#METABOLOMICS WORKBENCH ryant_20201211_114704 DATATRACK_ID:2350 STUDY_ID:ST001624 ANALYSIS_ID:AN002659 PROJECT_ID:PR001042 VERSION 1 CREATED_ON December 15, 2020, 11:09 am #PROJECT PR:PROJECT_TITLE Tissue-specific 1H-NMR metabolomic profiling in mice with adenine-induced PR:PROJECT_TITLE chronic kidney disease PR:PROJECT_TYPE Metabolomic profiling of the heart, liver, kidney, and skeletal muscles obtained PR:PROJECT_TYPE from mice with and without CKD via 1H NMR PR:PROJECT_SUMMARY This project is focused on a metabolomic analyses of the heart, liver, kidney, PR:PROJECT_SUMMARY and skeletal muscles obtained from mice with and without CKD. To accomplish this PR:PROJECT_SUMMARY objective, we extracted tissues from mice with CKD induced by long-term (24 PR:PROJECT_SUMMARY week) adenine-supplemented diet as well as their control-diet fed counterparts PR:PROJECT_SUMMARY with normal kidney function. Metabolites were extracted from tissues and 1H PR:PROJECT_SUMMARY nuclear magnetic resonance (NMR) was performed and coupled with multivariate PR:PROJECT_SUMMARY statistical analysis. PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiology and Kinesiology PR:LABORATORY Rm 42 and Rm 43 PR:LAST_NAME Ryan PR:FIRST_NAME Terence PR:ADDRESS 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA PR:EMAIL ryant@ufl.edu PR:PHONE 352-294-1700 PR:FUNDING_SOURCE This research was funded by grants from the National Institutes of Health and PR:FUNDING_SOURCE the National Heart, Lung, and Blood, Institute numbers R01-HL149704 (to T.E.R.) PR:FUNDING_SOURCE and the American Heart Association grant number 18CDA34110044 (to T.E.R.). PR:CONTRIBUTORS Ram B. Khattri, Trace Thome, and Terence E. Ryan #STUDY ST:STUDY_TITLE Tissue-specific 1H-NMR metabolomic profiling in mice with adenine-induced ST:STUDY_TITLE chronic kidney disease - aqueous phase Kidney (part-I) ST:STUDY_TYPE Metabolomic profiling of the heart, liver, kidney, and skeletal muscles obtained ST:STUDY_TYPE from mice with and without CKD via 1H NMR ST:STUDY_SUMMARY This project is focused on a metabolomic analyses of the heart, liver, kidney, ST:STUDY_SUMMARY and skeletal muscles obtained from mice with and without CKD. To accomplish this ST:STUDY_SUMMARY objective, we extracted tissues from mice with CKD induced by long-term (24 ST:STUDY_SUMMARY week) adenine-supplemented diet as well as their control-diet fed counterparts ST:STUDY_SUMMARY with normal kidney function. Metabolites were extracted from tissues and 1H ST:STUDY_SUMMARY nuclear magnetic resonance (NMR) was performed and coupled with multivariate ST:STUDY_SUMMARY statistical analysis. ST:INSTITUTE University of Florida ST:DEPARTMENT Applied Physiology and Kinesiology ST:LABORATORY Rm 42 and Rm 43 ST:LAST_NAME Ryan ST:FIRST_NAME Terence ST:ADDRESS 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA ST:EMAIL ryant@ufl.edu ST:PHONE 352-294-1700 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 17 ST:NUM_MALES All ST:STUDY_COMMENTS CKD metabolomic study via NMR using mice model ST:PUBLICATIONS MDPI #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 13 months SU:WEIGHT_OR_WEIGHT_RANGE (32.86±1.21 g (control mice) vs 23.57±1.27 g (CKD mice), P < 0.0001, SU:WEIGHT_OR_WEIGHT_RANGE N=7/group). SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs (Stock # 000664) SU:ANIMAL_HOUSING Housed in a temperature of 22 oC SU:ANIMAL_LIGHT_CYCLE 12-hour light/12-hour dark SU:ANIMAL_FEED Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD) SU:ANIMAL_WATER free access to food and water (3-5 animals per cage). SU:ANIMAL_INCLUSION_CRITERIA (3-5 animals per cage). #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 - CKD1Kidney_Aq Group:CKD RAW_FILE_NAME=CKD1Kidney_Aq SUBJECT_SAMPLE_FACTORS - CKD2Kidney_Aq Group:CKD RAW_FILE_NAME=CKD2Kidney_Aq SUBJECT_SAMPLE_FACTORS - CKD4Kidney_Aq Group:CKD RAW_FILE_NAME=CKD4Kidney_Aq SUBJECT_SAMPLE_FACTORS - CKD5Kidney_Aq Group:CKD RAW_FILE_NAME=CKD5Kidney_Aq SUBJECT_SAMPLE_FACTORS - CKD6Kidney_Aq Group:CKD RAW_FILE_NAME=CKD6Kidney_Aq SUBJECT_SAMPLE_FACTORS - CKD7Kidney_Aq Group:CKD RAW_FILE_NAME=CKD7Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con1Kidney_Aq Group:Control RAW_FILE_NAME=Con1Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con2Kidney_Aq Group:Control RAW_FILE_NAME=Con2Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con3Kidney_Aq Group:Control RAW_FILE_NAME=Con3Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con4Kidney_Aq Group:Control RAW_FILE_NAME=Con4Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con5Kidney_Aq Group:Control RAW_FILE_NAME=Con5Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con6Kidney_Aq Group:Control RAW_FILE_NAME=Con6Kidney_Aq SUBJECT_SAMPLE_FACTORS - Con7Kidney_Aq Group:Control RAW_FILE_NAME=Con7Kidney_Aq #COLLECTION CO:COLLECTION_SUMMARY While under isoflurane anesthesia, tissues were rapidly dissected and snap CO:COLLECTION_SUMMARY frozen in liquid nitrogen and stored at -80°C until metabolite extraction. The CO:COLLECTION_SUMMARY following tissues were used in this study: kidney, liver, heart (left CO:COLLECTION_SUMMARY ventricle), skeletal muscle (quadriceps). Euthanasia was carried out by CO:COLLECTION_SUMMARY thoracotomy followed by cervical dislocation. CO:SAMPLE_TYPE Kidney CO:COLLECTION_METHOD While under isoflurane anesthesia, tissues were rapidly dissected and snap CO:COLLECTION_METHOD frozen in liquid nitrogen and stored at -80°C until metabolite extraction CO:COLLECTION_LOCATION University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, CO:COLLECTION_LOCATION Gainesville, FL 32611 CO:STORAGE_CONDITIONS -80℃ CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY To induce CKD, we utilized an established adenine-diet model. Briefly, mice were TR:TREATMENT_SUMMARY assigned to a casein-base chow for 7-days, followed by induction of renal TR:TREATMENT_SUMMARY tubular injury by supplementing the diet with 0.2% adenine for 7-days, and TR:TREATMENT_SUMMARY subsequently maintained on a 0.15% adenine diet for 5 months and two weeks. CKD TR:TREATMENT_SUMMARY mice were then placed back on casein control diet for 2-weeks prior to TR:TREATMENT_SUMMARY euthanasia and terminal experiments, allowing for a washout period of adenine TR:TREATMENT_SUMMARY based chow. Control mice received casein diet for the duration of the study. TR:TREATMENT_SUMMARY Total duration of CKD encompassed 6-months. TR:ANIMAL_VET_TREATMENTS none TR:ANIMAL_ANESTHESIA isoflurane TR:ANIMAL_FASTING non-fasted TR:ANIMAL_ENDP_EUTHANASIA Euthanasia was carried out by