sphingosyl-beta-glucoside and Leukodystrophy--Globoid-Cell

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine

Topics: Acid Ceramidase; Administration, Oral; Animals; Benzoxazoles; Brain; Cell Line, Tumor; Enzyme Inhibitors; Female; Gaucher Disease; Humans; Leukodystrophy, Globoid Cell; Male; Mice; Molecular Structure; Psychosine; Structure-Activity Relationship

Glucosylsphingosine (GlcS) in plasma is considered to be a reliable biomarker of Gaucher disease. The detection difficulty of GlcS is that it is difficult to achieve simultaneous separation and quantification with its isomer galactosylsphingosine (GalS), a biomarker of Krabbe disease. In this work, a multiplexed stable isotope labeling absolute quantization strategy coupled with magnetic dispersive solid phase extraction using new prepared dummy magnetic molecularly imprinted polymers (DMMIPs) has been developed for this purpose by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). 8-Plex Amine-reactive Mass Difference Tags (M360/361/362/363/373/375/376/378-AMDTs), were designed, synthesized and used to label GalS and GlcS in different 8 plasma samples, respectively. Synchronously, M359-AMDTs was prepared and used to label mixed standards of GalS and GlcS, which served as internal standards in UHPLC-MS/MS quantitation. Then DMMIPs possessing dual recognition function were applied for specific enrichment and purification of all GlcS and GalS derivatives from a combined solution of labeled 8-plex plasma samples and mixed standards before UHPLC-MS/MS injection. The labeling efficiency, chromatographic retention and mass spectrometry responses of all the 9 AMDTs reagents were consistent for GlcS and GalS. The established and validated method enabled 8-plex plasma samples quantification in a single UHPLC-MS/MS run (<2.0 min). Good linearity of AMDTs-GlcS/GalS derivatives was obtained in the range of 0.02-800 nM. LODs of GlcS and GalS were both 0.005 nM. The recoveries were in the range of 96.1-107.2%. The method was successfully applied for multiplex quantitative analysis of GlcS and GalS in human plasma samples. The results indicated that this method was capable of better realizing the simultaneous separation and quantification of GalS and GlcS compared to reported methods.

Topics: Biomarkers; Chromatography, High Pressure Liquid; Humans; Isotope Labeling; Leukodystrophy, Globoid Cell; Molecularly Imprinted Polymers; Psychosine; Solid Phase Extraction

Glucosylsphingosine (GluSph) has emerged as a biomarker for the inherited metabolic disorder, Gaucher disease (GD). We developed a simple laboratory test to measure plasma GluSph and show that elevated GluSph is diagnostic for GD as well as informing on disease burden for monitoring patients on treatment.. GluSph was measured from a single-phase total lipid extraction of 0.01 mL of plasma by liquid chromatography-electrospray ionisation-tandem mass spectrometry and concentrations extrapolated from a seven point standard curve (0.04 to 20 pmoL). A total of 1464 samples were tested and longitudinal assessment of an additional 20 GD patients.. All patients with GD had elevated GluSph compared to unaffected controls and 16 other metabolic disorders. GluSph was also slightly elevated in three patients with Krabbe disease but not at concentrations to confuse a GD diagnosis. GluSph correlated with chitotriosidase in the majority of GD patients on treatment who were informative for this marker.. GluSph can be easily measured from 0.01 mL of plasma and is useful as a diagnostic marker for GD with the current platform suited to high-throughput screening. It outperforms other GD biomarkers for biochemical monitoring of patients receiving enzyme replacement therapy for all individuals.

Topics: Adult; Biomarkers; Chromatography, Liquid; Gaucher Disease; High-Throughput Screening Assays; Humans; Leukodystrophy, Globoid Cell; Psychosine; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Time Factors

To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.

Topics: Adolescent; Adult; Animals; Brain Chemistry; Child; Child, Preschool; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dogs; Gaucher Disease; Glycosphingolipids; Humans; Infant; Leukodystrophy, Globoid Cell; Macaca mulatta; Mice; Mice, Mutant Strains; Psychosine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spleen

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an approximately 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

Topics: Animals; Cations; Chromatography; Chromatography, Ion Exchange; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Gaucher Disease; Leukodystrophy, Globoid Cell; Lipid Metabolism; Lipids; Mice; Neutral Glycosphingolipids; Psychosine; Sphingosine

Globoid cell leukodystrophy (GLD) is an autosomal recessive disorder of infants, caused by deficient activity of cerebroside-beta-galactosidase resulting in loss of myelin accompanied by loss of oligodendrocytes. The loss of oligodendrocyte population is accompanied by accumulation of psychosine, which is considered as the molecule responsible for the observed pathophysiology of GLD. We were able to detect apoptotic cells by terminal dUTP nick-end labeling assay and nuclear localization of p53 in postmortem brain tissue of Krabbe's disease patients, which were not detected in the control brain. To study the role of psychosine in cell death, we investigated the effect of psychosine on C6 glial cell survival by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Similar to ceramide (43.8% loss) the galactopsychosine and glucopsychosine treatment killed up to 46.3 and 48.75% of cells, respectively. On the other hand, sphingosine had no effect. DNA laddering assay confirmed these results. Moreover, psychosine-induced detection of annexin-V positive cells supports a role for psychosine in C6 glial cell death via the apoptotic pathway. These results indicate that psychosine may play a role in apoptotic cell loss observed in GLD brain.

Topics: Animals; Annexin A5; Apoptosis; Brain; Cell Survival; Ceramides; DNA Fragmentation; Dose-Response Relationship, Drug; Glioma; Glutathione S-Transferase pi; Glutathione Transferase; Humans; In Situ Nick-End Labeling; Isoenzymes; Leukodystrophy, Globoid Cell; Psychosine; Rats; Sphingosine; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.

Topics: Actins; Cell Division; Humans; Leukodystrophy, Globoid Cell; Phagocytosis; Phosphorylcholine; Psychosine; Sphingosine; Tumor Cells, Cultured; U937 Cells