title: An optimized method for measuring fatty acids and cholesterol in stable isotope-labeled cells.
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.
authors.affiliation: Departments of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Argus
authors.affiliation: Departments of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Amy K
authors.initials: AK
authors.lastname: Yu
authors.affiliation: Departments of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Wang
authors.affiliation: Departments of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Williams
authors.affiliation: Departments of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095 sbensinger@mednet.ucla.edu.|Departments of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Bensinger
mesh_terms: D002460:Cell Line; D002784:Cholesterol / Q000302:isolation & purification* / Q000378:metabolism; D005227:Fatty Acids / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007554:Isotopes / Q000494:pharmacology; D050356:Lipid Metabolism
chemical_list: D005227:Fatty Acids; D007554:Isotopes; D002784:Cholesterol
keywords: lipids/chemistry; mass spectrometry; neutral lipids; sphingolipids; stable isotope labeling