Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

13C-isotope-based protocol for prenyl lipid metabolic analysis in zebrafish embryos

Nature Protocols volume 8, pages 2337–2347 (2013)Cite this article

Subjects

Abstract

Metabolism has a decisive role in many fundamental biological processes, including organism development and tissue homeostasis. Here we describe a protocol for fast and reliable 13C-isotope-based in vivo metabolic profiling. This protocol covers the loading of isotope precursor; extraction, preparation and quantification of the labeled lipid metabolites (e.g., the prenyl lipid CoQ10) by the means of HPLC-MS; and its analysis in zebrafish embryos. This protocol can be applied to different types of experimental settings, including tissue-specific metabolic analyses or dynamic metabolic changes that occur during vertebrate embryogenesis. The protocol takes 5–7 d to complete, requiring minimal equipment and analytical expertise, and it represents a unique alternative to the existing ex vivo (e.g., cell lines) isotope-based metabolic methods. This procedure represents a valuable approach for researchers interested in studying the effect of gene manipulation on lipid metabolism in zebrafish and in understanding the genetic conditions that result in metabolism dysfunction.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of PPHB formation and CoQ10 synthesis mediated by UbiA domain–containing prenyltransferase enzymes.
Figure 2: Representative scheme of the workflow for prenyl lipid 13C-based analysis in zebrafish embryos.
Figure 3: HPLC-MS analysis of zebrafish lipid extracts after delivery of 13C6-4-hydroxy-benzoate.

Similar content being viewed by others

References

  1. Crown, S.B. & Antoniewicz, M.R. Parallel labeling experiments and metabolic flux analysis: past, present and future methodologies. Metab. Eng. 16C, 21–32 (2012).

    Google Scholar 

  2. Klein, S. & Heinzle, E. Isotope labeling experiments in metabolomics and fluxomics. Wiley Interdiscip. Rev. Syst. Biol. Med. 4, 261–272 (2012).

    Article  CAS  Google Scholar 

  3. Kalen, A., Appelkvist, E.L., Chojnacki, T. & Dallner, G. Nonaprenyl-4-hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in the endoplasmic reticulum-Golgi system of rat liver. J. Biol. Chem. 265, 1158–1164 (1990).

    CAS  PubMed  Google Scholar 

  4. Bequette, B.J., Sunny, N.E., El-Kadi, S.W. & Owens, S.L. Application of stable isotopes and mass isotopomer distribution analysis to the study of intermediary metabolism of nutrients. J. Anim. Sci. 84 (suppl.), E50–E59 (2006).

    Article  Google Scholar 

  5. Mugoni, V. et al. Ubiad1 is an antioxidant enzyme that regulates eNOS activity by CoQ10 synthesis. Cell 152, 504–518 (2013).

    Article  CAS  Google Scholar 

  6. Fahy, E. et al. Update of the LIPID MAPS comprehensive classification system for lipids. J. Lipid Res. 50 (suppl.), S9–S14 (2009).

    Article  Google Scholar 

  7. Fahy, E. et al. A comprehensive classification system for lipids. J. Lipid Res. 46, 839–861 (2005).

    Article  CAS  Google Scholar 

  8. Raamsdonk, L.M. et al. A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations. Nat. Biotechnol. 19, 45–50 (2001).

    Article  CAS  Google Scholar 

  9. Zamboni, N. 13C metabolic flux analysis in complex systems. Curr. Opin. Biotechnol. 22, 103–108 (2011).

    Article  CAS  Google Scholar 

  10. Trethewey, R.N. Gene discovery via metabolic profiling. Curr. Opin. Biotechnol. 12, 135–138 (2001).

    Article  CAS  Google Scholar 

  11. Lv, H. Mass spectrometry-based metabolomics towards understanding of gene functions with a diversity of biological contexts. Mass Spectrom. Rev. 32, 118–128 (2013).

    Article  CAS  Google Scholar 

  12. Fiehn, O. Metabolomics--the link between genotypes and phenotypes. Plant Mol. Biol. 48, 155–171 (2002).

    Article  CAS  Google Scholar 

  13. Adamski, J. & Suhre, K. Metabolomics platforms for genome wide association studies-linking the genome to the metabolome. Curr. Opin. Biotechnol. 24, 39–47 (2013).

    Article  CAS  Google Scholar 

  14. Crane, F.L. Biochemical functions of coenzyme Q10. J. Am. Coll. Nutr. 20, 591–598 (2001).

    Article  CAS  Google Scholar 

  15. Bentinger, M., Dallner, G., Chojnacki, T. & Swiezewska, E. Distribution and breakdown of labeled coenzyme Q10 in rat. Free Radic. Biol. Med. 34, 563–575 (2003).

