Sphingomyelinase (SMase) is a hydrolase enzyme that is involved in sphingolipid metabolism. SMase is a member of the DNase I superfamily of enzymes and is responsible for the breakdown of sphingomyelin into phosphorylcholine and ceramide. The activation of SMase has been suggested as a major route for the production of ceramide in response to cellular stress.¹ The five distinct types of SMases are classified according to their cation requirements, cellular localization, and pH optima. The types are: Lysosomal Acid SMase, secreted zinc-dependent Acid SMase, a membrane-bound magnesium-dependent Neutral SMase, a cytosolic magnesium-independent Neutral SMase, and an Alkaline SMase.^2,3,4,5,6 Loss of acidic SMase activity due to a mutation in the acid SMase (ASM) gene results in type A and B Niemann-Pick disease.⁴ SMases are important in many physiological and pathophysiological processes, including: 1) lysosomal digestion of sphingomyelin, which is important for normal neuronal and vascular function; 2) ceramide-mediated signal transduction, leading to cytokine-induced apoptosis, cellular differentiation, and various immune and inflammatory responses; 3) lipoprotein aggregation within the vessel wall, which is a key event in atherogenesis and 4) intracellular cholesterol trafficking and metabolism.^7,8,9,10 Cayman’s Sphingomyelinase Assay provides a simple, reproducible, and sensitive tool for assaying neutral and acidic sphingomyelinase activity from tissue homogenates, cell lysates, serum, saliva, and urine. The SMase Assay utilizes a coupled enzymatic reaction to monitor SMase activity. SMase hydrolyzes sphingomyelin to yield ceramide and phosphorylcholine. Alkaline phosphatase hydrolyzes phosphorylcholine forming choline. Choline is then oxidized by choline oxidase to yield betaine and H₂O₂. Finally, H₂O₂, in the presence of horseradish peroxidase (HRP), reacts with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in a 1:1 stoichiometry to generate the highly fluorescent product resorufin.^11,12 Resorufin fluorescence is analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

1 Hannun, Y.A., and Obeid, L.M. The ceramide-centric universe of lipid-mediated cell regulation: Stress encounters of the lipid kind. J Biol Chem 277(29) 25847-25850 (2002).

2 Schuchman, E.H., Suchi, M., Takahashi, T., et al. Human acid sphingomyelinase: Isolation, nucleotide sequence, and expression of the full-length and alternatively spliced cDNAs. J Biol Chem 266(13) 8531-8539 (1991).

3 Schissel, S.L., Schuchman, E.H., Williams, K.J., et al. Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J Biol Chem 271(31) 18431-18436 (1996).

4 Bernardo, K., Krut, O., Wiegmann, K., et al. Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain. J Biol Chem 275(11) 7641-7647 (2000).

5 Yamaguchi, S., and Suzuki, K. A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system myelin. J Biol Chem 253(12) 4090-4092 (1978).

6 Duan, R., Bergman, T., Xu, N., et al. Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family. J Biol Chem 278(40) 38528-38536 (2003).

7 Kolesnick, R.N. Sphingomyelin and derivatives as cellular signals. Prog Lipid Res 30(1) 1-38 (1991).

8 Tabas, I., Li, Y., Brocia, R.W., et al. Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix: A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation. J Biol Chem 268(27) 20419-20432 (1993).

9 Pörn, M.I., and Slotte, J.P. Localization of cholesterol in sphingomyelinase-treated fibroblasts. Biochem J 308 269-274 (1995).

10 Smith, E.L., and Schuchman, E.H. The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases. FASEB J 22 3419-3431 (2008).

11 Mohanty, J.G., Jaffe, J.S., Schulman, E.S., et al. A highly sensitive fluorescent micro-assay of H₂O₂ release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods 202 133-141 (1997).

12 Zhou, M., Diwu, Z., Panchuk-Voloshina, N., et al. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253 162-168 (1997).

Pörn, M.I., and Slotte, J.P. Localization of cholesterol in sphingomyelinase-treated fibroblasts. Biochem J 308 269-274 (1995).

Zhou, M., Diwu, Z., Panchuk-Voloshina, N., et al. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253 162-168 (1997).

Hostetler, K.Y., and Yazaki, P.J. The subcellular localization of neutral sphingomyelinase in rat liver. J Lipid Res 20 456-463 (1979).

Yamaguchi, S., and Suzuki, K. A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system myelin. J Biol Chem 253(12) 4090-4092 (1978).

Tabas, I., Li, Y., Brocia, R.W., et al. Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix: A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation. J Biol Chem 268(27) 20419-20432 (1993).

Bernardo, K., Krut, O., Wiegmann, K., et al. Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain. J Biol Chem 275(11) 7641-7647 (2000).

Duan, R., Bergman, T., Xu, N., et al. Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family. J Biol Chem 278(40) 38528-38536 (2003).

Spence, M.W., Clarke, J.T.R., and Cook, H.W. Pathways of sphingomyelin metabolism in cultured fibroblasts from normal and sphingomyelin lipidosis subjects. J Biol Chem 258(14) 8595-8600 (1983).

Schissel, S.L., Schuchman, E.H., Williams, K.J., et al. Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J Biol Chem 271(31) 18431-18436 (1996).

Schuchman, E.H., Suchi, M., Takahashi, T., et al. Human acid sphingomyelinase: Isolation, nucleotide sequence, and expression of the full-length and alternatively spliced cDNAs. J Biol Chem 266(13) 8531-8539 (1991).

Kolesnick, R.N. Sphingomyelin and derivatives as cellular signals. Prog Lipid Res 30(1) 1-38 (1991).

Mohanty, J.G., Jaffe, J.S., Schulman, E.S., et al. A highly sensitive fluorescent micro-assay of H₂O₂ release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods 202 133-141 (1997).

Spence, M.W. Sphingomyelinases.. Adv Lipid Res 26 3-23 (1993).

Hannun, Y.A., and Obeid, L.M. The ceramide-centric universe of lipid-mediated cell regulation: Stress encounters of the lipid kind. J Biol Chem 277(29) 25847-25850 (2002).

Smith, E.L., and Schuchman, E.H. The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases. FASEB J 22 3419-3431 (2008).

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