| Autor: |
Dei Cas M; Department of Health Sciences, San Paolo Hospital, Università degli Studi di Milano, 20142 Milano, Italy., Montavoci L; Department of Health Sciences, San Paolo Hospital, Università degli Studi di Milano, 20142 Milano, Italy., Casati S; Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, 20133 Milan, Italy., Malagolini N; Department of Medical and Surgical Sciences (DIMEC), Università di Bologna, 40126 Bologna, Italy., Dall'Olio F; Department of Medical and Surgical Sciences (DIMEC), Università di Bologna, 40126 Bologna, Italy., Trinchera M; Department of Medicine and Surgery (DMC), University of Insubria, 21100 Varese, Italy. |
| Abstrakt: |
Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals. |