thoracotomy followed by cervical dislocation. TR:ANIMAL_ENDP_TISSUE_COLL_LIST kidney, liver, heart (left ventricle), skeletal muscle (quadriceps) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A modified form of FOLCH extraction protocol was used to extract metabolites SP:SAMPLEPREP_SUMMARY from the tissues. Wet weights of all tissue samples were recorded prior to SP:SAMPLEPREP_SUMMARY extraction. Tissue samples were immediately homogenized to prevent any possible SP:SAMPLEPREP_SUMMARY enzymatic action using 1 mL of ice-cold methanol in a PowerLyzer 24 Homogenizer SP:SAMPLEPREP_SUMMARY (QIAGEN Group, Hilden, Germany). The mixture was centrifuged using 13,200 rpm at SP:SAMPLEPREP_SUMMARY 4oC for 30 minutes and the resulting supernatant was transferred to a new glass SP:SAMPLEPREP_SUMMARY vial consisting 3 mL of ice cold chloroform:methanol (2:1, v/v) mixture. The SP:SAMPLEPREP_SUMMARY homogenate was vortexed and left in an ice bath for 15 minutes to allow for SP:SAMPLEPREP_SUMMARY phase separation. Next, 1 mL of 0.9% of saline was added, vortexed it for couple SP:SAMPLEPREP_SUMMARY of minutes followed by a second incubation in an ice bath for 30-45 min for SP:SAMPLEPREP_SUMMARY complete phase separation. The upper aqueous layer was transferred to a new SP:SAMPLEPREP_SUMMARY falcon tube. To the remaining organic phase sample, 1 mL of 0.9% of saline was SP:SAMPLEPREP_SUMMARY added again followed by vigorous mixing and letting it stand in ice bath (15 SP:SAMPLEPREP_SUMMARY minutes) for a second phase separation. This second aqueous phase was combined SP:SAMPLEPREP_SUMMARY with the first. The resulting aqueous and organic layers were dried separately. SP:SAMPLEPREP_SUMMARY The aqueous layer was dried overnight with a Labconco freezer dryer (Labconco SP:SAMPLEPREP_SUMMARY Corporation, MO, USA) and the organic layer was dried via inert nitrogen gas. SP:SAMPLEPREP_SUMMARY These two dried powders (aqueous and organic phases) were stored at -80oC until SP:SAMPLEPREP_SUMMARY performing NMR experiments. SP:PROCESSING_METHOD Lyophilization and Homogenization SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Modified FOLCH extraction SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION In 50 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS SP:SAMPLE_RESUSPENSION and 0.2% sodium azide for aqueous phase samples. SP:SAMPLE_SPIKING 0.5 mM of DSS for aqueous phase samples #ANALYSIS AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker Avance Neo 600 MHz/54mm console NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:STANDARD_CONCENTRATION 0.5 mM DSS NM:SPECTROMETER_FREQUENCY 600.2328273 MHz NM:NMR_PROBE 1.7 mm TXI CryoProbe NM:NMR_SOLVENT Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in NM:NMR_SOLVENT deuterated environment NM:NMR_TUBE_SIZE 1.7 mm O.D. NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 101 NM:OFFSET_FREQUENCY 2827.31 Hz NM:CHEMICAL_SHIFT_REF_CPD DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) NM:TEMPERATURE 25 o C NM:NUMBER_OF_SCANS 128 scans NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 4 s NM:RELAXATION_DELAY 1 s NM:SPECTRAL_WIDTH 7142.9 Hz NM:NUM_DATA_POINTS_ACQUIRED 28571 NM:REAL_DATA_POINTS 65536 NM:LINE_BROADENING 0.22 Hz NM:ZERO_FILLING 65,536 points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Spline NM:CHEMICAL_SHIFT_REF_STD 0 ppm for DSS #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS A.U. NMR_METABOLITE_DATA_START Samples CKD1Kidney_Aq CKD2Kidney_Aq CKD4Kidney_Aq CKD5Kidney_Aq CKD6Kidney_Aq CKD7Kidney_Aq Con1Kidney_Aq Con2Kidney_Aq Con3Kidney_Aq Con4Kidney_Aq Con5Kidney_Aq Con6Kidney_Aq Con7Kidney_Aq Factors Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Isocaproate 9.683602755 8.29349982 4.745361154 13.01075926 3.888010368 8.009215731 11.47617311 20.66492472 10.69258557 36.75338604 24.80784134 15.98524614 17.1362385 Leucine 13.27399873 11.92590815 7.778541898 17.50286218 8.933183381 16.08358924 17.67675853 21.43535642 13.86787943 23.23210754 19.9280389 24.1888152 24.48005667 Valine1 5.570766552 5.152986876 3.500566971 8.103186209 4.244401241 6.976735678 7.214633157 9.950012641 5.356281111 10.39609531 8.475982685 10.09514907 10.65297962 Isoleucine 3.473816323 2.952940346 2.033218717 4.66616518 2.149786782 4.149502856 4.338791083 6.10890184 3.224872742 6.996529955 5.122196882 5.942119503 6.259048449 Valine2 5.634644571 4.81520252 3.303159433 7.77174602 4.092894274 6.759723224 7.275887308 9.677509225 4.883500375 9.824572676 8.073518721 9.31537701 9.781558473 Isobutyrate 2.644509927 1.968583051 1.435113157 3.036680974 1.26665114 2.362583144 2.819925533 3.455504159 1.73726236 5.253228054 3.64516176 3.254544358 3.379622504 Lactate1 156.4193712 159.9923236 117.8920274 282.6286911 76.72953474 237.0665924 212.3838656 261.0791275 168.4431844 305.0831008 295.3134279 229.9761577 275.3626179 Alanine 4.867655269 2.706745856 2.51227014 4.855150226 1.829170535 4.749195172 6.30061491 6.359962951 3.857126708 6.945433682 3.500828735 5.993950544 6.183183619 Lysine 16.70372335 9.984820551 9.544000844 19.50792879 9.397965056 20.54354428 25.18460368 19.9252455 16.6758753 19.26610524 8.489229574 27.15080889 26.96971568 Acetate 7.950489479 3.100914646 2.527984002 6.825978944 3.294067886 4.457128746 9.394826741 5.181422347 3.2911044 5.296803041 4.780470554 6.040819312 6.094590145 Glutamate1 25.80188039 20.08851544 14.99875729 47.35464397 16.91433033 42.17885574 64.24915306 55.35192389 46.54895265 74.46491209 51.45839056 73.23260599 73.10317601 Glutamine1+Glutamate+Methionine+O-Acetylcholine 40.2136126 31.38278353 25.49340956 74.67037295 27.21770642 69.79072797 100.3155406 79.77522017 63.26545715 88.30251637 69.04225512 105.6634768 94.4576982 Glutamate 22.52661997 15.07241586 11.8469388 52.8309695 14.23168339 41.94719156 78.68424193 65.94587103 53.10735298 75.97481822 65.48857465 87.03165999 83.46584863 Pyruvate 7.153893784 3.481881399 2.910936119 4.058650934 2.749213881 4.482470645 7.82817468 5.845584706 3.837978749 4.468928318 3.182691644 5.116623547 4.67369062 Succinate 4.999044692 3.558641277 2.296654953 12.8415997 2.791190047 10.03018272 23.36080178 19.76559599 16.52578582 24.96418649 24.87232943 27.58171997 26.66674198 Citrate-1 15.01857778 14.69001391 9.9092125 6.820275434 12.94157763 9.15000885 8.603625834 4.262200719 5.189770571 4.489845799 1.886067456 6.065778916 5.712684935 Aspartate 5.437652513 1.752193102 3.336696307 9.746111545 3.623453367 9.54669697 13.35905164 12.42160729 12.86531423 16.96590078 7.301137482 16.8465665 19.42317834 Creatine1 12.4860875 