Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Mass Spectrometry Analysis of Globotriaosylsphingosine and Its Analogues in Dried Blood Spots.

Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired α-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer®B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3-52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD.

Multiplex Quantification of Plasma Biomarkers for Patients with Gaucher Disease Type 1.

Gaucher disease (GD) is a lysosomal storage disorder caused by a deficiency of the enzyme beta-glucocerebrosidase. This leads to the accumulation of glycolipids in macrophages and ultimately results in tissue damage. Recent metabolomic studies highlighted several potential biomarkers in plasma specimens. In hopes of better understanding the distribution, importance, and clinical significance of these potential markers, a UPLC-MS/MS method was developed and validated to quantify lyso-Gb1 and six related analogs (with the following modifications on the sphingosine moiety: -C2 H4 (-28 Da), -C2 H4 +O (-12 Da), -H2 (-2 Da), -H2 +O (+14 Da), +O (+16 Da), and +H2 O (+18 Da)), sphingosylphosphorylcholine, and N-palmitoyl-O-phosphocholineserine in plasma specimens of treated and untreated patients. This 12-min UPLC-MS/MS method involves a purification step via solid-phase extraction followed by evaporation under nitrogen flow and resuspension in an organic mix compatible with HILIC chromatography. This method is currently used for research purposes and might be used for monitoring, prognostics, and follow-up. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Quantitation of a Urinary Profile of Biomarkers in Gaucher Disease Type 1 Patients Using Tandem Mass Spectrometry.

Gaucher disease is a rare inherited disorder caused by a deficiency of the lysosomal acid beta-glucocerebrosidase enzyme. Metabolomic studies by our group targeted several new potential urinary biomarkers. Apart from lyso-Gb1, these studies highlighted lyso-Gb1 analogs -28, -26, -12 (A/B), +2, +14, +16 (A/B), +30, and +32 Da, and polycyclic lyso-Gb1 analogs 362, 366, 390, and 394 Da. The main objective of the current study was to develop and validate a robust UPLC-MS/MS method to study the urine distribution of these biomarkers in patients. METHOD: Urine samples were purified using solid-phase extraction. A 12 min UPLC-MS/MS method was developed. RESULTS: Validation assays revealed high precision and accuracy for creatinine and lyso-Gb1. Most lyso-Gb1 analogs had good recovery rates and high intra- and interday precision assays. Biomarker-estimated LOD and LOQ levels ranged from 56-109 pM to 186-354 pM, respectively. Comparison between GD patients and healthy controls showed significant differences in most biomarker levels. Typically, treated GD patients presented lower biomarker levels compared to untreated patients. CONCLUSIONS: These data suggest that the metabolites investigated might be interesting GD biomarkers. More studies with a larger cohort of patients will be needed to better understand the clinical significance of these GD biomarkers.

Methylmalonic acid analysis using urine filter paper samples to screen for metabolic vitamin B12 deficiency in older adults.

Aim: Methylmalonic acid (MMA) analysis in urine represents a noninvasive approach to screening for vitamin B12 deficiency in older adults. A method allowing the analysis of MMA/creatinine in fasting urine collected on filter paper was developed/validated. Method: Dry urine specimens were eluted using a solution containing internal standards, filtrated and analyzed by ultra-performance LC-MS/MS. Results: The method allowed the chromatographic separation of MMA from succinic acid. Dried urine samples were stable for 86 days at room temperature. The MMA/creatinine ratios measured in urine collected on filter paper were highly correlated with values derived from the corresponding liquid specimens. Conclusion: This robust filter paper method might greatly improve the accessibility and cost-effectiveness of vitamin B12 deficiency screening in older adults.

Metabolomic Study Using Time-of-Flight Mass Spectrometry Reveals Novel Urinary Biomarkers for Gaucher Disease Type 1.

