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
复制标题
DOI：
10.1194/jlr.d069336
复制
发表时间：
2017-02-01
影响因子：
6.5
通讯作者：
Bensinger, Steven J.
中科院分区：
文献类型：
--
作者：
Argus, Joseph P.;Yu, Amy K.;Bensinger, Steven J.
稳定同位素标记已成为测定正常和肿瘤细胞脂质代谢参数的重要方法。用于脂肪酸和胆固醇分析的常规方法具有一个或多个限制其用于体外稳定同位素标记研究的效用的问题。为了解决这个问题，我们开发了一种优化的方法，用于从少量稳定的同位素标记的培养细胞中测量脂肪酸和胆固醇。我们证明了定量衍生和提取脂肪酸从广泛的脂质类使用这种方法。重要的是，胆固醇也被回收，尽管产率略低，这提供了从同一样品中定量胆固醇和脂肪酸的机会。虽然我们发现背景污染会干扰少量起始材料中某些脂肪酸的定量，但我们的数据表明，这种优化方法可用于准确测量胆固醇和从少量培养细胞中分离的许多脂肪酸的质量同位素异构体分布。该方法的应用将有助于获得量化流量所需的脂质参数，并更好地了解脂质代谢如何影响细胞功能。
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.