#METABOLOMICS WORKBENCH javiarranz_20250707_063749 DATATRACK_ID:6139 STUDY_ID:ST004061 ANALYSIS_ID:AN006720 PROJECT_ID:PR002549 VERSION 1 CREATED_ON July 22, 2025, 12:01 pm #PROJECT PR:PROJECT_TITLE In Vitro Method Demonstrating Trained Immunity as a Distinctive Functional PR:PROJECT_TITLE Program: Implications for Biomarker Discovery, Adaptive Immune Responses, Sample PR:PROJECT_TITLE Cryopreservation, and Mouse Genetics PR:PROJECT_SUMMARY Trained immunity is a distinct program of macrophages, enabling them to secrete PR:PROJECT_SUMMARY varying levels of inflammatory cytokines according to their functional state PR:PROJECT_SUMMARY during sequential stimulation. Experimental models that accurately replicate the PR:PROJECT_SUMMARY defining steps of trained immunity are urgently needed. Here, we developed an in PR:PROJECT_SUMMARY vitro methodology to study trained immunity using murine bone marrow-derived PR:PROJECT_SUMMARY macrophages stimulated with β-glucan and lipopolysaccharide (LPS). Longitudinal PR:PROJECT_SUMMARY analysis of interleukin 6 (IL6) and tumor necrosis factor (TNF) production PR:PROJECT_SUMMARY demonstrated that trained macrophages secrete higher cytokine levels following PR:PROJECT_SUMMARY primary stimulation with β-glucancompared to unstimulated macrophages (step 1). PR:PROJECT_SUMMARY After a resting period, trained macrophages return to basal levels of cytokine PR:PROJECT_SUMMARY production (step 2) but rapidly produce enhanced levels of IL6 and TNF after PR:PROJECT_SUMMARY secondary stimulation with LPS, compared to macrophages individually stimulated PR:PROJECT_SUMMARY with either β-glucan (step 3) or LPS (step 4) alone. The combined cytokine PR:PROJECT_SUMMARY production of macrophages after single stimulation with βglucan (stimulus 1) PR:PROJECT_SUMMARY and LPS (stimulus 2) was significantly lower than the cytokine levels produced PR:PROJECT_SUMMARY by trained macrophages sequentially stimulated with both β-glucan and LPS PR:PROJECT_SUMMARY (stimulus 1+2) (step 5). These results experimentally reproduce, for the first PR:PROJECT_SUMMARY time, the distinctive functional stages that macrophages undergo during the PR:PROJECT_SUMMARY training process. Using this methodology, we identified serum amyloid A3 protein PR:PROJECT_SUMMARY (SAA3) as a novel biomarker of trained immunity and revealed that trained PR:PROJECT_SUMMARY macrophages induce T-cell proliferation via a contact dependent mechanisms. PR:PROJECT_SUMMARY Additionally, our results uncovered adverse effects of cell cryopreservation, PR:PROJECT_SUMMARY strain-specific differences, and the impact of mTOR genetic ablation in the PR:PROJECT_SUMMARY induction of trained immunity. Standardization of methodologies to study trained PR:PROJECT_SUMMARY immunity is critical for advancing our understanding of the fundamental PR:PROJECT_SUMMARY mechanisms that regulate innate immune memory responses and develop therapies PR:PROJECT_SUMMARY that specifically target trained immunity in multiple immune-mediated PR:PROJECT_SUMMARY pathological conditions. PR:INSTITUTE Instituto de Salud Carlos III PR:LAST_NAME Arranz Herrero PR:FIRST_NAME Javier PR:ADDRESS Dalia 52, Mostoles, Madrid, 