#METABOLOMICS WORKBENCH juliehaines_20240719_102536 DATATRACK_ID:5026 STUDY_ID:ST003332 ANALYSIS_ID:AN005458 PROJECT_ID:PR002070 VERSION 1 CREATED_ON July 19, 2024, 3:03 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 - media 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. The data in this study are from the assessment of the conditioned ST:STUDY_SUMMARY culture media at the end of the 24h incubation period. 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 AMC A S7 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-25 SUBJECT_SAMPLE_FACTORS A AMC A S8 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-26 SUBJECT_SAMPLE_FACTORS A AMC A S9 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-27 SUBJECT_SAMPLE_FACTORS B AMC B S7 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-28 SUBJECT_SAMPLE_FACTORS B AMC B S8 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-29 SUBJECT_SAMPLE_FACTORS B AMC B S9 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-30 SUBJECT_SAMPLE_FACTORS C AMC D S7 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-34 SUBJECT_SAMPLE_FACTORS C AMC D S8 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-35 SUBJECT_SAMPLE_FACTORS C AMC D S9 Disease state:AMC | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-36 SUBJECT_SAMPLE_FACTORS D PMS A S7 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-37 SUBJECT_SAMPLE_FACTORS D PMS A S8 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-38 SUBJECT_SAMPLE_FACTORS D PMS A S9 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-39 SUBJECT_SAMPLE_FACTORS E PMS B S7 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-40 SUBJECT_SAMPLE_FACTORS E PMS B S8 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-41 SUBJECT_SAMPLE_FACTORS E PMS B S9 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-42 SUBJECT_SAMPLE_FACTORS F PMS C S7 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-43 SUBJECT_SAMPLE_FACTORS F PMS C S8 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-44 SUBJECT_SAMPLE_FACTORS F PMS C S9 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-45 SUBJECT_SAMPLE_FACTORS G PMS D S7 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-46 SUBJECT_SAMPLE_FACTORS G PMS D S8 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-47 SUBJECT_SAMPLE_FACTORS G PMS D S9 Disease state:PMS | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-48 SUBJECT_SAMPLE_FACTORS NA NC 7 Disease state:no cell ctrl | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-49 SUBJECT_SAMPLE_FACTORS NA NC 8 Disease state:no cell ctrl | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-50 SUBJECT_SAMPLE_FACTORS NA NC 9 Disease state:no cell ctrl | Sample source:cell culture supernatant RAW_FILE_NAME(raw file name)=DN47-51 #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 Culture Media #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. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from thawed media aliquots (20 uL) were extracted with 480 uL of 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 for 10 min at 18,000 g, 4°C and stored at SP:SAMPLEPREP_SUMMARY −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 20 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 AMC A S7 AMC A S8 AMC A S9 AMC B S7 AMC B S8 AMC B S9 AMC D S7 AMC D S8 AMC D S9 PMS A S7 PMS A S8 PMS A S9 PMS B S7 PMS B S8 PMS B S9 PMS C S7 PMS C S8 PMS C S9 PMS D S7 PMS D S8 PMS D S9 NC 7 NC 8 NC 9 Factors Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:AMC | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:PMS | Sample source:cell culture supernatant Disease state:no