Effects of urinary macromolecules on hydroxyapatite crystal formation

Particle size analysis was combined with titration data obtained in constan t-composition, hydroxyapatite (HA)seeded. crystal growth assays. With addit ion of large amounts of HA (250 mug), titration rates were linear, new crys tal formation was minimal, and aggregation effects could be detected. With addition of small amounts of HA (62.5 mug), nucleation of new HA was observ ed. The effects of urinary macromolecules, i.e., osteopontin (OPN), recombi nant glutathione-S-transferase-OPN (G-OPN), Tamm-Horsfall protein, chondroi tin sulfate, 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 sys tem, was observed with PA. G-OPN, OPN, SFU1, and NU1. All of the macromolec ules tested inhibited aggregation, including Tamm-Horsfall protein, which d id not inhibit growth. As reflected by the ratio of the final number of par ticles to the initial number in the 62.5-mug seed addition, the macromolecu les that were most effective in inhibiting growth, i.e., OPN, G-OPN, PA, SF U1, and NU1, actually increased secondary nucleation. Recombinant G-OPN dem onstrated less inhibitory activity than did OPN isolated from cell culture. Chondroitin sulfate and human serum albumin exhibited no significant effec ts on the various components of HA crystallization under these conditions. SFU1 and NU1 slowed growth and increased secondary nucleation to similar de grees, and neither exhibited any measurable effect on aggregation. Therefor e, crystal surface sites that participate in nucleation, growth, and aggreg ation processes are affected independently by macromolecules, presumably be cause of differences in their structural features. These results illustrate the utility of combining these techniques to provide a much greater unders tanding of crystallization behavior than that possible with either analysis alone.