Revealing Metabolic Perturbation Following Heavy Methamphetamine Abuse by Human Hair Metabolomics and Network Analysis.

Methamphetamine (MA) is a highly addictive central nervous system stimulant. Drug addiction is not a static condition but rather a chronically relapsing disorder. Hair is a valuable and stable specimen for chronic toxicological monitoring as it retains toxicants and metabolites. The primary focus of this study was to discover the metabolic effects encompassing diverse pathological symptoms of MA addiction. Therefore, metabolic alterations were investigated in human hair following heavy MA abuse using both targeted and untargeted mass spectrometry and through integrated network analysis. The statistical analyses (t-test, variable importance on projection score, and receiver-operator characteristic curve) demonstrated that 32 metabolites (in targeted metabolomics) as well as 417 and 224 ion features (in positive and negative ionization modes of untargeted metabolomics, respectively) were critically dysregulated. The network analysis showed that the biosynthesis or metabolism of lipids, such as glycosphingolipids, sphingolipids, glycerophospholipids, and ether lipids, as well as the metabolism of amino acids (glycine, serine and threonine; cysteine and methionine) is affected by heavy MA abuse. These findings reveal crucial metabolic effects caused by MA addiction, with emphasis on the value of human hair as a diagnostic specimen for determining drug addiction, and will aid in identifying robust diagnostic markers and therapeutic targets.

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.

The Fusarium mycotoxin, 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) induces vacuolization in HepG2 cells.

The mycotoxin 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) has been isolated from cultures of the fungus Fusarium avenaceum, one of the most prevalent Fusarium species. AOD is an analogue of sphinganine and 1-deoxysphinganine, important intermediates in the de novo biosynthesis of cellular sphingolipids. Here we studied cellular effects of AOD using the human liver cell line HepG2 as a model system. AOD (10 µM) induced a transient accumulation of vacuoles in the cells. The effect was observed at non-cytotoxic concentrations and was not linked to cell death processes. Proteomic analyses indicated that protein degradation and/or vesicular transport may be a target for AOD. Further studies revealed that AOD had only minor effects on the initiation rate of macropinocytosis and autophagy. However, the AOD-induced vacuoles were lysosomal-associated membrane protein-1 (LAMP-1) positive, suggesting that they most likely originate from lysosomes or late endosomes. Accordingly, both endosomal and autophagic protein degradation were inhibited. Further studies revealed that treatment with concanamycin A or chloroquine completely blocked the AOD-induced vacuolization, suggesting that the vacuolization is dependent of acidic lysosomes. Overall, the results strongly suggest that the increased vacuolization is due to an accumulation of AOD in lysosomes or late endosomes thereby disturbing the later stages of the endolysosomal process.

Markhasphingolipid A, new phytosphingolipid from the leaves of Markhamia stipulata var. canaense V.S. Dang.

From the leaves of Markhamia stipulata var. canaense V.S. Dang, one new phytosphingolipid, named markhasphingolipid A (6) together with five known compounds, 4',7-O-dimethylapigenin (1), narigenin (2), tectoquinone (3), mollic acid (4), 1-hexadecanoyl-sn-glycerol (5) were classified by various chromatographic methods. Their structures were designated by IR, UV, HR-ESI-MS, HR-ESI-MS/MS and NMR experiments. All compounds were recognized for the first time from this species. The cytotoxicity of all n-hexane fractions and isolated compounds (5 & 6) against three human cancer cell lines (HeLa, HepG2, and MCF-7) were evaluated by SRB assay. All n-hexane fractions expressed cytotoxic effect on three tested cancer cell lines (at the concentration of 100 µg/mL, percent of cytotoxicity ranged from 55.81% to 95.83%) as well as compound 5 (IC50 ranged from 48.51 to 63.30 µM) whereas fraction H.I and compound 6 did not show activity.[Formula: see text].

Diagnostic strategy for females suspected of Fabry disease.

A total of 11 948 females suspicious of Fabry disease were tested by a combined biochemical and genetic approach. The enzyme activity, together with the concentration of lyso-GL-3 (lyso-Gb3) biomarker in dried blood spots (DBS), substantially improved the diagnostic detection of Fabry disease in females compared to the enzyme activity alone. Abnormal values for both were highly suspicious of Fabry disease (97% positive predictive value [PPV], similar to PPV in males). In cases with one abnormal biochemical value, elevated lyso-GL-3 is a far more important indicator than low enzyme activity (39% PPV vs 6% PPV). Cases with clearly negative results for both biochemical parameters are unlikely to have Fabry disease, even in clinically highly suspicious cases.

Iridoids and sfingolipids from Hedyotis diffusa.

Seven new compounds were isolated from the aerial part of Hedyotis diffusa, including three iridoid glycosides, hedyoiridoidside A - C (1-3), two cerebrosides, hedyocerenoside F (4) and G (5), and two new ceramides, hedyoceramide A (6) and B (7). And six known iridoid glycosides (8-13) were also obtained. Their structures were established by their physico-chemical constants and spectroscopic analysis. The cytotoxicity of all compounds against tumor cell lines of human cervical cancer HeLa, human leukemia HL-60, human lung cancer A459, human hepatoma HepG2, human gastric gland carcinoma BCG-823, human nasopharyngeal cancer CNE-2, human colon cancer HCT15, and human prostate cancer PC-3 were also evaluated in vitro. As a result, new compound 1 exhibited evident cytotoxicity to all tumor cell lines, and the IC50 values are from 9.5µM to 28.2µM, while new compound 2 exhibited evident cytotoxicity to Hela, HL-60, A459, HepG2, BGC-823, CNE-2, and HCT15, and the IC50 values are from 15.8µM to 26.2µM. Known compound 11 also exhibited evident cytotoxicity to HL-60, A459, HepG2, BGC-823, CNE-2, and HCT15, and the IC50 values are from 16.5µM to 40.4µM. New compounds 4-7 and known compounds 12 and 13 showed moderate cytotoxicity to some tumor cell lines.

Milk fat components with potential anticancer activity-a review.

During many years, the milk fat has been unfairly undervalued due to its association with higher levels of cardiovascular diseases, dyslipidaemia or obesity, among others. However, currently, this relationship is being re-evaluated because some of the dairy lipid components have been attributed potential health benefits. Due to this, and based on the increasing incidence of cancer in our society, this review work aims to discuss the state of the art concerning scientific evidence of milk lipid components and reported anticancer properties. Results from the in vitro and in vivo experiments suggest that specific fatty acids (FA) (as butyric acid and conjugated linoleic acid (CLA), among others), phospholipids and sphingolipids from milk globule membrane are potential anticarcinogenic agents. However, their mechanism of action remains still unclear due to limited and inconsistent findings in human studies.

Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients.

The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-ß isoforms: TGF-ß1 (T1), TGF-ß2 (T2), and TGF-ß3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-ß1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-ß pathway.

Structure of Sphingolipids From Sea Cucumber Cucumaria frondosa and Structure-Specific Cytotoxicity Against Human HepG2 Cells.

To investigate the relationship between structure and activity, three glucocerebroside series (CFC-1, CFC-2 and CFC-3), ceramides (CF-Cer) and long-chain bases (CF-LCB) of sea cucumber Cucumaria frondosa (C. frondosa) were isolated and evaluated in HepG2 cells. The molecular species of CFC-1, CFC-2 and CFC-3 and CF-Cer were identified using reversed-phase liquid chromatography with heated electrospray ionization coupled to high-resolution mass spectrometry (RPLC-HESI-HRMS), and determined on the basis of chemical and spectroscopic evidence: For the three glucocerebroside series, fatty acids (FA) were mainly saturated (18:0 and 22:0), monounsaturated (22:1, 23:1 and 24:1) and 2-hydroxyl FA (2-HFA) (23:1 h and 24:1 h), the structure of long-chain bases (LCB) were dihydroxy (d17:1, d18:1 and d18:2) and trihydroxy (t16:0 and t17:0), and the glycosylation was glucose; For CF-Cer, FA were primarily saturated (17:0) and monounsaturated (16:1 and 19:1), the structure of LCB were dihydroxy (d17:1 and d18:1), and trihydroxy (t16:0). The results of cell experiment indicated that all of three glucocerebroside series, CF-Cer and CF-LCB exhibited an inhibitory effects on cell proliferation. Moreover, CFC-3 was most effective in three glucocerebrosides to HepG-2 cell viability. The inhibition effect of CF-LCB was the strongest, and the inhibition effect of CF-Cer was much stronger than glucocerebrosides.

Adaptation of Skyline for Targeted Lipidomics.

