#METABOLOMICS WORKBENCH juliehaines_20210702_093259 DATATRACK_ID:2737 STUDY_ID:ST001864 ANALYSIS_ID:AN003022 PROJECT_ID:PR001177 VERSION 1 CREATED_ON July 17, 2021, 11:15 am #PROJECT PR:PROJECT_TITLE Targeting host glycolysis as a strategy for antimalarial development PR:PROJECT_SUMMARY Glycolysis controls cellular energy, redox balance, and biosynthesis. PR:PROJECT_SUMMARY Antiglycolytic therapies are under investigation for treatment of obesity, PR:PROJECT_SUMMARY cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is PR:PROJECT_SUMMARY highly valued as a therapeutic strategy, because glycolytic disruption is PR:PROJECT_SUMMARY generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting PR:PROJECT_SUMMARY side effect of these inhibitors and presents a major caveat to development of PR:PROJECT_SUMMARY antiglycolytic therapies. We developed specific inhibitors of enolase – a PR:PROJECT_SUMMARY critical enzyme in glycolysis – and validated their metabolic and cellular PR:PROJECT_SUMMARY effects on human erythrocytes. Enolase inhibition increases erythrocyte PR:PROJECT_SUMMARY susceptibility to oxidative damage and induces rapid and premature erythrocyte PR:PROJECT_SUMMARY senescence, rather than direct hemolysis. We apply our model of red cell PR:PROJECT_SUMMARY toxicity to address questions regarding erythrocyte glycolytic disruption in the PR:PROJECT_SUMMARY context of Plasmodium falciparum malaria pathogenesis. Our study provides a PR:PROJECT_SUMMARY framework for understanding red blood cell homeostasis under normal and disease PR:PROJECT_SUMMARY states and clarifies the importance of erythrocyte reductive capacity in malaria PR:PROJECT_SUMMARY parasite growth. PR:INSTITUTE University of Colorado Anschutz Medical Campus 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 Targeting host glycolysis as a strategy for antimalarial development ST:STUDY_SUMMARY Glycolysis controls cellular energy, redox balance, and biosynthesis. ST:STUDY_SUMMARY Antiglycolytic therapies are under investigation for treatment of obesity, ST:STUDY_SUMMARY cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is ST:STUDY_SUMMARY highly valued as a therapeutic strategy, because glycolytic disruption is ST:STUDY_SUMMARY generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting ST:STUDY_SUMMARY side effect of these inhibitors and presents a major caveat to development of ST:STUDY_SUMMARY antiglycolytic therapies. We developed specific inhibitors of enolase – a ST:STUDY_SUMMARY critical enzyme in glycolysis – and validated their metabolic and cellular ST:STUDY_SUMMARY effects on human erythrocytes. Enolase inhibition increases erythrocyte ST:STUDY_SUMMARY susceptibility to oxidative damage and induces rapid and premature erythrocyte ST:STUDY_SUMMARY senescence, rather than direct hemolysis. We apply our model of red cell ST:STUDY_SUMMARY toxicity to address questions regarding erythrocyte glycolytic disruption in the ST:STUDY_SUMMARY context of Plasmodium falciparum malaria pathogenesis. Our study provides a ST:STUDY_SUMMARY framework for understanding red blood cell homeostasis under normal and disease ST:STUDY_SUMMARY states and clarifies the importance of erythrocyte reductive capacity in malaria ST:STUDY_SUMMARY parasite growth. ST:INSTITUTE University of Colorado Anschutz Medical Campus 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 #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 - 2 group:Control, 0h RAW_FILE_NAME=2 SUBJECT_SAMPLE_FACTORS - 4 group:Control, 0h RAW_FILE_NAME=4 SUBJECT_SAMPLE_FACTORS - 6 group:Control, 0h RAW_FILE_NAME=6 SUBJECT_SAMPLE_FACTORS - 20 group:POMSF, 0h RAW_FILE_NAME=20 SUBJECT_SAMPLE_FACTORS - 22 group:POMSF, 0h RAW_FILE_NAME=22 SUBJECT_SAMPLE_FACTORS - 24 group:POMSF, 0h RAW_FILE_NAME=24 SUBJECT_SAMPLE_FACTORS - 26 group:POMHEX, 0h RAW_FILE_NAME=26 SUBJECT_SAMPLE_FACTORS - 28 group:POMHEX, 0h RAW_FILE_NAME=28 SUBJECT_SAMPLE_FACTORS - 30 group:POMHEX, 0h RAW_FILE_NAME=30 SUBJECT_SAMPLE_FACTORS - 32 group:Control, 0.5h RAW_FILE_NAME=32 SUBJECT_SAMPLE_FACTORS - 34 group:Control, 0.5h RAW_FILE_NAME=34 SUBJECT_SAMPLE_FACTORS - 36 