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

J. Enriqué Bayón, Lorella Pascolo, José M. Gonzalo-Orden, Jose R. Altonaga, Javier González-Gallego, Cecile Webster, W. Geoffrey Haigh, Matthias Stelzner, Cyndy Pekow, Claudio Tiribelli, J. Donald Ostrow

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Abstract

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.

Original languageEnglish
Pages (from-to)313-321
Number of pages9
JournalThe Journal of Laboratory and Clinical Medicine
Volume138
Issue number5
DOIs
Publication statusPublished - 2001

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Aminolevulinic Acid
Bilirubin
Bile
Dogs
Tritium
Radioactivity
Taurocholic Acid
Hypnotics and Sedatives
General Anesthesia
Anesthetics
Drainage
High Pressure Liquid Chromatography
Urine
Injections

ASJC Scopus subject areas

  • Medicine(all)
  • Pathology and Forensic Medicine

Cite this

Pitfalls in preparation of 3H-unconjugated bilirubin by biosynthetic labeling from precursor 3H-5-aminolevulinic acid in the dog. / Bayón, J. Enriqué; Pascolo, Lorella; Gonzalo-Orden, José M.; Altonaga, Jose R.; González-Gallego, Javier; Webster, Cecile; Haigh, W. Geoffrey; Stelzner, Matthias; Pekow, Cyndy; Tiribelli, Claudio; Ostrow, J. Donald.

In: The Journal of Laboratory and Clinical Medicine, Vol. 138, No. 5, 2001, p. 313-321.

Research output: Contribution to journalArticle

Bayón, JE, Pascolo, L, Gonzalo-Orden, JM, Altonaga, JR, González-Gallego, J, Webster, C, Haigh, WG, Stelzner, M, Pekow, C, Tiribelli, C & Ostrow, JD 2001, 'Pitfalls in preparation of 3H-unconjugated bilirubin by biosynthetic labeling from precursor 3H-5-aminolevulinic acid in the dog', The Journal of Laboratory and Clinical Medicine, vol. 138, no. 5, pp. 313-321. https://doi.org/10.1067/mlc.2001.118746
Bayón, J. Enriqué ; Pascolo, Lorella ; Gonzalo-Orden, José M. ; Altonaga, Jose R. ; González-Gallego, Javier ; Webster, Cecile ; Haigh, W. Geoffrey ; Stelzner, Matthias ; Pekow, Cyndy ; Tiribelli, Claudio ; Ostrow, J. Donald. / Pitfalls in preparation of 3H-unconjugated bilirubin by biosynthetic labeling from precursor 3H-5-aminolevulinic acid in the dog. In: The Journal of Laboratory and Clinical Medicine. 2001 ; Vol. 138, No. 5. pp. 313-321.
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abstract = "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.",
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AU - Gonzalo-Orden, José M.

AU - Altonaga, Jose R.

AU - González-Gallego, Javier

AU - Webster, Cecile

AU - Haigh, W. Geoffrey

AU - Stelzner, Matthias

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AU - Tiribelli, Claudio

AU - Ostrow, J. Donald

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