In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes.

Journal of Lipid Research
Regina EnsenauerAdelbert A Roscher

Abstract

Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relev...Continue Reading

References

Mar 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·S V Pande
Oct 1, 1968·Archives of Biochemistry and Biophysics·S PasseronI Harary
Sep 28, 1984·Biochemical and Biophysical Research Communications·R J WandersJ M Tager
Nov 24, 1982·Clinica Chimica Acta; International Journal of Clinical Chemistry·S KølvraaN Hobolth
Dec 1, 1996·The Biochemical Journal·S EatonM Pourfarzam
Oct 18, 2005·The British Journal of Dermatology·F CasparyJ D Nieland
Jul 27, 2007·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Malcolm A KingDerek C Davies
Jan 18, 2008·Nature Reviews. Molecular Cell Biology·Deborah M Muoio, Christopher B Newgard
Feb 21, 2009·The New England Journal of Medicine·Jean-Paul BonnefontFatima Djouadi
Jul 25, 2009·Journal of Lipid Research·Kellen BrunaldiJames A Hamilton
Feb 17, 2010·Endocrine Reviews·Mary-Elizabeth Patti, Silvia Corvera

❮ Previous
Next ❯

Citations

Mar 7, 2013·Analytical Biochemistry·Fang Ye, Charles L Hoppel
Sep 15, 2004·Molecular Aspects of Medicine·Rona R Ramsay, Victor A Zammit
Apr 25, 2018·Toxicological Sciences : an Official Journal of the Society of Toxicology·David GrünigStephan Krähenbühl
Jun 21, 2020·Rapid Communications in Mass Spectrometry : RCM·Yang ZhouSiqi Liu
May 10, 2018·Frontiers in Pharmacology·David GrünigStephan Krähenbühl

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.