group:Control, 0.5h RAW_FILE_NAME=36 SUBJECT_SAMPLE_FACTORS - 50 group:POMSF, 0.5h RAW_FILE_NAME=50 SUBJECT_SAMPLE_FACTORS - 52 group:POMSF, 0.5h RAW_FILE_NAME=52 SUBJECT_SAMPLE_FACTORS - 54 group:POMSF, 0.5h RAW_FILE_NAME=54 SUBJECT_SAMPLE_FACTORS - 56 group:POMHEX, 0.5h RAW_FILE_NAME=56 SUBJECT_SAMPLE_FACTORS - 58 group:POMHEX, 0.5h RAW_FILE_NAME=58 SUBJECT_SAMPLE_FACTORS - 60 group:POMHEX, 0.5h RAW_FILE_NAME=60 SUBJECT_SAMPLE_FACTORS - 62 group:Control, 1h RAW_FILE_NAME=62 SUBJECT_SAMPLE_FACTORS - 64 group:Control, 1h RAW_FILE_NAME=64 SUBJECT_SAMPLE_FACTORS - 66 group:Control, 1h RAW_FILE_NAME=66 SUBJECT_SAMPLE_FACTORS - 80 group:POMSF, 1h RAW_FILE_NAME=80 SUBJECT_SAMPLE_FACTORS - 82 group:POMSF, 1h RAW_FILE_NAME=82 SUBJECT_SAMPLE_FACTORS - 84 group:POMSF, 1h RAW_FILE_NAME=84 SUBJECT_SAMPLE_FACTORS - 86 group:POMHEX, 1h RAW_FILE_NAME=86 SUBJECT_SAMPLE_FACTORS - 88 group:POMHEX, 1h RAW_FILE_NAME=88 SUBJECT_SAMPLE_FACTORS - 90 group:POMHEX, 1h RAW_FILE_NAME=90 SUBJECT_SAMPLE_FACTORS - 122 group:Control, 6h RAW_FILE_NAME=122 SUBJECT_SAMPLE_FACTORS - 124 group:Control, 6h RAW_FILE_NAME=124 SUBJECT_SAMPLE_FACTORS - 126 group:Control, 6h RAW_FILE_NAME=126 SUBJECT_SAMPLE_FACTORS - 140 group:POMSF, 6H RAW_FILE_NAME=140 SUBJECT_SAMPLE_FACTORS - 142 group:POMSF, 6H RAW_FILE_NAME=142 SUBJECT_SAMPLE_FACTORS - 144 group:POMSF, 6H RAW_FILE_NAME=144 SUBJECT_SAMPLE_FACTORS - 146 group:POMHEX, 6H RAW_FILE_NAME=146 SUBJECT_SAMPLE_FACTORS - 148 group:POMHEX, 6H RAW_FILE_NAME=148 SUBJECT_SAMPLE_FACTORS - 150 group:POMHEX, 6H RAW_FILE_NAME=150 #COLLECTION CO:COLLECTION_SUMMARY Banked blood (Saint Louis Children’s Hospital) and fresh blood [healthy CO:COLLECTION_SUMMARY donors] was washed and stored at 50% hematocrit at 4ºC up to 1 month past the CO:COLLECTION_SUMMARY clinical expiration date and collection date, respectively. Parasite strains CO:COLLECTION_SUMMARY provided by the Malaria Research and Reference Reagent Resource Center (MR4) as CO:COLLECTION_SUMMARY follows: 3D7 (MRA-102); K1 (MRA-159); D10 (MRA-201); and IPC-5202 (MRA-1240). CO:COLLECTION_SUMMARY Unless indicated, erythrocytes were cultured as previously described at 2% CO:COLLECTION_SUMMARY hematocrit in complete media (incomplete media supplemented with 27mM sodium CO:COLLECTION_SUMMARY bicarbonate, 11mM glucose, 5mM HEPES, 1mM sodium pyruvate, 0.37mM hypoxanthine, CO:COLLECTION_SUMMARY 0.01mM thymidine, 10ug/mL gentamicin, and 0.5% Albumax (Thermo-Fisher) under 5% CO:COLLECTION_SUMMARY O2/5% CO2/90% N2 atmosphere at 37ºC (71). CO:SAMPLE_TYPE Blood (whole) #TREATMENT TR:TREATMENT_SUMMARY Freshly collected erythrocytes were washed, resuspended to 10% hematocrit in TR:TREATMENT_SUMMARY buffer [25mM HEPES (pH 7.4), 120mM NaCl, 5.4mM KCl, 1.8mM CaCl2, and 1mM TR:TREATMENT_SUMMARY NaH2PO4], incubated for 1 hour at 37ºC, then centrifuged at 2000xg for 5 TR:TREATMENT_SUMMARY minutes at 4ºC, resuspended in fresh wash buffer to 30-40% hematocrit, and TR:TREATMENT_SUMMARY split into 210uL aliquots of packed erythrocytes. Erythrocytes were resuspended TR:TREATMENT_SUMMARY in 253uL of RPMI containing 11.9mM D-[1,2,3-13C] glucose (Millipore-Sigma) and TR:TREATMENT_SUMMARY either treated with POM-SF or POM-HEX at five times the EC50s for MetHb TR:TREATMENT_SUMMARY formation, or an untreated solvent control with the balance volume to 550uL TR:TREATMENT_SUMMARY using washing buffer. All samples were incubated at 37ºC shaking at 500 RPM. TR:TREATMENT_SUMMARY Triplicate samples of supernatants and packed erythrocytes were collected at 0, TR:TREATMENT_SUMMARY 0.5, 1, and 6-hour intervals and snap-frozen in liquid N2. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY RBC and media samples were extracted at 1:10 and 1:20 dilutions, respectively, SP:SAMPLEPREP_SUMMARY in ice-cold 5:3:2 MeOH:MeCN:H2O v/v/v, with vigorous vortexing at 4 degrees C SP:SAMPLEPREP_SUMMARY for 30 min. The extraction buffer was supplemented with 40uM 3-13C lactate to SP:SAMPLEPREP_SUMMARY allow absolute quantification of lactate isotopologues, including lactate SP:SAMPLEPREP_SUMMARY generated via glycolysis ([1,2,3-13C3] enriched) vs. the pentose phosphate SP:SAMPLEPREP_SUMMARY pathway ([2,3-13C2] enriched). Supernatants were clarified via centrifugation SP:SAMPLEPREP_SUMMARY (10,000 g, 10 min, 4 C) then analyzed by UHPLC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 10 μl of RBC extracts were injected and separated through a 3 min isocratic CH:CHROMATOGRAPHY_SUMMARY elution on a Kinetex XB-C18 column (150 × 2.1 mm i.d., 1.7 μm particle size CH:CHROMATOGRAPHY_SUMMARY – Phenomenex, Torrance, CA, USA) at 250 μl/min (mobile phase: 5% CH:CHROMATOGRAPHY_SUMMARY acetonitrile, 95% 18 mΩ H2O, 0.1% formic acid; column temperature: 25°C). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Vanquish CH:COLUMN_NAME Kinetex XB-C18 2.1 x 150 mm, 1.7 um CH:FLOW_GRADIENT isocratic CH:FLOW_RATE 250 ul/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, MS:MS_COMMENTS San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution MS:MS_COMMENTS from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, MS:MS_COMMENTS operated in positive ion mode. Calibration was performed before each analysis MS:MS_COMMENTS using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, MS:MS_COMMENTS USA). Limits of detection (LOD) were characterized by determining the smallest MS:MS_COMMENTS injected amino acid amount required to provide a signal to noise (S/N) ratio MS:MS_COMMENTS greater than three using < 5 ppm error on the accurate intact mass. Based on a MS:MS_COMMENTS conservative definition for Limit of Quantitation (LOQ), these values were MS:MS_COMMENTS calculated to be three fold higher than determined LODs. MS data acquired from MS:MS_COMMENTS the QExactive was converted from .raw file format to.mzXML format using MS:MS_COMMENTS MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MS:MS_COMMENTS MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak MS:MS_COMMENTS picking, feature detection and metabolite assignment against the KEGG pathway MS:MS_COMMENTS database. Assignments were further confirmed using a process for chemical MS:MS_COMMENTS formula determination using isotopic patterns and accurate intact mass (Clasquin MS:MS_COMMENTS et al. 2012). Analyte retention times were confirmed by comparison with external MS:MS_COMMENTS standard retention times, as indicated above. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 2 4 6 20 22 24 26 28 30 32 34 36 50 52 54 56 58 60 62 64 66 80 82 84 86 88 90 122 124 126 140 142 144 146 148 150 Factors group:Control, 0h group:Control, 0h group:Control, 0h group:POMSF, 0h group:POMSF, 0h group:POMSF, 0h group:POMHEX, 0h group:POMHEX, 0h group:POMHEX, 0h group:Control, 0.5h group:Control, 0.5h group:Control, 0.5h group:POMSF, 0.5h group:POMSF, 0.5h group:POMSF, 0.5h group:POMHEX, 0.5h group:POMHEX, 0.5h group:POMHEX, 0.5h group:Control, 1h group:Control, 1h group:Control, 1h group:POMSF, 1h group:POMSF, 1h group:POMSF, 1h group:POMHEX, 1h group:POMHEX, 1h group:POMHEX, 1h group:Control, 6h group:Control, 6h group:Control, 6h group:POMSF, 6H group:POMSF, 6H group:POMSF, 6H group:POMHEX, 6H group:POMHEX, 6H group:POMHEX, 6H L-alanine 7242486 8912985 7158126 6401872 7707776 9548117 7276286 6965748 9276854 7480886 11017300 8467653 8488872 9986075 10053640 11022420 9016195 11613360 11535820 9231328 11538390 8346966 9138009 11150540 11237570 9082003 10491410 10969840 11443560 11424540 8349172 9189227 8309714 7742060 6765194 7989718 L-arginine 3645470 3443846 9090948 10991800 7604968 2766940 7336580 15088100 4282606 13723380 8684725 19371740 8178514 5942294 8911512 8785005 13727590 10864640 0 13744450 0 10190230 10068300 0 4177597 11876080 3039427 5252256 3209683 0 2946548 0 2669604 4957038 6708256 3717685 L-asparagine 1955497 1718105 1780944 1890954 2563879 2415541 3068902 3142219 2945099 3395971 3729742 3916355 3697817 4013357 4488063 5661364 6039170 5522350 7475794 8221690 7636048 7511366 6724548 7843522 7529444 8128166 7162482 8130102 8022632 7240662 5765030 5602932 5223490 5500968 5875878 6245446 L-aspartate 11091010 13810000 14237900 17090460 22648230 20645570 35189230 32183950 35373890 40054810 31004290 41877040 33760500 36995630 41818210 50653890 46697620 45450190 42812370 34213380 