Hyaluronans: Crystallization-promoting activity and HPLC analysis of urinary excretion

Abstract

This work follows from our earlier report of a crystallization-promoting population in the leading electrophoretic fractions of pooled urinary glycosaminoglycan extracts of renal stone formers (GAG(SF)), but not in those of healthy individuals (GAG(H)). Preliminary HPLC analysis of disaccharide products of the fractions indicated the presence of chondroitin sulfates (CS) and hyaluronan (HA). Because both commercial CS and urinary CS of healthy individuals showed a basal crystallization-promoting property, we hypothesized that the observed property was due to HA excreted by SF and not by healthy individuals. Defined quantities of a commercial preparation of reference HA (pig skin, 40 to 60 kD) were first tested in urine ultrafiltrate (prepared at pH 5.3, 1250 mosmol/kg and nominal cutoff of 10 kD) by a freezing procedure to assess the crystallization-promoting property. The reference HA moderately promoted crystallization, yielding fewer crystals (5 to 10 times those in neat urine ultrafiltrate) than similar hexuronate concentrations of the HA-containing GAG(SF) population (25 to 30 times those in neat urine ultrafiltrate) reported earlier. Urinary GAG were then recovered individually from random urine samples of SF and healthy individuals. Products of sequential digestion by Streptomyces hyaluronidase and then chondroitinase ABC were analyzed by HPLC. Both GAG(SF) and GAG(H) indicated the presence of HA. Taken together, these results suggest that the crystallization-promoting property of HA(SF) does not generally apply to all HA preparations and that although healthy individuals also excrete HA, HA(H) does not share the property of HA(SF).