Establishment of a Measurement System for Sphingolipids in the Cerebrospinal Fluid Based on Liquid Chromatography-Tandem Mass Spectrometry, and Its Application in the Diagnosis of Carcinomatous Meningitis.

OBJECTIVES: Sphingolipids have been demonstrated to be involved in many human diseases. However, measurement of sphingolipids, especially of sphingosine 1-phosphate (S1P) and dihydro-sphingosine 1-phosphate (dhS1P), in blood samples requires strict sampling, since blood cells easily secrete these substances during sampling and storage, making it difficult to introduce measurement of sphingolipids in clinical laboratory medicine. On the other hand, cerebrospinal fluid (CSF) contains few blood cells. Therefore, we attempted to establish a system based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of sphingolipids in the CSF, and applied it for the diagnosis of carcinomatous meningitis. METHODS: We developed and validated a LC-MS/MS-based measurement system for S1P and dhS1P and for ceramides and sphingosines, used this system to measure the levels of these sphingolipids in the CSF collected from the subjects with cancerous meningitis, and compared the levels with those in normal routine CSF samples. RESULTS: Both the measurement systems for S1P/dhS1P and for ceramides/sphingosines provided precision with the coefficient of variation below 20% for sphingolipids in the CSF samples. We also confirmed that the levels of S1P, as well as ceramides/sphingosines, in the CSF samples did not increase after the sampling. In the CSF samples collected from patients with cancerous meningitis, we observed that the ratio of S1P to ceramides/sphingosine and that of dhS1P to dihydro-sphingosine were higher than those in control samples. CONCLUSIONS: We established and validated a measurement system for sphingolipids in the CSF. The system offers promise for being introduced into clinical laboratory testing.

Isolation and Quantification of Sphingosine and Sphinganine from Rat Serum Revealed Gender Differences.

Sphingolipids are an important group of lipids that play crucial roles in living cells, facilitating cell recognition, signal transduction and endocytosis. The concentration of sphingosine and some of its derivatives like sphinganine may serve as a biomarker for the diagnosis of sphingolipidoses or be used for further research into similar diseases. In this study, a sphingolipid extraction and a high resolution detection method specific for sphingosine and sphinganine was adapted and tested. Lipids were extracted from rats' serum, coupled to o-phthalaldehyde and detected with a fluorescence detector after running through a silica gel column in a high performance liquid chromatography system. With this method, we analysed 20 male and 20 female rat serum samples and compared the concentrations of sphingosine and sphinganine. The results showed a significant difference between the sphingosine concentrations in the male and female rats. The sphingosine concentration in female rats was 805 ng/mL (standard deviation, SD ± 549), while that in males was significantly lower at (75 ng/mL (SD ± 40)). Furthermore, the sphingosine:sphinganine ratio was almost 15-fold higher in the females' samples. The method presented here facilitates the accurate quantification of sphingosine and sphinganine concentrations down to 2.6 ng and 3.0 ng, respectively, and their ratio in small amounts of rat serum samples to study the sphingolipid metabolism and its potential modulation due to gene mutations or the effect of prevalent toxins.

A Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia.

Giardia lamblia, a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a "minimalistic" life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes-and glycosphingolipids are critical for encystation-we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

Linear ion-trap MSn with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis.

1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H - H2O - (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H - H2O - (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes.

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.

Aptamer-based trapping of phytosphingosine in urine samples.

Usually, small molecules like single metabolites used in clinical diagnostic can be quantified by instrumental approaches like LC-MS or bioanalytical techniques using antibodies or aptamers as selective receptors. The present work comprises the generation of aptamers with an affinity towards the medically relevant metabolite phytosphingosine via the previously reported just in time-Selection approach (Hünniger et al., 2014). The whole approach could be seen as a proof of concept to extend the existing just in time-Selection protocol for selection towards small molecules with dissociation constants in the low nanomolar range. Moreover it is conceivable that the shown methods could be quickly adapted to further scopes. Aptamers could be applied for clean-up or concentration processes prior to further analysis. As an example, we used the selected aptamers towards phytosphingosine bound to magnetic particles for affinity enrichment in both selection buffer and urine samples. As an outcome, enrichment factors of up to 9-fold (selection buffer)/4-fold (urine samples) were achieved by this approach.

Effects of Asterias amurensis-derived Sphingoid Bases on the de novo Ceramide Synthesis in Cultured Normal Human Epidermal Keratinocytes.

Asterias amurensis starfish provide several bioactive species in addition to being fishery waste. Glucosyl ceramides (GlcCers) were extracted from the viscera of these starfish and were isolated by silica gel column chromatography. Degraded GlcCers generated A. amurensis sphingoid bases (ASBs) that mainly consisted of the triene-type bases d18:3 and 9-methyl-d18:3. The effect of these bases on ceramide synthesis and content were analyzed using normal human epidermal keratinocytes (NHEKs). The bases significantly enhanced the de novo ceramide synthesis and gene expression in NHEKs for proteins, such as serine-palmitoyltransferase and ceramide synthase. Total ceramide, GlcCer, and sphingomyelin contents increased dramatically upon ASB treatment. In particular, GlcCer bearing very-long-chain fatty acids (≥C28) exhibited a significant content increase. These ASB-induced enhancements on de novo ceramide synthesis were only observed in undifferentiated NHEKs. This stimulation of the de novo sphingolipid synthesis may improve skin barrier functions.

Quantitative Profiling of Long-Chain Bases by Mass Tagging and Parallel Reaction Monitoring.

Long-chain bases (LCBs) are both intermediates in sphingolipid metabolism and potent signaling molecules that control cellular processes. To understand how regulation of sphingolipid metabolism and levels of individual LCB species impinge upon physiological and pathophysiological processes requires sensitive and specific assays for monitoring these molecules. Here we describe a shotgun lipidomics method for quantitative profiling of LCB molecules. The method employs a "mass-tag" strategy where LCBs are chemically derivatized with deuterated methyliodide (CD3I) to produce trimethylated derivatives having a positively charged quaternary amine group. This chemical derivatization minimizes unwanted in-source fragmentation of LCB analytes and prompts a characteristic trimethylaminium fragment ion that enables sensitive and quantitative profiling of LCB molecules by parallel reaction monitoring on a hybrid quadrupole time-of-flight mass spectrometer. Notably, the strategy provides, for the first time, a routine for monitoring endogenous 3-ketosphinganine molecules and distinguishing them from more abundant isomeric sphingosine molecules. To demonstrate the efficacy of the methodology we report an in-depth characterization of the LCB composition of yeast mutants with defective sphingolipid metabolism and the absolute levels of LCBs in mammalian cells. The strategy is generic, applicable to other types of mass spectrometers and can readily be applied as an additional routine in workflows for global lipidome quantification and for functional studies of sphingolipid metabolism.

Lyso-sphingomyelin is elevated in dried blood spots of Niemann-Pick B patients.

Niemann-Pick disease type B (NPD-B) is caused by a partial deficiency of acid sphingomyelinase activity and results in the accumulation of lysosomal sphingomyelin (SPM) predominantly in macrophages. Notably, SPM is not significantly elevated in the plasma, whole blood, or urine of NPD-B patients. Here, we show that the de-acylated form of sphingomyelin, lyso-SPM, is elevated approximately 5-fold in dried blood spots (DBS) from NPD-B patients and has no overlap with normal controls, making it a potentially useful biomarker.

New long chain bases in lipophosphonoglycan of Acanthamoeba castellanii.

