TY - JOUR
T1 - A fluorescence-based, high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease
AU - He, Xingxuan
AU - Chen, Fei
AU - Dagan, Ari
AU - Gatt, Shimon
AU - Schuchman, Edward H.
N1 - Funding Information:
This work was supported by a United States–Israel Binational Grant (96-00095), a grant from the Fogart International Center (5R03 TW 01372), and a grant from the National Institutes of Health (R01 HD 28607).
PY - 2003/3/1
Y1 - 2003/3/1
N2 - 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 4min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0μl of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36pmol/ml/h) was only 2.7% of that in normal plasma (1334pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3pmol/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 1030-2124pmol/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.
AB - 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 4min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0μl of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36pmol/ml/h) was only 2.7% of that in normal plasma (1334pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3pmol/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 1030-2124pmol/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.
UR - http://www.scopus.com/inward/record.url?scp=0037334121&partnerID=8YFLogxK
U2 - 10.1016/S0003-2697(02)00629-2
DO - 10.1016/S0003-2697(02)00629-2
M3 - Article
C2 - 12633609
AN - SCOPUS:0037334121
SN - 0003-2697
VL - 314
SP - 116
EP - 120
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -