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

Publication date: Available online 27 September 2017
Source:Clinical Biochemistry
Author(s): Jaqueline Cé, Melissa Tôrres Rodrigues, Eduarda Tassoni Käfer, Vitória da Costa Moraes, Janice Carneiro Coelho
IntroductionMucopolysaccharidoses (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).ObjectivesThis 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.MethodsThe 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.ResultsThe correlations among the techniques were significant. Although the correlation coefficient was similar, fourfold reduction was selected. pH4.4 had the highest enzyme activity. GUSB's 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.ConclusionsThis 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.



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