A Rapid and Simple UHPLC-MS/MS Method for Quantification of Plasma Globotriaosylsphingosine (lyso-Gb3).

Alessandro Perrone,Manuela Contin,Susan Mohamed,Vincenzo Donadio,Rocco Liguori

doi:10.3390/molecules26237358

Abstract

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25–100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as <10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects (p < 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group (p = 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, p-value < 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.

Highlights

Fabry disease (FD) is an X-linked lysosomal disease caused by mutations in the αgalactosidase A gene (GLA), encoding for the homodimeric glycoprotein α-galactosidaseA (α-Gal A)
Affected males exhibit early manifestations such as acroparesthesias, angiokeratoma, corneal opacities, and hypohidrosis starting in childhood or adolescence, which develop into cardiomyopathy, kidney failure, and premature strokes in adulthood, due to the progressive accumulation of glycosphingolipids
Female Fabry phenotypes are more heterogeneous than male Fabry phenotypes, even among patients carrying the same genotype; they range from asymptomatic to severe manifestations of the disease, depending on random X-chromosomal inactivation during early embryonic development [6,7,8]

Summary

Introduction

Fabry disease (FD) is an X-linked lysosomal disease caused by mutations in the αgalactosidase A gene (GLA), encoding for the homodimeric glycoprotein α-galactosidaseA (α-Gal A). The “classic” phenotype is characterized by more severe symptoms in male patients, due to the reduced or absent activity of α-Gal A [4]. Affected males exhibit early manifestations such as acroparesthesias, angiokeratoma, corneal opacities, and hypohidrosis starting in childhood or adolescence, which develop into cardiomyopathy, kidney failure, and premature strokes in adulthood, due to the progressive accumulation of glycosphingolipids. The late-onset phenotype shows less severe manifestations and significant residual α-Gal A activity, typically lacking early symptoms but presenting with cardiomyopathy and chronic kidney disease in adulthood [5]. Female Fabry phenotypes are more heterogeneous than male Fabry phenotypes, even among patients carrying the same genotype; they range from asymptomatic to severe manifestations of the disease, depending on random X-chromosomal inactivation during early embryonic development [6,7,8]

Methods

Results

Conclusion