HIGH-RESOLUTION SEPARATION AND QUANTIFICATION OF NEUTRAL LIPID AND PHOSPHOLIPID SPECIES IN MAMMALIAN-CELLS AND SERA BY MULTI-ONE-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY

An improvement of current methods is needed for simple, rapid, and pre cise quantification of cellular lipids, including rare species of biol ogically active cellular lipids, such as phosphatidic acid (PA) and di radylglycerol (DG). In addition, further analysis of hydrolyzed acyl c hains from these species by methods such as gas chromatography require s complete separations. Methods have been developed for the quantifica tion of neutral lipids and several phospholipids extracted from mammal ian 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 designa ted as multi-one-dimensional thin-layer chromatography (MOD-TLC). Reso lved lipid bands were visualized by the lipophilic dye primulin (direc t 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 modifi cations in solvent mixtures and length of separation times, the majori ty of biological lipids could be resolved and quantified with MOD-TLC methods. Since the detection method is nondestructive, purified lipids could then be recovered by scraping the visualized bands and extracti ng the lipids from the silica. The structural identities of the recove red lipids were confirmed by fast-atom bombardment and electrospray ma ss spectrometry. Extracted lipids were also hydrolyzed to release acyl chains and acyl chain species were determined in comparison to authen tic standards by gas chromatography. PA and DG levels in ECV.304 cells were found to be 4.6 and 3.3%, respectively, of PC levels, with a PA/ DG ratio of 1.4, which is in accord with published experience using ot her methods and different cell types. PA in human serum was detected a t 0.6% of PC, indicating the sensitivity of the technique. In contrast to two-dimensional thin-layer chromatography, which allows for good r esolution of some lipid species, but cannot be used to analyze more th an a single experimental point per plate, MOD-TLC allows for direct co mparative analysis of multiple samples on a single TLC plate, while st ill providing good resolution for the quantification of most major cla sses of lipid species. (C) 1998 Academic Press.