An improved method for isolation of unconjugated bilirubin from rat and dog bile

Radiolabeled unconjugated bilirubin (UCB) is currently prepared by biosynthetic labeling of bilirubin in fistula bile from precursor-labeled δ-aminolevulinic acid (ALA) in rats or dogs. With existing methods, yields of labeled UCB from the bile are generally less than 50%. We here report modifications of the original method of Ostrow et al (Ostrow JD, Hammaker L, Schmid R. The preparation of crystalline bilirubin-C14. J Clin Invest 1961;40:1442-52) that result in improvement of yields to 72% from both dog and rat bile. The modifications include the initial deproteination of bile with a reverse-phase C18 cartridge, removal of ethanol before alkaline hydrolysis to avoid esterification of UCB, and adjustments for the high proportion of non-glucuronide UCB conjugates in dog bile not precipitated as lead salts. These improvements should save significantly on both costs and animal usage.