Quantification of sulfated polysaccharides in mouse and rat plasma by the Heparin Red mix-and-read fluorescence assay

TL;DR

This study adapts the Heparin Red fluorescence assay for quantifying sulfated polysaccharides like heparin and fucoidan in mouse and rat plasma, enabling sensitive pharmacokinetic analysis with small sample volumes.

Contribution

The paper introduces a modified Heparin Red protocol for detecting sulfated polysaccharides in rodent plasma, expanding its application beyond human samples.

Findings

Effective detection within 0-10 μg/mL range

Limits of quantification: 1.1-2.3 μg/mL for heparin

Sample volume required: 20 μL

Abstract

Sulfated polysaccharides constitute a large and complex group of macromolecules which possess a wide range of important biological properties. Many of them hold promise as new therapeutics, but determination of their blood levels during pharmacokinetic studies can be challenging. Heparin Red, a commercial mix-and-read fluorescence assay, has recently emerged as a tool in clinical drug development and pharmacokinetic analysis for the quantification of sulfated polysaccharides in human plasma. The present study describes the application of Heparin Red to the detection of heparin, a highly sulfated polysaccharide, and fucoidan, a less sulfated polysaccharide, in spiked mouse and rat plasmas. While the standard assay protocol for human plasma matrix gave less satisfactory results, a modified protocol was developed that provides within a detection range 0 to 10 micrograms per mL better limits of quantification, 1.1 to 2.3 micrograms per mL for heparin, and 1.7 to 3.4 micrograms per mL for fucoidan. The required plasma sample volume of only 20 microliters is advantegous in particular when blood samples need to be collected from mice. Our results suggest that Heparin Red is a promising tool for the preclinical evaluation of sulfated polysaccharides with varying sulfation degrees in mouse and rat models.