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An Optimized Method for Measuring Fatty Acids and Cholesterol in Stable Isotope Labeled Cells.

An Optimized Method for Measuring Fatty Acids and Cholesterol in Stable Isotope Labeled Cells.
Author Information (click to view)

Argus JP, Yu AK, Wang ES, Williams KJ, Bensinger SJ,


Argus JP, Yu AK, Wang ES, Williams KJ, Bensinger SJ, (click to view)

Argus JP, Yu AK, Wang ES, Williams KJ, Bensinger SJ,

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Journal of lipid research 2016 12 14() pii jlr.D069336
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 acid 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 indicates 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.

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