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. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.

Quantification of globotriaosylsphingosine in plasma and urine of fabry patients by stable isotope ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker. METHODS: We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] (13)C5-lysoGb3 (internal standard). After addition of the internal standard to 25 µL plasma or 400 µL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H](+) for lysoGb3, m/z 791.4 > 287.3 [M+H](+) for [5,6,7,8,9] (13)C5-lysoGb3, and 784.4 > 280.3 [M+H](+) for lyso-ene-Gb3. RESULTS: The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7-136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1-23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3-0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%-25% of total lysoGb3. The urine concentration of lysoGb3 was 40-480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations. CONCLUSIONS: This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.

Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies.

A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.

Synthesis of hyaluronic acid oligomers using chemoselective and one-pot strategies.

An efficient synthetic strategy toward a hyaluronic acid (HA) tri-, penta-, and heptamer having a glucosamine-reducing end is reported. The synthesis is based on a glucuronate ester thioglycoside and a trifluoro-N-phenylimidate glucosamine building block. The HA-fragments are synthesized using an S-phenyl GlcN-GluA building block through a combination of chemoselective and one-pot condensation strategies.

Synthesis of sugar nucleotides by application of phosphoramidites.

A new method for the construction of pyrophosphates is reported based on the coupling of a sugar phosphate and a nucleoside phosphoramidite. The in situ formed phosphate-phosphite intermediate was subsequently oxidized with tBuOOH. Three UDP-N-acetylglucosamine derivatives were prepared using this one-pot procedure in good yields.

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. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice. We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8+ T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.

A practical synthesis of capped 4-methylumbelliferyl hyaluronan disaccharides and tetrasaccharides as potential hyaluronidase substrates.

The synthesis of hyaluronan dimers and tetramers equipped with a 4-methylumbelliferyl group at the reducing end to potentially allow monitoring of hyaluronidase activities is described. The 4-OH at the non-reducing glucuronate in the presented series is either removed or methylated to prohibit transglycosylase reactions, leading to a total of four probes.