[Separation and quantification of glycosaminoglycans using HPLC].

Biochemical studies of proteoglycans (PGs) involve the characterization of their polysaccharide chains. In fact, PGs display a considerable heterogeneity with respect to type and size of the saccharide chains, to the ratio of iduronic to glucuronic acid and to the degree of sulphation. Several HPLC methods have been described for separation and identification of glycosaminoglycans (GAGs), which usually employ molecular sieving and disaccharide analysis, after specific enzyme digestion of GAGs. In order to separate intact GAGs, we utilized both high performance gel permeation and ion exchange chromatography, using a Spherogel TSK 4000SW and a Spherogel TSK DEAE 25W respectively. HPLC gel permeation chromatography makes it possible to separate only HA from other GAGs. This procedure can be useful to purify biological preparations from HA, so allowing GAG study in non-aggregating conditions. HPLC ion exchange chromatography was performed on GAG standard mixtures and the suitability of the method was tested on GAGs extracted from intima and media preparations of human thoracic aorta. In our chromatographic conditions HA eluted at 0.5 M NaCl, HS at 0.54 M NaCl, DS at 0.61 M NaCl and C6S at 0.65 M. C4S coeluted with DS and C6S. To determine DS concentration the samples were reanalyzed after chondroitinase AC treatment; the differences in uronic acid content between the original samples and the digests represented the total amount of C4S and C6S. Our data indicate a good reproducibility of the method that allows a rapid and accurate determination of intact GAGs in biological samples.