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<p>For liver metabolites, livers were perfused, frozen in liquid nitrogen, and then shattered in liquid nitrogen with a mortar and pestle at a later date. Several pieces of shattered liver totalling ~100 milligrams were collected then homogenized in 1mL 70% Ethanol at -20°C. Metabolites were extracted by adding 7mL 70% Ethanol at 75°C for 2 min. Extracts were centrifuged for 10 minutes at 4,000 rpm at 4°C. Clean metabolites extracts were dried in a vacuum centrifuge and re-suspended in double-distilled H2O with volume according to the weight of the extracted liver piece. Quantification of metabolites was performed on an Agilent 6550 QTOF instrument by flow injection analysis time-of-flight mass spectrometry (see: PMID 21830798). All samples were injected in duplicates. Ions were annotated based on their accurate mass and the Human Metabolome Database reference list (see: PMID 23161693) allowing a tolerance of 0.001 Da. Unknown ions and those annotated as adducts were discarded. Theoretical m/z ratios—beyond the significant digits from the measurement sensitivity—are used as the unique index in the online data on GeneNetwork. For example, deprotonated fumarate corresponds to 115.0036897_MZ, malate to 133.0142794_MZ, α-ketoglutarate to 145.0141831_MZ, and D2HG to 147.0298102_MZ.</p>
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