#METABOLOMICS WORKBENCH lgafeira_20220714_113333 DATATRACK_ID:3341 STUDY_ID:ST002277 ANALYSIS_ID:AN003721 PROJECT_ID:PR001458 VERSION 1 CREATED_ON September 1, 2022, 8:19 pm #PROJECT PR:PROJECT_TITLE Skin-to-blood pH shift triggers metabolome and proteome global remodelling in PR:PROJECT_TITLE Staphylococcus epidermidis PR:PROJECT_SUMMARY Staphylococcus epidermidis (SE) is one of the most common bacteria of the human PR:PROJECT_SUMMARY skin microbiota. Despite its role as a commensal, SE has emerged as an PR:PROJECT_SUMMARY opportunistic pathogen, associated with 80% of medical devices related PR:PROJECT_SUMMARY infections. Moreover, these bacteria are extremely difficult to treat due to PR:PROJECT_SUMMARY their ability to form biofilms and accumulate resistance to almost all classes PR:PROJECT_SUMMARY of antimicrobials developed so far. Thus new preventive and therapeutic PR:PROJECT_SUMMARY strategies are urgently needed. In spite of its clinical importance, the PR:PROJECT_SUMMARY molecular mechanisms associated with SE colonisation and disease are still PR:PROJECT_SUMMARY poorly understood. A deeper understanding of the metabolic and cellular PR:PROJECT_SUMMARY processes associated with response to environmental factors characteristic of SE PR:PROJECT_SUMMARY ecological niches in health and disease might provide new clues on colonisation PR:PROJECT_SUMMARY and disease processes. Here we studied the impact of pH conditions, mimicking PR:PROJECT_SUMMARY the skin pH (5.5) and blood pH (7.4), in a S. epidermidis commensal strain, PR:PROJECT_SUMMARY belonging to the B clonal lineage, by means of next-generation proteomics and 1H PR:PROJECT_SUMMARY NMR-based metabolomics. Moreover, we evaluated the metabolic changes occurring PR:PROJECT_SUMMARY when a sudden pH change arise, simulating the skin barrier break produced by a PR:PROJECT_SUMMARY catheter. We found that exposure of S. epidermidis to skin pH induced oxidative PR:PROJECT_SUMMARY phosphorylation and biosynthesis of peptidoglycan, lipoteichoic acids and PR:PROJECT_SUMMARY betaine. In contrast, at blood pH, the incorporation of monosaccharides and its PR:PROJECT_SUMMARY oxidation by glycolysis and fermentation was promoted. Additionally, several PR:PROJECT_SUMMARY proteins related to virulence and immune evasion, namely extracellular proteases PR:PROJECT_SUMMARY and membrane iron transporters were more abundant at blood pH. In the situation PR:PROJECT_SUMMARY of an abrupt skin-to-blood pH shift we observed the decrease in the osmolyte PR:PROJECT_SUMMARY betaine and changes in the levels of several metabolites and proteins involved PR:PROJECT_SUMMARY in redox cell homeostasis. Our results suggest that at the skin pH S. PR:PROJECT_SUMMARY epidermidis cells are metabolically more active and adhesion is