#METABOLOMICS WORKBENCH juliehaines_20240719_102536 DATATRACK_ID:5026 STUDY_ID:ST003332 ANALYSIS_ID:AN005459 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 Positive 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 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% 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 POSITIVE 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-alanine 3284612 2216189 2768536 2681530 2229748 3213628 3866803 3438853 3910888 1412645 1915648 2147244 2176144 2046095 2193545 1642490 1472006 1318878 2478558 1583079 1837423 141054 69557 90347 L-arginine 52974636 35023120 41679636 24445790 48068900 33245888 33734604 35302764 47450276 18072616 45439920 45267424 19112290 27542022 40549504 8035442 8659089 13774848 14074679 26255074 15589736 56134192 27794502 26291336 L-asparagine 376470 228135 210098 372097 404294 390539 141965 134770 101657 461430 526322 514977 294981 248907 270579 250551 244479 216074 234182 329934 167142 431188 142428 142431 L-glutamate 197305 208892 158701 207014 169708 225188 142888 175647 129392 213229 148469 145416 262035 167352 126822 214901 209163 220266 191466 252203 163735 148500 243518 158637 L-glutamine 36686076 22700408 31165416 21298830 27076840 23588506 26937974 23002874 24921066 16421100 33033154 31447154 15763577 20395352 20078274 10595136 10020347 15105333 16437600 19952570 13364051 19739046 9892077 6935428 L-histidine 3582886 2477137 3278412 2469673 3408388 3769994 3057738 2841734 4119848 1952420 4127839 4361335 2235334 2607206 3624770 2079013 2156525 2276374 2457002 2996515 2435386 3671083 2192653 2248182 L-leucine 69092256 122082936 51038160 52132812 52110496 65558368 93105336 65153184 75378440 127562816 47097716 47092308 56155056 63079712 56263572 68878256 76094344 89917824 53240172 51645020 46295252 67422864 187278928 69767576 L-lysine 6756737 9230212 8377266 10661327 5458807 11398411 12011945 12024635 11231464 8155295 5394720 6398394 10209153 8450333 7440862 7401596 7917284 5739116 7793686 5820547 7573097 6971644 10390411 12384781 L-methionine 9659761 5376911 4756581 9159017 9009951 7304860 7665083 8904736 6832334 7267710 4593681 3826751 8291178 8181054 6070850 3419674 4020451 3053529 3150267 5940760 5143763 8384578 6815856 11620281 L-proline 27670726 23690432 24883400 18328978 22129806 19537418 14926267 18407558 23025368 21938962 29081920 25007600 26558466 32823610 39222580 52000852 44499616 60742540 45451008 65787180 47375868 24342342 14345309 15290043 L-serine 228449 172682 219104 168636 194701 205931 258814 327400 268882 86093 70653 67600 107158 76613 77392 45515 49669 12186 35882 77152 52325 356040 245883 239944 L-threonine 3613479 2667850 3389222 2248712 3343141 2869691 2799060 2358139 2537234 2475637 3763996 4204774 2345140 3001524 3519408 2053951 1882778 2356908 2897305 2759756 2369608 3285600 1974915 1717495 L-tryptophan 36779460 33267294 35042292 29700290 37298948 23800522 29626720 27690442 25138688 24811408 23028880 22777322 26877226 35056468 31018902 21210844 18014916 21437642 16631276 24108226 21729856 38051904 28960778 32059978 L-tyrosine 33920628 40871356 21927716 25598840 26137050 24663030 16659664 19669568 10855159 18787106 17663358 15050563 18400000 31855422 9797698 10684481 15550435 32496010 13206936 13184515 21523494 26933320 18063870 26116450 L-valine 22968112 20920248 19293360 26546982 18622830 25460570 22407558 23395850 24968826 17023414 18990198 18958006 19296312 21415166 23055200 17234384 20464170 20267938 16198935 22855846 18169242 26099890 21574536 22248422 dAMP 293300 194156 207694 205183 192862 196169 205237 203441 185676 164460 277300 247085 133082 132548 152740 123480 131886 142080 129195 167038 180643 11076 0 0 Cytidine 196206 178790 214451 168321 214453 166692 224222 224759 260440 153228 240665 232006 186686 178704 235072 180344 195530 240238 267206 253767 238875 252007 215148 214118 UMP 1030476 1086871 1066257 1247546 664296 1366680 1356425 1307188 1262065 913117 874898 1026443 1324769 1043040 731970 1464751 1349007 1461753 1377863 1476261 729417 344913 687245 747325 Hypoxanthine 2060307 911266 1313730 1324385 1318771 1440559 2453269 2605014 1151020 574501 426863 423232 300784 269114 304498 147854 115632 195096 195731 241114 201271 2267232 1939226 3010731 Allantoate 435973 462503 437205 791824 547989 619181 266949 262873 409244 925284 649261 776601 1391187 1328726 1045533 2107604 1969008 1945822 1820265 1483588 1780773 141180 177970 205499 (S)(+)-Allantoin 87827 72848 65606 74407 54985 60886 64094 72932 82343 55024 39948 39367 60124 59115 41193 9266 19921 26583 39571 34792 38509 58556 70395 111530 5-Hydroxyisourate 1799395 1164107 1303719 1184280 1449112 1579930 1216020 1318992 1563854 802551 1343894 1325214 862967 883783 1179648 733703 706221 762856 928123 803340 968372 188948 40274 61931 Urate 868000 242643 451334 468829 285313 323159 407659 400687 147207 194437 330537 291376 222531 307661 369576 342242 377450 301177 353652 257702 438668 314695 507883 183658 4-Pyridoxate 5438 2010 3624 10306 29397 15995 86639 43948 18588 225170 482821 530091 418165 564696 1054110 123100 85608 170896 5945 0 4570 5359648 2383594 1902560 Pyridoxamine 5'-phosphate 288259 346634 449229 457195 267755 710465 542317 682841 1000686 360649 393603 653867 426281 269802 297708 386946 289752 206766 585945 269897 450983 319602 381378 460137 Nicotinamide 18469194 11316957 5110811 18202858 17104424 14769300 14781917 15397383 10763603 19557020 11635499 11489256 19963570 17796452 16360827 15505216 17777800 7237914 12298465 19091258 17044430 13861187 12300017 16661657 Nicotinate ribonucleotide 69613 57004 117913 38956 88761 52304 63712 89118 94250 46302 117219 155897 41722 54182 89758 54189 62603 80611 64004 56252 48947 76127 62985 71843 5-Phosphoribosylamine 24707 25112 30028 27469 20304 11859 21439 27621 41651 21092 31151 29717 24812 15802 27926 39826 25363 24248 27166 29132 28621 18014 25339 25947 Phosphate 1309066 856534 937382 578530 692266 347605 759788 815442 2205806 799436 905335 1103307 733013 766310 787213 784204 936611 894775 782355 811673 811687 771112 817875 752859 D-Glucose 6-phosphate 40606 17073 7759 46659 29446 15666 22561 27573 13551 22923 31792 26934 40683 43084 25768 35206 24542 8634 19821 36876 34719 0 3769 0 Ascorbate 1308487 380814 663601 691391 457720 463748 607852 598199 199739 367060 535860 455602 277736 443951 620470 534059 557745 442924 588670 358390 641752 460824 823307 270268 gamma-Glutamyl-Se-methylselenocysteine 225045 204125 196775 343628 227688 251099 112256 111851 148827 369268 353166 371853 604493 609189 507112 844483 909160 987819 797088 693965 776688 74001 70032 98587 5-L-Glutamyl-taurine 52339 23394 56578 21073 64512 48350 70270 58841 107884 22802 59842 84800 15897 35179 49626 41127 9739 38846 