14.81609421 10.33655569 12.30870461 5.864268638 12.49840787 28.08579782 15.57535295 8.437013964 11.62933308 10.62336688 23.01143636 24.20186727 Creatinine 2.27805682 1.984905546 1.446974301 2.11146863 1.559205071 2.400546814 2.873168514 1.725520513 2.195656831 2.361944625 1.316992336 2.95220764 3.395081337 O-phosphocholine 24.30019303 29.65362268 28.82353892 66.26627927 16.57894485 61.3568242 53.9220987 51.37851136 32.01419933 66.0373512 46.30389937 60.30320174 57.67741598 Sn-Glycero-3-Phosphocholine 118.7133589 112.6916546 84.14267831 171.7680075 71.86381332 143.5522169 111.3353544 152.6934882 118.0938993 195.3603325 263.6943562 196.3529399 208.2701128 Trimethyl-N-oxide 16.5317733 15.50824589 16.22915935 135.3842164 12.65642755 108.2804277 254.3183901 246.7887956 163.5103419 109.3921763 149.4440061 117.2540195 128.805767 Taurine1 175.5389263 158.8078181 112.9556279 201.3262838 111.4938611 206.1148581 177.8127094 207.4613093 155.6116553 189.9338961 224.1036364 201.1368608 234.4106388 Myo-inositol1 34.56948995 27.04989361 20.26301878 47.06119526 20.26906516 50.57899797 32.32741682 33.55514862 32.35764231 48.70177719 51.02896976 43.64718928 52.85662937 Taurine-2 148.7099216 124.32832 93.53643407 149.7267051 99.48347293 157.7907085 121.5941149 159.7679958 118.5435677 144.3497859 155.6336347 168.1792528 175.8903325 Myo-Inositol2 57.55266837 56.12210701 33.50009364 79.04596748 37.91386724 85.05956419 40.85033824 52.39811522 60.21709693 80.76811619 92.44661061 74.02369245 97.6478242 Glycine 8.729070245 7.654549603 4.870653843 20.61774331 6.321724156 18.38488729 34.72557287 35.44912101 31.79229388 42.24185425 37.71158532 49.58793007 47.33818168 Creatine2 11.0844147 12.43920654 8.874494372 12.39596305 5.437620051 13.09227813 23.08696873 14.5344368 9.027306137 8.15939392 13.12977845 21.01452186 22.08186509 Myo-inositol3 23.17712316 22.51558179 12.28369379 35.51442267 14.60582889 38.47359751 22.92173736 24.99686378 28.64635335 25.69891838 42.71968699 33.77101841 46.05002133 Lactate2 42.43221402 38.40936051 31.32602531 65.47819213 22.73795357 72.69673103 66.1668481 63.840088 53.2853185 43.71024855 66.46485232 61.58424127 71.33933879 Sn-Glycero-3-Phosphocholine2 26.3973128 23.48733614 15.30229191 36.43603401 17.09837346 37.05901233 26.57528259 32.65303159 29.89247321 34.12856601 55.6834799 44.74543554 48.83109548 ATP+AMP1 3.536662496 1.669017792 2.158562207 9.246097928 1.902835172 13.52388439 17.08353099 14.24053849 10.96106172 9.836809331 11.60021562 15.76118318 13.63198633 Inosine1 1.441600575 0.418520343 1.397181082 1.81941212 1.648279713 6.127334702 6.113140851 4.023527169 4.9255846 1.18308372 1.028119888 5.204732767 5.05396307 ATP+AMP2 0.547575775 -0.23176172 0.276277115 1.553474947 0.266643155 3.793364145 4.926283229 3.936990131 2.68877949 2.691430695 2.445248313 4.00408205 3.290663476 Glucose1 0.754463913 0.677419368 0.996871326 0.045000899 1.041621843 1.431362851 0.15386201 0.148003755 0.483176325 0.999736208 0.371893561 0.791747631 0.531231233 Glucose2 3.870948593 4.778976036 4.43265876 1.719678577 4.591414419 4.472715466 0.922417693 1.808307175 1.912886494 5.502935309 2.179678498 3.782105611 2.760240592 Allantoin 0.500784608 0.672430021 0.322092428 0.160103401 0.253299305 0.271559456 0.069278 0.096211236 0.249814884 