#METABOLOMICS WORKBENCH juliehaines_20240719_093451 DATATRACK_ID:5025 STUDY_ID:ST003331 ANALYSIS_ID:AN005457 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 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 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 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 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 L-arginine 717550 611773 664935 564380 529202 656517 493908 685994 574131 908439 1008665 1168166 1014439 1485593 679376 889309 935937 1053403 1329120 1197089 1384847 L-histidine 444374 204938 201756 230373 208001 238514 123704 198999 169822 404800 665525 528451 333205 1045116 467403 1083157 730670 1144359 1066367 835147 1146564 L-leucine/isoleucine 8113312 3911519 5118566 3568232 4014092 5221297 2974655 3487913 3262997 14239337 19497106 14761119 9385053 20342554 12964005 32513942 25892770 31436954 35777332 31983520 38577052 L-lysine 1285962 610053 654226 832008 639495 738455 665003 783633 890067 1473042 1426374 1858047 1198197 2472541 1547758 3842804 3028170 3620681 3990575 3340991 3742765 L-methionine 1338230 554609 638729 502939 529339 806419 512093 900935 733178 1670837 2192819 1892379 719872 2616941 1536464 2396079 2405483 2913343 3613430 2828932 3707321 L-phenylalanine 2468934 902909 1128228 1140689 1653275 1881901 896611 1083727 1477662 3239717 5594190 3988638 2808373 7567876 3845538 11967625 8546533 10313005 10278221 8846199 11518836 L-proline 39454060 30210782 34792632 23437432 20324630 18513670 20646074 18624878 18087934 57521500 63389940 56067836 77025335 103358763 98721408 156779264 208928000 163883616 201582800 171309072 203579344 L-serine 457781 324249 328491 286001 290196 272240 179314 223524 160772 312869 231610 327938 344772 490749 288311 352887 353896 391278 333171 412951 402498 L-tryptophan 1282012 585131 873946 944569 622973 822126 746464 1193731 1573371 1921773 2871082 2490902 1963525 2552822 2880190 6371548 5601689 7013410 8044466 7027866 10506373 L-tyrosine 2037328 1061413 1098707 1272214 1246444 1567663 897880 1178275 1894756 3414687 5651582 4534293 3326787 7554127 5231262 11312993 10333695 9331108 12412836 11226343 13151401 L-valine 4695606 2692270 3279378 3613651 3597688 4347600 2354017 2500823 2389315 8900581 11537493 9610325 6656881 13082657 8443100 17581688 15920189 16689760 19586330 18542328 20908656 AMP 860446 336665 271773 630369 451980 466671 439476 406210 587969 6409579 5445622 4599911 996035 2428195 1770936 4592380 5994554 3584523 4306714 4555776 6060747 dAMP 3613261 2563459 2681258 2395627 2606435 2759197 1922151 1462237 2335833 2373944 2629270 1981122 2820080 4527750 1978434 2172921 1861556 2415155 3012967 3088548 3601963 Guanine 101856 70412 80799 167594 136586 147980 95811 95276 94149 483469 430199 453221 378501 523752 381463 510860 639768 552857 561788 770849 663513 Cytosine 497409 421441 401943 366818 333330 469195 290457 450488 409012 451109 594435 580399 592654 1166391 947395 788768 1181536 698163 1001950 1203040 1427775 IMP 107641 40179 51328 14822 13260 43164 0 5951 15231 146053 197226 105264 0 45657 14575 153342 141153 72611 129510 159756 201231 Hypoxanthine 150250 76412 112194 184487 237044 218307 124944 119178 219700 517614 816015 500597 164965 709869 573404 976826 1063767 751227 638946 865001 1225947 Xanthine 825534 484233 567826 623160 609043 638761 336641 374131 710534 1175015 1517726 1072632 541427 843806 563468 894505 900533 1193140 1396249 1162182 1148687 Allantoate 433479 266012 230050 271257 266480 289397 206809 177710 228919 157471 188259 127413 168686 280974 211876 294843 205226 304274 313691 336085 340635 (S)(+)-Allantoin 198817 146866 188458 152337 172099 174935 124349 141530 145279 78022 118310 106007 168180 126722 111490 106869 124181 123938 198383 205889 172157 5-Hydroxyisourate 628556 462489 478663 331541 253715 384592 218576 284448 247482 271127 323583 281447 106656 815169 237131 2964 0 0 65066 40321 65365 5-6-Dihydrothymine 34483068 23693958 26608506 28659574 20079546 18359934 16728652 12933572 15613344 21911384 22546366 22737262 15053636 16790354 16092063 14067848 19015554 14470289 16226844 14807575 21095214 5-Phosphoribosylamine 63197 25258 43043 32839 51914 50521 19418 13253 21519 31139 35685 35135 32329 99335 28472 10852 10017 20430 23738 16805 30326 NADP+ 8746 5308 5411 3611 2286 3535 2415 2358 2064 12721 19689 5280 0 4347 2521 7212 5266 7309 7934 3923 5420 NAD+ 363124 387708 398125 357932 267224 229844 280618 270073 219235 800543 818706 744664 343505 358602 343804 493049 783663 437169 366194 304284 369183 Glutathione 54107968 35608836 35735488 32366330 29995442 36582620 26114960 22536474 32550454 37553480 52326560 35421036 34734837 90444640 47796016 42198804 44958556 43964852 53895744 53797900 61683712 Glutathione disulfide 151311 66800 62666 75027 30974 92307 78101 136747 114568 13905 45127 29697 48282 174858 19402 0 14187 32642 41360 0 0 Cys-Gly 2055869 1390323 1396685 1406967 1404327 1422290 911547 702804 1081459 1686030 2464798 1651000 1505232 3874947 1880743 1743020 1892662 2004216 2193128 2597672 2766490 gamma-L-Glutamyl-L-cysteine 90062 43133 43661 30528 25206 29684 23775 40845 66892 85797 93018 77990 63083 226820 139698 180124 166245 124125 277306 258891 244770 gamma-Glutamyl-gamma-aminobutyrate 69496 33009 56306 32104 18224 25150 24133 48172 22079 42659 66628 41628 33261 79925 50484 47225 67713 37590 23232 53104 29941 (5-L-Glutamyl)-L-glutamine 19632 26071 13323 5848 4736 6659 16012 47558 5478 15827 10212 15124 0 12782 14690 17326 17845 7086 14117 14549 25864 Phosphoserine 269569 141071 165169 178531 136779 115609 91545 122101 66406 146062 248119 148463 112258 632252 217706 335340 259234 219733 345834 355190 508183 S-Adenosyl-L-homocysteine 2727410 1850251 2240237 1546308 1355769 2965705 1677931 2274584 3218634 1811111 1896572 1909163 666773 1153087 773050 2748906 2338689 2959707 2334424 2419189 3506954 S-Adenosyl-L-methionine 0 16840 30709 25819 8780 22128 6462 9678 0 103742 90809 69884 0 24845 0 71194 120557 70545 72104 40073 80594 Dihydrofolate 30070 35671 42632 28607 24210 53812 6438 12280 0 139495 214260 142390 15259 874251 91175 135080 173767 136403 149338 90601 122226 Ornithine 64887 47814 18112 90255 53215 57694 63273 71084 73314 64745 67798 94997 184577 235415 175328 468147 364008 459044 506469 371127 562718 Putrescine 606486 515700 496577 874997 306939 594818 814395 912910 1009799 287494 290328 307062 1334588 632975 498619 108880 179910 258260 368008 351322 496581 Spermidine 2592205 2078570 2301700 2896634 1713079 1052333 1858549 1126325 2645965 1998309 2022264 1312572 3188935 1542878 1649131 2426671 2132823 2184467 2858271 3142191 4888222 Spermine 4314714 3422262 4138145 5542582 2973267 2284490 2164615 1855854 3083762 3492935 4700182 2319662 4031264 1602813 1892784 4042008 3491443 3320949 3728513 4790674 6382138 5-Guanidino-2-oxopentanoate 38470 29384 25971 15023 9471 36434 7299 20139 16223 68235 101100 90709 45737 44878 38929 69070 15533 57855 33663 24498 47902 Phosphocreatine 85444 89993 66096 28339 22365 9247 29648 43386 34496 7145 15724 7908 157982 336709 131591 228529 276568 175498 408549 212011 158744 Creatine 6003539 4347734 5135205 5256154 3876405 3625198 5650521 3218442 6181631 4138414 4178757 4802675 32230127 33753233 27108680 14374964 19464638 14957600 33778812 28889046 40062540 L-Methionine S-oxide 314681 137637 192974 238040 98039 160338 88660 9888 343759 253039 240881 270449 242242 202629 199171 224993 320056 272326 322393 204047 429402 5-Hydroxyindoleacetate 2267699 1545081 1931648 3170149 3343764 2474135 1604854 1953398 3671892 5985373 8728687 4126271 3992547 4815531 2782639 4176237 3586664 5909432 6916318 4681550 5520661 3-Methyleneoxindole 107152 53963 80183 90348 65176 66368 66550 97720 165493 179306 213356 225837 157531 256274 223542 426576 577015 544423 668848 585054 733651 Indole-3-acetaldehyde 16258 7863 9158 8043 9806 5592 6661 8227 16198 16153 15138 18248 19292 13365 15308 30359 39583 37011 48443 40275 57197 Anthranilate 107896 51466 63549 88223 105277 59003 88888 113121 87590 68884 83339 54532 236184 188246 145491 261268 187132 342358 368243 405951 560916 Picolinic acid 7872763 3668165 5027524 3564273 3213988 5641978 2960037 2169949 3646190 4704573 5876007 3552649 3578794 11460950 3955850 8905713 6026351 9767065 11789083 9029519 9589800 L-Adrenaline 438167 487918 445723 966813 457088 276066 982530 1251190 396720 776485 815965 948643 533204 369765 681661 695726 911563 531667 846062 626179 1093487 Sphingosine 252452 178119 228912 238124 249803 246517 255952 214015 238006 272476 223271 267933 254720 495664 282165 447232 417038 514180 846276 1007126 1242237 Acetylcholine 232137 99083 82606 146539 91054 81238 98927 173683 241862 172022 209535 165273 148845 169254 161001 229717 231247 225247 334111 332073 509935 Choline 42550316 24341138 23126848 20301534 22292150 24356130 16052569 19331306 23546426 29097272 33566392 26658618 17547937 33783149 19616054 37964412 33402758 45059636 42786596 32522038 46812380 L-Carnitine 35601692 35143088 35953980 26288738 19670018 18709806 20403746 14272360 21267682 48911716 45193472 44014180 35952091 44926368 25597474 32310130 38277276 27101302 21069590 18081840 19688474 acetyl-carnitine (acyl-C2) 12931132 9787483 10282974 8563092 8352750 8851627 5720001 6747540 8211040 10547805 12423560 10187423 30886889 42305862 26406226 20471078 19231012 20476626 25231086 23505686 26389558 propionyl-carnitine (acyl-C3) 2320681 1711420 1611952 1187577 1466204 1444592 508449 419951 468947 2763054 2996341 2432156 6004714 9353307 5072132 5022698 5368174 5021399 4058268 4143886 4619148 butanoyl-l-carnitine (acyl-C4) 4600712 3034695 3215976 5449521 3366058 2383139 4352835 5862236 5208194 4684447 4377768 4699226 2130299 2036941 2493717 2014827 2497099 1883018 1936108 1418187 1842924 acyl-C4-OH 161407 102993 97623 22487 29389 38581 82342 57780 79476 58604 65862 42683 156791 206878 129864 36169 79823 27462 64992 48122 115073 acyl-C4-DC 23979 20794 15515 38314 31402 35491 31125 23960 34798 37753 26738 31279 23615 58277 37453 76318 59911 81416 98859 86582 98933 acyl-C5:1 49347 39139 40404 20727 23068 19842 17095 31946 29426 26434 30207 30062 81231 99715 79856 73068 55988 72303 94992 81110 103966 acyl-C5-OH 59007 45337 65631 2783 4083 3319 288265 338516 352599 171042 232015 220465 168150 259131 158732 88575 87081 92250 72262 97132 118087 hexanoyl-L-carnitine (acyl-C6) 38285 22585 20241 73636 49207 37440 73027 65691 80330 36414 37629 34345 18652 25009 28510 28910 20853 29483 40692 37013 63029 O-dodecanoyl-carnitine (acyl-C12) 76501 23583 25409 57623 45418 37901 13984 4909 12739 76220 83955 68352 12555 52577 12167 38062 33692 46575 32292 43267 61086 O-tetradecanoyl-L-carnitine (acyl-C14) 42046 22155 11031 22848 19482 15379 17784 22572 34733 79066 114063 98876 61461 81721 73981 136653 107078 133231 211427 208204 240115 Tetradecenoyl Carnitine (acyl-C14:1) 41003 32526 19070 12709 13991 8505 18969 9533 16265 85106 120939 82293 35034 61333 67014 123349 143116 161229 206327 188329 269990 L-Palmitoylcarnitine (acyl-C16) 126240 37286 50158 40054 32679 34047 49903 53863 78130 176216 246848 175338 134555 179814 160236 246834 226018 274325 332063 319688 452637 Hexadecenoyl-carnitine (acyl-C16:1) 131603 67375 68360 30986 28222 28726 25790 30837 57658 221093 307297 277538 112914 223920 193518 365150 314543 310051 356344 426453 426186 Octadecanoyl-L-carnitine (acyl-C18) 13101 27260 5035 2366 12990 5798 9758 477 6505 5235 27841 9471 19784 29806 11161 19739 13257 61939 54394 68800 24960 O-octadecenoyl-L-carnitine (acyl-C18:1) 159995 52836 65251 37025 26354 29980 9897 14274 25803 268507 207237 250188 131698 109009 184765 292750 278401 295880 330346 424023 524810 acyl-C18:2 (Linoleoyl-CoA) 33537 15294 17078 7105 6262 6973 701 2875 5307 83410 78715 108954 30419 35758 53066 92987 76913 110107 122501 133578 164349 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID mz rt L-arginine C00062 175.1192 0.6620 L-histidine C00135 156.0771 0.6350 L-leucine/isoleucine C00123 132.1023 0.7650 L-lysine C00047 147.1131 0.6310 L-methionine C00073 150.0577 0.7240 L-phenylalanine C00079 166.0865 0.9150 L-proline C00148 116.0712 0.7070 L-serine C00065 106.0506 0.7040 L-tryptophan C00078 205.0976 1.7700 L-tyrosine C00082 182.0813 0.7230 L-valine C00183 118.0867 0.7100 AMP C00020 348.0707 0.7100 dAMP C00360 332.0797 0.7130 Guanine C00242 152.0569 0.7160 Cytosine C00380 112.0511 0.7120 IMP C00130 349.0574 0.7130 Hypoxanthine C00262 137.0460 0.7340 Xanthine C00385 153.0396 0.6170 Allantoate C00499 177.0598 0.7110 (S)(+)-Allantoin C02350 159.0487 0.7180 5-Hydroxyisourate C11821 185.0323 0.6860 5-6-Dihydrothymine C00906 129.0662 0.6960 5-Phosphoribosylamine C03090 230.0418 0.6740 NADP+ C00006 744.0825 0.7920 NAD+ C00003 664.1168 0.7070 Glutathione C00051 308.0909 0.7200 Glutathione disulfide C00127 613.1593 0.7170 Cys-Gly C01419 179.0484 0.7170 gamma-L-Glutamyl-L-cysteine C00669 251.0705 0.7190 gamma-Glutamyl-gamma-aminobutyrate C15767 233.1137 0.7080 (5-L-Glutamyl)-L-glutamine C05283 276.1192 0.7050 Phosphoserine C02532 186.0170 0.6870 S-Adenosyl-L-homocysteine C00021 385.1299 1.8110 S-Adenosyl-L-methionine C00019 399.1450 0.6760 Dihydrofolate C00415 444.1546 0.6800 Ornithine C01602 133.0975 0.6350 Putrescine C00134 89.1082 0.6390 Spermidine C00315 146.1655 0.6320 Spermine C00750 203.2234 0.6340 5-Guanidino-2-oxopentanoate C03771 174.0873 0.7130 Phosphocreatine C02305 212.0433 0.7110 Creatine C00300 132.0771 0.6970 L-Methionine S-oxide C02989 166.0528 0.9020 5-Hydroxyindoleacetate C05635 192.0620 0.6140 3-Methyleneoxindole C02796 146.0604 1.7740 Indole-3-acetaldehyde C00637 160.0761 1.8060 Anthranilate C00108 138.0549 0.7160 Picolinic acid C10164 124.0412 0.6290 L-Adrenaline C00788 184.0972 1.7520 Sphingosine C00319 300.2901 2.0610 Acetylcholine C01996 146.1179 1.7760 Choline C00114 104.1077 0.6780 L-Carnitine C00318 162.1127 0.6890 acetyl-carnitine (acyl-C2) C02571 204.1234 0.7170 propionyl-carnitine (acyl-C3) C03017 218.1391 0.7180 butanoyl-l-carnitine (acyl-C4) C02862 232.1548 1.7660 acyl-C4-OH HMDB13127 248.1495 0.7170 acyl-C4-DC HMDB13133 262.1292 1.8200 acyl-C5:1 HMDB02366 244.1548 1.8040 acyl-C5-OH ac107 262.1652 0.7250 hexanoyl-L-carnitine (acyl-C6) HMDB00756 260.1862 1.8260 O-dodecanoyl-carnitine (acyl-C12) HMDB02250 344.2801 1.9920 O-tetradecanoyl-L-carnitine (acyl-C14) HMDB05066 372.3108 2.0770 Tetradecenoyl Carnitine (acyl-C14:1) HMDB13329 370.2961 2.0230 L-Palmitoylcarnitine (acyl-C16) C02990 400.3427 2.2880 Hexadecenoyl-carnitine (acyl-C16:1) HMDB06317 398.3274 2.0950 Octadecanoyl-L-carnitine (acyl-C18) HMDB00848 428.3728 2.3250 O-octadecenoyl-L-carnitine (acyl-C18:1) HMDB06351 426.3584 2.3100 acyl-C18:2 (Linoleoyl-CoA) HMDB01064 424.3427 2.1680 METABOLITES_END #END