#METABOLOMICS WORKBENCH juliehaines_20240719_093451 DATATRACK_ID:5025 STUDY_ID:ST003331 ANALYSIS_ID:AN005456 PROJECT_ID:PR002070 VERSION 1 CREATED_ON July 19, 2024, 2:42 pm #PROJECT PR:PROJECT_TITLE Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem PR:PROJECT_TITLE Cell-Derived Model of Multiple Sclerosis PR:PROJECT_SUMMARY Senescent neural progenitor cells have been identified in brain lesions of PR:PROJECT_SUMMARY people with progressive multiple sclerosis (PMS). However, their role in disease PR:PROJECT_SUMMARY pathobiology and contribution to the lesion environment remains unclear. By PR:PROJECT_SUMMARY establishing directly induced neural stem/progenitor cell (iNSC) lines from PMS PR:PROJECT_SUMMARY patient fibroblasts, we studied their senescent phenotype in vitro. Senescence PR:PROJECT_SUMMARY was strongly associated with inflammatory signaling, hypermetabolism, and the PR:PROJECT_SUMMARY senescence associated secretory phenotype (SASP). PMS-derived iNSCs displayed PR:PROJECT_SUMMARY increased glucose-dependent fatty acid and cholesterol synthesis, which resulted PR:PROJECT_SUMMARY in the accumulation of cholesteryl ester-enriched lipid droplets. An HMG-CoA PR:PROJECT_SUMMARY reductase-mediated lipogenic state was found to induce secretion of the SASP in PR:PROJECT_SUMMARY PMS iNSC conditioned media via transcriptional regulation by PR:PROJECT_SUMMARY cholesterol-dependent transcription factors. SASP from PMS iNSCs induced PR:PROJECT_SUMMARY neurotoxicity. Chemical targeting of HMG-CoA reductase using the PR:PROJECT_SUMMARY cholesterol-lowering drug simvastatin (SV) reprogrammed the SASP and rescued PR:PROJECT_SUMMARY neurotoxicity. Our findings suggest a disease-associated, cholesterol-related, PR:PROJECT_SUMMARY hypermetabolic phenotype of PMS iNSCs that leads to neurotoxic signaling and is PR:PROJECT_SUMMARY rescuable pharmacologically. PR:INSTITUTE University of Colorado Denver PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Stefano Pluchino (Univ PR:LABORATORY of Cambridge) 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 Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem ST:STUDY_TITLE Cell-Derived Model of Multiple Sclerosis - cellular metabolomics ST:STUDY_SUMMARY Fibroblasts from skin biopsies from patients with progressive multiple sclerosis ST:STUDY_SUMMARY (PMS) and age-matched controls (AMC) were derived and reprogrammed to yield ST:STUDY_SUMMARY induced neural stem cells (iNSCs). The resulting iNSCs were cultured in the ST:STUDY_SUMMARY presence of 13C6 glucose for 24h to assess changes in the intracellular ST:STUDY_SUMMARY metabolome, in particular changes in the flux of glucose-derived carbon into the ST:STUDY_SUMMARY TCA cycle along with steady state levels of compounds in other areas of the ST:STUDY_SUMMARY metabolome. 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 Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Female #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 A DN47-01 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-01_r20- SUBJECT_SAMPLE_FACTORS A DN47-02 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-02_r19- SUBJECT_SAMPLE_FACTORS A DN47-03 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-03_r15- SUBJECT_SAMPLE_FACTORS B DN47-04 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-04_r8- SUBJECT_SAMPLE_FACTORS B DN47-05 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-05_r10- SUBJECT_SAMPLE_FACTORS B DN47-06 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-06_r13- SUBJECT_SAMPLE_FACTORS C DN47-10 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-10_r16- SUBJECT_SAMPLE_FACTORS C DN47-11 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-11_r3- SUBJECT_SAMPLE_FACTORS C DN47-12 Disease factor:AMC | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-12_r12- SUBJECT_SAMPLE_FACTORS D DN47-13 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-13_r11- SUBJECT_SAMPLE_FACTORS D DN47-14 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-14_r1- SUBJECT_SAMPLE_FACTORS D DN47-15 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-15_r17- SUBJECT_SAMPLE_FACTORS E DN47-16 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-16_r2- SUBJECT_SAMPLE_FACTORS E DN47-17 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-17_r5- SUBJECT_SAMPLE_FACTORS E DN47-18 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-18_r4- SUBJECT_SAMPLE_FACTORS F DN47-19 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-19_r9- SUBJECT_SAMPLE_FACTORS F DN47-20 