40853 65080 18458 27105 14465 42812 S-Adenosyl-L-homocysteine 20341548 14947477 23687594 15855453 21476706 15306456 15030443 17399738 19528834 10084112 26533386 24490944 17668702 17580318 22685242 15347229 17525446 15725789 19438474 14631133 16763632 19336922 17400012 15008355 Ornithine 170036 358565 177801 312156 180027 203942 269989 164096 211470 344300 255781 253290 574735 501288 298187 1036495 801739 756429 853697 604520 733551 158398 476833 168508 Putrescine 167847 219009 147129 256727 168306 155633 169572 176166 325378 220529 174145 162392 187450 147392 217572 232750 238612 246810 170956 194918 221895 195739 143681 192575 Spermine 61273 87874 53350 40361 32887 61998 135931 112701 164991 39476 62513 110806 27553 42385 29433 155883 26629 46844 277736 43581 173873 8521 15355 15084 D-Glucosamine 15878 48288 29140 12540 33599 11538 4180 5176 2623 39954 74069 77317 18679 46829 43901 73140 58609 115346 48257 140848 52229 0 0 0 Pantothenate 20159432 24606250 16406915 10151368 18231110 9546397 10611811 9444185 9637505 10629591 14794332 14160941 10995488 19789018 18401246 10513827 10597324 20511834 10069116 23473040 9390864 16230799 11552039 8526536 L-Methionine S-oxide 390649 293617 285328 186422 325575 158859 126689 186441 151223 133704 355739 376239 218287 261007 445746 183160 162987 344910 199262 461152 202576 444357 258056 189925 3-Methyldioxyindole 22018824 23127914 22582728 15414860 21928880 14863535 18123476 17819750 12616595 14259196 14040996 14414069 16542584 21039926 19766982 12565644 11242429 14537561 10628588 16984924 13202595 19678074 19493978 20268280 Indole 18586910 19576602 18883380 10821281 16053892 10687985 13254221 12746391 8952063 9487115 12205861 11941401 11131625 16944182 16671995 8876478 7646095 13072572 7668597 15401896 9483269 16648329 13893595 14764013 Indole-3-acetaldehyde 1076116 1121234 1793405 1669401 927672 1895009 890274 1495233 623029 1062554 742672 690874 1473978 1015521 1287601 929227 563285 1165003 1314245 897863 1769682 983552 992732 968924 Indole-3-acetate 183850 183816 323680 112489 503024 221800 22628 22690 22542 147823 865505 595603 440957 488904 645979 86748 21181 43332 166363 181252 173599 93729 90735 113823 Indoxyl 2517592 2670345 1878735 2977391 1620219 2598507 2417115 2372774 2462249 3175207 2010629 2732451 3225530 2262732 2150559 3623492 3001186 1615625 3036705 2245498 3188406 2441244 1942304 2383177 g-Oxalo-crotonate 480369 408399 399580 316326 277758 359573 710768 773053 625400 80082 63503 74124 22405 24266 26047 6359 0 0 2546 0 0 536982 559640 595454 Sphingosine 14136 12465 9738 10417 10528 14909 10768 7281 10098 19195 8596 9068 7075 7219 8442 5784 19111 8194 8316 7593 7264 8245 5833 5186 Acetylcholine 390694 399453 482972 259413 531880 336760 261056 282417 331577 262870 375853 334225 315459 426710 424429 443023 438127 424857 380079 467813 363478 448768 360213 259815 Choline 59645312 41257324 61757492 64271796 41889876 82084368 97762912 105288856 143506608 28749162 39585804 51786764 56213156 43441476 41781824 2542880 3546702 3386067 20507146 18517286 24595610 64118580 71104248 74340216 Sphinganine 1-phosphate 676073 5883 10050 96808 5337 237401 76669 60616 6090 12977 9669 11162 3363 75833 10291 31721 4741 7781 2650 3351 77945 55566 164974 40608 L-Carnitine 30679026 30648016 40574004 37494300 32917334 41785252 39335840 35503144 57635292 46199180 62923308 71391288 45130976 28381750 42571532 55147888 60457252 38363284 62521692 37997132 52892900 24910224 27000142 27483642 acetyl-carnitine (acyl-C2) 542680 281077 270953 151441 117155 247086 43787 75874 138334 810286 611390 647855 1118779 887562 733593 2167520 2053744 1499407 2156759 1226936 1982727 0 4889 907 butanoyl-l-carnitine (acyl-C4) 317935 317080 369004 180910 215824 164594 130450 102906 96458 825251 664527 784712 397074 426668 354313 400187 466879 601304 332730 440920 271397 0 2843 4556 L-octanoylcarnitine (acyl-C8) 13262 11949 5926 9914 16441 16208 4637 14252 3699 8806 11996 6734 7155 10340 13706 6411 20986 13212 12833 10308 9443 19542 5270 8730 O-Decenoyl-L-carnitine (acyl-C10:1) 8532 10468 8209 10708 11182 11430 9344 7463 11824 9657 9584 8676 9034 9466 10062 10095 8602 10181 9352 8874 8275 8677 12893 7870 O-dodecenoyl-carnitine (acyl-C12:1) 239866 241641 106214 225946 87737 226927 226537 145030 311700 151183 196943 138227 115885 92045 237114 256508 269362 135432 99335 209078 172423 323424 116412 103567 Tetradecenoyl Carnitine (acyl-C14:1) 5078 4728 4408 3798 4244 16672 4144 4889 3415 5176 3999 5320 4657 4725 4276 4316 18566 5669 4505 3191 8029 13699 3835 3421 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. L-alanine C00041 90.0547 0.71 L-arginine C00062 175.1183 0.69 L-asparagine C00152 133.0592 0.70 L-glutamate C00025 148.0601 1.70 L-glutamine C00064 147.0760 0.70 L-histidine C00135 156.0763 0.69 L-leucine C00123 132.1015 0.91 L-lysine C00047 147.1124 0.63 L-methionine C00073 150.0579 0.87 L-proline C00148 116.0702 0.71 L-serine C00065 106.0493 0.75 L-threonine C00188 120.0652 0.70 L-tryptophan C00078 205.0965 1.70 L-tyrosine C00082 182.0806 0.97 L-valine C00183 118.0858 0.72 dAMP C00360 332.0792 0.71 Cytidine C00475 244.0903 1.82 UMP C00105 325.0371 0.72 Hypoxanthine C00262 137.0454 0.89 Allantoate C00499 177.0606 0.66 (S)(+)-Allantoin C02350 159.0500 0.68 5-Hydroxyisourate C11821 185.0315 0.70 Urate C00366 169.0342 1.85 4-Pyridoxate C00847 184.0592 0.76 Pyridoxamine 5'-phosphate C00647 249.0602 1.16 Nicotinamide C00153 123.0549 0.95 Nicotinate ribonucleotide C01185 337.0515 1.74 5-Phosphoribosylamine C03090 230.0460 1.81 Phosphate C00009 98.9839 1.89 D-Glucose 6-phosphate C02965 261.0374 0.99 Ascorbate C00072 177.0394 1.98 gamma-Glutamyl-Se-methylselenocysteine C05695 313.0346 0.66 5-L-Glutamyl-taurine C05844 255.0644 0.74 S-Adenosyl-L-homocysteine C00021 385.1280 1.74 Ornithine C01602 133.0972 0.71 Putrescine C00134 89.1070 0.64 Spermine C00750 203.2223 0.71 D-Glucosamine C00329 180.0869 1.69 Pantothenate C00864 220.1172 1.69 L-Methionine S-oxide C02989 166.0528 0.72 3-Methyldioxyindole C05834 146.0596 1.70 Indole C00463 118.0647 1.71 Indole-3-acetaldehyde C00637 160.0752 1.56 Indole-3-acetate C00954 176.0722 1.72 Indoxyl C05658 134.0596 0.72 g-Oxalo-crotonate C03453 159.0274 0.77 Sphingosine C00319 300.2889 2.64 Acetylcholine C01996 146.1172 1.25 Choline C00114 104.1066 0.70 Sphinganine 1-phosphate C01120 382.2785 1.93 L-Carnitine C00318 162.1119 0.70 acetyl-carnitine (acyl-C2) C02571 204.1223 0.72 butanoyl-l-carnitine (acyl-C4) C02862 232.1534 1.70 L-octanoylcarnitine (acyl-C8) C02838 288.2157 1.74 O-Decenoyl-L-carnitine (acyl-C10:1) CID57357170 314.2374 2.34 O-dodecenoyl-carnitine (acyl-C12:1) HMDB13326 342.2630 1.73 Tetradecenoyl Carnitine (acyl-C14:1) HMDB13329 370.3000 2.96 METABOLITES_END #END