Quantification of Globotriaosylsphingosine in Plasma and Urine of Fabry Patients by Stable Isotope Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

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] 13C5-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] 13C5-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.

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

“…Briefly, 25 lL of plasma were extracted and GSLs were determined in the lower phase by HPLC using C17-sphinganine as internal standard (Avanti Polar Lipids, Alabaster, AL, USA) [21]. Lyso-GSLs were analysed in the upper-phase by LC-ESI-MS/MS using 13 C 5 -LysoGb3 and 13 C 5 -GlcSph as internal standard [5,22]. 13 C 5 -sphingoid bases contain five 13 C atoms in the sphingosine moiety.…”

Uncertain Diagnosis of Fabry Disease in Patients with Neuropathic Pain, Angiokeratoma or Cornea Verticillata: Consensus on the Approach to Diagnosis and Follow-Up

“…Interestingly, most male patients with a GVUS demonstrate residual enzyme activity, in contrast to the absent or near absent enzyme activity in classically affected males. Also, previous studies have shown that patients with a nonclassical phenotype often only show a slight increase of lysoGb3 in plasma, while classically affected males invariably have very high levels (Aerts et al 2008;Rombach et al 2010;Gold et al 2013;Lukas et al 2013).…”

A Fast Determination of Globotriaosylsphingosine in Plasma for Screening Fabry Disease Using UPLC-ESI-MS/MS

“…[3][4][5] Recently, researcher have paid attention to the excretion of plasma lyso-Gb3 level and published it's significant correlation with clinical severity of disease manifestations. 6,7,[9][10][11] Traditionally analytical method of lyso-Gb3 was based on O-phthalaldehyde derivatization of amine in the lysoglycolipid followed by HPLC-fluorescence detection. 11-14 LC-MS/MS has it's own strengths such as high sensitivity, specificity and rapidity of analysis, thus the paradigm of the analysis is getting shifted to the usage of LC-MS/MS including lyso-Gb3.…”

“…11-14 LC-MS/MS has it's own strengths such as high sensitivity, specificity and rapidity of analysis, thus the paradigm of the analysis is getting shifted to the usage of LC-MS/MS including lyso-Gb3. 9,[15][16][17][18][19] This study is devised to develop and validate a fast and simple analytical method for quantification of lyso-Gb3 in human plasma using UPLC-ESI-MS/MS.…”