Biomarker for liver ischemia/reperfusion-induced injury

项目摘要/摘要：肝脏缺血再灌注所致的肝损伤是多发创伤、热损伤、出血性和败血性休克、肝移植过程中肝功能衰竭的主要致病原因，是导致多器官功能衰竭和死亡的重要原因。目前的血清ALT或LDH酶促检测要么没有特异性，要么不够敏感，无法检测早期肝损伤，评估实质细胞死亡的程度，或预测肝脏恢复。基于肝脏蛋白质组学降解组学方法，我们正在开发一组新的肝缺血性损伤生物标志物。迄今为止发现的最有希望的生物标志物候选者是尿素/一氧化氮循环精氨酸琥珀酸合成酶（ASS）酶。本提案的目标是设计、生产和验证一种夹心（SW） ELISA试剂盒，用于定量测定生物体液中用于诊断缺血性肝损伤的ASS。目的1：设计并建立精氨酸琥珀酸合成酶（ASS）的夹心ELISA定量检测方法。特定目标1：配置用于定量检测生物液体中ASS的原型ELISA试剂盒。我们将开发一种SW酶联免疫吸附测定试剂盒，优化其用于血清或血浆中ASS的敏感检测和定量，并生产该酶联免疫吸附测定试剂盒用于Specific Aim 2的初步验证。特异性目的2：初步验证ASS夹心ELISA在大鼠肝I/R损伤模型中的诊断效用。本特定目的的目的是检查三明治ELISA原型的效用，通过测量血液中的ASS水平来评估肝缺血损伤的程度和结果。特异性目标2：用于测量血清中ASS水平的诊断ELISA试剂盒，用于非侵入性诊断和监测肝脏I/R损伤。这些努力的总体结果将是产生一种有效的用于检测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实用性。该ELISA试剂盒作为人类缺血性肝损伤可靠诊断工具的进一步验证将在II期SBIR期间对腹部和多发性创伤、出血性和感染性休克以及肝移植患者进行。项目简介：各种形式的肝损伤引起的肝功能衰竭是总死亡率的一个重要来源。肝损伤的病因包括枪伤和爆炸造成的多发和腹部创伤、药物和酒精毒性、出血性和感染性休克以及肝移植后移植物衰竭。肝酶的酶分析传统上被用作肝损伤的标志物。然而，这些诊断测试要么不敏感，要么非特异性，不能评估肝损伤的程度，也不能预测结果。因此，临床明确需要更好的非侵入性缺血性肝损伤生物标志物，这可以导致早期诊断和更好的治疗。免疫分析技术，如酶联免疫吸附试验（ELISA）可用于开发一种简单、快速、无创和可靠的肝I/R损伤诊断测试。除其他用途外，它将在军事医疗或对国家灾难、败血症和多器官衰竭的紧急反应中发挥重要作用。此外，它可能为肝损伤的生化和分子机制，功能恢复和/或新治疗策略的潜在靶点提供更多信息。我们已经为I/ r诱导的肝损伤开发了一种特异性和敏感的生物标志物，从最初的实验来看，它似乎与损伤的严重程度相关，并且可能对治疗干预敏感。此外，该生物标志物提供了参与肝脏一氧化氮合成和尿素循环调节的病理生物学机制的直接评估，这可能代表了脓毒症期间肝脏I/R损伤、肝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）是一种重要的肝酶。在缺血期间，ASS迅速上调（在一小时内）并释放到循环中。本提案的总体目标是开发一种原型ELISA试剂盒，用于定量测定包括血清或血浆在内的生物液体中作为新型诊断生物标志物的ASS。

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.