promoted, while PR:PROJECT_SUMMARY at blood pH, metabolism is tuned down and cells have a more virulent profile. pH PR:PROJECT_SUMMARY increase during commensal-to-pathogen conversion appears to be a critical PR:PROJECT_SUMMARY environmental signal to the remodelling of the S. epidermidis metabolism towards PR:PROJECT_SUMMARY a more pathogenic state. Targeting S. epidermidis proteins induced by a low PR:PROJECT_SUMMARY alkaline pH and local acidification of medical devices microenvironment might be PR:PROJECT_SUMMARY new strategies to treat and prevent S. epidermidis infections. PR:INSTITUTE ITQB NOVA PR:LAST_NAME Gonçalves PR:FIRST_NAME Luís PR:ADDRESS Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal PR:EMAIL lgafeira@itqb.unl.pt PR:PHONE 214469464 #STUDY ST:STUDY_TITLE Skin-to-blood pH shift triggers metabolome and proteome global remodelling in ST:STUDY_TITLE Staphylococcus epidermidis ST:STUDY_TYPE NMR Metabolomics combine with proteomics to study pH adaptation of ST:STUDY_TYPE Staphylococcus epidermidis 19N ST:STUDY_SUMMARY Staphylococcus epidermidis (SE) is one of the most common bacteria of the human ST:STUDY_SUMMARY skin microbiota. Despite its role as a commensal, SE has emerged as an ST:STUDY_SUMMARY opportunistic pathogen, associated with 80% of medical devices related ST:STUDY_SUMMARY infections. Moreover, these bacteria are extremely difficult to treat due to ST:STUDY_SUMMARY their ability to form biofilms and accumulate resistance to almost all classes ST:STUDY_SUMMARY of antimicrobials developed so far. Thus new preventive and therapeutic ST:STUDY_SUMMARY strategies are urgently needed. In spite of its clinical importance, the ST:STUDY_SUMMARY molecular mechanisms associated with SE colonisation and disease are still ST:STUDY_SUMMARY poorly understood. A deeper understanding of the metabolic and cellular ST:STUDY_SUMMARY processes associated with response to environmental factors characteristic of SE ST:STUDY_SUMMARY ecological niches in health and disease might provide new clues on colonisation ST:STUDY_SUMMARY and disease processes. Here we studied the impact of pH conditions, mimicking ST:STUDY_SUMMARY the skin pH (5.5) and blood pH (7.4), in a S. epidermidis commensal strain, ST:STUDY_SUMMARY belonging to the B clonal lineage, by means of next-generation proteomics and 1H ST:STUDY_SUMMARY NMR-based metabolomics. Moreover, we evaluated the metabolic changes occurring ST:STUDY_SUMMARY when a sudden pH change arise, simulating the skin barrier break produced by a ST:STUDY_SUMMARY catheter. We found that exposure of S. epidermidis to skin pH induced oxidative ST:STUDY_SUMMARY phosphorylation and biosynthesis of peptidoglycan, lipoteichoic acids and ST:STUDY_SUMMARY betaine. In contrast, at blood pH, the incorporation of monosaccharides and