Gaucher disease (GD) is a lysosomal storage disorder resulting from a biallelic mutation in the gene GBA1, leading to deficiencies in the enzyme ß-glucocerebrosidase (Gcase). Inabilities of the Gcase to catabolize its substrate result in the accumulation of sphingolipids in macrophages, which impairs the cell functions and ultimately leads to multisystemic clinical manifestations. Important variability in symptoms and manifestations may lead to challenging diagnosis and patient care. Plasma glucosylsphingosine (lyso-Gb1) is a biomarker frequently used for prognosis, monitoring, and patient follow-up. While lyso-Gb1 appears to be a valid biomarker, few studies have investigated other matrices for potential GD biomarkers. The main objective of this study was to investigate the urine matrix as a potential source of new GD biomarkers by performing a metabolomic study using time-of-flight mass spectrometry. Our study highlighted a significant increase of eight urinary lyso-Gb1 analogues. Moreover, a novel class of biomarkers, named polycyclic lyso-Gb1 analogues, was identified. These four new molecules were more elevated than lyso-Gb1 and related analogues in urine specimens of GD patients. Further investigations are warranted to validate the efficiency of these newly found biomarkers on a larger cohort of Gaucher patients and to compare them with plasma biomarkers currently quantified in clinical laboratories.

Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.

Aim: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid ß-glucocerebrosidase. Recent metabolomic studies highlighted several new metabolites increased in the plasma of GD patients. We aimed to develop and validate a UPLC-MS/MS method allowing a relative quantitation of lyso-Gb1 and lyso-Gb1 analogs -28, -12, -2, +14, +16 and +18 Da in addition to sphingosylphosphorylcholine, N-palmitoyl-O-phosphocholine to study potential correlations with clinical manifestations. Methodology & results: Following solid-phase extraction, plasma samples were evaporated and resuspended in 100 µl of resuspension solution. Three microliter is injected into the UPLC-MS/MS for analysis. Conclusion: All biomarkers studied were increased in GD patients. Significant correlations were observed between specific analogs and hematological, and visceral complications, as well as overall disease severity.

Lysosphingolipid urine screening test using mass spectrometry for the early detection of lysosomal storage disorders.

Background: Sphingolipidoses are caused by a defective sphingolipid catabolism, leading to an accumulation of several glycolipid species in tissues and resulting in neurotoxicity and severe systemic manifestations. Methods & results: Urine samples from controls and patients were purified by solid-phase extraction prior to the analysis by ultra-high-performance liquid chromatography (UPLC) combined with MS/MS. A UPLC-MS/MS method for the analysis of 21 urinary creatinine-normalized biomarkers for eight diseases was developed and validated. Conclusion: Considering the growing demand to identify patients with different sphingolipidoses early and reliably, this methodology will be applied for high-risk screening to target efficiently patients with various sphingolipidoses.

Vitamin B-12 Intake from Dairy but Not Meat Is Associated with Decreased Risk of Low Vitamin B-12 Status and Deficiency in Older Adults from Quebec, Canada.

BACKGROUND: Vitamin B-12 deficiency can result in irreversible neurologic damages. It is most prevalent among older adults (∼5%-15%), mainly due to impaired absorption. Vitamin B-12 bioavailability varies between food sources, so their importance in preventing deficiency may also vary. OBJECTIVES: Using the NuAge Database and Biobank, we examined the associations between vitamin B-12 intake (total and by specific food groups) and low vitamin B-12 status and deficiency in older adults. METHODS: NuAge-the Quebec Longitudinal Study on Nutrition and Successful Aging-included 1753 adults aged 67-84 y who were followed 4 y. Analytic samples comprised 1230-1463 individuals. Dietary vitamin B-12 intake was assessed annually using three 24-h dietary recalls. Vitamin B-12 status was assessed annually as low serum vitamin B-12 (<221 pmol/L), elevated urinary methylmalonic acid (MMA)/creatinine ratio (>2 µmol/mmol), and a combination of both (deficiency). Vitamin B-12 supplement users were excluded. Multilevel logistic regressions, adjusted for relevant confounders, were used. RESULTS: Across all study years, 21.8%-32.5% of participants had low serum vitamin B-12, 12.5%-17.0% had elevated urine MMA/creatinine, and 10.1%-12.7% had deficiency. Median (IQR) total vitamin B-12 intake was 3.19 µg/d (2.31-4.37). Main sources were "dairy" and "meat, poultry, and organ meats." The ORs (95% CIs) in the fifth quintile compared with the first of total vitamin B-12 intake were as follows: for low serum vitamin B-12, 0.52 (0.37, 0.75; P-trend < 0.0001); for elevated urine MMA/creatinine, 0.63 (0.37, 1.08; P-trend = 0.091); and for vitamin B-12 deficiency, 0.38 (0.18, 0.79; P-trend = 0.006). Similarly, ORs (95% CIs) in the fourth quartile compared with the first of dairy-derived vitamin B-12 intake were 0.46 (0.32, 0.66; P-trend < 0.0001), 0.51 (0.30, 0.87; P-trend = 0.006), and 0.35 (0.17, 0.73; P-trend = 0.003), respectively. No associations were observed with vitamin B-12 from "meat, poultry, and organ meats." CONCLUSIONS: Higher dietary vitamin B-12 intake, especially from dairy, was associated with decreased risk of low vitamin B-12 status and deficiency in older adults. Food groups might contribute differently at reducing risk of deficiency in older populations.

