decrease in the function of insulin to lower blood sugar levels. When insulin resistance is high, the body produces too much insulin, which can lead to high blood pressure and dyslipidemia, as well as heart disease and diabetes. In particular, in type 2 diabetes, the increase in insulin in muscle and fat tissue is not noticed, so the action of insulin does not occur.
본 명세서에서 용어 “당뇨병성 신병증(diabetic nephropathy, DN)”은 제1형 또는 제2형 당뇨병 환자에서 신장 내 혈관들의 손상으로 인해 사구체의 기능 이상이 발생하고, 이로 인해 신장 기능이 만성적으로 상실되는 질환을 의미한다. As used herein, the term “diabetic nephropathy (DN)” refers to a condition in which glomerular dysfunction occurs due to damage to blood vessels within the kidney in patients with type 1 or type 2 diabetes, resulting in chronic loss of kidney function. It means a disease that occurs.
당뇨병성 신병증은 심혈관 합병증을 포함한 여러 당뇨혈관 합병증의 발병 뿐 아니라 환자의 생존률 및 예후 등과 밀접한 관련이 있으므로, 당뇨병성 신병증에 대한 신속한 진단은 당뇨 환자에 대한 종합적인 치료 전략을 조기에 수립하는 데에 있어 매우 중요하다.Diabetic nephropathy is closely related to the development of various diabetic vascular complications, including cardiovascular complications, as well as the patient's survival rate and prognosis. Therefore, prompt diagnosis of diabetic nephropathy is essential for early establishment of a comprehensive treatment strategy for diabetic patients. It is very important in
본 명세서에서 용어 “진단”은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는 지 여부를 판정하는 것(예컨대, 당뇨병 또는 그 합병증의 동정), 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 특정 약물의 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 모두 포괄하는 의미이다.As used herein, the term “diagnosis” refers to determining an object's susceptibility to a specific disease or disorder, determining whether an object currently has a specific disease or condition (e.g., diabetes or its complications). identification), determining the prognosis of a subject with a particular disease or condition, or therametrics (e.g., monitoring the condition of a subject to provide information about the therapeutic efficacy of a particular drug) ) is meant to encompass all of them.
본 발명자들은 공복 혈당, 혈청 HbA1c, 혈청 당화 알부민 농도 또는 혈청 크레아티닌 수준과 같은 당뇨병성 신병증에 대한 혈청 표지자(serum indicator)가 소변 내의 당화 알부민 농도와도 그 경향성에서 높은 일치도를 보일 뿐 아니라 신장 기능을 반영하는 추정 사구체 여과율(eGFR)과 유의한 음의 상관관계를 나타낸다는 사실을 처음으로 규명하고, 소변 내의 당화 알부민 농도를 비침습적 바이오 마커로 최초로 제안하였다. 소변과 혈액은 모두 다양한 대사체를 포함하는 체액으로서 내분비/대사 질환에 대한 정보를 제공하는 중요한 생체 시료이나, 전신을 순환하면서 다양한 대사 산물을 함유하는 혈액과 달리 소변은 신장에서 혈액을 여과한 뒤의 노폐물을 포함하고 있어 필연적으로 혈액과는 상이한 대사체 조성을 가진다. 이에, 혈액에서 나타나는 특정 대사체의 존부나 증감은 소변에서 동일하게 재연되지 않는 것이 일반적이며, 이 때문에 정확한 대사체 진단을 위해서는 불가피하게 침습적인 혈액 검사가 시행되어 왔다. 그러나, 본 발명의 새로운 발견을 통해 종래 혈액 검사로만 얻어지던 당뇨병성 신병증 및 관련 증상에 대한 진단 정보를 간단한 소변 검사만으로 신속하고 정확하게 수득할 수 있게 되었다. The present inventors have found that serum indicators for diabetic nephropathy, such as fasting blood sugar, serum HbA1c, serum glycated albumin concentration, or serum creatinine level, not only show high agreement in their tendency with urine glycated albumin concentration, but also show renal function. It was first established that there was a significant negative correlation with the estimated glomerular filtration rate (eGFR), which reflects , and the concentration of glycated albumin in urine was first proposed as a non-invasive biomarker. Both urine and blood are body fluids containing various metabolites and are important biological samples that provide information on endocrine/metabolic diseases. However, unlike blood, which contains various metabolites while circulating throughout the body, urine is used after blood is filtered by the kidneys. It contains waste products and inevitably has a different metabolite composition from blood. Accordingly, the presence or increase or decrease of a specific metabolite in the blood is generally not replicated in the same way in urine, and for this reason, invasive blood tests have been inevitably performed for accurate metabolite diagnosis. However, through the new discovery of the present invention, diagnostic information on diabetic nephropathy and related symptoms, which was previously obtained only through blood tests, can be quickly and accurately obtained through a simple urine test.
본 명세서에서 용어“당뇨병성 신병증의 진단용 조성물”은 대상체의 당뇨병성 신병증 발병 여부를 예측하기 위해 소변 내 당화 알부민 수준의 측정 수단을 포함하는 통합적인 혼합물(mixture) 또는 장비(device)를 의미하며, 이에“당뇨병성 신병증의 진단용 키트”로 표현될 수도 있다.As used herein, the term “diagnostic composition for diabetic nephropathy” refers to an integrated mixture or device that includes a means for measuring the level of glycated albumin in urine to predict whether a subject will develop diabetic nephropathy. It can also be expressed as a “diagnostic kit for diabetic nephropathy.”
본 발명의 구체적인 구현예에 따르면, 본 발명에서 진단하고자 하는 당뇨병성 신병증은 알부민뇨를 동반한 2형 당뇨병으로 인한 당뇨병성 신병증이다. According to a specific embodiment of the present invention, the diabetic nephropathy to be diagnosed in the present invention is diabetic nephropathy caused by type 2 diabetes accompanied by albuminuria.
본 명세서에서 용어“알부민뇨(albuminuria)”는 단백뇨(proteinuria)의 일종으로, 신장 기능의 저하로 인해 알부민이 신장에서 여과되지 않고 소변을 통해 체외로 배출되는 증상을 의미한다. 소변 내에서 하루 30mg 초과의 알부민이 검출되는 경우 알부민뇨(혹은 미세알부민뇨)로 정의된다. As used herein, the term “albuminuria” is a type of proteinuria and refers to a symptom in which albumin is not filtered by the kidneys and is excreted out of the body through urine due to decreased renal function. If more than 30 mg of albumin is detected in urine per day, albuminuria (or microalbuminuria) is defined.
본 발명의 구체적인 구현예에 따르면, 상기 당뇨병성 신병증은 만성신부전(Chronic renal disease, CKD)이다. According to a specific embodiment of the present invention, the diabetic nephropathy is chronic renal disease (CKD).
본 명세서에서 용어“만성신부전(Chronic renal disease, CKD)”는 수개월 또는 수년에 걸쳐 신장 기능이 점진적으로 소실되는 질환을 의미한다. 만성 심부전은 심혈관 질환, 고혈압, 골질환 및 빈혈과 같은 합병증을 유발하며, 원인질환 중 가장 큰 부분을 차지하는 것이 당뇨병으로, 만성신부전에서 투석이나 이식이 필요한 말기신질환(End-stage renal disease, ESRD)으로 진행된 환자 중 약 40%가 당뇨병을 가지고 있는 것으로 보고되었다.As used herein, the term “chronic renal disease (CKD)” refers to a disease in which kidney function is gradually lost over several months or years. Chronic heart failure causes complications such as cardiovascular disease, high blood pressure, bone disease, and anemia, and the largest cause of the disease is diabetes, and end-stage renal disease (ESRD) that requires dialysis or transplantation in chronic renal failure. It was reported that approximately 40% of patients who progressed to diabetes had diabetes.
