The lipids characterization has become an emergent and fundamental issue for many omics sciences, and it plays an important role especially in metabolomics. Against this background, liquid chromatography coupled with an atmospheric pressure source to a mass spectrometer analyzer represents a key instrumentation in order to understand biochemical pathways. The partnership between our mass spec and the biotech labs was established few years ago, to investigate the UBIAD1 enzyme function during zebrafish embryos development. The point was how to demonstrate the activity of the non-mitochondrial enzyme Ubiad1 in the biosynthesis of the Coenzyme Q10 (ubiquinone or CoQ10) by using isotopomers. CoQ10 is synthesized from 4-hydroxy-benzoate and trans-decaprenyl-pyrophosphate, through the intermediate 4-hydroxy--3-polyprenyl benzoate (PPHB). CoQ10 is an important prenyl lipid-soluble antioxidant, available in the membranes of Golgi, which has been demonstrated to play a critical role in protecting the cardiovascular system from oxidative stress. The LC-HRMS method developed was based on the quantitation of the13C-isotope-labeled CoQ10 in zebrafish (Danio rerio) embryos after isotopomeric 4-hydroxy-benzoate administration. The study involved wild-type animals and mutants (called barolo, a null allele of zebrafish Ubiad1), used as metabolomics analysis model. Extraction and liquid chromatography separation protocols were examined and studied. A liquid-liquid extraction of 13C-labeled CoQ10 from zebrafish embryos using n-heptane was selected, followed by a liquid chromatography separation performed with a reverse phase C4 column and finally a mass spectrometry analysis carried out with a LTQ-Orbitrap mass analyzer equipped with an ESI source. This protocol was developed and optimized to analyze the CoQ10, in zebrafish tissue, but it is adaptable to the investigation of many others metabolic lipids classes, such as sterols, fatty acids, triglycerides and so on. Moreover this procedure represents an innovative in vivo fluxomics-suited approach to study and understand the metabolic pathways of several lipids classes involved in physiological pathways and metabolic dysfunctions.

13C-Phenyl Lipid Metabolic Analysis in Zebrafish Embryos by LC-HRMS. From biotech to mass spec.

DAL BELLO, FEDERICA;MARTANO, CHIARA
2014-01-01

Abstract

The lipids characterization has become an emergent and fundamental issue for many omics sciences, and it plays an important role especially in metabolomics. Against this background, liquid chromatography coupled with an atmospheric pressure source to a mass spectrometer analyzer represents a key instrumentation in order to understand biochemical pathways. The partnership between our mass spec and the biotech labs was established few years ago, to investigate the UBIAD1 enzyme function during zebrafish embryos development. The point was how to demonstrate the activity of the non-mitochondrial enzyme Ubiad1 in the biosynthesis of the Coenzyme Q10 (ubiquinone or CoQ10) by using isotopomers. CoQ10 is synthesized from 4-hydroxy-benzoate and trans-decaprenyl-pyrophosphate, through the intermediate 4-hydroxy--3-polyprenyl benzoate (PPHB). CoQ10 is an important prenyl lipid-soluble antioxidant, available in the membranes of Golgi, which has been demonstrated to play a critical role in protecting the cardiovascular system from oxidative stress. The LC-HRMS method developed was based on the quantitation of the13C-isotope-labeled CoQ10 in zebrafish (Danio rerio) embryos after isotopomeric 4-hydroxy-benzoate administration. The study involved wild-type animals and mutants (called barolo, a null allele of zebrafish Ubiad1), used as metabolomics analysis model. Extraction and liquid chromatography separation protocols were examined and studied. A liquid-liquid extraction of 13C-labeled CoQ10 from zebrafish embryos using n-heptane was selected, followed by a liquid chromatography separation performed with a reverse phase C4 column and finally a mass spectrometry analysis carried out with a LTQ-Orbitrap mass analyzer equipped with an ESI source. This protocol was developed and optimized to analyze the CoQ10, in zebrafish tissue, but it is adaptable to the investigation of many others metabolic lipids classes, such as sterols, fatty acids, triglycerides and so on. Moreover this procedure represents an innovative in vivo fluxomics-suited approach to study and understand the metabolic pathways of several lipids classes involved in physiological pathways and metabolic dysfunctions.
2014
1st IMaSS NetWork
Roma
26-27/05/2014
1st IMaSS NetWork. Innovation, food analysis and health care research
20
20
http://www.imass.it/?q=node/38
F. Dal Bello; C. Martano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/153709
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