Diagnosing and monitoring NAFLD using unbound FFA profiles

描述（由申请人提供）：NAFLD项目摘要/摘要本研究将利用非酒精性脂肪性肝炎临床研究网络（NASH CRN）收集的样本和数据来开发一种快速，非侵入性和廉价的诊断方法，以检测和可能分期非酒精性脂肪性肝病（NAFLD）。NAFLD是一种进行性疾病，其中单纯性脂肪变性可导致脂肪性肝炎、肝硬化、肝衰竭和肝细胞癌。NAFLD的患病率与肥胖和肥胖相关的合并症如胰岛素抵抗、糖尿病和血脂异常密切相关。目前，需要肝活检来确定患者诊断的NAFLD进展阶段。这极大地阻碍了NAFLD的研究，因为活检相关的费用和医疗并发症的风险使得对高危人群的广泛筛查变得不切实际。拟议的诊断将利用一套荧光标记的脂肪酸结合蛋白，以检测血清水平的疏水代谢物，特别是游离脂肪酸。通过使用具有不同结合和信号传导特异性的多个探针，获得了表征患者代谢状态的探针响应谱。这种新的基于探针的方法测量血清中游离脂肪酸（FFA）的未结合浓度，而不是总浓度，其中包括与细胞膜和载体蛋白结合的FFA。未结合FFA的测量是优选的，因为它们揭示了有多少FFA容易用于受体结合和其他相互作用或反应。未结合FFA浓度的曲线与总FFA的曲线不同，因为不同的FFA与细胞膜和载体蛋白达到不同的结合/未结合平衡。在利用来自NASH-CRN的138个样品和一套具有不同脂肪酸结合特异性的12个探针的初步研究中，我们已经表明探针-响应谱可以提供NAFLD患者与糖尿病患者和健康对照的极好分离，并提供简单脂肪变性和严重NASH之间的区分。拟议的研究将利用高通量方法对血清样本进行分析，并通过生产和表征新的探针来改进诊断测定。具体地，活检验证的NAFLD患者和来自其他来源的样品的NASH-CRN储存库将用于1）验证血清代谢物的一套探针可以区分NAFLD和非NAFLD患者和对照，2）开发和优化用于区分NAFLD的医学公认阶段的一套探针，3）测试谱分析是否可用于监测在NASH-CRN试验中接受治疗的成人（PIVENS）和儿童（PIVENS）患者的改善，和4）完成一套可以定量测量血清未结合FFA谱的探针的开发。鉴于肥胖在美国的高患病率，NAFLD预计将造成巨大的社会经济负担，因此开发用于监测普通人群中NASH的非侵入性诊断至关重要。非酒精性脂肪性肝病（NAFLD）可导致肝硬化和肝癌，与肥胖密切相关，预计将影响美国15%至30%的普通人群，包括儿童。由于确定NAFLD严重程度的唯一手段是肝活检，这涉及增加的成本和医疗并发症的风险，许多处于风险中的患者没有被诊断出来，并且很难在一般人群中研究疾病的严重程度。拟议中的研究项目将开发一种快速、非侵入性和廉价的诊断方法，仅用一滴血就能检测出非酒精性脂肪肝的存在和严重程度。

DESCRIPTION (provided by applicant): NAFLD Project Summary/Abstract This research will utilize samples and data collected by the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) to develop a rapid, non-invasive and inexpensive diagnostic assay to detect and possibly stage non-alcoholic fatty liver disease (NAFLD). NAFLD is a progressive disease in which simple steatosis can lead to steatohepatitis, cirrhosis, liver failure and hepatocellular carcinoma. The prevalence of NAFLD is closely linked to obesity and obesity-related co-morbidities such as insulin resistance, diabetes, and dyslipidemia. Currently, liver biopsies are required to identify NAFLD stages of progression for patient diagnosis. This has greatly hindered NAFLD research because biopsy-related expenses and the risk of medical complications make the widespread screening of at-risk populations impractical. The proposed diagnostic will utilize a suite of fluorescently labeled fatty acid binding proteins to detect serum levels of hydrophobic metabolites, especially free fatty acids. By using multiple probes with distinct binding and signaling specificities, a probe-response profile that is characteristic of the patient's metabolic state is obtained. This novel, probe-based approach measures the unbound concentration of free fatty acids (FFA) in serum rather than the total concentration, which includes FFA bound to cell membranes and carrier proteins. Measurements of unbound FFA are preferable because they reveal how much of the FFA is readily available for receptor binding and other interactions or reactions. The profile of unbound FFA concentrations is distinct from the profile for total FFA, because different FFA reach different bound/unbound equilibriums with cell membranes and carrier proteins. In preliminary study utilizing 138 samples from the NASH-CRN and a suite of 12 probes with distinct fatty acid binding specificities we have shown that probe-response profiles can provide excellent separation of NAFLD patients from diabetics and healthy controls and provide discrimination between simple steatosis and severe NASH. The proposed studies will utilize high throughput methods for the profiling of serum samples and for the improvement of the diagnostic assay through the production and characterization of new probes. Specifically, the NASH-CRN repository of biopsy verified NAFLD patients and samples from other sources will be used to 1) verify that a suite of probes for serum metabolites can distinguish between NAFLD and non-NAFLD patients and controls, 2) develop and optimize a suite of probes for distinguishing between the medically recognized stages of NAFLD, 3) test whether profiling can be used to monitor the improvement of patients receiving therapy in NASH-CRN trials on adult (PIVENS) and pediatric (PIVENS) patients, and 4) complete the development of a suite of probes that can quantitatively measure a serum unbound FFA profile. Given the high prevalence of obesity in the United States, NAFLD is expected to create a substantial socioeconomic burden and it is critical that a noninvasive diagnostic for monitoring NASH in the general population be developed. Narrative Non-alcoholic fatty liver disease (NAFLD), which can lead to cirrhosis and liver cancer, is closely linked to obesity and is expected to affect 15% to 30% of the general population of the United States, including children. Because the only means of identifying the severity of NAFLD is a liver biopsy, which involves increased cost and the risk of medical complications, many at risk patients are not diagnosed and it is difficult to study the severity of the disease in the general population. The proposed research project will develop a rapid, non- invasive and inexpensive diagnostic assay to detect the presence and severity of NAFLD using only a drop of blood.