Tritiated bilirubin: preparation and physiological studies

Abstract

A procedure is described for the preparation of tritiated bilirubin by the Wilzbach technique, either directly (preparation W) or in conjunction with irradiation from a Co⁶⁰ source (preparation C-W). After equilibration of the preparations with polar and nonpolar solvents to remove labile tritium and repeated precipitation, the constant specific activities were 110 µc/mg (W) and 30 µc/mg (C-W), respectively. Radiochemical purity of the bilirubin-H³ preparations and their azobilirubin-H³ derivatives was demonstrated by paper chromatography. Bilirubin-H³ and a high dose of carrier bilirubin were injected simultaneously into rats and the comparative bilirubin levels in serum and bile were determined by radioactivity and colorimetric methods. After 75 min, 20–30% of the initial label remained in the serum as nonbilirubin radioactivity. Pre-equilibration in vitro of bilirubin-H³ with albumin removed the protein-labile tritium. It was concluded that the adsorption of bilirubin to serum protein may involve hydrogen binding at an aliphatic group on the bilirubin molecule. In serum and bile the distribution of the bilirubin-H³ (pre-equilibrated with albumin in vitro) was identical to the distribution of bilirubin measured colorimetrically. The half-life for disappearance of bilirubin from serum in these rats was 25–35 min.