Multiplex Analysis of Novel Urinary Lyso-Gb₃‑Related Biomarkers for Fabry Disease by Tandem Mass Spectrometry

Fabry disease is a lysosomal storage disorder caused by the absence or reduction of α-galactosidase A enzyme activity. The enzymatic deficiency results in the impaired catabolism of neutral sphingolipids with terminal α-galactosyl residues and subsequent accumulation in several tissues. Biomarkers reflecting disease severity and progression, the response to therapeutic intervention, and details of molecular pathogenesis are needed. Until now, two sphingolipids were targeted as biomarkers in urine and plasma of Fabry patients: globotriaosylceramide (Gb₃) and globotriaosylsphingosine (lyso-Gb₃). Using metabolomic approaches, our group recently discovered seven novel urinary lyso-Gb₃-related Fabry disease biomarkers with mass-to-charge ratios (<i>m</i>/<i>z</i>) of 758, 774, 784, 800, 802, 820, and 836. All these biomarkers exhibited modifications of the lyso-Gb₃ sphingosine moiety. The aims of the present study were to devise and validate a specific tandem mass spectrometry multiplex methodology for the relative quantification of these seven analogues and to evaluate their urinary excretion levels in samples from 164 Fabry patients and 94 healthy controls. We found no detectable analogues in healthy controls, except for trace amounts of the analogue with <i>m</i>/<i>z</i> 836. Significant correlations were established between lyso-Gb₃ analogue levels in urine and gender (<i>p</i> < 0.001). Fabry males had higher excretion levels compared to females with the disease. Lyso-Gb₃ analogue levels correlated well with enzyme replacement therapy (ERT) status in males (<i>p</i> < 0.05). The urinary analogue distributions varied among Fabry patients. However, the analogues with <i>m</i>/<i>z</i> 802, 820, and 836 were generally more abundant in the majority of patients. Lyso-Gb₃ analogues are promising urinary biomarkers for Fabry disease

Urinary Globotriaosylsphingosine-Related Biomarkers for Fabry Disease Targeted by Metabolomics

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in glycosphingolipid accumulation in organs and tissues, including plasma and urine. Two disease-specific Fabry biomarkers have been identified and quantified in plasma and urine: globotriaosylceramide (Gb₃) and globotriaosylsphingosine (lyso-Gb₃). The search continues for biomarkers that might be reliable indicators of disease severity and response to treatment. The main objective of this study was to target other urinary biomarkers using a time-of-flight mass spectrometry metabolomic approach. Urinary metabolites of 63 untreated Fabry patients and 59 controls were analyzed. A multivariate statistical analysis performed on a subset of male samples revealed seven novel Fabry biomarkers in urine, all lyso-Gb₃ analogues having modified sphingosine moieties. The empirical formulas of the sphingosine modifications were determined by exact mass measurements (− C₂H₄, – C₂H₄ + O, – H₂, – H₂ + O, + O, + H₂O₂, + H₂O₃). We evaluated the relative concentration of lyso-Gb₃ and its seven analogues by measuring area counts for each analogue in all Fabry patients. All samples were normalized to creatinine. We found higher concentrations for males with Fabry disease compared to females. None of these biomarkers were detected in controls. To our knowledge, this is the first time that lyso-Gb₃-related Fabry disease biomarkers are detected in urine

Urinary Globotriaosylsphingosine-Related Biomarkers for Fabry Disease Targeted by Metabolomics

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in glycosphingolipid accumulation in organs and tissues, including plasma and urine. Two disease-specific Fabry biomarkers have been identified and quantified in plasma and urine: globotriaosylceramide (Gb₃) and globotriaosylsphingosine (lyso-Gb₃). The search continues for biomarkers that might be reliable indicators of disease severity and response to treatment. The main objective of this study was to target other urinary biomarkers using a time-of-flight mass spectrometry metabolomic approach. Urinary metabolites of 63 untreated Fabry patients and 59 controls were analyzed. A multivariate statistical analysis performed on a subset of male samples revealed seven novel Fabry biomarkers in urine, all lyso-Gb₃ analogues having modified sphingosine moieties. The empirical formulas of the sphingosine modifications were determined by exact mass measurements (− C₂H₄, – C₂H₄ + O, – H₂, – H₂ + O, + O, + H₂O₂, + H₂O₃). We evaluated the relative concentration of lyso-Gb₃ and its seven analogues by measuring area counts for each analogue in all Fabry patients. All samples were normalized to creatinine. We found higher concentrations for males with Fabry disease compared to females. None of these biomarkers were detected in controls. To our knowledge, this is the first time that lyso-Gb₃-related Fabry disease biomarkers are detected in urine