Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

Opinion article on lipidomics: Inherent challenges of lipidomic analysis of sphingolipids.

A challenge for sphingolipidomic analysis is the vast number of subspecies, including a large number of isomers-a complication that was even appreciated by the original discoverer of sphingolipids J. L. W. Thudichum (The Chemistry of the Brain, p. x, 1884): "In the course of my researches many unforeseen complications arose, prominent amongst which were those caused by the occurrence of chemical principles having the same atomic or elementary composition, but differing in other chemical, or in physical properties, varieties producing the phenomenon which in chemistry is termed isomerism." Therefore, it is essential to choose the appropriate method(s) for the goal of the analysis, to know the assumptions and limitations of method(s) used, and to temper interpretation of the data accordingly. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages.

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS).

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or "sphingolipidomic" methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.

Myo-inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early-stage somatic embryos.

• Myo-inositol hexakisphosphate (InsP(6)), abundant in animals and plants, is well known for its anticancer activity. However, many aspects of InsP(6) function in plants remain undefined. We now report the first evidence that InsP(6) can inhibit cellular proliferation in plants under growth conditions where phosphorus is not limited. • A highly anionic molecule inhibitory to early-stage somatic embryo growth of loblolly pine (LP) was purified chromatographically from late-stage LP female gametophytes (FGs), and then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. • Exact mass and mass spectrometry-mass spectrometry (MS-MS) fragmentation identified the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo-isomer (i.e. InsP(6)) on the basis of (1)H-, (31)P- and (13)C-NMR, (1)H-(1)H correlation spectroscopy (COSY), (1)H-(31)P heteronuclear single quantum correlation (HSQC) and (1)H-(13)C HSQC. Topical application of InsP(6) to early-stage somatic embryos indeed inhibits embryonic growth. • Recently evidence has begun to emerge that InsP(6) may also play a regulatory role in plant cells. We anticipate that our findings will help to stimulate additional investigations aimed at elucidating the roles of inositol phosphates in cellular growth and development in plants.

(Glyco)sphingolipidology: an amazing challenge and opportunity for systems biology.

Sphingolipids are found in essentially all eukaryotes and in some prokaryotes and viruses, where they influence cell structure, signaling and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, the sphingolipidome comprises untold thousands of species that encompass bioactive backbones and complex phospho- and glycolipids. Mass spectrometry is able to analyze a growing fraction of the sphingolipidome and is beginning to provide information about localization. Use of these structure specific, quantitative methods is producing insights, and surprises, regarding sphingolipid structure, metabolism, function and disease. Dealing with such large data sets poses special challenges for systems biology, but the intrinsic and elegant interrelationships among these compounds might provide a key to dealing with the complexity of the sphingolipidome.

Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry.

This study describes the use of a stable-isotope labeled precursor ([U-¹³C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (¹³C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-¹³C]palmitic acid, ∼60% of the total palmitoyl-CoA was ¹³C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ∼1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.

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.

A mouse macrophage lipidome.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

Elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry.

BACKGROUND: Sulfatides (ST) are a category of sulfated galactosylceramides (GalCer) that are elevated in many types of cancer including, possibly, ovarian cancer. Previous evidence for elevation of ST in ovarian cancer was based on a colorimetric reagent that does not provide structural details and can also react with other lipids. Therefore, this study utilized mass spectrometry for a structure-specific and quantitative analysis of the types, amounts, and tissue localization of ST in ovarian cancer, and combined these findings with analysis of mRNAs for the relevant enzymes of ST metabolism to explore possible mechanisms. RESULTS: Analysis of 12 ovarian tissues graded as histologically normal or having epithelial ovarian tumors by liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) established that most tumor-bearing tissues have higher amounts of ST. Because ovarian cancer tissues are comprised of many different cell types, histological tissue slices were analyzed by matrix-assisted laser desorption ionization-tissue-imaging MS (MALDI-TIMS). The regions where ST were detected by MALDI-TIMS overlapped with the ovarian epithelial carcinoma as identified by H & E staining and histological scoring. Furthermore, the structures for the most prevalent species observed via MALDI-TIMS (d18:1/C16:0-, d18:1/C24:1- and d18:1/C24:0-ST) were confirmed by MALDI-TIMS/MS, whereas, a neighboring ion(m/z 885.6) that was not tumor specific was identified as a phosphatidylinositol. Microarray analysis of mRNAs collected using laser capture microdissection revealed that expression of GalCer synthase and Gal3ST1 (3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase) were approximately 11- and 3.5-fold higher, respectively, in the ovarian epithelial carcinoma cells versus normal ovarian stromal tissue, and they were 5- and 2.3-fold higher in comparison with normal surface ovarian epithelial cells, which is a likely explanation for the higher ST. CONCLUSIONS: This study combined transcriptomic and lipidomic approaches to establish that sulfatides are elevated in ovarian cancer and should be evaluated further as factors that might be important in ovarian cancer biology and, possibly, as biomarkers.

