Effects of Urinary Macromolecules on Hydroxyapatite Crystal Formation

Particle size analysis was combined with titration data obtained in constant-composition, hydroxyapatite (HA)- seeded, crystal growth assays. With addition of large amounts of HA (250 g), titration rates were linear, new crystal forma- tion was minimal, and aggregation effects could be detected. With addition of small amounts of HA (62.5g), nucleation of new HA was observed. The effects of urinary macromolecules, i.e., osteopontin (OPN), recombinant glutathione-S-trans- ferase-OPN (G-OPN), Tamm-Horsfall protein, chondroitin sul- fate, human serum albumin, mixed urinary macromolecules from a stone-former (SFU1), mixed urinary macromolecules from a normal individual (NU1), and polyaspartic acid (PA), were examined in this system. Crystal growth inhibition, as measured by the slope of linear titration curves in this system, was observed with PA, G-OPN, OPN, SFU1, and NU1. All of the macromolecules tested inhibited aggregation, including Tamm-Horsfall protein, which did not inhibit growth. As re- flected by the ratio of the final number of particles to the initial number in the 62.5-g seed addition, the macromolecules that were most effective in inhibiting growth, i.e., OPN, G-OPN, PA, SFU1, and NU1, actually increased secondary nucleation. Recombinant G-OPN demonstrated less inhibitory activity than did OPN isolated from cell culture. Chondroitin sulfate and human serum albumin exhibited no significant effects on the various components of HA crystallization under these conditions. SFU1 and NU1 slowed growth and increased sec- ondary nucleation to similar degrees, and neither exhibited any measurable effect on aggregation. Therefore, crystal surface sites that participate in nucleation, growth, and aggregation processes are affected independently by macromolecules, pre- sumably because of differences in their structural features. These results illustrate the utility of combining these tech- niques to provide a much greater understanding of crystalliza- tion behavior than that possible with either analysis alone.