The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Abstract

Glycosaminoglycans (GAGs) are linear polysaccharides that are found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. Thus, simple, sensitive, and robust GAG quantification methods are needed for the development of biomarkers. We have systematically compared three available GAG quantification assays including an HPLC-based assay, a simplified Alcian Blue assay, and a miniaturized carbazole assay. The carbazole and Alcian Blue assays were reproducible and simple to perform in general lab settings, but had important limitations: The carbazole assay could not detect KS and it overestimated GAGs that were contaminated with salts or dissolved in PBS. The Alcian Blue assay detected only those GAGs that were sulfated. In contrast, while the HPLC method was time-consuming, it was a robust and sensitive assay that not only detected all GAGs but also quantified glucosamine-GAGs and galactosamine-GAGs simultaneously. The HPLC assay was not affected by salt or level of GAG sulfation and it yielded reproducible values for all types of GAGs tested. These results suggest that an automated HPLC assay would be generally useful for the routine measurement of a panel of GAG-based biomarkers while the carbazole assay and the Alcian Blue assays could prove valuable for more specific purposes.