41737220 31242800 32609190 28977270 22781500 22855950 17495780 15131190 16100970 15237630 13088800 12512050 11753010 10782740 11882530 11221290 L-cysteine 164335 15456 21691 753697 333364 73529 410844 441704 192847 372697 617454 876904 561229 255438 157519 389729 984356 384315 839938 1901536 857355 1799691 1833489 569716 256841 1283310 621093 182424 450465 327922 396722 113805 220393 497907 597171 1046938 L-glutamate 11577210 14549840 17442730 18758910 22203190 23167270 39111810 36415790 41567720 47836980 36828700 44181400 32566450 34313870 36771310 36589290 35517710 35454460 37446450 28754190 33860430 31818100 26790660 24948110 21648810 21005850 17337580 15451160 16259690 13287320 7502046 6145646 6743000 5993644 6138418 6664012 L-glutamine 18782980 19098040 20496370 26240550 25889110 22819750 35105340 36487550 27667520 40028650 43453770 53001990 37675980 40447630 51119000 60019230 67716420 70323830 87982600 89650900 87960390 84990440 90075740 82648600 69670420 88069180 65724080 80429540 86487620 75386690 62602440 57636720 54822440 63653390 62558980 63527520 glycine 916092 1229423 1076504 991120 929621 1128718 1292235 1209066 1296391 1330217 1501164 1186888 1559153 1612522 1747246 2207561 2319184 2352487 2332139 2494888 2354494 1774210 2277206 2530172 2528167 2516934 2977812 2173097 1698986 1578259 1742885 1714175 1543826 1509661 1660320 1671999 L-histidine 1033545 1232930 1449159 1619643 1234682 1630573 1769342 1855329 1637418 1876483 1470164 1546089 1391019 1382119 1348738 1306931 1354423 1303530 1393839 1557032 1666253 1586777 1542917 1566890 1220038 1285775 1073028 1638212 1590312 1558378 1455397 1380310 1442633 1601860 1469214 1874259 L-leucine 84864420 98450840 113613900 98293860 121621000 95003460 113213700 129158500 101707700 176780100 145869600 209409200 170340900 183069700 203282700 215707300 281365200 204740400 269772900 174288200 286553600 227143600 253522000 230609800 197049200 284880100 135540700 236095600 182780800 234542100 126729300 158261800 214992900 146707300 146636200 127084400 L-isoleucine 27213850 31808080 18323510 30400470 29503980 22626360 0 27936670 20057970 26536840 18821960 28306620 17875020 16703940 16900170 17042920 15478430 16072940 15411240 9726021 15039160 12721400 12448730 10936780 9186887 13060150 8518843 13770660 14295000 16570240 11665480 13137060 14932190 12783540 14155870 11402200 L-lysine 3740801 3855109 4049775 3758556 3805278 3275392 3764694 3909943 3344321 3449402 2802153 3558222 2697722 2387239 2589775 2545018 2495028 1992931 2587068 2170731 2252804 2401434 2033049 1892300 1462514 1532769 1053325 1685797 1672237 1650726 1431318 1332538 1515191 1470367 1239543 1446493 L-methionine 9200364 11001770 12370150 10420790 12949040 9062012 11956280 12811740 9765501 17111030 13409880 19758990 15214200 16647130 18540080 19536960 25744730 18473870 25072570 16441860 26839330 22400440 23539650 22203540 18061030 29116580 15850100 21473350 19221410 21242210 12699880 14782820 18305190 15566940 16613260 12651970 L-phenylalanine 4000974 4370876 5234788 4951308 5411315 4321240 3891675 5354106 4427160 6541264 5620058 7398644 8078942 8858083 11243140 13667980 17993540 12454130 16507440 11649700 16523760 15787740 14930370 14305940 12185220 15606000 8200482 13037470 11015450 9639966 6889194 7467530 8392055 7055430 6501138 4974045 L-proline 25905060 31503310 37904130 38608690 36174930 32050070 38653320 40424470 27828480 44150680 42732580 61939070 42189960 34470820 39593780 48674620 52245730 38873150 52767020 54651640 76282780 65842720 59039420 53928540 41362770 62461570 46346440 73253990 77275160 85140290 72463830 66349620 67761050 63860800 54607500 59644550 L-serine 1668785 1634940 1541094 1543712 1815681 2047142 2274197 2299246 2071490 2664274 3157992 2801792 2932647 2658852 3792309 4555184 4840247 4454737 5539202 6387166 6063134 4641730 5259884 5997134 6193948 5885706 5910584 7095122 6610254 5793606 4639024 4683864 4281188 4503508 4599316 4861448 L-threonine 2399336 2632629 2622006 2655535 3056179 2691288 3337686 3502021 3234011 4629332 6548448 5786486 5712476 5049590 5980532 6272120 7919814 7084572 9317557 8192858 8955097 8463630 8550655 