title: Analysis of acid sphingomyelinase activity in dried blood spots using tandem mass spectrometry.
abstract: BACKGROUND
  Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening.
  
  
  METHODS
  ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C(29)H(59)N(2)O(6)P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C(22)H(43)NO(3))]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product).
  
  
  RESULTS
  ASM activities were stable for up to 2 months at or below 4℃. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 µmol/h/L (mean 10.6) with a SD of 5.06 µmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 µmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 µmol/h/L).
  
  
  CONCLUSIONS
  This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.
authors.affiliation: Women's and Children's Health Department, University of Padua, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Legnini
authors.forename: Joe J
authors.initials: JJ
authors.lastname: Orsini
authors.forename: Adolf
authors.initials: A
authors.lastname: Mühl
authors.forename: Britt
authors.initials: B
authors.lastname: Johnson
authors.forename: Angela
authors.initials: A
authors.lastname: Dajnoki
authors.forename: Olaf A
authors.initials: OA
authors.lastname: Bodamer
mesh_terms: D059788:Dried Blood Spot Testing; D006400:Hematocrit; D006801:Humans; D007231:Infant, Newborn; D012015:Reference Standards; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis* / Q000592:standards; D013109:Sphingomyelins / Q000378:metabolism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D013109:Sphingomyelins; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase