In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by C -sphingosine precursor feeding and mass spectrometry.

Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope-labelled precursor combined with liquid chromatography-mass spectrometry (MS)/MS analysis allows accurate determination of the IC values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells.