For cell culture, polar metabolites and fatty acids were extracted using methanol/water/chloroform and analyzed as previously described7 (link). For tissue and plasma, metabolites and total fatty acids were extracted from tissues and plasma using a Folch-based methanol/chloroform/saline extraction at a ratio of 1:2:1 with inclusion of [2H31] palmitate and norvaline as lipid and polar internal standards respectively. Briefly, 250 μl MeOH, 500 μls CHCL3, 250 μls saline and fatty acid isotope internal standards were added to weighed pre-ground tissue. This was vortexed for 10 minutes followed by centrifugation at 10,000 g for 5 minutes. Polar metabolites were derivatized in 2% (w/v) methoxyamine hydrochloride (Thermo Scientific) in pyridine and incubated at 37°C for 60 minutes. Samples were then silylated with N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (tBDMS) (Regis Technologies) at 37°C for 30–45 minutes. Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x 0.25 μm, Agilent J&W Scientific) installed in an Agilent 7890A gas chromatograph (GC) interfaced with an Agilent 5975C mass spectrometer (MS). The lower chloroform phase was dried and then derivitised to form fatty acid methyl esters(FAMES) via addition of 500 μls 2% H2SO4 in MeOH and incubation at 50°C for 2 hours. FAMES were extracted via addition of 100 μl saturated salt solution and 500 μl hexane and these were analyzed using a Select FAME column (100m x 0.25mm i.d.) installed in an Aglient 7890A GC interfaced with an Agilent 5975C MS using the following temperature program: 80 °C initial, increase by 20 °C/min to 170 °C, increase by 1 °C/min to 204 °C, then 20 °C/min to 250 °C and hold for 10 min. The % isotopologue distribution of each fatty acid and polar metabolite was determined and corrected for natural abundance using in-house algorithms adapted from Fernandez et al55 (link). The metabolite ion fragments used are summarized in methods table 4. Mole percent enrichment (MPE) was calculated via the following equation:
where n is the number of carbon atoms in the metabolite and Mi is the relative abundance of the ith mass isotopologue. Molar enrichment was determined by multiplying the MPE by the abundance.
YSI (yellow springs instrument) was used to quantify glucose, lactate, glutamine and glutamate in cell culture media.
where n is the number of carbon atoms in the metabolite and Mi is the relative abundance of the ith mass isotopologue. Molar enrichment was determined by multiplying the MPE by the abundance.
YSI (yellow springs instrument) was used to quantify glucose, lactate, glutamine and glutamate in cell culture media.