Direct analysis of cellulose in poplar stem by matrix-assisted laser desorption/ionization imaging mass spectrometry.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was applied to the analysis of the spatial distribution of cellulose on a cross-section of juvenile poplar (Populus deltoids) stems. Microcrystalline cellulose (MCC) was used to optimize matrix (2,5-dihydroxybenzoic acid) application and instrument parameters for the detection of low hexose oligomers, which originated from cellulose in the solid phase. A section of poplar cellulose isolated from juvenile poplar stem which consisted primarily of glucose (∼95%) and minor components such as xylose and lignin was used for the MALDI-IMS studies. The mass spectrum of poplar cellulose consisted of a series of evenly spaced signals having a difference of 162 m/z units, which was similar to that of MCC in linear and reflectron positive ion modes. MS images of cellulose compounds with sodium ion adducts were generated and illustrated the distribution of cellulose on the surface of the poplar stem.

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.

Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.

Rapid identification of Vibrio parahaemolyticus by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.

Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrix coating system and its application to analysis of lipids in brain from a mouse model of Tay-Sachs/Sandhoff disease.

The quality of tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) depends on the effectiveness of the matrix deposition, especially for lipids that may dissolve in the solvent used for the matrix application. This article describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This system was then applied to the analysis of histological slices of brains from mice with homozygous disruption of the hexb gene (hexb-/-), a model of Tay-Sachs and Sandhoff disease, versus the functionally normal heterozygote (hexb+/-) by imaging MALDI-MS. This allowed profiling and localization of many different lipid species, and of particular interest, ganglioside GM2, asialo-GM2 (GA2), and sulfatides (ST). The presence of these compounds was confirmed by analysis of brain extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS). The major fatty acid of the ceramide backbone of both GM2 and GA2 was identified as stearic acid (18:0) versus nervonic acid (24:1) for ST by both tissue-imaging MS and ESI-MS/MS. GM2 and GA2 were highly elevated in hexb-/- and were both localized in the granular cell region of the cerebellum. ST, however, was localized mainly in myelinated fiber (white matter) region of the cerebellum as well as in the brain stem with a relatively uniform distribution and had similar relative signal intensity for both hexb+/- and hexb-/- brain. It was also observed that there were distinct localizations for numerous other lipid subclasses; hence, imaging MALDI-MS could be used for "lipidomic" studies. These results illustrate the usefulness of tissue-imaging MALDI-MS with matrix deposition by OCN for histologic comparison of lipids in tissues such as brains from this mouse model of Tay-Sachs and Sandhoff disease.

Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.

Sphingolipids are comprised of a backbone sphingoid base that may be phosphorylated, acylated, glycosylated, bridged to various headgroups through phosphodiester linkages, or otherwise modified. Organisms usually contain large numbers of sphingolipid subspecies and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, "sphingolipidomic" analysis is becoming feasible (at least for important subsets such as all of the backbone "signaling" subspecies: ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry, and these profiles are revealing many surprises, such as that under certain conditions cells contain significant amounts of "unusual" species: N-mono-, di-, and tri-methyl-sphingoid bases (including N,N-dimethylsphingosine); 3-ketodihydroceramides; N-acetyl-sphingoid bases (C2-ceramides); and dihydroceramides, in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4-hydroxyphenylretinamide). The elevation of DHceramides by fenretinide is befuddling because the 4,5-trans-double bond of ceramide has been thought to be required for biological activity; however, DHceramides induce autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.

Structure-specific, quantitative methods for analysis of sphingolipids by liquid chromatography-tandem mass spectrometry: "inside-out" sphingolipidomics.

