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LCMS Profiling of Phosphorylated and Free Sphingoid Bases in Gliomas, Peritumoral Tissues and Serums

Introduction Development and progression of gliomas are accompanied by aberrations in sphingolipid (SL) metabolism. SL metabolites and phosphorylated sphingoid bases (S1Ps) are important signalling molecules involved in the pathogenesis of gliomas. The aim of this study was to analyse free and phosphorylated sphingoid bases in the glioblastoma multiforme (GBM) and different grade gliomas, corresponding peritumoral tissues (PTs), patients’ serums and healthy control serums, as well as to establish the method of preparation and LCMS analysis for each sample type. Methods Free sphingoid bases (SBs) and sphingosine- and sphinganine-1-phosphates (S1Ps) were extracted and purified by base hydrolysis from the tissue homogenates and serum samples by Sullards et al [1] modified in our laboratory. d14:1 was added to samples prior to extraction as internal standard and samples were analysed by Agilent 6550 iFunnel Q-TOF LC/MS. MRM analysis protocol was developed via optimization of ionization, CID conditions and retention time for each analyte of interest. Quantification was performed using the calibration curve obtained by external standard cocktail of SBs and S1Ps. Results Five SBs and two S1Ps were detected and characterized as d16:1, d18:1, d18:0, d20:1, 20:0, d18:1-P and d18:0-P in all tissue samples (diffuse astrocytoma gr. II, glioma gr. III, two GBMs and four corresponding PT tissues), corresponding patients’ serums (PS, 4) and serums of healthy controls (HS, 4). d16:1 was quantified only in GBM samples, while it was detected in all other samples. d18:1 was quantified in all samples with the highest concentration in tumoral tissues and significantly higher concentrations in HSs than in PSs. d18:0 and d20:0 were detected in all samples. d20:1 was quantified in tissue samples and HSs, but not detected in PSs. d18:1-P was detected in GBMs, but not in low grade glioma, while quantified in HSs in significantly higher concentrations than in PSs. d18:0-P was quantified only in serum samples with approx. 20 times higher concentrations in PSs than in HSs, while it was not detected in tissue samples. Also, concentrations of d18:0-P in PSs were proportional to the malignancy grade of the glioma type, with the grade II glioma serum having the lowest conc. of d18:0-P. Innovative aspects • Certain sphingoid bases were determined for the first time in these samples. • d18:0-P conc. in patients’ serums was significantly higher than in healthy controls’ serums. • d18:0-P conc. in patients’ serums was proportional to the grade of the glioma type. Acknowledgements This research was supported by Adris Foundation to D.F. and by the European Fund for Reg. Development to the Institute for Anthropological Research (BIOANT, KK.01.1.1.02.0002).

LCMS ; Sphingoid Bases ; Glioma

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