Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry.
10.3343/alm.2012.32.5.319
- Author:
Elisa LEGNINI
1
;
Joe J ORSINI
;
Adolf MUHL
;
Britt JOHNSON
;
Angela DAJNOKI
;
Olaf A BODAMER
Author Information
1. Women's and Children's Health Department, University of Padua, Italy.
- Publication Type:Original Article
- Keywords:
Tandem mass spectrometry;
Dried blood spot;
Lysosomal enzyme;
Acid sphingomyelinase
- MeSH:
*Dried Blood Spot Testing;
Hematocrit;
Humans;
Infant, Newborn;
Reference Standards;
Sphingomyelin Phosphodiesterase/*analysis/standards;
Sphingomyelins/metabolism;
Substrate Specificity;
*Tandem Mass Spectrometry/standards
- From:Annals of Laboratory Medicine
2012;32(5):319-323
- CountryRepublic of Korea
- Language:English
-
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 (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). 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 4degrees C. 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 micromol/h/L (mean 10.6) with a SD of 5.06 micromol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 micromol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 micromol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.