본 발명에서 이용되는 당화 알부민 측정용 제제는, 체액 시료, 구체적으로는 소변 시료 내에서 당화 알부민과의 선택적인 반응을 통해 이를 정량적으로 검출할 수 있는 여하한 화합물, 효소, 항체, 앱타머 등을 모두 포괄하는 의미이다. 예를 들어, 단백질 분해효소를 이용한 효소적 방법을 이용하거나, 당화 알부민에 대한 항체를 이용한 면역 어세이(예를 들어 효소 면역측정법(ELISA), 효소-보론산 면역측정법(ELIBA), 측면유동면역발색법(lateral flow immunochromatography))을 이용하거나, 당화 알부민 특이적 앱타머를 이용한 분석법(예를 들어 SPR(surface plasmon resonance) 측정법)을 이용하거나, 또는 당화 알부민의 질량값을 이용한 질량 분석법(예를 들어 MALDI-TOF, SELDI-TOF, LC-MS, LC-MS/MS 등)을 이용할 수 있으나, 이에 제한되지 않고 당업계에 공지된 단백질 검출/정량 수단이 모두 이용될 수 있다. The agent for measuring glycated albumin used in the present invention contains any compound, enzyme, antibody, aptamer, etc. that can quantitatively detect glycated albumin through a selective reaction with glycated albumin in a body fluid sample, specifically a urine sample. It is meant to encompass everything. For example, using enzymatic methods using proteolytic enzymes, or immunoassays using antibodies against glycated albumin (e.g. enzyme-linked immunosorbent assay (ELISA), enzyme-boronic acid immunosorbent assay (ELIBA), lateral flow immunoassay) lateral flow immunochromatography), an analysis method using a glycated albumin-specific aptamer (e.g., SPR (surface plasmon resonance) measurement), or a mass spectrometry method using the mass value of glycated albumin (e.g. For example, MALDI-TOF, SELDI-TOF, LC-MS, LC-MS/MS, etc.) can be used, but is not limited thereto, and all protein detection/quantification methods known in the art can be used.
본 발명의 구체적인 구현예에 따르면, 본 발명은 시료 내에 이미 존재하는 당화 아미노산과 당화 펩타이드를 제거하기 위한 당화 아미노산 산화효소(glycated amino acid oxidase) 및 알부민을 아미노산 또는 펩타이드로 분해하기 위한 알부민-특이적 프로티네이즈(proteinase)를 이용한 효소적 방법을 통해 수행될 수 있다. 이때 프로티네이즈에 의해 생성된 당화 아미노산/ 펩타이드는 다시 당화 아미노산 산화효소에 의해 산화되어 과산화수소를 생성하며, 이는 다양한 방법으로 정량되어 시료 내 당화 알부민 양에 대한 정확한 정보를 제공한다. According to a specific embodiment of the present invention, the present invention provides a glycated amino acid oxidase for removing glycated amino acids and glycated peptides already present in the sample and an albumin-specific enzyme for decomposing albumin into amino acids or peptides. It can be performed through an enzymatic method using proteinase. At this time, the glycated amino acid/peptide produced by proteinase is again oxidized by glycated amino acid oxidase to produce hydrogen peroxide, which is quantified in various ways to provide accurate information about the amount of glycated albumin in the sample.
보다 구체적으로는, 본 발명에서 이용되는 당화 아미노산 산화효소(glycated amino acid oxidase)는 케토아민 옥시다제(ketoamine oxidase) 또는 프룩토실-아미노 애시드 옥시다제(Fructosyl-amino acid oxidase)일 수 있으며, 가장 구체적으로는 케토아민 옥시다제이다. More specifically, the glycated amino acid oxidase used in the present invention may be ketoamine oxidase or fructosyl-amino acid oxidase, and the most specific It is ketoamine oxidase.
보다 구체적으로는, 본 발명에서 이용되는 알부민-특이적 프로티네이즈(proteinase)는 프로티네이즈 N(Proteinase N), 중성 프로티네이즈(Neutral proteinase) 또는 프로티네이즈 K(Proteinase K)일 수 있으며, 가장 구체적으로는 프로티네이즈 K이다. More specifically, the albumin-specific proteinase used in the present invention may be Proteinase N, Neutral proteinase, or Proteinase K. , most specifically proteinase K.
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물은 당뇨병성 신병증의 조기 진단용 조성물이다.According to a specific embodiment of the present invention, the composition of the present invention is a composition for early diagnosis of diabetic nephropathy.
