LIPID MAPS Research Articles

Mass spectrometry tools and metabolite-specific databases for molecular identification in metabolomics

The chemical identification of mass spectrometric signals in metabolomic applications is important to provide conversion of analytical data to biological knowledge about metabolic pathways. The complexity of electrospray mass spectrometric data acquired from a range of samples (serum, urine, yeast intracellular extracts, yeast metabolic footprints, placental tissue metabolic footprints) has been investigated and has defined the frequency of different ion types routinely detected. Although some ion types were expected (protonated and deprotonated peaks, isotope peaks, multiply charged peaks) others were not expected (sodium formate adduct ions). In parallel, the Manchester Metabolomics Database (MMD) has been constructed with data from genome scale metabolic reconstructions, HMDB, KEGG, Lipid Maps, BioCyc and DrugBank to provide knowledge on 42,687 endogenous and exogenous metabolite species. The combination of accurate mass data for a large collection of metabolites, theoretical isotope abundance data and knowledge of the different ion types detected provided a greater number of electrospray mass spectrometric signals which were putatively identified and with greater confidence in the samples studied. To provide definitive identification metabolite-specific mass spectral libraries for UPLC-MS and GC-MS have been constructed for 1,065 commercially available authentic standards. The MMD data are available at http://dbkgroup.org/MMD/.

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.

Abstract B102: Fenretinide induces lethal autophagy via a novel ensemble of life and death regulators: dihydroceramide and sphinganine versus sphinganine 1-phosphate

Abstract B102 Fenretinide (4-hydroxyphenylretinamide, 4HPR) is a promising anticancer agent that is being evaluated in human clinical trials. One of the proposed mechanisms of action for 4HPR is to increase the biosynthesis of ceramide, an inducer of apoptosis. Using liquid chromatography, electrospray ionization tandem mass spectrometry (LC ESI MS/MS) for “sphingolipidomic” analysis, we have found (H Wang et al., Mol. Cancer Ther. 7: 2967-2976, 2008) that 4HPR elevates dihydroceramides instead of ceramides due to the inhibition of dihydroceramide desaturase, and also increases the amounts of sphinganine and sphinganine 1-phosphate. In exploring how these metabolites could mediate 4HPR cytotoxicity, we have additionally discovered that dihydroceramides induce autophagy, and whether this is cytotoxic depends on the relative amounts of sphinganine 1-phosphate versus sphinganine--a highly toxic compound due to its effects on multiple signaling pathways as well as being lysosomotrophic. These findings reveal new facets of sphingolipid biology and autophagy, and suggest that the efficacy of 4HPR_and presumably other inhibitors of dihydroceramide desaturase--might be increased by concomitant inhibition of sphingosine kinase. (Acknowledgement: Some of these studies were conducted in collaboration with Dr. Myles Cabot of the John Wayne Cancer Institute, and funded by the Lipid MAPS Consortium grant GM069338.) Citation Information: Cancer Prev Res 2008;1(7 Suppl):B102.

Lipid imaging with cluster time-of-flight secondary ion mass spectrometry

This brief article provides an overview of the current state of the art in biological imaging mass spectrometry using cluster time-of-flight secondary ion mass spectrometry (TOF-SIMS). Recent and spectacular improvements in terms of sensitivity of TOF-SIMS imaging methods have allowed many biological applications to recently be successfully tested, such as mapping of lipid disorders in human muscles of a patient suffering from dystrophy, localization of surfactins after the swarming of bacteria on a surface, or lipid mapping over whole-body animal sections.

Lipidomics analysis of essential fatty acids in macrophages

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.

Thematic Review Series: Sphingolipids. Biodiversity of sphingoid bases (“sphingosines”) and related amino alcohols

“Sphingosin” was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of “lipides derived from sphingosine as sphingolipides.” This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the α,ω-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds.

