Abstract
We report problems that were encountered during preparation of labeled 3H-unconjugated bilirubin (3H-UCB) from precursor 3H-4-aminolevulinic acid (3H-ALA), in two dogs with external biliary drainage installed under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of 3H-ALA was administered i.v. in two divided doses and bile 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, using the improved method of Webster, et al. (1).  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 sp. activities of the crystalline 3H-UCB from the two pools were only 39.5 and 30.0 x 103 dpm/µg.  HPLC analysis revealed that only 4% of ALA degraded during 5 min in injection solution at pH 6.8.  The low incorporation of 3H-ALA and low sp. act. of 3H-UCB was apparently due mainly to prior degradation and exchange of labile tritium of the 3H-precursor, and probably to 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 sp. act. of the crystalline 3H-UCB to 122.0 and 50.8 x 103 dpm/µg, but urinary excretion increased to 28.5%.  These experiences emphasize possible pitfalls in preparing 3H-UCB by biosynthetic labeling of from 3H-ALA administered to dogs.

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