Due to the large number of highly bioactive subspecies, elucidation of the roles of sphingolipids in cell structure, signaling, and function is beginning to require that one perform structure-specific and quantitative (i.e., "sphingolipidomic") analysis of all individual subspecies, or at least of those are relevant to the biologic system of interest. As part of the LIPID MAPS Consortium, methods have been developed and validated for the extraction, liquid chromatographic (LC) separation, and identification and quantitation by electrospray ionization (ESI), tandem mass spectrometry (MS/MS) using an internal standard cocktail that encompasses the signaling metabolites (e.g., ceramides, ceramide 1-phosphates, sphingoid bases, and sphingoid base 1-phosphates) as well as more complex species (sphingomyelins, mono- and di-hexosylceramides). The number of species that can be analyzed is growing rapidly with the addition of sulfatides and other complex sphingolipids as more internal standards become available. This review describes these methods as well as summarizes others from the published literature. Sphingolipids are an amazingly complex family of compounds that are found in all eukaryotes as well as some prokaryotes and viruses. The size of the sphingolipidome (i.e., all of the individual molecular species of sphingolipids) is not known, but must be immense considering mammals have over 400 headgroup variants (for a listing, see http://www.sphingomap.org), each of which is comprised of at least a few-and, in some cases, dozens-of lipid backbones. No methods have yet been developed that can encompass so many different compounds in a structurally specific and quantitative manner. Nonetheless, it is possible to analyze useful subsets of the sphingolipidome, such as the backbone sphingolipids involved in signaling (sphingoid bases, sphingoid base 1-phosphates, ceramides, and ceramide 1-phosphates) and metabolites at important branchpoints, such as the partitioning of ceramide into sphingomyelins, glucosylceramides, galactosylceramides, and ceramide 1-phosphate versus turnover to the backbone sphingoid base. This review describes methodology that has been developed as part of the LIPID MAPS Consortium (www.lipidmaps.org) as well as other methods that can be used for sphingolipidomic analysis to the extent that such is currently feasible. The focus of this review is primarily mammalian sphingolipids; hence, if readers are interested in methods to study other organisms, they should consult the excellent review by Stephen Levery in another volume of Methods in Enzymology (Levery, 2005), which covers additional species found in plants, fungi, and other organisms. It should be noted from the start that although many analytical challenges remain in the development of methods to analyze the full "sphingolipidome," the major impediment to progress is the limited availability of reliable internal standards for most of the compounds of interest. Because it is an intrinsic feature of mass spectrometry that ion yields tend to vary considerably among different compounds, sources, methods, and instruments, an analysis that purports to be quantitative will not be conclusive unless enough internal standards have been added to correct for these variables. Ideally, there should be some way of standardizing every compound in the unknown mixture; however, that is difficult, if not impossible, to do because the compounds are not available, and the inclusion of so many internal standards generates a spectrum that may be too complex to interpret. Therefore, a few representative internal standards are usually added, and any known differences in the ion yields of the analytes of interest versus the spiked standard are factored into the calculations. Identification of appropriate internal standards has been a major focus of the LIPID MAPS Consortium, and the methods described in this review are based on the development of a certified (i.e., compositionally and quantitatively defined by the supplier) internal standard cocktail that is now commercially available (Avanti Polar Lipids, Alabaster, AL). For practical and philosophical reasons, an internal standard cocktail was chosen over the process of an investigator adding individual standards for only the analytes of interest. On the practical level, addition of a single cocktail minimizes pipetting errors as well as keeping track of whether each internal standard is still usable (e.g., has it degraded while in solution?). Philosophically, the internal standard cocktail was chosen because an underlying premise of systems analysis asserts that, due to the high relevancy of unexpected interrelationships involving more distant components, one can only understand a biological system when factors outside the primary focus of the experiment have also been examined. Indeed, the first payoffs of "omics" and systems approaches involve the discoveries of interesting compounds in unexpected places when a "sphingolipidomic" analytical method was being used as routine practice instead of a simpler method that would have only measured the compound initially thought to be important (Zheng et al., 2006). Thus, routine addition of a broad internal standard cocktail at the outset of any analysis maximizes the opportunity for such discoveries, both at the time the original measurements are made and when one decides to return to the samples later, which can fortunately be done for many sphingolipids because they remain relatively stable in storage.

Hydrolyzed fumonisins HFB1 and HFB2 are acylated in vitro and in vivo by ceramide synthase to form cytotoxic N-acyl-metabolites.