Aggregation Behavior of an Ultra-Pure Lipopolysaccharide that Stimulates TLR-4 Receptors

The innate immune systems of humans and other animals are activated by lipopolysaccharides (LPS), which are glucosamine-based phospholipids that form the outer leaflet of the outer membranes of Gram-negative bacteria. Activation involves interactions of LPS with the innate immunity-receptor comprised of toll-like receptor 4 in complex with so-called MD-2 protein and accessory proteins, such as CD14 and LPS binding protein. The Lipid Metabolites and Pathways Strategy (LIPID MAPS) Consortium has isolated in large amounts a nearly homogeneous LPS, Kdo 2-Lipid A, and demonstrated that it activates macrophages via toll-like receptor 4. The active form of LPS, monomer or aggregate, is controversial. We have therefore examined the aggregation behavior and other physical properties of Kdo 2-Lipid A. Differential scanning calorimetry of Kdo 2-Lipid A suspensions revealed a gel-to-liquid crystalline phase transition at 36.4°C ( T m). The nominal critical aggregation concentration, determined by dynamic light scattering, was found to be 41.2 ± 1.6 nM below the T m (25°C), but only 8.1 ± 0.3 nM above the T m (37°C). The specific molecular volume of Kdo 2-Lipid A, obtained by densitometry measurements was found to be 3159 ± 71 Å 3 at 25°C, from which the number of molecules in each aggregate was estimated to be 5.8 × 10 5. The aggregation behavior of Kdo 2-Lipid A in the presence of lipoprotein-deficient serum suggests that Re LPS monomers and multimers are the active units for the immune system in the CD14-dependent and -independent pathways, respectively.

Profiling of lipid mediators released spinally in response to peripheral painful inflammation

A facilitated processing of noxious input at the spinal level contributes to tissue injury and inflammation‐induced hyperalgesia. Lipid metabolism in the spinal cord is of particular interest because of the high concentration of lipids in the central nervous system, where lipid mediators such as eicosanoids, ethanolamines and free fatty acids may play important roles in the processing of pain signals at the spinal level. Previous work has demonstrated that lipid mediators can have both hyperalgesic and anti‐hyperalgesic properties. To study the production of lipid mediators during hyperalgesia, we employed a model of inflammatory pain in which carrageenan is injected to the hind paw of rats, and cerebrospinal fluid and spinal cords were harvested at different time points over a 24 hour period. Using GC‐MS and LC‐MS/MS methodology capable of analyzing over 150 different lipid mediators, we have detected over 40 different metabolites derived from each of these classes. Application of this method may allow for the detection of lipid mediators with previously unappreciated functions, as well as determining unidentified connections between these classes of signaling molecules. This work was supported by the LIPID MAPS Large Scale Collaborative Grant GM069338 (MWB, DLS, EAD), NIH NS16541 (TLY), DA021654 (CIS), 2T32DK007202‐32 (MWB), and the Arthritis Foundation (CIS).

Temporal eicosanoid profiling of the inflammatory response to infection by the Lyme disease bacterium

Lyme disease is caused by infection with the spirochete B. burgdorferi. If untreated, 60% of infected individuals will develop an arthritis characterized by the infiltration of macrophages and neutrophils into the infected joint. Eicosanoids play a critical role in recruiting innate immune cells during the development of arthritis; however, their role in the response to infection is poorly understood. To study the contribution of eicosanoids to infection response, we utilized a murine model of Lyme disease that recapitulates many of the pathological hallmarks of the human disease before spontaneously resolving. Susceptible C3H/HeJ mice were infected with B. burgdorferi, and joint tissue was removed at days 0, 3, 7, 10, 14, 17, 21, 24, 28 and 35 for histological and molecular analysis. Using LC‐MS/MS methodology that has the capability to identify and quantitate over 100 different eicosanoids, we detected over 30 different metabolites from COX, 5‐LO, 12‐LO, 15‐LO and CYP450s in the infected joints. Furthermore, we have identified temporal eicosanoid patterns that correlate with the pathological progression of infection. Application of this method may allow for the identification of lipid mediators with previously unappreciated functions in this as well as other disease models. This work was supported by the LIPID MAPS Large Scale Collaborative Grant GM069338, NIH AR052748 and 2T32DK007202‐32.

Finding the way with LIPID MAPS

Finding the way with LIPID MAPS

Lipid mapping in human dystrophic muscle by cluster-time-of-flight secondary ion mass spectrometry imaging

Human striated muscle samples, from male control and Duchenne muscular dystrophy-affected children, were subjected to cluster-time-of-flight secondary ion mass spectrometry (cluster-ToF-SIMS) imaging using a 25 keV Bi(3)(+) liquid metal ion gun under static SIMS conditions. Spectra and ion density maps, or secondary ion images, were acquired in both positive and negative ion mode over several areas of 500 x 500 microm(2) (image resolution, 256 x 256 pixels). Characteristic distributions of various lipids were observed. Vitamin E and phosphatidylinositols were found to concentrate within the cells, whereas intact phosphocholines accumulated over the most damaged areas of the dystrophic muscles, together with cholesterol and sphingomyelin species. Fatty acyl chain composition varied depending on the region, allowing estimation of the local damage extent.

A rapid approach to lipid profiling of mycobacteria using 2D HSQC NMR maps

Mycobacteria, including Mycobacterium tuberculosis, are characterized by a unique cell wall rich in complex lipids, glycolipids, polyketides, and terpenoids. Many of these metabolites have been shown to play important roles in mycobacterial virulence and their inherent resistance to many antibiotics. Here, we report the development of a new simple method for global analysis of these metabolites using two-dimensional (1)H-(13)C heteronuclear single quantum coherence nuclear magnetic resonance. The major advantages of this method are as follows: the small amount of sample and the minimal sample manipulation required; a relatively short procedural time; and the ability to rapidly attain a qualitative and quantitative lipid profile of a mycobacterial sample in which the majority of the clinically relevant lipids can be observed simultaneously. The effectiveness of this method is demonstrated in four different areas of major concern to the mycobacterial research community: i) adaptive changes in cell wall lipids as a result of drug treatment; ii) analysis of gene function; iii) characterization of new mycobacterial species; and iv) analysis of the production of virulence factors in clinical isolates of M. tuberculosis. This method is complementary to mass spectrometry-based lipidomic technologies and provides an urgently needed tool to gain a better understanding of the role of lipids in mycobacteria pathogenesis.

A Laboratory Information Management System for High-Throughput Experimental Lipidomics: Minimal Information Required for the Analysis of Lipidomics Experiments (MIALE)

Sequencing of the human genome has opened the way and provided the impetus for building a comprehensive picture of a mammalian cell. Significant efforts are underway in the fields of genomics and proteomics to identify all genes and proteins in a given organism. The goal is a complete map of the genes, gene products, and their interaction networks in a functioning cell. The next step in establishing a comprehensive picture of a cell will be to integrate the cell's metabolome with the rapidly developing genomic and proteomic maps. A cell's metabolome, however, is such an enormous and complex entity that characterizing it can only be approached in sections. Our group of laboratories, the LIPID MAPS consortium, has focused on the lipid section of the metabolome. We have implemented a Lipid Metabolites and Pathways Strategy, termed LIPID MAPS, that applies a global integrated approach to the study of lipidomics in cells and tissues. This paper describes key aspects of the design, implementation, and accessibility features of a Laboratory Information Management System (LIMS) which serves the LIPID MAPS consortium. This software serves as a model system for integrating experimental information obtained by laboratories participating in metabolomics studies. (JALA 2007;12:230–8)

LIPID MAPS online tools for lipid research

The LIPID MAPS consortium has developed a number of online tools for performing tasks such as drawing lipid structures and predicting possible structures from mass spectrometry (MS) data. A simple online interface has been developed to enable an end-user to rapidly generate a variety of lipid chemical structures, along with corresponding systematic names and ontological information. The structure-drawing tools are available for six categories of lipids: (i) fatty acyls, (ii) glycerolipids, (iii) glycerophospholipids, (iv) cardiolipins, (v) sphingolipids and (vi) sterols. Within each category, the structure-drawing tools support the specification of various parameters such as chain lengths at a specific sn position, head groups, double bond positions and stereochemistry to generate a specific lipid structure. The structure-drawing tools have also been integrated with a second set of online tools which predict possible lipid structures from precursor-ion and product-ion MS experimental data. The MS prediction tools are available for three categories of lipids: (i) mono/di/triacylglycerols, (ii) glycerophospholipids and (iii) cardiolipins. The LIPID MAPS online tools are publicly available at www.lipidmaps.org/tools/.

Up‐regulation of de novo sphingolipid biosynthesis through serine palmitoyltransferease in RAW 264.7 cells upon Kdo2‐Lipid A treatment

Utilizing the uniform protocols established by the LIPID MAPS Consortium (www.lipidmaps.org), RAW 264.7 cells were treated with Kdo2-Lipid A, a defined endotoxin that activates macrophages via TLR-...

Lipid raft and map kinase interaction play a key role in angiotensin-II preconditioning of the heart

Lipid raft and map kinase interaction play a key role in angiotensin-II preconditioning of the heart

Geometry of hydrogen bonds formed by lipid bilayer nitroxide probes: a high-frequency pulsed ENDOR/EPR study.

