MetaboLights

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spectrometry data was analyzed using El-Maven (v0.12.0, Elucidata) with the compound database search detection method and the following parameters: EIC extraction window 10 ppm, minimum peak intensity 10,000, minimum quality 0.5, minimum signal/blank ratio 2.0, minimum signal/baseline ratio 2.0, minimum peak width 3 scans. Detected peaks were manually inspected and metabolites annotated based on match to in-house metabolite library established by measuring retention time on column and accurate mass of authentic analytical standards and by match to MS2 libraries.MetaboLightsPublicLiquid Chromatography MS - alternating - hilicLC separation was performed on an XBridge BEH Amide column (2.1 x 150 mm, 2.5 µm particle size, 130 Å pore size; Waters Corporation) using a gradient of solvent A (95:5 water:acetonitrile with 20 mM of ammonium acetate and 20 mM of ammonium hydroxide, pH 9.45) and solvent B (acetonitrile). Flow rate was 150 µL/min. The LC gradient was: 0.0 min, 90% B; 2.0 min, 90% B; 3.0 min, 75% B; 7.0 min, 75% B; 8.0 min, 70% B; 9.0 min, 70% B; 10.0 min, 50% B; 12.0 min, 50% B; 13.0 min, 25% B; 14.0 min, 25% B; 16.0 min, 0.5% B; 20.5 min, 0.5% B; 21.0 min, 90% B; 25.0 min, 90% B.Microbiome metabolism of dietary phytochemicals controls the anticancer activity of PI3K inhibitors.Asael RoichmanPrinceton UniversityblankDietserumfecesmass spectrometry assayFor serum samples, 3 µL serum was mixed with 117 µL methanol pre-cooled to -20°C, vortexed for 30 s and centrifuged at 17,000g for 20 min at 4°C. The supernatants were transferred to another Eppendorf tube, centrifuged again at 17,000g for 10 min at 4°C, and the clear supernatant was transferred to MS vials and loaded onto the mass spectrometer autosampler. For fecal samples, freeze-clamped material was transferred into 2 mL Eppendorf tubes, kept on dry ice, containing a 6.5 mm ceramic bead (product 4039GM-S065, Inframat Advanced Materials LLC, Manchester, CT) and then pulverized under liquid nitrogen using CryoMill (Restch, Newtown, PA). Next, 10-20 mg of powdered sample were weighed, 40x volume (40 µL extraction solvent per 1 mg sample material) of 40:40:20 methanol: acetonitrile: water with 0.5% formic acid pre-cooled to -20°C was added, vortexed and incubated on ice for 10 min. Acid was then neutralized by adding 15% (w/v) ammonium bicarbonate aqueous solution in amount of 8.75% v/v of extraction buffer, and samples were vortexed and centrifuged at 17,000 g for 20 min at 4°C. The supernatants were transferred into a new Eppendorf tube, centrifuged again at 17,000 g for 10 min at 4°C, and clear supernatant was transferred to MS vials and loaded onto the mass spectrometer autosampler. For diets, we used the same extraction method as for fecal samples, except that we used cold methanol as extraction solvent without neutralization. Mus musculusblankDiethttps://www.ebi.ac.uk/metabolights/MTBLS10944Joshua Rabinowitz. Joshr@Princeton.edu.Asael Roichman. Asaelr@princeton.edu.Thermo raw data files were analyzed using either Xcalibur Qual Browser or Freestyle (Thermo Scientific). Alternatively, Thermo raw data files were converted to mzXML format using the msconvert utility bundled with ProteoWizard.DietTimepointasaelr@princeton.eduTail snipping was used for systemic blood collection. Blood was allowed to clot for 15 min at room temperature and then put on ice until further processing. Serum was separated by centrifuging blood at 2000 x g for 10 min at 4 °C. For fecal sample collection, mice were restrained and gently massaged on the belly to facilitate defecation, and fecal pellets were immediately placed on dry ice. All samples were stored at -80 °C until further analysis.Metabolomicsuntargeted metabolomicsmetabolomic profilingAlpelisibFecesSerum global profilingDietary Interventionuntargeted metabolomicsmetabolomic profilingAlpelisibFecesSerum global profilingDietary InterventionMetabolite measurements were obtained by running samples on Q Exactive HF Orbitrap (serum samples) or orbitrap Exploris 480 (fecal samples) mass spectrometer (Thermo Scientific) coupled with hydrophilic interaction chromatography (HILIC) with electrospray ionization. Injection volume was 5-10 µL and autosampler temperature was 4 °C. MS scans were in negative and positive ion switching mode with a resolution of 120,000 at m/z 200 and scan range of m/z 70-1000 for negative mode and m/z 58-116.5 and m/z 120-1000 for positive mode. Diet samples were also analyzed at a scan range of 1000-2000 m/z in negative ionization mode. Other scan parameters are AGC target, 1e7 for Exploris 480, and 5e6 for Q Exactive HF, ITmax, 200 ms. Additional instrument parameters are: spray voltage 3000 V, sheath gas 35 (Arb), aux gas 10 (Arb), sweep gas 0.5 (Arb), ion transfer tube temperature 300 °C, vaporizer temperature 35 °C, internal mass calibration on, RF lens 60.falseMicrobiome Metabolism of Dietary Phytochemicals Controls the Anticancer Activity of PI3K InhibitorsPhosphatidylinositol 3-kinase (PI3K) signaling is both the effector pathway of insulin and among the most frequently activated pathways in human cancer. In murine cancer models, the efficacy of PI3K inhibitors is dramatically enhanced by a ketogenic diet, with a proposed mechanism involving dietary suppression of insulin. Here we confirm profound diet-PI3K anticancer synergy but show that it is, surprisingly, unrelated to diet macronutrient composition. Instead, the diet-PI3K interaction involves microbiome metabolism of ingested phytochemicals. Specifically, murine ketogenic diet lacks the complex spectrum of phytochemicals found in standard chow, including the soy phytochemicals soyasaponins. We find that soyasaponins are converted by the microbiome into inducers of the hepatic cytochrome P450 enzyme CYP3A11, and thereby lower PI3K inhibitor blood levels and anticancer activity. A high-carbohydrate, low-phytochemical diet synergizes with PI3K inhibition to treat cancer in mice, as do antibiotics that curtail the gut microbiome. Thus, diet impacts anticancer drug activity through phytochemical-microbiome-liver interactions.2025-03-262024-08-23MTBLS10944