A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Potential role of acid sphingomyelinase in environmental health / 中南大学学报(医学版)

Acid sphingomyelinase (ASM) is one enzyme responsible for the production of ceramide via the hydrolysis of sphingomyelin.Recent findings have revealed the important role of ASM in the initiation of ceramide-induced cell apoptosis,as well as in the pathophysiology of many common diseases (e.g.cardiovascular diseases,diabetes,pulmonary diseases,and neurological diseases).Other studies have also shown that ASM activation may occur through the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS),as well as by inflammatory mechanisms that may be induced by environmental and occupational stresses.ASM activation,in turn,may create excess or abnormally distributed ceramides,which could lead to tissue and organ injuries,including to the pulmonary,liver,kidney,and nervous systems.This review will discuss the basic biology of ASM and focus on the role and regulation of ASM in environmental stress responses.We propose that ASM activation is an important factor in environmental health,and that ASM-based therapeutics may have a key role in preventing environmental-induced tissue injury.