Document Detail

Liquid chromatography-high resolution mass spectrometry analysis of fatty acid metabolism.
MedLine Citation:
PMID:  22004349     Owner:  NLM     Status:  MEDLINE    
We present a liquid chromatography/mass spectrometry (LC/MS) method for long-chain and very-long-chain fatty acid analysis and its application to (13)C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C(8) reversed-phase chromatography using a water-methanol gradient with tributylamine as ion pairing agent, ionized by electrospray and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/mL with a linear dynamic range of 100-fold. Ratios of unlabeled to (13)C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of (13)C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC/MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.
Jurre J Kamphorst; Jing Fan; Wenyun Lu; Eileen White; Joshua D Rabinowitz
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-11-04
Journal Detail:
Title:  Analytical chemistry     Volume:  83     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2011 Dec 
Date Detail:
Created Date:  2011-11-30     Completed Date:  2012-03-21     Revised Date:  2014-09-22    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  9114-22     Citation Subset:  IM    
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MeSH Terms
Carbon Isotopes / chemistry
Cell Line
Chromatography, High Pressure Liquid*
Chromatography, Reverse-Phase
Fatty Acids / analysis,  metabolism*
Glucose / pharmacology
Glutamine / pharmacology
Spectrometry, Mass, Electrospray Ionization*
Grant Support
1RC1CA147961-02/CA/NCI NIH HHS; P50 GM071508/GM/NIGMS NIH HHS; P50 GM071508-09/GM/NIGMS NIH HHS; P50GM071508/GM/NIGMS NIH HHS; R01 CA130893/CA/NCI NIH HHS; RC1 CA147961/CA/NCI NIH HHS; RC1 CA147961-02/CA/NCI NIH HHS; //Howard Hughes Medical Institute
Reg. No./Substance:
0/Carbon Isotopes; 0/Fatty Acids; 0RH81L854J/Glutamine; IY9XDZ35W2/Glucose

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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