Optimization of a rapid assay to quantify circulating glycosaminoglycans and identify vascular endotypes of sepsis

摘要 内皮糖萼是一种富含糖胺聚糖（GAG）的层，衬在所有血管的管腔中。 内皮糖萼的完整性是血管稳态所必需的;因此，脓毒症相关的糖萼 降解导致脓毒性器官损伤的微血管功能障碍特征。升高 循环GAG片段的浓度（通过金标准质谱法定量 由我们的小组开发）因此与脓毒症患者的不良结局相关。 考虑到糖萼降解和脓毒性器官损伤之间的关联， 循环GAG是内皮损伤和脓毒症严重性的有吸引力的生物标志物。然而，质量 光谱测定法昂贵且不适合床边使用。为了满足快速，廉价，点- 在可溶性GAG的护理前测定中，我们开发了二甲基亚甲基蓝（DMMB）测定， 可以在未经处理的人类尿液和空气流体中轻松进行的硫酸化GAG。但 血液中DMMB的准确性尚不清楚，因为这种比色测定不仅可能受到基线的干扰， 血液/血浆的颜色，而且与血液中常见的循环无细胞DNA的交叉反应性， 败血症患者或者，由于血浆GAG迅速排泄到尿液中，因此尿DMMB可能是一种代谢物。 血浆GAG的非侵入性指数，这将避免血浆DMMB的潜在缺陷。但 尿DMMB与血浆GAG的一致性还没有研究。 在本申请的R21部分中，我们提出优化DMMB作为血浆GAG的量度，使用 正常人血液的离体测试以及DMMB与金标准质谱法的比较， 从已建立的脓毒症患者生物库中提取的血浆。此外，我们将前瞻性地收集匹配的 血液和尿液样品以确定尿DMMB是否与血浆GAG相关。 在本申请的R33部分，我们将在波士顿的三个学术机构中进行前瞻性研究。 医学中心以确定血浆GAG的DMMB指数（在治疗期间的各个时间点测量）是否 脓毒症进展）可以作为内皮损伤的生物标志物，由一组蛋白质生物标志物定义 与内皮激活有关。最后，我们将确定DMMB指标的糖萼降解 与脓毒症严重程度的临床指标相关，证明了该测定的翻译相关性。 如果成功，我们的R21/R33将确定一种快速、廉价的即时检测方法，用于评估内皮细胞 脓毒症患者的糖萼降解和内皮损伤。这种分析不仅可以预测 脓毒症中的生物标志物，但可能可以识别脓毒症“内型”，适用于未来的内皮靶向 治疗，允许在重症患者的护理中采用精确的医学方法。

ABSTRACT The endothelial glycocalyx is a glycosaminoglycan (GAG)-enriched layer lining the lumen of all blood vessels. Endothelial glycocalyx integrity is necessary for vascular homeostasis; as such, sepsis-associated glycocalyx degradation contributes to the microvascular dysfunction characteristic of septic organ injury. Elevated concentrations of circulating GAG fragments (quantified by gold-standard mass spectrometry approaches developed by our group) are accordingly associated with poor outcomes in septic humans. Given the association between glycocalyx degradation and septic organ injury, point-of-care quantification of circulating GAGs is an attractive biomarker of endothelial injury and thus sepsis severity. However, mass spectrometry is expensive and ill-suited for bedside use. To address the need for a rapid, inexpensive, point- of-care assay of soluble GAGs, we developed the dimethylmethylene blue (DMMB) assay, a colorimetric assay of sulfated GAGs that can be performed easily in unprocessed human urine and airspace fluid. However, the accuracy of DMMB in blood is unknown, as this colorimetric assay may be confounded by not only the baseline color of blood/plasma but also cross-reactivity with circulating cell-free DNA commonly found in the blood of septic patients. Alternatively, as plasma GAGs are rapidly excreted into the urine, urinary DMMB could be a noninvasive index of plasma GAGs that would avoid the potential pitfalls of plasma DMMB. However, the concordance of urine DMMB with plasma GAGs has not been explored. In the R21 section of this application, we propose to optimize DMMB as a measure of plasma GAGs, using both ex vivo testing of normal human blood as well comparing DMMB with gold-standard mass spectrometry in plasma from an established biobank of septic patients. Furthermore, we will prospectively collect matched blood and urine samples to determine if urine DMMB correlates with plasma GAGs. In the R33 section of this application, we will perform a prospective study across three Boston academic medical centers to determine if DMMB indices of plasma GAGs (measured at various timepoints during the progression of sepsis) can serve as a biomarker of endothelial injury, defined by a panel of protein biomarkers associated with endothelial activation. Finally, we will determine if DMMB indices of glycocalyx degradation correlate with clinical indices of sepsis severity, demonstrating the translational relevance of this assay. If successful, our R21/R33 will identify a rapid, inexpensive, point-of-care assay for assessing endothelial glycocalyx degradation and endothelial injury in septic patients. This assay would not only serve as a predictive biomarker in sepsis, but potentially could identify sepsis “endotypes” amenable to future endothelial-targeted therapies, allowing for precision medicine approaches in the care of critically ill patients.