Michel Boutin

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 (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.

High-sensitivity nanoLC-MS/MS analysis of urinary desmosine and isodesmosine.

Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium-based cross-linker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D(4)-DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 microL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 +/- 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 +/- 3.1 ng/mg crea), (c) healthy smokers (13.2 +/- 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 +/- 2.9 ng/mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.

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 (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). Using metabolomic approaches, our group recently discovered seven novel urinary lyso-Gb(3)-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-Gb(3) 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-Gb(3) analogue levels in urine and gender (p < 0.001). Fabry males had higher excretion levels compared to females with the disease. Lyso-Gb(3) 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-Gb(3) analogues are promising urinary biomarkers for Fabry disease.

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.

LC-MS/MS analysis of plasma lyso-Gb3 in Fabry disease.

BACKGROUND Fabry disease is a complex, multisystemic and clinically heterogeneous disease, with elevated excretion of globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)) accumulating in biological fluids caused by deficiency of the enzyme, lysosomal α-galactosidase A. Our aims were to propose a tandem mass spectrometry fragmentation mechanism for lyso-Gb(3), to develop and validate a simple, and robust methodology for the measurement of plasma lyso-Gb(3) using LC-MS/MS in large Fabry cohorts and in controls. Response to treatment was also evaluated. METHOD A solid-phase extraction procedure was used to process plasma samples. The 1-β-D-glucosylsphingosine (GSG) internal standard was chosen for its commercial availability. A liquid chromatography method was devised to allow the co-elution of the GSG internal standard with lyso-Gb(3), thus compensating for system variability and reducing the matrix effect. A multiple reaction monitoring method was developed, working in positive electrospray ionization. RESULTS The validation of the method provided good accuracy and precision: intraday and interday biases of less than 8% and 5%, respectively, and intraday and interday CVs of <12% and 7%, respectively. Limit of detection was 0.7 nmol/l and limit of quantification was 2.5 nmol/l. Plasma samples were stable for up to 6h at room temperature, 48 h at 4 °C, and 20 weeks at -20 °C. Regarding untreated Fabry patients, the mean lyso-Gb(3) concentrations were 170 nmol/l for males and 9.7 nmol/l for females, and for treated patients, 40.2 nmol/l for males and 7.5 nmol/l for females. CONCLUSION A robust LC-MS/MS methodology is presented for plasma lyso-Gb(3) quantification.

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.

Urinary biomarker investigation in children with Fabry disease using tandem mass spectrometry.

BACKGROUND Fabry disease is an X-linked lysosomal storage disorder affecting both males and females with tremendous genotypic/phenotypic variability. Concentrations of globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3)/related analogues were investigated in pediatric and adult Fabry cohorts. The aims of this study were to transfer and validate an HPLC-MS/MS methodology on a UPLC-MS/MS new generation platform, using an HPLC column, for urine analysis of treated and untreated pediatric and adult Fabry patients, to establish correlations between the excretion of Fabry biomarkers with gender, treatment, types of mutations, and to evaluate the biomarker reliability for early detection of pediatric Fabry patients. METHOD A UPLC-MS/MS was used for biomarker analysis. RESULTS Reference values are presented for all biomarkers. Results show that gender strongly influences the excretion of each biomarker in the pediatric Fabry cohort, with females having lower urinary levels of all biomarkers. Urinary distribution of lyso-Gb3/related analogues in treated Fabry males was similar to the untreated and treated Fabry female groups in both children and adult cohorts. Children with the late-onset p.N215S mutation had normal urinary levels of Gb3, and lyso-Gb3 but abnormal levels of related analogues. CONCLUSIONS In this study, Fabry males and most Fabry females would have been diagnosed using the urinary lyso-Gb3/related analogue profile.

Novel gb(3) isoforms detected in urine of fabry disease patients: a metabolomic study.

