Determination of Acid β-Galactosidase Activity: Methodology and Perspectives

Early, accurate diagnosis of lysosomal storage disorders is a major challenge, even for trained specialists. Finding innovative, accurate diagnostic methods, and high throughput, cost-effective tools are crucial to medical progress and will contribute to improved quality of life. The goal of this work was to improve currently used protocols to determine activity of acid β-galactosidase, and discuss the possibility analysing lysosomal enzymes with microfluidic systems. A principle of the determination of β-galactosidase activity was fluorometric measurement of a deprotonated form of 4-methylumbelliferone released in the enzymatic reaction. Measurements were performed using Jurkat T cells as a source of the enzyme. We observed the temperature-dependent substrate inhibition effect and determined the substrate (4-MU-β-d-galactopyranoside) concentration which should be used to determine acid β-galactosidase activity at 37 °C (0.8 mM) and at room temperature (0.6 mM). We proved that the sample incubation time may be significantly reduced to only a few minutes. We also showed that the amount of alkaline buffer used to stop the enzymatic reaction may be minimized and even, in some cases, eliminated. The presented results show how the sensitivity of the available methods to diagnose patients suffer from gangliosidosis GM1 or Morquio B disease can be improved. The proposed method may be easily implemented with microfluidic systems, which currently are promising tools for point-of-care applications.     

A Rare Case of Mucopolysaccharidosis

Mucopolysaccharidosis are a group of rare metabolic disorders of the lysosomal storage disease family caused by the absence or malfunctioning of lysosomal enzymes responsible for their breakdown. It encompasses disorders in which undegraded or partly degraded glycosaminoglycans accumulate in the lysosomes of many tissues owing to a deficiency of specific lysosomal enzymes. Here we report a case of a 7 years old child displaying the symptoms of Morquio’s disease (Mucopolysaccharidosis type IV). Urine screening tests were performed which gave contrasting results.     

Picrotoxin-induced convulsions and lysosomal function in the rat brain

The effect of picrotoxin-induced convulsions on lysosomal function in rat brain were evaluated by measuring the free as well as total acid phosphatase, cathepsin D, acid ribonuclease (RNAse II) and acid deoxyribonuclease (DNAse II) activities. Following picrotoxin treatment the free RNAse II activity increased whereas the total activities of practically all the other enzymes decreased. Paradoxically, the cathepsin D activity, free as well the total was completely abolished. In case of all the enzymes the ratio of Total activity/Free activity decreased indicating increased lysosomal membrane fragility which could lead to process of neurodegeneration in the epileptic animals.     

The use of lysosomal enzymuria in the early detection and monitoring of the progression of diabetic nephropathy

Recent acquisitions on the early detection and monitoring of the progression of diabetic complications (nephropathy) using the techniques of enzymology (lysosomal enzymes) are reviewed. it appears that the kidney is the principal source of urinary lysosomal enzymes. Urinary samples for lysosomal enzyme determination can be either 24-hour or spot-collection. The use of synthetic substrates (4-methylumbelliferyl substrates) provides an easy, inexpensive, sensitive and highly reproducible method of lysosomal enzyme assay. It is recommended that more than one enzyme be assayed in the process. The use of fractional enzyme excretion (FEE) ratios is further recommended. The urinary lysosomal glycosidases investigated and found to be of particular diagnostic value in the early detection of diabetic nephropathy include N-acetyl-β-D-glucosaminidase (β-hexosaminidase, NAG), β-glucuronidase and β-galactosidase, with NAG being the most useful indicator. Urinary NAG can be used in monitoring the progression of diabetic nephropathy. The fluorimetric assay of lysosomal glycosidases is particuarly recommended in developing countries since it is simple, sensitive and inexpensive.