Accumulation of unconjugated bilirubin in cholesterol pellets implanted in swine gallbladders

Background and Aims: Most cholesterol gallstones have a pigmented center, but it is unclear whether its presence is primary or secondary. This study was performed to determine if bilirubin would accumulate in a gallstone model consisting of cholesterol pellets. Methods: Cholesterol was compressed into pellets at 2500 psi, producing a pellet that behaved like human cholesterol gallstones in regard to penetration of solutes into the stone. Pellets were implanted into gallbladders of pigs and harvested after 4 weeks. Bilirubin species were measured by high-performance liquid chromatography. Results: The proportions of bilirubin species in bile were not changed by the presence of pellets, i.e., diconjugates (mean ± SD, 1.9% ± 1.0% vs. 0.7% ± 0.8%), monoconjugates (83.8% ± 5.5% vs. 87.8% ± 6.6%), and unconjugated bilirubin (14.2% ± 5.3% vs. 11.5% ± 5.6%) were similar at the time of implantation and removal. The cut surfaces of the pellets were pigmented. Pellets contained 5.46 ± 1.38 μg bilirubin/g sample at harvesting, and 98.6% ± 2.3% of bilirubin in pellets was unconjugated. In in vitro studies, there was a large increase in unconjugated bilirubin in the bile. Pellets also became pigmented in vitro, but there was considerable variability in the bilirubin species present in the pellets. Conclusions: Unconjugated bilirubin accumulates in cholesterol pellets and pigments them. This provides a mechanism by which cholesterol gallstones could become secondarily pigmented.