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

• Published in: Clinical biochemistry Vol. 50; no. 18; pp. 1243 - 1248
• Main Authors: Cé, Jaqueline, Rodrigues, Melissa Tôrres, Käfer, Eduarda Tassoni, da Costa Moraes, Vitória, Coelho, Janice Carneiro
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2017
• Subjects: Beta-glucuronidase; Dried blood spots; Mucopolyssaccharidoses; Sly syndrome; MPS; MPS VII; Km; DBS; ANOVA; Dried blood spots; Vmax; GAGs; Beta-glucuronidase; Mucopolyssaccharidoses; GUSB; Sly syndrome
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2017.09.022

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). 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. 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. The 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. 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. •1.2mm DBS and reduced 4 fold the volume of reagents in beta-glucuronidase (GUSB) activity measure•Optimal pH was 4.4 and GUSB’s Km in DBS of healthy individuals was 1.25mM while Vmax was 594.48nmol/h/mL.•At different temperatures and storage times, GUSB activity remained stable.•The normality range for GUSB in 1.2mm DBS may facilitate the identification of individuals with milder mutations.