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 (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). 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-Gb(3) analogues having modified sphingosine moieties. The empirical formulas of the sphingosine modifications were determined by exact mass measurements (- C(2)H(4), - C(2)H(4) + O, - H(2), - H(2) + O, + O, + H(2)O(2), + H(2)O(3)). We evaluated the relative concentration of lyso-Gb(3) 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-Gb(3)-related Fabry disease biomarkers are detected in urine.
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 (m/z)
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 m/z 836. Significant
correlations were established between lyso-Gb3 analogue
levels in urine and gender (p < 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 (p < 0.05). The
urinary analogue distributions varied among Fabry patients. However,
the analogues with m/z 802, 820,
and 836 were generally more abundant in the majority of patients.
Lyso-Gb3 analogues are promising urinary biomarkers for
Fabry disease.
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