In response to the urgent need for analysis software that is capable of handling data from targeted high-throughput lipidomics experiments, we here present a systematic workflow for the straightforward method design and analysis of selected reaction monitoring data in lipidomics based on lipid building blocks. Skyline is a powerful software primarily designed for proteomics applications where it is widely used. We adapted this tool to a "Plug and Play" system for lipid research. This extension offers the unique capability to assemble targeted mass spectrometry methods for complex lipids easily by making use of building blocks. With simple yet tailored modifications, targeted methods to analyze main lipid classes such as glycerophospholipids, sphingolipids, glycerolipids, cholesteryl-esters, and cholesterol can be quickly introduced into Skyline for easy application by end users without distinct bioinformatics skills. To illustrate the benefits of our novel strategy, we used Skyline to quantify sphingolipids in mesenchymal stem cells. We demonstrate a simple method building procedure for sphingolipids screening, collision energy optimization, and absolute quantification of sphingolipids. In total, 72 sphingolipids were identified and absolutely quantified at the fatty acid scan species level by utilizing Skyline for data interpretation and visualization.

Rapid and comprehensive 'shotgun' lipidome profiling of colorectal cancer cell derived exosomes.

There is an increasing recognition of the role that cancer cell derived exosomes play in intercellular signaling upon fusion or uptake with a target cell, including immune system evasion, tumor growth and metastasis. To date, however, although exosomal membrane and cargo lipids are expected to play a pivotal role in exosome biogenesis and secretion, as well as in fusion or uptake and target cell functional response, the detailed characterization of cancer cell derived exosome lipids across a range of different cancers has not yet been broadly explored. Here, a simple and straightforward lipidome analysis strategy consisting of optimized sample extraction and novel sample derivatization techniques, coupled with high-resolution 'shotgun' mass spectrometry and 'targeted' tandem mass spectrometry methods, is demonstrated for the rapid identification of >520 individual lipids in 36 lipid classes and sub classes from exosomes secreted by the colorectal cancer cell line, LIM1215. Relative quantification and comparison of exosome versus cellular lipid profiles reveals significant enrichment of certain lipid classes, as well as substantial lipid subclass remodeling and changes in abundance of individual lipids, including sphingolipids, sterol lipids, glycerolipids and glycerophospholipids, and particularly plasmalogen- and alkyl ether-containing glycerophospholipids. This analysis strategy therefore provides a platform for comprehensive lipidome profiling across a wide range of cancer cell or tissue derived exosomes, that will facilitate subsequent functional studies aimed at elucidating the role of specific cellular or exosome lipids in the onset and progression of colorectal cancer, or to identify specific lipid(s) that could serve as effective diagnostic or prognostic disease biomarkers.

Leishmanial sphingolipid induces apoptosis in Sarcoma 180 cancer cells through regulation of tumour growth via angiogenic switchover.

Sphingolipids are membrane and intracellular lipids that typically modulate cellular processes to cause cell death. Exogenous administration of sphingolipids may cause restriction of tumour growth and several alternative strategies are being used to control the cell growth. The microbes, their cellular component(s) or metabolites like DHA, EPA and also FTY720 have been employed as new therapeutic entities to regulate the disease condition. The therapeutic efficacy of lipids from Leishmania donovani in rheumatoid arthritis and also in sepsis condition associated with inflammatory diseases is well established. In this study, we explored the apoptotic effect of LSPL-1 (leishmanial sphingolipid-1) in Sarcoma 180 cells towards the regulation of tumour growth. The study using a panel of cancer cell lines revealed that LSPL-1 induces cell death in Sarcoma 180. The apoptotic changes were assessed by annexin exposure and DNA content analysis using flow cytometry. LSPL-1 appears to activate several pro- and anti-apoptotic molecules through reactive oxygen species (ROS) generation and also caspase activation, as determined by Western blot and ELISA analyses. Simultaneously, it may improve the survival rate of mice bearing tumour induced by Sarcoma 180 cells, with pathological changes. LSPL-1 may also suppress the cancer-associated inflammatory responses with the expression of matrix metalloproteinase having inhibitory role. It may regulate several angiogenic factors including VEGF, Ang-2 and CD34 in angiogenic events generated in Sarcoma 180 cell-induced tumour. These studies underline the significance of anti-neoplastic potential of LSPL-1 through apoptosis induction and abrogation of angiogenic responses in Sarcoma 180 cell-associated tumour.

Two new sphingolipids from the leaves of Piper betle L.

Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis.

Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Differential induction of apoptosis of leukemic cells by rhizochalin, two headed sphingolipids from sponge and its derivatives.

Two-headed sphingolipids bear a certain similarity with sphingolipids in the structure and but differing from classical sphingolipids in alpha,omega-position of the basic groups. We analyzed the apoptotic effects of some two-headed sphingolipids including rhizochalin (Rhz) and its derivatives isolated from sponge Rhizochalina incrustata on human leukemia HL-60 cells. Direct addition of Rhz induced apoptosis of HL-60 cells. The aglycon of Rhz, which has no galactosyl residue, showed a stronger ability to induce apoptotic activity than Rhz. Rhz congeners with acetate derivatives only weakly induced apoptosis. The usual mitochondrial membrane permeability changes and the decrease of protein levels of procaspases-8, -9, and -3 correlated with the apoptotic activity of Rhzs. These results suggest that derivatives of two-headed sphingolipids potently induce apoptosis in mammalian cells when administered exogenously and this cell death was dependent on caspase activation pathways.

