Tuen-Jen Wang

A modified liquid chromatography/tandem mass spectrometry method for predominant disaccharide units of urinary glycosaminoglycans in patients with mucopolysaccharidoses

BackgroundThe identification of acid mucopolysaccharide by the liquid chromatography/tandem mass spectrometry method (LC-MS/MS) of the predominant disaccharide units of glycosaminoglycans (GAGs) (chondroitin sulfate, CS; dermatan sulfate, DS; heparan sulfate, HS) after methanolysis is validated and applicable for mucopolysaccharidosis (MPS) type determination.MethodsA total of 76 urine samples were collected and analyzed, from nine MPS I patients, 13 MPS II patients, seven MPS III patients, eight MPS VI patients, and 39 normal controls. Urinary GAG was first precipitated by the Alcian blue method followed by a treatment of 3 N HCl methanol. The protonated species of the methylated disaccharide products were detected by using a multiple reaction monitoring experiment. Internal standards, [2H6] CS, [2H6] DS and [2H6] HS, were prepared in-house by deuteriomethanolysis of CS, DS and HS.ResultsOne particular disaccharide for each GAG was selected, in which the parent ion and its daughter ion after collision were m/z 426.1 → 236.2 for DS (m/z 432 → 239 for dimers derived from [2H6] CS and [2H6] DS) and m/z 384.2 → 161.9 for HS (m/z 390.4 → 162.5 for the [2H6] HS dimer). The quantities of DS and HS were determined, which varied from one MPS type to the other. The results can be used to evaluate the severity of MPS subgroups, as well as urinary GAG amelioration at follow-up after enzyme replacement therapy (ERT).ConclusionsThe modified LC–MS/MS method for MPS type determination is specific, sensitive, validated, accurate, and applicable for simultaneous quantifications of urinary DS and HS. This method can help to make correct diagnosis of MPS patients and evaluate the effectiveness of ERT.

CNS-targeted AAV5 gene transfer results in global dispersal of vector and prevention of morphological and function deterioration in CNS of globoid cell leukodystrophy mouse model

Globoid cell leukodystrophy (GLD) is a devastating lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). Currently, there is no definite cure for GLD. Several attempts with CNS-directed gene therapy in twitcher mice (a murine model of GLD) demonstrated restricted expression of GALC activity in CNS and failure of therapeutic efficacy in cerebellum and spinal cord, resulting in various degrees of correction of biochemical, pathological and clinical phenotype. More recently, twitcher mice receiving a combination of hematopoietic and viral vector gene transfer therapies were not protected from neurodegeneration and axonopathy in both cerebellum and spinal cord. This evidence indicates the requirement of sufficient and widespread GALC expression in CNS and rescue of cerebellum and spinal cord in the therapeutic intervention of murine model of GLD. In this study, we have optimized intracranial delivery of AAV2/5-GALC to the neocortex, hippocampus and cerebellum, instead of the thalamus as was previously conducted, of twitcher mice. The CNS-targeted AAV2/5 gene transfer effectively dispersed GALC transgene along the neuraxis of CNS as far as the lumbar spinal cord, and reduced the accumulation of psychosine in the CNS of twitcher mice. Most importantly, the treated twitcher mice were protected from loss of oligodendrocytes and Purkinje cells, axonopathy and marked gliosis, and had significantly improved neuromotor function and prolonged lifespan. These preclinical findings with our approach are encouraging, although a more robust response in the spinal cord would be desirable. Collectively, the information in this study validates the efficacy of this gene delivery approach to correct enzymatic deficiency, psychosine accumulation and neuropathy in CNS of GLD. Combining cell therapy such as bone marrow transplantation with treatment with the aim of reducing inflammation, replacing dead or dying oligodendrocytes and targeting PNS may provide a synergistic and more complete correction of this disease.

Serum p-cresyl sulfate predicts cardiovascular disease and mortality in elderly hemodialysis patients

Introduction Previous studies have shown that serum p-cresyl sulfate (PCS) and indoxyl sulfate (IS) were significantly related to clinical outcomes in patients on hemodialysis (HD). However, evidence for the relationship in elderly HD patients remains scarce. We explore whether the two toxins can predict clinical outcomes in elderly HD patients. Material and methods Fifty stable HD patients more than 65 years old were enrolled from a single medical center. Serum total and free PCS, IS levels and biochemistry were measured concurrently. The clinical outcomes including cardiovascular events and all-cause mortality were analyzed after 38-month follow-up. Results Univariate Cox proportional hazard ratio analysis revealed that cardiovascular events were associated with gender (p = 0.02), diabetes (p < 0.01), calcium (p = 0.01), total PCS (p < 0.01), free PCS (p < 0.01) and total IS (p = 0.05). Multivariate analysis showed that diabetes (p = 0.01), total PCS (p = 0.01) and free PCS (p = 0.04) were related to cardiovascular events. For all-cause mortality, only total PCS (p = 0.01) reached significance after adjusting other confounding factors. However, Kaplan-Meier analysis indicated that free PCS (p = 0.02) and total PCS (p < 0.01) were significantly associated with cardiovascular events and total PCS (p = 0.048) was related to all-cause mortality during 38-month follow-up. Conclusions Our results indicate that total PCS is a valuable marker in predicting cardiovascular event and all-cause mortality in elderly HD patients.

A method for lactate and pyruvate determination in filter-paper dried blood spots

Lactic acidemia is commonly associated with severe diseases in pediatric patients. Quantitation of blood lactate and pyruvate is important for the diagnosis and clinical management. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using dried blood spots (DBS) was developed and could be used for simultaneous quantification of blood lactate and pyruvate. The applicability of the developed method was tested and confirmed by the regression analysis between LC-MS/MS method and enzymatic assay. Lactate and pyruvate were extracted from DBS obtained from 580 full-term, 120 pre-term infants (gestations ranging from 24 to 36 weeks), and 65 patients with suspected lactic acidemia, with methanolic internal standard (IS) solutions of sodium L-lactate-(13)C(3) and pyruvate-(13)C(3). An API-2000 LC-MS/MS system with multiple reaction monitoring (MRM) mode was applied. The within-run and between-run precisions (CV%) were determined and the results were 1.9% and 3.9% for lactate (n=20) and 5.7% and 7.3% for pyruvate (n=20). The linearity of lactate (r=0.9986) and pyruvate (r=0.9973) based on the IS was excellent. The parameter r squared (r(2)) of linear regression between LC-MS/MS method and enzymatic assay was 0.9405 for lactate and 0.9447 for pyruvate, respectively, and the agreement between these methods was consistent and acceptable. The stability of lactate and pyruvate on DBS was also confirmed. The LC-MS/MS method we developed is a specific, sensitive, and reproducible method for measuring blood lactate and pyruvate concentrations. The use of DBS in this method makes it particularly attractive for pediatric patients.

Mitigation of cerebellar neuropathy in globoid cell leukodystrophy mice by AAV-mediated gene therapy

Globoid cell leukodystrophy (GLD) is an autosomal recessive, lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). The absence of GALC activity leads to the accumulation of the toxic substance psychosine and the preferential loss of myelinating cells in the central and peripheral nervous systems. Profound demyelination, astrogliosis and axonopathy are the hallmarks of the pathogenesis of GLD, and cerebellar ataxia is one of the dominant manifestations in adolescents and adults affected with GLD. To date, studies regarding cerebellar degeneration in GLD are limited. In this study, the efficacy of cerebellum-targeted gene therapy on the cerebellar neuropathology in twitcher mice (a murine model of GLD) has been validated. We observed degeneration of Purkinje cells, Bergmann glia, and granule cells in addition to astrocytosis and demyelination in the cerebellum of the twitcher mice. Ultrastructural analysis revealed dark cell degeneration and disintegration of the cellular composition of Purkinje cells in untreated twitcher mice. In addition, the expressions of neurotrophic factors CNTF, GDNF and IGF-I were up-regulated and the expression of BDNF was down-regulated. Intracerebellar-mediated gene therapy efficiently corrected enzymatic deficiency by direct transduction to Purkinje cells and cross-correction in other cell types in the cerebellum, leading to the amelioration of both neuroinflammation and demyelination. The population, dendritic territory, and axonal processes of Purkinje cells remained normal in the cerebellum of treated twitcher mice, where radial fibers of Bergmann glia spanned the molecular layer and collateral branches ensheathed the dendritic processes of Purkinje cells. Moreover, the aberrant expressions of neurotrophic factors were mitigated in the cerebellum of treated twitcher mice, indicating the preservation of cellular function in addition to maintaining the neuronal architecture. The life span of the treated twitcher mice was significantly prolonged and their neurobehavioral performance was improved. Taken together, our findings underscore the complexity of cerebellar neurodegeneration in GLD and highlight the potential effectiveness of gene therapy in mitigating neuropathological deficits in GLD and other neurodegenerative disorders in which Purkinje cells are involved.