cell ctrl | Sample source:cell culture supernatant Disease state:no cell ctrl | Sample source:cell culture supernatant Disease state:no cell ctrl | Sample source:cell culture supernatant L-cystine 543988 666069 507098 602448 595044 589439 644583 586727 562947 86836 80571 63833 24575 13803 30475 492 556 570 2555 530 591 607986 544020 501124 Guanosine 18424 22073 18258 23773 22853 30353 40988 1679 12788 20113 45016 13917 11921 14492 34783 15004 4739 15430 17612 4890 17818 2875 4369 0 dCMP 24197 13505 14617 14130 19202 21408 21819 14476 12670 9434 11921 9395 16447 19314 16548 12499 9545 11804 6517 9829 12567 14591 22056 25929 dTMP 21255 19703 28701 12841 21223 35009 18331 31109 15700 15829 32796 13822 19480 39450 29223 15948 26478 20669 17200 31043 30264 27449 46234 29249 Thymine 136567 122661 100152 133465 147571 116952 126090 165535 139293 119790 128339 135219 89261 117052 111151 61994 60355 55541 87440 71192 67766 73102 103111 63928 UDP 70188 93933 81570 68647 55408 84916 91846 109408 101235 69152 87911 81254 86301 63786 94775 67882 66348 54604 68517 64830 67855 110243 69789 90707 Inosine 19434 19912 28179 14515 31613 18884 71043 44758 48888 14955 21458 15456 27182 24286 40810 16733 8992 15554 11679 24885 23458 98637 67662 62921 Xanthine 141067 146822 123073 75675 71756 90449 40740 27361 44746 122515 72781 89946 83876 59551 61211 91937 167016 114677 135327 127358 154066 1882 1126 1657 5-6-Dihydrothymine 2310689 2301084 2116038 1994035 2097311 2042173 2093962 2483348 2381644 1596728 1401039 1571460 1252157 1395835 1326254 797713 861573 942996 1062369 940805 985438 632875 561410 595111 Pyridoxal 337723 311357 287384 300468 291042 278600 272464 265876 249933 473199 479114 562375 407490 389080 405529 587309 560598 562779 599873 518973 580955 343554 289342 290319 Diphosphate 9410 9671 12934 7194 12491 10841 11630 7452 10791 1603 7971 10562 9328 15006 9653 2794 11330 9240 14387 7516 7331 11245 10065 13573 D-Glucose 193976 147025 213545 132252 174680 202115 243306 223730 185530 103139 109843 123037 91876 146868 96323 139509 108077 113185 102559 145947 118660 262711 225075 217748 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 3012509 3067078 1490528 2706964 852760 2879889 2188349 1343444 2397187 2866507 1225419 828155 1472804 960079 1734007 3115128 1805792 1812203 770578 1341353 2178932 2360111 1563260 1070770 Pyruvate 719964 601209 578374 575460 503055 366499 628066 565713 630818 403048 316496 380914 514232 578899 539754 374876 369951 376087 447008 451704 503355 4282819 4293666 4246774 Lactate 19271856 17477776 17993454 16513952 17187426 15614813 12638568 11261656 12159712 25881550 24298530 27253600 21008646 23606744 19995938 33736024 31475946 33743192 35621868 33647740 35256804 382772 324333 279346 Mannitol 945968 925601 691837 881952 817338 825185 919946 820812 829277 703820 591820 701805 839917 719792 711846 616078 669487 730650 605953 607680 652237 1024063 886345 985795 D-Ribose 129387 89695 108750 133492 111901 120862 65849 87628 101000 71131 87381 64033 74585 89071 95110 65248 52979 42857 20260 49429 83561 98782 108759 133744 D-Arabitol 31692 55104 76635 63318 67958 58408 42115 93972 77258 32622 62639 65593 38987 46692 58807 55892 42843 52648 47286 24262 83981 101021 61082 85268 Citrate 1245369 80169 92156 92512 84001 88431 71792 72557 79036 719373 91270 77099 80504 99291 81883 81937 118019 73798 69584 97825 101019 59992 96871 98425 2-Oxoglutarate 273194 234408 230898 322596 282823 274405 86983 60683 58578 1068295 911477 1025583 318017 266806 302593 388678 408646 495857 319604 361330 396286 1932 4524 5311 Succinate 79123 50285 40176 82653 44513 47545 35448 