High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and HPLC

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur to make possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in Subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high-molecular mass and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ~10 min and Reverse Phase (RP)-HPLC in fluorescence in ~21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in Patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes as regards both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment