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. 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. Fatty acids and cholesterol perform essential structural, energetic, and signaling roles in all animal cells (1.Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes..5th edition. Elsevier B.V, Oxford, UK2008Google Scholar). Renewed interest in understanding how cholesterol and fatty acid homeostasis is dynamically modulated in normal cellular states and during pathophysiologic processes (e.g., oncogenic signaling in cancer) has led to increased demand for methodologies that can accurately interrogate these lipid classes (2.Currie E. Schulze A. Zechner R. Walther T.C. Farese R.V. Cellular fatty acid metabolism and cancer.Cell Metab. 2013; 18: 153-161Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, 3.Cyster J.G. Dang E.V. Reboldi A. Yi T. 25-Hydroxycholesterols in innate and adaptive immunity.Nat. Rev. Immunol. 2014; 14: 731-743Crossref PubMed Scopus (216) Google Scholar, 4.Lee S.D. Tontonoz P. Liver X receptors at the intersection of lipid metabolism and atherogenesis.Atherosclerosis. 2015; 242: 29-36Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). One approach that has gained significant favor is the use of stable isotope labeling to quantify how cellular requirements for cholesterol and fatty acid pool sizes are achieved in normal and neoplastic cells (5.Buescher J.M. Antoniewicz M.R. Boros L.G. Burgess S.C. Brunengraber H. Clish C.B. DeBerardinis R.J. Feron O. Fr