16327 23390 69126 47795 59057 76359 68797 69912 186973 174269 192505 165658 172548 186454 7121 8357 10803 Fumarate 125284 82722 94726 101624 67850 77620 52274 30349 42268 176121 99035 85331 170583 168999 140417 253718 257303 280920 257012 379215 295753 118602 114384 115981 Malate 386606 278241 303269 330432 254507 257454 157370 140103 125824 608568 300631 377618 604032 564466 582778 880351 1049531 942266 933258 1104450 1165666 17662 18122 23556 2-Hydroxyglutarate/Citramalate 123181 88620 95543 461419 456229 409664 75338 39763 45684 778555 596643 667030 105097 96644 88921 83899 101416 104743 81915 92984 116831 18649 29277 14600 Sedoheptulose 1-phosphate 24998 43150 29614 33717 26819 31170 40726 52061 53629 1599 3815 1931 6566 13684 3658 1342 1062 848 1101 2082 1092 4417 1740 3566 5-Phospho-alpha-D-ribose 1-diphosphate 28598 42137 33384 28683 28749 32615 49664 56083 34051 5938 4318 8871 18807 15339 11689 6106 4716 7863 4966 4547 4517 57783 56998 62608 Glutathione 297643 251014 256205 259812 347906 351853 236826 266359 277180 200256 193522 194377 239906 237781 245444 198675 218518 202018 182513 256781 177624 402915 395040 377449 5-Oxoproline 31166752 31241350 29912186 29015758 28938448 28746970 33622864 34230644 34105872 21471842 21954870 23400590 25670806 23320160 25175758 22942072 20256862 20412452 21654560 21603272 21881896 36694916 37938364 34903856 S-Glutathionyl-L-cysteine 353938 267189 240454 293683 345092 294299 143354 166960 145241 268062 157293 168521 107263 85523 159353 32341 36071 40923 15576 45823 32712 90554 125336 102333 Dehydroascorbate 48467 43223 48348 44097 38716 34666 44305 42666 44875 40482 35659 35635 38227 41624 47557 37417 40581 38415 42472 42611 53106 47743 51834 43517 gamma-L-Glutamyl-L-cysteine 116376 75442 46500 55121 93760 76625 60125 106919 81664 78142 35516 55090 108132 105298 94295 24076 71906 71649 53204 78058 19215 115136 86996 127526 gamma-L-Glutamyl-D-alanine 210149 183598 171019 402568 381673 464888 473557 507909 473643 368417 335272 380366 670074 546003 650964 318567 276402 311016 145726 148082 170457 1207432 1060163 1200640 gamma-Glutamyl-gamma-aminobutyrate 3640178 3223580 3038221 3467427 3608247 2659262 2081858 1832596 1856229 4827048 4411359 4411974 4624769 4688201 3964699 4460285 4735890 4076922 4244493 4165454 4434045 17042 16215 2030 Bis-gamma-glutamylcystine 4435451 3551833 121037 831068 315673 205440 459413 293110 99383 3301111 170614 313754 753349 594409 111988 144010 5139582 2440915 337731 1802625 401844 173376 996842 382396 (5-L-Glutamyl)-L-glutamine 171710 158474 166061 212834 207152 167389 186911 165887 169649 180229 175856 155759 170342 166761 178907 177952 147216 166520 198423 191722 220529 205987 186679 205365 Dimethylglycine 18139 20017 22292 31837 24756 21957 14405 14926 18417 8149 6085 9736 21090 21531 27705 13606 5913 8335 15274 17480 16434 434344 389972 395771 Phosphoserine 11847 10792 10459 8054 7416 6579 10125 4292 10033 8519 5884 8273 4618 11130 10820 9001 8380 8405 14344 8105 8872 7620 9382 7867 10-Formyltetrahydrofolate 84105 160232 136957 119614 163596 140810 151902 170458 188180 53487 59933 85451 127839 94563 108141 91541 74424 48548 81383 75736 67416 266599 234425 262459 N-Acetylneuraminate 3112 2173 0 1103 0 2240 0 0 0 4094 2626 4982 21452 21183 19751 91764 61413 85044 57282 72060 72131 0 0 0 alpha-D-Glucosamine 1-phosphate 31228 17926 18121 14724 18844 33442 21322 30965 24336 21959 23107 18103 32027 24908 27141 12057 25474 15818 10856 19487 26590 16798 23222 35554 1-4-beta-D-Xylan 10069 2440 3756 42030 19690 25054 12184 5868 3224 39861 28948 27823 4988 2510 1469 17708 17900 11421 14418 9832 14029 3042 1728 840 4-Acetamidobutanoate 12944 15154 12901 14731 20037 7873 6609 10630 7946 15842 17108 14753 21027 13636 16295 17755 18184 23341 15171 13582 18720 5307 7128 8754 N-Acetyl-L-citrulline 127712 77734 99466 423543 521078 1023296 2169442 2045300 1301474 5088774 6069507 7881173 13793724 14364701 18044718 3559659 3031546 2550056 379926 157581 212164 126903848 120690968 115223912 N-Acetylornithine 10197 11512 6999 23006 23173 23357 22645 30923 26576 15312 13974 29740 47235 40837 41601 17835 13711 25523 7562 7803 9251 71539 77370 75964 Guanidinoacetate 1072812 1180239 881998 1058066 880701 812319 1038586 960777 912075 694672 586039 709282 847042 856781 756933 605066 549680 536087 602295 582755 641567 1061783 928294 1168724 trans-4-Hydroxy-L-proline 102010 130189 119319 104835 122615 100007 55861 54941 57908 134247 117057 113226 122693 93996 107889 160066 177287 218304 189807 182735 214526 5151 9367 9353 N-Succinyl-L-glutamate 5-semialdehyde 675894 534468 518251 619921 650059 439468 218295 198434 239653 1624178 1329350 1510489 814859 713265 711390 649670 714597 682014 893353 715796 819807 2171 1782 2217 D-4--Phosphopantothenate 12908 12307 10064 12099 11388 11951 11526 12437 11908 7976 7467 7520 10973 5793 9434 7750 5801 6595 7530 7450 5623 11971 13134 13156 Mercaptopyruvate 46204 66220 24326 37765 38196 31600 61049 62839 55562 7405 8575 22612 22109 28855 33857 13279 17399 4909 9746 17527 5641 83095 74414 93033 Thiocysteine 4721238 4577221 4742237 5583605 4744267 4352529 3011099 2435556 2335776 8418432 7077099 8870066 7659821 7486842 7066673 10279092 8132018 8649691 9081496 8580473 8499724 89132 71711 74600 Thiosulfate 9210 10070 8555 13411 7820 8063 9910 7078 5276 9272 15916 15923 17097 16642 21065 17268 14283 12820 15108 18652 15238 6571 4851 6880 2S-5S-Methionine sulfoximine 193976 147025 69770 132252 174680 103282 170765 149800 185530 103139 109843 76106 73630 146868 96323 74199 108077 78460 83082 130550 99638 262711 225075 217748 5-Hydroxyindoleacetate 201165 284863 282609 329052 232822 238062 194157 141189 149101 370725 392417 426082 364464 292690 304961 542866 462535 465361 494353 486080 509467 23114 0 16102 Indolepyruvate 750645 726603 718922 721325 640500 697700 885341 758261 700162 480139 456400 490043 676989 606998 618065 464918 478327 513818 463786 450364 506151 951710 926352 945401 N-formyl kynurenine 694786 1132830 480976 775466 655746 727105 675096 558530 859835 684161 463713 512303 562794 709524 577975 558356 572459 718926 482330 426644 464049 889953 955107 874513 2-Aminomuconate 28910 24263 37117 14285 10681 10734 8328 8613 7502 672026 507915 622766 15783 11050 10028 55974 44406 69216 42654 45852 41099 10565 16171 2629 Hydroxyindole-acetylglycine 52981 84438 101222 76827 102108 124163 90365 88340 165297 36008 87314 102464 96105 27935 56784 50872 69071 43480 71252 66143 63010 194058 148882 134776 Glycerol 3-phosphate 80464 63403 39867 63924 123390 25880 31856 26474 21492 181027 35482 89790 23470 18763 31987 35381 111803 42422 22453 112738 22929 29821 36659 20172 Butanoic/Butyric acid 135478 89968 116295 185516 136936 114745 114004 113290 70086 130299 156320 187190 153094 103600 114985 144978 156038 206602 217306 201237 165980 80650 40329 18671 Pentanoate (valerate) 77838 84600 90623 92200 61611 74755 80377 94579 70527 122169 131439 127688 103237 76533 79186 165741 162472 148382 136608 110871 122616 77418 44150 52362 Hexanoic acid (caproate) 614841 327447 314972 510431 315969 326804 328733 297088 250368 580675 405554 426461 470246 411069 413329 655976 749900 574430 671992 619459 705294 356693 232563 148060 Heptanoic acid 415112 151456 156082 228556 