28933, Spain PR:EMAIL j.arranz3@usp.ceu.es PR:PHONE 655653445 #STUDY ST:STUDY_TITLE In Vitro Method Demonstrating Trained Immunity as a Distinctive Functional ST:STUDY_TITLE Program: Implications for Biomarker Discovery, Adaptive Immune Responses, Sample ST:STUDY_TITLE Cryopreservation, and Mouse Genetics ST:STUDY_SUMMARY Trained immunity is a distinct program of macrophages, enabling them to secrete ST:STUDY_SUMMARY varying levels of inflammatory cytokines according to their functional state ST:STUDY_SUMMARY during sequential stimulation. Experimental models that accurately replicate the ST:STUDY_SUMMARY defining steps of trained immunity are urgently needed. Here, we developed an in ST:STUDY_SUMMARY vitro methodology to study trained immunity using murine bone marrow-derived ST:STUDY_SUMMARY macrophages stimulated with β-glucan and lipopolysaccharide (LPS). Longitudinal ST:STUDY_SUMMARY analysis of interleukin 6 (IL6) and tumor necrosis factor (TNF) production ST:STUDY_SUMMARY demonstrated that trained macrophages secrete higher cytokine levels following ST:STUDY_SUMMARY primary stimulation with β-glucancompared to unstimulated macrophages (step 1). ST:STUDY_SUMMARY After a resting period, trained macrophages return to basal levels of cytokine ST:STUDY_SUMMARY production (step 2) but rapidly produce enhanced levels of IL6 and TNF after ST:STUDY_SUMMARY secondary stimulation with LPS, compared to macrophages individually stimulated ST:STUDY_SUMMARY with either β-glucan (step 3) or LPS (step 4) alone. The combined cytokine ST:STUDY_SUMMARY production of macrophages after single stimulation with βglucan (stimulus 1) ST:STUDY_SUMMARY and LPS (stimulus 2) was significantly lower than the cytokine levels produced ST:STUDY_SUMMARY by trained macrophages sequentially stimulated with both β-glucan and LPS ST:STUDY_SUMMARY (stimulus 1+2) (step 5). These results experimentally reproduce, for the first ST:STUDY_SUMMARY time, the distinctive functional stages that macrophages undergo during the ST:STUDY_SUMMARY training process. Using this methodology, we identified serum amyloid A3 protein ST:STUDY_SUMMARY (SAA3) as a novel biomarker of trained immunity and revealed that trained ST:STUDY_SUMMARY macrophages induce T-cell proliferation via a contact dependent mechanisms. ST:STUDY_SUMMARY Additionally, our results uncovered adverse effects of cell cryopreservation, ST:STUDY_SUMMARY strain-specific differences, and the impact of mTOR genetic ablation in the ST:STUDY_SUMMARY induction of trained immunity. Standardization of methodologies to study trained ST:STUDY_SUMMARY immunity is critical for advancing our understanding of the fundamental ST:STUDY_SUMMARY mechanisms that regulate innate immune memory responses and develop therapies ST:STUDY_SUMMARY that specifically target trained immunity in multiple immune-mediated ST:STUDY_SUMMARY pathological conditions. ST:INSTITUTE Instituto de Salud Carlos III ST:LAST_NAME Arranz Herrero ST:FIRST_NAME Javier ST:ADDRESS Dalia 52, Mostoles, Madrid, 28933, Spain ST:EMAIL j.arranz3@usp.ceu.es ST:PHONE 655653445 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - Naive_01 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_1-r001.d