Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry

Background: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achi eved hv measurement of the lysosomal enzymes acid sphingomyelinase (ASR-I) and galactocerebroside beta -galactosidase (GCG), respectively. Conventiona l assays use radiolabeled or fluorescent substrates and do not allow simult aneous determination of two or more enzymes in the sample. Methods: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidinagarose beads and, following release, were analyzed by electrospray ionization mass spec trometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions. Results: The method demonstrated excellent linearity of ASM and GCG enzymat ic product formation with the amount of cellular protein and incubation tim e. The range of ASM activities in fibroblast lysates from six healthy patie nts was 39-70 nmol.mg(-1).h(-1) compared with 3.7-5.1 nmol.mg(-1).h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GC G activities toward the corresponding substrate conjugate were 4.0-6.8 nmol .mg(-1).h(-1) in cell lysates from healthy patients compared with 0.1-0.2 n mol.mg(-1).h(-1) in cell lysates from two patients affected with Krabbe dis ease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 mug) for both ASM and GCG. Conclusions: Electrospray mass spectrometry combined with the use of biotin ylated substrate conjugates and bioaffinity purification represents a new a pproach for the diagnosis of lysosomal storage disease as demonstrated for Niemann-Pick A and Krabbe diseases, No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and G CG in one cell sample, (C) 2001 American Association for Clinical Chemistry .