Rapid association of unconjugated bilirubin with amorphous calcium phosphate

The association of unconjugated bilirubin (UCB) with amorphous calcium phosphate was studied in vitro. To this end UCB, solubilized in different micellar bile salt solutions, was incubated with freshly prepared calcium phosphate precipitate. It was demonstrated that amorphous calcium phosphate (ACP) rapidly binds and precipitates UCB in a dose-dependent way. The results indicate that binding of UCB to ACP is specific: binding to barium phosphate was negligible and addition of low amounts of Mg2+ before formation of the calcium phosphate precipitate (Ca:Mg = 5:1) inhibited binding by 80%. Free Ca2+ stimulated binding, whereas free phosphate ions inhibited binding of UCB in taurocholate solutions and to a lesser extent in glycocholate solutions. The apparent affinity of UCB for amorphous calcium phosphate was different in the various bile salt solutions. Binding of UCB decreased at pH > 8.5 in taurocholate solutions, but not in glycocholate solutions where binding of UCB was constant from pH 7.5-10.5. We propose a model in which UCB directly binds to amorphous calcium phosphate in the presence of bile salts that weakly interact with ACP, like taurocholate. In the presence of bile salts that strongly interact with ACP, such as glycochenodeoxycholate, binding of UCB may also occur via the bile salt. In conditions of unconjugated hyperbilirubinemia, such as the Crigler-Najjar syndrome, neonatal jaundice, and in the Gunn rat, considerable amounts of UCB diffuse across the intestinal mucosa. Binding of UCB to calcium phosphate in the intestine may stimulate its excretion and thereby constitute a relevant mechanism of excretion.