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.

Effect of u18666a on beta-glucosidase, sphingomyelinase, and beta-galactosidase activities in astrocytes of young rats.

Niemann-Pick type C disease (NPC) is a neurodegenerative genetic disorder caused by accumulation of lipids, especially cholesterol, in the perinuclear space. U18666A is a cholesterol transport-inhibiting agent, being used to mimic NPC, mainly in fibroblasts. The objective of this study was to observe the effect of the drug U18666A, which causes the accumulation of cholesterol in the cytoplasm of astrocytes from newborn rats, on some lysosomal hydrolase activities. Filipin staining and fluorescence microscopy, through CellM software, were used for visualization and quantification of cholesterol. The dose of U18666A that provided the greatest accumulation of cholesterol was that of 0.25 µg/mL in incubation for 48 h. Primary rat astrocytes incubated with the drug (NPC) showed a significantly higher amount of cholesterol than those without U18666A (controls). The measurement of activity of enzymes sphingomyelinase and beta-glucosidase in astrocytes of rats with NPC was significantly lower than that of control astrocytes, which is consistent with the disease in humans. The activity of the enzyme beta-galactosidase showed no significant difference between both groups. We concluded that U18666A appears to be an excellent intracellular cholesterol transport-inhibiting agent affecting some metabolic pathways in astrocytes of young rats, which mimics NPC in these animals. Just like the change in the activity of lysosomal enzymes has been demonstrated, other biochemical parameters of the cell can be tested with this animal model, thus contributing to a better understanding 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 ß-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.

Comparison between alpha-galactosidase A activity in blood samples collected on filter paper, leukocytes and plasma.

OBJECTIVES: To compare alpha-galactosidase A activity in dried blood spots on filter paper, plasma, and leukocytes of Fabry disease patients and healthy controls, and to develop a miniaturization approach of the techniques to measure activity using plasma and leukocytes. DESIGN AND METHODS: Blood was collected from healthy controls and Fabry disease patients. Two drops were spotted on filter paper. Plasma and leukocytes were separated from the remaining sample. Enzyme activity was assessed by fluorometry. RESULTS: Significant positive correlation between standard and miniaturized techniques was observed. Alpha-galactosidase activity differed for male and female subjects when analyzed using filter paper and plasma. New reference and cutoff values were established based on the differences in alpha-galactosidase activity between genders. A good correlation was observed across biological materials assessed. CONCLUSIONS: The establishment of specific values for men and women increases reliability of commonly used techniques to screen and diagnose Fabry disease.