Jaqueline Cé

Determination of the lysosomal hydrolase activity in blood collected on filter paper, an alternative to screen high risk populations.

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.

Screening of high-risk Gaucher disease patients in Brazil using miniaturized dried blood spots and leukocyte techniques.

This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and β-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis.

The modulation of inflammatory parameters, Brain-derived neurotrophic factor levels and global histone H4 acetylation status in peripheral blood of patients with Gaucher disease type 1

OBJECTIVES Gauchers disease type 1 (GD1) pathophysiology includes an imbalance on brain-derived neurotrophic factor (BDNF) levels and in the inflammatory system. However, the pathways involved remain poorly understood. The hypothesis of this study is that epigenetic mechanisms might be involved, at least partially, in this phenomenon. DESIGN AND METHODS This study investigated the BDNF modulation, global histone H4 acetylation and pro- and anti-inflammatory cytokines levels in the peripheral blood of GD1 patients (n=10) when compared with control samples (CS) (n=11). RESULTS The results showed a significant increase in Chitotriosidase (CT) (p=0.019) and decreased β-glucosidase (GBA) activities (p=0.001) in GD1 samples when compared to CS, for GD1 diagnostic confirmation. Reduced levels of BDNF (p=0.004) and elevated levels of TNF-α (p=0.017) and IL-4 (p=0.035) were also found in the GD group. No significant differences were observed in IL-6 or IL-17a levels between groups (p>0.05). Finally, a trend on higher global histone H4 acetylation levels (p=0.054) was observed in the control group when compared to GD1 individuals. CONCLUSIONS Combined, these results suggest inflammatory cytokines imbalance, reduced BDNF levels and global histone H4 hypoacetylation status in GD type 1 physiopathology. These preliminary findings may open new avenues to introduce therapies and strategies in the preventive management and treatment of this population.

Comparing the alpha-galactosidase A biochemical properties from healthy individuals and Fabry disease patients

BACKGROUND Due to the importance and the difficulty still present in determining the biochemical diagnosis of Fabry disease (FD), the aim of this study was to establish and compare the biochemical and kinetic properties of alpha-galactosidase A (GLA) in dried blood spots (DBS), plasma and leukocyte samples of FD patients and healthy subjects to evaluate the possible use of these parameters as an auxiliary tool in the diagnosis of this disease. METHODS GLA activity in DBS, plasma and leukocyte samples from Fabry disease patients and healthy subjects was compared and characterized in terms of optimal pH, Km and Vmax and heat stability. RESULTS A difference was observed between the Km and Vmax of FD patients and healthy controls using DBS, plasma and leukocyte samples. In leukocytes, pre-incubation at 50°C for 60 min was effective to differentiate FD patients from healthy controls. CONCLUSION These results can be used as an auxiliary method to the FD diagnosis, especially in cases of patients whose GLA activity is within normal range.

ÃÂ-Glucosidase Deficiency Promotes Increasing Protein Oxidative Damagein Pompe Disease Patients

Objective: Pompe disease is an autosomal recessive disorder of lysosomal storage, caused by the deficiency of α-glucosidase lysosomal enzymes. Several studies have demonstrated the involvement of oxidative stress in numerous pathophysiological changes. To assess parameters of oxidative stress in patients with Pompe’s disease (PD) and in normal controls, establishing a possible analysis of the differences between both groups. Methods: Evaluation, in plasma samples and leukocytes, of the enzyme activities of α-glucosidase, of antioxidants SOD1 and CAT enzymes, as well as the levels of lipid peroxidation (TBARS), protein damage (carbonyl) and non-enzymatic antioxidant (sulphydryl) defenses on samples of 10 individuals with PD (4 women and 6 men) and 10 healthy individuals. Results: There was a reduction in the enzymatic activity of α-glucosidase in samples of leukocytes of patients with PD compared to samples from normal subjects, confirming the deficiency of this enzyme. With respect to oxidative stress, there was an increase of carbonyl groups in the plasma of the PD patients studied relative to controls, suggesting oxidative damage to proteins. No differences were observed between the two groups for the remaining oxidative stress parameters evaluated. Discussion: We conclude, therefore, that the presence of PD is a significant factor to increase the oxidative stress levels, with no change in levels of antioxidant enzymes. It is suggested that further studies with other lysosomal storage diseases be carried out in order to propose, in the future, antioxidant therapies to prevent protein damage.

Beta-glucuronidase activity in dried blood spots: Reduced technique with biochemical parameters determined

INTRODUCTION Mucopolysaccharidoses (MPS) occur due to deficiency in the activity of enzymes that catalyze the breakdown of glycosaminoglycans. MPS VII is caused by deficiency of the beta-glucuronidase enzyme (GUSB). OBJECTIVES This study aimed to enhance the technique to measure GUSB activity by reducing the amount of reagents and the size of the DBS, as well as to determine some biochemical parameters of enzyme of healthy individuals. METHODS The measurement of GUSB in 3 and 1.2mm DBS (with reagents reduced 2.5- and fourfold) was correlated and the precision of the technique was tested. Optimal pH, Km and Vmax, and thermostability parameters were determined and time and temperature of sample storage were established. RESULTS The correlations among the techniques were significant. Although the correlation coefficient was similar, fourfold reduction was selected. pH4.4 had the highest enzyme activity. GUSBs Km was 1.25mM, while Vmax was 594.48nmol/h/mL. After pre-incubation of the sample at 60°C, its activity dropped from 100% to 15.8% at 120min. GUSB activity significantly decreased after 45days of storage at 4, 25, and 37°C. CONCLUSIONS This research allowed a previously described technique for MPS VII diagnosis to be adapted for smaller amounts of sample and reagents. That will facilitate the use of smaller amounts of samples, which may be used for other techniques and to save material. Given the importance of early MPS VII diagnosis due to the severity of the disease, using reliable diagnostic techniques in DBS is essential.

Alpha-l-iduronidase and arylsulfatase B in dried blood spots on filter paper: Biochemical parameters and time stability

BACKGROUND The goal of this study was to assess the biochemical parameters of the enzymes α-l-iduronidase (IDUA) and arylsulfatase B (ASB), which are deficient in mucopolysaccharidosis (MPS) I and VI, respectively, in dried blood spot (DBS) samples impregnated on filter paper. METHODS AND RESULTS The optimal pH, Km, and Vmax of IDUA and ASB in DBS are hereby presented. After these analyses, the reference values for the activities of these enzymes in DBS with cutoff of 3.65nmol/h/mL for IDUA and 6.80nmol/h/mL for ASB were established. The research also showed that the stability (21days) of the IDUA activity is lower than ASB, which maintained its enzymatic activity stable up until 60days of analysis, after impregnating the filter paper with blood. CONCLUSION Currently, DBS ensures important advantages in handling storage and transportation of samples with respect to neonatal screening programs. This study contributes to characterizing and differentiating the biochemistry of deficient enzymes in MPSs I and VI of DBS samples.