its ST:STUDY_SUMMARY oxidation by glycolysis and fermentation was promoted. Additionally, several ST:STUDY_SUMMARY proteins related to virulence and immune evasion, namely extracellular proteases ST:STUDY_SUMMARY and membrane iron transporters were more abundant at blood pH. In the situation ST:STUDY_SUMMARY of an abrupt skin-to-blood pH shift we observed the decrease in the osmolyte ST:STUDY_SUMMARY betaine and changes in the levels of several metabolites and proteins involved ST:STUDY_SUMMARY in redox cell homeostasis. Our results suggest that at the skin pH S. ST:STUDY_SUMMARY epidermidis cells are metabolically more active and adhesion is promoted, while ST:STUDY_SUMMARY at blood pH, metabolism is tuned down and cells have a more virulent profile. pH ST:STUDY_SUMMARY increase during commensal-to-pathogen conversion appears to be a critical ST:STUDY_SUMMARY environmental signal to the remodelling of the S. epidermidis metabolism towards ST:STUDY_SUMMARY a more pathogenic state. Targeting S. epidermidis proteins induced by a low ST:STUDY_SUMMARY alkaline pH and local acidification of medical devices microenvironment might be ST:STUDY_SUMMARY new strategies to treat and prevent S. epidermidis infections. ST:INSTITUTE ITQB NOVA ST:LABORATORY Proteomics of Non-Model Organisms ST:LAST_NAME Gonçalves ST:FIRST_NAME Luís ST:ADDRESS Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal ST:EMAIL lgafeira@itqb.unl.pt ST:PHONE 214469464 ST:NUM_GROUPS 3 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Staphylococcus epidermidis SU:TAXONOMY_ID 1282 SU:GENOTYPE_STRAIN Staphylococcus epidermidis 19N #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 - SE_12 class:N55 RAW_FILE_NAME=SE_12 SUBJECT_SAMPLE_FACTORS - SE_16 class:N57 RAW_FILE_NAME=SE_16 SUBJECT_SAMPLE_FACTORS - SE_18 class:N57 RAW_FILE_NAME=SE_18 SUBJECT_SAMPLE_FACTORS - SE_19 class:N57 RAW_FILE_NAME=SE_19 SUBJECT_SAMPLE_FACTORS - SE_2 class:N77 RAW_FILE_NAME=SE_2 SUBJECT_SAMPLE_FACTORS - SE_20 class:N57 RAW_FILE_NAME=SE_20 SUBJECT_SAMPLE_FACTORS - SE_22 class:N55 RAW_FILE_NAME=SE_22 SUBJECT_SAMPLE_FACTORS - SE_23 class:N57 RAW_FILE_NAME=SE_23 SUBJECT_SAMPLE_FACTORS - SE_27 class:N57 RAW_FILE_NAME=SE_27 SUBJECT_SAMPLE_FACTORS - SE_28 class:N57 RAW_FILE_NAME=SE_28 SUBJECT_SAMPLE_FACTORS - SE_29 class:N55 RAW_FILE_NAME=SE_29 SUBJECT_SAMPLE_FACTORS - SE_30 class:N77 RAW_FILE_NAME=SE_30 SUBJECT_SAMPLE_FACTORS - SE_31 class:N55 RAW_FILE_NAME=SE_31 SUBJECT_SAMPLE_FACTORS - SE_34 class:N57 RAW_FILE_NAME=SE_34 SUBJECT_SAMPLE_FACTORS - SE_37 class:N77 RAW_FILE_NAME=SE_37 SUBJECT_SAMPLE_FACTORS - SE_39 class:N55 RAW_FILE_NAME=SE_39 SUBJECT_SAMPLE_FACTORS - SE_4 class:N77 RAW_FILE_NAME=SE_4 SUBJECT_SAMPLE_FACTORS - SE_43 class:N77 RAW_FILE_NAME=SE_43 SUBJECT_SAMPLE_FACTORS - SE_44 class:N55 RAW_FILE_NAME=SE_44 