Analysis of Urinary Glycosaminoglycans to Predict Outcome in COVID-19 and Community-Acquired Pneumonia-A Proof-of-Concept Study

Abstract

Although coronavirus disease 2019 (COVID-19) is considered a systemic disease associated with vascular inflammation and eventual destruction of the protective endothelial glycocalyx (eGC), biomarkers of eGC damage are not yet available in the clinic. The most prominent components of eGC are sulphated glycosaminoglycans (sGAGs) attached to core proteoglycans. We hypothesised that the amount of sGAG fragments shed in urine (as a surrogate for systemic eGC damage) would correlate with disease severity and outcome. Total urinary sGAG concentration was measured using an in-house optimised 1,9-dimethylmethylene blue (DMMB) assay, which is highly accurate and insensitive to interferences. The median urinary sGAG concentration was significantly higher in 67 hospitalised patients with COVID-19 compared to 72 hospitalised patients with community-acquired pneumonia (CAP). In both groups, urinary sGAG concentrations predicted a combined endpoint (including intubation and death) with an area under the receiver operator characteristic curve of 0.72 (95% CI 0.55-0.88, p = 0.01) and 0.70 (95% CI 0.57-0.83, p = 0.007), respectively. In conclusion, the inexpensive and easy-to-perform DMMB assay provides a surrogate parameter for eGC damage that may be useful for risk stratification of patients with COVID-19 and CAP.

Keywords: 1,9-dimethylmethylene blue (DMMB); COVID-19; community-acquired pneumonia; endothelial dysfunction; endothelial glycocalyx; endotheliopathy; glycosaminoglycans.

Figure 1

Improving analytical sensitivity by adoption of a wavelength difference method. (A) Spectral scan of the 1,9-dimethylmethylene blue (DMMB) assay from 500 to 700 nm in 5 nm steps showing changes in absorbance (optical density (OD)) depending on concentration of sulfated glycosaminoglycans (sGAG). Dotted lines indicate α peak at 645 nm, β peak at 590 nm and μ peak at 525 nm, respectively. (B) Assay sensitivity for wavelength 525, 590 and 645 nm as well as wavelength difference 525–590 and 525–645 nm. Higher slope of the standard curve corresponds to higher sensitivity.

Figure 2

Pre-analytics of the DMMB assay using urine samples. (A–D): DMMB assay characterization with urinary samples from septic shock patients and healthy controls. Same samples are coded by same colours. (A) Influence of possible interfering urinary constituents. Native samples (na) of septic shock patients or healthy controls (n = 2 each) were spiked with supra-physiologic concentrations of albumin (al), glucose (gl), acetone (ac) or calcium chloride (ca). (B) Samples were spiked with 62.5 µg/mL Chondroitinsulfate (CS) solution. Calculated concentrations (calc.) of sulfated glycosaminoglycans (sGAG) were compared to concentrations measured with DMMB assay (meas.). Urine samples from healthy subjects (n = 3) were spiked with 250 µg/mL CS solution (spike) and subsequently digested with chondroitinase (enzyme). (C,D) Linear regression with 95% confidence interval between (C) centrifugated and non-centrifugated samples, as well as (D) manual and semi-automatic-operated DMMB assay.

Figure 3

Clinical application of the DMMB assay in COVID-19 and CAP. (A) Boxplots showing urinary sGAG concentration COVID-19 patients (n = 67) and patients with community-acquired pneumonia (CAP, n = 72). Healthy controls (HC, n = 10) served to establish reference values. Groups were compared by the Mann–Whitney test (* p < 0.05, *** p < 0.0001). Receiver-operator characteristic (ROC) curve analysis of sGAG concentration for prediction of the combined outcome in (B) COVID-19 patients (moderate-to-severe ARDS, intubation, or death) and (C) CAP (intubation or death).