Pitfalls in preparation of H-3-unconjugated bilirubin by biosynthetic labeling from precursor H-3-5-aminolevulinic acid in the dog

We report problems encountered during preparation of tritium-labeled unconj ugated bilirubin (H-3-UCB) from precursor H-3-5-aminolevulinic acid (H-3-AL A) in 2 dogs with external biliary drainage installed into the animals unde r general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of H-3-ALA was administered intravenously in two divided doses, and bile was collecte d for 9 hours. In one animal, taurocholate (TC) infusion was needed to main tain bile flow. H-3-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelii! C, Ostrow JD. J Lab Clin Med 2001; 137:370-3). Based on radioactivity and pigment content , hourly bile collections were pooled to optimize specific activities. Surp risingly, in the first dog, only 2.9% of injected radioactivity was recover ed in bile and only 14.1% in urine, and the specific activities of the crys talline H-3-UCB from the two pools were only 39.5 and 30.0 x 10(3) dpm/mug. High-performance liquid chromatography analysis revealed that only 4% of A LA degraded during 5 minutes in injection solution at pH 6.8. The low incor poration of H-3-ALA and low specific activity of H-3-UCB was apparently cau sed mainly by prior degradation and exchange of labile tritium of the H-3-A LA and probably by enhanced endogenous ALA synthesis caused by the anesthet ic/sedative agents. Revised procedures in the second dog improved the incor poration of H-3-ALA to 11.9% excreted in bile and the specific activity of the crystalline H-3-UCB to 122.0 and 50.8 x 10(3) dpm/mug, while urinary ex cretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing H-3-UCB by biosynthetic labeling from H-3-ALA admini stered to dogs.