Biomarker for liver ischemia/reperfusion-induced injury

DESCRIPTION (provided by applicant): 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. 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.