Sphingolipid-mediated restoration of Mitf expression and repigmentation in vivo in a mouse model of hair graying.

Recent advances in the identification and characterisation of stem cell populations has led to substantial interest in understanding the precise triggers that would operate to induce activation of quiescent stem cells. Melanocyte stem cells (MSCs) reside in the bulge region of the hair follicles and are characterised by reduced expression of the microphthalmia-associated transcription factor (Mitf) and its target genes implicated in differentiation. Vitiligo is characterised by progressive destruction of differentiated melanocytes. However, therapies using UV irradiation therapy can induce a degree of repigmentation, suggesting that MSCs may be activated. As Mitf is implicated in control of proliferation, we have explored the possibility that inducing Mitf expression via lipid-mediated activation of the p38 stress-signalling pathway may represent a re-pigmentation strategy. Here we have isolated from placental extract a C18:0 sphingolipid able to induce Mitf and tyrosinase expression via activation of the p38 stress-signalling pathway. Strikingly, in age-onset gray-haired C57BL/6J mice that exhibit decaying Mitf expression, topical application of placental sphingolipid leads to increased Mitf in follicular melanocytes and fresh dense black hair growth. The results raise the possibility that lipid-mediated activation of the p38 pathway may represent a novel approach to an effective vitiligo therapy.

Biochemistry of inositol lipids.

Nature has created an immense combinatorial and structural heterogeneity among lipids. It is becoming increasingly accepted that the vast range of unique chemical entities encodes for distinct functions within biological systems. A unique group of lipids which stands out in terms of diversity as well as biological activity are inositol-containing lipids. The most well characterized inositol lipids are the phosphoinositides, phosphorylated derivatives of glycerophosphoinositol, which play a wide variety of cellular roles in many eukaryotic cells. Less well understood are ceramides containing inositol in fungi, and inositol glycolipids in pathogens. Here we review biochemical aspects of inositol-containing lipids with a focus on novel analytical procedures for their characterization.

Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection.

In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.

Sphingolipids with neuritogenic activity from Euphorbia sororia.

Two groups of sphingolipids 1 and 2 were isolated from the aerial parts of Euphorbia sororia. On the basis of spectroscopic data, chemical methods and GC-MS analysis, the structures of 1 and 2 were characterized as 1-O-beta-D-glucopyranosyl-(2S,3S,4R,8Z)-2-[(2'R)-2'-hydroxydocosanoyl approximately hexacosanoyl, octacosanoyl amino]-1,3,4-octadecanetriol-8-ene and (2S,3S,4R,8E)-2-[(2'R)-2'-hydroxyeicosanoyl approximately hexacosanoyl amino]-1,3,4-octadecanetriol-8-ene, respectively. Both of them exhibited marked neuritogenic activity on the rat pheochromocytoma PC12 cell line.

A sphingolipid rich lipid fraction isolated from attenuated Leishmania donovani promastigote induces apoptosis in mouse and human melanoma cells in vitro.

Lipids, especially sphingolipids, are emerging as inducer of apoptosis in a wide range of immortal cells, potentiating their therapeutic application in cancer. In the present study, a sphingolipid rich lipid fraction (denoted here as ALL), isolated from an attenuated strain of Leishmania donovani promastigote, was tested for its tumoricidal activity taking melanoma, the dreaded form of skin cancer cells, as model. ALL was found to induce chromatin condensation, internucleosomal DNA fragmentation and phosphatidylserine externalization with enhanced cell population in sub-G1 region in both mouse and human melanoma systems, namely B16F10 and A375 respectively. These are the hallmarks of cells undergoing apoptosis. Further analysis demonstrated that ALL treated melanoma cells showed significant increase in ROS generation, mitochondrial membrane potential depolarization, release of cytochrome c, and caspase-3 activation, which are the events closely involved in apoptosis. These findings indicate that one or more bioactive sphingolipid(s)/ceramide(s) present in ALL could be the causative agent(s) for the induction of apoptosis in melanoma cells. Further studies are thus necessary to identify these specific bioactive sphingolipid(s)/ceramide(s) and to establish their mechanism of action, in order to explore their use as anticancer agents.