Received for publication March 29, 2005.
Revised May 31, 2005.
Accepted for publication June 9, 2005.

Canalicular and sinusoidal disposition of bilirubin mono- and diglucuronides in sandwich-cultured human and rat primary hepatocytes

Abstract

Due to cholestasis or adverse drug effects, the excretion of bilirubin conjugates can decrease therefore the level of bilirubin (B) and bilirubin glucuronides (BG) increases in the serum with the concomitant shift of bilirubin di- versus monoglucuronide (BDG/BMG) equilibrium. The aim was to utilize the collagen-sandwich culture of hepatocytes as an in vitro model for studying B conjugation and canalicular versus sinusoidal disposition of BG. Canalicular and sinusoidal efflux of BMG and BDG obtained in sandwich-cultured rat primary hepatocytes was compared with that measured in human hepatocyte cultures. The BMG and BDG were separated by HPLC and identified by MS. Biliary excretion index (BEI) was estimated by measuring disposition of BG into standard and Ca²⁺, Mg²⁺ free medium. Significantly more BG were excreted into the canalicular networks than into the medium in 96-h sandwich culture of both human and rat hepatocytes (BEI: 62.5 and 80.6, respectively). The BDG/BMG ratio in the medium versus in the canalicular networks was 0.55/1.48, which is similar to the serum/bile values (0.6/1.5) observed in vivo by Mesa et al (1997). In contrast BEI for p-nitrophenol glucuronide (pNPG) was 5.2. The low BEI value is in agreement with empirical observations, which suggest that molecules with low molecular weight are preferably excreted by the kidney. In conclusion, sandwich-cultured primary hepatocytes provide a useful in vitro method to differentiate between sinusoidal and canalicular disposition of BG. Since the normal BDG/BMG ratio changes in hyperbilirubinemia, this model could be used to predict drug effects leading to hyperbilirubinemia.

Key words: ABC transporters, glucuronidation, hepatobiliary disposition, HPLC, mass spectrometry, MRP, UDP glucuronyltransferases