HIGH-RESOLUTION SEPARATION OF DISACCHARIDE AND OLIGOSACCHARIDE-ALDITOLS FROM CHONDROITIN SULFATE, DERMATAN SULFATE AND HYALURONAN USING CARBOPAC PA1 CHROMATOGRAPHY

Recent literature indicates that specific glycosaminoglycan structures are involved in various biological processes, such as anticoagulation, growth factor activation and viral infection. The initial step in the structural analysis of glycosaminoglycans is a definitive compositional analysis of its characteristic disaccharide repeat structures. Current chromatographic or electrophoretic procedures may have limitations in analysing glycosaminoglycan samples that are in low abundance, contain novel structures that need to be further characterized, or are metabolically labelled from radioactive precursors as a result of biosynthetic experiments. This study presents a new methodology for analysing disaccharides and oligosaccharides derived from chondroitin sulphate, dermatan sulphate and hyaluronan that fulfils the above criteria. The procedure involves the separation of reduced forms of these glycoconjugates on a CarboPac PA1 column using alkaline eluants. This study adopted a strategy which uses specific enzymes to release these disaccharides from their glycosaminoglycan forms. A borohydride reduction reaction was modified to be compatible with the buffer conditions commonly used with these enzymes in order to quantitatively reduce the disaccharides to their alditol forms (thereby stabilizing them to alkaline pH). Chromatography conditions were established which separated all known disaccharide alditol structures from chondroitin sulphate, dermatan sulphate and hyaluronan with extremely high resolution in a single run. Integrated pulsed amperometry was compared to UV absorbance measurement at 232 nm as two sensitive methods for detecting these reduced disaccharides; most of them could be routinely detected in the range of 50-500 ng. Data are presented applying this method to quantify hyaluronan in a biological sample which contains similar to 5000 cells and only similar to 10 ng of hyaluronan. Additional data are presented to demonstrate that this procedure will also separate oligosaccharide alditols derived from hyaluronan.