Sterols

Brain tumor tissue (4mg) was homogenized in extraction solution by mixing acetonitrile, isopropanol and water in proportions 3:3:2 (JT Baker, Center Valley PA), then vortexed for 45 seconds and then 5 minutes at 4C. Following centrifugation for 2 minutes at 14,000 rcf, two aliquots of the supernatant (500 μL each aliquot) were made for analysis and one for backup. One aliquot was dried via evaporation overnight in the Labconco Centrivap cold trap concentrator (Labconco, Kansas City MO). The dried aliquot was then resuspended with 500μL 50% acetonitrile (degassed as given), then centrifuged for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. The supernatant was moved to a new Eppendorf tube and again evaporated to dryness. Internal standards (C08-C30, fatty acid methyl esters) were then added and the sample was derivatized by methoxyamine hydrochloride in pyridine and subsequently by N-methyl-N-trimethylsilyltrifluoroacetamide for trimethylsilylation of acidic protons. Data were acquired as previously described.^{45 (link)} Briefly, metabolites were measured using a Restek corporation rtx5Sil-MS column (Restek Corporation; Bellefonte PA; 30 m length x 0.25 mm internal diameter with 0.25μm film made of 95% dimethyl/5%diphenylpolysiloxane) protected by a 10m long empty guard column which is cut by 20cm intervals whenever the reference mixture QC samples indicate problems caused by column contaminations. This sequence of column cuts has been validated by UC Davis Metabolomics Core with no detrimental effects detected with respect to peak shapes, absolute or relative metabolite retention times or reproducibility of quantifications. This chromatography method yields excellent retention and separation of primary metabolite classes (amino acids, hydroxyl acids, carbohydrates, sugar acids, sterols, aromatics, nucleosides, amines and miscellaneous compounds) with arrow peak widths of 2–3 seconds and very good within-series retention time reproducibility of better than 0.2s absolute deviation of retention times. The mobile phase consisted of helium, with a flow rate of 1 mL/min, and injection volume of 0.5 μL. The following mass spectrometry parameters were used: a Leco Pegasus IV mass spectrometer with unit mass resolution at 17 spectra s-1 from 80-500 Da at −70 eV for elution of metabolites. As a quality control, for each sequence of sample extractions, one blank negative control was performed by applying the total procedure (i.e. all materials and plastic ware) without biological sample. Result files were transformed by calculating the sum intensities of all structurally identified compounds for each sample (i.e. those signals that had been positively identified in the data pre-processing schema outlined above), and subsequently dividing all data associated with a sample by the corresponding metabolite sum. The resulting data were multiplied by a constant factor in order to obtain values without decimal places. Intensities of identified metabolites with more than one peak (e.g. for the syn- and anti-forms of methoximated reducing sugars) were summed to only one value in the transformed data set. The original nontransformed data set was retained. The general concept of this data transformation is to normalize data to the ‘total metabolite content’, but disregarding unknowns that might potentially comprise artifact peaks or chemical contaminants.