Multiplex Tandem Mass Spectrometry Analysis of Novel Plasma Lyso-Gb₃‑Related Analogues in Fabry Disease

Fabry disease is a multisystemic, X-linked lysosomal storage disorder caused by a deficit in α-galactosidase A enzyme activity leading to glycosphingolipid accumulation, mainly globotriaosylceramide (Gb₃) and globotriaosylsphingosine (lyso-Gb₃). Recent metabolomic studies have led to the discovery of novel biomarkers related to lyso-Gb₃ in plasma and urine. These biomarkers show modifications of the sphingosine moiety of the lyso-Gb₃ molecule. The objectives of this study were to develop and validate a liquid chromatography-tandem mass spectrometry method for the relative quantification of novel plasma lyso-Gb₃-related analogues, to evaluate their levels in plasma of 74 Fabry patients and 41 healthy controls and to correlate these results with patient gender, enzyme replacement therapy treatment, and lyso-Gb₃ analogue levels previously measured in urine for the same patients. As expected, the concentrations of lyso-Gb₃ and its related analogues in plasma are higher in Fabry males compared to Fabry females and higher for untreated males compared to treated males. The concentration of lyso-Gb₃ and its related analogues in plasma decrease significantly after the beginning of enzyme replacement therapy (ERT) treatment and remain stable for 30 months of monitored therapy in a Fabry male. In plasma, lyso-Gb₃ is significantly more abundant than its related analogues, which differs from urine where the majority of the lyso-Gb₃ analogues are more increased than lyso-Gb₃ itself. In contrast to urine, the relative distribution of lyso-Gb₃ and its analogues in plasma is similar from one individual to another in the same group of Fabry patients, irrespective of ERT. This study revealed a large discrepancy between the relative abundance of lyso-Gb₃ and its analogues in urine and plasma. Further studies will thus be needed to better understand the metabolic relationship between plasma and urine lyso-Gb₃-related biomarkers

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

A Metabolomic Study To Identify New Globotriaosyl­ceramide-Related Biomarkers in the Plasma of Fabry Disease Patients

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A, which results in the progressive accumulation of glycosphingolipids. In addition to the two biomarkers, globotriaosyl­ceramide (Gb₃) and globotriaosyl­sphingosine (lyso-Gb₃), which are routinely used for detection and high-risk screening of Fabry disease patients, novel urinary Gb₃-related isoforms/analogues as well as newly defined lyso-Gb₃ analogues in plasma and urine from Fabry patients have recently been described by our group. The aim of this study was to extend our recent analyses to identify and evaluate new potential Gb₃-related biomarkers in the plasma of untreated male Fabry disease patients using a mass spectrometry metabolomic approach. A multivariate statistical analysis revealed five Gb₃-related novel biomarkers in the plasma of male Fabry patients. Three of these new biomarkers correspond to Gb₃, which has an extra double bond on the sphingosine with C16:0, C18:0, and C22:1 fatty acid chains. The fourth biomarker corresponds to a mixture of two structural isomers, the first with a d16:1 sphingosine and a C16:0 fatty acid and the second with a d18:1 sphingosine and a C14:0 fatty acid. To our knowledge, it is the first time that a Gb₃ analogue with a d16:1 sphingosine moiety has been reported. In addition, this Gb₃ analogue was also present in its methylated form. These biomarkers are part of a metabolic profile that may provide insight into the pathophysiology of Fabry disease

Tandem Mass Spectrometry Multiplex Analysis of Glucosylceramide and Galactosylceramide Isoforms in Brain Tissues at Different Stages of Parkinson Disease

Previous studies demonstrated that Parkinson disease (PD) is associated with a decreased activity of the glucocerebrosidase (GCase) enzyme in brain tissues. The objective of this study was to determine if GCase deficiency is associated with the accumulation of its glucosylceramide (GluCer) substrate in PD brain tissues. An ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed, optimized, and validated for the multiplex analysis of GluCer isoforms (C18:0, C20:0, C22:0, C24:1, and C24:0) in brain tissue samples. These molecules were chromatographically separated from their isobaric galactosylceramide (GalCer) counterparts using normal phase chromatography. The analysis was performed by tandem mass spectrometry in the multiple reaction monitoring (MRM) acquisition mode. Limits of detection ranging from 0.4 to 1.1 nmol/g brain tissue were established for the different GluCer isoforms analyzed. For the first time, GluCer isoform levels were analyzed in temporal cortex brain tissue samples from 26 PD patients who were divided into three PD disease stages (IIa, III, and IV) according to the Unified Staging System for Lewy Body Disorders. These specimens were compared with brain tissue samples from 12 controls and 6 patients with Incidental Lewy Body Disease. No significant GluCer concentration differences were observed between the 5 sample groups. The GluCer isoform levels were also normalized with their matching GalCer isoforms. The normalized results showed a trend for GluCer levels which increased with PD severity. However, the differences observed between the groups were not significant, owing likely to the high standard deviations measured

Separation and Analysis of Lactosylceramide, Galabiosylceramide, and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients

Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga₂), globotriaosylceramide (Gb₃), and globotriaosylsphingosine (lyso-Gb₃) by hydrolyzing the terminal α-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring, and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga₂ is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga₂ isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb₃ isoforms/analogues having the same fatty acid moieties as those of Ga₂ for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT) and 54 untreated and 19 ERT-treated Fabry females, along with 34 male and 25 female healthy controls. The chromatographic separation of Ga₂ from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga₂ but normal levels of Gb₃, supporting the importance of analyzing Ga_2, in addition to Gb₃. Our results show that urine LacCer levels from females were significantly higher than those from males. Moreover, LacCer levels were not affected by Fabry disease for both males and females

Separation and Analysis of Lactosylceramide, Galabiosylceramide, and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients

Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga₂), globotriaosylceramide (Gb₃), and globotriaosylsphingosine (lyso-Gb₃) by hydrolyzing the terminal α-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring, and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga₂ is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga₂ isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb₃ isoforms/analogues having the same fatty acid moieties as those of Ga₂ for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT) and 54 untreated and 19 ERT-treated Fabry females, along with 34 male and 25 female healthy controls. The chromatographic separation of Ga₂ from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga₂ but normal levels of Gb₃, supporting the importance of analyzing Ga_2, in addition to Gb₃. Our results show that urine LacCer levels from females were significantly higher than those from males. Moreover, LacCer levels were not affected by Fabry disease for both males and females

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

Additional file 1: of Diagnosis of late-onset Pompe disease and other muscle disorders by next-generation sequencing

Supplemental information. (DOCX 157 kb