0.564583974 0.391509908 0.174116608 0.475669983 Sucrose 0.473927369 0.672357089 0.508928807 0.312156645 0.699830974 0.25686388 0.150015697 0.06307424 0.026277345 0.91183632 0.176268007 0.002776924 0.133649234 Uracil 0.45374644 0.842863544 0.420083168 0.771337555 0.68601097 0.540289056 0.42395334 1.182248154 0.239815934 1.008029567 0.292356615 1.026754732 1.061438454 Uridine Triphosphate 0.026643854 0.717080179 0.405613026 0.960522539 0.596919405 0.660542501 0.581362135 1.61931847 0.798061281 1.706640549 0.620978287 2.015106487 2.095498547 NAD+1 0.246799841 0.10840205 0.168475418 0.376163279 0.018510005 0.08024204 1.591405071 2.046878246 0.65349541 1.998178477 1.168440454 2.017989894 1.689042287 NAD+2 0.570220859 1.11646102 0.832466344 1.910077925 1.063354926 1.458553399 3.454765199 4.360620577 2.933705189 3.82731211 3.111688658 4.459012476 4.854936907 ATP+AMP3 0.843826785 1.218504941 0.526193008 3.564182981 0.523114768 3.0022762 5.425639826 5.770390569 3.140511031 5.414136098 5.582101463 5.806205434 5.300143491 Fumarate 0.489369989 0.545445879 0.335875849 0.710182675 0.289375469 0.677359283 2.15711571 1.451889843 0.9220434 0.873121108 1.177710176 1.136558757 1.122576326 P-Cresol1? 0.185402054 0.183131546 0.095189423 0.124469095 0.079994639 0.113406333 0.273251424 0.149387969 0.1127204 0.030675636 0.072995312 0.026389129 0.081585225 p-Cresol2? 0.212348447 0.447505698 0.242503471 0.150292733 0.198332366 0.159992292 0.039447287 0.114438495 0.04954347 0.085828871 0.058449079 0.059824963 0.08418311 Tyrosine1 0.957641203 0.933274774 0.73776951 1.786085525 1.047250306 1.671182422 3.557400095 3.260457036 1.71174895 2.006070072 2.494811593 3.586892734 4.649068448 Histidine 0.021373545 0.404100598 0.397228127 0.613197761 0.218545449 0.413352034 0.863705563 0.732921815 0.636697944 0.731201075 0.824172117 0.879984213 0.88769445 Unknown7.14to7.11ppm 0.139030348 0.287600465 0.299782592 0.511912064 0.201007751 0.211673887 1.776188138 1.786630853 2.252420753 1.979225495 2.481832058 2.359453401 2.706991972 Tyrosine2 1.044694844 1.437678037 1.004934182 2.610553059 1.422958878 2.175876686 4.582077086 4.279845232 2.39614073 2.679440675 3.350381843 4.619695453 5.996024982 Tryptophan1 0.4720186 0.905768902 0.670646774 1.282330953 0.678870199 0.729461565 2.459964881 2.579797302 2.899994907 2.649011673 3.329072606 3.125878826 3.594054732 Phenylalanine 0.930649503 1.490032964 1.199723651 2.280072574 1.299154995 1.781256446 2.363238124 2.33476096 1.614874892 1.193915629 2.454758654 1.79515254 2.251380846 Benzoate 4.084846271 3.064998124 3.031784621 6.975546637 2.797224664 2.334734903 3.420913845 3.352344325 1.83638775 0.723283029 2.124638726 2.168984261 2.322911788 Nicotinurate2 0.672819428 1.217516449 1.039259381 1.978788376 0.877561026 1.203109188 2.528623223 2.163329586 2.124741391 1.689609173 2.18379683 2.056404008 2.308947294 Tyramine? 0.015457235 0.224952975 0.253622557 0.8678805 0.164746971 0.15221019 0.573455562 0.575900025 0.559284501 0.165335316 0.445236434 0.394273082 0.293323972 Tryptophan2 2.845527063 1.699856407 1.33474656 1.993197629 1.117324942 1.260891325 2.905083241 2.539621467 1.287048572 1.466668573 1.398482041 1.312419468 0.93844078 Xanthine? 