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.

Identification of Chemically Sulfated/desulfated Glycosaminoglycans in Contaminated Heparins and Development of a Simple Assay for the Detection of Most Contaminants in Heparin

“…Heparin/heparan sulfate consists of repeating glucosamine-and uronic acid-containing disaccharides whereas chondroitin/dermatan sulfate consists of repeating galactosamine-and uronic acid-containing disaccharides. Analysis of 26 lots of contaminated heparin from different sources showed similar amounts of uronic acid per gram dry weight compared to authentic heparin by a carbazole assay, 12 indicating that heparin was most likely contaminated with GAGs. By using a glucosamine and galactosamine quantification assay, 12,13 we found variable amounts of galactosamine-containing contaminants in the 26 heparin lots (from 2-37%, Table 1) compared to 0.23% in the 26 authentic heparin lots.…”

“…Analysis of 26 lots of contaminated heparin from different sources showed similar amounts of uronic acid per gram dry weight compared to authentic heparin by a carbazole assay, 12 indicating that heparin was most likely contaminated with GAGs. By using a glucosamine and galactosamine quantification assay, 12,13 we found variable amounts of galactosamine-containing contaminants in the 26 heparin lots (from 2-37%, Table 1) compared to 0.23% in the 26 authentic heparin lots. Although the glucosamine/galactosamine quantification assay quantifies the level of chondroitin/dermatan sulfate contamination, the assay could not exclude the possibility that heparin was also contaminated with heparan sulfate and/or oversulfated heparan sulfate.…”

“…12 In brief, a PCR instrument was set at 100 °C for the hydrolysis and carbazole reactions. The reagents used in the carbazole assay were 0.025M sodium tetraborate⋅10 H 2 O in sulfuric acid (H 2 SO 4 +Borex) and 0.125% carbazole in absolute ethanol (W/V).…”

A Comparison of the Histochemical and Image-Derived Proteoglycan Content of Articular Cartilage

“…However, these methods are either limited in their sensitivity, time-consuming, or expensive [8,9]. Direct micro-volume quantification of protein via UV-Vis absorbance was employed in this study using the NanoDrop™ spectrophotometer.…”

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

“…26 More detrimental to the selectivity, both mono-and disaccharides react with the carbazole, producing a high level of background noise that requires the purification of targeted polysaccharides.…”