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 th e structural analysis of glycosaminoglycans is a definitive compositio nal analysis of its characteristic disaccharide repeat structures. Cur rent chromatographic or electrophoretic procedures may have limitation s in analysing glycosaminoglycan samples that are in low abundance, co ntain novel structures that need to be further characterized, or are m etabolically labelled from radioactive precursors as a result of biosy nthetic experiments. This study presents a new methodology for analysi ng disaccharides and oligosaccharides derived from chondroitin sulphat e, dermatan sulphate and hyaluronan that fulfils the above criteria. T he procedure involves the separation of reduced forms of these glycoco njugates on a CarboPac PA1 column using alkaline eluants. This study a dopted a strategy which uses specific enzymes to release these disacch arides from their glycosaminoglycan forms. A borohydride reduction rea ction was modified to be compatible with the buffer conditions commonl y used with these enzymes in order to quantitatively reduce the disacc harides to their alditol forms (thereby stabilizing them to alkaline p H). Chromatography conditions were established which separated all kno wn disaccharide alditol structures from chondroitin sulphate, dermatan sulphate and hyaluronan with extremely high resolution in a single ru n. Integrated pulsed amperometry was compared to UV absorbance measure ment at 232 nm as two sensitive methods for detecting these reduced di saccharides; most of them could be routinely detected in the range of 50-500 ng. Data are presented applying this method to quantify hyaluro nan in a biological sample which contains similar to 5000 cells and on ly similar to 10 ng of hyaluronan. Additional data are presented to de monstrate that this procedure will also separate oligosaccharide aldit ols derived from hyaluronan.