Abstract：
Measurements of sphingolipid metabolism are most accurately performed by liquid chromatography-mass spectrometry. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-Ceramide to NBD-C6-sphingomyelin, NBD-C6-Hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that PDMP, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.
摘要：
鞘脂代谢的测量最准确地通过液相色谱-质谱进行。然而,这项技术很昂贵，无法广泛访问，并且不使用特定的探针，它不能提供对通过该途径的代谢通量的洞察。使用荧光神经酰胺类似物NBD-C6-神经酰胺作为完整细胞中的示踪剂，我们开发了一种基于HPLC的综合方法，可以同时测量高尔基体中神经酰胺代谢的主要节点。因此，通过定量NBD-C6-神经酰胺向NBD-C6-鞘磷脂的转化，NBD-C6-己糖神经酰胺，和NBD-C6-神经酰胺-1-磷酸（NBD-C1P），高尔基常驻酶鞘磷脂合酶1，葡萄糖神经酰胺合酶的活性，可以同时测量神经酰胺激酶（CERK）。重要的是,NBD-C1P的检测使我们能够量化细胞中的CERK活性，通常是困难的任务。通过应用此方法，我们评估了常用鞘脂抑制剂的特异性，发现PDMP，靶向葡萄糖神经酰胺合成酶，和fenretinide(4HPR)，二氢神经酰胺去饱和酶的抑制剂，也抑制了CERK的活动。这项研究证明了对高尔基体中神经酰胺代谢进行扩展分析的好处，它提供了一种定性且易于实现的方法。
