Effect of Intravenous Bilirubin Infusion on Biliary Phospholipid Secretion, Hepatic P-Glycoprotein Expression, and Biliary Cytotoxicity in Pigs

Abstract

Background: Infusion of large intravenous bilirubin loads in bile acid-depleted pigs reduces P-glycoprotein-dependent biliary phospholipid secretion and increases the cytotoxicity of bile. The reasons for the diminution of biliary phospholipid secretion and the increase in biliary cytotoxicity are not known. This study was undertaken to determine whether the bilirubin-induced lowering of biliary phospholipid secretion is associated with alterations in hepatic P-glycoprotein (P-gp) expression and to determine why bilirubin infusions increase biliary cytotoxicity. Methods: Hepatic bile was collected from bile acid-depleted pigs before and during intravenous bilirubin infusion. Hepatic P-gp expression was measured with protein blot analysis, using the P-gp-specific antibody C219. Biliary cytotoxicity was assayed against erythrocytes. The biliary phospholipid fatty acid profile was determined by means of gas chromatography. Results: Bilirubin infusions lowered biliary phospholipid secretion by 69% without changing hepatic P-gp expression, suggesting that bilirubin infusions have an inhibitory effect on hepatic P-gp activity. Bilirubin infusions did not cause P-gp losses into bile. An unequivocal, proportional relationship (r² = 0.80) pertained between cytotoxicity and the bile acid to phospholipid ratio in bile secreted before and during bilirubin infusion and in phosphatidylcholine-supplemented bile. Unconjugated bilirubin in bile did not contribute to biliary cytotoxicity. Biliary phospholipids were always phosphatidylcholine > phosphatidylethanolamine, mainly of C_{16:0, 18:2} and C_{16:0, 18:1} fatty acid configuration. Conclusions: Intravenous bilirubin loads reduce biliary phospholipid secretion without changing hepatic P-gp expression. Bilirubin infusions increase biliary cytotoxicity by augmenting the biliary bile acid to phospholipid ratio.