An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease

Sphingomyelin is an important lipid component of cell membranes and lipopro teins which can be hydrolyzed by sphingomyelinases into ceramide and phosph orylcholine. The type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingo myelinase, and the resultant accumulation of sphingomyelin in cells and tis sues. In this paper we report a new, enzyme-based method to quantify the le vels of sphingomyelin in tissues and plasma of normal individuals and NPD p atients. The method utilizes sphingomyelinase from Bacillus cereus to compl etely hydrolyze the sphingomyelin into ceramide, Quantification of the sphi ngomyelin-derived ceramide is accomplished using Escherichia coli diacylgly cerol (DAG) kinase and [gamma-P-32]ATP. The resulting [P-32]ceramide is qua ntified using a phosphor-imager system following TLC separation. This proce dure allowed quantification of sphingomyelin over a broad range from 10 pmo l to 1 nmol. To validate this assay we quantified sphingomyelin in plasma a nd tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma wa s significantly elevated compared to that of normal mice (up to twofold), M oreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also we re significantly elevated compared to normal individuals of the same age. B ased on these results we propose that this new, enzyme based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful t ool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method p rovides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract. (C) 2001 Academic Press.