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J Lipid Res. 2017 Feb;58(2):460-468. doi: 10.1194/jlr.D069336. Epub 2016 Dec 14.

An optimized method for measuring fatty acids and cholesterol in stable isotope-labeled cells.

Author information

1
Departments of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
2
Departments of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
3
Departments of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095 sbensinger@mednet.ucla.edu.

Abstract

Stable isotope labeling has become an important methodology for determining lipid metabolic parameters of normal and neoplastic cells. Conventional methods for fatty acid and cholesterol analysis have one or more issues that limit their utility for in vitro stable isotope-labeling studies. To address this, we developed a method optimized for measuring both fatty acids and cholesterol from small numbers of stable isotope-labeled cultured cells. We demonstrate quantitative derivatization and extraction of fatty acids from a wide range of lipid classes using this approach. Importantly, cholesterol is also recovered, albeit at a modestly lower yield, affording the opportunity to quantitate both cholesterol and fatty acids from the same sample. Although we find that background contamination can interfere with quantitation of certain fatty acids in low amounts of starting material, our data indicate that this optimized method can be used to accurately measure mass isotopomer distributions for cholesterol and many fatty acids isolated from small numbers of cultured cells. Application of this method will facilitate acquisition of lipid parameters required for quantifying flux and provide a better understanding of how lipid metabolism influences cellular function.

KEYWORDS:

lipids/chemistry; mass spectrometry; neutral lipids; sphingolipids; stable isotope labeling

PMID:
27974366
PMCID:
PMC5282944
DOI:
10.1194/jlr.D069336
[Indexed for MEDLINE]
Free PMC Article

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