Fabry disease is characterized by the accumulation of globotriaosylsphingosine (lyso-Gb(3)) and globotriaosylceramide (Gb(3)) in biological fluids and tissues. Metabolomic studies recently undertaken by our group, showed the presence of novel plasma and urine lyso-Gb(3)-related analogs in male and female Fabry patients. These analogs are distinguished by differences in structure of the sphingosine moiety. The principal aim of this study was to evaluate the possibility of detecting other Fabry disease biomarkers structurally related to Gb(3). A time-of-flight mass spectrometry metabolomic approach, focusing on mass-to-charge (m/z) ratios from 1000 to 1200 Da, was devised. This m/z window corresponds to the isoforms and potential analogs of Gb(3). Five different categories of Gb(3)- related isoforms/analogs were detected: Gb(3)-related isoforms with saturated fatty acids, methylated Gb(3)-related isoforms, Gb(3)-related isoforms/analogs with one double bond, Gb(3) analogs with hydrated sphingosine, and Gb(3)-related isoforms/analogs with two double bonds. A secondary objective was to elucidate the relationship between Gb(3) and lyso-Gb(3). The methylation observed on Gb(3)-related analogs was not detected on lyso-Gb(3). We speculate that the methylated Gb(3) may be an intermediate compound in the deacylation of Gb(3) to generate the lyso-Gb(3) molecule. We are in the process of devising a quantification methodology for these methylated Gb(3)-related analogs in Fabry patients to try to understand the underlying biochemical mechanisms involved in this complex disease.

A Metabolomic Study Reveals Novel Plasma Lyso-Gb3 Analogs As Fabry Disease Biomarkers

Fabry disease is an X-linked, multisystemic lysosomal storage disorder due to alpha-galactosidase A deficiency. It is characterized by the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb(3)), in biological fluids, vascular endothelium, heart, and kidneys. Treatment by enzyme replacement therapy has been shown to be beneficial in both males and females affected with the disease. In addition to Gb(3), increased concentrations of globotriaosylsphingosine (lyso-Gb(3)) have recently been reported in urine and plasma of Fabry patients. The overall objective of this metabolomic study was to identify and characterize new potential plasma biomarkers in treated and untreated males and females affected with Fabry disease which might better reflect disease severity and progression. We employed a time-of-flight mass spectrometry metabolomic approach using plasma samples of Fabry patients compared to age-matched controls. We found three new lyso-Gb(3) analogs in Fabry patients presenting m/z ratios at 802, 804, and 820. As previously detected by our group, we also found a m/z ratio of 784 corresponding to the lyso-Gb(3) molecule minus two hydrogen atoms. Using exact mass measurements and tandem mass spectrometry, we confirmed that these analogs result from modifications of the lyso-Gb(3) sphingosine moiety. We evaluated the relative plasma concentration by measuring area counts for each lyso-Gb(3) analog. None of these analogs was detected in the majority of healthy controls. The relative concentration of each analog was higher in males compared to female Fabry patients. We demonstrated that mass spectrometry combined to a metabolomic approach is a powerful tool to detect and identify new potential biomarkers.

Metabolomic Discovery of Novel Urinary Galabiosylceramide Analogs as Fabry Disease Biomarkers

AbstractFabry disease is an X-linked, complex, multisystemic lysosomal storage disorder presenting marked phenotypic and genotypic variability among affected male and female patients. Glycosphingolipids, mainly globotriaosylceramide (Gb3) isoforms/analogs, globotriaosylsphingosine (lyso-Gb3) and analogs, as well as galabiosylceramide (Ga2) isoforms/analogs accumulate in the vascular endothelium, nerves, cardiomyocytes, renal glomerular and tubular epithelial cells, and biological fluids. The search for biomarkers reflecting disease severity and progression is still on-going. A metabolomic study using quadrupole time-of-flight mass spectrometry has revealed 22 galabiosylceramide isoforms/analogs in urine of untreated Fabry patients classified in seven groups according to their chemical structure: (1) Saturated fatty acid; (2) one extra double bond; (3) two extra double bonds; (4) hydroxylated saturated fatty acid; (5) hydroxylated fatty acid and one extra double bond; (6) hydrated sphingosine and hydroxylated fatty acid; (7) methylated amide linkage. Relative quantification of both Ga2 and Gb3 isoforms/analogs was performed. All these biomarkers are significantly more abundant in urine samples from untreated Fabry males compared with healthy male controls. A significant amount of Ga2 isoforms/analogs, accounting for 18% of all glycosphingolipids analyzed (Ga2 + Gb3 and respective isoforms/analogs), were present in urine of Fabry patients. Gb3 isoforms containing saturated fatty acids are the most abundant (60.9%) compared with 26.3% for Ga2. A comparison between Ga2 isoforms/analogs and their Gb3 counterparts also showed that the proportion of analogs with hydroxylated fatty acids is significantly greater for Ga2 (35.8%) compared with Gb3 (1.9%). These results suggest different biological pathways involved in the synthesis and/or degradation of Gb3 and Ga2 metabolites. Graphical Abstractᅟ