A new fluorimetric enzyme assay for the diagnosis of Niemann-Pick A/B, with specificity of natural sphingomyelinase substrate

J Inherit Metab Dis. 2005;28(5):733-41. doi: 10.1007/s10545-005-0105-y.

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.

MeSH terms

  • Blood Chemical Analysis / methods*
  • Ceramides / chemistry
  • Chemistry, Clinical / methods*
  • Clinical Enzyme Tests
  • Diagnosis, Differential
  • Dose-Response Relationship, Drug
  • Fibroblasts / enzymology
  • Fibroblasts / metabolism
  • Fluorometry / methods*
  • Gene Expression Regulation, Enzymologic
  • Hexosaminidases / chemistry
  • Humans
  • Hydrolysis
  • Leukocytes / enzymology
  • Leukocytes / metabolism
  • Mutation
  • Niemann-Pick Diseases / diagnosis*
  • Niemann-Pick Diseases / enzymology
  • Phospholipid Ethers / chemistry
  • Phosphorylcholine / analogs & derivatives
  • Phosphorylcholine / chemistry
  • Protein Binding
  • Reproducibility of Results
  • Skin / metabolism
  • Sphingomyelin Phosphodiesterase / chemistry*
  • Sphingomyelins / chemistry
  • Sphingosine / analogs & derivatives
  • Sphingosine / chemistry
  • Substrate Specificity
  • Time Factors

Substances

  • Ceramides
  • Phospholipid Ethers
  • Sphingomyelins
  • sphingosine phosphorylcholine
  • Phosphorylcholine
  • 4-methylumbelliferyl phosphorylcholine
  • Sphingomyelin Phosphodiesterase
  • Hexosaminidases
  • chitotriosidase
  • Sphingosine