Sphingomyelin is an important
lipid component of cell membranes and
lipoproteins that can be hydrolyzed by sphingomyelinases into
ceramide and
phosphorylcholine. The Type A and B forms of
Niemann-Pick disease (
NPD) are
lipid storage disorders due to the deficient activity of the
enzyme acid sphingomyelinase and the resultant accumulation of
sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of
sphingomyelin in plasma, urine, or tissues from
NPD patients and mice. In this assay, bacterial
sphingomyelinase is first used to hydrolyze
sphingomyelin to
phosphorylcholine and
ceramide.
Alkaline phosphatase then generates
choline from the
phosphorylcholine, and the newly formed
choline is then used to generate
hydrogen peroxide in a reaction catalyzed by
choline oxidase. Finally, with
peroxidase as a catalyst,
hydrogen peroxide reacts with the
Amplex Red reagent to generate a highly fluorescent product,
resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of
sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using
diacylglycerol kinase and 50 times more sensitive than a colorimetric,
aminoantipyrine/
phenol-based assay. To validate this new assay method, we quantified
sphingomyelin in plasma, urine, and tissues from normal individuals and from
NPD mice and patients. The
sphingomyelin content in adult homozygous or heterozygous
NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated
sphingomyelin in the tissues of
NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The
sphingomyelin levels in plasma from several Type B
NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible
sphingomyelin quantification in tissues and fluids from normal individuals and
NPD patients. It could also be a useful tool for the study of other
sphingomyelin-related diseases and in a variety of research settings where
sphingomyelin quantification is required.