#METABOLOMICS WORKBENCH juliehaines_20211122_115205 DATATRACK_ID:2938 STUDY_ID:ST001997 ANALYSIS_ID:AN003257 PROJECT_ID:000000 VERSION 1 CREATED_ON November 22, 2021, 12:06 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-1or 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 2) 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-1or 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 - 4h 1 category:4h unexposed RAW_FILE_NAME=37 SUBJECT_SAMPLE_FACTORS - 4h 2 category:4h unexposed RAW_FILE_NAME=40 SUBJECT_SAMPLE_FACTORS - 4h 3 category:4h unexposed RAW_FILE_NAME=43 SUBJECT_SAMPLE_FACTORS - 4h 1- category:4h non-engulfing RAW_FILE_NAME=38 SUBJECT_SAMPLE_FACTORS - 4h 2- category:4h non-engulfing RAW_FILE_NAME=41 SUBJECT_SAMPLE_FACTORS - 4h 3- category:4h non-engulfing RAW_FILE_NAME=44 SUBJECT_SAMPLE_FACTORS - 4h 1+ category:4h engulfing RAW_FILE_NAME=39 SUBJECT_SAMPLE_FACTORS - 4h 2+ category:4h engulfing RAW_FILE_NAME=42 SUBJECT_SAMPLE_FACTORS - 4h 3+ category:4h engulfing RAW_FILE_NAME=45 SUBJECT_SAMPLE_FACTORS - 8h 1 category:8h unexposed RAW_FILE_NAME=46 SUBJECT_SAMPLE_FACTORS - 8h 2 category:8h unexposed RAW_FILE_NAME=49 SUBJECT_SAMPLE_FACTORS - 8h 3 category:8h unexposed RAW_FILE_NAME=52 SUBJECT_SAMPLE_FACTORS - 8h 1- category:8h non-engulfing RAW_FILE_NAME=47 SUBJECT_SAMPLE_FACTORS - 8h 2- category:8h non-engulfing RAW_FILE_NAME=50 SUBJECT_SAMPLE_FACTORS - 8h 3- category:8h non-engulfing RAW_FILE_NAME=53 SUBJECT_SAMPLE_FACTORS - 8h 1+ category:8h engulfing RAW_FILE_NAME=48 SUBJECT_SAMPLE_FACTORS - 8h 2+ category:8h engulfing RAW_FILE_NAME=51 SUBJECT_SAMPLE_FACTORS - 8h 3+ category:8h engulfing RAW_FILE_NAME=54 #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. Human tissue This study CO:COLLECTION_SUMMARY used deidentified donor peripheral blood mononuclear cells (PBMC) collected and CO:COLLECTION_SUMMARY isolated (Young et al., 2011) by the National Jewish Blood Prep Core (IRB No. CO:COLLECTION_SUMMARY HS-1285). Age and sex of deidentified blood donors in this study is not known. CO:COLLECTION_SUMMARY Cell Lines Jurkat (human T lymphocytes, clone E6-1) were obtained from ATCC and CO:COLLECTION_SUMMARY cultured in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal CO:COLLECTION_SUMMARY bovine serum (v/v) (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium CO:COLLECTION_SUMMARY Pyruvate. Cells were cultured in a humidified CO2 incubator at 37C. The Jurkat CO:COLLECTION_SUMMARY cell line was established from peripheral blood of a 14-year-old male diagnosed CO:COLLECTION_SUMMARY with T cell leukemia. Murine primary cell cultures Peritoneal cells were CO:COLLECTION_SUMMARY isolated from naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA. CO:COLLECTION_SUMMARY Lavage was estimated to contain 50% macrophages. Peritoneal macrophages were CO:COLLECTION_SUMMARY isolated by adhesion purification of lavage cells in RPMI-10 (RPMI-1640 CO:COLLECTION_SUMMARY containing 10% heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM CO:COLLECTION_SUMMARY HEPES, and 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well CO:COLLECTION_SUMMARY plates for FACS sorting or 4x106 macrophage/well in 24-well plates for flow CO:COLLECTION_SUMMARY cytometry. Non-adherent cells were removed by washing after 3-5 hours, and CO:COLLECTION_SUMMARY macrophages were cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli CO:COLLECTION_SUMMARY O111:B4 LPS (List Biological Laboratories Inc.) was added to generate CO:COLLECTION_SUMMARY inflammatory macrophages 12h before the addition of target cells. Bone CO:COLLECTION_SUMMARY marrow-derived macrophages were generated by 8-day culture of murine bone marrow CO:COLLECTION_SUMMARY cells grown in media containing M-CSF (High glucose DMEM containing 10% v/v FBS, CO:COLLECTION_SUMMARY 20% v/v L929-conditioned media, 1X Pen/Strep/Glut, 100M Sodium Pyruvate, CO:COLLECTION_SUMMARY 55M beta-Mercaptoethanol). All macrophages were removed from culture dishes CO:COLLECTION_SUMMARY by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY LPS-primed murine peritoneal macrophages were fed fluorescently-tagged TR:TREATMENT_SUMMARY IgG-opsonized live Jurkat cells for 1h. Unengulfed targets were washed off and TR:TREATMENT_SUMMARY macrophages were left to degrade targets for a further 3 or 7 hours. Macrophages TR:TREATMENT_SUMMARY were then collected and sorted into non-engulfing or engulfing populations, TR:TREATMENT_SUMMARY alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and TR:TREATMENT_SUMMARY dry pellets were snap frozen and stored at -80C until metabolite extraction. #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. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Isocratic flow of 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in acetonitrile) at 250 uL/min. 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 POSITIVE MS:MS_COMMENTS Global metabolomics analyses were performed using a 3 min isocratic run in MS:MS_COMMENTS positive and negative ion modes (separate runs) as described previously (Nemkov MS:MS_COMMENTS et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed MS:MS_COMMENTS using a 5 min C18 gradient in positive and negative ion modes (separate runs) as MS:MS_COMMENTS described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS MS:MS_COMMENTS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in MS:MS_COMMENTS conjunction with the KEGG database), integrated, and quality control performed MS:MS_COMMENTS using Maven (Princeton University) as described. Stable isotope tracing results MS:MS_COMMENTS were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and MS:MS_COMMENTS 15N2 (Nemkov et al., 2017). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 4h 1 4h 2 4h 3 4h 1- 4h 2- 4h 3- 4h 1+ 4h 2+ 4h 3+ 8h 1 8h 2 8h 3 8h 1- 8h 2- 8h 3- 8h 1+ 8h 2+ 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 L-arginine 1.15E+04 3.79E+04 2.33E+04 2.04E+04 3.04E+04 3.68E+04 5.43E+05 4.23E+05 4.42E+05 2.69E+04 3.87E+04 3.32E+04 3.23E+04 2.10E+04 3.61E+04 2.61E+05 2.31E+05 2.70E+05 L-cysteine 6.20E+04 4.66E+04 4.19E+04 3.45E+04 3.45E+04 6.42E+04 1.59E+05 1.94E+05 2.40E+05 4.36E+04 8.09E+04 4.90E+04 4.81E+04 7.27E+04 4.35E+04 1.88E+05 1.53E+05 1.52E+05 L-glutamate 3.69E+05 4.36E+05 2.59E+05 2.93E+05 2.98E+05 5.50E+05 6.12E+05 4.85E+05 5.19E+05 4.14E+05 5.77E+05 4.06E+05 4.08E+05 5.36E+05 4.85E+05 7.93E+05 6.81E+05 5.74E+05 L-glutamine 1.60E+05 1.85E+05 1.28E+05 1.03E+05 1.26E+05 1.93E+05 4.35E+05 3.99E+05 4.83E+05 1.18E+05 1.58E+05 1.42E+05 1.51E+05 1.21E+05 1.87E+05 5.47E+05 4.11E+05 4.61E+05 glycine 1.41E+06 1.24E+06 1.42E+06 1.35E+06 1.29E+06 1.35E+06 1.59E+06 1.29E+06 1.28E+06 1.41E+06 1.29E+06 1.37E+06 1.30E+06 1.36E+06 1.17E+06 1.31E+06 1.46E+06 1.50E+06 L-leucine 5.39E+05 1.72E+06 5.41E+05 4.61E+05 5.93E+05 8.80E+05 2.25E+06 2.44E+06 3.34E+06 5.86E+05 5.93E+05 5.44E+05 6.40E+05 5.37E+05 7.73E+05 1.99E+06 2.02E+06 2.42E+06 L-lysine 4.36E+04 6.37E+04 5.81E+04 5.96E+04 4.62E+04 6.87E+04 1.28E+06 8.60E+05 1.06E+06 4.56E+04 7.18E+04 4.37E+04 6.90E+04 6.33E+04 5.63E+04 6.91E+05 5.79E+05 7.75E+05 L-methionine 1.22E+04 1.34E+05 2.05E+04 1.41E+04 2.23E+04 2.60E+04 1.44E+05 1.21E+05 1.65E+05 2.01E+04 1.93E+04 1.63E+04 2.68E+04 1.43E+04 2.54E+04 1.43E+05 1.02E+05 1.52E+05 L-phenylalanine 8.89E+04 2.98E+05 8.77E+04 9.70E+04 1.16E+05 1.69E+05 2.34E+05 2.63E+05 3.42E+05 1.14E+05 1.23E+05 1.14E+05 1.16E+05 8.65E+04 1.29E+05 3.13E+05 2.47E+05 3.00E+05 L-proline 6.42E+05 8.51E+05 4.67E+05 5.09E+05 5.85E+05 8.45E+05 1.34E+06 1.19E+06 1.67E+06 5.57E+05 6.61E+05 4.62E+05 7.20E+05 6.75E+05 7.05E+05 1.73E+06 1.52E+06 1.42E+06 L-threonine 4.58E+04 3.77E+04 2.80E+04 1.49E+04 3.71E+04 5.18E+04 1.03E+05 8.97E+04 1.32E+05 2.74E+04 4.84E+04 2.37E+04 3.36E+04 3.93E+04 5.17E+04 1.32E+05 1.03E+05 1.16E+05 L-tryptophan 3.03E+05 4.17E+05 2.74E+05 2.30E+05 2.72E+05 4.99E+05 1.15E+06 1.32E+06 1.50E+06 3.64E+05 5.22E+05 3.43E+05 3.60E+05 2.62E+05 4.40E+05 1.48E+06 1.39E+06 1.65E+06 L-tyrosine 7.59E+04 2.58E+05 5.67E+04 5.99E+04 7.10E+04 1.11E+05 1.93E+05 1.95E+05 2.80E+05 7.30E+04 8.11E+04 7.30E+04 8.18E+04 5.09E+04 1.12E+05 2.53E+05 2.16E+05 2.76E+05 L-valine 9.92E+05 9.33E+05 8.32E+05 7.77E+05 8.03E+05 1.22E+06 1.00E+06 9.99E+05 1.38E+06 8.61E+05 9.98E+05 7.69E+05 7.80E+05 8.07E+05 1.01E+06 1.13E+06 7.98E+05 1.06E+06 Adenosine 1.23E+06 6.37E+06 1.24E+06 9.14E+05 1.19E+06 2.33E+06 7.25E+05 6.02E+05 1.29E+06 1.91E+06 8.46E+05 1.35E+06 1.99E+06 1.01E+06 2.42E+06 8.44E+05 1.64E+06 1.36E+06 Adenine 3.33E+04 2.48E+04 3.08E+04 3.28E+04 3.32E+04 4.43E+04 1.94E+04 3.28E+04 2.59E+04 2.94E+04 2.77E+04 5.08E+04 2.81E+04 3.17E+04 3.05E+04 2.08E+04 2.90E+04 4.07E+05 Guanosine 7.25E+03 7.01E+04 5.60E+03 0.00E+00 2.88E+03 2.16E+04 1.40E+04 9.41E+03 1.85E+04 1.12E+04 2.94E+03 4.15E+03 1.19E+04 2.00E+03 8.73E+03 2.02E+04 1.26E+04 2.72E+04 Guanine 1.90E+03 2.02E+04 2.31E+03 2.16E+03 1.89E+03 3.85E+03 1.09E+05 1.05E+05 1.11E+05 2.42E+03 2.04E+03 2.38E+03 4.37E+03 0.00E+00 2.55E+03 3.99E+04 3.51E+04 5.45E+04 Cytosine 5.19E+03 9.69E+03 2.38E+03 1.41E+04 7.55E+03 1.95E+04 4.61E+05 2.97E+05 5.78E+05 7.00E+03 9.83E+03 7.81E+03 1.32E+04 1.72E+04 1.25E+04 4.01E+05 2.31E+05 4.01E+05 Thymidine 5.98E+03 5.01E+04 1.07E+04 0.00E+00 4.17E+04 3.76E+04 2.36E+04 7.93E+03 8.99E+03 3.68E+03 1.37E+05 3.45E+03 2.14E+04 5.73E+03 7.91E+03 1.46E+04 2.34E+05 1.20E+04 Thymine 3.05E+04 3.05E+04 2.33E+04 2.66E+04 2.92E+04 3.28E+04 1.08E+05 9.65E+04 1.35E+05 2.72E+04 2.47E+04 2.69E+04 2.74E+04 3.46E+04 9.29E+04 6.59E+04 7.24E+04 7.25E+04 Uracil 1.68E+05 1.97E+05 8.41E+04 8.21E+04 1.30E+05 1.56E+05 1.67E+05 1.23E+05 1.28E+05 9.75E+04 1.13E+05 6.18E+04 1.93E+05 1.11E+05 5.76E+04 9.73E+04 4.82E+04 7.75E+04 Inosine 7.94E+03 1.03E+05 1.46E+04 2.15E+03 1.65E+04 9.38E+03 2.80E+04 3.62E+04 2.41E+04 1.68E+04 5.53E+03 4.16E+03 9.29E+03 2.51E+03 1.15E+04 3.13E+04 2.18E+04 3.30E+04 Hypoxanthine 5.58E+04 1.84E+05 6.57E+04 6.31E+04 6.90E+04 6.92E+04 4.38E+05 4.69E+05 4.52E+05 5.35E+04 6.35E+04 5.83E+04 6.43E+04 7.27E+04 7.09E+04 2.29E+05 2.09E+05 2.85E+05 Xanthine 7.95E+03 5.10E+04 1.10E+04 5.71E+03 9.38E+03 1.02E+04 2.75E+04 3.79E+04 4.06E+04 1.31E+04 7.10E+03 5.36E+03 8.29E+03 4.52E+03 1.22E+04 2.87E+04 3.82E+04 3.12E+04 3',5'-Cyclic IMP 1.23E+05 1.43E+05 1.49E+05 1.32E+05 1.38E+05 1.05E+05 1.24E+05 1.25E+05 1.12E+05 1.00E+05 7.97E+04 1.13E+05 1.12E+05 8.85E+04 1.08E+05 9.10E+04 8.64E+04 9.96E+04 Nicotinamide 3.76E+06 1.28E+07 3.80E+06 3.63E+06 3.65E+06 4.43E+06 4.90E+06 4.15E+06 5.19E+06 7.04E+06 4.51E+06 4.14E+06 5.80E+06 4.63E+06 5.02E+06 5.36E+06 5.76E+06 5.31E+06 Glutathione disulfide 3.21E+04 2.77E+05 3.92E+04 3.76E+04 5.07E+04 4.18E+04 6.90E+04 4.55E+04 5.82E+04 8.86E+04 5.49E+04 6.00E+04 8.92E+04 6.15E+04 6.14E+04 8.91E+04 7.15E+04 7.69E+04 5-Oxoproline 1.52E+05 2.40E+05 1.50E+05 1.28E+05 1.44E+05 2.03E+05 2.81E+05 2.35E+05 2.87E+05 1.52E+05 1.76E+05 1.69E+05 1.83E+05 1.61E+05 2.55E+05 2.67E+05 2.48E+05 2.42E+05 Phosphoserine 2.27E+05 3.64E+05 2.59E+05 2.09E+05 2.41E+05 2.92E+05 2.47E+05 2.92E+05 2.97E+05 2.33E+05 3.49E+05 1.56E+05 2.56E+05 2.20E+05 2.48E+05 2.39E+05 1.40E+05 