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.

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.

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.

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.

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.

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.

Accumulation of fingolimod (FTY720) in lymphoid tissues contributes to prolonged efficacy.

The immunomodulator fingolimod (FTY720) induces lymphopenia by inhibiting lymphocyte egress from thymus and secondary lymphoid organs (SLOs). It is phosphorylated mainly by sphingosine kinase (SK) 2 in vivo. FTY720-phosphate (FTY-P) activated and rapidly internalized S1P(1), which is the major sphingosine 1-phosphate (S1P) receptor for mediating lymphocyte egress. Although FTY-P is thought to be the active metabolite for triggering the onset of lymphopenia, nonphosphorylated FTY720 was much more potent in inhibiting cellular calcium flux and splenocyte chemotaxis via S1P(1) than FTY-P after preincubation. Determination of both compounds by liquid chromatography coupled to mass spectrometry revealed efficient uptake and accumulation of FTY720 but not FTY-P by splenocytes. Coculture experiments of B and T cells with and without FTY720 pretreatment led to rapid cellular transfer and phosphorylation by mouse lymphocytes. The presence of FTY720 in lymphoid tissues of FTY720-treated SK2-deficient mice without onset of lymphopenia excluded a potential role of the nonphosphorylated compound for lymphocyte egress. Local concentrations of both phosphorylated and nonphosphorylated FTY720 were much higher in lymphoid tissues than in blood. Therefore, we conclude that cellular accumulation of FTY720 generates a reservoir in thymus and SLOs, leading to sustained FTY-P production and activation of S1P(1) within tissues.

Glycosylceramides obtain from the starfish Asterias amurensis Lütken.

Complex lipids in the starfish Asterias amurensis were characterized and the influence of sphingoid bases on human colon carcinoma Caco-2 cells was also investigated. Lipid content of gonad and viscera were 3.3% and 6.8%, respectively, in wet basis. The main lipid class in gonad was ceramide monohexoside (CMH) while triglyceride (TG) was predominant in the viscera. The most abundant fatty acid in the polar lipid was eicosapentaenoic acid (EPA, C20:5n-3), with the gonad and viscera samples having the highest proportion of 41.5% and 32.7%, respectively, of total fatty acids. Starfish internal organ contained enormous amount (0.7% in wet base) of glycosylceramide. Sphingoid bases of the glycosylceramide were mainly consisted of d22:2, d22:1 and d18:3. This sphingoid base exerted an apoptotic activity on Caco-2 cells. Thus, starfish could be used as a potential source of precious and useful complex lipids.

FTY720, a synthetic compound from Isaria sinclairii, inhibits proliferation and induces apoptosis in pancreatic cancer cells.

FTY720, a synthetic compound produced by modification of a metabolite from Isaria sinclairii, is known as a unique immunosuppressive agent that exerts its activity by inducing apoptosis of lymphocytes [S. Suzuki, FTY720: Mechanisms of action and its effect on organ transplantation, Transplant. Proc. 31 (1999) 2779-2782]. Additionally, it has been found that FTY720 has inhibitory effects on various cancer growth and metastasis [J.D. Wang, S. Takahara, N. Nonomura, Early induction of apoptosis in androgen-independent prostate cancer cell line by FTY720 requires caspase-3 activation, Prostate 40 (1999) 50-55]. To investigate its effect on the growth and metastasis of pancreatic cancer, FTY720 was used to treat three pancreatic cancer cell lines (BxPC-3, AsPC-1, and PANC-1). The MTT assay and flow cytometry were used to evaluate the cell death after FTY720 treatment; the wound closure assay, three-dimensional (3D) Matrigel assay, and invasive assay were used to evaluate the migration, colony formation and invasion abilities after FTY720 treatment, respectively. Protein expression in BxPC-3, AsPC-1, and PANC-1 cells after FTY720 treatment was detected by Western blotting. The MTT assay indicated that the growth of pancreatic cancer cells could be inhibited by FTY720 at various concentrations between 0 and 17 microM in a dose-dependent manner, which was also confirmed by flow cytometry. The wound closure assay, 3D Matrigel assay and cell invasion assay all showed that FTY720 significantly suppressed migration, colony formation and invasion ability of cancer cells at concentrations from 5 to 17 microM. After FTY720 treatment, the phospho-Akt, Bcl-2, pro-caspase-3 expression were down-regulated while the caspase-9 protein expression was increased. In conclusion, FTY720 can inhibit the growth, migration and invasion of pancreatic cancer cells. Our study provides a preclinical support for chemotherapeutic approach with FTY720 for the treatment of pancreatic cancer.

