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

We report problems encountered during preparation of tritium-labeled unconjugated bilirubin ( ³H-UCB) from precursor ³H-5-aminolevulinic acid ( ³H-ALA) in 2 dogs with external biliary drainage installed into the animals under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCl of ³H-ALA was administered intravenously in two divided doses, and bile was collected for 9 hours. In one animal, taurocholate (TC) infusion was needed to maintain bile flow. ³H-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelli 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. Surprisingly, in the first dog, only 2.9% of injected radioactivity was recovered in bile and only 14.1% in urine, and the specific activities of the crystalline ³H-UCB from the two pools were only 39.5 and 30.0 × 10 ³ dpm/μg. High-performance liquid chromatography analysis revealed that only 4% of ALA degraded during 5 minutes in injection solution at pH 6.8. The low incorporation of ³H-ALA and low specific activity of ³H-UCB was apparently caused mainly by prior degradation and exchange of labile tritium of the ³H-ALA and probably by enhanced endogenous ALA synthesis caused by the anesthetic/sedative agents. Revised procedures in the second dog improved the incorporation of ³H-ALA to 11.9% excreted in bile and the specific activity of the crystalline ³H-UCB to 122.0 and 50.8 × 10 ³ dpm/μg, while urinary excretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing ³H-UCB by biosynthetic labeling from ³H-ALA administered to dogs.