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

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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.