Figure 4. SAX HPLC analysis of fluorescently labeled HS disaccharides purified from OCM and SCM indicate that SCs and OECs secrete distinct HS structures. A, B, HS purified from OCM (A) and SCM (B) was digested with heparinase enzymes to generate HS disaccharides, which were fluorescently labeled with BODIPY and separated by SAX HPLC over a 45 min, 0–1.5 m NaCl gradient (black lines). HS disaccharide standards were separated over the same gradient as a reference guide (dashed lines). C, Relative abundances of the eight HS standard disaccharides in each conditioned medium sample were calculated as a percentage of total HS. D, Summary table detailing the composition of OCM HS and SCM HS. SCM HS was found to be more highly sulfated than OCM HS, with an average of 1.02 sulfates per disaccharide compared with 0.75 sulfates per disaccharide in OCM HS. SCM HS contained a higher proportion of disulfated and trisulfated disaccharides (disaccharides 4, 5, 8, and 6) and the singly 6-O-sulfated disaccharide (disaccharide 2) compared with OCM HS (C), which contained a higher proportion of the unsulfated disaccharide (disaccharide 1) (C, D). UA, uronic acid; GlcN, glucosamine; NAc, N-acetyl; NS, N-sulfate; 6OS, 6-O-sulfate; 2OS, 2-O-sulfate.