#METABOLOMICS WORKBENCH cwalker_bcm_20200127_072952 DATATRACK_ID:1909 STUDY_ID:ST001309 ANALYSIS_ID:AN002178 PROJECT_ID:PR000890 VERSION 1 CREATED_ON January 29, 2020, 2:04 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling after early-life exposure to an endocrine disrupting PR:PROJECT_TITLE chemical in the liver. PR:PROJECT_TYPE Targeted MS analysis PR:PROJECT_SUMMARY Metabolic profiling in the liver (240 days post-natal) after early-life exposure PR:PROJECT_SUMMARY to an endocrine disrupting chemical. PR:INSTITUTE Baylor College of Medicine PR:LAST_NAME Walker PR:FIRST_NAME Cheryl PR:ADDRESS 1 Baylor Plaza, Houston, TX, 77030, USA PR:EMAIL Cheryl.walker@bcm.edu PR:PHONE 713-798-8219 #STUDY ST:STUDY_TITLE Metabolite expression in liver after early life exposure to an endocrine ST:STUDY_TITLE disruptor at 240 days postnatal (part-I) ST:STUDY_TYPE Metabolite expression after chemical exposure versus control. ST:STUDY_SUMMARY Our early-life environment has a profound influence on developing organs that ST:STUDY_SUMMARY impact metabolic function and determines disease susceptibility across the ST:STUDY_SUMMARY life-course. Using a rat model for exposure to an endocrine disrupting chemical ST:STUDY_SUMMARY (EDC), we show that early-life exposure causes metabolic dysfunction in ST:STUDY_SUMMARY adulthood and reprograms histone marks in the developing liver to accelerate ST:STUDY_SUMMARY acquisition of an adult epigenomic signature. This epigenomic reprogramming ST:STUDY_SUMMARY persists long after the initial exposure, but many reprogrammed genes remain ST:STUDY_SUMMARY transcriptionally silent with their impact on metabolism not revealed until a ST:STUDY_SUMMARY later life exposure to a Western-style diet. Diet-dependent metabolic disruption ST:STUDY_SUMMARY was largely driven by reprogramming of the Early Growth Response 1 (EGR1) ST:STUDY_SUMMARY transcriptome and production of metabolites in pathways linked to cholesterol, ST:STUDY_SUMMARY lipid and one-carbon metabolism. These findings demonstrate the importance of ST:STUDY_SUMMARY epigenome: environment interactions, which early in life accelerate epigenomic ST:STUDY_SUMMARY aging, and later in adulthood unlock metabolically restricted epigenetic ST:STUDY_SUMMARY reprogramming to drive metabolic dysfunction. ST:INSTITUTE Baylor College of Medicine ST:DEPARTMENT Molecular and Cellular Biology ST:LABORATORY Center for Precision Environmental Health ST:LAST_NAME Walker ST:FIRST_NAME Cheryl ST:ADDRESS 1 Baylor Plaza, Houston, TX, 77030, USA ST:EMAIL Cheryl.walker@bcm.edu ST:PHONE 713-798-8219 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 10 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENOTYPE_STRAIN Sprague Dawley SU:AGE_OR_AGE_RANGE 240 days SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Harlan SU:ANIMAL_HOUSING polycarbonate-free caging SU:ANIMAL_LIGHT_CYCLE 14-hr light and 10-hr dark SU:ANIMAL_FEED Phytoestrogen Reduced II 18-5 (Ziegler Bros, Inc) or D09100301 (Research Diets, SU:ANIMAL_FEED Inc) #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 - HFD-VEH13 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH15 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH18 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH12 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH14 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA12 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA13 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA14 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA16 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA18 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. #COLLECTION CO:COLLECTION_SUMMARY Liver tissue was harvested on post-natal day 240 after challenge with CO:COLLECTION_SUMMARY Western-style diet.Tissue was snap-frozen in liquid nitrogen. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Neonatal rats were treated with vehicle (sesame oil) or bisphenol A (BPA; 50 TR:TREATMENT_SUMMARY µg/kg dissolved in sesame oil) orally via pipette tip on post-natal days 1, 3, TR:TREATMENT_SUMMARY and 5. Littermates were randomly assigned to the treatment groups. BPA was TR:TREATMENT_SUMMARY obtained from the National Institute of Environmental Health Sciences (NIEHS). TR:TREATMENT_SUMMARY The dose and route of administration recapitulates human exposure to BPA. At day TR:TREATMENT_SUMMARY 180, adult rats in both treatment groups were fed a diet high in fat (40% kcal), TR:TREATMENT_SUMMARY fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days TR:TREATMENT_SUMMARY (D09100301, Research Diets, Inc). Rats were fasted overnight prior to tissue TR:TREATMENT_SUMMARY collection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from crushed liver samples and a mouse liver pool was SP:SAMPLEPREP_SUMMARY used for quality control. Twenty-five mg of crushed liver was used for the SP:SAMPLEPREP_SUMMARY metabolic extraction. The extraction step started with the addition of 750 µL SP:SAMPLEPREP_SUMMARY ice-cold methanol:water (4:1) containing 20 µL spiked internal standards to SP:SAMPLEPREP_SUMMARY each tissue sample. Ice-cold chloroform and water were added in a 3:1 ratio for SP:SAMPLEPREP_SUMMARY a final proportion of 1:4:3:1 water:methanol:chloroform:water. The organic SP:SAMPLEPREP_SUMMARY (methanol and chloroform) and aqueous layers were mixed, dried and resuspended SP:SAMPLEPREP_SUMMARY with 50:50 methanol: water. The extract was deproteinized using a 3kDa molecular SP:SAMPLEPREP_SUMMARY filter (Amicon ultracel-3K Membrane; Millipore Corporation, Billerica, MA) and SP:SAMPLEPREP_SUMMARY the filtrate was dried under vacuum (Genevac EZ-2plus; Gardiner, Stone Ridge, SP:SAMPLEPREP_SUMMARY NY). Prior to mass spectrometry, the dried extracts were re-suspended in SP:SAMPLEPREP_SUMMARY identical volumes of injection solvent composed of 1:1 water: methanol and were SP:SAMPLEPREP_SUMMARY subjected to liquid chromatography-mass spectrometry. Fifty µl of sample was SP:SAMPLEPREP_SUMMARY used for preparation. Internal standards were spiked into the samples. Then it SP:SAMPLEPREP_SUMMARY was processed through a 3 kDa filter. After that, 50 µl of sample was diluted SP:SAMPLEPREP_SUMMARY with 450 µl solvent (methanol: water = 50:50 v/v) and subjected to LC/MS SP:SAMPLEPREP_SUMMARY analysis. The injection volume was 10 µl. For internal standards, SP:SAMPLEPREP_SUMMARY high-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and SP:SAMPLEPREP_SUMMARY water were procured from Burdick & Jackson (Morristown, NJ). Mass SP:SAMPLEPREP_SUMMARY spectrometry-grade formic acid was purchased from Sigma-Aldrich (St Louis, MO). SP:SAMPLEPREP_SUMMARY Calibration solution containing multiple calibrants in a solution of SP:SAMPLEPREP_SUMMARY acetonitrile, trifluroacetic acid, and water was purchased from Agilent SP:SAMPLEPREP_SUMMARY Technologies (Santa Clara, CA). Metabolites and internal standards, including SP:SAMPLEPREP_SUMMARY N-acetyl Aspartic acid-d3, Tryptophan-15N2, Sarcosine-d3, Glutamic acid-d5, SP:SAMPLEPREP_SUMMARY Thymine-d4, Gibberellic acid, Trans-Zeatine, Jasmonic acid, 15N Anthranilic SP:SAMPLEPREP_SUMMARY acid, and Testosterone-d3, were purchased from Sigma-Aldrich (St. Louis, MO). SP:SAMPLEPREP_SUMMARY Three LC- MS methods were used to separate metabolites. Method A: In ESI SP:SAMPLEPREP_SUMMARY positive mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 mm SP:SAMPLEPREP_SUMMARY (Waters, Milford, MA). Mobile phase A and B were 0.1% formic acid in water and SP:SAMPLEPREP_SUMMARY acetonitrile, respectively. Gradient flow: 0-3 min 85% B; 3-12 min 30% B, 12-15 SP:SAMPLEPREP_SUMMARY min 2% B, 16 min 95%B, followed by re-equilibration till the end of the gradient SP:SAMPLEPREP_SUMMARY 23 min to the initial starting condition of 85% B. Flow rate of the solvents SP:SAMPLEPREP_SUMMARY used for the analysis is 0.3 ml/min. Injection volume was 10 µL. Method B: In SP:SAMPLEPREP_SUMMARY ESI negative mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 SP:SAMPLEPREP_SUMMARY mm (Waters, Milford, MA). Mobile phase A and B were 20 mM ammonium acetate in SP:SAMPLEPREP_SUMMARY water with pH 9.0 and 100% acetonitrile, respectively. Gradient flow: 0-3 min SP:SAMPLEPREP_SUMMARY 85% B, 3-12 min 30% B, 12-15 min 2% B, 15-16 min 85% B followed by SP:SAMPLEPREP_SUMMARY re-equilibration till the end of the gradient 23 min to the initial starting SP:SAMPLEPREP_SUMMARY condition of 85% B. Flow rate of the solvents used for analysis is 0.3 ml/min. SP:SAMPLEPREP_SUMMARY Injection volume was 10 µL. Method C: In ESI positive mode the HPLC column was SP:SAMPLEPREP_SUMMARY Luna 3 µM NH2 100 A0 Chromatography column (Phenomenex, Torrance, CA). Mobile SP:SAMPLEPREP_SUMMARY phase A and B were 20 mM ammonium acetate in water with pH 9.0 and 100% SP:SAMPLEPREP_SUMMARY acetonitrile, respectively. Gradient flow: 0-3 min 85% B, 3-12 min 30% B, 12-15 SP:SAMPLEPREP_SUMMARY min 2% B, 15-16 min 85% B followed by re-equilibration till the end of the SP:SAMPLEPREP_SUMMARY gradient 23 min to the initial starting condition of 85% B. Flow rate of the SP:SAMPLEPREP_SUMMARY solvents used for analysis is 0.3 ml/min. Injection volume was 10 µL. For data SP:SAMPLEPREP_SUMMARY acquisition through LC/MS analysis, 10 µL of suspended samples were injected SP:SAMPLEPREP_SUMMARY and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent SP:SAMPLEPREP_SUMMARY Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, SP:SAMPLEPREP_SUMMARY Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were SP:SAMPLEPREP_SUMMARY as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; SP:SAMPLEPREP_SUMMARY Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V SP:SAMPLEPREP_SUMMARY positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V SP:SAMPLEPREP_SUMMARY negative. Approximately 8–11 data points were acquired per detected SP:SAMPLEPREP_SUMMARY metabolite. The data acquired using Agilent mass hunter software and data was SP:SAMPLEPREP_SUMMARY analyzed using mass hunter quantitative analysis software. SP:SAMPLEPREP_PROTOCOL_FILENAME Targeted.MS.method.pdf;unbiased.liver.MS.method.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6495 QQQ CH:COLUMN_NAME Waters XBridge Amide (100 x 4.6mm, 3.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS For data acquisition through LC/MS analysis, 10 µL of suspended samples were MS:MS_COMMENTS injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent MS:MS_COMMENTS Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, MS:MS_COMMENTS Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were MS:MS_COMMENTS as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; MS:MS_COMMENTS Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V MS:MS_COMMENTS positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V MS:MS_COMMENTS negative. Approximately 8–11 data points were acquired per detected MS:MS_COMMENTS metabolite. The data acquired using Agilent mass hunter software and data was MS:MS_COMMENTS analyzed using mass hunter quantitative analysis software. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak intensity MS_METABOLITE_DATA_START Samples HFD-VEH13 HFD-VEH15 HFD-VEH18 HFD-VEH12 HFD-VEH14 HFD-BPA12 HFD-BPA13 HFD-BPA14 HFD-BPA16 HFD-BPA18 Factors Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:BPA Treatment:BPA Treatment:BPA Treatment:BPA Treatment:BPA 2-Aminooctanoic acid 278549.519 664617.777 340761.8695 299286.0273 301848.8526 170741.0364 234069.0918 189203.2978 133268.9852 366496.1754 Octonyl Carnitine 472930.985 294675.0353 344135.9308 556394.2531 321294.3685 210611.022 379924.3923 358481.9373 413154.3906 259047.6114 1-Stearoyl-sn-glycero-3-phosphocholine 10932955.53 10633554.09 7693075.196 8748290.86 12338814.35 8143491.015 12213536.54 13218814.83 9371530.926 19276929.02 Oleic acid 6604079.717 5062332.038 6743628.44 7195186.73 7575174.317 6764715.643 5605824.425 4783489.752 4712699.616 4896811.127 Acetyl Carnitine 199777374.7 151663536.7 167611336.2 135597588.5 165053765.9 135297866.1 166465986.4 142179576.2 131513503.9 110091970.8 Propeonyl Carnitine 28493905.35 33630241.8 25684275.33 27770720.71 16283386.83 23109113.36 15493295.98 19428314.74 24877452.43 31649932.55 4 Acetamidobutanoic acid 237352.0134 140081.904 168704.9081 211174.2497 102250.3728 148115.2663 122925.945 110682.1135 121667.2705 138838.53 2-Hydroxypyridine 1217558.322 1543523.492 1406751.619 1279497.802 1436948.087 1520877.201 1129003.028 1270017.682 1293388.707 1245434.931 Nicotinamide 184284954 219215778.1 216476372.6 178029224.3 199380459 249971821.6 171891888.5 200002255 208586928.5 183733884.2 1-Methyl tryptophan 117126.1448 121351.4007 101278.1108 108606.2595 117658.777 143210.7124 117157.642 133640.0576 164010.8213 71401.20183 Acetylcholine 2526493.283 2288019.318 1772835.861 1960317.519 1739310.505 1302389.218 1625964.477 1241679.172 1415504.634 1552121.167 Aspartame 180436.7753 170770.4815 164347.1847 122275.4996 189068.7616 196341.2124 144511.8321 116521.2717 163003.1448 146871.3876 Carnitine 34613808.58 36887932.16 37784065.91 32477309.44 41282446.53 27227535.94 32538391 30053293.41 34127772.06 26586000.7 Glutamic acid 104249.4113 139176.1082 6504622.836 83681.28903 82679.03327 77850.19281 70587.57958 83444.81641 82647.74446 88435.82137 Serine 192401.7772 189681.8136 221891.2698 247744.8661 204692.8605 237971.2817 190180.2168 250585.6659 201738.892 235308.7268 Gly-Gly 326243.7576 325199.9896 334162.3193 298338.2189 321833.1274 373439.5573 294773.7462 272276.0933 277527.78 271282.5192 Kynurenic acid 76127.60909 110588.0993 110274.4747 78363.50458 93560.06533 96490.93889 91617.79413 72824.44736 91827.02018 78243.69705 Isoleucine 69818372.22 70670320.8 74165997.88 67054973.3 70635413.83 76914483.46 63643730.31 61242302.45 63729152.01 62002696.46 Phenyl alanine 32705425.04 29298937.19 34765654.96 27894699.42 30847071.94 30076756.37 31753443.58 27209894.69 29333848.04 28020224.49 purine 58299.87087 42005.06711 61228.86505 56515.30028 60472.94397 50650.00602 48944.60827 54715.37169 47377.64024 43503.22651 Urea 15315393.6 19644080.69 13441898.24 11424964.72 11906196.86 14970218.96 22145926.93 14228086.04 11770153.16 14289947.84 4 Hydroxybutyric acid 1515529.061 1356784.906 1554386.156 1701335.818 1356712.113 1662776.677 1286278.972 1713418.018 1449364.446 1632233.276 5-Hydroxyindoleacetic acid 18985.4084 35516.12481 27304.40018 18487.28437 15718.77198 58678.13536 24439.0998 52021.47566 24747.80837 25751.8214 Aminobutyric acid 13442434 13662317.25 14184593.31 15765298.46 13187913.79 15130211.81 12245285.69 15742738.85 12760988.62 15719077.78 Choline 9076224.518 8730056.569 9442366.635 10755323.72 8639829.471 10277971.09 8168409.064 10771603.19 8236308.723 9436485.812 Kynurenine 215333.1746 240282.0759 221470.1324 215880.5196 177855.4513 311300.7129 152946.9515 225865.7116 178441.7364 178039.9145 Hypoxanthine 77452925.16 74320049.71 79761750.19 65360656.89 71572692.71 84739374.32 64483287.48 77751715.62 72256526.1 66415624.45 Creatinine 6727901.918 6933413.591 5666988.715 5513563.415 6279322.988 6426114.437 8766222.156 5480249.441 6118568.338 5831279.371 Gly-Leu 1925102.984 1330296.701 1699166.298 1557218.623 1747869.693 857422.7661 1118317.289 660513.8343 998062.4359 839235.901 Tryptophan 4592615.421 3699323.41 4006414.956 3189527.993 3797550.323 3013047.57 3136303.516 2796037.906 2973152.001 2565350.293 Valine 20240772.16 19316793.96 22733504.16 21149310.98 20180867.32 24207927.65 18614007.06 18203285.2 16249576.23 19416131.9 2-Methyl glutamic acid 413090.1537 465120.8703 392620.5899 416205.0704 465780.3488 341498.4009 415058.8881 385841.4202 380678.587 436752.7927 Xanthine 5424478.395 5111632.332 5064203.647 5183808.492 4582585.076 5827105.965 4321111.584 4286763.917 4250289.214 5252042.343 Guanidine acetic acid 12279274.3 11079159.95 7737469.586 10994907.7 10311905.61 8662454.195 16797731.77 12326222.53 10764725.2 10522305.08 N-Acetyl Lysine 319433.5085 378025.3918 290874.7747 253800.7318 235091.2527 389818.6613 308203.1012 232691.8444 283127.9826 406307.3178 Tyrosine 1399435.183 1399520.364 1439807.296 1499905.084 1321291.354 1856708.473 1235083.656 1090927.315 959039.971 1280225.078 Homocysteine 367450.7515 367135.1045 283856.7807 337066.0449 268226.1005 364230.4078 368162.6884 233144.1775 218338.4968 272959.8912 Betaine 137408906.2 128662684.9 123722596 118171690.5 113152608.4 132970502.1 136700515.9 133096664.4 138645393.8 126077915.9 Methyl Nicotinamide 2004586.645 1745367.835 2113669.637 1753786.114 2304486.869 1941021.689 1802439.952 2031393.481 1742916.875 1779955.831 Proline 4331215.055 4340633.037 4049893.286 4410807.095 3574375.431 5045184.007 3631487.759 3373840.181 3779756.123 4069363.57 Sarcosine/Alanine 1071077.128 894241.0442 1022489.586 974527.6984 956780.0652 1347980.763 1032811.498 743874.5592 919434.2274 867660.8684 Guanosine 42232.73243 39634.2594 31902.7711 26909.99657 29991.64849 18639.57828 46323.34639 48624.48259 31317.54563 24222.96447 N, N Dimethyl Glycine 3545532.537 3405308.965 4278795.386 4788711.496 3886666.662 3191320.421 4215886.179 4434488.952 3353475.864 3080378.975 Dimethyl Arginine 228114.2541 295697.8286 241182.1822 120962.3343 188622.7053 532426.3817 173022.9883 116017.4527 124584.2259 122444.4913 Glycine 88791.66113 95741.54298 89658.982 93693.48155 75321.91376 86618.15343 93981.58279 117498.2959 86174.75707 104268.788 Ergothioneine 53086.87006 42615.21106 67049.03308 62922.48954 48310.59 71529.1823 75643.38731 94463.71384 71682.69401 49537.38564 1-Methyl-Histidine 629894.3426 837321.6483 688717.1332 569238.5054 566450.9125 615015.6162 856003.6449 720160.4315 560989.378 568539.6967 Spermidine 26436.21861 49286.86427 19278.95208 18136.59591 13408.55842 8809.739113 27720.90881 15709.83979 10391.04528 14355.88144 Allo-Threonine 946637.0257 964646.3176 1079572.821 1090240.454 1143276.65 1184924.506 1182415.049 1125083.063 942958.7396 1136376.98 Homoserine 946637.0257 964646.3176 1079572.821 1090240.454 1143276.65 1184924.506 1182415.049 1125083.063 942958.7396 1136376.98 N-Acetylputrescine 105017.3638 83677.09934 115325.8823 81415.0168 87099.67107 103419.4714 78270.77869 89009.84516 73611.10496 72818.5916 citrulline 41650.52585 42554.56067 45926.75052 47264.29635 36132.76131 49571.43707 41548.52321 71924.99791 51865.44213 80717.06143 Lysine 13934602.01 13724201.97 15543811.4 16197041.26 16526530.11 16380193.9 14679282.6 16035784.39 14981738 15752010.26 Methylglutaric acid 532842.178 465030.3626 530054.4528 479709.6929 533848.5146 470201.4135 564188.3997 559368.7153 524864.911 481789.1081 5-oxo-proline 54223270.17 55030576.38 56599334.53 61565002.64 63270918.03 59431643.81 56428693.44 55219922.65 51147315.28 53329841.41 DL-Pipecolic acid 54223270.17 55030576.38 56599334.53 61565002.64 63270918.03 59431643.81 56428693.44 55219922.65 51147315.28 53329841.41 SAH 1389382.221 1543236.076 1744539.82 1517493.077 1509874.228 1646979.937 1319156.259 1638059.585 929834.5941 1851616.798 dCMP 116202.0617 151065.3363 131082.8721 124897.1084 93296.00893 116619.2212 70641.26398 130889.7035 116412.606 140408.6118 Arginine 323911.4205 392149.8403 234268.511 250297.4312 262669.3405 363432.1708 295308.6418 287690.2494 195040.9656 302337.1234 2-Aminobutyraldehyde 111019.0864 110413.7895 123677.7648 145670.4804 137463.3031 116327.0815 131817.9125 109797.7166 97703.18325 153786.4725 Ethylmalonate 122160.4449 160076.4898 83844.59556 122239.9024 97286.73756 113256.3247 159972.4694 109103.1059 134841.8858 156915.5794 Glutamyl alanine 194996.133 105572.8651 166204.3768 146617.5576 166937.0088 171923.479 158139.0351 243345.9481 143190.3205 220488.2103 Phospho Choline 4747335.787 2288498.621 3392731.617 3578291.131 2560301.907 1812204.898 3522761.42 2573573.582 2847246.724 3168470.154 3-Hydroxykynurenine 1046036.812 1275335.928 1075152.087 1099791.305 992652.8363 1092445.166 1009589.451 1224381.405 935701.6376 748600.9278 Deoxy carnitine 11088329.54 11330718.32 6895616.663 8992318.97 6776040.888 5280820.984 6364028.974 5706458.745 5727537.135 6721819.455 3-phospho-serine 374821.0354 211219.1398 273217.34 293989.7815 241919.3346 200541.4655 325199.9465 205460.1354 254688.1245 277524.4985 Glu-Tyr 351520.3704 358009.3262 327618.4733 323286.2387 263151.2322 337492.0923 264667.0053 348326.7584 302770.5192 251952.3321 Asparagine 633676.9846 568317.6997 713734.6295 720992.3567 742730.2478 805917.8008 680779.3554 501997.4513 525022.4457 584017.926 MYRISTATE (14:0)+Na 93993.26973 77478.84256 116575.4893 95414.76666 94712.37323 106824.7954 114656.6757 134043.5277 94511.42493 99007.12187 S-ribosyl-L-homocysteine 50250.43869 55473.58725 46784.84302 43691.18426 41205.72423 53213.5723 66993.03444 50475.21626 54358.44328 51521.85814 Nicotinamide ribotide 3798283.779 3106430.551 6732885.777 6162627.429 6309741.614 9169815.858 5099274.535 5392148.087 4474689.709 6609341.667 AMP 92531424.43 105792766.3 76008605.07 75518668.12 53000371.95 52993455.78 71802851.09 86410481.61 80394793.04 85555594.53 acadesine 175882.0857 146945.3154 