Ann Lab Med.  2012 Sep;32(5):319-323. 10.3343/alm.2012.32.5.319.

Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry

• Legnini E
• Orsini JJ
• Muhl A
• Johnson B
• Dajnoki A
• Bodamer OA

• KMID: 1387345
• DOI: http://doi.org/10.3343/alm.2012.32.5.319

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.

Keyword

Tandem mass spectrometry; Dried blood spot; Lysosomal enzyme; Acid sphingomyelinase

MeSH Terms

*Dried Blood Spot Testing
Hematocrit
Humans
Infant, Newborn
Reference Standards
Sphingomyelin Phosphodiesterase/*analysis/standards
Sphingomyelins/metabolism
Substrate Specificity
*Tandem Mass Spectrometry/standards

Figure

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  Fig. 1 Stability of ASM (median +/- SD) in DBS for 7, 14, 21, 28 days and 2 months at -20℃, 4℃, room temperature and at 37℃. Abbreviations: ASM, acid sphingomyelinase; DBS, dried blood spots.

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  Fig. 2 Correlation between activity of ASM (median +/-SD) and hematocrit and location of the punch (peripheral versus central) respectively. Abbreviation: ASM, acid sphingomyelinase.

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  Fig. 3 ASM activity in anonymous newborn screening filter cards (N=2,085). Abbreviation: ASM, acid sphingomyelinase.

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