1.69E+05 3-Phosphonooxypyruvate 3.55E+07 3.21E+07 3.99E+07 2.93E+07 2.87E+07 4.26E+07 3.58E+07 3.34E+07 3.20E+07 3.14E+07 2.94E+07 3.54E+07 3.20E+07 2.78E+07 3.29E+07 3.47E+07 3.36E+07 3.20E+07 Ornithine 4.14E+04 4.67E+04 3.52E+04 2.55E+04 3.60E+04 5.29E+04 1.96E+05 1.67E+05 1.24E+05 2.96E+04 4.40E+04 2.66E+04 3.47E+04 2.72E+04 3.93E+04 1.67E+05 1.64E+05 1.48E+05 L-Citrulline 2.37E+05 2.20E+05 2.02E+05 1.96E+05 1.89E+05 4.64E+05 4.03E+05 2.61E+05 4.06E+05 7.62E+04 1.36E+05 1.47E+05 1.23E+05 1.02E+05 1.96E+05 2.38E+05 1.61E+05 2.37E+05 Putrescine 1.18E+06 1.30E+06 9.59E+05 1.08E+06 8.85E+05 1.12E+06 1.07E+06 9.16E+05 9.23E+05 8.85E+05 8.98E+05 7.96E+05 8.65E+05 9.21E+05 6.82E+05 1.22E+06 1.11E+06 8.39E+05 Spermidine 8.33E+06 7.04E+06 9.05E+06 1.45E+07 7.97E+06 6.71E+06 8.53E+06 8.96E+06 1.00E+07 8.95E+06 8.52E+06 7.74E+06 1.20E+07 8.67E+06 6.42E+06 1.04E+07 1.47E+07 1.17E+07 Spermine 6.50E+05 6.07E+05 9.01E+05 1.29E+06 7.61E+05 5.99E+05 9.59E+05 1.17E+06 1.32E+06 6.09E+05 6.55E+05 5.64E+05 7.53E+05 5.80E+05 3.99E+05 1.24E+06 1.32E+06 2.24E+06 D-Glucosamine 4.81E+04 1.44E+05 6.85E+04 8.07E+04 8.65E+04 7.48E+04 5.85E+04 7.69E+04 4.54E+04 4.52E+04 3.86E+04 6.80E+04 5.99E+04 4.90E+04 5.36E+04 6.06E+04 7.15E+04 8.82E+04 Creatine 1.05E+06 1.36E+06 1.01E+06 8.20E+05 7.24E+05 1.72E+06 8.62E+05 9.31E+05 1.28E+06 2.57E+06 2.70E+06 2.80E+06 2.58E+06 2.75E+06 3.31E+06 3.46E+06 3.41E+06 3.55E+06 4-Acetamidobutanoate 9.62E+04 1.51E+05 9.00E+04 9.76E+04 9.51E+04 1.26E+05 1.91E+05 1.01E+05 1.84E+05 8.60E+04 9.83E+04 1.04E+05 1.34E+05 1.03E+05 1.31E+05 1.61E+05 1.72E+05 2.41E+05 trans-4-Hydroxy-L-proline 1.38E+05 1.22E+05 9.88E+04 9.45E+04 1.03E+05 1.37E+05 1.22E+05 1.12E+05 9.43E+04 1.15E+05 1.30E+05 1.04E+05 1.28E+05 1.21E+05 1.07E+05 1.26E+05 1.13E+05 1.22E+05 Pantothenol 3.14E+04 8.65E+04 2.39E+04 2.75E+04 2.64E+04 2.65E+04 3.41E+04 2.93E+04 3.48E+04 3.19E+04 3.27E+04 2.92E+04 3.82E+04 3.48E+04 3.45E+04 6.00E+04 4.72E+04 4.69E+04 L-Methionine S-oxide 4.07E+03 4.71E+04 8.80E+03 1.14E+04 1.39E+04 1.52E+04 5.13E+04 7.47E+04 8.82E+04 1.43E+04 6.77E+03 9.78E+03 1.07E+04 1.24E+04 1.27E+04 4.92E+04 5.39E+04 5.48E+04 Picolinic acid 6.52E+04 1.59E+05 5.17E+04 4.41E+04 1.22E+05 5.32E+04 5.34E+04 6.73E+04 5.45E+04 6.27E+04 7.01E+04 6.40E+04 5.60E+04 5.45E+04 6.24E+04 6.00E+04 6.80E+04 1.61E+05 Sphingosine 1.43E+04 1.06E+04 4.31E+04 1.03E+04 3.06E+04 3.40E+04 8.11E+03 1.49E+04 3.37E+04 2.33E+04 2.98E+04 1.34E+04 1.71E+04 1.26E+04 1.96E+04 2.06E+04 1.27E+04 2.69E+04 Acetylcholine 5.47E+05 4.40E+05 3.79E+05 4.11E+05 3.81E+05 4.83E+05 4.47E+05 4.05E+05 4.82E+05 3.21E+05 4.52E+05 3.34E+05 4.84E+05 5.64E+05 4.13E+05 6.41E+05 4.49E+05 3.85E+05 Choline 6.76E+06 7.43E+06 6.68E+06 6.09E+06 5.05E+06 8.95E+06 8.38E+06 6.48E+06 7.88E+06 1.03E+07 1.12E+07 