Nonchromatographic method for acid sphingomyelinase in WBC lysate using modified Dole solvent

Abstract

Correspondence: Mohammad Zouheir Habbal Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, PO Box 11-0236, Riad El Solh 110-72020, Beirut, Lebanon Tel +96 11 350 000 ext 5220 Fax +96 11 370 845 Email mh03@aub.edu.lb 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.