    Article  CAS  Google Scholar 

  16. Huang, S.M., Xu, F., Lam, S.H., Gong, Z. & Ong, C.N. Metabolomics of developing zebrafish embryos using gas chromatography- and liquid chromatography-mass spectrometry. Mol. Biosyst. 9, 1372–1380 (2013).

    Article  CAS  Google Scholar 

  17. Nath, A.K. et al. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure. FASEB J. 27, 1928–1938 (2013).

    Article  CAS  Google Scholar 

  18. Hayashi, S. et al. A novel application of metabolomics in vertebrate development. Biochem. Biophys. Res. Commun. 386, 268–272 (2009).

    Article  CAS  Google Scholar 

  19. Ong, E.S., Chor, C.F., Zou, L. & Ong, C.N. A multi-analytical approach for metabolomic profiling of zebrafish (Danio rerio) livers. Mol. Biosyst. 5, 288–298 (2009).

    Article  CAS  Google Scholar 

  20. Mootha, V.K. & Hirschhorn, J.N. Inborn variation in metabolism. Nat. Genet. 42, 97–98 (2010).

    Article  CAS  Google Scholar 

  21. Weiss, R.H. & Kim, K. Metabolomics in the study of kidney diseases. Nat. Rev. Nephrol. 8, 22–33 (2012).

    Article  CAS  Google Scholar 

  22. Jin, S.W. et al. A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos. Dev. Biol. 307, 29–42 (2007).

    Article  CAS  Google Scholar 

  23. Bentinger, M. et al. Polyisoprenoid epoxides stimulate the biosynthesis of coenzyme Q and inhibit cholesterol synthesis. J. Biol. Chem. 283, 14645–14653 (2008).

    Article  CAS  Google Scholar 

  24. Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M. & Altman, D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  Google Scholar 

  25. Rosen, J.N., Sweeney, M.F. & Mably, J.D. Microinjection of zebrafish embryos to analyze gene function. J. Vis. Exp. 2009, 1115 (2009).

    Google Scholar 

  26. Singleman, C. & Holtzman, N.G. Heart dissection in larval, juvenile and adult zebrafish, Danio rerio. J. Vis. Exp. 2011, 3165 (2011).

    Google Scholar 

  27. Gupta, T. & Mullins, M.C. Dissection of organs from the adult zebrafish. J. Vis. Exp. 2011, 2839 (2010).

    Google Scholar 

  28. Turunen, M., Olsson, J. & Dallner, G. Metabolism and function of coenzyme Q. Biochim. Biophys. Acta 1660, 171–199 (2004).

    Article  CAS  Google Scholar 

  29. Link, V., Shevchenko, A. & Heisenberg, C.P. Proteomics of early zebrafish embryos. BMC Dev. Biol. 6, 1 (2006).

    Article  Google Scholar 

  30. United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the welfare of animals in experimental neoplasia (second edition). Br. J. Cancer 77, 1–10 (1998).

Download references

Acknowledgements

M.M.S.'s laboratory was supported by research grants from the Marie Curie Action IRG 247852, Telethon GGP10195 and AIRC MFAG-8911. We thank E.J. Corcoran for editorial assistance.

Author information

Authors and Affiliations

  1. Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy

    Vera Mugoni, Claudio Medana & Massimo M Santoro

  2. Laboratory of Endothelial Molecular Biology, Vesalius Research Center, Vlaams Instituut voor Biotechnologie (VIB), Leuven, Belgium

    Massimo M Santoro

  3. Department of Oncology, Laboratory of Endothelial Molecular Biology, Vesalius Research Center, Katholieke Universiteit (KU) Leuven, Leuven, Belgium

    Massimo M Santoro

Authors
  1. Vera Mugoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudio Medana
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Massimo M Santoro
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.M. and M.M.S. designed the study. V.M., C.M. and M.M.S. performed the experiments, analyzed the data and discussed this study. V.M., C.M. and M.M.S. wrote the manuscript.

Corresponding author

Correspondence to Massimo M Santoro.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mugoni, V., Medana, C. & Santoro, M. 13C-isotope-based protocol for prenyl lipid metabolic analysis in zebrafish embryos. Nat Protoc 8, 2337–2347 (2013). https://doi.org/10.1038/nprot.2013.139

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.139

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing