Regulation and Implication of Hemoglobin Clearance in Subarachnoid Hemorrhagic Patients

急性蛛网膜下腔出血（aSAH）是一种复杂的多方面疾病， 几天到几周。由于脑血管痉挛（CV）、迟发性脑缺血 (DCI)，以及SAH结果较差，已经做出了不成功的努力来建立治疗， 降低CV的发生率。鉴定早期癌症的易感遗传因素和/或生物标志物 CV的预测将作为aSAH患者重症监护管理的临床有用工具。 知道CV在SAH后7-9天达到峰值，我们在这里有一个独特的治疗窗口-只要我们 正确的预测工具。在aSAH后，蛛网膜下腔的溶血释放出大量游离的 血红蛋白（Hb）。这种有毒的促氧化和促炎血红蛋白和代谢产物（铁，胆红素和胆红素 氧化产物）然后直接与供应大脑的主动脉血管外接触。几 研究表明蛛网膜下腔（即CSF）内Hb浓度的变化倾向于 反映CV的演变;尽管游离Hb可能导致这种延迟CV的机制是 不太了解。结合珠蛋白，血红素结合蛋白，sCD 163，转铁蛋白和铁蛋白是一些主要的解毒 针对有毒游离Hb和代谢物的结合蛋白。事实上，我们最近在PNAS上报道说， 结合珠蛋白2-2基因型可能是CV、DCI和长期功能性SAH不良的独立危险因素 结果，可能是由于无效的管理和从蛛网膜下腔清除游离Hb。 在这里，我们计划通过详细的机理分析来验证这一假设，同时扩展这些结果 使用两种互补的分析方法开发预后和诊断生物标志物：1） 使用基于iTRAQ nanoflow LC-MS/MS的方法进行无偏倚的广泛分析，以及2）在 基于luminex的Multi-Analyte Profile平台。包括这种蛋白质组分析方法提供了强有力的 该R21建议的探索性方面最有可能提供额外的无偏倚的新途径。 目的1：研究有毒血红蛋白和代谢物在预测关键aSAH中的生物标志物潜力 临床事件。目的2：研究保护性血红蛋白及其代谢产物的生物标志物潜力。 结合蛋白在预测关键aSAH临床事件中的作用。成对的血清和CSF血红蛋白、代谢物和 保护性结合蛋白将在入院时测量，此后每隔6小时测量一次，直至术后14天。 流血。这些时间曲线将与CV的发生率、严重程度和动力学（上升、峰值、 消退）、DCI发生率、死亡率和出院时、出血后6周和12个月的功能结局。我们 已经以标准化的方式收集了大部分临床数据。总之，这种方法将 允许候选预后和诊断生物标志物的潜在发展，并可提供 对aSAH后血液清除动力学的更好/新的机制理解。因此，我们希望 能够确定这些途径中最合适的关键参与者，以用于未来的治疗靶向。

Acute aneurysmal subarachnoid hemorrhage (aSAH) is a complex and multifaceted disorder that plays out over days to weeks. Because of this relationship between cerebrovasospasm (CV), delayed cerebral ischemia (DCI), and poor SAH outcomes, there have been unsuccessful efforts made to establish treatments that decrease the incidence of CV. Identifying a predisposing genetic factor and/or biomarker for the early prediction of CV would serve as a clinically useful tool in the critical care management of aSAH patients. Knowing that CV peaks at 7-9 days post SAH, we have a unique therapeutic window here–as long as we have the right predictive tools. Following aSAH, hemolysis in the subarachnoid space releases large amounts of free hemoglobin (Hb). This toxic pro-oxidative and pro-inflammatory Hb and metabolites (iron, bilirubin, and bilirubin oxidation products) are then directly in extravascular contact with the main arteries supplying the brain. A few studies have suggested that changes in the Hb concentrations within the subarachnoid space (i.e. CSF) tend to mirror the evolution of CV; although, the mechanisms by which free Hb may cause this delayed CV are poorly understood. Haptoglobin, hemopexin, sCD163, transferrin, and ferritin are some of the main detoxifying binding proteins against toxic free Hb and metabolites. Indeed, we have recently reported in PNAS that haptoglobin 2-2 genotype could be an independent risk factor for CV, DCI, and poor long-term functional SAH outcomes, likely as a result of ineffective management and clearance of free Hb from the subarachnoid space. Here, we plan to test this hypothesis by detailed mechanistic analyses and simultaneously extend these results to prognostic and diagnostic biomarker development using two complementary analytical methods: 1) unbiased broad profiling with an iTRAQ nanoflow LC-MS/MS-based approach, and 2) targeted profiling on a luminex-based Multi-Analyte Profile platform. Inclusion of such proteome profiling approach provides a strong exploratory aspect to this R21 proposal that is most likley to provide additional unbiased novel pathways. Aim 1: To investigate the biomarker potential of toxic hemoglobin and metabolites in predicting key aSAH clinical events. Aim 2: To investigate the biomarker potential of the protective hemoglobin and metabolite- binding proteins in predicting key aSAH clinical events. Paired serum and CSF levels of Hb, metabolites, and protective binding proteins will be measured at admission and at 6h intervals thereafter for up to 14d post- bleed. These temporal profiles will be correlated to the incidence, severity, and dynamics of CV (rise, peak, resolution), incidence of DCI, mortality, and functional outcomes at discharge, 6wk, and 12mo post-bleed. We have already collected the majority of this clinical data in a standardized manner. Together, this approach will allow the potential development of candidate prognostic and diagnostic biomarkers and may provide a better/novel mechanistic understanding of the dynamics of blood clearance after aSAH. As such, we expect to be able to identify the most opportune key players in these pathways for future therapeutic targeting.