Diagnosing and monitoring NAFLD using unbound FFA profiles

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，开发一种快速、无创和廉价的诊断方法，以检测和分期非酒精性脂肪性肝病（NAFLD）。NAFLD是一种进行性疾病，单纯性脂肪变性可导致脂肪性肝炎、肝硬化、肝功能衰竭和肝细胞癌。NAFLD的流行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NAFLD的进展阶段。这极大地阻碍了NAFLD的研究，因为与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不切实际。建议的诊断将利用一套荧光标记的脂肪酸结合蛋白来检测血清中疏水代谢物的水平，特别是游离脂肪酸。通过使用具有不同结合和信号特异性的多个探针，可以获得具有患者代谢状态特征的探针响应谱。这种新颖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，其中包括结合细胞膜和载体蛋白的游离脂肪酸。未结合的FFA的测量是可取的，因为它们揭示了多少FFA容易用于受体结合和其他相互作用或反应。未结合的游离脂肪酸浓度谱与总游离脂肪酸谱不同，因为不同的游离脂肪酸与细胞膜和载体蛋白达到不同的结合/未结合平衡。在利用NASH- crn的138个样本和12个具有不同脂肪酸结合特异性的探针的初步研究中，我们已经表明探针反应图谱可以很好地将NAFLD患者与糖尿病患者和健康对照者分开，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探针来改进诊断分析。具体来说，NASH-CRN活检证实的NAFLD患者和其他来源的样本库将用于1)验证一套血清代谢物探针可以区分NAFLD和非NAFLD患者及对照组，2)开发和优化一套探针来区分医学上公认的NAFLD阶段。3)在成人（PIVENS）和儿童（PIVENS） NASH-CRN试验中，测试谱分析是否可用于监测接受治疗的患者的改善情况；4)完成一套可定量测量血清游离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，因此在普通人群中开发一种监测NASH的无创诊断方法至关重要。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，预计将影响美国总人口的15%至30%，包括儿童。由于确定NAFLD严重程度的唯一手段是肝活检，这增加了成本和医疗并发症的风险，许多高危患者没有得到诊断，很难研究一般人群中疾病的严重程度。拟议的研究项目将开发一种快速、无创和廉价的诊断方法，仅使用一滴血就能检测NAFLD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.