Protocol for the study of hepatic bilirubin uptake in the isolated perfused rat liver

We present a protocol for the study of bilirubin uptake in the isolated and perfused rat liver. The liver is perfused with an albumin-free saline buffered solution supplemented with glucose, pyruvate and lactate, in the absence of oxygen, at a physiologically low flow rate. Fractions of the venous effluent are collected and analyzed for bilirubin, bilirubin glucuronide and biomarkers of liver integrity. The liver preparation is viable and intact for 1 h after isolation from the general circulation, with constant levels of both bilirubin and bilirubin glucuronide (< 2 nM) in the effluent. Up to 12 boli of 10 nmol bilirubin can be sequentially injected into the portal vein without and with molecules that target sinusoidal membrane transporters of organic anions. Selective inhibition of bilirubin or bilirubin glucuronide uptake is detected as transient peaks in the effluent (Cmax up 6 to 60 nM). This protocol allows collecting repeated observations in the same liver, thus reducing the animal number by a factor of 10.