0.251380304 0.236903443 0.229679828 0.902113131 0.160925432 0.344436525 0.833163274 0.705730054 0.572273422 0.603708099 0.551364476 0.557824156 0.562073188 Unknown7.96to7.94 0.56661232 0.681276962 0.539666179 2.328175678 0.376987803 1.145349566 2.036548788 2.144848427 1.242492434 1.163242138 1.562298806 1.687013277 1.259211942 Inosine_or_Adenosine 1.136464871 1.903106832 1.371927505 2.635445404 1.600319699 1.681006334 3.25976492 3.730150429 3.070903892 2.847243767 2.986907815 3.980847061 4.477344936 NAD+3 0.049008322 0.122075542 0.079471933 0.921572411 0.000114615 0.205971516 1.920096011 2.068426088 0.989532769 1.386951237 1.697616631 1.865096223 1.387258554 Formate 2.46688326 1.309806986 0.733113047 1.732546199 0.766218686 0.747009512 2.56519885 1.759335085 1.087664537 0.573402772 1.017656695 0.712319952 0.785144894 NADH 0.010901619 0.065400682 0.043613631 0.255717158 0.045716936 0.071022495 0.293679511 0.226448116 0.235247501 0.26540466 0.168766034 0.321759874 0.291243064 NADH2 0.016233312 0.075905529 0.054877862 0.370280878 0.041191551 0.108691092 0.573642568 0.363263451 0.349781108 0.331929541 0.395290303 0.502615568 0.411990818 ATP2 0.019230929 0.111549857 0.020208849 0.384849029 0.032184625 0.080511042 0.468437125 0.271757011 0.196080395 0.303915415 0.148858805 0.493996665 0.208423518 Unknown8.61to8.59 0.060664095 0.11318953 0.058215311 0.719994395 0.071393867 0.327414732 1.558910308 1.428215972 0.727302674 1.048943045 1.212422437 1.199109142 0.952123843 ATP+AMP4 0.75136545 1.088016144 0.430452938 3.337888354 0.411149351 2.417164511 3.844715044 4.359433672 2.31212709 3.445647433 4.440017902 3.988270983 3.566030913 Nicotinurate 3 0.610825332 0.661736332 0.519962619 1.251339431 0.442010129 0.69509091 1.65419965 1.34511576 1.301202114 1.380380746 1.390577968 1.492486627 1.654327595 NAD+4 0.140819272 0.05601847 0.023464654 0.772814995 0.052473861 0.140871808 1.699464742 1.675046438 0.737323884 0.935309508 1.329489563 1.463811234 1.014017296 Nicotinurate4 0.523890076 0.495002818 0.442654267 0.853274465 0.361438567 0.587791045 1.477010951 1.21129369 1.012989119 1.175055582 1.097095255 1.32662493 1.353064662 NAD+5 0.064104557 0.080306208 0.067129993 0.317063557 0.045174629 0.245151028 1.888747058 1.92401364 0.741790122 1.101489816 1.546897038 1.80053657 1.296714181 NAD+6 0.052184246 0.145075244 0.065407292 0.664256797 -0.000164615 0.406320056 2.076853452 2.103386268 1.007605147 1.649170378 1.894488059 2.086250024 1.798287306 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Isocaproate Leucine Valine1 Isoleucine Valine2 Isobutyrate Lactate1 Alanine Lysine Acetate Glutamate1 Glutamine1+Glutamate+Methionine+O-Acetylcholine Glutamate Pyruvate Succinate Citrate-1 Aspartate Creatine1 Creatinine O-phosphocholine Sn-Glycero-3-Phosphocholine Trimethyl-N-oxide Taurine1 Myo-inositol1 Taurine-2 Myo-Inositol2 Glycine Creatine2 Myo-inositol3 Lactate2 Sn-Glycero-3-Phosphocholine2 ATP+AMP1 Inosine1 ATP+AMP2 Glucose1 Glucose2 Allantoin Sucrose Uracil Uridine Triphosphate NAD+1 NAD+2 ATP+AMP3 Fumarate P-Cresol1? p-Cresol2? Tyrosine1 Histidine Unknown7.14to7.11ppm Tyrosine2 Tryptophan1 Phenylalanine Benzoate Nicotinurate2 Tyramine? Tryptophan2 Xanthine? Unknown7.96to7.94 Inosine_or_Adenosine NAD+3 Formate NADH NADH2 ATP2 Unknown8.61to8.59 ATP+AMP4 Nicotinurate 3 NAD+4 Nicotinurate4 NAD+5 NAD+6 METABOLITES_END #END