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Detection and quantification of stable isotope incorporation by high-resolution mass spectrometry and 13C{2H,1H} nuclear magnetic resonance difference spectroscopy. Utilisation of carbon and hydrogen atoms of ethanol in the biosynthesis of bile acids

  • D. M. Wilson
  • A. L. Burlingame
  • S. Evans
  • T. Cronholm
  • J. Sjövall

Abstract

We have been interested for some time in developing reliable and sensitive methods of detecting stable isotope incorporation in steroids. The focus of this interest has been the transfer of deuterium and 13C atoms from labelled ethanol to bile acids, which can be isolated in rather large amounts using bile fistula rats. Initially, low-resolution GC—MS—computer techniques were used to calculate heavy isotope excess at certain characteristic m/es (Cronholm, Burlingame and Sjövall, 1974). For example, Figure 18.1 shows the differently labelled molecular species of cholesterol and chenodeoxycholic acid that result from continuous administration of [2,2,2-2 H3]-ethanol. The filled bars show the distributions of labelled species actually found. However, the sites and extents of deuterium incorporation along the steroid skeleton cannot be directly determined by GC—MS. Instead, using a number of assumptions and known incorporation sites of the atoms transferred from ethanol to the steroid, one can construct a mathematical model to generate theoretical distributions, which tend to support or disprove the initial premises. The open bars in Figure 18.1 show a theoretical distribution, arrived at by such an approach. However, the method is severely underdetermined, particularly in the case of [1,1-2 H2] -ethanol metabolism. In this case, deuterium is lost immediately and can be incorporated only via NADPH-dependent reductions later on in steroid biosynthesis. There may, for example, be different NADPH pools for mevalonate synthesis and for later reductions in the completed steroid skeleton, leading to different levels of incorporation per site.

Keywords

Bile Acid Difference Spectrum Chenodeoxycholic Acid Deuterium Incorporation Lithium Aluminium Hydride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Biemann, K. (1962). Mass Spectrometry: Organic Chemical Applications, McGraw-Hill, New YorkGoogle Scholar
  2. Cronholm, T., Burlingame, A. L. and Sjövall, J. (1974). Europ. J. Biochem., 49, 497CrossRefGoogle Scholar
  3. Cronholm, T., Makino, I. and Sjövall, J. (1972). Europ. J. Biochem., 24, 507CrossRefGoogle Scholar
  4. Watson, J. A., Fang, M. and Lowenstein, J. M. (1969). Arch. Biochem. Biophys., 135, 209CrossRefGoogle Scholar
  5. Wilson, D. M., Burlingame, A. L., Cronholm, T. and Sjövall, J. (1974a). Biochem. Biophys. Res. Commun., 56, 828CrossRefGoogle Scholar
  6. Wilson, D. M., Burlingame, A. L., Cronholm, T. and Sjövall, J. (1975). Proceedings of the Second International Conference on Stable Isotopes (ed. E. R. Klein and P. D. Klein), Oak Brook, Illinois, U.S.A., p. 485. National Technical Information Service Document CONF-751027Google Scholar
  7. Wilson, D. M., Olsen, R. W. and Burlingame, A. L. (1974b). Rev. Sci. Instrum., 45, 1095CrossRefGoogle Scholar

Copyright information

© The Contributors 1978

Authors and Affiliations

  • D. M. Wilson
    • 1
  • A. L. Burlingame
    • 1
  • S. Evans
    • 2
  • T. Cronholm
    • 3
  • J. Sjövall
    • 3
  1. 1.Space Sciences LaboratoryUniversity of CaliforniaBerkeleyUSA
  2. 2.AEI Scientific Apparatus LtdManchesterUK
  3. 3.Kemiska InstitutionenKarolinska InstitutetStockholmSweden

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