1.03E+07 1.23E+07 1.13E+07 1.09E+07 1.24E+07 1.15E+07 1.14E+07 Triacanthine 9.09E+05 6.46E+06 8.55E+05 9.68E+05 9.77E+05 1.17E+06 1.20E+06 7.96E+05 9.43E+05 9.10E+05 6.91E+05 8.14E+05 1.12E+06 1.03E+06 9.48E+05 8.49E+05 9.54E+05 6.73E+05 6-Lactoyl-5-6-7-8-tetrahydropterin 3.33E+04 8.48E+04 3.01E+04 4.48E+04 4.17E+04 5.05E+04 5.06E+04 4.96E+04 8.08E+04 4.56E+04 3.18E+04 2.47E+04 4.26E+04 4.40E+04 3.51E+04 5.58E+04 5.71E+04 5.50E+04 Fe(III)dicitrate 1.29E+05 1.64E+05 1.49E+05 1.42E+05 1.47E+05 1.54E+05 1.52E+05 1.13E+05 1.47E+05 1.21E+05 1.55E+05 1.69E+05 1.46E+05 1.40E+05 1.55E+05 1.54E+05 1.75E+05 1.23E+05 Pyridoxamine 0.00E+00 7.54E+03 1.34E+04 1.10E+04 1.40E+04 3.63E+03 1.21E+05 1.24E+05 1.64E+05 1.50E+04 1.13E+04 1.72E+04 7.89E+03 1.64E+04 7.48E+03 1.01E+05 9.94E+04 5.27E+04 L-Noradrenaline 1.48E+04 1.89E+04 1.46E+04 1.67E+04 7.00E+03 1.32E+04 1.60E+04 1.61E+04 1.69E+04 1.32E+04 1.78E+04 1.80E+04 1.74E+04 1.40E+04 1.68E+04 1.09E+04 1.36E+04 2.49E+04 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. L-arginine C00062 175.1190 0.64 L-cysteine C00097 122.0272 0.68 L-glutamate C00025 148.0604 0.67 L-glutamine C00064 147.0764 0.67 glycine C00037 76.0402 1.87 L-leucine C00123 132.1020 0.99 L-lysine C00047 147.1128 0.61 L-methionine C00073 150.0584 0.89 L-phenylalanine C00079 166.0864 1.39 L-proline C00148 116.0710 0.68 L-threonine C00188 120.0658 0.67 L-tryptophan C00078 205.0972 1.72 L-tyrosine C00082 182.0813 0.92 L-valine C00183 118.0866 0.68 Adenosine C00212 268.1039 0.91 Adenine C00147 136.0618 0.26 Guanosine C00387 284.0988 0.94 Guanine C00242 152.0567 0.97 Cytosine C00380 112.0509 0.69 Thymidine C00214 243.0991 1.74 Thymine C00178 127.0504 1.27 Uracil C00106 113.0361 0.59 Inosine C00294 269.0879 0.95 Hypoxanthine C00262 137.0459 1.05 Xanthine C00385 153.0407 0.89 3',5'-Cyclic IMP C00943 331.0447 0.97 Nicotinamide C00153 123.0555 0.91 Glutathione disulfide C00127 613.1587 0.89 5-Oxoproline C01879 130.0500 0.69 Phosphoserine C02532 186.0186 0.59 3-Phosphonooxypyruvate C03232 184.9855 3.79 Ornithine C01602 133.0972 0.61 L-Citrulline C00327 176.1029 0.67 Putrescine C00134 89.1080 0.61 Spermidine C00315 146.1652 0.61 Spermine C00750 203.2230 0.60 D-Glucosamine C00329 180.0866 0.90 Creatine C00300 132.0769 0.67 4-Acetamidobutanoate C02946 146.0812 1.08 trans-4-Hydroxy-L-proline C01157 132.0656 0.71 Pantothenol C00864 220.1180 1.65 L-Methionine S-oxide C02989 166.0533 0.88 Picolinic acid C10164 124.0395 0.23 Sphingosine C00319 300.2896 2.74 Acetylcholine C01996 146.1175 0.72 Choline C00114 104.1075 0.66 Triacanthine C08435 204.1231 0.89 6-Lactoyl-5-6-7-8-tetrahydropterin C04244 240.1090 0.89 Fe(III)dicitrate C06229 245.9450 0.86 Pyridoxamine C00534 169.0961 0.60 L-Noradrenaline C00547 170.0811 0.95 METABOLITES_END #END