LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population.

Background Gaucher disease (GD), caused by a deficiency in acid ß-glucosidase, leads to the accumulation of glucosylsphingosine (GluSph), which has been used as a powerful biomarker for the diagnosis and follow-up of GD. Our aim was to perform the first retrospective study of GluSph in Spanish patients, analyzing its relationship with classical biomarkers and other parameters of disease and its utility regarding treatment monitoring. Methods Classical biomarkers were evaluated retrospectively by standard methods in a total of 145 subjects, including 47 GD patients, carriers, healthy controls and patients suffering from other lysosomal lipidoses. GluSph was also measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed as part of the present study. Results The optimized method presented intra- and inter-assay variations of 3.1 and 11.5%, respectively, overall recovery higher than 96% and linearity up to plasma concentrations of 1000 ng/mL with 100% specificity and sensitivity. Only GD patients displayed GluSph levels above 5.4 ng/mL at diagnosis and this was significantly correlated with the classical biomarkers chitotriosidase (r = 0.560) and the chemokine CCL18/PARC (CCL18/PARC) (ρ = 0.515), as well as with the Spanish magnetic resonance imaging index (S-MRI, r = 0.364), whereas chitotriosidase correlated with liver volume (r = 0.372) and CCL18/PARC increased in patients with bone manifestations (p = 0.005). GluSph levels decreased with treatment in naïve patients. Conclusions Plasma GluSph is the most disease-specific biomarker for GD with demonstrated diagnostic value and responsiveness to therapy. GluSph in the present series of patients failed to demonstrate better correlations with clinical characteristics at onset than classical biomarkers.

Evaluation of reversed-phase nano liquid chromatography conditions by using reversed-phase thin layer chromatography based on Hansen solubility parameters for the analysis of amphiphilic glycosylsphingolipid transformations.

Pulse chase analysis is often used in investigating dynamics of cellular substances. Fluorescently labeled lactosyl sphingosine molecule is useful in chasing its transformation, however the analysis of such metabolites in attomole level is of extreme difficult due to the presence of large amount of endogenous amphiphilic molecules such as glycosphingolipids, sphingomyerin, and glycerophospholipids. Nano LC suites for analyzing the attomole scale metabolites, therefore removal of endogenous substances prior to nano LC and finding appropriate nano LC conditions are necessary. Thus, we focused on the solubility of fluorescent BODIPY-labeled lactosylsphingosine (Lac-Sph-BODIPY) to identify suitable solvents to remove endogenous compounds. In this study, we evaluated solvents by using C18 thin layer chromatography (RP TLC). The mobility (Rf) of Lac-Sph-BODIPY against several solvent mixtures on RP TLC were plotted against polarity and hydrogen bonding capability followed by Hansen solubility parameters (HSPs). The optimum solvent mixture with Rf = 0.3 ±â€¯0.1 was chosen for elimination of endogenous phospholipids on a ZrO2-SiO2 cartridge column and subsequent separation by nano LC. Efficient removal of endogenous phospholipids was demonstrated, and good resolution in nano LC analysis of Lac-Sph-BODIPY extracted from Chinese hamster ovary (CHO)-K1 cells was achieved. It was also shown that the amount of exogenously added compound was important in the investigation of metabolites using cultured cells.

Rapid, single-phase extraction of glucosylsphingosine from plasma: A universal screening and monitoring tool.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Selective extraction and effective separation of galactosylsphingosine (psychosine) and glucosylsphingosine from other glycosphingolipids in pathological tissue samples.

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.

A microwave-mediated saponification of galactosylceramide and galactosylceramide I3-sulfate and identification of their lyso-compounds by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Small amounts of galactosylceramide (cerebroside) and galactosylceramide I3-sulfate (sulfatide) obtained from porcine spinal cord and equine kidney were deacylated by a rapid method of microwave-mediated saponification to prepare their lyso-compounds. Mass spectra of their protonated or deprotonated molecular ion peaks were detected by recently developed new technology of a delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometer with reflector detector in positive or negative ion mode. Long chain bases of lysocerebroside and lysosulfatide were different between porcine spinal cord and equine kidney, but similar to each other in the same organ, suggesting their common synthetic pathway. It is noted that the new rapid method can be similarly applied to the deacylation of both cerebroside and sulfatide in contrast to our classical method which was able to be applied to cerebroside, but not to sulfatide.

