Elevation of the serum bilirubin diconjugate fraction provides an early marker for cholestasis in the rat

Aims/Methods: During cholestasis, components normally excreted into bile, e.g. bilirubin, accumulate in liver cells and biliary passages. In order to assess the conjugation of bilirubin retained in the hepatocyte during cholestasis, we analyzed the pattern of bilirubin pigments in rat serum and bile, using reversed phase alkaline methanolysis-HPLC. Cholestasis was induced by bile flow interuption for 1 to 2 h. Results: One hour after initiation of cholestaiss, the serum concentration of total bilirubin rose 2-fold due to increase in bilirubin di-(BDC) and monoconjugate (BMC), while unconjugated bilirubin (UCB) decreased by 33%. As a result, the BDC/BMC ratio increased to 1.67±0.20 vs 0.60±0.10 in controls (p<0.01) and the BMC/UCB ratio to 1.0±0.2 vs 0.1±0.1 (p<0.01). After relief of biliary obstruction, biliary output rose to 8.0±0.5 vs 5.5±0.3 μmol • min−1 • kg (p<0.01), and the biliary BDC/BMC ratio to 4.0±0.3 vs 1.5±0.2 (p<0.01). In contrast, the biliary BMC/UCB ratio remained unchanged throughout. Increasing the duration of obstruction to 2 h led to a further increase in the serum BMC/UCB ratio to 2.2±0.3 (p<0.01), but not in the BDC/BMC ratio. Serum aminotransferase activity and the concentration of total bile acids increased 3- and 100-fold above their respective control values. Alkaline phosphatase activity remained unaltered, and electron microscopical features of cholestasis became apparent only after 2 h of biliary obstruction. Conclusions: We suggest that one of the initial events of cholestasis is a more efficient conjugation of bilirubin retained in the hepatocyte. This results in a shift of the equilibrium among bilirubin pigments towards BDC, the end-product of conjugation. Such a shift provides an early marker for cholestasis.