High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography

Analytical Biochemistry
T WhiteE Nudelman

Abstract

An improvement of current methods is needed for simple, rapid, and precise quantification of cellular lipids, including rare species of biologically active cellular lipids, such as phosphatidic acid (PA) and diradylglycerol (DG). In addition, further analysis of hydrolyzed acyl chains from these species by methods such as gas chromatography requires complete separations. Methods have been developed for the quantification of neutral lipids and several phospholipids extracted from mammalian cells and sera. Lipid masses were determined for the major classes of the neutral, nonpolar lipids, and of the phospholipids. The lipid classes were separated by a multistep thin-layer chromatography (TLC) procedure in different solvent systems, a method which we have designated as multi-one-dimensional thin-layer chromatography (MOD-TLC). Resolved lipid bands were visualized by the lipophilic dye primulin (direct yellow 59) and scanned by an automated laser-fluorescence detector. The mass of each band was determined by comparing band intensities of unknown samples to dilution curves of authentic standards. With modifications in solvent mixtures and length of separation times, the majority of biological lipids could be resolved and quantified ...Continue Reading

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