#METABOLOMICS WORKBENCH sven_haange_20200602_042949 DATATRACK_ID:2034 STUDY_ID:ST001388 ANALYSIS_ID:AN002317 PROJECT_ID:PR000952 VERSION 1 CREATED_ON June 2, 2020, 11:14 am #PROJECT PR:PROJECT_TITLE Enteric microbiota and liver metabolomics during the postnatal period PR:PROJECT_SUMMARY Following birth, the neonatal intestine is exposed to maternal and environmental PR:PROJECT_SUMMARY bacteria that successively form a dense and highly dynamic intestinal PR:PROJECT_SUMMARY microbiota. Whereas the effect of exogenous factors has been extensively PR:PROJECT_SUMMARY investigated, endogenous, host-mediated mechanisms have remained largely PR:PROJECT_SUMMARY unexplored. Concomitantly with microbial colonization, the liver undergoes PR:PROJECT_SUMMARY functional transition from a hematopoietic organ to a central organ of metabolic PR:PROJECT_SUMMARY regulation and immune surveillance. The aim of the present study was to analyze PR:PROJECT_SUMMARY the influence of the developing hepatic function and liver metabolism on the PR:PROJECT_SUMMARY early intestinal microbiota. Using metabolomic and microbial profiling in PR:PROJECT_SUMMARY combination with multivariate analysis we characterized the colonization PR:PROJECT_SUMMARY dynamics and liver metabolism in the murine gastrointestinal tract (n=6-10 per PR:PROJECT_SUMMARY age group). We observed major age-dependent microbial and metabolic changes and PR:PROJECT_SUMMARY identified bile acids as potent drivers of the early intestinal microbiota PR:PROJECT_SUMMARY maturation. Consistently, oral administration of tauro-cholic acid or PR:PROJECT_SUMMARY β-tauro-murocholic acid to newborn mice (n= 7-14 per group) accelerated PR:PROJECT_SUMMARY postnatal microbiota maturation. PR:INSTITUTE Helmholtz Centre for Environmental Research - UFZ PR:LAST_NAME Haange PR:FIRST_NAME Sven PR:ADDRESS Permoserstrasse 1, Leipzig, Saxony, 04318, Germany PR:EMAIL sven.haange@ufz.de PR:PHONE 0049 341 2351099 #STUDY ST:STUDY_TITLE Quantitative bile acids study on total murine liver tissue from mice at ST:STUDY_TITLE different age ST:STUDY_TYPE Liver tissue/Primary tissue ST:STUDY_SUMMARY Bile acids in total liver tissue from healthy C57BL/6 mice at 1, 7, 14, 21, 28 ST:STUDY_SUMMARY and 56 day after birth was analyzed. ST:INSTITUTE Helmholtz Centre for Environmental Research - UFZ ST:DEPARTMENT Department of Molecular Systems biology ST:LABORATORY Functional Metabolomics ST:LAST_NAME Rolle-Kampczyk ST:FIRST_NAME Ulrike ST:ADDRESS Permoserstrasse 15, 04318 Leipzig, Germany ST:EMAIL ulrike.rolle-kampczyk@ufz.de ST:PHONE 0049 341 235 1537 #SUBJECT SU:SUBJECT_TYPE Mammal 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 - L_K9D1 Day after birth:1 Litter=9; RAW_FILE_NAME=L_K9D1.wiff SUBJECT_SAMPLE_FACTORS - L_K10D1 Day after birth:1 Litter=10; RAW_FILE_NAME=L_K10D1.wiff SUBJECT_SAMPLE_FACTORS - L_K7D7 Day after birth:7 