Fumonisins B1 and B2 (FB1 and FB2) are the most abundant members of the fumonisins--mycotoxins that are produced by Fusarium verticillioides and are natural inhibitors of ceramide synthase. Their hydrolyzed forms, HFB1 and HFB2 (also called AP1 and AP2) are found in some foods, and they are not only inhibitors of ceramide synthase but also undergo acylation by this enzyme. This study characterized the conversion of HFB1 and HFB2 by ceramide synthase to their respective N-acylated metabolites using rat liver microsomes and palmitoyl-CoA or nervonoyl-CoA as cosubstrates, and examined animals that had been dosed with hydrolyzed fumonisins to ascertain if acylation occurs in vivo. Using an HPLC-MS/MS method that allowed the sensitive and selective detection of the acylation products, both HFB1 and HFB2 were found to be metabolized in vitro to nervonoyl- or palmitoyl-HFB1 and -HFB2 (i.e. C24:1-HFB1/2 and C16-HFB1/2, respectively). The apparent vmax was considerably higher for formation of C24:1HFB1 (157 pmol/min/mg protein) than for formation of C16HFB1 (8.7 pmol/min/mg protein). The acylation products also inhibited ceramide synthase and significantly reduced the number of viable cells in an in vitro [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay using a human colonic cell line (HT29). Furthermore, HPLC-MS/MS analysis of tissues from rats given intraperitoneal doses of HFB1 confirmed that formation of N-acyl-HFB1 occurs in vivo to produce metabolites with fatty acids of various chain lengths. The contribution of acylated HFB1 and HFB2 metabolites to fumonisin toxicity in vivo warrants further investigation.

Fumonisins and fumonisin analogs as inhibitors of ceramide synthase and inducers of apoptosis.

Sphingoid bases are growth inhibitory and pro-apoptotic for many types of cells when added to cells exogenously, and can be elevated to toxic amounts endogenously when cells are exposed to inhibitors of ceramide synthase. An important category of naturally occurring inhibitors are the fumonisins, which inhibit ceramide synthase through structural similarities with both the sphingoid base and fatty acyl-CoA co-substrates. Fumonisins cause a wide spectrum of disease (liver and renal toxicity and carcinogenesis, neurotoxicity, induction of pulmonary edema, and others), and most-possibly all-of the pathophysiologic effects of fumonisins are attributable to disruption of the sphingolipid metabolism. The products of alkaline hydrolysis of fumonisins (which occurs during the preparation of masa flour for tortillas) are aminopentols that also inhibit ceramide synthase, but more weakly. Nonetheless, the aminopentols (and other 1-deoxy analogs of sphinganine) are acylated to derivatives that inhibit ceramide synthase, perhaps as product analogs, elevate sphinganine, and kill the cells. Somewhat paradoxically, fumonisins sometimes stimulate growth and inhibit apoptosis, possibly due to elevation of sphinganine 1-phosphate, which is known to have these cellular effects. These findings underscore the complexity of sphingolipid metabolism and the difficulty of identifying the pertinent mediators unless a full profile of the potentially bioactive species is evaluated.

Ceramide content is increased in skeletal muscle from obese insulin-resistant humans.

Increased intramyocellular lipid concentrations are thought to play a role in insulin resistance, but the precise nature of the lipid species that produce insulin resistance in human muscle are unknown. Ceramides, either generated via activation of sphingomyelinase or produced by de novo synthesis, induce insulin resistance in cultured cells by inhibitory effects on insulin signaling. The present study was undertaken to determine whether ceramides or other sphingolipids are increased in muscle from obese insulin-resistant subjects and to assess whether ceramide plays a role in the insulin resistance of Akt in human muscle. Lean insulin-sensitive and obese insulin-resistant subjects (n = 10 each) received euglycemic-hyperinsulinemic clamps with muscle biopsies basally and after 30, 45, or 60 min of insulin infusion. The rate of glucose infusion required to maintain euglycemia (reflecting glucose uptake) was reduced by >50%, as expected, in the obese subjects at each time point (P < 0.01). Under basal conditions, total muscle ceramide content was increased nearly twofold in the obese subjects (46 +/- 9 vs. 25 +/- 2 pmol/2 mg muscle, P < 0.05). All species of ceramides were increased similarly in the obese subjects; in contrast, no other sphingolipid was increased. Stimulation of Akt phosphorylation by insulin in the obese subjects was significantly reduced after 30 min (0.96 +/- 0.11 vs. 1.84 +/- 0.38 arbitrary units) or 45-60 min (0.68 +/- 0.17 vs. 1.52 +/- 0.26) of insulin infusion (P < 0.05 for both). Muscle ceramide content was significantly correlated with the plasma free fatty acid concentration (r = 0.51, P < 0.05). We conclude that obesity is associated with increased intramyocellular ceramide content. This twofold increase in ceramide may be involved in the decrease in Akt phosphorylation observed after insulin infusion and could theoretically play a role in the reduced ability of insulin to stimulate glucose uptake in skeletal muscle from obese subjects.