[Show abstract][Hide abstract] ABSTRACT: Glycerophosphocholines are the major building blocks of biological membranes. They are also precursors of the low molecular weight second messengers with mass to charge ratios of 450-600. These messengers include lysophosphatidylcholines (LPCs) and lyso-platelet activating factors (PAFs) that can be further processed into PAFs. Often conceptualized as a single species, LPCs, PAFs, and lyso-PAFs are, in fact, rich families of glycerophosphocholine-derived lipids distinguished by the linkage of their sn-1 carbon chains to the phosphoglyceride backbone (ester or ether), their sn-1 carbon chain length and degree of unsaturation, and the identity of their sn-2 constituents (a hydroxyl or acetyl group). Each LPC and PAF species exhibits a different affinity for their cognate G-protein coupled receptors and each species can elicit receptor-independent actions, all of which play critical signalling roles. Targeted mass spectrometry-based lipidomics approaches are enabling the molecular identification and quantification of these low-abundance second messengers. Variations between datasets map the temporal landscape of second messengers available for signalling and provide us with snapshots of the state of structural membrane compositional remodeling at the time of extraction. Here, we review a number of advances, from extraction to detection, in lipidomic methodologies used to identify LPCs, lyso-PAFs, and PAFs, and we highlight how these targeted approaches are providing valuable insight into the roles played by the cellular lipidome in cell function and disease susceptibility. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Investigations of complex metabolic mechanisms and networks have become a focus of research in the post-genomic area, thereby creating an increasing demand for sophisticated analytical approaches. One such tool are lipidomics analyses that provide a detailed picture of the lipid composition of a system at a given time. Introducing stable isotopes into the studied system can additionally provide information on the synthesis, transformation and degradation of individual lipid species. Capturing the entire dynamics of lipid networks, however, is still a challenge. We developed and evaluated a novel strategy for the in-depth analysis of the dynamics of lipid metabolism with the capacity for high molecular specificity and network coverage. The general workflow consists of stable isotope-labeling experiments, ultra high-performance liquid chromatography (UHPLC)-high resolution Orbitrap-MS lipid profiling and data processing by a software tool for global isotopomer filtering and matching. As a proof of concept, this approach was applied to the network-wide mapping of dynamic lipid metabolism in primary human skeletal muscle cells cultured for 4, 12 and 24 h with [U-13C]-palmitate. In the myocellular lipid extracts 692 isotopomers were detected that could be assigned to 203 labeled lipid species spanning 12 lipid (sub-) classes. Interestingly, some lipid classes showed high turnover rates but stable total amounts while the amount of others increased in the course of palmitate treatment. The novel strategy presented here has the potential to open new detailed insights into the dynamics of lipid metabolism that may lead to a better understanding of physiological mechanisms and metabolic perturbations.
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