Abstract
Liquid chromatography-electrospray ionization mass spectrometry (LC-MS) is an effective and popular technique used in lipid metabolomic studies. Although many LC-MS methods enabling the determination of sphingolipid molecular species have been reported, they do not cover a broad range of sphingolipid metabolites with expanding glycerophospholipids (GPLs) and diacylglycerol (DAG). In this study, we developed an approach for the comprehensive analysis of sphingolipids, GPLs and DAG molecular species in a biological sample, without alkaline hydrolysis. After validating the reliability of this approach, we analyzed tissue lipids of sphingomyelin synthase 2-knockout mice and found that changes in sphingolipid metabolism in the liver affect the level of docosahexaenoic acid-containing GPLs. Our method analyzes GPLs and DAG, as well as sphingolipids within biological samples and, thus, will facilitate more comprehensive studies of sphingolipid metabolism in pathology and diagnostics.
Highlights
Sphingolipids, which have a characteristic sphingoid base structure, include several classes of molecules, such as sphingosine (Sph), sphingosine-1-phosphate (S1P), ceramide (Cer), glucosylceramide (GlcCer), sphingomyelin (SM) and ganglioside [1]
Sphingolipid metabolism is linked to GPL metabolism via PtdCho and DAG, which are involved in an interchange reaction between Cer and SM
Membrane microdomains that are enriched in sphingolipids are thought to play an important role in membrane receptor function [24,25,26]; analyzing the relative levels of the various sphingolipids and GPLs is critical for understanding biological systems
Summary
Sphingolipids, which have a characteristic sphingoid base structure, include several classes of molecules, such as sphingosine (Sph), sphingosine-1-phosphate (S1P), ceramide (Cer), glucosylceramide (GlcCer), sphingomyelin (SM) and ganglioside [1] These lipids are interconvertible in biological systems. We employed diethylaminoethyl (DEAE)-cellulose chromatography to separate the acidic lipids from the large amounts of basic and neutral lipids present in biological samples [22,23] These extracts were analyzed by LC-MS/MS, to determine the molecular species level of each lipid class with minimization of the ion-suppression that may occur when a lipid extract from a complex biological sample is subjected to an electrospray ionization MS analysis. Our results indicated that this method would be useful for comprehensive studies of sphingolipid metabolism in cellular responses and pathology
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