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-20_r21- SUBJECT_SAMPLE_FACTORS F DN47-21 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-21_r18- SUBJECT_SAMPLE_FACTORS G DN47-22 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-22_r7- SUBJECT_SAMPLE_FACTORS G DN47-23 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-23_r24- SUBJECT_SAMPLE_FACTORS G DN47-24 Disease factor:PMS | Sample source:induced neural stem cell RAW_FILE_NAME(raw file name)=DN47-24_r6- #COLLECTION CO:COLLECTION_SUMMARY 3-5 mm skin biopsies were collected in Biopsy Collection Medium (RPMI 1460 CO:COLLECTION_SUMMARY [Thermo Fisher] with 1X Antibiotic-Antimycotic [Thermo Fisher]), cut into CO:COLLECTION_SUMMARY smaller pieces (<1 mm) and plated onto a TC-treated 35 mm dish in Biopsy Plating CO:COLLECTION_SUMMARY Medium, composed by Knockout DMEM (Thermo Fisher), 2 mM GlutaMax (Thermo CO:COLLECTION_SUMMARY Fisher), 0.1 mM non-essential amino acids (Thermo Fisher), 0.1 mM CO:COLLECTION_SUMMARY β-Mercaptoethanol (Thermo Fisher), 10% Fetal Bovine Serum (FBS) (Thermo CO:COLLECTION_SUMMARY Fisher), 1X Penicillin-Streptomycin (P/S) (Thermo Fisher) and 1% Nucleosides CO:COLLECTION_SUMMARY (Millipore). Once the first fibroblasts migrated out of the biopsies, the CO:COLLECTION_SUMMARY cultures were maintained in growth medium (DMEM Glutamax I [Thermo Fisher] CO:COLLECTION_SUMMARY supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1 mM CO:COLLECTION_SUMMARY sodium pyruvate (Thermo Fisher) at 37°C with 5 % CO2 and fed every 3-4 days. CO:COLLECTION_SUMMARY After reaching 90% confluency the fibroblasts were detached with trypsin-EDTA CO:COLLECTION_SUMMARY 0.05% for 5 min followed by neutralization in DMEM and spun down at 300xg for 5 CO:COLLECTION_SUMMARY min. They were split 1:4 into growth media. Fibroblasts at passage 3-5 were CO:COLLECTION_SUMMARY reprogrammed to iPSCs using the integration free technology based on CO:COLLECTION_SUMMARY non-modified RNA plus microRNA (kit from REPROCELL, formerly Stemgent), CO:COLLECTION_SUMMARY following manufacturer’s instructions. About 25x10^3 fibroblasts/well were CO:COLLECTION_SUMMARY plated onto Matrigel-coated 12-well plates in culture medium for 24 hours and CO:COLLECTION_SUMMARY then in NuFF-conditioned Pluriton reprogramming medium with B18R. Cells were CO:COLLECTION_SUMMARY transfected for 11 consecutive days using Stemfect as following: day 0 microRNA CO:COLLECTION_SUMMARY only, days 1-3 RNA only, day 4 microRNA plus RNA, days 5-11 RNA only. From day CO:COLLECTION_SUMMARY 11, TRA-1-60+ colonies (live stained) were manually picked and re-plated on CO:COLLECTION_SUMMARY mouse embryonic fibroblasts in HUESM medium (Knockout-DMEM, 20% knock-out serum, CO:COLLECTION_SUMMARY glutamax 2mM, NEAA 0.1mM, 1X P/S and β-mercaptoethanol 0.1mM [Thermo Fisher]). CO:COLLECTION_SUMMARY Pluripotent colonies were passaged and adapted to feeder-free conditions with CO:COLLECTION_SUMMARY hESC matrigel (Corning) and mTeSR1 (STEMCELL Technologies) medium. The WIBJ iPSC CO:COLLECTION_SUMMARY line, obtained from Cambridge BioResource as a gift from Alessandra Granada, was CO:COLLECTION_SUMMARY reprogrammed from fibroblasts using the CytoTune 1 non-integrating kit (Thermo CO:COLLECTION_SUMMARY Fisher). iPSC media was changed every day. When confluent, cells were lifted CO:COLLECTION_SUMMARY using accutase (Thermo Fisher), spun at 300xg for 5 min, and split 1:10-1:20 CO:COLLECTION_SUMMARY onto hESC-matrigel coated plates with Y-27632 (10 uM) (Thermo Fisher) in mTeSR1 CO:COLLECTION_SUMMARY media. To generate directly induced neural stem cells (iNSCs) from fibroblasts CO:COLLECTION_SUMMARY we used a nonintegrating Sendai virus-based direct conversion strategy, as CO:COLLECTION_SUMMARY previously described (https://doi.org/10.3791/52831). Briefly, fibroblasts were CO:COLLECTION_SUMMARY seeded at 75,000/well in fibroblast media in a non-coated 12-well. On the next CO:COLLECTION_SUMMARY day the fibroblasts were transduced using the CytoTune-iPS 2.0 Sendai CO:COLLECTION_SUMMARY Reprogramming Kit (Thermo Fisher) with hKOS (MOI: 3), hc-Myc (MOI: 3), hKlf4 CO:COLLECTION_SUMMARY (MOI: 3). The day following transfection, the medium was switched to neural CO:COLLECTION_SUMMARY induction medium (NIM) [DMEM:F12 and Neurobasal (1:1), supplemented with N2 CO:COLLECTION_SUMMARY supplement (1x, ThermoFisher), 1% glutamax, B27 supplement (1x, ThermoFisher), CO:COLLECTION_SUMMARY CHIR99021 (3 µM, Cell Guidance Systems), SB-431542 (2 µM, Cayman Chemicals) CO:COLLECTION_SUMMARY and hLIF (10 ng/ml, Cell Signaling Technology)], and cells were moved to 39°C CO:COLLECTION_SUMMARY with 5% CO2 to achieve viral clearance over 14 days. A few samples were CO:COLLECTION_SUMMARY collected here to generate positive controls for quality control assays. Medium CO:COLLECTION_SUMMARY changes were performed every other day. Following 25 days of transfection, iNSC CO:COLLECTION_SUMMARY colonies were manually selected, seeded onto Growth Factor Reduced (GFR) CO:COLLECTION_SUMMARY Matrigel Matrix (1:20 in DMEM/F12) coated plates for expansion, and subjected to CO:COLLECTION_SUMMARY quality control assays. iNSCs were maintained in NIM media until 70% confluent, CO:COLLECTION_SUMMARY then lifted using accutase (ThermoFisher), spun at 300xg for 3 mins, and plated CO:COLLECTION_SUMMARY onto GFR-Matrigel coated plates with Y-27632 (10 µM, Miltenyi Biotec) between CO:COLLECTION_SUMMARY 1:3-1:5 in NIM media. Media was changed every second day as needed. Experiments CO:COLLECTION_SUMMARY were performed on cells from passages 20-40. CO:SAMPLE_TYPE Stem cells #TREATMENT TR:TREATMENT_SUMMARY iNSCs were seeded in NIM with Y-27632 (10 µM) at a density of 100,000 cells/cm2 TR:TREATMENT_SUMMARY GFR-matrigel coated wells, in technical triplicates per line. Medium was changed TR:TREATMENT_SUMMARY the next day. Glucose tracer experiments were run on day 2 after seeding by TR:TREATMENT_SUMMARY removing the culture medium and adding glucose-free NIM [DMEM:F12 w/o glucose TR:TREATMENT_SUMMARY and Neurobasal (1:1), supplemented with N2 supplement (1x, ThermoFisher), TR:TREATMENT_SUMMARY Glutamax (Thermo Fisher, 3.2 mM), pyruvate (Agilent, 0.35 mM), B27 supplement TR:TREATMENT_SUMMARY (1x, ThermoFisher), CHIR99021 (3 µM, Cell Guidance Systems), SB-431542 (2 µM, TR:TREATMENT_SUMMARY Cayman Chemicals) and hLIF (10 ng/ml, Cell Signaling Technology)] supplemented TR:TREATMENT_SUMMARY with D-Glucose-13C6 (ThermoFisher, 20.4 mM). Samples were collected after 24 TR:TREATMENT_SUMMARY hours for optimal resolution into TCA cycle and anabolic metabolic pathways. TR:TREATMENT_SUMMARY Sample collection was performed on ice. Conditioned culture medium was TR:TREATMENT_SUMMARY collected, spun at 300 x g for 5 minutes to remove cellular debris and 100 µL TR:TREATMENT_SUMMARY was stored at -80 oC until sample processing. Cells were then lifted using TR:TREATMENT_SUMMARY accutase and spun at 300 x g for 5 mins. Cell pellets were resuspended in PBS TR:TREATMENT_SUMMARY for counting and spun once more at 300 x g for 5 mins. Supernatants were TR:TREATMENT_SUMMARY aspirated and cell pellets were stored at -80oC until sample processing. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from frozen pellets were extracted at 2e6 cells per mL using ice SP:SAMPLEPREP_SUMMARY cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 SP:SAMPLEPREP_SUMMARY degrees C followed by centrifugation as described for 10 min at 18,000 g at 4°C SP:SAMPLEPREP_SUMMARY and stored at −80°C until analysis. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Negative C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 95% water/5% acetonitrile; 1 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water; 1 mM ammonium acetate CH:FLOW_GRADIENT 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min CH:FLOW_GRADIENT 100-0% B, 3-5 min hold at 0% B CH:FLOW_RATE 450 uL/min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 6 uL #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 Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, MS:MS_COMMENTS microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, MS:MS_COMMENTS capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas MS:MS_COMMENTS 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples DN47-01 DN47-02 DN47-03 DN47-04 DN47-05 DN47-06 DN47-10 DN47-11 DN47-12 DN47-13 DN47-14 DN47-15 DN47-16 DN47-17 DN47-18 DN47-19 DN47-20 DN47-21 DN47-22 DN47-23 DN47-24 Factors Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:AMC | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell Disease factor:PMS | Sample source:induced neural stem cell (R)-Mevalonate 274664 155872 200378 304880 197026 202644 47485 93119 96538 319364 247064 398360 106605 83078 208851 212549 300067 309747 147024 123640 208106 phosphomevalonate M-2H 188000 118848 129328 82799 79941 87512 26372 12088 9124 203387 122473 247897 126209 242001 432644 316305 464092 449987 433846 571710 442554 p-mevalonate 13C4 M-2H 57928 32483 40008 36657 29802 33761 40898 11309 37058 81930 