Litter=7; RAW_FILE_NAME=L_K7D7.wiff SUBJECT_SAMPLE_FACTORS - L_K8D7 Day after birth:7 Litter=8; RAW_FILE_NAME=L_K8D7.wiff SUBJECT_SAMPLE_FACTORS - L_K9D7 Day after birth:7 Litter=9; RAW_FILE_NAME=L_K9D7.wiff SUBJECT_SAMPLE_FACTORS - L_K10D7 Day after birth:7 Litter=10; RAW_FILE_NAME=L_K10D7.wiff SUBJECT_SAMPLE_FACTORS - L_K11D7 Day after birth:7 Litter=11; RAW_FILE_NAME=L_K11D7.wiff SUBJECT_SAMPLE_FACTORS - L_K7D14 Day after birth:14 Litter=7; RAW_FILE_NAME=L_K7D14.wiff SUBJECT_SAMPLE_FACTORS - L_K8D14 Day after birth:14 Litter=8; RAW_FILE_NAME=L_K8D14.wiff SUBJECT_SAMPLE_FACTORS - L_K9D14 Day after birth:14 Litter=9; RAW_FILE_NAME=L_K9D14.wiff SUBJECT_SAMPLE_FACTORS - L_K10D14 Day after birth:14 Litter=10; RAW_FILE_NAME=L_K10D14.wiff SUBJECT_SAMPLE_FACTORS - L_K11D14 Day after birth:14 Litter=11; RAW_FILE_NAME=L_K11D14.wiff SUBJECT_SAMPLE_FACTORS - L_K7D21 Day after birth:21 Litter=7; RAW_FILE_NAME=L_K7D21.wiff SUBJECT_SAMPLE_FACTORS - L_K8D21 Day after birth:21 Litter=8; RAW_FILE_NAME=L_K8D21.wiff SUBJECT_SAMPLE_FACTORS - L_K9D21 Day after birth:21 Litter=9; RAW_FILE_NAME=L_K9D21.wiff SUBJECT_SAMPLE_FACTORS - L_K10D21 Day after birth:21 Litter=10; RAW_FILE_NAME=L_K10D21.wiff SUBJECT_SAMPLE_FACTORS - L_K11D21 Day after birth:21 Litter=11; RAW_FILE_NAME=L_K11D21.wiff SUBJECT_SAMPLE_FACTORS - L_K7D28 Day after birth:28 Litter=7; RAW_FILE_NAME=L_K7D28.wiff SUBJECT_SAMPLE_FACTORS - L_K8D28 Day after birth:28 Litter=8; RAW_FILE_NAME=L_K8D28.wiff SUBJECT_SAMPLE_FACTORS - L_K9D28 Day after birth:28 Litter=9; RAW_FILE_NAME=L_K9D28.wiff SUBJECT_SAMPLE_FACTORS - L_K10D28 Day after birth:28 Litter=10; RAW_FILE_NAME=L_K10D28.wiff SUBJECT_SAMPLE_FACTORS - L_K11D28 Day after birth:28 Litter=11; RAW_FILE_NAME=L_K11D28.wiff SUBJECT_SAMPLE_FACTORS - L_K7D56 Day after birth:56 Litter=7; RAW_FILE_NAME=L_K7D56.wiff SUBJECT_SAMPLE_FACTORS - L_K8D56 Day after birth:56 Litter=8; RAW_FILE_NAME=L_K8D56.wiff SUBJECT_SAMPLE_FACTORS - L_K9D56 Day after birth:56 Litter=9; RAW_FILE_NAME=L_K9D56.wiff SUBJECT_SAMPLE_FACTORS - L_K10D56 Day after birth:56 Litter=10; RAW_FILE_NAME=L_K10D56.wiff SUBJECT_SAMPLE_FACTORS - L_K11D56 Day after birth:56 Litter=11; RAW_FILE_NAME=L_K11D56.wiff #COLLECTION CO:COLLECTION_SUMMARY Animals were sacrificed and the liver tissue was removed, snap frozen in liquid CO:COLLECTION_SUMMARY nitrogen and stored at -80°C until further analysis CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY All C57BL6J wildtype mice were bred locally and held under specific TR:TREATMENT_SUMMARY pathogen-free conditions at the Institute of Laboratory Animal Science at RWTH TR:TREATMENT_SUMMARY Aachen University Hospital. The day of birth was considered day 0, i.e. animals TR:TREATMENT_SUMMARY screened at day 1 were approximately 24 h old and verified to have ingested TR:TREATMENT_SUMMARY breast milk (abdominal milk spot). Mice were weaned at PND21. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Solvents: Acetonitril (Merck KGaA, Darmstadt, Germany hypergrade for LC-MS) SP:SAMPLEPREP_SUMMARY Water MiliQ, Extracting agent - ACN / H2O (1:1) Equipment 4 steel balls size M SP:SAMPLEPREP_SUMMARY Eppendorf Tubes 2mL Tissue slicer (Rettberg, Germany) Centrifuge (Sigma) Work SP:SAMPLEPREP_SUMMARY steps To approximately 100 mg of sample four steel balls of size M rae added in SP:SAMPLEPREP_SUMMARY Eppendorf tubes. Then add for each mg of sample 5 µL of extracting agent. Shake SP:SAMPLEPREP_SUMMARY the samples for 10 minutes at 30 Hz in the tissue slicer and centrifuge for 2 SP:SAMPLEPREP_SUMMARY minutes at 14000 rpm. The supernatant is used for targeted analytics as SP:SAMPLEPREP_SUMMARY described in the kit manufactures manual. Kit measurement The analysis was SP:SAMPLEPREP_SUMMARY performed using the validated Bile Acid Kit (Biocrates Life Sciences, Inssbruck, SP:SAMPLEPREP_SUMMARY Austria) as described in Pham et al. 2017 [1]. Data processing is carried out SP:SAMPLEPREP_SUMMARY with the provided quantitation method Kit (Biocrates Life Sciences AG, SP:SAMPLEPREP_SUMMARY Innsbruck, Austria). 1 Pham HT, Arnhard K, Asad YJ, Deng L, Felder TK, St SP:SAMPLEPREP_SUMMARY John-Williams L, et al. Inter-Laboratory Robustness of Next-Generation Bile Acid SP:SAMPLEPREP_SUMMARY Study in Mice and Humans: International Ring Trial Involving 12 Laboratories. SP:SAMPLEPREP_SUMMARY The Journal of Applied Laboratory Medicine 2019;1:129-42. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME UPLC (Waters Acquity, Waters Corporation, Milford, USA) CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Analyst version 1.6 Software from SCIEX. For Validation METIDQ Software version MS:MS_COMMENTS Boron from Biocrates #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS µM MS_METABOLITE_DATA_START Samples L_K9D1 L_K10D1 L_K7D7 L_K8D7 L_K9D7 L_K10D7 L_K11D7 L_K7D14 L_K8D14 L_K9D14 L_K10D14 L_K11D14 L_K7D21 L_K8D21 L_K9D21 L_K10D21 L_K11D21 L_K7D28 L_K8D28 L_K9D28 L_K10D28 L_K11D28 L_K7D56 L_K8D56 L_K9D56 L_K10D56 L_K11D56 Factors Day after birth:1 Day after birth:1 Day after birth:7 Day after birth:7 Day after birth:7 Day after birth:7 Day after birth:7 Day after birth:14 Day after birth:14 Day after birth:14 Day after birth:14 Day after birth:14 Day after birth:21 Day after birth:21 Day after birth:21 Day after birth:21 Day after birth:21 Day after birth:28 Day after birth:28 Day after birth:28 Day after birth:28 Day after birth:28 Day after birth:56 Day after birth:56 Day after birth:56 Day after birth:56 Day after birth:56 Cholic Acid 0.007541899 0.003636364 0.004019769 0.003741935 0.004700974 0.003605016 0.001565996 0.001710376 0.001551584 0.001843972 0.001844262 0.003228553 0.0004375 0.001105207 0.000754717 0.007213579 0.002618557 0.003275942 0.006813417 0.007401247 0.003425016 0.004799768 0.002642783 