van Echten-Deckert G, Klein A, Linke T, Heinemann T, Weisgerber J, Sandhoff K
Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Germany.
J Lipid Res. 1997 Dec;38(12):2569-79.
De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.
通过用[14C]丝氨酸对细胞鞘脂进行生物合成标记，研究了法伯脂肪肉芽肿病患者培养的皮肤成纤维细胞中内源性神经酰胺的从头合成和周转。正常和法伯成纤维细胞中[14C]丝氨酸的细胞摄取以及掺入从头合成的神经酰胺中的情况相似，正常细胞和患病细胞中新合成神经酰胺的半衰期均为2.7小时。发现新合成的神经酰胺直接进入复杂鞘脂的生物合成途径，而不是在法伯成纤维细胞中积累的神经酰胺池中发挥作用。与对照成纤维细胞相比，法伯细胞中复杂鞘脂分解代谢产生的神经酰胺的降解大大延迟，在6小时的追踪时间后，放射性标记的细胞神经酰胺量的差异变得明显。不同的法伯细胞系在积累的神经酰胺量上彼此不同；然而，在体外测定的神经酰胺积累与残余酸性神经酰胺酶活性之间未发现相关性。此外，法伯成纤维细胞中放射性标记的鞘磷脂量显著增加，表明在这种神经酰胺储存障碍中该化合物的降解延迟。我们建议，除其他既定方法外，用[14C]丝氨酸对内源性神经酰胺进行生物合成标记，作为诊断法伯病的一种高度敏感和可靠的方法，能够对法伯脂肪肉芽肿病患者培养的皮肤成纤维细胞中的神经酰胺积累进行半定量测量。
