Glycosphingolipids and oxidative stress: Evaluation of hydroxyl radical oxidation of galactosyl and lactosylceramides using mass spectrometry

• C24:1GalCer and C24:1LacCer free radical oxidation was studied by C5–HPLC–MS and MS/MS.
• Oxidation in fatty acyl chains, loss of sugar moiety with formation of C24:1Cer and oxidized C24:1Cer were observed in MS.
• Keto, hydroxy and hydroperoxy derivatives of C24:1GalCer and C24:1LacCer were identified.
• C24:1GlcCer was identified as an oxidation product of C24:1LacCer.
• Radical oxidation of C16:0GalCer leaded only to the formation of C16:0Cer.

Galactosylceramide (GalCer) and lactosylceramide (LacCer) are structural and signaling lipids, playing important roles in signal transduction and cell adhesion. They are especially abundant in the nervous system and in important components of the myelin sheath. Although neurodegenerative disorders are associated with increased oxidative stress and lipid oxidation, the connection between oxidative stress and glycosphingolipid modification has been scarcely addressed.In this study, we aimed to characterize the structural changes caused by the hydroxyl radical to GalCer and LacCer molecular species using electrospray ionization mass spectrometry (ESI–MS and MS/MS) and high performance liquid chromatography–tandem mass spectrometry (HPLC-MSn). ESI–MS and LC–MS spectra of 24:1GalCer and 24:1LacCer after free radical oxidation showed the formation of new species, which were identified as keto, hydroxyl and hydroperoxy derivatives, arising from modification in the mono unsaturated fatty acyl chain. Formation of ceramide and oxidized ceramides was also observed as a result of 24:1GalCer and 24:1LacCer radical oxidation. 24:1GlcCer (glucosylceramide) was detected after LacCer oxidation, probably due to oxidative cleavage of lactosyl moiety. This study shows that glycosphingolipids are prone to radical induced oxidation, which can be one of the causes of the increased ceramides content and pro apoptotic events during oxidative conditions and neurodegeneration. This MS study will support the future identification of oxidized galactosyl- and lactosylceramide species in sphingolipidomic studies applied to biological samples related with oxidative conditions.