8853418 7846008 8675493 7029224 10112340 10771460 9588840 6950698 7993730 7078666 8822238 8151734 8771327 L-tryptophan 3875911 2971389 4179942 5640244 3842866 3636155 4610792 3916344 4513774 3918519 5951456 5314482 5933300 3776525 4714087 5024426 4783172 4620149 4634684 5083126 3705227 4996572 4414736 4348400 3997264 3893585 2668211 2242726 2249158 1770914 1426287 1207282 1712412 1850765 1630310 1266923 L-tyrosine 9819055 12384870 13186980 11105300 12673510 8942085 13109670 14429420 10609080 21223430 17103670 24344380 19447910 20923130 21612620 23678360 30120250 23727170 31843290 22086390 34124320 25777940 28736400 27511530 23887520 36199650 22133130 33006780 27436980 30090640 17352420 21653340 24970450 21338880 23044650 16856950 L-valine 94790780 120532000 111798200 91523130 114851800 138536000 124533000 119934100 119285300 130492700 140741900 146753700 133793600 145416600 141754600 170500400 166747300 160342600 154491000 126128600 170886600 153296800 146531900 151010300 159184700 133927300 166485600 143683400 163899700 181770300 173139900 188680000 172792700 159161400 162102000 147152300 AMP 848440 743273 969475 1480470 1217078 1210007 2427259 4082492 2384482 970419 2246575 1143026 2219571 1694654 1241942 2038929 4362031 1738786 506136 866012 921815 2138458 1657126 1771637 1959322 2601012 1218053 399988 294786 274563 502118 408555 585272 372148 497296 488108 Cytosine 50318 41932 36597 44987 51506 127229 85457 238140 474432 468114 353620 577592 561985 581079 575038 636887 552688 672873 584383 473589 416615 367906 333341 322381 475667 342725 266982 129029 96772 48291 80549 41630 51936 0 0 0 Xanthine 353816 331585 325432 284515 363070 338429 386500 378072 391009 337165 337079 288908 366141 357245 328598 339786 331695 309317 273730 191378 224273 230597 191415 189689 187881 142585 148391 120820 114168 93736 82895 98148 92447 78196 72041 83053 Allantoate 215979 291440 395496 591935 419994 147691 362687 439892 225726 384391 359695 572779 271115 203970 255959 252451 384609 239029 433968 494383 499867 665229 503563 314641 127648 405965 101548 555548 781967 678656 544744 305564 473965 578160 591710 852104 5-Hydroxyisourate 3520943 4484736 3342449 2986779 4004029 4097905 4576981 4137426 5159640 5484614 5767334 5158496 5173060 5159936 5684584 6716168 4602454 5109334 3484308 1241391 2658584 1498294 2098099 4214002 4866014 2622047 4928044 3002117 3287091 4557006 4596630 7026874 4569560 4805142 4493092 3269531 Nicotinamide 17695020 22932120 22942710 15687080 23502230 26625030 25644780 26082850 28934970 28186300 29146560 32267610 36853290 39379030 37216570 37370020 42312260 38969670 40452660 30969620 48213360 34616800 37797060 48575360 48045310 50795930 41800080 36360780 28538400 31589450 25434410 31762010 26684450 20164270 21607350 18233590 NAD+ 16014 14017 48961 42594 268418 234083 320487 325026 410885 415784 59048 217944 77617 198747 413750 523908 246839 445534 189261 98690 134427 87330 41062 43998 15669 44600 21461 19745 63968 110660 56510 40010 93017 52874 59460 98539 Glutathione 90790380 91277530 105289400 166742000 141376100 96583620 128866300 141526000 145170900 150883600 130681400 184168600 164095600 151838000 144792000 145879400 138207200 122325600 143605500 83234780 124156000 153599500 149582800 91690660 50645600 77735670 57434140 42323210 71690820 67953900 41436760 33306830 33673380 45890000 38679130 43347650 Cys-Gly 1265812 1298274 1476999 2173179 2001217 1458611 1911857 2026439 2129311 2041609 1874078 2805206 2554519 2282035 2127208 2174782 2132634 1970652 2184732 1201590 1961014 2317702 2136714 1316489 757079 1195785 808783 547757 1012563 914813 706261 527098 456492 780780 557222 770028 gamma-Glutamyl-gamma-aminobutyrate 72229 110308 101415 86917 89110 106730 80783 58981 110469 111527 89519 102344 99270 131652 100094 98433 113168 104677 77835 61285 113317 76316 89259 105686 90001 58860 72574 57841 99247 73181 58894 66443 59972 58716 52619 33564 5-L-Glutamyl-taurine 185262 163911 137646 127849 93753 174081 113217 105442 156925 118524 106527 100855 145472 153750 136048 165468 174022 86803 91788 120877 146223 111131 118202 115541 172503 93789 210974 163411 147519 160627 168545 169693 166476 149356 128339 134607 Dimethylglycine 1158315 1119542 1148019 887867 1007648 1451348 1126342 1121192 1220684 1023901 1313678 1124184 1143787 1172701 1056851 1057688 1081250 974504 922661 817410 997550 885609 1019251 895514 1121919 860458 1044726 733963 870545 893963 746152 819752 597643 687291 686462 597891 Phosphoserine 34901 75142 89006 48678 238263 225470 131097 159138 151921 185437 63095 115299 54285 259964 71349 51746 36270 35436 0 0 83926 0 0 34569 0 0 17697 0 0 68283 54102 59064 27359 23248 25682 10410 5-10-Methenyltetrahydrofolate 306445 253138 240917 232563 306028 371155 351552 349097 347523 334051 343353 264651 353605 416941 323701 306631 318900 347029 324880 73862 214135 105857 79596 106137 180627 174125 110128 93634 195933 199820 107596 127336 127304 153752 137489 105432 Dihydrofolate 683383 962222 1366938 2520762 2249420 796018 2172015 2079941 1275623 1771559 1713174 2183656 1241337 672423 1265272 1716039 1969456 2050220 2016746 1704250 1928295 3002905 2068950 984928 693795 1828188 654660 1128063 2025175 1963669 1854582 970433 1180881 1440768 1739643 1870007 Ornithine 2990241 2787284 2895834 2194669 2531277 2486040 2514672 2562544 2473310 3033119 2643493 2824507 2340844 2336708 2322689 2166915 2000352 1645857 2348722 1675975 2047026 1574158 1551047 1569893 1572732 1304313 1157778 2059251 1991110 1916218 1633977 1677024 1772457 1751728 1634916 1640906 L-Citrulline 1767039 1748466 1436237 1147182 1839881 2357700 2184273 2130999 2774189 2587890 3444301 2509326 3175462 3524434 3478369 4163411 4124376 4141121 3855932 3071037 3688271 2741721 3233512 3991063 5239894 3996319 4923732 3510993 3217070 2882749 2425838 3346727 2922783 2707606 3040124 2480167 Putrescine 73032 123540 85455 47345 62064 72670 50371 62643 78725 64359 65002 76024 82958 71188 67992 78162 78667 88250 78102 20499 67097 49616 28683 42348 40436 30431 26254 31370 38905 44683 37752 45487 44557 42404 26967 31081 Spermidine 9669072 10973220 11595560 11591200 9798829 10462710 9432251 10076880 9634518 8995980 8291308 8658556 9648868 9194941 9183828 10982440 8207350 9509229 10787930 6066270 9713023 8368386 7923916 6823004 7322232 6779582 6254818 5520509 6985576 7388376 6370488 6429104 6269272 6663330 5528966 5684254 Spermine 7537668 8315000 9062967 9695125 8727847 7987754 8859790 9580675 9081707 9078657 7557918 11298180 9735713 8895951 10009370 11024820 9130809 8602339 11898880 6319848 10199840 10875730 11047970 7995280 7575438 7096642 6916938 6482126 9569657 9562685 8120150 8005472 7754088 8482888 7611702 6886198 alpha-D-Glucosamine 1-phosphate 1754212 2294460 2059948 1104679 1629167 3066858 1717910 1655192 2323316 1832612 1921680 1978100 2085126 2400531 2178979 2318962 1610935 1743297 1454972 622140 1390976 445390 615148 857838 885785 447527 1206572 556250 483712 891138 854331 1674888 1259018 963190 822304 547638 D-Glucosamine 100599 71465 43228 101862 80725 148024 65635 126447 110609 155612 243234 114112 172754 202140 187680 257111 305071 216461 157444 275806 172217 229347 126798 204138 603082 334369 1047878 387256 240532 315223 284479 282166 201156 333472 252679 270924 Carnosine 292183 368499 318063 173913 221355 349476 222104 172612 252196 243178 248919 175830 264054 293412 291329 265699 153550 251971 223936 122431 204551 106274 94538 202780 208897 171191 189571 130503 138071 155862 167635 225552 166394 145710 152171 115481 Creatine 234414600 318146100 270408200 221715200 262051900 327596100 263908300 263017300 295904900 263065500 302022600 310500600 298583300 328117300 301746400 382087200 313305400 315468600 300368000 203992800 328406900 246046800 226030900 223187200 257064200 212314400 230439100 179072800 180543300 159443500 160293200 166273000 156512900 136700500 141876800 127181700 Creatinine 3432043 4822214 4392838 3206365 3745552 4224918 3898479 3592886 3780629 3547669 3718582 4339918 3458522 3247964 2949527 3989326 3093836 3733526 3069319 2187872 3433499 2765076 2618535 2869582 2384491 2514293 2494148 2233818 2653736 2681164 2213653 2245138 2283341 2180963 2317730 1846364 4-Acetamidobutanoate 482838 605201 515242 500313 569969 792565 704801 681390 853011 805159 863213 716805 793028 854494 872988 1131192 1109252 904544 987170 815404 974823 890785 978190 1036717 1121744 927219 1254033 994681 1057988 1143814 1112732 1240005 1028027 1031214 975830 893186 Pantothenol 368701 281903 343141 350304 466036 286176 272508 350689 334964 289268 337690 207834 268863 246874 208466 274857 189366 158193 200947 178337 188249 234083 240826 190643 193157 132865 188660 126092 191566 182721 224320 254855 264480 282305 218752 217982 Taurine 299653 291009 258042 271240 361676 429164 424303 376886 531447 464208 756434 657622 739618 1034483 1053512 1393084 1120399 1329468 1206362 1526766 1547151 1135181 1164582 1538401 1773471 1312687 2028976 1075145 1189036 1262806 1345898 1220096 977457 964803 907633 1249708 Hypotaurine 0 0 33466 0 0 30993 34868 15679 30015 15560 29297 0 52271 41610 51476 36888 40751 42898 30815 60127 40631 62413 52968 51661 103467 51099 99880 54838 51221 40166 77569 35710 37590 70983 33421 59442 2S-5S-Methionine sulfoximine 25579 46253 41861 30311 36891 33168 36947 36744 42463 38795 35672 32821 34776 57898 31125 48843 36047 28976 33465 34188 63216 34471 36948 47274 57074 27488 38058 57584 18547 62312 68125 137515 68219 58641 68236 51953 L-Selenomethionine 66877 74609 62170 72186 99674 71547 74652 90598 93395 76412 85441 55979 67041 62951 59433 48320 56506 52817 54028 63222 70785 39314 65738 66273 89063 83908 77833 63030 64754 59376 44531 47659 64186 55508 66985 58627 3-Methyleneoxindole 289631 330826 395037 444247 372500 445986 689466 554842 558429 488045 715096 678767 767893 557601 624170 812286 632503 552382 661965 650956 303637 615007 629784 552821 639362 437321 382851 183625 283678 224255 123957 118613 108916 181389 61098 50112 Indole 0 45254 34280 0 0 7879 51743 110000 110694 129912 54871 134092 72125 70760 98240 245698 170484 200535 255474 141892 287735 84224 251938 138234 237965 297789 60205 179564 136399 201487 91036 171349 135763 119936 69563 13546 kynurenine 44778 47750 49273 91755 52583 23225 66973 72882 34793 56844 48807 71503 43185 24759 42470 43153 65784 51345 68870 60470 56763 94770 68226 46634 26487 49009 35126 38857 58373 63085 46718 35945 32710 46369 48343 57205 Anthranilate 2136706 2394115 1993240 1609968 2248408 2823925 2409012 2342804 2495756 2773476 2563938 2833630 2715907 2568390 2830827 3080599 3621297 2837360 2855851 2371713 3720293 2678246 3053952 3113083 3955486 3806074 4460602 3330015 3210149 3300941 3566868 3548797 3472605 3134830 3002063 2686948 L-Adrenaline 450330 684016 689073 768482 753492 533812 608148 768103 706670 595860 553183 663994 791391 625721 740050 763337 598169 620457 460741 352261 650787 531125 581400 535752 396648 439686 218937 303330 354834 367846 389723 346161 294346 362105 283502 363803 N-Methylethanolamine phosphate 49882480 65965330 47142250 28547390 46289080 58875180 46241220 39764690 46690290 46505040 43754470 40622520 39859450 46928930 42756600 41564330 35555920 34358640 24728300 10244570 21553970 12536960 10400600 10270420 19899090 7832400 11939380 7516694 9019291 9632585 8371394 16564570 12875210 10021600 9425862 7505018 L-Carnitine 21211810 31360660 23751390 20900810 21878360 28486170 22589830 20440680 26911520 20644770 22396530 23855580 26751310 28286090 26577670 29186070 22497960 27169700 23243610 15685010 23770330 18625500 18824590 19828420 19317950 14256860 12675050 11624360 12525140 13794300 14206890 14256750 13816500 12795580 11640490 11256240 Triacanthine 22164610 30042780 30998680 27036160 25951270 30706750 26886270 26795550 29032470 25269550 21375150 37561630 34400670 28139860 31260250 37119930 34166360 36829210 48913950 32952940 63466050 42589900 46588470 42131970 39231420 44387530 38153160 37865450 37399170 40524370 31742420 32096140 33392830 27578160 23469700 20526440 2-Methyleneglutarate 154078 186555 146330 81073 138201 272465 303800 277260 215683 298268 424735 225674 413694 345612 471790 555029 651401 370575 442526 463875 377695 400929 379619 409462 1025708 656045 1454474 617559 483273 516957 523402 522880 467619 571287 707295 431368 6-Lactoyl-5-6-7-8-tetrahydropterin 16996110 20905530 26160860 45772780 38416290 16698790 39367820 39099040 25926470 