SUBJECT_SAMPLE_FACTORS - Naive_02 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_1-r002.d SUBJECT_SAMPLE_FACTORS - Naive_03 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_2-r001.d SUBJECT_SAMPLE_FACTORS - Naive_04 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_2-r002.d SUBJECT_SAMPLE_FACTORS - Naive_05 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_3-r001.d SUBJECT_SAMPLE_FACTORS - Naive_06 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_3-r002.d SUBJECT_SAMPLE_FACTORS - Naive_07 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_4-r001.d SUBJECT_SAMPLE_FACTORS - Naive_08 Sample source:macrophages | Condition:Naive RAW_FILE_NAME(Raw file name)=N_4-r002.d SUBJECT_SAMPLE_FACTORS - LPS_01 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_1-r001.d SUBJECT_SAMPLE_FACTORS - LPS_02 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_1-r002.d SUBJECT_SAMPLE_FACTORS - LPS_03 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_2-r001.d SUBJECT_SAMPLE_FACTORS - LPS_04 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_2-r002.d SUBJECT_SAMPLE_FACTORS - LPS_05 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_3-r001.d SUBJECT_SAMPLE_FACTORS - LPS_06 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_3-r002.d SUBJECT_SAMPLE_FACTORS - LPS_07 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_4-r001d SUBJECT_SAMPLE_FACTORS - LPS_08 Sample source:macrophages | Condition:LPS RAW_FILE_NAME(Raw file name)=LPS_4-r002.d SUBJECT_SAMPLE_FACTORS - Trained_01 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_1-r001.d SUBJECT_SAMPLE_FACTORS - Trained_02 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_1-r002.d SUBJECT_SAMPLE_FACTORS - Trained_03 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_2-r001.d SUBJECT_SAMPLE_FACTORS - Trained_04 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_2-r002.d SUBJECT_SAMPLE_FACTORS - Trained_05 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_3-r001.d SUBJECT_SAMPLE_FACTORS - Trained_06 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_3-r002.d SUBJECT_SAMPLE_FACTORS - Trained_07 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_4-r001d SUBJECT_SAMPLE_FACTORS - Trained_08 Sample source:macrophages | Condition:Trained RAW_FILE_NAME(Raw file name)=T_4-r002.d #COLLECTION CO:COLLECTION_SUMMARY Bone marrow-derived macrophages (BMDMs) were obtained from C57BL/6 mice and CO:COLLECTION_SUMMARY cultured under standard conditions. Naive macrophages were either left CO:COLLECTION_SUMMARY untreated, activated with LPS, or trained with β-glucan before metabolite CO:COLLECTION_SUMMARY extraction. After stimulation, cells were washed, collected, and lysed in CO:COLLECTION_SUMMARY methanol for metabolomic analysis. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY Murine bone marrow monocytes were isolated by negative selection and cultured in TR:TREATMENT_SUMMARY DMEM supplemented with 10% FBS, antibiotics, amino acids, and 30 ng/mL M-CSF. TR:TREATMENT_SUMMARY Naive macrophages were cultured for 6 days without additional stimulation. TR:TREATMENT_SUMMARY Activated macrophages were stimulated with 10 ng/mL LPS for 6 hours on day 6. TR:TREATMENT_SUMMARY Trained macrophages were primed with 10 µg/mL β-glucan on day 0, followed by a TR:TREATMENT_SUMMARY media change on day 3 and a 3-day resting period. On day 6, trained macrophages TR:TREATMENT_SUMMARY were re-stimulated with LPS (10 ng/mL) for 6 hours before metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After culture and treatment, macrophages were resuspended in 60 μL of methanol SP:SAMPLEPREP_SUMMARY and ultrasonicated using a UP200S device with an S2 probe (Hielscher SP:SAMPLEPREP_SUMMARY Ultrasonics). Organic and aqueous fractions were separated by centrifugation. SP:SAMPLEPREP_SUMMARY Supernatants were collected and stored at -80°C until further analysis. Samples SP:SAMPLEPREP_SUMMARY were then subjected to LC-MS/MS for metabolomic profiling. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6470 CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 HILIC-Z (150 x 2.1mm, 2.7um) CH:SOLVENT_A 100% water; 10 mM ammonium acetate; 2.5 μM InfinityLab Deactivator Additive (pH CH:SOLVENT_A 9) CH:SOLVENT_B 15% water/85% acetonitrile; 10 mM ammonium acetate; 2.5 μM InfinityLab CH:SOLVENT_B Deactivator Additive (pH 9) CH:FLOW_GRADIENT Gradient elution from 96% to 65% B, followed by re-equilibration to 96% B: CH:FLOW_GRADIENT Linear gradient steps: 0.0–2.0 min at 96% B (isocratic), 2.0–5.5 min: linear CH:FLOW_GRADIENT decrease from 96% to 88% B, 5.5–8.5 min at 88% B, 8.5–9.0 min: linear CH:FLOW_GRADIENT decrease to 86% B, 9.0–14.0 min at 86% B, 14.0–17.0 min: linear decrease to CH:FLOW_GRADIENT 82% B, 17.0–22.0 min at 82% B, 22.0–23.0 min: linear decrease to 65% B, CH:FLOW_GRADIENT 23.0–24.0 min at 65% B, 24.5–39.0 min: re-equilibration at 96% B. CH:FLOW_RATE 0.250 mL/min CH:COLUMN_TEMPERATURE 50℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6470 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Agilent 6470 triple quadrupole system was operated in negative ion mode, MS:MS_COMMENTS with gas temperature at 225°C, gas flow 13 L/min, nebulizer pressure 60 psi, MS:MS_COMMENTS sheath gas temperature 250°C, sheath gas flow 12 L/min, capillary voltage MS:MS_COMMENTS –3500 V, and nozzle voltage 0 V. MS/MS acquisition was performed in dynamic MS:MS_COMMENTS Multiple Reaction Monitoring (MRM) mode, using optimized transitions for each MS:MS_COMMENTS compound. Data processing Comments: Data were processed using MS:MS_COMMENTS transition-specific MRM settings previously validated at the CEMBIO laboratory MS:MS_COMMENTS to ensure accurate quantification and compound identification. Chromatographic MS:MS_COMMENTS peaks were manually inspected and integrated. Software/procedures used for MS:MS_COMMENTS feature assignments: Feature assignment and quantification were performed using MS:MS_COMMENTS Agilent MassHunter Workstation software, with compound transitions and MS:MS_COMMENTS parameters optimized and validated internally at the CEMBIO laboratory. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Relative intensity MS_METABOLITE_DATA_START Samples Naive_01 Naive_02 Naive_03 Naive_04 Naive_05 Naive_06 Naive_07 Naive_08 LPS_01 LPS_02 LPS_03 LPS_04 LPS_05 LPS_06 LPS_07 LPS_08 Trained_01 Trained_02 Trained_03 Trained_04 Trained_05 Trained_06 Trained_07 Trained_08 Factors Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:Naive Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:LPS Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Sample source:macrophages | Condition:Trained Mevalonolactone 7 7.6 12.5 12.1 8.1 9.3 9.5 13.6 6.1 6.8 7.8 7.8 11.4 11.2 8 8.5 12.1 12.9 14.2 13 7.5 8.1 13.1 12 5-Deoxy-5-(methylthio)adenosine 0.83 0.89 0.85 1.64 0.86 1.12 1.21 1.02 0.4 0.42 0.54 0.44 0.7 0.55 0.63 0.36 2.91 3.54 2.35 2.76 1.71 1.96 4.15 4.12 Adenine 3.5 3.4 3.2 2.8 2.6 2.7 2.5 2.3 1.4 1.8 4.3 4.5 5.6 5.2 1.2 1.3 5.6 5.5 17 16.8 4.7 4.9 6.7 6.3 Pyridoxine 0.3 0.6 1.7 1.5 1.1 1.4 1.3 1.2 0.8 0.9 0.8 1 1 1 1.7 1.7 1.5 1.7 1.4 1.4 0.9 1 1.1 1 Adenosine 12.1 13.2 13.4 12.4 12.1 12.4 10.9 10.7 8.6 9 18.8 18.4 23.8 23.3 5.4 5.3 24.8 24.7 78.7 83.3 17.5 20 25.4 25.7 Uridine 1.6 1.5 2.3 2.8 2.2 2.5 4 3.9 2 1.8 3.2 2.7 1.7 2.2 2.2 1.9 5.2 3.7 8.3 7.1 4 3.3 5.9 5.6 Nicotinic acid 1.6 1.8 1.9 2.2 1.1 1.4 2.3 2.2 1.4 1 1.1 1.3 1.8 1.7 1.7 1.4 2.2 2.3 2.9 2.7 1.7 1.4 2.3 2.2 Pyruvic acid 24.7 26.8 13.9 24.3 19 16.1 37 30.5 24.4 17 7.1 7.2 19.3 13.8 29.6 35.9 23 36 17.4 23.8 29.6 29.8 23.2 15.9 Maleic acid 3.3 4.1 3.1 3.5 2.3 2.2 1.8 2.6 1.3 2.7 1.6 1 2.1 2.1 3.5 4.2 2.3 2.3 3.4 2.8 1.4 1.4 2.5 2.2 Xanthine 4.1 4.2 18.4 18.1 17.8 17 15.4 27.6 1.9 1.4 6.8 5.7 4.8 4.3 4.5 5.1 23 22.2 27.3 26.9 12.1 11.2 39.1 40 4-Hydroxybenzoic acid 380.8 380 672.2 631.6 597.6 588.3 817.9 831.3 569.4 578 293.4 279.3 293.4 277.9 584.6 591.3 655.6 674.9 388.3 395.4 668.5 689.3 349.1 331 L-Methionine 14.1 15.9 18.7 18.7 14.9 16 28.6 26.5 2.4 2.5 2.6 3.2 3.2 3.1 2.9 3.1 9.7 8.4 15.1 16.5 5.7 6.5 8.5 8.1 Glyceric acid 2.5 2.9 3.3 3.9 1.8 2.4 4 3.8 2.9 2.7 0.5 0.8 1.1 1.3 4.6 4.6 1.2 1.3 2.3 2.7 3.4 3.8 1.9 1.7 Uric acid 1.8 2.1 4.6 4.5 4 3.8 7 7.2 61.2 63.4 1.2 1 2.2 2.1 3.3 3.5 8.7 9.3 6.7 6.7 19.4 20.4 6 6.3 L-Tyrosine 18.2 19.9 31.4 28.2 21.6 21.8 49.7 51.4 8.6 10.2 11.8 11.4 10.6 10.3 12.6 12.7 14.2 14.8 26.9 29.1 13.6 14 18.3 18.9 Creatine 87.1 94.1 20 17.8 35.9 22.3 249.2 231.5 27.5 33.5 38.3 26.8 3.6 3.7 38.5 34.1 42.2 25.4 136.5 127.3 300.5 290 36.4 28.4 L-Serine 3 3.1 4.1 4.2 3.5 3.6 8 8 2 2.1 1.1 1.1 1 1 2.1 2.1 2.4 2.4 3.7 3.8 3.3 3.3 1.9 1.8 L-asparagine 24.5 26.3 28.7 28 26.2 29.3 65.3 63.8 12.9 14.4 8.7 8.4 6.2 7.3 12.6 13 17 16.9 25.1 27 21.9 20.9 14.8 13.2 Itaconic acid 2.9 3 2.4 2.2 2 2.3 6.8 6.7 10.5 10.5 6.6 5.2 5.1 5 11.1 11.3 66.5 68.5 95.7 93.5 131.3 131.3 54.3 51.8 L-Aspartic Acid 140.7 142.1 135.8 135.7 106.4 113.2 217.1 221.5 46.7 47.5 26.6 22.1 19.3 20.6 77.6 76.5 43.1 45.5 62.4 64.2 104.5 107.5 71.5 71.8 UDP-N-acteylglucosamine 253.3 230.5 107.6 115.3 70.3 66.4 285.3 374.6 36.9 32 nd nd nd nd 40.6 67.3 70.7 89 157.3 196.7 450.7 464.9 130.2 161.9 Mevalonic acid 4.7 4.9 3.5 3.6 3.1 3.3 6.5 6.8 4 4.2 2.6 2.5 2.6 2.6 4.2 4.2 3.2 3.3 4.6 4.8 7.1 7.2 3.8 3.9 beta-Nicotinamide adenine dinucleotide (NAD) 137.9 177.4 71.5 88.3 83.4 94.9 177.3 178 nd nd nd nd nd nd 23.1 31.9 nd nd 38.1 37.5 65 48.5 31.9 27.1 L-Malic acid 27.2 27.9 17.9 17.8 15.1 15.9 30.6 30.5 17.6 17.6 13.5 12.7 12.5 12.5 20.1 19.7 14.7 14.9 17.7 17.6 36.1 37.7 18.7 18.7 Adipic acid 18.7 19.2 33.2 28.3 16.9 15.8 24.9 25.2 20.1 20 19.1 19.4 20.6 18.4 24.1 24.5 16.1 16.5 13 13 16.3 17.3 17.5 17.5 cis-Aconitic acid 0.69 0.78 0.38 0.38 0.26 0.26 0.77 0.88 0.24 0.24 nd nd nd nd 0.31 0.31 nd nd 0.32 0.32 0.91 0.87 0.29 0.3 N-Acetyl-alpha-D-glucosamine 1-phosphate 41.9 40.6 40.2 33.6 27.6 31.4 53.8 47.2 nd nd nd nd nd nd nd nd 30.6 31.1 39.7 44.4 46.5 54.4 36.8 37.6 Inosine 5-monophosphate 104.2 85.7 70.2 75.5 51.9 65 339.8 352.4 57.5 40.1 55.4 48.4 63.3 50.1 74.9 82.2 58 64.1 120.5 113.3 193.8 178 201.9 183.7 L-Arginine 66.9 64.3 120.3 116.5 76.2 81.6 149.1 155 18.6 18.2 14.9 15.5 16.8 19.5 30.1 30.7 51.2 52 71.8 71.5 36.7 38.9 43.7 45.5 Citric and isocitric acid 58.1 58.7 20 20.3 11.6 12.3 44.5 44.3 13.4 13 9.1 7.9 10.5 11 29.3 29.1 10.7 11.9 13.2 13.1 31.2 31.5 14.5 14.7 L-Glutathione oxdized 187.6 180.6 39.7 51.6 24.1 24.3 153.9 157.2 57.4 48.9 44.9 38 23.4 22.6 62.9 50 28.2 25.5 38.7 37.1 217.2 212.4 37.6 44.2 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB PubChem ChEBI KEGG METLIN SMILES Mevalonolactone HMDB0006024 5325923 NA NA NA C[C@]1(O)CCOC(=O)C1 5-Deoxy-5-(methylthio)adenosine NA NA NA NA NA NA Adenine HMDB0000034 190 16708 C00147 85 NC1=C2NC=NC2=NC=N1 Pyridoxine HMDB0000239 1054 16709 C00314 5245 CC1=C(O)C(CO)=C(CO)C=N1 Adenosine HMDB0000050 60961 16335 C00212 86 NC1=C2N=CN([C@@H]3O[C@H](CO)[C@@H](O)[C@H]3O)C2=NC=N1 Uridine HMDB0000296 6029 16704 C00299 90 OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)N1C=CC(=O)NC1=O Nicotinic acid HMDB0001488 938 15940 C00253 6272 OC(=O)C1=CN=CC=C1 Pyruvic acid HMDB0000243 1060 32816 C00022 117 CC(=O)C(O)=O Maleic acid HMDB0000176 444266 18300 C01384 4198 OC(=O)C=C/C(O)=O Xanthine HMDB0000292 1188 17712 C00385 82 O=C1NC2=C(NC=N2)C(=O)N1 4-Hydroxybenzoic acid HMDB0000500 135 30763 C00156 5486 OC(=O)C1=CC=C(O)C=C1 L-Methionine HMDB0000696 6137 16643 C00073 5664 CSCC[C@H](N)C(O)=O Glyceric acid HMDB0000139 439194 32398 C00258 280 OC[C@@H](O)C(O)=O Uric acid HMDB0000289 1175 17775 C00366 88 O=C1NC2=C(N1)C(=O)NC(=O)N2 L-Tyrosine HMDB0000158 6057 17895 C00082 34 N[C@@H](CC1=CC=C(O)C=C1)C(O)=O Creatine HMDB0000064 586 16919 C00300 7 CN(CC(O)=O)C(N)=N L-Serine HMDB0000187 5951 17115 C00065 5203 N[C@@H](CO)C(O)=O L-asparagine HMDB0000168 6267 17196 C00152 14 N[C@@H](CC(N)=O)C(O)=O Itaconic acid HMDB0002092 811 30838 C00490 6483 OC(=O)CC(=C)C(O)=O L-Aspartic Acid HMDB0000191 5960 17053 C00049 5206 N[C@@H](CC(O)=O)C(O)=O UDP-N-acteylglucosamine HMDB0000290 C00043 445675 16264 5281 NA Mevalonic acid HMDB0000227 439230 17710 C00418 127 C[C@@](O)(CCO)CC(O)=O beta-Nicotinamide adenine dinucleotide (NAD) HMDB0000902 C00003 5892 44215 5858 NA L-Malic acid HMDB0000156 222656 30797 C00149 NA O[C@@H](CC(O)=O)C(O)=O Adipic acid HMDB0000448 196 30832 C06104 115 OC(=O)CCCCC(O)=O cis-Aconitic acid HMDB0000072 643757 32805 C00417 5130 OC(=O)CC(=CC(O)=O)C(O)=O N-Acetyl-alpha-D-glucosamine 1-phosphate HMDB0001367 440272 7125 C04501 6194 CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OP(O)(O)=O Inosine 5-monophosphate HMDB0000175 C00130 8582 17202 5196 NA L-Arginine HMDB0000517 6322 16467 C00062 5502 N[C@@H](CCCNC(N)=N)C(O)=O Citric and isocitric acid NA NA NA NA NA NA L-Glutathione oxdized HMDB0003337 C00127 65359 17858 6893 NA METABOLITES_END #END