J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.M700521-JLR200 on January 25, 2008

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Journal of Lipid Research, Vol. 49, 804-813, April 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry

Patrick M. Hutchins, Robert M. Barkley and Robert C. Murphy1

Department of Pharmacology, University of Colorado Denver, Aurora, CO 80045-6511

Published, JLR Papers in Press, January 25, 2008.

1 To whom correspondence should be addressed. e-mail: robert.murphy{at}uchsc.edu

Neutral lipids are an important class of hydrophobic compounds found in all cells that play critical roles from energy storage to signal transduction. Several distinct structural families make up this class, and within each family there are numbers of individual molecular species. A solvent extraction protocol has been developed to efficiently isolate neutral lipids without complete extraction of more polar phospholipids. Normal-phase HPLC was used for the separation of cholesteryl esters (CEs), monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids such as ubiquinone-9 could be detected in RAW 264.7 cells. Molecular species that make up each neutral lipid class can be analyzed both qualitatively and quantitatively by on-line LC-MS and LC-MS/MS strategies. The quantitation of >20 CE molecular species revealed that challenging RAW 264.7 cells with a Toll-like receptor 4 agonist caused a >20-fold increase in the content of CEs within cells, particularly those CE molecular species that contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain CE molecular species did not change in response to the activation of these cells.

Supplementary key words cholesteryl esters • triacylglycerol • monoalkyl diacylglycerol • ubiquinone-9 • high-performance liquid chromatography • molecular species • RAW 264.7 • Kdo2-lipid A

Abbreviations: CE, cholesteryl ester; DAG, diacylglycerol; DPBS, Dulbecco's phosphate-buffered saline; MeDAG, monoalkylether diacylglycerol; MTBE, methyl tert-butyl ether; NP, normal-phase; TAG, triacylglycerol


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