title: A new fluorimetric enzyme assay for the diagnosis of Niemann-Pick A/B, with specificity of natural sphingomyelinase substrate.
abstract: 6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required.
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Centre, Rotterdam, The Netherlands. o.vandiggelen@erasmusmc.nl
authors.forename: O P
authors.initials: OP
authors.lastname: van Diggelen
authors.forename: Ya V
authors.initials: YV
authors.lastname: Voznyi
authors.forename: J L M
authors.initials: JL
authors.lastname: Keulemans
authors.forename: K
authors.initials: K
authors.lastname: Schoonderwoerd
authors.forename: J
authors.initials: J
authors.lastname: Ledvinova
authors.forename: E
authors.initials: E
authors.lastname: Mengel
authors.forename: M
authors.initials: M
authors.lastname: Zschiesche
authors.forename: R
authors.initials: R
authors.lastname: Santer
authors.forename: K
authors.initials: K
authors.lastname: Harzer
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D002518:Ceramides / Q000737:chemistry; D002624:Chemistry, Clinical / Q000379:methods*; D004796:Clinical Enzyme Tests; D003937:Diagnosis, Differential; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic; D006596:Hexosaminidases / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007962:Leukocytes / Q000201:enzymology / Q000378:metabolism; D009154:Mutation; D009542:Niemann-Pick Diseases / Q000175:diagnosis* / Q000201:enzymology; D010742:Phospholipid Ethers / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding; D015203:Reproducibility of Results; D012867:Skin / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*; D013109:Sphingomyelins / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D002518:Ceramides; D010742:Phospholipid Ethers; D013109:Sphingomyelins; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; C046258:4-methylumbelliferyl phosphorylcholine; D013108:Sphingomyelin Phosphodiesterase; D006596:Hexosaminidases; C086400:chitotriosidase; D013110:Sphingosine