#METABOLOMICS WORKBENCH juliehaines_20211122_073825 DATATRACK_ID:2937 STUDY_ID:ST001996 ANALYSIS_ID:AN003256 PROJECT_ID:PR001268 VERSION 1 CREATED_ON November 22, 2021, 1:42 pm #PROJECT PR:PROJECT_TITLE Polyamine import and accumulation causes immunomodulation in macrophages PR:PROJECT_TITLE engulfing apoptotic cells PR:PROJECT_SUMMARY Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue PR:PROJECT_SUMMARY homeostasis and drives anti-inflammatory programming in engulfing macrophages. PR:PROJECT_SUMMARY Here, we assess metabolites in naïve and inflammatory macrophages following PR:PROJECT_SUMMARY engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to PR:PROJECT_SUMMARY unique increases in the arginine-derived polyamines, spermidine and spermine, in PR:PROJECT_SUMMARY vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does PR:PROJECT_SUMMARY not arise from retention of apoptotic cell metabolites or de novo synthesis, but PR:PROJECT_SUMMARY from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. PR:PROJECT_SUMMARY Blocking polyamine import prevents efferocytosis from suppressing macrophage PR:PROJECT_SUMMARY IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine PR:PROJECT_SUMMARY import and accumulation, and imported polyamines as mediators of PR:PROJECT_SUMMARY efferocytosis-induced immune reprogramming. PR:INSTITUTE University of Colorado Denver PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE Polyamine import and accumulation causes immunomodulation in macrophages ST:STUDY_TITLE engulfing apoptotic cells (Part 1) ST:STUDY_SUMMARY Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue ST:STUDY_SUMMARY homeostasis and drives anti-inflammatory programming in engulfing macrophages. ST:STUDY_SUMMARY Here, we assess metabolites in naïve and inflammatory macrophages following ST:STUDY_SUMMARY engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to ST:STUDY_SUMMARY unique increases in the arginine-derived polyamines, spermidine and spermine, in ST:STUDY_SUMMARY vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does ST:STUDY_SUMMARY not arise from retention of apoptotic cell metabolites or de novo synthesis, but ST:STUDY_SUMMARY from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. ST:STUDY_SUMMARY Blocking polyamine import prevents efferocytosis from suppressing macrophage ST:STUDY_SUMMARY IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine ST:STUDY_SUMMARY import and accumulation, and imported polyamines as mediators of ST:STUDY_SUMMARY efferocytosis-induced immune reprogramming. ST:INSTITUTE University of Colorado Denver ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - NecJ 4h 1 category:4h unexposed RAW_FILE_NAME=18 SUBJECT_SAMPLE_FACTORS - NecJ 4h 2 category:4h unexposed RAW_FILE_NAME=21 SUBJECT_SAMPLE_FACTORS - NecJ 4h 3 category:4h unexposed RAW_FILE_NAME=24 SUBJECT_SAMPLE_FACTORS - NecJ 4h 1- category:4h non-engulfing RAW_FILE_NAME=19 SUBJECT_SAMPLE_FACTORS - NecJ 4h 2- category:4h non-engulfing RAW_FILE_NAME=22 SUBJECT_SAMPLE_FACTORS - NecJ 4h 3- category:4h non-engulfing RAW_FILE_NAME=25 SUBJECT_SAMPLE_FACTORS - NecJ 4h 1+ category:4h engulfing RAW_FILE_NAME=20 SUBJECT_SAMPLE_FACTORS - NecJ 4h 2+ category:4h engulfing RAW_FILE_NAME=23 SUBJECT_SAMPLE_FACTORS - NecJ 4h 3+ category:4h engulfing RAW_FILE_NAME=26 SUBJECT_SAMPLE_FACTORS - NecJ 8h 1 category:8h unexposed RAW_FILE_NAME=27 SUBJECT_SAMPLE_FACTORS - NecJ 8h 2 category:8h unexposed RAW_FILE_NAME=30 SUBJECT_SAMPLE_FACTORS - NecJ 8h 3 category:8h unexposed RAW_FILE_NAME=33 SUBJECT_SAMPLE_FACTORS - NecJ 8h 1- category:8h non-engulfing RAW_FILE_NAME=28 SUBJECT_SAMPLE_FACTORS - NecJ 8h 2- category:8h non-engulfing RAW_FILE_NAME=31 SUBJECT_SAMPLE_FACTORS - NecJ 8h 3- category:8h non-engulfing RAW_FILE_NAME=34 SUBJECT_SAMPLE_FACTORS - NecJ 8h 1+ category:8h engulfing RAW_FILE_NAME=29 SUBJECT_SAMPLE_FACTORS - NecJ 8h 2+ category:8h engulfing RAW_FILE_NAME=32 SUBJECT_SAMPLE_FACTORS - NecJ 8h 3+ category:8h engulfing RAW_FILE_NAME=35 #COLLECTION CO:COLLECTION_SUMMARY Experimental animals This study was approved and performed in accordance with CO:COLLECTION_SUMMARY the ethical guidelines of the Institutional Animal Care and Use Committee at CO:COLLECTION_SUMMARY National Jewish Health. C57BL/6J (000664), B6-CD45.1 (002014) and CCR2KO CO:COLLECTION_SUMMARY (004999) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). CO:COLLECTION_SUMMARY DsRed.T3 mice (available as 006051 at Jackson Labs) were generously shared by CO:COLLECTION_SUMMARY the Nagy laboratory (Vintersten et al., 2004). CCR2KO mice were crossed with CO:COLLECTION_SUMMARY DsRed mice until double homozygous. Mice used in experiments were between 8-16 CO:COLLECTION_SUMMARY weeks of age. Both male and female sex were used in experiments except bone CO:COLLECTION_SUMMARY marrow chimera experiments, which used only male mice. Cell Lines Jurkat (human CO:COLLECTION_SUMMARY T lymphocytes, clone E6-1) were obtained from ATCC and cultured in RPMI-1640 CO:COLLECTION_SUMMARY (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (v/v) CO:COLLECTION_SUMMARY (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium Pyruvate. Cells CO:COLLECTION_SUMMARY were cultured in a humidified CO2 incubator at 37C. The Jurkat cell line was CO:COLLECTION_SUMMARY established from peripheral blood of a 14-year-old male diagnosed with T cell CO:COLLECTION_SUMMARY leukemia. Murine primary cell cultures Peritoneal cells were isolated from CO:COLLECTION_SUMMARY naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA. Lavage was CO:COLLECTION_SUMMARY estimated to contain 50% macrophages. Peritoneal macrophages were isolated by CO:COLLECTION_SUMMARY adhesion purification of lavage cells in RPMI-10 (RPMI-1640 containing 10% CO:COLLECTION_SUMMARY heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM HEPES, and CO:COLLECTION_SUMMARY 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well plates for CO:COLLECTION_SUMMARY FACS sorting or 4x106 macrophage/well in 24-well plates for flow cytometry. CO:COLLECTION_SUMMARY Non-adherent cells were removed by washing after 3-5 hours, and macrophages were CO:COLLECTION_SUMMARY cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli O111:B4 LPS (List CO:COLLECTION_SUMMARY Biological Laboratories Inc.) was added to generate inflammatory macrophages 12h CO:COLLECTION_SUMMARY before the addition of target cells. Bone marrow-derived macrophages were CO:COLLECTION_SUMMARY generated by 8-day culture of murine bone marrow cells grown in media containing CO:COLLECTION_SUMMARY M-CSF (High glucose DMEM containing 10% v/v FBS, 20% v/v L929-conditioned media, CO:COLLECTION_SUMMARY 1X Pen/Strep/Glut, 100M Sodium Pyruvate, 55M beta-Mercaptoethanol). All CO:COLLECTION_SUMMARY macrophages were removed from culture dishes by 3-minute incubation with 1X CO:COLLECTION_SUMMARY Trypsin-EDTA (Sigma) plus gentle scraping. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY Apoptotic target cells Live Jurkat cells in RPMI-10 were exposed to 60,000 J TR:TREATMENT_SUMMARY of UV energy on a UV Stratalinker 2400 (Stratagene), then incubated at 37C for TR:TREATMENT_SUMMARY 4h to generate apoptotic Jurkat cells (ApoJ). Thymocytes, obtained by crushing TR:TREATMENT_SUMMARY WT murine thymi and filtering for a single cell suspension, were converted to TR:TREATMENT_SUMMARY apoptotic thymocytes in the same manner. For TAMRA-labeling, apoptotic targets TR:TREATMENT_SUMMARY were stained during the final hour by 15 minute incubation at 37C with 10M TR:TREATMENT_SUMMARY 5(6)-TAMRA,SE (Invitrogen) in PBS followed by two washes with RPMI-10 to remove TR:TREATMENT_SUMMARY excess TAMRA. Apoptotic targets were resuspended in fresh RPMI-10 and added to TR:TREATMENT_SUMMARY macrophages in culture at a ratio of 5:1 and co-cultured for 1h. Unengulfed TR:TREATMENT_SUMMARY targets were removed by washing macrophages three times with cold 1X PBS. To TR:TREATMENT_SUMMARY assess in vivo uptake of exogenous targets, 2x106 ApoJ were injected IP. Other TR:TREATMENT_SUMMARY cell targets Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior TR:TREATMENT_SUMMARY to opsonization or killing using the procedure described above. Labeled cells TR:TREATMENT_SUMMARY were incubated for 30 minutes on ice with 25g/mL purified anti-human CD3 TR:TREATMENT_SUMMARY (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to TR:TREATMENT_SUMMARY generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added TR:TREATMENT_SUMMARY to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added TR:TREATMENT_SUMMARY to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were TR:TREATMENT_SUMMARY removed by washing macrophages three times with cold 1X PBS. To assess in vivo TR:TREATMENT_SUMMARY uptake of exogenous targets, 2x106 IgGJ were injected IP. Non-cell targets TR:TREATMENT_SUMMARY 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and TR:TREATMENT_SUMMARY 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids Inc.) TR:TREATMENT_SUMMARY were used to generate PS-containing liposomes. Lipids suspended in chloroform TR:TREATMENT_SUMMARY were mixed at 70:30 POPC:POPS, then chloroform was evaporated under nitrogen. TR:TREATMENT_SUMMARY Dry lipids were resuspended in RPMI for a final concentration of 500M lipid TR:TREATMENT_SUMMARY and sonicated for 15 minutes until liposomes were formed and solution TR:TREATMENT_SUMMARY transitioned from opaque to clear. Liposomes were added to macrophages at a TR:TREATMENT_SUMMARY final concentration of 50M in RPMI-10. For other experiments, Flash Red 5m TR:TREATMENT_SUMMARY carboxylated polystyrene beads (PS/DVB-COOH·(660,690) carboxyl polystyrene TR:TREATMENT_SUMMARY beads, Bangs Laboratories Inc.) were added to macrophages at a final ratio of TR:TREATMENT_SUMMARY 1:2. Macrophages were co-cultured with non-cell targets for 1h and unengulfed TR:TREATMENT_SUMMARY targets were removed by washing macrophages three times with cold 1X PBS. TR:TREATMENT_SUMMARY Isotope-labeled metabolite use For some experiments, Jurkat T cells were TR:TREATMENT_SUMMARY cultured for 4 days in media containing [13C,15N]cell-free amino acid mixture TR:TREATMENT_SUMMARY (Sigma-Aldrich) (RPMI-1640 medium for SILAC, 10% v/v dyalized FBS, 1X Pen/Strep, TR:TREATMENT_SUMMARY 1mM HEPES, 4% v/v amino acid mixture). For some experiments, macrophages were TR:TREATMENT_SUMMARY cultured for 7 hours in media containing [13C,15N]Arginine (Cambridge Isotope TR:TREATMENT_SUMMARY Laboratories): RPMI-1640 medium for SILAC, 10% dyalized FBS v/v, 1X Pen/Strep, TR:TREATMENT_SUMMARY 1mM HEPES, 100M Sodium Pyruvate, 0.2 g/L [13C,15N]Arginine. FACS sorting TR:TREATMENT_SUMMARY engulfing and non-engulfing cells Single cell suspensions were incubated with TR:TREATMENT_SUMMARY unlabeled CD16/CD32 for 5 min to block non-specific FcR-mediated binding. TR:TREATMENT_SUMMARY Cells were stained with surface antibody panels for 20 min, and then washed. TR:TREATMENT_SUMMARY Cells were protected from light and incubations were performed on ice. HBSS TR:TREATMENT_SUMMARY containing 0.3% BSA and 0.3 mM EDTA was used as a buffer for all incubations. TR:TREATMENT_SUMMARY DAPI was added to cells immediately prior to sort to distinguish live and dead TR:TREATMENT_SUMMARY cells. For all in vitro target cell engulfment assays, live macrophages (murine TR:TREATMENT_SUMMARY peritoneal macrophages: CD45+F4/80+DAPI-; murine BMDM: CD45+CD88+DAPI-; HMDM: TR:TREATMENT_SUMMARY CD45+HLA-DR+DAPI-) were separated into engulfing (TAMRA+) and non-engulfing TR:TREATMENT_SUMMARY (TAMRA-) populations. For in vivo IP ApoJ or IgGJ engulfment assays, live TR:TREATMENT_SUMMARY macrophages were identified as CD45+Ly6G-CD88+CD11b+F4/80hiDAPI- and TAMRA+ or TR:TREATMENT_SUMMARY TAMRA-. For bead engulfment assays, live macrophages (CD45+F4/80+DAPI-) were TR:TREATMENT_SUMMARY sorted into engulfing (Flash Red+) and non-engulfing (Flash Red-) populations. TR:TREATMENT_SUMMARY Liposomes had no label; liposome-fed macrophages were only sorted for viability. TR:TREATMENT_SUMMARY Primary antibodies used (source/clone): unlabeled CD16/32 (eBioscience/93), Ly6G TR:TREATMENT_SUMMARY (BD/IA8), F4/80 (ebioscience/BM8), CD45 (BD/30-F11), CD11b (eBioscience/M1/70), TR:TREATMENT_SUMMARY CD88 (BioLegend/20/70), CD64 (Biolegend/X54-5/7.1). All primary antibodies were TR:TREATMENT_SUMMARY used at a 1:400 dilution. All macrophages were sorted into 0.06% BSA-coated TR:TREATMENT_SUMMARY tubes on a Synergy cell sorter (Sony Biotech). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To process cells for assessment of intracellular metabolites, cells were SP:SAMPLEPREP_SUMMARY pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and SP:SAMPLEPREP_SUMMARY discarded; residual liquid was carefully wicked away from the pellet with a SP:SAMPLEPREP_SUMMARY kimwipe. Dry pellets were immediately snap frozen and stored at -80C until SP:SAMPLEPREP_SUMMARY processing. To process culture supernatants for assessment of metabolites, SP:SAMPLEPREP_SUMMARY supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant SP:SAMPLEPREP_SUMMARY was then transferred to a fresh tube, snap frozen, and stored at -80C until SP:SAMPLEPREP_SUMMARY processing. Ultra-high pressure liquid chromatography-mass spectrometry SP:SAMPLEPREP_SUMMARY (UHPLC-MS) was performed by the University of Colorado School of Medicine SP:SAMPLEPREP_SUMMARY Metabolomics Core. Metabolites from frozen cell pellets were extracted at 2e6 SP:SAMPLEPREP_SUMMARY cells/mL in ice cold 5:3:2 MeOH:acetonitrile:water (v/v/v). Media was thawed on SP:SAMPLEPREP_SUMMARY ice and a 10 L aliquot treated with 240 L of the same extraction solution. SP:SAMPLEPREP_SUMMARY Extractions were carried out using vigorous vortexing for 30 min at 4C. SP:SAMPLEPREP_SUMMARY Supernatants were clarified by centrifugation (10 min, 18,000 g, 4C) and SP:SAMPLEPREP_SUMMARY analyzed using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass SP:SAMPLEPREP_SUMMARY spectrometer. Global metabolomics analyses were performed using a 3 min SP:SAMPLEPREP_SUMMARY isocratic run in positive and negative ion modes (separate runs) as described SP:SAMPLEPREP_SUMMARY previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing SP:SAMPLEPREP_SUMMARY samples were analyzed using a 5 min C18 gradient in positive and negative ion SP:SAMPLEPREP_SUMMARY modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). SP:SAMPLEPREP_SUMMARY For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. SP:SAMPLEPREP_SUMMARY Peaks were annotated (in conjunction with the KEGG database), integrated, and SP:SAMPLEPREP_SUMMARY quality control performed using Maven (Princeton University) as described. SP:SAMPLEPREP_SUMMARY Stable isotope tracing results were isotopically corrected for the natural SP:SAMPLEPREP_SUMMARY abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 10 uL of each sample was injected. The LC was operated under isocratic CH:CHROMATOGRAPHY_SUMMARY conditions using phase A (95% water, 5% acetonitrile, 10 mM ammonium acetate). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) CH:FLOW_GRADIENT isocratic CH:FLOW_RATE 250 ul/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. MS:MS_COMMENTS Peaks were annotated (in conjunction with the KEGG database), integrated, and MS:MS_COMMENTS quality control performed using Maven (Princeton University) as described. MS:MS_COMMENTS Stable isotope tracing results were isotopically corrected for the natural MS:MS_COMMENTS abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples NecJ 4h 1 NecJ 4h 2 NecJ 4h 3 NecJ 4h 1- NecJ 4h 2- NecJ 4h 3- NecJ 4h 1+ NecJ 4h 2+ NecJ 4h 3+ NecJ 8h 1 NecJ 8h 2 NecJ 8h 3 NecJ 8h 1- NecJ 8h 2- NecJ 8h 3- NecJ 8h 1+ NecJ 8h 2+ NecJ 8h 3+ Factors category:4h unexposed category:4h unexposed category:4h unexposed category:4h non-engulfing category:4h non-engulfing category:4h non-engulfing category:4h engulfing category:4h engulfing category:4h engulfing category:8h unexposed category:8h unexposed category:8h unexposed category:8h non-engulfing category:8h non-engulfing category:8h non-engulfing category:8h engulfing category:8h engulfing category:8h engulfing UTP 2.40E+03 4.98E+03 3.92E+03 4.09E+03 3.07E+03 6.08E+03 2.67E+03 1.30E+03 3.99E+03 3.66E+03 3.08E+03 5.93E+03 1.67E+03 3.64E+03 3.64E+03 3.16E+03 5.39E+03 3.54E+03 Uracil 2.00E+05 2.04E+05 2.01E+05 2.06E+05 2.30E+05 2.25E+05 1.94E+05 1.91E+05 1.64E+05 1.77E+05 1.66E+05 2.29E+05 1.98E+05 1.83E+05 2.58E+05 1.97E+05 1.95E+05 2.03E+05 Urate 1.37E+05 1.54E+05 1.80E+05 2.64E+05 1.42E+05 4.09E+05 2.09E+05 2.62E+05 4.24E+05 1.03E+05 6.92E+04 9.51E+04 3.26E+04 9.69E+04 4.80E+04 1.38E+05 2.65E+05 1.69E+05 3',5'-Cyclic IMP 1.47E+05 2.93E+04 2.76E+04 1.62E+05 3.81E+04 1.74E+05 7.02E+04 1.80E+04 3.27E+05 1.26E+05 1.94E+04 3.13E+05 7.72E+02 5.13E+04 0.00E+00 1.52E+05 2.10E+05 3.06E+04 Phosphate 9.91E+07 1.07E+08 1.05E+08 8.93E+07 8.34E+07 1.28E+08 1.05E+08 1.02E+08 1.13E+08 8.67E+07 8.49E+07 1.10E+08 1.06E+08 1.03E+08 1.10E+08 1.01E+08 8.55E+07 9.69E+07 Diphosphate 9.71E+06 1.61E+07 1.10E+07 1.04E+07 1.05E+07 2.25E+07 1.17E+07 1.11E+07 1.60E+07 1.12E+07 8.09E+06 1.68E+07 1.65E+07 1.28E+07 1.36E+07 1.48E+07 1.14E+07 1.38E+07 D-Glucose 2.45E+06 1.67E+06 2.00E+06 2.23E+06 1.85E+06 1.86E+06 2.14E+06 1.77E+06 2.77E+06 2.20E+06 1.99E+06 2.07E+06 2.39E+06 1.75E+06 1.64E+06 2.64E+06 2.72E+06 1.64E+06 D-Glucose 6-phosphate 3.89E+05 3.22E+05 4.59E+05 4.68E+05 3.62E+05 4.05E+05 7.12E+05 5.56E+05 5.70E+05 5.41E+05 4.71E+05 3.89E+05 3.22E+05 4.37E+05 2.33E+05 5.96E+05 6.39E+05 4.69E+05 D-Fructose 1-6-bisphosphate 9.77E+04 1.14E+05 1.73E+05 2.28E+05 1.33E+05 4.04E+05 1.46E+05 1.38E+05 2.77E+05 9.15E+04 5.79E+04 1.13E+05 2.40E+04 9.02E+04 1.95E+04 8.01E+04 1.48E+05 1.23E+05 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 2.80E+05 1.68E+05 2.51E+05 2.42E+05 1.91E+05 2.86E+05 2.29E+05 3.28E+05 3.05E+05 2.32E+05 1.43E+05 1.84E+05 1.59E+05 1.40E+05 4.37E+04 1.13E+05 2.06E+05 2.80E+05 Pyruvate 2.95E+05 2.74E+05 3.82E+05 3.10E+05 2.60E+05 2.64E+05 3.45E+05 2.88E+05 3.66E+05 3.74E+05 3.08E+05 2.70E+05 3.57E+05 2.76E+05 3.52E+05 3.67E+05 3.65E+05 2.86E+05 Lactate 1.69E+06 2.21E+06 3.15E+06 1.89E+06 1.79E+06 2.50E+06 2.82E+06 2.23E+06 1.80E+06 4.71E+06 2.02E+06 1.97E+06 1.41E+06 1.71E+06 3.48E+06 1.74E+06 1.43E+06 2.67E+06 Maltose 2.82E+04 5.03E+04 9.80E+04 4.44E+04 1.60E+04 6.52E+04 7.03E+04 4.61E+04 2.71E+04 1.45E+05 7.37E+03 1.79E+04 3.39E+04 1.07E+04 2.13E+05 3.44E+04 2.18E+04 4.71E+04 Citrate 5.41E+05 8.18E+05 1.05E+06 3.95E+05 4.67E+05 3.92E+05 8.93E+05 7.81E+05 6.74E+05 1.40E+06 4.28E+05 9.83E+05 5.81E+05 9.24E+05 9.44E+05 1.55E+06 7.15E+05 1.23E+06 Succinate 9.49E+04 1.02E+05 1.85E+05 1.03E+05 5.83E+04 6.55E+04 1.30E+05 1.37E+05 1.68E+05 9.96E+04 1.28E+05 1.31E+05 1.06E+05 9.30E+04 1.25E+05 2.07E+05 1.78E+05 2.21E+05 Fumarate 9.97E+03 1.18E+04 6.43E+03 2.14E+04 8.40E+03 1.21E+04 1.59E+04 1.35E+04 9.41E+03 9.98E+03 5.91E+03 6.36E+03 6.83E+02 1.16E+04 4.69E+02 5.88E+03 7.05E+03 1.41E+04 Malate 2.84E+05 7.41E+05 6.49E+05 1.91E+05 3.66E+05 2.93E+05 5.09E+05 8.54E+05 3.95E+05 5.10E+05 3.65E+05 3.40E+05 2.73E+05 3.58E+05 7.00E+05 4.91E+05 3.35E+05 7.10E+05 Oxaloacetate 1.75E+05 1.66E+05 1.77E+05 1.70E+05 1.81E+05 2.37E+05 1.74E+05 1.54E+05 1.90E+05 1.65E+05 1.50E+05 2.03E+05 1.41E+05 1.28E+05 1.76E+05 1.91E+05 1.93E+05 1.99E+05 Itaconate 2.00E+06 2.16E+06 7.04E+05 1.58E+06 1.85E+06 2.65E+06 4.60E+06 4.94E+06 5.50E+06 2.39E+06 2.08E+06 2.99E+06 2.18E+06 2.58E+06 2.01E+06 2.52E+06 6.30E+06 6.55E+06 2-Hydroxyglutarate/Citramalate 2.33E+05 3.49E+05 2.66E+05 1.63E+05 2.15E+05 1.77E+05 2.18E+05 2.50E+05 1.91E+05 2.26E+05 2.87E+05 2.56E+05 1.52E+05 2.43E+05 2.17E+05 2.20E+05 1.98E+05 2.78E+05 Sedoheptulose 1-phosphate 7.13E+04 6.20E+04 1.01E+05 9.71E+04 8.26E+04 8.52E+04 1.13E+05 1.28E+05 1.16E+05 8.95E+04 8.11E+04 6.71E+04 6.15E+04 8.03E+04 5.67E+04 9.87E+04 1.09E+05 9.43E+04 alpha-D-Ribose 1-phosphate 8.82E+04 4.42E+04 9.34E+04 8.90E+04 7.10E+04 7.97E+04 1.78E+05 1.54E+05 1.21E+05 5.87E+04 5.45E+04 4.19E+04 5.09E+04 7.14E+04 3.82E+04 8.86E+04 8.77E+04 8.34E+04 Glutathione 1.43E+05 1.44E+05 1.35E+05 2.47E+05 1.68E+05 2.29E+05 1.68E+05 1.57E+05 2.14E+05 2.77E+05 2.36E+05 2.47E+05 1.41E+05 1.88E+05 1.32E+05 2.04E+05 3.41E+05 2.50E+05 Ascorbate 1.22E+05 1.21E+05 1.68E+05 1.33E+05 1.17E+05 1.16E+05 1.33E+05 1.23E+05 1.80E+05 1.54E+05 1.40E+05 1.16E+05 1.59E+05 1.36E+05 1.37E+05 1.83E+05 1.63E+05 1.19E+05 Ornithine 9.14E+04 4.72E+04 6.50E+04 8.32E+04 6.85E+04 6.47E+04 1.58E+05 1.17E+05 1.00E+05 5.68E+04 2.55E+04 3.81E+04 4.39E+04 3.41E+04 5.91E+04 7.14E+04 7.91E+04 7.14E+04 N-Glycoloyl-neuraminate 3.02E+04 4.14E+04 4.19E+04 3.16E+04 3.83E+04 2.33E+04 3.76E+04 4.89E+04 3.24E+04 6.30E+04 6.79E+04 7.23E+04 4.04E+04 4.70E+04 3.44E+04 4.15E+04 5.41E+04 5.21E+04 1-4-beta-D-Xylan 7.91E+04 4.47E+04 6.74E+04 7.54E+04 5.88E+04 6.79E+04 6.57E+04 6.59E+04 9.26E+04 8.54E+04 7.19E+04 8.18E+04 9.22E+04 5.15E+04 6.82E+04 9.73E+04 1.01E+05 5.97E+04 N-formyl kynurenine 5.36E+04 5.19E+04 5.64E+04 4.05E+04 5.04E+04 4.19E+04 8.19E+04 7.87E+04 5.80E+04 7.09E+04 7.29E+04 4.72E+04 5.95E+04 6.55E+04 7.71E+04 1.02E+05 9.66E+04 9.26E+04 2-Oxoadipate 1.09E+05 9.05E+04 1.16E+05 1.20E+05 1.07E+05 8.49E+04 1.28E+05 1.10E+05 1.37E+05 1.05E+05 1.21E+05 1.01E+05 1.39E+05 1.03E+05 1.11E+05 1.65E+05 1.39E+05 9.42E+04 Glycerol 3-phosphate 3.97E+04 3.15E+04 4.07E+04 4.46E+04 3.77E+04 5.58E+04 5.45E+04 5.98E+04 6.79E+04 4.37E+04 3.62E+04 3.68E+04 2.61E+04 4.25E+04 3.26E+04 3.68E+04 3.77E+04 4.41E+04 Ethanolamine phosphate 8.59E+05 7.38E+05 5.73E+05 7.53E+05 7.66E+05 5.80E+05 7.58E+05 7.27E+05 5.92E+05 1.23E+06 1.10E+06 1.21E+06 1.04E+06 1.04E+06 1.21E+06 6.18E+05 1.06E+06 8.03E+05 2-Methyleneglutarate 2.73E+05 1.76E+05 2.43E+05 2.03E+05 2.06E+05 1.64E+05 1.77E+05 2.02E+05 2.59E+05 2.29E+05 2.05E+05 2.08E+05 2.60E+05 1.90E+05 2.11E+05 2.94E+05 2.83E+05 1.83E+05 D-glucono-1,5-lactone 2.68E+05 2.40E+05 3.30E+05 3.12E+05 2.85E+05 2.39E+05 2.79E+05 2.40E+05 3.45E+05 2.96E+05 2.82E+05 2.15E+05 3.44E+05 2.43E+05 2.57E+05 3.93E+05 3.54E+05 2.40E+05 9-Oxononanoic acid 2.98E+04 6.44E+04 8.52E+04 6.06E+04 4.77E+04 6.24E+04 3.13E+04 5.50E+04 2.92E+04 4.37E+04 4.51E+04 2.89E+04 3.32E+04 4.47E+04 3.77E+04 5.27E+04 3.55E+04 2.82E+04 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. UTP C00075 482.9600 2.16 Uracil C00106 111.0185 1.17 Urate C00366 167.0196 1.21 3',5'-Cyclic IMP C00943 329.0299 1.26 Phosphate C00009 96.9680 1.15 Diphosphate C00013 176.9346 1.12 D-Glucose C00031 179.0552 1.19 D-Glucose 6-phosphate C00092 259.0215 1.14 D-Fructose 1-6-bisphosphate C00354 338.9883 1.27 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.9886 1.14 Pyruvate C00022 87.0071 1.20 Lactate C01432 89.0228 1.30 Maltose C00208 341.1084 1.22 Citrate C00158 191.0187 1.09 Succinate C00042 117.0178 1.15 Fumarate C00122 115.0023 1.47 Malate C00149 133.0128 1.12 Oxaloacetate C00036 131.0003 1.17 Itaconate C00490 129.0177 1.76 2-Hydroxyglutarate/Citramalate C02630 147.0286 1.10 Sedoheptulose 1-phosphate C06222 289.0324 1.12 alpha-D-Ribose 1-phosphate C00620 229.0110 1.14 Glutathione C00051 306.0761 1.26 Ascorbate C00072 175.0242 1.19 Ornithine C01602 131.0811 1.22 N-Glycoloyl-neuraminate C03410 324.0929 1.13 1-4-beta-D-Xylan C02352 131.0337 1.20 N-formyl kynurenine C02700 235.0750 1.21 2-Oxoadipate C00322 159.0289 1.20 Glycerol 3-phosphate C00093 171.0054 1.14 Ethanolamine phosphate C00346 140.0103 1.13 2-Methyleneglutarate C02930 143.0336 1.20 D-glucono-1,5-lactone C00198 177.0397 1.19 9-Oxononanoic acid C16322 171.1015 1.32 METABOLITES_END #END