Untargeted metabolomics between ALDOB KO or WT mice exposed to control or 10% fructose solution
Description
Hydrophilic Interaction Liquid Chromatography (HILIC) was used to successfully separate isobaric hexose phosphate species, as previously described. Our data revealed that F1P was the most significantly altered metabolite in ALDOB KO mice upon fructose exposure. Interestingly, sorbitol levels were also increased suggesting that the polyol pathway (formation of fructose from glucose via sorbitol) is active in this setting.
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Polar metabolite extraction was performed as previously described. Cells at 80% confluence or homogenized tissue samples were collected and extracted with 1 mL extraction solvent (50% methanol, 20% Milli-Q water, 30% acetonitrile) containing internal standards (5-Fu ). After sonication and -20°C extraction for 30 min, samples were centrifuged (16,100g, 4°C, 10 min). Supernatants were freeze-dried, reconstituted in mobile phase, and stored at -80°C for LC-MS analysis. Protein concentration was determined by BCA assay. Metabolomics analysis was performed using QTOF mass spectrometer (AB 6600 TripleTOF, SCIEX) coupled with UHPLC (Nexera UHPLC LC-30A, SHIMADZU). Separation was achieved on Waters ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm) using mobile phase A (water with 25 mM NH₄OH and NH₄OAc) and B (ACN). MS was operated in IDA mode with TOF mass range m/z 60-1200 Da. Data analysis was conducted using Metabolomics and Limma in R (v.4.0.3).