A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction.

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.

Avanti lipid tools: connecting lipids, technology, and cell biology.

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein-lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.

Sphingosine induces the aggregation of imine-containing peroxidized vesicles.

Lipid peroxidation plays a central role in the pathogenesis of many diseases like atherosclerosis and multiple sclerosis. We have analyzed the interaction of sphingosine with peroxidized bilayers in model membranes. Cu(2+) induced peroxidation was checked following UV absorbance at 245nm, and also using the novel Avanti snoopers®. Mass spectrometry confirms the oxidation of phospholipid unsaturated chains. Our results show that sphingosine causes aggregation of Cu(2+)-peroxidized vesicles. We observed that aggregation is facilitated by the presence of negatively-charged phospholipids in the membrane, and inhibited by anti-oxidants e.g. BHT. Interestingly, long-chain alkylamines (C18, C16) but not their short-chain analogues (C10, C6, C1) can substitute sphingosine as promoters of vesicle aggregation. Furthermore, sphinganine but not sphingosine-1-phosphate can mimic this effect. Formation of imines in the membrane upon peroxidation was detected by (1)H-NMR and it appeared to be necessary for the aggregation effect. (31)P-NMR spectroscopy reveals that sphingosine facilitates formation of non-lamellar phase in parallel with vesicle aggregation. The data might suggest a role for sphingosine in the pathogenesis of atherosclerosis.

Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy.

Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo(2)-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 10(9) molecules per cell in 24 h, based on the sum of subspecies analyzed by "lipidomic" mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-(13)C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.

Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry.

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture ( approximately 10(6)-10(7)), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 +/- 1.0 pmol/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.