0.001691109 0.00190372 0.004055767 0.039579968 Chenodeoxycholic Acid 0.00175 0.001625 0.001875 0.002125 0.0025 0.00225 0.000875 0.002125 0.002625 0.001875 0.002375 0.0025 0.0015 0.003375 0.002875 0.006375 0.005625 0.002 0.004875 0.003125 0.004 0.002375 0.003 0.001125 0.0035 0.0045 0.012875 Deoxycholic Acid 0.000726257 0.000438871 0.000313015 0.000156682 0.000222531 0.000297806 0.000380313 0.000148233 0.000121852 0.000248227 0.000114754 0.000117878 0.00009375 7.44E-05 0.000210243 0.00029703 0.000103093 0.000130293 0.000162474 0.000120582 0.000150622 8.13E-05 0.000129803 7.79E-05 0.000153173 0.000215463 0.001195477 Glcyocholic Acid 0.000726257 9.40E-05 0 1.84E-05 0.000305981 3.13E-05 0 0.000114025 0 0 0 1.31E-05 0.000416667 0.00088204 0.000242588 0.002057992 0.000810997 0.00023732 0.001278826 0.000640333 0.000641781 0.000829948 0.000145379 4.58E-05 0.000166302 0.000564005 0.000726979 Glycochenodeoxycholic Acid 0.000558659 0.000219436 0.000115321 6.45E-05 9.74E-05 0.000109718 0.000178971 7.98E-05 5.69E-05 0.000124113 5.74E-05 4.58E-05 0.000046875 4.78E-05 5.39E-05 6.36E-05 8.93E-05 3.26E-05 3.67E-05 2.91E-05 4.58E-05 4.64E-05 3.63E-05 3.21E-05 3.50E-05 5.70E-05 4.31E-05 Glycodeoxycholic Acid 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9.31E-06 6.81E-05 0 0 0 1.04E-05 0 4.38E-06 3.17E-05 3.23E-05 Glycolithocholic Acid 0 0 1.65E-05 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glycoursodeoxycholic Acid 0 0 0 0 0 0 0 1.14E-05 0 0 8.20E-06 0 2.60E-05 2.66E-05 2.70E-05 5.66E-05 6.19E-05 4.65E-06 2.62E-05 4.16E-06 6.55E-06 2.32E-05 5.19E-06 4.58E-06 8.75E-06 1.90E-05 0 Hyodeoxycholic Acid 0.000335196 0.000188088 0.000214168 0.000147465 0.000236439 0.000203762 0.000201342 6.84E-05 4.06E-05 0.000106383 4.92E-05 6.55E-05 6.77E-05 0.00021254 0.000264151 0.001407355 2.75E-05 0.000544439 0.001100629 0.000465696 0.000235756 0.000481718 0.000539979 0.000288726 0.000809628 0.000633714 0.000813139 Lithocholic Acid 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000921833 0.000190948 0 0 0.000214885 0 0 9.87E-05 0 0 0 0 0 a-Muricholic Acid 0.003463687 0.001943574 0.002965404 0.002064516 0.00201669 0.002852665 0.001230425 0.001664766 0.001356621 0.00070922 0.001098361 0.002003929 0.000640625 0.002008502 0.001148248 0.005636492 0.005285223 0.001596091 0.003647799 0.001197505 0.002632613 0.001828207 0.002009346 0.000756187 0.002175055 0.003105196 0.004270328 b-Muricholic Acid 0.020558659 0.017084639 0.054859967 0.041474654 0.053963839 0.045611285 0.031543624 0.04686431 0.046872461 0.041312057 0.03852459 0.048395547 0.004307292 0.017587673 0.019730458 0.053182461 0.065498282 0.011865984 0.039832285 0.024781705 0.033529797 0.039988392 0.012097612 0.007882676 0.014179431 0.028580482 0.029617663 o-Muricholic Acid 0 0.000438871 0.000197694 0.001225806 5.56E-05 0.00015674 2.24E-05 2.28E-05 0.000722989 0 8.20E-06 9.82E-05 0.0003125 0.001524973 