156919 164844 184505 172133 146638 270964 184305 208072 181398 159560 167128 263766 396425 220158 203885 213772 232465 146673 185931 120117 Octanoic acid (caprylic acid) 7308770 7679290 13242495 7041250 7384447 6820792 5856424 19570098 5906866 6291963 5872712 7739200 6176297 6247499 5016958 6316338 7587181 8165383 6548363 5213900 4374060 4592624 6236115 4007883 Nonanoic acid (pelargonic acid) 3449244 1610047 1387992 1561285 1192277 1208238 1923235 1455034 1066096 1547925 1195683 1583349 1269005 1331707 1417869 1285661 2536838 1472164 1402394 1679248 1428760 1734188 1373086 1090236 Decanoic acid (capric acid) 1503479 1044470 1104088 1116036 845394 896712 1149746 1026290 1044747 997238 1011711 1000558 1041188 913839 931130 1061433 1220124 1034742 1101460 1037084 1047664 1019288 862389 933544 Dodecanoic acid (lauric acid) 1764396 1406412 803625 1262714 822215 968769 1090863 949587 850446 1345458 794370 838697 878172 893371 846016 920280 2033133 1294256 1135246 895116 1576580 830735 985461 1113601 Tetradecanoic acid (myristic acid) 12730608 2758830 2320362 6382981 2293211 2494615 4480960 2872797 2240863 2253983 2075774 2449289 2311517 3253774 2349709 3229744 2917080 2679616 3364737 2217713 2818688 2397826 4937678 3475419 Hexadecanoic acid (palmitic acid) 122155360 63663604 61342732 101493080 61486148 57013188 100702336 54320892 78913024 63432048 44403908 78560744 40862780 91396408 54900320 83144760 61117280 71966728 102596864 48955824 70918928 56093836 71628688 80231192 Octadecanoic acid (stearic acid) 448261088 240360912 177543936 294411904 213587440 165375392 329728864 114367392 272126944 186479360 118602240 246352128 98931928 275451648 172334080 294937824 234705616 275532672 400760736 193403728 224618304 174250160 265474768 263272240 Tetradecenoic acid (myristoleic acid) 123906 80746 53819 98403 73564 71332 103144 78711 64492 46347 48266 56789 150534 113174 136851 48910 56223 90962 117402 112755 117553 45428 52394 57016 Hexadecenoic acid (Palmitoleic acid) 2198209 552928 478804 1272218 479105 503161 1036360 527530 506180 377383 447895 521505 595420 800554 610949 496931 458157 602295 700639 543349 750241 418592 770249 575479 Octadecenoic acid (Oleic acid) 7037636 6720059 3765384 4946126 4746689 3228955 8701780 2051571 6156784 4233882 2091776 5347955 2707814 5789650 3082365 6160348 4444576 6680188 10807227 4594931 5586250 1679470 4419956 4660301 Linoleic acid ((9Z,12Z)-Octadecadienoic acid) 991519 791146 474433 589256 630200 1053432 1537266 1480802 1681491 507871 260426 819560 415859 664397 591924 661268 569813 726349 1211131 481704 535913 8170816 7468808 8568231 a-Linolenic acid (Octadecatrienoic acid) 46197 60934 39014 43716 45241 240751 231787 408370 405456 17959 14486 81263 44085 39154 79838 13497 17315 14926 22379 23493 24911 2716901 2216488 2461814 Arachidonic acid (Eicosatetraenoic acid) 155321 137431 108259 35979 36872 72645 554440 661127 514318 23949 16560 23598 28915 26413 32870 14814 16065 13279 20221 22450 22537 10668 9881 9986 Docosahexaenoic acid 338005 349918 264652 142965 122674 213356 540374 591237 500322 47100 44973 75439 41902 36714 53944 13375 16369 17972 15540 19929 15916 3132 1300 1962 Eicosapentaenoic acid 13691 16685 12156 19793 10496 12415 76940 109070 96920 11034 9197 12153 9879 10298 9787 20145 14214 11927 13013 10786 10384 11959 11490 9014 Dihomo-gamma-linolenic acid ((8Z-11Z-14Z)-Icosatrienoic acid) 110608 132263 115988 31275 32901 47545 170219 131952 127632 29568 28219 46078 35768 41725 47671 28905 29101 33625 51080 50391 58719 2991 7698 6090 Docosapentaenoic acid ((7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid) 111566 