Submitted on November 13, 2007
Revised on January 24, 2008
Accepted on January 25, 2008
Separation of cellular non-polar neutral lipids by normal phase chromatography and analysis by electrospray ionization mass spectrometry
Patrick M. Hutchins, Robert M. Barkley, and Robert C. Murphy
Pharmacology, University of Colorado Denver, Aurora, CO 80045-0511
Corresponding Author: robert.murphy{at}uchsc.edu
Neutral lipids are an important class of hydrophobic compounds found in all cells which 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 cholesterol esters, monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids could be detected such as ubiquinone–9 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 over 20 cholesterol ester molecular species revealed that challenging RAW 264.7 cells with a Toll–like receptor 4 agonist caused over a 20–fold increase in the content of cholesterol esters within cells and uniquely those cholesterol ester molecular species which contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain cholesterol ester molecular species did not change in response to activation of these cells.
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