title: 13C-isotope-based protocol for prenyl lipid metabolic analysis in zebrafish embryos.
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 (13)C-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.
authors.affiliation: Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.
authors.forename: Vera
authors.initials: V
authors.lastname: Mugoni
authors.forename: Claudio
authors.initials: C
authors.lastname: Medana
authors.forename: Massimo M
authors.initials: MM
authors.lastname: Santoro
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D004122:Dimethylallyltranstransferase / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism; D013058:Mass Spectrometry; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D054876:Prenylation; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D029961:Zebrafish Proteins; D014451:Ubiquinone; D004122:Dimethylallyltranstransferase; C579268:Ubiad1 protein, zebrafish; C024989:coenzyme Q10