85700 76365 43881 80298 103722 172149 160701 145747 199326 203629 184850 D-Glucose 10432650 4269266 12625460 3701224 9785642 11264890 2490162 2112911 2504775 12784740 10361170 18595260 2926517 11955980 8846607 12373390 15840700 16287500 14278690 11205940 4467104 Glucose 13C6 1274698 537206 1579403 686010 1828382 2921663 375672 319067 396969 1983640 1750889 3112036 322627 1388814 960203 2067853 2569074 2593382 1825257 1421538 1115650 Citrate 3173608 2508989 2679444 2801346 2184873 1737625 1765746 2802835 1539488 3581959 2790820 3472414 3171600 3331233 5306492 4096498 3860084 3812635 4975650 5106736 5371030 Citrate 13C2 2306282 1985132 2101457 2241574 1539150 1299593 551833 486092 420877 3187049 2845866 3372750 3559409 4646384 8092642 6903208 6908136 7189344 10206740 9969467 10997580 Citrate 13C3 657953 613930 604470 586008 413848 322117 204029 180154 200766 1467324 1219332 1394600 743636 914309 1618342 2016310 1840246 1985825 2129697 2329399 2245234 Citrate 13C4 675599 488122 545200 615609 404252 362288 199793 255913 110145 1485834 1185946 1641198 731277 1076380 1933404 3087334 2671944 2926776 3308978 3034544 3310677 Citrate 13C5 548074 416388 445456 454405 296607 258469 82533 63510 94702 1266158 1132933 1260062 705417 839564 1549837 2459884 2192921 2281295 2402578 2272561 2385476 2-Oxoglutarate 2572072 1649446 1329410 1910474 1340522 738119 739995 465448 655702 3053020 3163578 2991175 388159 390405 773533 1486550 1557358 1373636 1001182 960581 357800 2-Oxoglutarate 13C2 746839 477518 372390 526796 350567 216256 54521 73389 118950 1051488 1117527 1147646 87254 102157 166967 450912 527248 322082 260264 311529 98903 2-Oxoglutarate 13C3 207119 78450 117523 125655 82695 60228 10937 10624 12953 499687 611097 540254 33906 12389 44719 183424 214706 191533 104010 91501 46595 2-Oxoglutarate 13C4 272554 145208 95894 179452 91954 39560 9244 7088 13271 635804 693162 701623 38322 30363 52847 285404 242182 228135 127745 90591 51997 2-Oxoglutarate 13C5 152708 161680 90684 98961 62888 42121 14199 12975 26917 380601 461907 421155 13724 10360 24399 145789 171104 114685 42902 77734 19486 Itaconate 956563 621735 615860 3647438 2339418 2220223 1089145 859790 747548 1666886 1329467 1640058 302114 254641 450569 250939 214931 228372 256152 245080 357355 Itaconate 13C2 291832 242992 170078 785340 501952 471938 139256 141328 131146 932218 673358 877788 92339 83396 133505 97931 120458 143208 158125 151797 155975 cis-Aconitate 138743 70865 110589 258764 213166 176915 83583 76861 103248 456104 444264 509603 72173 63478 101383 68328 64032 71048 73876 127806 120307 cis-Aconitate 13C2 65726 14848 31478 55082 23165 28171 10486 11433 14166 53588 45038 74150 83791 96780 146183 119481 114541 128593 197891 204468 237932 cis-Aconitate 13C3 61373 39315 14159 82357 56930 55080 11594 12839 31734 113595 134515 163899 17246 11777 19365 35735 40771 41487 33104 35506 114062 cis-Aconitate 13C4 12301 7301 6502 7728 0 429 4508 0 5483 26633 12159 42081 11014 8103 23662 44326 50909 36885 66380 68389 63845 Succinate 3127445 900285 592365 938050 1578727 465206 590546 464218 892259 1250036 783049 1214046 456045 811251 1223549 2071242 2596413 1763906 2083841 6064066 3346627 Succinate 13C2 546903 346528 138898 215422 235742 58124 30120 20787 79336 396313 213238 281352 93865 79441 193288 472383 589728 575957 438246 1336502 708781 Succinate 13C3 201704 175601 41014 92351 99015 27651 9360 10236 41555 288517 133865 258752 12924 32039 48501 334020 345124 300061 297601 640625 356573 Succinate 13C4 263110 209097 67718 88730 108897 48653 27728 17416 41756 261501 232740 237951 87467 74065 76976 331964 289268 281493 216706 519632 263672 Fumarate 4117905 2704513 2499136 2583038 1661160 1705854 1914305 1895191 2111304 791904 662113 1045328 1462324 1342247 2255266 1313627 1308192 1564104 1929015 2666976 2573122 Fumarate 13C2 832807 566434 404798 399379 350446 355608 96750 125490 143492 223606 193181 315832 329196 319791 519554 451312 424580 517936 505628 793930 707695 Fumarate 13C3 748130 581009 500965 465447 303077 325797 236291 256567 283571 242455 229266 344687 301403 177222 392450 371564 347420 377402 416401 509650 535269 Fumarate 13C4 170216 106159 89793 88392 46830 62633 11634 8529 14375 77865 102765 117602 60925 53425 106948 122420 144060 155632 134126 184961 87975 (S)-Malate 25151870 18070070 18186100 16483920 11446820 11083990 11425800 10727080 11823200 4453860 4427052 6588070 10985710 9423269 16509000 8558718 8900613 9647045 12840780 17389900 19009950 Malate 13C2 5434674 4007521 3947985 3455190 2197543 2444403 732264 800169 911825 1468971 1397725 2212841 2360513 1973637 3508219 2805281 3039213 3332356 3844052 5154968 5479750 Malate 13C3 6153680 4217794 4274576 3236372 2147799 2188569 1676561 1760346 1982498 1785916 1863566 2601557 2037048 1634925 3000955 2718714 2594276 2957440 3116376 4092078 4323704 Malate 13C4 1413705 1039774 1037163 825704 624928 588163 147152 149898 157881 852853 886159 1152431 635876 592804 974933 1580893 1436956 1526427 1225506 1610335 1778319 L-alanine 4917993 2388743 4256220 1623625 2968536 3559480 2195390 3090799 2903502 2308341 1789150 3483460 3611014 6729916 3608402 6725414 6750295 5263099 8080342 6439522 6654048 L-asparagine 1129420 386436 495142 312143 515742 436290 203762 297789 194380 764478 790771 809901 725084 1709881 1009651 1337964 1499089 1485955 2476193 1841503 2231971 L-aspartate 6392714 4276461 4275224 4584278 3984714 4216389 5867527 6584828 5569970 1335167 1342785 1650361 5719948 12167117 6465356 2863204 3468609 3118067 5761640 5268778 5272421 L-cysteine 48205 25024 42465 13930 42344 37501 4980 5183 18103 34008 47671 54744 52901 156427 53511 150101 146601 127929 177141 157050 148510 L-glutamate 71528024 52053284 55269648 56893396 55573152 48039412 45823296 51052516 44306728 53822144 57307956 60246896 61819045 140652619 83625112 61934484 67263880 63886976 81941192 69772152 72829864 L-glutamine 20596890 18082774 31656078 5041599 20498422 24830570 11217211 12913688 4953186 9625748 9513490 12967288 10429495 20469440 11621943 158508 375661 414624 2044330 1733470 1774583 L-threonine 6310332 2700249 3992036 3277713 2951456 3095992 1558085 2259319 2910243 3455424 3311211 5184218 4259994 9521212 5327664 5951000 8033090 7615378 9530417 9661571 9032260 ATP 1956613 1484100 1570387 1772035 1652690 1680294 1490680 2232308 1209154 3042576 5404737 3483957 1857287 2546764 1556501 2496372 2048824 2065932 2488118 1824440 1889947 ADP 1456950 941426 992575 1008839 1100518 1192845 876536 1180043 657129 3289363 4165356 4577515 1359739 2548741 1644581 2698963 2517110 2779884 3853957 3168533 4374900 Adenosine 38994 35966 74674 37427 76735 105131 34305 45892 18711 131119 136521 142850 77826 197722 80952 88509 94015 105004 108956 130788 103468 GDP 268566 164204 138359 257411 194173 175160 150436 249284 228691 547746 688994 507303 206761 349117 218141 410284 317981 390590 328273 224605 296658 GMP 28258 26595 12145 14081 22559 33656 17698 76786 8245 562122 359266 438511 52616 104148 131469 410471 515475 333428 267883 269406 263230 CDP 591820 373929 365344 483707 434965 410122 183862 271749 260893 197735 271799 290378 338878 558414 397041 547720 491821 501270 587190 570198 610751 CMP 66882 11565 8838 40418 20893 25043 12975 25266 13979 59734 54086 78778 28878 119880 105871 155250 172284 127851 151667 220702 280180 Thymidine 66259 57216 55352 22576 50568 54278 25284 39513 24534 49012 51187 74928 42432 130909 48407 60902 87429 92727 75531 56079 45301 Thymine 457703 157037 92226 192176 160480 85718 71451 123199 190378 198277 98840 101501 307003 95632 132769 69995 168545 57684 149269 216930 133103 UTP 528610 398226 384081 428422 507416 450663 199435 285513 270498 64768 118719 89854 233590 250446 224037 198757 213452 221700 388931 324368 312638 UMP 95289 23555 12041 78875 43838 38993 33952 68677 41923 70804 35461 81094 138155 156415 175725 208710 336669 136801 342860 344045 613887 Inosine 60235 28509 45946 28749 41781 39567 35403 30615 15510 77295 109233 75909 17477 20720 24871 86763 100135 82095 54612 60353 74176 3',5'-Cyclic AMP 713598 183311 146848 612093 525587 281589 198708 363891 319920 432927 351475 446752 1541976 1751154 2453040 1380107 1494933 1151205 1866059 2152826 2846591 Adenylyl sulfate 5022 7317 7897 17542 2055 5177 0 0 0 10147 13946 17008 17673 27895 13673 39030 22210 41494 36946 35202 45873 Adenylosuccinic acid 23398 11861 15946 10827 5462 9614 39980 32786 52800 4595 2411 2741 4003 3618 3114 5199 1968 0 6493 4495 7893 UDP-glucose 83604 55804 44437 99855 63435 80080 25305 40096 39996 9233 23506 11180 18693 72562 45274 55109 38414 54726 130349 55431 143646 NADPH 23822 24537 6288 9108 11396 10969 3368 11082 20278 34561 44399 34064 26177 51929 35187 29876 34241 44286 43369 43457 58209 Phosphate 181169424 175637360 170610096 196134928 163863168 151167312 197881776 182841488 239745552 129147432 130114664 167359536 424576365 256774877 142346640 134951568 153860016 163690176 146091600 160864048 157773056 Diphosphate 22936710 26467230 25475960 27461986 22331208 19584922 35527116 26072134 42389604 18120398 14398549 20850974 79291475 29671814 20509066 20866864 22211912 22993248 20116058 25256250 22760110 D-Glucose 6-phosphate 417123 551528 859557 1406643 631606 991571 386507 567458 505031 629269 480728 813666 506798 816737 593156 544901 440916 529208 622190 669521 724383 D-Fructose 1-6-bisphosphate 653807 610144 509232 754240 660366 558334 480481 546132 661811 648109 639093 910493 640057 1051420 512280 488875 443530 538031 748155 723645 683753 2/3-Phospho-D-glycerate 333753 180214 167366 185715 139869 79386 145000 112686 290350 218504 148952 234643 189814 300451 237266 368434 355577 305018 222212 221724 261264 Pyruvate 212398 243965 264921 160647 189739 215730 127503 126732 117164 279063 275205 319776 286026 335051 224090 277269 342261 252411 272041 204514 269351 D-Arabitol 4038121 2747534 3011614 2696003 2135221 2299912 716442 849824 756083 7374903 8112482 8171836 2404392 5756238 3303315 7911246 7821672 6923732 5242051 4462836 4594169 2-Oxoglutaramate 298674 308635 190786 163080 224654 209708 198256 216936 117805 406397 398137 375016 534577 483866 473018 381578 345006 338959 468323 435827 435746 2-Hydroxyglutarate/Citramalate 4321345 2754247 2588430 12847177 9595982 10124495 4522706 3236879 3786675 7145740 5786492 6289150 2483247 1743693 1422977 521511 460105 551553 785889 820964 916856 Sedoheptulose 1-phosphate 59805 43221 29471 52270 59959 62431 60645 83791 89469 44651 73298 81195 99554 122121 94836 70475 49981 59060 72703 92771 73560 alpha-D-Ribose 1-phosphate 97359 46941 33322 92015 59466 77005 58490 87218 50077 164713 139143 180091 64687 72128 37229 67174 96779 36998 86452 99754 80561 5-Oxoproline 28980950 11184027 14532657 7706534 11671641 11385089 7869068 10138307 5984323 42877580 41859020 47506740 27430357 35043825 27509994 48752512 45434540 46904352 39633132 40069412 40561476 Ascorbate 68876 33073 40092 77226 77013 70332 44762 31788 15288 75184 58652 76550 81638 103731 71554 63031 110676 68202 120127 129178 108326 Dehydroascorbate 138743 59713 110589 118687 100320 176915 54250 71922 91045 292843 49898 509603 166923 139276 101383 62554 64032 71048 73876 127806 120307 gamma-Glutamyl-Se-methylselenocysteine 21488 13966 21462 26620 10166 13103 33640 21600 17566 12427 3167 9609 13903 17535 18275 11054 19056 19788 19865 15231 14830 gamma-L-Glutamyl-D-alanine 78280 34921 71293 21082 47649 25904 22364 30500 4677 162616 159572 146708 176516 176792 84833 119603 133623 153621 201329 163826 292076 Cystathionine 482494 232618 398924 602191 822005 682350 172918 209130 102935 497819 648913 461191 2393246 5965431 2853198 3387780 3715247 3804054 6444630 6251147 6628708 Dimethylglycine 1806717 981535 1359025 1446481 1284131 1161051 1063200 991629 1089110 1277811 1328019 1552883 1400918 3689072 1972111 1271286 1678657 1468684 1893495 1469322 1943757 5-10-Methenyltetrahydrofolate 0 18601 26061 0 51956 38379 0 0 0 56909 84619 86612 0 26922 21898 168029 217176 199288 226663 172291 46887 Argininosuccinate 14493 11390 18393 10115 7785 6243 13321 9781 11935 0 0 0 19204 57492 23080 2965 7659 2273 52318 18882 33613 N-Acetylneuraminate 297948 237729 203710 249941 180836 189716 89620 117147 104386 230648 235887 196605 338083 632280 448615 427174 453759 475481 476108 575920 521013 alpha-D-Glucosamine 1-phosphate 117980 61148 86204 48962 29989 25465 52040 33114 14829 113846 127242 130845 26544 99826 66487 39573 117234 87484 86746 103323 100156 UDP-N-acetyl-D-glucosamine 46452 37927 37744 62925 46933 50939 21631 26581 57955 14601 19536 23358 30395 54118 31297 269168 282165 256660 602089 400183 527488 CMP-N-acetylneuraminate 117781 67119 32663 29570 19206 13387 49311 43965 50149 10361 6529 47151 0 23654 10041 7974 13218 15550 16703 13119 14210 4-Acetamidobutanoate 40215 39536 33783 78269 50319 51713 57850 64527 64879 43062 34984 47873 28702 26410 48457 29159 13746 30817 37670 40981 32005 Guanidinoacetate 13926 6899 38198 10878 13663 37921 7519 15624 0 34912 17785 9627 57240 34817 67315 94795 76605 144806 166578 197586 197849 trans-4-Hydroxy-L-proline 2197949 1670330 1413228 1520792 1202305 1308451 2292801 2199607 2276697 772719 540839 880220 1460828 850824 752083 905700 721865 926654 1013587 1210262 1256026 Pantothenol 18935446 16720013 24559786 14681585 36349504 35707428 12411976 12342633 12552013 46242292 42740340 49250540 16279767 45200559 37410548 50102852 56231952 53775500 71816456 69083160 63533932 5-6-Dihydrouracil 154740 105214 188328 104092 133815 163305 102743 135941 30583 109215 127294 130833 324106 260227 277871 287809 279903 310137 559114 603214 658664 D-4--Phosphopantothenate 91891 48205 52337 53261 42235 18563 82334 103268 72485 127119 96240 96584 122165 239845 150258 222683 217256 185423 217399 209329 267009 Hypotaurine 57564 60060 96576 53708 130279 117922 15129 21225 7798 252023 203369 341467 304061 845115 340783 426184 796498 663246 781765 516227 580057 L-Selenomethionine 65961 83528 83010 97995 114193 73501 114749 161645 107461 100003 132965 67479 354438 224875 153857 103133 78488 82833 108931 48772 108161 L-Cysteate 202733 172153 157831 161048 131312 141576 275819 398950 328267 75417 79796 73045 117566 167426 121972 71691 90322 65516 72184 81757 73595 Indole 8230 3891 5029 6714 4314 3170 4333 7391 9665 12076 11574 12403 13996 13228 16527 36663 38610 41259 48206 45116 55958 Indolepyruvate 65903 60587 92732 11697 71896 94220 20968 27375 14090 127670 75599 84422 46042 45196 76067 62502 55455 80146 337798 67003 132984 Indoxyl 1282213 444954 448794 361371 383829 319353 125837 147281 242399 1058204 1069606 1018118 457941 859895 510215 893407 790016 841305 832294 1185775 798649 quinolinic acid 53973 137349 38854 19720 37289 10272 21342 11738 15121 10208 21025 11615 38088 20333 21766 41290 35676 32915 53266 27391 21640 N-formyl kynurenine 119753 151789 275696 78895 284945 336432 41031 74569 42631 696222 493329 622094 164327 590592 324090 780696 1015015 941191 1369374 869515 868789 g-Oxalo-crotonate 27358 15610 10098 61968 23164 2760 65387 32976 44509 56446 59034 63635 52541 132486 79963 243243 239896 300837 353254 335514 333900 2-Aminomuconate 119979 62890 81499 54308 66164 95817 38671 29961 37684 100437 115553 105231 64998 145978 111058 215787 194565 163552 292320 324778 344122 Hydroxyindole-acetylglycine 10260 3500 5934 11237 2163 3230 0 0 0 7421 13841 17813 6252 18989 10148 30320 42424 37166 15851 27211 23273 Glycerol 3-phosphate 1881099 1201326 1058487 3138743 2271515 2108282 999866 882598 1016119 6118760 5678511 8371876 131166 124550 94654 834263 724991 809315 791827 663292 704849 Ethanolamine phosphate 36675100 23059578 31599930 21767040 31006998 33504946 25434106 27575546 15928119 18486634 19762104 18721794 22080539 36334300 22409174 5817100 6950648 5960812 13273477 14239345 11001775 Butanoic/Butyric acid 109312 86548 134062 130470 103849 107675 80934 113990 112548 101309 63239 163804 130386 211228 36481 79773 178179 171913 136870 110315 79878 Hexanoic acid (caproate) 650790 679701 663302 838987 561545 580997 723356 884677 843633 516890 635352 600049 1395326 1255925 599875 644870 679253 525082 576811 514475 602729 Heptanoic acid 1118826 1095061 936841 1337638 901382 992157 1148716 1360199 1286259 1088207 746990 1076902 1768066 1639819 884984 1121271 1047006 1011529 956850 975045 1008858 Octanoic acid (caprylate) 10313857 11012428 6799019 11331092 5370724 6481051 13024955 15378600 18918750 7654006 4051740 12312000 12612841 16152531 5147678 10638077 9465912 12197381 8491666 11870497 5710282 Hexadecenoic acid (Palmitoleic acid) 7914762 9673236 7600875 7190008 8346995 7210622 10445981 6382424 9952547 7462791 6049834 8146598 15058969 17394350 7323496 7429327 9538373 6950574 8467703 10076334 7640632 Octadecenoic acid (Oleic acid) 14393319 8578557 8482829 6490374 5450572 7059899 8043037 7928184 9513078 8287714 5229919 10429156 10716567 14201453 9500631 8455090 10390453 10530511 11223485 15243412 16877776 Linoleic acid ((9Z,12Z)-Octadecadienoic acid) 1319290 1494499 1114861 966113 1094217 1123169 1531084 1112344 1742382 1239569 855822 1103199 2117611 2475160 1188702 925794 1376609 1047019 1119965 1650221 1277725 Arachidonic acid (Eicosatetraenoic acid) 489814 406761 423871 377241 145286 284701 829598 1510837 2162182 203172 178480 197834 239531 187907 197788 89704 130587 68330 126771 167497 191093 Docosahexaenoic acid 617278 383053 460674 362833 137640 277358 649246 902704 1421155 234553 186847 261542 117653 125221 162963 121165 141762 112768 134390 192013 184406 Dodecanedioic acid 272118 250479 268909 318045 276483 355753 244388 345943 246643 283684 441662 253347 816121 631965 317794 250473 246678 246154 279599 228589 311788 (8Z-11Z-14Z)-Icosatrienoic acid 448108 293043 438926 245943 112721 175615 202472 325225 456038 511039 316996 536278 422505 385883 470037 317112 359971 308636 788023 1073248 941821 (7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid 241801 232585 232960 79885 58444 83629 234991 430966 739912 62292 54849 73016 8580 0 16406 15029 18647 10161 13836 24954 20217 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID mz rt (R)-Mevalonate C00418 130.3542 0.7018297 phosphomevalonate M-2H C01107 113.0138 