Diurnal Variation of Urinary Fabry Disease Biomarkers during Enzyme Replacement Therapy Cycles.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the α-galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (-C2H4, -C2H4+O, -H2, -H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-naïve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.

Analysis of globotriaosylceramide (Gb3) isoforms/analogs in unfractionated leukocytes, B lymphocytes and monocytes from Fabry patients using ultra-high performance liquid chromatography/tandem mass spectrometry.

Fabry disease is an X-linked lysosomal storage disorder with marked variability in the phenotype and genotype. Glycosphingolipids such as globotriaosylceramide (Gb3) isoforms/analogs, globotriaosylsphingosine (lyso-Gb3) and analogs, and galabiosylceramide (Ga2) isoforms/analogs may accumulate in biological fluids and different organs. The aims of this study were to: 1) develop/validate a novel UHPLC-MS/MS method for relative quantitation of Gb3 in leukocytes (unfractionated white blood cells), B lymphocytes and monocytes; 2) evaluate these biomarkers in a cohort of Fabry patients and healthy controls; and 3) assess correlations between these biomarkers, treatment and genotype. Whole blood, plasma and urine samples from 21 Fabry patients and 20 healthy controls were analyzed. Samples were purified by liquid-liquid extraction and analyzed by UHPLC-MS/MS in positive electrospray ionization. Methylated Gb3 isoforms were detected, showing that a methylation process occurs at the cellular level. Our results show that there were no significant differences in the distribution of the different Gb3 isoforms/analogs in blood cells between Fabry patients and healthy controls. In leukocyte, Gb3[(d18:1)(C14:0)], Gb3[(d18:1)(C16:0)], Gb3 [(d18:1)(C16:0)]Me, Gb3 [(d18:1)(C16:1)], Gb3 [(d18:1)(C18:0)], Gb3 [(d18:1)(C18:1)], Gb3 [(d18:1)(C20:1)], Gb3 [(d18:1)(C24:2)], Gb3 [(d18:1)(C26:1)] and total Gb3 allowed good discrimination between male Fabry patients and male controls, patients having higher biomarker levels than controls. Regarding B lymphocytes and monocytes, the same tendency was observed without reaching statistical significance. A positive concordance between mutation types and biomarker levels in white blood cells was established. Our results might provide a deeper mechanistic comprehension of the underlying biochemical processes of Gb3 biomarkers in white blood cells of Fabry patients.