Association of Indoxyl Sulfate With Fibroblast Growth Factor 23 in Patients With Advanced Chronic Kidney Disease

Background:Protein-bound uremic toxins—indoxyl sulfate (IS) and p-cresyl sulfate (PCS)—can not only predict clinical outcomes but also may relate to bone-mineral disorders in patients with chronic kidney disease (CKD). However, the relationship between protein-bound uremic toxins and fibroblast growth factor 23 (FGF23) has not been studied before. The objective of this study was to explore the association of IS and PCS with FGF23 in a CKD-based cohort. Methods:This is a cross-sectional study that enrolled 80 stable CKD stage 3 to 5 patients who met the inclusion criteria in a single medical center. Serum levels of IS, PCS and FGF23 were measured concurrently. General biochemistry and patient background were also investigated. Results:Serum FGF23 and IS concentrations were elevated commensurately with deteriorating renal function. Pearsons analysis showed that FGF23 levels were significantly associated with blood urea nitrogen (r = 0.381, P < 0.05), creatinine (r = 0.632, P < 0.01), estimated glomerular filtration rate (r = −0.447, P < 0.05), phosphate (r = 0.543, P < 0.01), intact parathyroid hormone (r = 0.543, P < 0.01), IS (r = 0.432, P < 0.01) and PCS (r = 0.318, P < 0.05). After adjusting other confounding factors by stepwise multiple linear regression analysis, only creatinine (&bgr; = 0.82, P < 0.01), phosphate (&bgr; = 0.28, P = 0.02) and IS (&bgr; = 0.39, P = 0.04) retained statistically significant associations with FGF23. Moreover, serum levels of IS were higher in patients with high FGF23 concentration (>90 pg/mL, median value) than those with lower FGF23 (P < 0.01). Conclusions:Results indicated that only IS but not PCS correlated independently with FGF23 in worsening CKD. IS may be an independent factor involved in regulation of bone-mineral metabolism.

Gastrointestinal-related Uremic Toxins in Peritoneal Dialysis: A Pilot Study with a 5-year Follow-up

BACKGROUND AND AIMS P-cresyl sulfate (PCS) and indoxyl sulfate (IS) were not only novel but essential factors associated with cardiovascular disease and mortality in patients with chronic kidney disease and hemodialysis. However, little evidence exams the effect in peritoneal dialysis (PD) patients. METHODS This pilot study recruited 46 stable PD patients in a single medical center. Serum levels of IS, PCS and biochemistry were measured concurrently. Clinical outcomes including cardiovascular, all-cause mortality and PD failure event were recorded during a 5-year follow-up. RESULTS Serum levels of free and total PCS were lower in patients with residual renal function (11.67 ± 6.92, p = 0.014, 0.77 ± 0.48, p = 0.046, respectively). Multivariate Cox regression analysis showed age (HR: 1.07, p = 0.01), serum CO2 (HR: 0.67, p = 0.02) and total PCS (HR: 1.05, p <0.01) were independently associated with cardiovascular events; only free PCS (HR: 1.42, p <0.01) reached significant correlation with all-cause mortality. Total IS (HR: 1.27, p = 0.03) significantly correlated with PD failure event after adjusting other confounding factors. Kaplan-Meier analysis revealed that patients with higher total and free PCS levels had higher cardiovascular events (log rank p <0.01, log rank p = 0.05, respectively) and mortality event (log rank p = 0.02, log rank p = 0.03, respectively) than those with lower levels. In addition, total IS (log rank p = 0.04), total PCS (log rank p = 0.01) and free PCS (log rank p <0.01) could independently predict PD failure event during the study period. CONCLUSIONS Our findings suggest PCS and IS may be a valuable surrogate in predicting poor clinical outcomes in PD patients.