Radiocarbon tracers provide the specificity for revealing the biological fate of a drug, xenobiotic, or endogenous molecule. However, long-lived isotopes as 14C are inefficiently detected by decay counting methods, and this inefficiency (ie limited sensitivity) detracts from the value afforded by high specificity. Accelerator Mass Spectrometry (AMS) solves the sensitivity limitations through direct counting of 14C atom without regards to its decay properties. As a results, AMS can be used to conduct metabolic studies at the picomole to the attomole level or used to identify macromolecular targets for drugs and toxic compounds. Objectives: We characterized the metabolism of [14C]testosterone in male Japanese Quail (Coturnix japonicus). Our aim was to record the dose retention/excretion behavior by mass balance, and establish metabolite profiles or “metabolic fingerprints” as endpoints of endocrine function in avian species. Experiment: Japanese Quail (n=4) were injected IM with 15 DPM (< 0.00001 microCi) of [14C]testosterone. Cumulative fecal/urates were collected at 2 hr intervals early and longer intervals over a three day study period; the excretory material was dried, digested and the 14C contents were determined by AMS. The 0-2 hr post dose fractions were analyzed (predigestion) by HPLC-AMS using a reversed phased gradient system that developed over 80 min. One-min eluent fractions were collected in a tributyrin carbon diluent and analyzed for 14C contents above background by AMS. Results: The balance data showed quantitative recovery of the dose after 72-hr along a biphasic pattern. At least seven chemically distinct metabolites were apparent in the radiochromatogram, with about 10% of the 14C associated with the intact parent compound. Conclusion: AMS quantifies 14C metabolites down to ~ 2 amol (10^-18), over a 10 amol background, representing a 5-order of magnitude improvement in sensitivity over traditional radiometric detectors. Balance data show that exogenously delivered testosterone is completely eliminated after 72 hr. The AMS measurement is precise to < 3% imprecision and is largely free of matrix effects, facilitating metabolite studies using ambient levels of radioactivity. Wastes qualify as ‘non-radioactive' and can be disposed of accordingly. Acknowledgement. This work was performed in part under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.
Web Page: www.vitaleascience.com
See more of AMS in Low Dose Biosciences Posters
See more of The 10th International Conference on Accelerator Mass Spectrometry (September 5-10, 2005)