Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels.

Biochemical response to substrate reduction therapy versus enzyme replacement therapy in Gaucher disease type 1 patients.

Background: We retrospectively compared biochemical responses in type 1 Gaucher disease patients to treatment with glycosphingolipid synthesis inhibitors miglustat and eliglustat and ERT.

Methods: Seventeen GD1 patients were included (n = 6 eliglustat, (two switched from ERT), n = 9 miglustat (seven switchers), n = 4 ERT (median dose 60U/kg/m). Plasma protein markers reflecting disease burden (chitotriosidase, CCL18) and lipids reflecting substrate accumulation (glucosylsphingosine, glucosylceramide) were determined.

Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids.

Lyso-glycosphingolipid abnormalities in different murine models of lysosomal storage disorders.

In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC–MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids.

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa.

Synthesis of 6-Hydroxysphingosine and α-Hydroxy Ceramide Using a Cross-Metathesis Strategy.

In this paper, a new synthetic route toward 6-hydroxysphingosine and α-hydroxy ceramide is described. The synthesis employs a cross-metathesis to unite a sphingosine head allylic alcohol with a long-chain fatty acid alkene that also bears an allylic alcohol group. To allow for a productive CM coupling, the sphingosine head allylic alcohol was protected with a cyclic carbonate moiety and a reactive CM catalyst system, consisting of Grubbs II catalyst and CuI, was employed.

Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard.

Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS.

Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8(+) T cell immune response.

Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co-hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300-400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro.

Gaucher disease and Fabry disease: new markers and insights in pathophysiology for two distinct glycosphingolipidoses.

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively.