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.
