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.

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

MUGONI, VERA;MEDANA, Claudio;SANTORO, Massimo Mattia
2013-01-01

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.
2013
8
2337
2347
http://www.nature.com/nprot/journal/v8/n12/full/nprot.2013.139.html
Vera Mugoni;Claudio Medana;Massimo M Santoro
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/147062
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