본 명세서에서 용어“조기 진단(early diagnosis)”은 당뇨병성 신병증의 초기 단계, 예를 들어 사구체 손상(glomerular injury) 이전 단계에 당뇨병성 신병증에 대한 임상적 예측이 가능할 정도의 정보를 수득하는 것을 의미한다.As used herein, the term “early diagnosis” refers to obtaining information sufficient to enable clinical prediction of diabetic nephropathy in the early stages of diabetic nephropathy, for example, before glomerular injury. means that
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물은 신세뇨관의 손상 정도에 대한 정보를 제공한다.According to a specific embodiment of the present invention, the composition of the present invention provides information on the degree of damage to renal tubules.
당뇨병성 신병증에서는 사구체 손상이 일어나기 이전에 신세뇨관의 손상이 발생하는데, 만성신부전 등의 당뇨병성 신병증에 대한 종래의 진단 표지자인 알부민뇨(albuminuria)나 신사구체 여과율과 달리, 본 발명의 조성물은 소변 내 당화 단백질을 측정함으로써 신세뇨관과 신사구체의 손상을 함께 예측할 수 있어, 당뇨병성 신병증의 발병을 조기에 검출할 수 있다. In diabetic nephropathy, damage to the renal tubules occurs before glomerular damage occurs. Unlike albuminuria or glomerular filtration rate, which are conventional diagnostic markers for diabetic nephropathy such as chronic renal failure, the composition of the present invention By measuring glycated proteins in urine, damage to renal tubules and glomeruli can be predicted, allowing early detection of the onset of diabetic nephropathy.
본 발명의 다른 양태에 따르면, 본 발명은 소변 내의 당화 알부민 측정용 제제를 유효성분으로 포함하는 대상체의 혈당 변화량 측정용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for measuring the change in blood sugar level of a subject, comprising as an active ingredient an agent for measuring glycated albumin in urine.
본 발명에서 이용되는 소변 내의 당화 알부민 측정용 제제의 구체적인 조성에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위해 그 기재를 생략한다. Since the specific composition of the agent for measuring glycated albumin in urine used in the present invention has already been described in detail, its description is omitted to avoid excessive duplication.
알부민은 글루코스와의 결합률이 매우 높아 당화혈색소보다 당화 알부민이 혈당 변화에 더욱 민감하게 반응하며, 적혈구 수명이 90일 정도인 것에 비하여 알부민은 15~20일 정도로 짧아서, 최근 2주 동안의 혈중 평균 글루코스 수준을 모니터링할 수 있다. 따라서, 혈당이 일정하게 유지되지 못하는 만성신부전 등의 당뇨병성 신병증 환자에서 당화 알부민의 농도는 매우 중요한 혈당 조절 지표로 이용될 수 있다. 따라서, 본 발명의 조성물은 혈청 내 당화 알부민 수준을 객관적으로 반영하는 소변 내의 당화 알부민 수준을 측정함으로써 대상체의 혈당 변화량에 대한 신로도 높은 정보를 제공할 수 있다.Albumin has a very high binding rate with glucose, so glycated albumin reacts more sensitively to changes in blood sugar than glycated hemoglobin. Compared to the lifespan of red blood cells, which is about 90 days, albumin's lifespan is shorter, about 15 to 20 days, so the average glucose in the blood over the past two weeks is Levels can be monitored. Therefore, the concentration of glycated albumin can be used as a very important indicator of blood sugar control in patients with diabetic nephropathy such as chronic renal failure in which blood sugar levels cannot be maintained at a constant level. Therefore, the composition of the present invention can provide highly reliable information about the amount of change in blood sugar in a subject by measuring the level of glycated albumin in urine, which objectively reflects the level of glycated albumin in serum.
본 발명의 또 다른 양태에 따르면, 본 발명은 대상체로부터 분리된 소변 내의 당화 알부민 농도를 측정하는 단계를 포함하는 당뇨병성 신병증(diabetic nephropathy)의 예측 또는 진단에 필요한 정보를 제공하는 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method of providing information necessary for prediction or diagnosis of diabetic nephropathy, comprising measuring the concentration of glycated albumin in urine isolated from a subject. .
본 발명에서 진단하고자 하는 당뇨병성 신병증 및 당화 알부민 농도의 일반적인 측정 방법에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위하여 그 기재를 생략한다.Since the general measurement method for diabetic nephropathy and glycated albumin concentration to be diagnosed in the present invention has already been described in detail, the description thereof is omitted to avoid excessive duplication.
본 명세서에서 용어“대상체”는 당화 알부민의 농도를 측정하기 위한 소변 시료를 제공하고, 당뇨병성 신병증 발병의 예측 대상이 되는 개체를 의미한다. 개체는 제한없이 인간, 마우스, 래트, 기니아 피그, 개, 고양이, 말, 소, 돼지, 원숭이, 침팬지, 비비 또는 붉은털 원숭이를 포함하며, 구체적으로는 인간이다. 본 발명의 조성물은 발병 여부를 예측하기 위한 정보를 제공하기 때문에, 본 발명의 대상체는 당뇨 또는 당뇨병성 신병증 환자일 수도 있고 아직 이들 질환으로 확진되지 않은 정상개체(healthy subject)일 수도 있다. As used herein, the term “subject” refers to an individual who provides a urine sample to measure the concentration of glycated albumin and is predicted to develop diabetic nephropathy. Subjects include, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys, and are specifically humans. Since the composition of the present invention provides information for predicting the onset of the disease, the subject of the present invention may be a patient with diabetes or diabetic nephropathy, or may be a healthy subject who has not yet been diagnosed with these diseases.
본 발명의 또 다른 양태에 따르면, 본 발명은 대상체로부터 분리된 소변 내의 당화 알부민 농도를 측정하는 단계를 포함하는 대상체 내의 혈당 변화량의 예측 또는 측정에 필요한 정보를 제공하는 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method of providing information necessary for predicting or measuring the amount of change in blood sugar in a subject, including the step of measuring the concentration of glycated albumin in urine separated from the subject.
본 발명에서 예측하고자 하는 혈당 변화 및 당화 알부민 농도의 일반적인 측정 방법에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위하여 그 기재를 생략한다.Since the general measurement method for blood sugar changes and glycated albumin concentration to be predicted in the present invention has already been described in detail, the description is omitted to avoid excessive duplication.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 당뇨병성 신병증(diabetic nephropathy)의 진단용 조성물 및 혈당 변화량 측정용 조성물을 제공한다.(a) The present invention provides a composition for diagnosing diabetic nephropathy and a composition for measuring change in blood sugar level.
(b) 본 발명은 소변 내 당화 알부민의 비침습적 측정만으로도 당뇨병성 신병증 및 혈당의 변화량에 대한 신뢰도 높은 진단 정보를 제공한다.(b) The present invention provides highly reliable diagnostic information on diabetic nephropathy and changes in blood sugar levels through non-invasive measurement of glycated albumin in urine.
(c) 본 발명은 당뇨병성 신병증에 있어서 사구체 손상에 선행하는 신세뇨관의 손상은 물론 혈당 변화에 대한 정보까지 제공함으로써, 질환의 조기 진단 뿐 아니라 질환의 진행 정도에 대한 효율적인 바이오마커로 유용하게 이용될 수 있다. (c) The present invention provides information on blood sugar changes as well as renal tubular damage that precedes glomerular damage in diabetic nephropathy, making it useful not only for early diagnosis of the disease but also as an efficient biomarker for the degree of disease progression. It can be used.
도 1은 효소적 방법을 이용한 당화 알부민의 분석 과정을 나타낸 모식도이다.
도 2는 소변 내 당화 알부민 수준과 혈청 내 공복혈당(도 2a), HbA1c (도 2b) 및 당화 알부민(도 2c)간의 관계를 보여주는 그림이다. GA, 당화 알부민; HbA1c, 당화 헤모글로빈 A1c.
도 3은 소변 당화 알부민과 혈청 크레아티닌(도 3a), 소변 ACR과 혈청 크레아티닌(도 3b), 소변 당화 알부민과 eGFR(도 3c) 및 소변 ACR과 eGFR(도 3d)간의 관계를 각각 보여주는 그림이다. ACR, 알부민-크레아티닌 비; GA, 당화 알부민; eGFR, 추정 사구체 여과율.Figure 1 is a schematic diagram showing the analysis process of glycated albumin using an enzymatic method.
Figure 2 is a diagram showing the relationship between the level of glycated albumin in urine and fasting blood sugar (Figure 2a), HbA1c (Figure 2b), and glycated albumin (Figure 2c) in serum. GA, glycated albumin; HbA1c, glycosylated hemoglobin A1c.
Figure 3 is a diagram showing the relationships between urine glycated albumin and serum creatinine (Figure 3a), urine ACR and serum creatinine (Figure 3b), urine glycated albumin and eGFR (Figure 3c), and urine ACR and eGFR (Figure 3d). ACR, albumin-creatinine ratio; GA, glycated albumin; eGFR, estimated glomerular filtration rate.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
실험방법Experiment method
소변 시료의 제작Preparation of urine samples
외래환자의 통상적인 소변검사 후 잔여 소변 샘플을 알부민뇨 환자 시료로부터 수득하였다. 시료를 원심분리 후 상등액 2 ml를 동결튜브(Cryotube)로 옮겼다. 이후 소변 내 당화 알부민(GA) 측정시까지 -80℃에서 보관하였다. 이후 보관된 시료를 상온으로 승온시킨 뒤 측정을 수행하였다.After routine outpatient urinalysis, residual urine samples were obtained from albuminuric patient samples. After centrifuging the sample, 2 ml of the supernatant was transferred to a cryotube. Afterwards, it was stored at -80°C until measurement of glycated albumin (GA) in urine. Afterwards, the stored sample was raised to room temperature and then measured.
효소적 방법을 이용한 소변 GA의 측정Measurement of urine GA using enzymatic methods
일반적인 시약 및 정제된 혈청 단백질은 Sigma-Aldrich(St. Louis, MO)에서 구입하였다. 당화 알부민의 정제는 종래 보고된 방법에 따라 수행하였다(Clin Chim Acta, 2002;324(1-2):61-71). 당화 알부민은 Lucica® GA-L 킷을 이용한 효소적 방법을 통해 종래에 보고된 방법(J Diabetes Sci Technol. 2011 Nov 1;5(6):1455-62)에 따라 측정하였다. 효소적 어세이에서, 1 단계로 먼저 내인성 당화 아미노산 및 과산화물은 케토아민 옥시다제 및 퍼옥시다제 반응에 의해 제거된다. 2 단계에서, GA는 알부민-특이적 프로티네이즈(proteinase)에 의해 아미노산 또는 펩타이드로 가수분해되며, 당화 아미노산 또는 펩타이드는 케토아민 옥시다제에 의해 산화되어 과산화수소를 생성하는데, 이는 정량적으로 측정될 수 있다. General reagents and purified serum proteins were purchased from Sigma-Aldrich (St. Louis, MO). Purification of glycated albumin was performed according to a previously reported method ( Clin Chim Acta , 2002;324(1-2):61-71). Glycosylated albumin was measured according to a previously reported method ( J Diabetes Sci Technol . 2011 Nov 1;5(6):1455-62) through an enzymatic method using the Lucica® GA-L kit. In the enzymatic assay, in one step, endogenous glycated amino acids and peroxides are first removed by ketoamine oxidase and peroxidase reactions. In step 2, GA is hydrolyzed by albumin-specific proteinases to amino acids or peptides, and the glycosylated amino acids or peptides are oxidized by ketoamine oxidase to produce hydrogen peroxide, which can be measured quantitatively. there is.
3 단계에서, BCP(bromocresol purple) 방법으로 알부민 농도를 측정하였으며, 마지막 4단계에서, GA 수준을 전체 알부민 대 GA의 상대적 비율로서 계산하였다(도 1).In step 3, albumin concentration was measured using the bromocresol purple (BCP) method, and in the final step 4, GA levels were calculated as the relative ratio of total albumin to GA (Figure 1).
실험결과Experiment result
알부민뇨 환자에서의 소변 당화 알부민 측정Measurement of urinary glycated albumin in patients with albuminuria
본 발명자들은 알부민뇨 환자들의 소변 시료로부터 당화 알부민 농도를 측정하였으며, 대상 환자들의 임상적 특성은 하기 표 1과 같다:The present inventors measured glycated albumin concentration from urine samples of patients with albuminuria, and the clinical characteristics of the patients are shown in Table 1 below:
데이터는 평균 ± 표준편차(SD) 또는 % 값으로 나타내었다. BMI, 체질량지수; HbA1c, 당화 헤모글로빈 A1c.Data are expressed as mean ± standard deviation (SD) or % values. BMI, body mass index; HbA1c, glycosylated hemoglobin A1c.
