Distribution of neuraminidase activity in fibroblasts from post-mortem samples

Abstract

Sialidosis (MIM 256550) is caused by α-N-acetyl neuraminidase (EC 3.2.1.18) deficiency resulting from a mutation in the neuraminidase gene (NEU1) located on chromosome 6p21.33. Currently, samples for the diagnosis of sialidosis were sent out overseas as there is no suitable test available in Malaysia. This study aimed to assess and establish the performance of neuraminidase assay using fibroblasts samples for laboratory diagnosis. Fluorometric measurements of 4-methylumbelliferone-α-D-acetylneuraminic acid (4-MuF-NueAc) were used as an artificial substrate to evaluate the neuraminidase activity. Carbonate buffer pH 10.7 was used as a stopping reagent. The fluorescence intensity of 4-MuF release was measured at a specific wavelength of 366nm excitation and 446 nm emission. Method verification was performed according to the IMR laboratory quality procedure (LQP) guideline. Linearity study showed 4MuF was linear up to 40,000 nmol. Limit of detection and limit of quantitation were 7.998 nmol/hr/mg and 26.66 nmol/hr/mg protein, respectively. Repeatability and reproducibility test results expressed as coefficient of variation (%CV) were 11.38% and 12.52%, respectively. The neuraminidase activity was measured in 8 normal controls and 18 postmortem patients’ samples. The median (min to max value) neuraminidase activities in normal and postmortem patients’ samples were 38.41 (15.21 to 97.34) and 24.28 (9.49 to 45.77) nmol/h/mg protein, respectively demonstrating a significant difference between both (p<0.05). In conclusion, study findings showed that the neuraminidase assay accomplished an appropriate method verification requirement. A new laboratory test for the diagnosis of sialidosis has been effectively established in Malaysia.