Biomarker for liver ischemia/reperfusion-induced injury

描述(申请人提供)：项目摘要/摘要：肝脏缺血再灌注导致的肝损伤是多发伤、热损伤、失血性和感染性休克以及肝移植中肝衰竭的主要致病原因，这对多器官衰竭和死亡率有很大影响。目前的血清ALT或LDH酶分析要么不具特异性，要么不够灵敏，不足以发现肝脏损伤的早期阶段，评估实质细胞死亡的程度，或预测肝脏恢复。基于肝脏蛋白质组降解的方法，我们正在开发一组新的肝脏缺血性损伤的生物标志物。迄今为止发现的最有希望的生物标志物候选是尿素/一氧化氮循环精氨酸琥珀酸合成酶(ASS)的一种酶。这项建议的目标是设计、生产和验证一种用于定量测定生物液中ASS的夹心(SW)ELISA试剂盒，用于诊断缺血性肝损伤。具体目的1：设计并建立检测精氨酸琥珀酸合成酶(ASS)的夹心ELISA法。具体目标1：配置用于定量检测生物体液中ASS的酶联免疫吸附试验原型试剂盒。我们将开发一种软件ELISA，对其进行优化以用于ASS的灵敏检测和定量，包括在血清或血浆中，并生产该试剂盒用于特定目的的初步验证2.特定目的2：初步验证ASS夹心ELISA法在大鼠肝I/R损伤模型中的诊断作用。这一特定目的的目的是通过测量血液中ASS水平来检验原型夹心ELISA法在评估肝脏缺血损伤的程度和预后方面的实用性。来自特定目标2：用于非侵入性诊断和监测肝脏I/R损伤的血清ASS水平的诊断酶联免疫吸附试验试剂盒。这些努力的总体结果将是产生一种有效的检测ASS的夹心ELISA试剂盒，并初步验证其在实验性缺血/再灌注肝损伤大鼠模型中的应用。在腹部和多发创伤、失血性和感染性休克以及肝移植患者的第二阶段SBIR期间，将进一步验证该试剂盒作为人类缺血性肝损伤的可靠诊断工具的有效性。项目简介：各种形式的肝损伤导致的肝功能衰竭是总死亡率的一个重要来源。肝损伤的病因包括由枪击和冲击伤引起的多发性和腹部创伤、药物和酒精中毒、失血性和感染性休克，以及肝移植后的移植失败。传统上，肝酶测定被用作肝损伤的标志物。然而，这些诊断试验要么不敏感，要么不具特异性，既不能评估肝脏损伤的程度，也不能预测结果。因此，临床上显然需要更好的非侵入性生物标记物来检测缺血性肝损伤，这可以导致早期诊断和更好的治疗。以酶联免疫吸附试验(ELISA)为代表的免疫分析技术可用于发展一种简便、快速、无创、可靠的肝I/R损伤诊断方法。在其他用途中，它将在军事医疗或对国家灾难、脓毒症和多器官衰竭的紧急反应中具有价值。此外，它还可能提供更多关于导致肝损伤、功能恢复和/或新治疗策略的潜在靶点的生化和分子机制的信息。我们已经为I/R诱导的肝损伤开发了一种特异和敏感的生物标记物，从最初的实验来看，它似乎与损伤的严重程度相关，并可能对治疗干预敏感。此外，该生物标志物还可以直接评估参与调节肝脏一氧化氮合成和尿素循环的病理生物学机制，这些机制可能代表脓毒症期间肝I/R损伤、肝功能衰竭和代谢性脑病之间的潜在联系。也就是说，精氨酸琥珀酸合成酶(ASS)是一种至关重要的肝酶。ASS在缺血期间迅速上调(在一小时内)，并被释放到循环中。这项建议的总体目标是开发一种用于定量测定ASS的原型ELISA试剂盒，作为包括血清或血浆在内的生物液中的新的诊断生物标志物。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Liver injury induced by hepatic ischemia followed by reperfusion is a major pathogenic cause of liver failure during multiple trauma, thermal injury, hemorrhagic and septic shock, and liver transplantation, which contributes significantly to multiple organ failure and mortality. Current enzymatic assays of serum ALT or LDH are either non-specific or not sensitive enough to detect early stages of liver damage, assess magnitude of parenchymal cell death, or to predict hepatic recovery. Based on liver proteomic degradomics approach, we are developing a panel of novel biomarkers for liver ischemic injury. The most promising biomarker candidate discovered to date is an enzyme of urea/nitric oxide cycle argininosuccinate synthase (ASS). The goals of this proposal are to design, produce, and validate a sandwich (SW) ELISA Kit for quantitative determination of ASS in biological fluids for diagnostics of ischemic liver injury. Specific Aim 1: Design and develop sandwich ELISA for the quantitative detection of argininosuccinate synthase (ASS). Deliverable from Specific Aim 1: Configuration of a prototype ELISA kit for the quantitative detection of ASS in biological fluids. We will develop a SW ELISA, optimize it for sensitive detection and quantification of ASS, including in serum or plasma, and produce this ELISA Kit for preliminary validation in Specific Aim 2. Specific Aim 2: Preliminary validation of the diagnostic utility of the ASS sandwich ELISA in a rat model of liver I/R injury. The objective of this Specific Aim is to examine the utility of the prototype sandwich ELISA for assessment of the magnitude of hepatic ischemic injury and outcome by measuring ASS levels in blood. Deliverable from Specific Aim 2: Diagnostic ELISA Kit for measuring ASS levels in serum for non-invasive diagnostics and monitoring liver I/R injury. The overall result of these efforts will be generation of a validated sandwich ELISA Kit for detection of ASS, and preliminary validation of its utility in experimental ischemia/reperfusion liver injury rat model. Further validation of this ELISA Kit as a reliable diagnostic tool for human ischemic liver Injury will be performed during Phase II SBIR in patients with abdominal and multiple trauma, hemorrhagic and septic shock, and liver transplantation. Project Narrative: Liver failure due to various forms of hepatic injury is a significant source of overall mortality. Etiologies of liver injury includes multiple and abdominal trauma from gunshot and blast injuries, drug and alcohol toxicity, hemorrhagic and septic shock, and graft failure after liver transplantation. Enzymatic assays of liver enzymes have traditionally been used as markers of liver injury. However, these diagnostic tests are either not sensitive or non-specific, do not allow for assessment of the magnitude of liver injury, nor do they predict outcome. Thus, there is an explicit clinical need for better non-invasive biomarkers of ischemic liver injury, which can lead to early diagnosis and better treatment. Immunoassay technologies, such as the enzyme-linked immunosorbent assay (ELISA) can be employed to develop a simple, rapid, noninvasive and reliable diagnostic test for liver I/R injury. Amongst other uses, it will be valuable for military medical or emergency responses to national disasters, sepsis and multiple organ failure. In addition, it may provide more information on biochemical and molecular mechanisms contributing to liver injury, recovery of function and/or potential targets for novel therapeutic strategies. We have developed a specific and sensitive biomarker for I/R-induced liver injury that appears, from initial experiments, to be correlated with the severity of damage and may be sensitive to therapeutic intervention. Moreover, this biomarker provides direct assessment of pathobiological mechanisms involved in regulation of nitric oxide synthesis and the urea cycle in the liver that may represent a potential link between liver I/R injury, liver failure, and metabolic encephalopathy during sepsis. Namely, argininosuccinate synthase (ASS) is a crucial liver enzyme. ASS is rapidly up-regulated (within an hour) during ischemia and is released into the circulation. The overall goal of this proposal is to develop a prototype ELISA kit for the quantitative determination of ASS as novel diagnostic biomarker in biological fluids including serum or plasma.