#METABOLOMICS WORKBENCH thflott_20200602_061913 DATATRACK_ID:2035 STUDY_ID:ST001390 ANALYSIS_ID:AN002319 PROJECT_ID:PR000953 VERSION 1 CREATED_ON June 2, 2020, 5:28 pm #PROJECT PR:PROJECT_TITLE Natural History of the Systemic Responses to a Renal Inoculation of PR:PROJECT_TITLE Uropathogenic E. coli in Swine PR:PROJECT_SUMMARY Metabolomics of whole blood samples from a novel large animal model of systemic PR:PROJECT_SUMMARY infection. PR:INSTITUTE University of Michigan PR:LAST_NAME Flott PR:FIRST_NAME Thomas PR:ADDRESS 428 Church St., Ann Arbor, MI, 48109 PR:EMAIL thflott@med.umich.edu PR:PHONE 7346604241 PR:CONTRIBUTORS Mohamad Hakam Tiba, Brendan M. McCracken, Robert P.Dickson, Jean A. Nemzek, PR:CONTRIBUTORS Carmen I. Colmenero, Danielle C. Leander, Thomas L. Flott, Rodney C. Daniels, PR:CONTRIBUTORS Kristine Konopka, J. Scott VanEpps, Kathleen A. Stringer, and Kevin R. Ward #STUDY ST:STUDY_TITLE Natural history of the systemic responses to a renal inoculation of ST:STUDY_TITLE uropathogenic E. coli in swine ST:STUDY_SUMMARY Background: The pathogenesis of systemic infection and its progression to sepsis ST:STUDY_SUMMARY remains poorly understood. Progress in the field has been stifled by the ST:STUDY_SUMMARY shortcomings of experimental models which include poor replication of the human ST:STUDY_SUMMARY condition. To address these challenges, we developed a novel large animal model ST:STUDY_SUMMARY of systemic infection that is capable of generating high-dimensional clinically ST:STUDY_SUMMARY relevant data. Methods: Male swine (n=5) were anesthetized, mechanically ST:STUDY_SUMMARY ventilated, and surgically instrumented for continuous hemodynamic monitoring ST:STUDY_SUMMARY and serial blood sampling. Animals were inoculated with uropathogenic E. coli by ST:STUDY_SUMMARY direct injection into the renal parenchyma and were maintained under anesthesia ST:STUDY_SUMMARY for up to 24 hours. The natural history of the infection was studied, animals ST:STUDY_SUMMARY were not resuscitated. Multi-dimensional data were collected hourly to every 6 ST:STUDY_SUMMARY hours; all animals were euthanized when at predetermined physiologic endpoints. ST:STUDY_SUMMARY Results: Core body temperature progressively increased from mean (SD) 37.9(0.8) ST:STUDY_SUMMARY ̊C at baseline to 43.0(1.2) ̊C at experiment termination (p=0.006). While mean ST:STUDY_SUMMARY arterial pressure did not begin to decline until 6h post inoculation, dropping ST:STUDY_SUMMARY from 86(9) mmHg at baseline to 28(5) mmHg (p=0.005) at termination. Blood ST:STUDY_SUMMARY glucose progressively declined but lactate levels did not elevate until the last ST:STUDY_SUMMARY hours of the experiment. There were also temporal changes in whole blood ST:STUDY_SUMMARY concentrations of a number of metabolites including increases in the ST:STUDY_SUMMARY catecholamine precursors, tyrosine (p=0.005) and phenylalanine (p=0.005). Lung, ST:STUDY_SUMMARY liver, and kidney function parameters worsened as infection progressed and at ST:STUDY_SUMMARY study termination there was histopathological evidence of injury in these ST:STUDY_SUMMARY end-organs. Conclusion: We demonstrate a versatile, multi-dimensional, ST:STUDY_SUMMARY longitudinal, swine model of systemic infection that could be used to further ST:STUDY_SUMMARY our understanding of the mechanisms that underlie infection-induced multi-organ ST:STUDY_SUMMARY dysfunction and failure, optimize