INSERM, U858, Toulouse, France.
J Lipid Res. 2010 Dec;51(12):3542-7. doi: 10.1194/jlr.D010033. Epub 2010 Sep 24.
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.
酸性神经酰胺酶（aCDase）是负责哺乳动物细胞内神经酰胺降解的几种酶之一。因此，aCDase 调节细胞内生物活性脂质神经酰胺的水平。aCDase 活性的遗传性缺乏导致法伯病（FD），也称为脂肉芽肿病，其特征是患者组织中神经酰胺的积累。FD 的诊断通过证明 aCDase 活性缺乏以及随后的神经酰胺储存来确认。现有的方法包括极其复杂的测定方法，其中许多方法使用放射性标记化合物。因此，aCDase 测定和 FD 的体外酶诊断仍然仅在极少数专门实验室中进行。在这里，合成并表征了新的荧光底物 Rbm14-12，作为测定 aCDase 活性的新工具。优化后的测定法在 96 孔板中进行，并用来自 FD 患者和对照的不同成纤维细胞和淋巴样细胞系进行测试以测量 aCDase 活性。结果，发现 FD 患者细胞中的活性非常低甚至为零。这种新的荧光方法为特异性和准确测定 aCDase 活性以及诊断 FD 提供了非常简单和快速的方法。
