Abstract
Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. As such, aCDase regulates the intracellular levels of the bioactive lipid ceramide. An inherited deficiency of aCDase activity results in Farber disease (FD), also called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. Diagnosis of FD is confirmed by demonstration of a deficient aCDase activity and the subsequent storage of ceramide. Existing methods include extremely complex assays, many of them using radiolabeled compounds. Therefore, the aCDase assay and the in vitro enzymatic diagnosis of FD are still performed in only a very limited number of specialized laboratories. Here, the new fluorogenic substrate Rbm14-12 was synthesized and characterized as a new tool to determine aCDase activity. The resulting optimized assay was performed in 96-well plates, and different fibroblast and lymphoid cell lines derived from FD patients and controls were tested to measure aCDase activity. As a result, the activity in cells of FD patients was found to be very low or even null. This new fluorogenic method offers a very easy and rapid way for specific and accurate determination of aCDase activity and, consequently, for diagnosis of FD.
Highlights
Acid ceramidase is one of several enzymes responsible for ceramide degradation within mammalian cells
The methods that have been used for Farber disease (FD) diagnostic purposes can be classified into three groups: i) acid ceramidase (aCDase) enzymatic assays [4,5,6,7,8,9,10,11,12,13,14,15,16], classically performed with radiolabeled substrates [4,5,6,7,8,9,10,11,12,13], which are rather water-insoluble and require at least one detergent; ii) loading tests, consisting of the addition of exogenous radiolabeled sphingolipids, e.g., ceramide [17, 18], sulfatide [19, 20], or sphingomyelin [21], on cultured living cells and the study of their metabolism; and iii) determination of accumulated ceramide, either by sophisticated chromatographic methods [22, 23] or through the use of the diacylglycerol kinase assay in the presence of ␥[32P]ATP to measure the radiolabeled ceramide 1-phosphate produced [24]
We present a novel method to determine aCDase activity that proved useful for FD diagnosis
Summary
Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. An inherited deficiency of aCDase activity results in Farber disease (FD), called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease. Farber disease (FD) is a rare inherited lipid storage disorder, known as lipogranulomatosis, which is characterized by accumulation of ceramide in the cells and tissues of patients [1, 2]. This accumulation is the consequence of a deficient intracellular activity of aCDase, a lysosomal. Some of the main drawbacks of these methods are the use of radiolabeled compounds, most of them being no longer commercially available; the laborious process of separation and identification of reaction products; the Abbreviations: aCDase, acid ceramidase; FD, Farber disease. 1 To whom correspondence should be addressed
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