40051310 37203440 54721780 34146880 23333240 33607280 36374720 58450240 55746290 66647440 101763500 83126740 100368300 101032200 53336450 25027300 57319660 18590220 49194560 59179680 48531360 43135110 25993480 32699620 38726790 38461850 51770350 Methylenediurea 2417236 3569764 3002049 2374217 2941613 1786621 2799328 2569735 3234268 2712193 3285142 3337051 1706986 3689554 3217221 1860148 3034831 3408143 3326286 2204778 0 2632574 2339792 2464966 2743227 1639654 2537452 964346 1409285 1713689 718505 0 0 0 273716 1395920 3-Oxalomalate 2244073 2744972 2255156 2192783 2168116 2454102 1932854 1738194 2117245 2039326 1714721 1684613 1605792 1948504 1490777 1067926 1056530 1175766 834036 429826 808154 682818 667434 783731 753229 593127 804119 652252 1063626 1682338 1736982 2345188 2250576 1731914 1814147 1731486 cis-p-Coumarate 2070246 2729943 3061878 2992060 2938005 2030771 3021448 3072764 2393429 5003258 3905261 6058574 4748520 4509256 5415018 5653550 6679384 5152054 7647230 5185008 7913792 6106428 7067380 6952470 5421210 6948648 5240592 7867638 6509826 7041522 4099038 5263296 6045816 5010038 5209538 3974754 Poly-gamma-D-glutamate 13284100 13261030 13833430 17194170 20423520 15869960 22832790 23839590 18601020 26900940 28301590 35631510 24894430 27716280 34294160 39495050 46113520 48594600 58838240 59054840 60230940 57794520 61893020 55385120 45514340 60061630 43103990 54711650 58140170 50556000 42833100 38871560 39133470 44525350 42536610 44643690 N-Acyl-D-aspartate 191192 239223 216349 250239 271767 272112 281773 323992 289207 279018 235901 256854 246531 300573 249991 231176 255874 258130 225203 160350 205986 157384 116413 131125 138665 104645 77190 54150 50213 55369 61930 72225 71820 61607 54953 52563 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID parent medRt L-alanine C00041 90.0557 1.16 L-arginine C00062 175.1191 1.11 L-asparagine C00152 133.0609 1.17 L-aspartate C00049 134.0449 1.35 L-cysteine C00097 122.0273 1.20 L-glutamate C00025 148.0604 1.28 L-glutamine C00064 147.0765 1.17 glycine C00037 76.0402 1.17 L-histidine C00135 156.0769 1.14 L-leucine C00123 132.1021 1.22 L-isoleucine C00407 132.1020 1.63 L-lysine C00047 147.1129 1.02 L-methionine C00073 150.0584 1.22 L-phenylalanine C00079 166.0863 1.24 L-proline C00148 116.0710 1.20 L-serine C00065 106.0504 1.17 L-threonine C00188 120.0658 1.18 L-tryptophan C00078 205.0973 1.27 L-tyrosine C00082 182.0811 1.22 L-valine C00183 118.0866 1.19 AMP C00020 348.0704 1.82 Cytosine C00380 112.0510 1.34 Xanthine C00385 153.0399 1.00 Allantoate C00499 177.0610 1.08 5-Hydroxyisourate C11821 185.0322 1.17 Nicotinamide C00153 123.0556 1.22 NAD+ C00003 664.1161 1.75 Glutathione C00051 308.0909 1.70 Cys-Gly C01419 179.0485 1.70 gamma-Glutamyl-gamma-aminobutyrate C15767 233.1132 2.25 5-L-Glutamyl-taurine C05844 255.0631 1.13 Dimethylglycine C01026 104.0711 1.19 Phosphoserine C02532 186.0165 1.27 5-10-Methenyltetrahydrofolate C00445 457.1687 1.18 Dihydrofolate C00415 444.1613 1.18 Ornithine C01602 133.0974 1.02 L-Citrulline C00327 176.1031 1.17 Putrescine C00134 89.1081 1.02 Spermidine C00315 146.1653 1.03 Spermine C00750 203.2232 1.03 alpha-D-Glucosamine 1-phosphate C06156 260.0533 1.20 D-Glucosamine C00329 180.0867 1.19 Carnosine C00386 227.1152 1.12 Creatine C00300 132.0769 1.18 Creatinine C00791 114.0666 1.18 4-Acetamidobutanoate C02946 146.0812 1.19 Pantothenol C00864 220.1181 0.26 Taurine C00245 126.0223 1.18 Hypotaurine C00519 110.0275 1.18 2S-5S-Methionine sulfoximine C03829 181.0626 1.11 L-Selenomethionine C05335 198.0015 0.98 3-Methyleneoxindole C02796 146.0602 1.27 Indole C00463 118.0655 1.24 kynurenine C00328 209.0903 1.11 Anthranilate C00108 138.0551 1.19 L-Adrenaline C00788 184.0946 1.15 N-Methylethanolamine phosphate C01210 156.0422 1.18 L-Carnitine C00318 162.1126 1.16 Triacanthine C08435 204.1231 1.22 2-Methyleneglutarate C02930 145.0497 1.19 6-Lactoyl-5-6-7-8-tetrahydropterin C04244 240.1093 1.16 Methylenediurea C06381 133.0739 1.19 3-Oxalomalate C01990 207.0142 1.15 cis-p-Coumarate C06738 165.0547 1.22 Poly-gamma-D-glutamate C05723 130.0501 1.17 N-Acyl-D-aspartate C06380 161.0297 0.99 METABOLITES_END #END