Neonatal Urine Screening Program in the Province of Quebec: Technological Upgrade from Thin Layer Chromatography to Tandem Mass Spectrometry.

The Quebec Neonatal Urine Screening Program was initiated in 1971 with overall screening inception of newborns in 1973. Forty-seven years later, over 3.5 million babies have been screened for up to 25 inborn errors of metabolism divided into two groups: (1) urea cycle disorders and organic acidurias; and (2) disorders of amino acid metabolism and transport. The main goal of this preventive genetic medicine program is the detection of treatable diseases before the onset of clinical symptoms. Urine specimens from 21-day-old babies are collected and dried on filter paper by parents at home. The participation is voluntary with a high compliance rate over the years (~90%). Specimens are analyzed by thin layer chromatography (TLC). The main objective of this evaluative research project was to assess the feasibility of a technological upgrade towards mass spectrometry. A 2.85-min flow injection method was devised, normal values established, and abnormal profiles confirmed using second-tier tests. The validated assays are sensitive, specific, and suitable for populational screening, as well as for high-risk screening laboratories. Triple H syndrome, which would not be detected in newborns by blood screening at two days of age was found to be positive in the urine of an affected patient.

Lentivirus-mediated gene therapy for Fabry disease.

Enzyme and chaperone therapies are used to treat Fabry disease. Such treatments are expensive and require intrusive biweekly infusions; they are also not particularly efficacious. In this pilot, single-arm study (NCT02800070), five adult males with Type 1 (classical) phenotype Fabry disease were infused with autologous lentivirus-transduced, CD34+-selected, hematopoietic stem/progenitor cells engineered to express alpha-galactosidase A (α-gal A). Safety and toxicity are the primary endpoints. The non-myeloablative preparative regimen consisted of intravenous melphalan. No serious adverse events (AEs) are attributable to the investigational product. All patients produced α-gal A to near normal levels within one week. Vector is detected in peripheral blood and bone marrow cells, plasma and leukocytes demonstrate α-gal A activity within or above the reference range, and reductions in plasma and urine globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are seen. While the study and evaluations are still ongoing, the first patient is nearly three years post-infusion. Three patients have elected to discontinue enzyme therapy.

Globotriaosylsphingosine (lyso-Gb3) and analogues in plasma and urine of patients with Fabry disease and correlations with long-term treatment and genotypes in a nationwide female Danish cohort.

INTRODUCTION: Recent studies showed the usefulness of globotriaosylsphingosine (lyso-Gb3) and related analogues, deacylated forms of globotriaosylceramide (Gb3), for high-risk screening, treatment monitoring and follow-up for patients with Fabry disease. METHODS: We evaluated Gb3, lyso-Gb3 and analogues using tandem mass spectrometry in 57 women with Fabry disease followed during a period of 15.4 years. Twenty-one women were never treated and 36 received treatment (agalsidase-beta, n=30; agalsidase-alfa, n=5; or migalastat, n=1). Lyso-Gb3 and analogues at m/z (-28), (-2), (+16), (+34) and (+50) were analysed in plasma and urine. Total Gb3 and lyso-Gb3 analogues at m/z (-12) and (+14) were evaluated in urine while the analogue at m/z (+18) was evaluated in plasma. RESULTS: A strong correlation between plasma and urine lyso-Gb3 and analogue levels was revealed. Plasma and urine lyso-Gb3 and analogue levels were not statistically different between patients carrying missense (n=49), nonsense (n=6) or deletion mutations (n=2). Never treated patients had lower plasma lyso-Gb3 and analogues at m/z (-28), (-2), (+16), (+34) and the seven urinary lyso-Gb3 analogues compared with pretreatment levels of the treated patients. A significant reduction of plasma lyso-Gb3 and five analogues, as well as urine Gb3 and six lyso-Gb3 analogues, but not lyso-Gb3 and lyso-Gb3 at m/z (+50), was observed post-treatment with agalsidase-beta. The same tendency was observed with agalsidase-alfa. CONCLUSION: Women with Fabry disease who started treatment based on clinical manifestations had higher lyso-Gb3 and analogue biomarker levels than never treated women. This indicates that a biomarker cut-off could potentially be a decision tool for treatment initiation in women with Fabry disease.

Identification of a Reliable Biomarker Profile for the Diagnosis of Gaucher Disease Type 1 Patients Using a Mass Spectrometry-Based Metabolomic Approach.

Gaucher disease (GD) is a rare autosomal recessive multisystemic lysosomal storage disorder presenting a marked phenotypic and genotypic variability. GD is caused by a deficiency in the glucocerebrosidase enzyme. The diagnosis of GD remains challenging because of the large clinical spectrum associated with the disease. Moreover, GD biomarkers are often not sensitive enough and can be subject to polymorphic variations. The main objective of this study was to perform a metabolomic study using an ultra-performance liquid chromatography system coupled to a time-of-flight mass spectrometer to identify novel GD biomarkers. Following the analysis of plasma samples from patients with GD, and age- and gender-matched control samples, supervised statistical analyses were used to find the best molecules to differentiate the two groups. Targeted biomarkers were structurally elucidated using accurate mass measurements and tandem mass spectrometry. This metabolomic study was successful in highlighting seven biomarkers associated with GD. Fragmentation tests revealed that these latter biomarkers were lyso-Gb1 (glucosylsphingosine) and four related analogs (with the following modifications on the sphingosine moiety: -C2H4, -H2, -H2+O, and +H2O), sphingosylphosphorylcholine, and N-palmitoyl-O-phosphocholineserine. Based on the plasma biomarker distribution, we suggest the evaluation of this GD biomarker profile, which might facilitate early diagnosis, monitoring, and follow-up of patients.

Assessing the role of glycosphingolipids in the phenotype severity of Fabry disease mouse model.

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb3 isoforms (various fatty acids) and lyso-Gb3 analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb3 and lyso-Gb3 levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb3 and particular lyso-Gb3 analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb3 and lyso-Gb3 isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb3 massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb3 accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb3 or lyso-Gb3 may play major roles in the pathogenesis of Fabry disease, and that Gb3 and lyso-Gb3 are not responsible for the pathology in all tissues or cell types.

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.

Therapeutic challenges in two adolescent male patients with Fabry disease and high antibody titres.

Enzyme replacement therapy (ERT) has been shown to stabilize certain aspects of Fabry disease (FD). However, in some patients on ERT, high antibody titres have been documented, with limited clinical improvement in systemic manifestations and often with significant adverse drug reactions. We present two related adolescent males with a 4.5 kb GLA deletion, not amenable to chaperone therapy, leading to profound reduction in α-galactosidase A (α-gal A) enzyme activity. Over a 3-year period of ERT, increasing IgG antibody titres against α-gal A were noted. After starting ERT serial urine globotriaosylceramide (Gb3) measurements showed an upward trend from 333 to 2260 µg/mmol creatinine for patient 1 and 1165 to 2260 µg/mmol creatinine for patient 2. Markedly increased levels of urine and plasma globotriaosylsphingosine (Lyso-Gb3) analogues were also found. The patients experienced recurrent infusion-associated reactions necessitating premedication and prolonged infusion times. Over the 3-year period of ERT, the patients experienced continued malaise, gastrointestinal symptoms and neuropathic pain. In addition, they had increasing anxiety related to their disease and apparent lack of response to ERT which led to a decision to ultimately stop ERT. No other approved treatment options are currently available for these patients. It is possible that the rapid development of the high antidrug neutralizing antibody (ADA) titres is related to the large GLA deletion leading to virtually absent enzyme activity. It remains unclear if their symptomatology during the period of receiving ERT is related to lack of its efficacy, the rising ADA titres, or both. These two patients highlight the need for further research into the management of antidrug antibodies and additional therapeutic approaches for FD. SYNOPSIS: The development of very high antidrug antibody titres in response to ERT in two related adolescent males with FD highlight the need for other therapeutic options for patients in whom ERT or other currently approved therapies does not meet their treatment needs.

