Abstract
Fabry disease (FD) is an X-linked lysosomal storage disorder, resulting from a deficiency of the enzyme α-galactosidase A, responsible for breaking down glycolipids such as globotriaosylceramide and its deacylated derivative, globotriaosylsphingosine (LysoGb3). Here, wecompare the levels of LysoGb3 in dried blood spots (DBS) and plasma in patients with classic and late-onset phenotypes. LysoGb3 measurements were performed in 104 FD patients, 39 males and 65 females. Venous blood was collected. A portion was spotted onto filter paper and another portion separated to obtain plasma. The LysoGb3 concentrations in DBS and plasma were determined by highly sensitive electrospray ionization liquid chromatography tandem mass spectrometry. Agreement between different matrices was assessed using linear regression and Bland Altman analysis. The method on DBS was validated by evaluating its precision, accuracy, matrix effect, recovery, and stability. The analytical performances were verified by comparison of a total of 104 paired DBS and plasma samples from as many FD patients (representing 46 GLA variants). There was a strong correlation between plasma and the corresponding DBS LysoGb3 concentrations, with few exceptions. Discrepancies were observed in anemic patients with typically low hematocrit levels compared to the normal range. The method proved to be efficient for the rapid analysis of LysoGb3. DBS provides a convenient, sensitive, and reproducible method for measuring LysoGb3 levels for diagnosis, initial phenotypic assignment, and therapeutic monitoring in patients with FD.
Highlights
Fabry disease (FD, OMIM 301500) is caused by an inborn error of glycosphingolipid metabolism, a genetic defectThis work is licensed under the Creative Commons Attribution 4.0Malvagia et al.: Comparison of LysoGb3 in dried blood spots (DBS) and plasma resulting in partial or complete deficiency of the lysosomal hydrolase alpha-galactosidase A [1]
Differences on LysoGb3 quantification related to DBS punch location that we observed were lower than 15% CV, negligible
The amount of solvent required for complete extraction of LysoGb3 from DBS was verified by performing a second extraction on the exhausted DBS
Summary
Fabry disease (FD, OMIM 301500) is caused by an inborn error of glycosphingolipid metabolism, a genetic defectThis work is licensed under the Creative Commons Attribution 4.0Malvagia et al.: Comparison of LysoGb3 in DBS and plasma resulting in partial or complete deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal A) [1]. Alpha-Gal A deficiency results in an inability to hydrolyze certain glycolipids, primarily globotriaosylceramide (Gb3) which accumulates in the lining of the blood vessels within the kidney, heart, skin, brain and other organs together with other glycoconjugates with α-galactosyl terminal moieties, including the deacylated derivative of Gb3, globotriaosylsphingosine [3]. The progressive accumulation of these lipids in the blood vessels is at the basis of clinical manifestations such as kidney failure, stroke, cardiovascular disease, severe pain and numbness [4]. In addition to the classical phenotype, an increasing number of patients with an attenuated late-onset phenotype are diagnosed. These patients have been described in the literature with symptoms affecting one or several organs, mainly the cardiovascular system
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