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.

Keywords: lipids/chemistry; mass spectrometry; neutral lipids; sphingolipids; stable isotope labeling.

Fig. 1.

The current method results in a significantly higher yield of VLC saturates and monounsaturates from cultured cells. H1299 cells were collected at 85% confluency from 60 mm plates in triplicate and were analyzed using the current method or a FEFA method (45°C, 16 h). Values represent micrograms on the plate normalized to a 19:0 methyl ester added before derivatization, then further normalized to the average result of the FEFA method. Data are presented as the mean ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01

Fig. 2.

FAME MIDs collected from stable isotope-labeled cells using the current method. H1299 cells were brought to isotopic steady state in DMEM containing 100% ¹³C₆-glucose and 5% FBS. Cells were collected at 85% confluency from 60 mm plates in triplicate and were analyzed using the current method. A: 22:4 n-6. B: 24:1 n-9. Data are presented as the mean ± SD (n = 3).