resuscitation protocols and test therapeutic ST:STUDY_SUMMARY interventions. Such a model could improve translation of findings from the bench ST:STUDY_SUMMARY to the bedside, circumventing a significant obstacle in sepsis research. ST:INSTITUTE University of Michigan ST:LAST_NAME Flott ST:FIRST_NAME Thomas ST:ADDRESS 428 Church St., Ann Arbor, MI, 48109 ST:EMAIL thflott@med.umich.edu ST:PHONE 7346604241 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Sus Scrofa SU:TAXONOMY_ID 9823 SU:GENDER Male #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 17x10022 224_003 Timepoint:6 Sample Collection Site=JV; RAW_FILE_NAME=224_003 SUBJECT_SAMPLE_FACTORS 17x10022 224_035 Timepoint:- Sample Collection Site=JV; RAW_FILE_NAME=224_035 SUBJECT_SAMPLE_FACTORS 17x10022 224_097 Timepoint:12 Sample Collection Site=JV; RAW_FILE_NAME=224_097 SUBJECT_SAMPLE_FACTORS 17x10039 224_013 Timepoint:- Sample Collection Site=JV; RAW_FILE_NAME=224_013 SUBJECT_SAMPLE_FACTORS 17x10039 224_025 Timepoint:12 Sample Collection Site=JV; RAW_FILE_NAME=224_025 SUBJECT_SAMPLE_FACTORS 17x10039 224_079 Timepoint:6 Sample Collection Site=JV; RAW_FILE_NAME=224_079 SUBJECT_SAMPLE_FACTORS 17x10055 224_009 Timepoint:- Sample Collection Site=JV; RAW_FILE_NAME=224_009 SUBJECT_SAMPLE_FACTORS 17x10055 224_010 Timepoint:12 Sample Collection Site=JV; RAW_FILE_NAME=224_010 SUBJECT_SAMPLE_FACTORS 17x10055 224_020 Timepoint:6 Sample Collection Site=JV; RAW_FILE_NAME=224_020 SUBJECT_SAMPLE_FACTORS 17x10064 224_001 Timepoint:12 Sample Collection Site=JV; RAW_FILE_NAME=224_001 SUBJECT_SAMPLE_FACTORS 17x10064 224_024 Timepoint:- Sample Collection Site=JV; RAW_FILE_NAME=224_024 SUBJECT_SAMPLE_FACTORS 17x10064 224_090 Timepoint:6 Sample Collection Site=JV; RAW_FILE_NAME=224_090 #COLLECTION CO:COLLECTION_SUMMARY Whole blood (WB) samples were collected from the indwelling line in the internal CO:COLLECTION_SUMMARY jugular vein. Blood was collected into 4ml Vacutainer tubes containing sodium CO:COLLECTION_SUMMARY heparin. Following collection, tubes were inverted several times to ensure CO:COLLECTION_SUMMARY adequate mixing and were immediately placed on ice. Each of two 600uL aliquots CO:COLLECTION_SUMMARY were added to screw-top cryogenic storage tubes and flash frozen in liquid CO:COLLECTION_SUMMARY nitrogen. Frozen samples were placed on dry ice until the completion of the CO:COLLECTION_SUMMARY experiment at which time they were transferred to storage in liquid nitrogen and CO:COLLECTION_SUMMARY stored until the time of assay. The remaining whole blood volume in the CO:COLLECTION_SUMMARY collection tube was centrifuged (1300g for 10 min at 4C). Aliquots of CO:COLLECTION_SUMMARY plasma(~600uL) were transferred into screw-top cryogenic storage tubes, flash CO:COLLECTION_SUMMARY frozen in liquid nitrogen, placed on dry ice, and stored in liquid nitrogen for CO:COLLECTION_SUMMARY future assays. CO:SAMPLE_TYPE Blood (whole) CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Male swine (n=5) were anesthetized, mechanically ventilated, and surgically TR:TREATMENT_SUMMARY instrumented for continuous hemodynamic monitoring and serial blood sampling. TR:TREATMENT_SUMMARY Animals were inoculated with uropathogenic E. coli by direct injection into the TR:TREATMENT_SUMMARY renal parenchyma and were maintained under anesthesia for up to 24 hours. The TR:TREATMENT_SUMMARY natural history of the infection was studied, animals were not resuscitated. TR:TREATMENT_SUMMARY Multi-dimensional data were collected hourly to every 6 hours; all animals were TR:TREATMENT_SUMMARY euthanized when at predetermined physiologic endpoints. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Whole blood samples were thawed on ice and subjected to a methanol-chloroform SP:SAMPLEPREP_SUMMARY precipitation. Briefly, samples were thawed in an ice-water bath after which 500 SP:SAMPLEPREP_SUMMARY μL of blood was transferred to a microcentrifuge tube and 1ml of a 1:1 SP:SAMPLEPREP_SUMMARY methanol-chloroform solution was added. Samples were sonicated for 2 minutes at SP:SAMPLEPREP_SUMMARY 4°C, then incubated at -20°C for 20 minutes, and then centrifuged (13,400g at SP:SAMPLEPREP_SUMMARY 4°C for 30 minutes). The aqueous supernatant was transferred to a new SP:SAMPLEPREP_SUMMARY microcentrifuge tube and dried by lyophilization. Samples were then resuspended SP:SAMPLEPREP_SUMMARY in 600 μL of 50mM sodium phosphate buffer in D2O for NMR analysis. SP:PROCESSING_STORAGE_CONDITIONS On ice #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Varian NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500 MHz #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS uM NMR_METABOLITE_DATA_START Samples 224_035 224_013 224_009 224_024 224_003 224_079 224_020 224_090 224_097 224_010 224_025 224_001 Factors Timepoint:- Timepoint:- Timepoint:- Timepoint:- Timepoint:6 Timepoint:6 Timepoint:6 Timepoint:6 Timepoint:12 Timepoint:12 Timepoint:12 Timepoint:12 Aminoadipic acid 37.07096774 52.572 55.7375 68.94097222 83.5456 222.9464286 2-Hydroxybutyric acid 22 23.6516129 19.175 Ketoleucine 26.95 27.5 16.26595745 30.88064 20.87962963 29.69032258 17.95918367 13.728 22.1 24.63541667 19.9104 35.49744898 ADP 212.74 193.0729167 106.7234043 144.97504 135.2083333 238.0258065 288.0952381 147.108 247.1625 194.21875 178.2176 219.4387755 AMP 113.08 73.33333333 69.27659574 135.41024 181.5509259 204.1419355 108.6904762 159.432 166.725 143.0381944 194.224 313.1632653 ATP 323.51 327.7083333 185.8297872 201.95392 261.6319444 301.7677419 480.0340136 150.696 370.825 369.9131944 166.1152 183.380102 Alanine 199.32 431.40625 248.2021277 553.52864 596.2152778 814.2193548 636.6156463 345.384 740.1875 1421.40625 845.216 1233.852041 Betaine 112.2 255.234375 157.9787234 347.47552 100.3240741 190.0516129 156.7687075 139.62 118.4625 194.7916667 168.2624 259.4260204 Carnitine 6.36 7.8 12.8 8.78 8.28 Choline 22.88 18.046875 14.62765957 17.35328 26.6087963 24.82580645 31.05442177 12.48 33.3125 38.19444444 19.3248 25.67602041 Creatine 94.82 100.2604167 174.9468085 237.89024 187.025463 238.3612903 332.4319728 233.532 397.8 519.0625 431.5872 618.0484694 Creatinine 68.2 61.015625 54.18085106 53.2896 75.37037037 79.50967742 142.3639456 43.992 185.9 233.9409722 98.7712 164.1581633 Dimethyl sulfone 8.36 22.20052083 13.69148936 22.27232 8.530092593 24.32258065 16.64965986 9.672 13.65 16.04166667 15.4208 16.97704082 Glucose 6417.62 4338.268229 2892.06383 4863.56416 2906.851852 3267.277419 2286.428571 2214.576 2254.6875 1009.861111 2061.8976 1678.622449 Glutamic acid 169.07 286.171875 150.2553191 292.54624 186.0069444 363.3290323 232.1598639 316.992 259.35 224.9652778 420.0704 597.1428571 Glutamine 228.03 325.1302083 200.3404255 316.73152 324.3981481 505.0709677 355.255102 236.34 571.35 636.7013889 503.8112 569.0816327 Glutathione 98.89 87.08333333 53.71276596 69.41312 133.1712963 134.3612903 