The polymer called lipophosphonoglycan (LPG) was isolated from Acanthamoeba castellanii membranes after exhaustive delipidation and butanol extraction. A novel extremely long phytosphingosine was revealed in glycoinositolphosphosphingolipid (GIPSL). All data obtained by gas-liquid chromatography coupled with MS analyses of products liberated during acid methanolysis and products of sodium metaperiodate and permanganate-periodate oxidations showed an unusual pattern of long chain bases (LCB) with branched bases (anteiso-C24, anteiso-C25, anteiso-C26, iso-C26, anteiso-C27, and anteiso-C28) and normal ones (C24, C25, C26, C27). The phytosphingosines with hexa-, hepta-, and octacosanoic chains have not been detected in Acanthamoeba cells up to now. Also, the isomer configuration of long chain bases in LPG of A. castellanii was not defined in earlier reports. In the GC-MS chromatograms, the component forming a peak corresponding to anteiso-C25 phytosphingosine was the most abundant and constituted more than 50 % of all LCB.

Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP.

Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser127 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S1P induces YAP nuclear localization through S1P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S1P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S1P and LPA might modulate cell proliferation and tumorigenesis through YAP activation.

Isoform-selective assays for sphingosine kinase activity.

Sphingosine kinases (SK) 1 and 2 are unique lipid kinases that phosphorylate sphingosine to form -sphingosine-1-phosphate (S1P). S1P is a bioactive molecule eliciting multiple effects both extracellularly via cell surface S1P receptors and intracellularly through a number of recently identified protein targets. The two enzymes arise from different genes, and differ in their cellular localisation, developmental expression, catalytic properties, and in at least some functional roles. Here, we describe methods for selectively detecting SK1 and SK2 activities in vitro, highlighting conditions that can discriminate between the activities of these two enzymes. The assays measure the production of (32)P-labelled S1P following the addition of exogenous sphingosine and [γ(32)P] adenosine-5'-triphosphate. The S1P product can be purified by Bligh-Dyer solvent extraction, separated by thin-layer chromatography (TLC), and the radiolabelled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 and SK2 activities in tissue, cell, and recombinant protein samples.

Bioactive lipids from the sponge Spirastrella abata.

Three sphingosine 4-sulfates (1-3) and a lysophosphatidylglycerol (4) were isolated from the Korean sponge Spirastrella abata. The structures of these compounds were determined based on the combined results of spectroscopic analyses. Based on the results of combined synthesis and comparison of specific rotation and circular dichroism, the absolute configurations of 1-3 were found to be enantiomeric to the previously isolated metabolites. The configurations of 4 were also partially determined by similar chemical and spectroscopic methods. The compounds exhibited significant cytotoxicity and weak antimicrobial activity (1), as well as weak-to-moderate inhibitory activity against isocitrate lyase and Na(+)/K(+)-ATPase. A structure-activity relationship was found for the sphingosine 4-sulfates.

Determination of sphingosine kinase 2 activity using fluorescent sphingosine by capillary electrophoresis.

The study of sphingosine and sphingosine-1-phosphate is now widespread due to their immense role as intra- and extracellular messenger molecules. The balance and interplay of these ceramide metabolites is dependent on the activities of kinase and phosphatase enzymes. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells; thus, they require highly sensitive techniques for quantitative analysis. In this study, we developed a quantitative assay for the determination of sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using CE-LIF. Sphingosine fluorescein was used as the substrate. The K(M) of SphK2 for sphingosine fluorescein was 2.8 ± 0.8 µM with a V(max) of 2490 ± 520 µM/min and a k(cat) of 1920 ± 402/s. The inhibition of SphK2 was also investigated using four different inhibitors for which 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole inhibitor was the most potent for the in vitro inhibition of SphK2 while N,N-dimethylsphingosine (DMS) did not inhibit but rather increased SphK2 activity. The fluorescence-based approach for the determination of the enzymatic activity of SphK2 proves to be useful for the quantitative determination of SphK2 activity in vitro and in cell lysates, and could be extended to single-cell analysis or applied in drug screening.

Identification and evaluation of neutral sphingomyelinase 2 inhibitors.

Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, D-lyxophytosphingosine and D-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the Mg(2+)-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on Mg(2+)-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.

Identification of specific inhibitors for a trypanosomatid RNA editing reaction.

