title: A fluorescence-based, high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease.
abstract: Acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is the lysosomal enzyme that hydrolyzes sphingomyelin (SPM) to phosphorylcholine and ceramide. An inherited deficiency of ASM activity results in Types A and B Niemann-Pick disease (NPD). In this study we report a new assay method to detect ASM activity and diagnose NPD using the fluorescent substrate BODIPY C12-SPM and reverse-phase high-performance liquid chromatography (HPLC). The reaction product, BODIPY C12-ceramide (B12Cer), could be clearly and efficiently separated from the substrate within 4 min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0 micro l of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36 pmol/ml/h) was only 2.7% of that in normal plasma (1334 pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3 pmol/ml/h) also was significantly reduced (19.5% of normal). The ranges of ASM plasma activities in NPD patients (N=19), NPD carriers (N=11), and normal subjects (N=15) were 2.5-97.3, 108-551, and 1030-2124 pmol/ml/h, respectively. Based on these results, we suggest that this fluorescence-based HPLC assay method is a reliable, rapid, and highly sensitive technique to determine ASM activity and that plasma is a very reliable and simple source for the accurate diagnosis of NPD patients and carriers based on ASM activity.
authors.affiliation: Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Xingxuan
authors.initials: X
authors.lastname: He
authors.forename: Fei
authors.initials: F
authors.lastname: Chen
authors.forename: Ari
authors.initials: A
authors.lastname: Dagan
authors.forename: Shimon
authors.initials: S
authors.lastname: Gatt
authors.forename: Edward H
authors.initials: EH
authors.lastname: Schuchman
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D009542:Niemann-Pick Diseases / Q000097:blood* / Q000175:diagnosis*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013108:Sphingomyelin Phosphodiesterase / Q000097:blood*
chemical_list: D013108:Sphingomyelin Phosphodiesterase