Solvent effects on magnetic parameters of nitroxide spin labels in combination with side-directed spin-labeling EPR methods provide very useful means for elucidating polarity profiles in lipid bilayers and mapping local electrostatic effects in complex biomolecular systems. One major contributor to these solvent effects is the hydrogen bonds that could be formed between the nitroxide moiety and water and/or the available hydroxyl groups. Here, formation of hydrogen bonds between a lipid bilayer spin probe5-doxyl stearic acid, 5DSAand hydrogen-bond donors has been studied using high-frequency (HF) pulsed ENDOR and EPR. A hydrogen-bonded deuteron was directly detected in HF ENDOR (130 GHz) spectra of 5DSA dissolved in several deuterated alcohols, while the characteristic signal was absent in nonpolar toluene-d8. The length of the hydrogen bond, 1.74 ± 0.06 Å, and its geometry were found to be essentially the same for all four alcohols studied, indicating that nearly identical hydrogen bonds have been formed regardless of the solvent dielectric constant. This strengthens a hypothesis that HF EPR spectra are exclusively sensitive to formation of hydrogen bonds and could be used for probing the hydrogen-bond network in complex biomolecular assemblies and lipid bilayers with site-directed spin-labeling methods.

The Edinburgh human metabolic network reconstruction and its functional analysis

A better understanding of human metabolism and its relationship with diseases is an important task in human systems biology studies. In this paper, we present a high-quality human metabolic network manually reconstructed by integrating genome annotation information from different databases and metabolic reaction information from literature. The network contains nearly 3000 metabolic reactions, which were reorganized into about 70 human-specific metabolic pathways according to their functional relationships. By analysis of the functional connectivity of the metabolites in the network, the bow-tie structure, which was found previously by structure analysis, is reconfirmed. Furthermore, the distribution of the disease related genes in the network suggests that the IN (substrates) subset of the bow-tie structure has more flexibility than other parts.

Fenretinide induces lethal autophagy via a novel ensemble of life and death regulators: dihydroceramide and sphinganine versus sphinganine 1‐phosphate.

Fenretinide (4-hydroxyphenylretinamide, 4HPR) is a promising anticancer agent that is being evaluated in human clinical trials. One of the proposed mechanisms of action for fenretinide is to increase the biosynthesis of ceramide, an inducer of apoptosis. Using LC ESI MS/MS for “sphingolipidomic” analysis of MCF7 cells, we find that fenretinide elevates dihydroceramides instead of ceramides (due to inhibition of dihydroceramide desaturase) and increases the amounts of sphinganine and sphinganine 1-phosphate. In exploring how these metabolites could mediate fenretinide cytotoxicity, we have discovered that dihydroceramides induce autophagy, and whether this is cytotoxic depends on the relative amounts of sphinganine 1-phosphate versus sphinganine--a highly toxic compound due to its effects on multiple signaling pathways as well as being lysosomotrophic. These findings reveal new facets of sphingolipid biology and autophagy, and suggest that the efficacy of fenretinide might be increased by concomitant inhibition of sphingosine kinase. (Acknowledgement: Studies were conducted in collaboration with Dr. Myles Cabot of the John Wayne Cancer Institute, and funded by the Lipid MAPS Consortium grant GM069338.)

LMSD: LIPID MAPS structure database

The LIPID MAPS Structure Database (LMSD) is a relational database encompassing structures and annotations of biologically relevant lipids. Structures of lipids in the database come from four sources: (i) LIPID MAPS Consortium's core laboratories and partners; (ii) lipids identified by LIPID MAPS experiments; (iii) computationally generated structures for appropriate lipid classes; (iv) biologically relevant lipids manually curated from LIPID BANK, LIPIDAT and other public sources. All the lipid structures in LMSD are drawn in a consistent fashion. In addition to a classification-based retrieval of lipids, users can search LMSD using either text-based or structure-based search options. The text-based search implementation supports data retrieval by any combination of these data fields: LIPID MAPS ID, systematic or common name, mass, formula, category, main class, and subclass data fields. The structure-based search, in conjunction with optional data fields, provides the capability to perform a substructure search or exact match for the structure drawn by the user. Search results, in addition to structure and annotations, also include relevant links to external databases. The LMSD is publicly available at