137640.7798 137759.9783 75903.20694 171526.5871 168501.822 135563.941 137525.7765 123670.4617 Cystathionine 451134.0832 494743.3149 568897.4248 454817.2515 391456.7047 821000.0794 355358.5627 308310.1012 276603.4254 469480.735 aminoimidazole carboxamide ribonucleotide 58719.40468 48008.49159 51937.47646 75330.13214 34900.37569 37290.57936 79654.56943 69270.05131 57718.81445 66896.77521 thiamine 21721.99763 14588.26763 20651.56555 11408.25356 22136.99814 27081.79919 8243.163126 17784.1462 13018.66396 26997.12811 Cytosine 1112103.173 1469541.672 1447006.718 1314588.5 1331404.737 1345178.057 863591.9153 1051794.254 1002470.274 1030745.621 Cytidine 5 -Monophosphate (5-CMP) 3721799.847 3425109.497 2811681.31 3056355.027 2451363.494 2489212.855 2777717.219 3785349.862 2913097.496 4494155.937 Inositol-1P 52710.75123 75430.91048 24179.93625 51684.28793 28051.76743 23138.83926 38621.93355 89522.98424 69278.14898 44901.77881 N-acetyl Glucosamine 6 Phos 49482.33237 58546.66893 61883.0748 63196.67733 47068.2074 99802.35244 64842.80735 50579.26062 41475.12027 77176.02028 Glutamine 25902314.65 25524329.7 29402360.27 29967813.11 32504656.94 24365420.37 26146145.27 26185023.08 25684730.95 29215402.58 Histidine 43982343.31 47997815.2 50619671.67 36107057.03 42032581.15 50851413.15 41974057.42 42585870.78 35275151.18 51706004.62 NAD 4200508.151 1417521.793 4711784.741 1643686.36 1884911.886 1769207.887 2552713.099 3048417.708 2242441.574 2981572.974 GMP 11613724.15 11262154.51 9562788.635 7978575.989 4588533.039 2956871.51 11054372.05 9547622.453 10987427.81 8188013.054 Glu-Glu 136004.9324 120148.7278 130917.9694 101138.3534 140947.9671 125599.1389 115567.6875 125247.0794 111110.6889 147061.6369 xanthosine-5-phosphate 168960.2284 202889.7 85284.26367 173375.9996 102086.6581 141192.4237 124892.6685 166735.6991 120047.5149 158318.4102 UMP 787321.0187 859021.7343 457266.9014 506216.6082 393032.05 248762.1454 534710.4159 813569.0385 634767.0153 461000.4277 L-Creatinine (internal standard) 6157139.722 5728782.935 5592526.099 5072963.773 5264991.607 5716845.615 4677357.851 4968603.857 5411106.852 4248299.067 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 2-Aminooctanoic acid Octonyl Carnitine 1-Stearoyl-sn-glycero-3-phosphocholine Oleic acid Acetyl Carnitine Propeonyl Carnitine 4 Acetamidobutanoic acid 2-Hydroxypyridine Nicotinamide 1-Methyl tryptophan Acetylcholine Aspartame Carnitine Glutamic acid Serine Gly-Gly Kynurenic acid Isoleucine Phenyl alanine purine Urea 4 Hydroxybutyric acid 5-Hydroxyindoleacetic acid Aminobutyric acid Choline Kynurenine Hypoxanthine Creatinine Gly-Leu Tryptophan Valine 2-Methyl glutamic acid Xanthine Guanidine acetic acid N-Acetyl Lysine Tyrosine Homocysteine Betaine Methyl Nicotinamide Proline Sarcosine/Alanine Guanosine N, N Dimethyl Glycine Dimethyl Arginine Glycine Ergothioneine 1-Methyl-Histidine Spermidine Allo-Threonine Homoserine N-Acetylputrescine citrulline Lysine Methylglutaric acid 5-oxo-proline DL-Pipecolic acid SAH dCMP Arginine 2-Aminobutyraldehyde Ethylmalonate Glutamyl alanine Phospho Choline 3-Hydroxykynurenine Deoxy carnitine 3-phospho-serine Glu-Tyr Asparagine MYRISTATE (14:0)+Na S-ribosyl-L-homocysteine Nicotinamide ribotide AMP acadesine Cystathionine aminoimidazole carboxamide ribonucleotide thiamine Cytosine Cytidine 5 -Monophosphate (5-CMP) Inositol-1P N-acetyl Glucosamine 6 Phos Glutamine Histidine NAD GMP Glu-Glu xanthosine-5-phosphate UMP L-Creatinine (internal standard) METABOLITES_END #END