Abstract
Due to cholestasis or adverse drug effects, the excretion of bilirubin conjugates can decrease; therefore, the level of bilirubin (B) and bilirubin glucuronides (BGs) increases in the serum with the concomitant shift of bilirubin diversus monoglucuronide (BDG/BMG) equilibrium. The aim of this study was to utilize the collagen-sandwich culture of hepatocytes as an in vitro model for studying B conjugation and canalicular versus sinusoidal disposition of BGs. 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 high-performance liquid chromatography and identified by mass spectrometry. The biliary excretion index (BEI) was estimated by measuring disposition of BGs into standard and Ca2+, Mg2+-free medium. Significantly more BGs 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 that 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. [Mesa VA, De Vos R, and Fevery J (1997) J Hepatol 27:912–916]. In contrast, the BEI for p-nitrophenol glucuronide 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 BGs. Since the normal BDG/BMG ratio changes in hyperbilirubinemia, this model could be used to predict drug effects leading to hyperbilirubinemia.
Footnotes
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This work was supported by National Research and Development Programme (NKFP) MediChem2 Grant 1/A/005/2004.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.004481.
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ABBREVIATIONS: B, bilirubin; BMG, bilirubin monoglucuronide; BDG, bilirubin diglucuronide; BG, bilirubin glucuronide; pNP, p-nitrophenol; pNPG, p-nitrophenol glucuronide; MRP, multidrug resistance-associated protein; UGT, UDP-glucuronosyltransferase; HBSS, Hanks' balanced salt solution; HPLC, high-performance liquid chromatography; BEI, biliary excretion index; Q1 scan, the first quadrupole is scanning.
- Received February 24, 2005.
- Accepted June 9, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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