106.0714286 62.556 127.5625 105.7986111 131.76 125.5739796 Glycine 1036.42 1050.15625 591.4255319 1234.40576 1225.405093 1379.006452 805.7312925 742.404 1446.575 831.1111111 1048.0288 1357.181122 Hippuric acid 72.58503401 61.75 109.2361111 71.13520408 Histidine 58.52 71.61458333 49.61702128 36.61952 70.02314815 92.09032258 101.207483 56.004 118.4625 107.1354167 89.5968 112.8061224 Hypoxanthine 27.24537037 48.07823129 36.2375 122.03125 42.65306122 IMP 12.87234043 34.12037037 42.60645161 49.57482993 23.712 34.9375 88.22916667 35.5264 43.63520408 Isoleucine 67.1 90.6640625 56.87234043 97.28768 70.40509259 99.97419355 64.3537415 71.292 80.275 80.20833333 117.7056 203.0229592 Lactic acid 1383.03 1456.783854 1099.06383 1792.58016 2291.666667 1503.806452 1394.829932 596.076 2600.4875 7598.784722 1075.7472 2767.959184 Leucine 145.2 161.4192708 101.106383 154.12992 132.7893519 184.516129 129.829932 120.588 177.6125 169.0104167 256.2976 372.5127551 Lysine 193.38 162.7083333 124.0425532 198.67456 295.2430556 327.2645161 302.1258503 234 422.5 359.21875 357.216 495.1403061 Malonic acid 174.35 145.3776042 104.5 159.45888 176.0763889 185.1870968 195.4931973 111.228 201.825 183.5243056 153.232 197.130102 Methionine 22.11 31.3671875 20.59574468 32.7936 37.04861111 49.31612903 39.09863946 28.704 48.9125 65.12152778 47.2384 72.25765306 CAR(2:0) 2.01 2.85 4.23 7.45 Ornithine 89.21 124.4661458 114.9148936 165.06112 117.2569444 183.1741935 185.2040816 131.508 146.4125 241.3888889 207.888 269.5280612 Phenylalanine 51.7 59.58333333 39.20212766 45.91104 78.04398148 110.2064516 90.54421769 70.824 136.825 124.3229167 141.9104 197.6913265 Proline 223.63 253.3723958 149.4361702 233.79104 188.8078704 354.1032258 328.3163265 164.58 267.15 281.4930556 258.4448 414.6045918 Pyruvic acid 47.19 73.90625 63.65957447 101.93344 127.6967593 142.7483871 104.2006803 59.748 107.4125 157.5520833 82.3744 121.7857143 Serine 52.03 132.34375 51.60638298 43.58816 95.3587963 114.5677419 162.755102 102.804 143.65 138.6458333 134.4928 186.0459184 Taurine 82.35677083 89.63829787 71.46272 158.125 166.5677419 217.9421769 218.712 163.15 189.8263889 120.048 409.1326531 Threonine 67.43 84.93489583 61.43617021 34.56992 142.8472222 121.4451613 127.0238095 121.992 206.7 129.0972222 162.6016 295.7653061 Trimethylamine N-oxide 51.04 31.08072917 21.53191489 42.63168 65.94907407 56.19354839 54.25170068 30.264 71.825 52.51736111 48.4096 55.14030612 Tyrosine 43.45 45.83333333 37.21276596 45.7744 62.25694444 70.95483871 82.68707483 57.408 87.75 87.65625 93.3056 125.7142857 Valine 170.17 230.8854167 155.4042553 239.8032 186.1342593 262.3483871 174.7278912 159.744 224.7375 178.5590278 291.6288 427.9336735 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID Aminoadipic acid C00956 2-Hydroxybutyric acid C05984 Ketoleucine C00233 ADP C00008 AMP C00020 ATP C00002 Alanine C00041 Betaine C00719 Carnitine C00318 Choline C00114 Creatine C00300 Creatinine C00791 Dimethyl sulfone C11142 Glucose C00221 Glutamic acid C00025 Glutamine C00064 Glutathione C00051 Glycine C00037 Hippuric acid C01586 Histidine C00135 Hypoxanthine C00262 IMP C00130 Isoleucine C00407 Lactic acid C00186 Leucine C00123 Lysine C00047 Malonic acid C00383 Methionine C00073 CAR(2:0) C02571 Ornithine C00077 Phenylalanine C00079 Proline C00148 Pyruvic acid C00022 Serine C00065 Taurine C00245 Threonine C00188 Trimethylamine N-oxide C01104 Tyrosine C00082 Valine C00183 METABOLITES_END #END