Quantitation of nanogram amounts of glycosaminoglycans in samples containing proteins by a new densitometric method

Glycosaminoglycans, separated electrophoretically and stained with Alcian Blue 8GX, were quantified by densitometry. The method allows quantitation of all classes of glycosaminoglycans in samples containing up to 10 mg/ml of serum proteins. Detection sensitivity is 40–300 ng and the optimum range of analysis is either 40–400 or 300–2100 ng, depending on the classes of glycosaminoglycans examined. The method is based on the densitometric comparison between a reference standard of 150 ng of hyaluronate and biological samples electrophoresed on the same sheet. By densitometry the total glycosaminoglycan concentration as well as the percentage and concentration of single fractions are determined. Each class of glycosaminoglycans presents different affinity to Alcian Blue, resulting in over‐ or underestimation in comparison with hyaluronate. Such error is constant over the whole range of determination and may be corrected by correction factors evaluated for each class. Both electrophoresis and densitometric quantitation may be optimized for an analysis of different glycosaminoglycans extracted from biological samples. For instance, heparin and heparan sulfate may be identified by nitrosation applied directly on the sheet; each class may be quantified by comparison with its own standard glycosaminoglycan, avoiding over‐ or underestimation; moreover, dermatan sulfate may be subdivided into fractions. The method is suitable for the quantitation of glycosaminoglycans present in low concentration in biological samples.