113290 100455 32191 27012 53953 237408 239368 209556 7977 8520 17362 10116 10003 14918 4516 4166 3527 5313 4083 4320 1940 1718 1035 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. L-cystine C00491 239.0156 0.69 Guanosine C00387 282.0880 0.70 dCMP C00239 306.0535 0.79 dTMP C00364 321.0519 0.71 Thymine C00178 125.0340 0.66 UDP C00015 402.9990 0.73 Inosine C00294 267.0779 0.71 Xanthine C00385 151.0246 0.79 5-6-Dihydrothymine C00906 127.0497 0.68 Pyridoxal C00250 166.0495 0.73 Diphosphate C00013 176.9321 0.62 D-Glucose C00031 179.0551 0.68 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.9879 1.73 Pyruvate C00022 87.0071 0.68 Lactate C01432 89.0227 0.68 Mannitol C00392 181.0692 0.83 D-Ribose C00121 149.0455 0.71 D-Arabitol C01904 151.0582 0.69 Citrate C00158 191.0184 0.63 2-Oxoglutarate C00026 145.0128 0.62 Succinate C00042 117.0176 0.63 Fumarate C00122 115.0020 0.63 Malate C00149 133.0126 0.63 2-Hydroxyglutarate/Citramalate C02630 147.0284 0.61 Sedoheptulose 1-phosphate C06222 289.0373 0.96 5-Phospho-alpha-D-ribose 1-diphosphate C00119 388.9388 0.75 Glutathione C00051 306.0814 0.69 5-Oxoproline C01879 128.0337 0.67 S-Glutathionyl-L-cysteine C05526 425.0795 0.63 Dehydroascorbate C05422 173.0062 0.63 gamma-L-Glutamyl-L-cysteine C00669 249.0550 0.69 gamma-L-Glutamyl-D-alanine C03738 217.0818 0.64 gamma-Glutamyl-gamma-aminobutyrate C15767 231.0945 0.70 Bis-gamma-glutamylcystine C03646 497.0916 2.45 (5-L-Glutamyl)-L-glutamine C05283 274.1036 0.64 Dimethylglycine C01026 102.0543 0.63 Phosphoserine C02532 184.0005 1.77 10-Formyltetrahydrofolate C00234 472.1696 0.70 N-Acetylneuraminate C00270 308.0982 0.63 alpha-D-Glucosamine 1-phosphate C06156 258.0452 0.74 1-4-beta-D-Xylan C02352 131.0336 0.63 4-Acetamidobutanoate C02946 144.0650 0.63 N-Acetyl-L-citrulline C15532 216.0979 0.65 N-Acetylornithine C00437 173.0917 0.64 Guanidinoacetate C00581 116.0488 1.70 trans-4-Hydroxy-L-proline C01157 130.0493 0.63 N-Succinyl-L-glutamate 5-semialdehyde C05932 230.0632 0.70 D-4--Phosphopantothenate C03492 298.0750 0.96 Mercaptopyruvate C00957 118.9775 0.71 Thiocysteine C01962 151.9879 0.67 Thiosulfate C00320 111.9277 0.63 2S-5S-Methionine sulfoximine C03829 179.0548 0.69 5-Hydroxyindoleacetate C05635 190.0504 0.64 Indolepyruvate C00331 202.0474 0.88 N-formyl kynurenine C02700 235.0747 0.67 2-Aminomuconate C02220 159.0285 0.63 Hydroxyindole-acetylglycine C05832 247.0771 0.70 Glycerol 3-phosphate C00093 171.0105 1.70 Butanoic/Butyric acid C00246 87.0434 0.71 Pentanoate (valerate) C00803 101.0591 1.09 Hexanoic acid (caproate) C01585 115.0748 1.77 Heptanoic acid C17714 129.0905 1.89 Octanoic acid (caprylic acid) C06423 143.1062 1.98 Nonanoic acid (pelargonic acid) C01601 157.1220 2.09 Decanoic acid (capric acid) C01571 171.1377 2.17 Dodecanoic acid (lauric acid) C02679 199.1691 2.35 Tetradecanoic acid (myristic acid) C06424 227.2007 2.61 Hexadecanoic acid (palmitic acid) C00249 255.2319 3.01 Octadecanoic acid (stearic acid) C01530 283.2633 3.61 Tetradecenoic acid (myristoleic acid) C08322 225.1850 2.40 Hexadecenoic acid (Palmitoleic acid) C08362 253.2167 2.68 Octadecenoic acid (Oleic acid) C00712 281.2479 3.06 Linoleic acid ((9Z,12Z)-Octadecadienoic acid) C01595 279.2323 2.72 a-Linolenic acid (Octadecatrienoic acid) C06427 277.2166 2.50 Arachidonic acid (Eicosatetraenoic acid) C00219 303.2324 2.62 Docosahexaenoic acid C06429 327.2329 2.51 Eicosapentaenoic acid C06428 301.2166 2.62 Dihomo-gamma-linolenic acid ((8Z-11Z-14Z)-Icosatrienoic acid) C03242 305.2480 2.89 Docosapentaenoic acid ((7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid) C16513 329.2468 2.64 METABOLITES_END #END