0.6291461 p-mevalonate 13C4 M-2H C01107 115.0208 0.6409334 D-Glucose C00031 179.0553 0.6720361 Glucose 13C6 C00031 185.0757 0.6733918 Citrate C00158 191.019 0.6208895 Citrate 13C2 C00158 193.0255 0.6200824 Citrate 13C3 C00158 194.0289 0.6222625 Citrate 13C4 C00158 195.0335 0.6330815 Citrate 13C5 C00158 196.0357 0.6234483 2-Oxoglutarate C00026 145.0131 0.6185809 2-Oxoglutarate 13C2 C00026 147.0197 0.6190657 2-Oxoglutarate 13C3 C00026 148.0232 0.6127177 2-Oxoglutarate 13C4 C00026 149.0256 0.6222812 2-Oxoglutarate 13C5 C00026 150.0298 0.6229495 Itaconate C00490 129.0179 0.6285515 Itaconate 13C2 C00490 131.0246 0.6291786 cis-Aconitate C00417 173.0089 0.6210903 cis-Aconitate 13C2 C00417 175.0147 0.6210964 cis-Aconitate 13C3 C00417 176.019 0.6316277 cis-Aconitate 13C4 C00417 177.0208 0.6105029 Succinate C00042 117.0179 0.6534518 Succinate 13C2 C00042 119.024 0.6860461 Succinate 13C3 C00042 120.028 0.6569047 Succinate 13C4 C00042 121.0314 0.6484977 Fumarate C00122 115.0022 0.6243475 Fumarate 13C2 C00122 117.0089 0.6135837 Fumarate 13C3 C00122 118.0124 0.6241509 Fumarate 13C4 C00122 119.0157 0.6181778 (S)-Malate C00149 133.0129 0.6212373 Malate 13C2 C00149 135.0194 0.6249124 Malate 13C3 C00149 136.023 0.616926 Malate 13C4 C00149 137.0257 0.6198658 L-alanine C00041 88.0391 0.6940 L-asparagine C00152 131.0448 0.6690 L-aspartate C00049 132.0292 0.6160 L-cysteine C00097 120.0115 0.6720 L-glutamate C00025 146.0450 0.6210 L-glutamine C00064 145.0610 0.6740 L-threonine C00188 118.0499 0.6830 ATP C00002 505.9896 0.5700 ADP C00008 426.0233 0.5970 Adenosine C00212 266.0892 0.6780 GDP C00035 442.0181 0.5680 GMP C00144 362.0526 0.6380 CDP C00112 402.0116 0.5610 CMP C00055 322.0456 0.6050 Thymidine C00214 241.0824 0.6910 Thymine C00178 125.0346 0.6970 UTP C00075 482.9624 0.5540 UMP C00105 323.0294 0.6260 Inosine C00294 267.0729 0.6410 3',5'-Cyclic AMP C00575 328.0463 0.6290 Adenylyl sulfate C00224 426.0089 0.5340 Adenylosuccinic acid C03794 462.0694 1.8030 UDP-glucose C00029 565.0490 0.5660 NADPH C00005 744.0860 0.5510 Phosphate C00009 96.9683 0.6180 Diphosphate C00013 176.9354 0.6110 D-Glucose 6-phosphate C02965 259.0230 0.5900 D-Fructose 1-6-bisphosphate C00354 338.9895 0.5560 2/3-Phospho-D-glycerate C00631 184.9852 0.5710 Pyruvate C00022 87.0074 0.6670 D-Arabitol C01904 151.0619 0.6240 2-Oxoglutaramate C00940 144.0293 0.6460 2-Hydroxyglutarate/Citramalate C02630 147.0291 0.5950 Sedoheptulose 1-phosphate C06222 289.0338 0.5910 alpha-D-Ribose 1-phosphate C00620 229.0120 0.5980 5-Oxoproline C01879 128.0343 0.6510 Ascorbate C00072 175.0244 0.5830 Dehydroascorbate C05422 173.0095 0.5970 gamma-Glutamyl-Se-methylselenocysteine C05695 311.0180 1.9300 gamma-L-Glutamyl-D-alanine C03738 217.0831 0.6530 Cystathionine C00542 221.0601 0.6590 Dimethylglycine C01026 102.0548 0.6200 5-10-Methenyltetrahydrofolate C00445 455.1583 0.6760 Argininosuccinate C03406 289.1158 0.6170 N-Acetylneuraminate C00270 308.1004 0.5670 alpha-D-Glucosamine 1-phosphate C06156 258.0390 0.6110 UDP-N-acetyl-D-glucosamine C00043 606.0757 0.5660 CMP-N-acetylneuraminate C00128 613.1512 0.5820 4-Acetamidobutanoate C02946 144.0657 0.5950 Guanidinoacetate C00581 116.0448 0.6550 trans-4-Hydroxy-L-proline C01157 130.0500 0.5990 Pantothenol C00864 218.1035 0.6860 5-6-Dihydrouracil C00429 113.0345 0.6420 D-4--Phosphopantothenate C03492 298.0703 0.5830 Hypotaurine C00519 108.0113 0.6790 L-Selenomethionine C05335 195.9918 0.7710 L-Cysteate C00506 167.9966 0.6160 Indole C00463 116.0497 1.7660 Indolepyruvate C00331 202.0481 0.6940 Indoxyl C05658 132.0472 0.6790 quinolinic acid C03722 166.0153 0.6770 N-formyl kynurenine C02700 235.0757 0.6730 g-Oxalo-crotonate C03453 157.0142 0.6650 2-Aminomuconate C00322 159.0295 0.6660 Hydroxyindole-acetylglycine C05832 247.0754 0.5450 Glycerol 3-phosphate C00093 171.0058 0.6050 Ethanolamine phosphate C00346 140.0109 0.6400 Butanoic/Butyric acid C00246 87.0438 0.6900 Hexanoic acid (caproate) C01585 115.0754 1.8010 Heptanoic acid C17714 129.0911 1.8850 Octanoic acid (caprylate) C06423 143.1069 1.9850 Hexadecenoic acid (Palmitoleic acid) C08362 253.2177 2.6840 Octadecenoic acid (Oleic acid) C00712 281.2492 3.0410 Linoleic acid ((9Z,12Z)-Octadecadienoic acid) C01595 279.2336 2.7160 Arachidonic acid (Eicosatetraenoic acid) C00219 303.2337 2.6190 Docosahexaenoic acid C06429 327.2339 2.5270 Dodecanedioic acid C02678 229.1448 1.7870 (8Z-11Z-14Z)-Icosatrienoic acid C03242 305.2494 2.8590 (7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid C16513 329.2504 2.7130 METABOLITES_END #END