Several mitochondrial mRNAs of the trypanosomatid protozoa are edited through the post-transcriptional insertion and deletion of uridylates. The reaction has provided insights into basic cellular biology and is also important as a potential therapeutic target for the diseases caused by trypanosomatid pathogens. Despite this importance, the field has been hindered by the lack of specific inhibitors that could be used as probes of the reaction mechanism or developed into novel therapeutics. In this study, an electrochemiluminescent aptamer-switch was utilized in a high-throughput screen for inhibitors of a trypanosomatid RNA editing reaction. The screen identified GW5074, mitoxantrone, NF 023, protoporphyrin IX, and D-sphingosine as inhibitors of insertion editing, with IC(50) values ranging from 1 to 3 µM. GW5074 and protoporphyrin IX are demonstrated to inhibit at or before the endonuclease cleavage that initiates editing and will be valuable biochemical probes for the early events of the in vitro reaction. Since protoporphyrin IX and sphingosine are both naturally present within the trypanosomatids, their effectiveness as in vitro inhibitors is also suggestive of the potential for in vivo modulatory roles.

Penasins A-E, long-chain cytotoxic sphingoid bases, from a marine sponge Penares sp.

Five sphingoid bases, penasin A (1), penasin B (2), and a mixture of penasins C-E (3-5), were identified from a marine sponge Penares sp. as cytotoxic constituents. The structure of the common polar head part was assigned by analysis of the NMR data, whereas the structures of the long aliphatic chains including the locations of double bond(s) and a branched methyl group were determined by analysis of tandem FABMS and (13)C NMR data together with the GC-MS analysis of ozonolysis products. The absolute configuration of the headgroup was defined for the mixture of 3-5 by the modified Mosher method. Penasins exhibit moderate cytotoxicity against HeLa and P388 cells.

Characterization of a germination-accelerating factor from the silkworm (Bombyx mori Linnaeus) of entomopathogenic fungus Nomuraea rileyi (Farlow) Samson.

The conidium of the entomopathogenic fungus, Nomuraea rileyi, has been found to germinate rapidly in the presence of a host insect-derived extract. This extract therefore appears to contain an important factor involved in host recognition by N. rileyi, although the substance (germination-accelerating factor, GAF) responsible for such unique germination behavior has yet to be identified. Our previous study was extended to the isolation of GAF from pupae of the silkworm, a host insect of N. rileyi. This present work subjects GAF to a structural analysis. The chemical structure of GAF is characterized as 2S-amino-tetradeca-4-ene-1,3R-diol (D-erythro-C(14)-sphingosine) based on spectroscopic data. An examination of the structure-activity relationship shows that the activity of D-erythro-C(14)-sphingosine was superior to that of sphingosines with shorter and longer carbon chains. It is suggested that the molecular species with a 14-carbon chain of a sphingosine is important for host recognition.

Saturated ceramides from the sponge Dysidea robusta.

Chemical investigation of the crude extract of a marine sponge Dysidea robusta led to the isolation of an inseparable mixture of saturated ceramides. These were identified from spectroscopic data as well as by hydrolysis followed by LC-MS analysis of the sphingosine moieties.

Ascotricins A and B, novel antagonists of sphingosine-1-phosphate receptor 1 from Ascotricha chartarum Berk. SANK 14186.

Ascotricins A and B were isolated as novel sphingosine-1-phosphate receptor 1 (S1P(1)) antagonists from a cultured broth of a fungus identified as Ascotricha chartarum Berk. SANK 14186. The two compounds were purified by solvent extraction, reversed-phase (RP) column chromatography and a preparative RP-HPLC. The structures were determined by various NMR experiments and by LC/MS and GC/MS analyses. The S1P(1) antagonist activities were measured by a cyclic AMP assay using S1P(1)-expressing cells and the IC(50) values were 8.2 and 1.8 microM, respectively. In a [(33)P]sphingosine-1-phosphate/S1P(1)-binding assay, those values were 120 and 39 microM, and in a migration assay using human umbilical vein endothelial cells (HUVECs), they were 94 and 28 microM, respectively. Thus, ascotricins A and B are novel S1P(1) antagonists showing an inhibition activity toward HUVEC migration.