소변 당화 알부민과 혈당 파라미터 간의 관계Relationship between urine glycated albumin and blood glucose parameters
본 발명자들은 혈청 내 공복 혈당, 당화 헤모글로빈 A1c(HbA1c) 및 당화 알부민을 포함하는 혈당 파라미터와 소변 당화 알부민 간의 관련성을 조사하였다. 도 2에 나타낸 바와 같이, 소변 내 당화 알부민 수준은 유용한 혈당 파라미터들과 양의 상관관계를 가지는 것으로 나타났다. 도 2에 나타난 이러한 명확한 경향성을 통해, 소변 내 당화 알부민은 혈당 조절 등에 대한 신뢰도 높은 바이오 마커로 기능할 수 있을 것으로 기대된다.We investigated the relationship between urinary glycated albumin and blood glucose parameters including fasting blood sugar, glycated hemoglobin A1c (HbA1c) and glycated albumin in serum. As shown in Figure 2, urinary glycated albumin levels were shown to have a positive correlation with useful blood glucose parameters. Through this clear trend shown in Figure 2, glycated albumin in urine is expected to function as a highly reliable biomarker for blood sugar control.
당화 알부민 및 소변 알부민-크레아티닌 비와 신장 기능과의 관계Relationship between glycated albumin and urine albumin-creatinine ratio and renal function.
본 발명자들은 소변 당화 알부민; 소변 알부민-크레아티닌 비(uACR); 혈청 크레아티닌 수준 및 추정 사구체 여과율(eGFR)을 포함하는 신장 기능 파라미터 간의 상관관계를 조사하였다. 그 결과, 도 3에서 보는 바와 같이 소변 당화 알부민 수준은 uACR 보다 혈청 크레아티닌 수준 및 eGFR과 보다 두드러진 상관 관계를 나타냄을 확인하였다. 이를 통해, 소변 당화 알부민 수준이 uACR에 비해 만성신부전의 진행 정도를 반영하는 보다 우수한 바이오마커로 기능할 수 있음을 알 수 있었다. The present inventors discovered that urine glycated albumin; urine albumin-creatinine ratio (uACR); The correlation between renal function parameters including serum creatinine levels and estimated glomerular filtration rate (eGFR) was investigated. As a result, as shown in Figure 3, it was confirmed that the urine glycated albumin level showed a more pronounced correlation with the serum creatinine level and eGFR than with the uACR. Through this, it was found that urine glycated albumin level can function as a better biomarker that reflects the progress of chronic renal failure compared to uACR.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (13)
상기 조성물은 신세뇨관의 손상 정도에 대한 정보를 제공하고;
소변 내 당화 알부민이 증가할 때 초기 당뇨병성 신병증이 있는 것으로 진단하는 것을 특징으로 하는 조성물.
A composition for early diagnosis of diabetic nephropathy comprising as an active ingredient an agent for measuring glycated albumin in urine,
The composition provides information about the degree of damage to the renal tubules;
A composition characterized in that early diabetic nephropathy is diagnosed when glycated albumin in urine increases.
The composition according to claim 1, wherein the diabetes is type 2 diabetes accompanied by albuminuria.
The composition of claim 1, wherein the diabetic nephropathy is chronic renal disease (CKD).
The composition according to claim 1, wherein the agent for measuring glycated albumin contains albumin-specific proteinase and glycated amino acid oxidase.
The composition according to claim 4, wherein the glycated amino acid oxidase is ketoamine oxidase.
상기 방법은 신세뇨관의 손상 정도에 대한 정보를 제공하고,
소변 내 당화 알부민이 증가하는 경우 초기 당뇨병성 신병증이 있는 것으로 진단하는 것을 특징으로 하는 방법.
A method of providing information necessary for prediction or diagnosis of early diabetic nephropathy, comprising measuring the concentration of glycated albumin in urine isolated from the subject,
The method provides information about the degree of damage to the renal tubules,
A method characterized in that diagnosis of early diabetic nephropathy occurs when glycated albumin in urine increases.
The method of claim 9, wherein the diabetic nephropathy is chronic renal disease (CKD).
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