Nonchromatographic method for acid sphingomyelinase in WBC lysate using modified Dole solvent
Mohammad Zouheir Habbal
Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon
Purpose: To use a modified fluorometric method to measure white blood cell acid sphingomyelinase activity.
Methods: White blood cell lysates were prepared and used as a source of the enzyme. Two tubes were used for each assay, the second as a blank. In each, N-(NBD-Aminolauroyl)sphingomyelin dissolved in chloroform-methanol (2:1) was added and the organic solvent was removed by nitrogen gas. Acetate buffer, 1% Triton™ X100 solution, and sonicated protein (the reaction tube only) were added to the residue. The mixture in each was then incubated at 37°C for 2 hours, which was followed by the addition of buffer, Dole solvent, heptane, and sodium chloride solution. The sonicated protein was added to the blank tube and NBD-ceramide was extracted by vortex for 5 minutes and brief centrifugation at 3000 rpm. The intensity of fluorescence of the upper phase was determined in a fluorometer at an excitation wavelength of 465 nm and emission wavelength at 530 nm.
Results: In 20 normal patients, the range of enzyme activity was 305–1008 pmol with a mean of 570 pmol. In a proven case of Niemann–Pick disease type A by molecular gene analysis, enzyme activity was undetectable.
Conclusion: The modified method is convenient in a biomedical genetics laboratory where a request for sphingomyelinase is very infrequent.
Keywords: acid sphingomyelinase, Niemann–Pick disease, fluorescent analysis, white blood cells
This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.Download Article [PDF]