SUBJECT_SAMPLE_FACTORS - SE_48 class:N55 RAW_FILE_NAME=SE_48 SUBJECT_SAMPLE_FACTORS - SE_50 class:N77 RAW_FILE_NAME=SE_50 SUBJECT_SAMPLE_FACTORS - SE_6 class:N77 RAW_FILE_NAME=SE_6 SUBJECT_SAMPLE_FACTORS - SE_8 class:N77 RAW_FILE_NAME=SE_8 #COLLECTION CO:COLLECTION_SUMMARY The Staphylococcus epidermidis 19N strain was collected from the anterior nares CO:COLLECTION_SUMMARY of a healthy person in Portugal in 2001. This strain was previously CO:COLLECTION_SUMMARY characterised by whole genome sequencing and belongs to clonal lineage B. A CO:COLLECTION_SUMMARY single colony from a S. epidermidis 19N strain culture grown O/N at 37ºC (TSA, CO:COLLECTION_SUMMARY BactoTM), was used to pre-inoculate Tryptic Soy Broth (TSB) medium with two CO:COLLECTION_SUMMARY different pH (5.5 and 7.4) that was incubated overnight at 37ºC under CO:COLLECTION_SUMMARY agitation. Pre-inoculums were adjusted either to pH 5.5 or pH 7.4, with CO:COLLECTION_SUMMARY hydrochloric acid. In this work, three pH transitions from pre-inoculum to CO:COLLECTION_SUMMARY inoculum were assayed. S. epidermidis pre-inoculums and the growth were CO:COLLECTION_SUMMARY performed at medium with pH 7.4, to mimic the blood pH; and pH 5.5, to mimic the CO:COLLECTION_SUMMARY skin pH. The pre-inoculum cellular density was adjusted to 0.06 (OD600) CO:COLLECTION_SUMMARY (aprox.1.5x108 CFU/mL) and used to inoculate fresh medium in the three CO:COLLECTION_SUMMARY conditions depicted in Figure 1, simulating S. epidermidis at skin and blood and CO:COLLECTION_SUMMARY a pH shock endured by S. epidermidis during the infection process from skin to CO:COLLECTION_SUMMARY blood transition. The cell cultures incubated at 37ºC with 225 rpm were CO:COLLECTION_SUMMARY followed by OD600 and recovered at mid-exponential phase for further analysis. CO:SAMPLE_TYPE Staphylococcus epidermidis intracellular CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY In this work, three pH transitions from pre-inoculum to inoculum were assayed. TR:TREATMENT_SUMMARY S. epidermidis pre-inoculums and the growth were performed at medium with pH TR:TREATMENT_SUMMARY 7.4, to mimic the blood pH; and pH 5.5, to mimic the skin pH. The pre-inoculum TR:TREATMENT_SUMMARY cellular density was adjusted to 0.06 (OD600) (aprox.1.5x108 CFU/mL) and used to TR:TREATMENT_SUMMARY inoculate fresh medium in the three conditions depicted in Figure 1, simulating TR:TREATMENT_SUMMARY S. epidermidis at skin and blood and a pH shock endured by S. epidermidis during TR:TREATMENT_SUMMARY the infection process from skin to blood transition. The cell cultures incubated TR:TREATMENT_SUMMARY at 37ºC with 225 rpm were followed by OD600 and recovered at mid-exponential TR:TREATMENT_SUMMARY phase for further analysis. Pre-inocula were prepared in TSB medium at pH 5.5 or TR:TREATMENT_SUMMARY 7.4. Inocula at pH 5.5 was used for the cultures grown at 5.5 (N55) and 7.4 TR:TREATMENT_SUMMARY (N57), and the inoculum at pH 7.4 for the culture at the same pH (N77). The TR:TREATMENT_SUMMARY cells were harvested at mid-exponential phase. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were recovered at mid-exponential phase from 100 mL cultures following a SP:SAMPLEPREP_SUMMARY protocol adapted from Somerville & Powers (Somerville and Powers 2014). Eight SP:SAMPLEPREP_SUMMARY biological replicates of each independent growth condition were obtained. Cells SP:SAMPLEPREP_SUMMARY were harvested by centrifugation at 5000 x g for 5 min at 4ºC. Cells were SP:SAMPLEPREP_SUMMARY washed with 20 mM phosphate buffer pH 7.2-7.4 and centrifuged for 1 min at SP:SAMPLEPREP_SUMMARY 13,000 rpm. Cell pellet was suspended in the same buffer with a final OD600 of SP:SAMPLEPREP_SUMMARY 20 and stored at -80ºC for further metabolite extraction. Cells were thawed in SP:SAMPLEPREP_SUMMARY a water bath at room temperature and 750 µL of 60% methanol were added and SP:SAMPLEPREP_SUMMARY subjected to three freeze-thaw cycles using liquid nitrogen. Extracted samples SP:SAMPLEPREP_SUMMARY were centrifuged at 21,000 g for 5 min at 4ºC. The extraction process on the SP:SAMPLEPREP_SUMMARY pellets was repeated twice. The supernatants were kept and stored together at SP:SAMPLEPREP_SUMMARY -20ºC overnight and dried in a SpeedVac. Dried samples were dissolved in: 750 SP:SAMPLEPREP_SUMMARY µL phosphate buffer (33 mM, pH 7.0 in D2O with 2 mM of sodium azide) with 0.21 SP:SAMPLEPREP_SUMMARY mM of 3-(trimethylsilyl)propionic-2,2,3,3-d4 (TSP). The suspensions were SP:SAMPLEPREP_SUMMARY centrifuged at 21,000 g for 5 min at 4ºC and the resulting supernatants were SP:SAMPLEPREP_SUMMARY then transferred to 5 mm NMR tubes. #ANALYSIS AN:SOFTWARE_VERSION Bruker TopSpin 3.2 #NMR NM:INSTRUMENT_NAME Bruker Avance II+ 800 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 800 NM:NMR_PROBE 5 mm TXI-Z H/C/N/-D NM:NMR_SOLVENT D2O NM:NMR_TUBE_SIZE 5 mm NM:SHIMMING_METHOD Topshim NM:TEMPERATURE 25 NM:NUMBER_OF_SCANS 256 NM:DUMMY_SCANS 4 NM:CHEMICAL_SHIFT_REF_STD TSP NM:NMR_RESULTS_FILE NMR_SE #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS nanomoles NMR_METABOLITE_DATA_START Samples SE_12 SE_16 SE_18 SE_19 SE_2 SE_20 SE_22 SE_23 SE_27 SE_28 SE_29 SE_30 SE_31 SE_34 SE_37 SE_39 SE_4 SE_43 SE_44 SE_48 SE_50 SE_6 SE_8 Factors class:N55 class:N57 class:N57 class:N57 class:N77 class:N57 class:N55 class:N57 class:N57 class:N57 class:N55 class:N77 class:N55 class:N57 class:N77 class:N55 class:N77 class:N77 class:N55 class:N55 class:N77 class:N77 class:N77 2-Hydroxyisobutyrate 0.7 0.9 1.8 0.7 1.3 0.7 4.9 2 1.3 1.5 4.6 2.1 1.5 1 2.8 2.3 1.3 2.5 1.5 2.2 3 1.2 1.5 3-Hydroxyisovalerate 4.6 5.5 4.7 4.6 3.7 4.5 4.7 5.3 5.6 4.9 5.1 2.9 13.3 2.8 2.9 13.4 4 4.1 2.8 2.9 13.3 3.7 3.9 AMP 4.1 1.7 3.5 14.6 15.5 9.7 3.7 1.3 12.4 7.3 9 6.4 6 3.8 1.6 5.6 9.6 2 5.1 6.2 2.9 11.8 9.9 Acetate 51.5 77.1 72.4 39 131 49.8 61.8 63.7 47.5 65.1 47.9 90.6 59.2 57.6 174.1 62.6 157.9 111.2 67.2 74.9 133 114.7 161.7 Adenine 3.4 13.8 4.3 5.3 9.5 6.3 3 8.8 5.8 6.5 5.2 18.7 16.7 15.5 23.6 17.2 8.8 19.1 13.2 19 20.7 13.7 9.5 Adenosine 10.8 11.2 11.5 20.6 18.8 28.6 14.7 6.6 9.6 16.9 14.5 35.6 31.4 46.9 37.6 33.5 17.3 43 28.5 37.5 45.7 21.4 18 Alanine 22.5 19.7 18.7 26.4 34.7 36.6 17.6 15.3 13.1 32.7 37.1 60 56 54.7 82.7 51 32.2 68.9 59.7 68.5 62.5 40 30.6 Arabinose 12.7 16.9 16.5 26.9 15.5 9.8 11.4 10.9 1.9 12.7 17.9 19 14.4 12.3 17.6 22.8 6.3 21.7 21.7 20.1 17.7 18.9 14.1 Asparagine 39.7 90.7 124.6 68.5 80.8 70.6 47.4 103.6 7.7 86.7 58.3 71.6 22.3 40.3 120.3 22.4 95 96.9 23.3 15.7 110.6 77 95.5 Aspartate 101.5 216.5 250.8 191.1 386.5 275.6 119.7 201.7 149.6 211.7 133.2 475.2 162.1 262.1 509.2 159 355.8 493.5 153.3 168.5 503.7 409.8 340.4 Betaine 1106.8 541.6 765.4 206.3 1297.3 305.8 1413.3 628.5 134.5 489.9 1647.1 1257.8 1331.8 148 1331.2 1289.2 1150.8 1382.5 1198.6 1301.1 1225 1433.1 955.6 Choline 19.5 5.2 5.5 3.2 13.7 3.6 24.2 5 6.7 4.7 32.4 15.8 39.6 2.4 17.3 37 15.6 16.3 32.1 38.9 14.8 15.6 12.5 Coenzyme A 7.2 4.8 18.9 22.7 21 13.8 6.1 7.5 11.7 14.3 7.7 2.9 3.4 3.3 4.3 6.9 7.8 9.7 4 3.4 3.9 26.8 18.5 Cytidine monophosphate 4.1 2.2 6 27.2 32.5 15.2 6 3.5 12.8 6.1 4.4 32.7 28.2 25.4 13.2 29.1 29 16.5 23 28.6 27.5 35.8 27.6 Formate 25.2 33.6 24 29.3 17.5 27.7 31.5 31.7 30.3 27.4 34.6 130.1 156.7 87.9 105.1 177 16 121.7 90.5 147.9 115.3 15.9 15.8 Glucose 22.3 4 270.7 5.23 6.8 7 114.1 6.7 3.4 4.7 1.9 Glutamate 689.7 512.7 638.2 406.8 726.2 464.2 895.7 510.2 294.4 464.6 965 647.2 757.2 247.6 635.3 796.7 610.1 662.3 709.6 837.5 677.1 666.5 583.6 Glutamine 101.5 50.3 50.6 39.2 13.9 138.9 51.1 18.8 41 165.9 49.9 53.1 57.2 32.2 95.7 50.6 34.1 35.4 59.5 69.4 75.1 Glycine 7.7 1.7 1.7 102.1 0.6 5.1 6.3 1.8 32.7 24.7 7.2 0.9 1 1 1 1 4.6 1.3 0.9 1.7 1.1 9 0.8 Guanosine 3.9 1.3 2.6 5 5.1 4.7 2.4 1.7 2.8 2.1 3.4 7.8 6 11.9 5.7 7.8 5.4 8.9 6.7 8.1 7.9 3.3 5.3 Histidine 2.2 3.7 3.8 3.1 4.9 4.4 3 3.8 2.6 3.8 3 6.4 5.4 3.7 6.4 4.9 4.3 5.8 5.5 5.1 6.3 5 4 Isoleucine 3.4 4.6 5.6 2.7 6.5 4.7 4.5 5.4 1.6 4 5.5 5.8 2.5 1.9 6.7 2.1 5.1 5.9 1.7 2.9 7.3 6.1 5 Isovalerate 1.1 1 1 1.3 4.5 0.7 1.1 0.7 1.7 1.2 1.1 3 5.2 3.3 3.7 5.2 5.5 3.2 5.3 5 3.9 4.9 4.4 Lactate 29.9 25.3 24.7 7 56.1 19.8 28.8 18.1 9 15 37.4 30 12.8 13.4 90.3 10.1 79.9 55.1 12.6 5.4 55.8 55.3 68.7 Leucine 16.4 17.1 24.5 11.8 21.2 19 19.3 21.7 3.3 20.7 25.1 26.4 15.2 10.4 31.3 13.6 21.1 25.9 11.6 16.6 34 22.3 21.1 Lysine 11.5 16.1 18.2 15.3 21 16 10.2 20.3 7.8 16 15 26 25.8 20.8 33.1 26.6 20.2 28.5 28.9 28 31.8 22.5 19.9 NAD+ 16.8 22.1 21.1 14.6 15.7 15.7 19.9 18.7 20.9 15.7 16.7 13.5 15 15.5 16.8 13.9 15.7 14.4 14 14.3 16 14.4 15.8 NADP+ 2.4 4.1 3.8 2.7 2.2 2.4 2.9 3.1 7.3 3 2.3 2.5 2.1 3.2 3.1 1.8 2.3 2.2 2.2 1.8 3 2.7 2.9 Nicotinate 1.5 0.8 1.5 0.6 0.7 1.6 3.4 1.2 2.9 1.1 2 1.4 1.1 1.7 2.3 2.7 1.8 1.7 Phenylalanine 6.8 9.8 13.2 8.3 13.1 10.2 8.8 10 5.2 9.1 10.6 13.8 9.9 6 14.5 9.9 12.5 12.9 8.6 10.2 13.7 12.8 12.1 Phosphoenolpyruvic acid 6.8 30.6 22 12.9 9.7 19.2 11.4 16.3 16.9 19.7 12.4 13.9 11.4 6.3 17.1 10.5 4.8 14.2 11.2 15 14.7 3.9 7.1 Succinate 7.6 8.3 11.2 8.8 16.8 9.5 9.7 9.2 13 8 12.7 20.3 24.4 16.5 22.8 24.3 15.5 20.2 24.9 26.8 18.8 18.2 15.5 Sucrose 18.8 180.8 30.2 1.2 1.9 66.2 143.5 1.6 4.8 2.8 3 0.9 3 21.4 2.2 Threonine 6.2 12.4 10.5 19.9 8.3 5.9 16.8 8.7 2.6 7 20.5 7.2 6.2 4 6.5 6.5 7.9 6.2 4.6 19.7 8.1 9.6 7.8 Tryptophan 2.1 1.2 2.5 1.2 0.8 2.7 1.6 2.2 3.8 1.7 1.8 3 3.3 1.8 0.6 Tyrosine 0.6 1.6 1.1 0.9 1.9 2.1 1.1 1.5 0.6 1.3 0.8 3.3 1.9 1.5 3.1 2 2.3 3.4 1.6 2.3 3.5 1.6 2.4 UMP 2 2.3 2.3 8.7 12.6 8.7 1.9 1.1 8.2 3.4 2.2 11.4 10.7 6.8 2.8 8.7 8.2 4 7.2 8.7 7.9 10.6 8.7 Uracil 1.4 1.3 1.4 4.2 8.3 4.5 1.5 1.7 2.8 1.8 1.2 13 14.9 12.1 14.2 16.1 7.9 13.4 12.9 17.3 14.7 8.7 8.4 Uridine 1.7 3.3 2.3 3.7 2.6 8.7 2.1 5.4 3 2.8 4.1 8.9 6.9 11.5 13.2 9.4 2.9 9.2 9.6 6.1 9 2.8 2.9 Valine 6.9 5.8 8.1 4.8 10.1 7.1 6.7 10.2 2.5 5.6 8.8 10.6 7.8 3.9 11.3 6.8 9.7 10 6.1 8 13.1 10.7 8.1 sn-Glycero-3-phosphocholine 117.5 35.2 45.4 16.5 29.3 17.3 137.4 38.9 12.1 30.1 151.4 32.8 94.7 13.7 34.2 95.1 28.9 32.3 85 107.4 36.3 30.7 26.1 beta-Alanine 2.8 5.3 6.6 4.7 10.6 10.5 5 7.6 2.1 4.1 11.3 13.5 15.9 9.6 16.5 10.6 7 15.9 9.8 13 12.7 7.7 5.6 Cystathionine 8.4 34.9 36.7 17.6 26.1 21.2 7.7 33.1 12.3 33.1 12 28.1 10 18.6 29 7.5 24.4 30 6.5 6.1 30 28.5 22.4 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem KEGG 2-Hydroxyisobutyrate 11671 NA 3-Hydroxyisovalerate 69362 C20827 AMP 6083 C00020 Acetate 176 C00033 Adenine 190 C00147 Adenosine 60961 C00212 Alanine 5950 C00041 Arabinose 439195 C02479 Asparagine 6267 C00152 Aspartate 5960 C00049 Betaine 247 C00719 Choline 305 C00114 Coenzyme A 87642 C00010 Cytidine monophosphate 6131 C00055 Formate 284 C00058 Glucose 5793 C00221 Glutamate 33032 C00025 Glutamine 5961 C00064 Glycine 750 C00037 Guanosine 6802 C00387 Histidine 6274 C00135 Isoleucine 6306 C00407 Isovalerate 10430 C08262 Lactate 107689 C00186 Leucine 6106 C00123 Lysine 5962 C00047 NAD+ 5893 C00003 NADP+ 5886 C00006 Nicotinate 938 C00253 Phenylalanine 6140 C00079 Phosphoenolpyruvic acid 1005 C00074 Succinate 1110 C00042 Sucrose 5988 C00089 Threonine 6288 C00188 Tryptophan 6305 C00078 Tyrosine 6057 C00082 UMP 6030 C00105 Uracil 1174 C00106 Uridine 6029 C00299 Valine 6287 C00183 sn-Glycero-3-phosphocholine 71920 C00670 beta-Alanine 239 C00099 Cystathionine 439258 C02291 METABOLITES_END #END