0.000873315 0.00669024 0.000109966 0.003941368 0.008647799 0.001322245 0.002213491 0.002356355 0.002403946 0.002140238 0.002017505 0.003403042 0.003457189 Taurocholic Acid 0 0 0 0 0 0.277429467 0.673378076 0.258836944 0.269699431 0.326241135 0.200819672 0 0 0 0 0 0 0.107026524 0 0.084823285 0 0 0.075804777 0.071494042 0.066083151 0.130544994 0.107700592 Taurochenodeoxycholic Acid 0.069273743 0.030376176 0.004200988 0.003253456 0.008386648 0.005846395 0.004787472 0.01299886 0.005605199 0.007624113 0.006131148 0.006457105 0.015208333 0.014930925 0.017250674 0.014497878 0.039381443 0.00233597 0.006079665 0.003189189 0.008120498 0.006210099 0.002481828 0.002667278 0.000652079 0.007541191 0.00543888 Taurodeoxycholic Acid 0.00027933 0.000188088 4.94E-05 5.53E-05 0.000125174 0.000125392 0.000134228 0.000102623 5.69E-05 0.000159574 0.001147541 2.62E-05 0.001578125 0.001992561 0.008463612 0.009052334 0 0.008562122 0.013469602 0.006070686 0.007531107 0.002855485 0.006282451 0.004532539 0.004161926 0.01400507 0.011416263 Taurolithocholic Acid 0.000502793 0.000219436 4.94E-05 7.37E-05 8.34E-05 3.13E-05 0.000111857 7.98E-05 4.87E-05 0.000106383 4.92E-05 5.24E-05 6.77E-05 6.91E-05 0.000199461 0.000148515 5.50E-05 9.31E-05 0.000246331 0.00010395 0.000124427 6.96E-05 0.000103842 8.71E-05 8.32E-05 0.000234474 0.000183091 Tauromuricholic Acid 0.472625698 0.322884013 0.146787479 0.084976959 0.118636996 0.138714734 0.16442953 0.078449259 0.08773355 0.145390071 0.051885246 0.05356909 0.0625 0.059511158 0.093261456 0.090523338 0.07628866 0.038250349 0.109538784 0.037962578 0.114603798 0.046256529 0.035669782 0.027772686 0.023632385 0.070342205 0.064081852 Tauroursodeoxycholic Acid 0.008603352 0.007836991 0.003871499 0.002258065 0.007579972 0.004184953 0.003534676 0.011197263 0.002599513 0.003687943 0.002959016 0.004348396 0.009635417 0.012167906 0.00851752 0.020721358 0.029690722 0.006281992 0.009800839 0.008523909 0.009495743 0.010562972 0.002923157 0.002887259 0.002726477 0.009569075 0.008131395 Ursodeoxycholic Acid 0 0 8.24E-05 0.000552995 0.000292072 0.000125392 0 0 0 0 0 0 0.0000625 0.00098831 0.001309973 0.008274399 0.000123711 0.002940903 0.006132075 0.002203742 0.001185331 0.002507255 0.001962617 0.000870761 0.001798687 0.003491762 0.003974152 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID Cholic Acid C00695 Chenodeoxycholic Acid C02528 Deoxycholic Acid C04483 Glcyocholic Acid C01921 Glycochenodeoxycholic Acid C05466 Glycodeoxycholic Acid C05464 Glycolithocholic Acid C15557 Glycoursodeoxycholic Acid - Hyodeoxycholic Acid C15517 Lithocholic Acid C03990 a-Muricholic Acid C17647 b-Muricholic Acid C17726 o-Muricholic Acid C17727 Taurocholic Acid C02258 Taurochenodeoxycholic Acid C05465 Taurodeoxycholic Acid C05463 Taurolithocholic Acid C02592 Tauromuricholic Acid - Tauroursodeoxycholic Acid C16868 Ursodeoxycholic Acid C07880 METABOLITES_END #END