Mass spectrometry analysis of urinary methylmalonic acid to screen for metabolic vitamin B12 deficiency in older adults.

Aim: Vitamin B12 deficiency is characterized metabolically by increased serum and urine methylmalonic acid (MMA). Urinary MMA/creatinine ratio is suggested for screening for metabolic vitamin B12 deficiency in older populations. Results: A UPLC-MS/MS method for the analysis of urinary MMA and creatinine was developed/validated. A good separation of MMA from succinic acid, its structural isomer, was achieved. Intra- and interday accuracy biases and precision coefficients were all ≤6.3% for MMA and creatinine. Urine and serum samples of 34 individuals of the NuAge Biobank were analyzed for technical comparisons showing that urinary MMA/creatinine ratios by UPLC-MS/MS strongly correlated with GC-MS values, and with serum MMA values. Conclusion: The UPLC-MS/MS method developed is rapid/reliable for the analysis of urinary MMA/creatinine ratios.

Mass Spectrometry Evaluation of Biomarkers in the Vitreous Fluid in Gaucher Disease Type 3 with Disease Progression Despite Long-Term Treatment.

Intraocular lesions have been infrequently reported in patients with Gaucher disease type 3 (GD3). We previously reported siblings with GD3 who responded well to the combination of enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Here we report progressive bilateral vitreous and preretinal deposits with declining visual acuity requiring bilateral vitrectomies in one of these siblings. These ocular manifestations had progressed despite combined ERT and SRT with improvement in visual acuity after vitrectomies. Vitrectomy fluid analysis performed for the first time by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identified a high concentration of glucosylceramide (GluCer) in the patient (262.842 nM) compared to a sample (0.428 nM from a patient without a lysosomal storage or known hereditary metabolic disorder). The GluCer detected in our patient was resolved into 12 different isoforms including two methylated ones. No evidence of galactosylceramide (GalCer) was detected. The development of these intraocular manifestations and their characterization by UPLC-MS/MS indicate a need for ongoing ophthalmologic evaluation of all GD patients and for new therapies that can cross the blood-retinal and blood-brain barriers for patients with GD and other neuropathic lysosomal storage disorders.

High-risk screening for Fabry disease in a Canadian cohort of chronic kidney disease patients.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder with a highly heterogeneous clinical presentation. This complex disease is caused by a deficient activity of the enzyme α-galactosidase A, which is involved in the catabolism of glycosphingolipids. The prevalence of Fabry disease is underestimated, due to the presence of atypical variants. High-risk screening protocols are particularly relevant for this disease due to the availability of treatments, such as enzyme replacement and chaperone therapies. As kidney manifestations are present in the majority of male and many female patients with Fabry disease, a high-risk screening protocol was performed for patients with chronic kidney disease of unknown etiology. METHODS: Recruitment of 397 participants took place in four centers across Canada from 2011 to 2017. Globotriaosylceramide (Gb3) was analyzed in dried urine spots by liquid chromatography/tandem mass spectrometry followed by globotriaosylsphingosine (lyso-Gb3) on the repeat analysis. RESULTS: The collection and shipment of urine specimens on filter paper resulted in easier handling/shipment and significant cost-saving. No Fabry patients were detected in this study. CONCLUSIONS: Increased concentrations of urinary Gb3 were observed in 13.6% of patients with chronic kidney disease suggesting that chronic kidney disease or other comorbidities might be associated with increased urinary Gb3 concentrations.