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

> **NIH NIH R21** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $231,535

## Abstract

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.

## Key facts

- **NIH application ID:** 10886117
- **Project number:** 5R21GM151565-02
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** NATHAN I SHAPIRO
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $231,535
- **Award type:** 5
- **Project period:** 2023-07-14 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10886117

## Citation

> US National Institutes of Health, RePORTER application 10886117, Optimization of a rapid assay to quantify circulating glycosaminoglycans and identify vascular endotypes of sepsis (5R21GM151565-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10886117. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*