Pachastrissamine, a cytotoxic anhydrophytosphingosine from a marine sponge, Pachastrissa sp.

An anhydrophytosphingosine named pachastrissamine (3) has been isolated as a cytotoxic principle of a sponge, Pachastrissa sp., and the structure including the absolute configuration determined by spectroscopic and chemical analysis.

Cytotoxic sphingosine 4-sulfates from the sponge Spirastrella abata.

Two new sphingosine 4-sulfates (1 and 2) have been isolated from the sponge Spirastrella abata as cytotoxic constituents. The gross structures of 1 and 2 were determined by spectroscopic analysis, and their stereochemistry was established by chemical conversion. The compounds exhibited significant cytotoxicity against a small panel of five human tumor cell lines.

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Improved fluorescent determination method of cellular sphingoid bases in high-performance liquid chromatography.

Precolumn orthophthaldehyde (OPA) labeling method of sphingoid bases, sphingosine and sphinganine, was investigated to obtain high fluorescent detectability. In order to improve the fluorescent yield, we investigated the optimal solubility of sphingoid bases for five pre-incubation solvents by incorporating the heating procedure before OPA derivatization. The pre-incubation in ethanol prominently increased the fluorescent peak height of OPA derivative for each sphingoid bases in high performance liquid chromatography. About ten-fold increase of detectability was archived by pre-incubating lipid extracts pellets in ethanol at 60 degrees C for 30 min. Optimal derivatization was performed in 30 min at ambient temperature and the fluorescent intensity of OPA derivative was stable for two weeks at 4 degrees C. The detection limit of sphingosine was 0.1 pmol as injected amount. This method was applied to the determination of cellular sphingosine and sphinganine in various human lung cancer cells. This OPA procedure was prospective to be useful for quantitating the amount of sphingoid bases in other cancer cells.

Formation of endogenous free sphingoid bases in cells induced by changing medium conditions.

Sphingoid bases are precursors and breakdown products of sphingolipids and may function as second messengers. Here we have tested the hypothesis that sphingoid bases are produced in cells in response to external stimuli. Using a high-performance liquid chromatography system, the pattern and the amounts of free sphingoid bases in various cell types (i.e., NIH-3T3, A431, NG108-15) were determined. The predominant sphingoid base in these mammalian cells was identified as C-18 sphingosine, followed by C-18 sphinganine (dihydrosphingosine). In all cells examined, the levels of endogenous sphingoid bases can be rapidly elevated by replacing cell-conditioned medium with Hepes-buffered saline or with fresh medium, causing a dramatic increase (up to 9.5-fold) in sphingosine levels within 60 min; sphinganine levels were raised to a lesser extent (up to 4.5-fold). Addition of ammonium ions inhibited the generation of sphingoid bases. These results suggest that the machinery for metabolizing sphingoid bases can be stimulated rapidly, although the exact nature of the stimulus remains obscure. Nevertheless, the ability to control sphingosine formation in cells by changing medium conditions provides a powerful tool for investigations of the physiological roles of endogenous sphingosine.

Enzymatic synthesis of N,N-dimethyl-sphingosine: demonstration of the sphingosine: N-methyltransferase in mouse brain.

Based on our recent finding that N,N-dimethyl-D-erythro-sphingenine strongly inhibits protein kinase C (PK-C) whereas D-erythro-sphingenine produces only weak inhibition, we have studied the presence of N-methyltransferase responsible for conversion of sphingosine to its N,N-dimethyl derivative. The enzyme activity was detected in crude mouse brain tissue homogenate but was hardly detectable in liver homogenate, in which N-methylation of phosphatidylethanolamine (PE) is predominant.

Symbioramide, a novel Ca2+-ATPase activator from the cultured dinoflagellate Symbiodinium sp.

A novel sphingosine derivative, symbioramide, has been isolated from the laboratory-cultured dinoflagellate Symbiodinium sp. as a sarcoplasmic reticulum (SR)Ca2+-ATPase activator, and its structure elucidated to be 1 on the basis of spectral and chemical means.