Plasmalopsychosine, a novel plasmal (fatty aldehyde) conjugate of psychosine with cyclic acetal linkage. Isolation and characterization from human brain white matter.

Through a systematic examination of basic (cationic) lipids separated on Folch's lower phase from extracts of human brain by cation exchange chromatography on carboxymethyl Sephadex in a chloroform/methanol mixture, followed by successive chromatographies on Florisil and Iatrobeads columns, five compounds of basic lipids were separated. Two major unknown compounds A and B and a minor unknown compound C were separated, in addition to minor compounds sphingosine and N,N-dimethylsphingosine. This paper describes the isolation and chemical characterization of major unknown compounds A and B, which were found only in the white matter but not in the gray matter of the human brain. Unmodified psychosine (galactosylsphingosine) was essentially undetectable under the experimental conditions. Unknown compounds A and B were identified as novel plasmal (fatty aldehyde) conjugates of psychosine with cyclic acetal linkage at the galactosyl residue of psychosine. Fatty aldehydes were identified as mainly palmital (16:0) and stearal (18:0). Sphingosine was identified as d18:1 sphingosine. Faster migrating compound A had 3,4-cyclic acetal linkage, and slower migrating compound B had 4,6-cyclic acetal linkage (where m is 14 or 16 and n is 12) as shown below. [formula: see text] Preliminary studies showed that compounds A, B, and C had a weak inhibitory effect on protein kinase C (PKC) and had no cytotoxic effect. In contrast, psychosine displayed a strong cytotoxicity and inhibitory effect on PKC. Therefore, the process controlling the addition or deletion of plasmal cyclic linkage to psychosine could be a crucial step in regulation of PKC, src, or other kinases susceptible to psychosine.

The occurrence of psychosine and other glycolipids in spleen and liver from the three major types of Gaucher's disease.

Glycolipid changes in spleen autopsy specimens were determined in four cases of Gaucher's disease type I, three cases of type II, and twelve cases of type III. These changes were also determined in liver autopsy specimens from three cases of type II and in nine cases of type III. The concentration of glucosylceramide in spleen was of the same magnitude in all three types, 36.3 +/- 11.7 mmol/kg in type I, 32.7 +/- 8.5 mmol/kg in type II, and 32.6 +/- 6.9 mmol/kg in type III. In liver there were large differences in the glucosylceramide concentration between splenectomized and non-splenectomized cases. Thus, in the non-splenectomized type III cases it was 9.9 +/- 3.0 mmol/kg, while in the splenectomized type III cases it was 24.1 +/- 6.1 mmol/kg. The accelerated deposition of glucosylceramide in liver after splenectomy was also demonstrated by analyses of liver biopsy specimens. A 2-6-fold increase of gangliosides was found in liver and spleen from the three types, with no significant differences between the types. The increase of gangliosides was limited almost exclusively to GM3. Glucosylsphingosine, never detected in normal tissue, was demonstrated in all samples from Gaucher's livers and spleens. The concentration in spleen was in type II, 0.16 +/- 0.05 mmol/kg, in type III, 0.19 +/- 0.05 mmol/kg, while in type I it was significantly lower, 0.07 +/- 0.03 mmol/kg. In liver, the highest concentrations occurred in the splenectomized type III subjects, 0.16 +/- 0.08 mmol/kg, while in the non-splenectomized type III cases it was 0.06 +/- 0.02 mmol/kg and in type II 0.09 +/- 0.02 mmol/kg. The demonstration of high concentrations of the cytotoxic compound glycosylsphingosine may be a contributing factor behind the tissue necrosis and fibrosis commonly seen in spleens and livers from Gaucher's patients.