Quantitation and molecular species determination of diacylglycerols, phosphatidylcholines, ceramides, and sphingomyelins with gas chromatography

In addition to the role of building block for biological membranes, phospholipids and their metabolites have been implicated in other important cellular functions, such as proliferation and apoptosis. Ceramides and their precursor, sphingomyelin, are thought to play a role in cellular apoptosis. In contrast, the metabolism of diacylglycerols and one of their precursors, phosphatidylcholine, is thought to be partly responsible for the opposite effect, cellular proliferation. Quantitative determination of these lipids in biological samples is important in investigating the complicated interactions between these molecules. In this report, we describe a capillary gas chromatographic procedure for the quantitative determination of molecular species of diacylglycerols, ceramides, phosphatidylcholines, and sphingomyelins. Lipid extracts are separated into these classes with a silica gel column. Diacylglycerols and ceramides are analyzed as trimethylsilyl derivatives. Phosphatidylcholines and sphingomyelins are converted to their diacylglycerol and ceramide components with sphingomyelinase hydrolysis. Internal standards for each analyzed fraction are used in the procedure. This method is used to determine the lipids in liver homogenate and subcellular fractions, including mitochondria, light mitochondria, and microsomes from young and old Fischer 344 rats. Our data show that the ceramide and sphingomyelin content is higher in the mitochondria of old rats. This relationship is consistent with the potential role of ceramide in mitochondria-induced apoptosis. More study is needed to substantiate this relationship. (C) 2003 Elsevier Inc. All rights reserved.