#METABOLOMICS WORKBENCH tarling_vallim_BA_20241223_091537 DATATRACK_ID:5491 STUDY_ID:ST003641 ANALYSIS_ID:AN005980 PROJECT_ID:PR002251 VERSION 1 CREATED_ON 12-30-2024 #PROJECT PR:PROJECT_TITLE The Spatial Transcriptional Activity of Hepatic TCF7L2 Regulates Zonated PR:PROJECT_TITLE Metabolic Pathways that Contribute to Liver Fibrosis PR:PROJECT_SUMMARY Study used single nuclei genomics techniques to examine the spatial PR:PROJECT_SUMMARY transcriptional function of the transcription factor 7-like 2 (TCF7L2) in rodent PR:PROJECT_SUMMARY liver. Research aimed to determine the consequences of TCF7L2 transcriptional PR:PROJECT_SUMMARY inactivation on the metabolic architecture of the liver, and on the function of PR:PROJECT_SUMMARY key zonated metabolic pathways that influence the development of fibrotic liver PR:PROJECT_SUMMARY diseases. Dietary stress was investigated using the Gubra Amylin Nash (GAN) and PR:PROJECT_SUMMARY choline-deficient amino acid-defined high fat (CDAHFD) diets to investigate the PR:PROJECT_SUMMARY susceptibility of liver-specific TCF7L2 mutant mice (Hep-TCF7L2ÎDBD) compared PR:PROJECT_SUMMARY to control (TCF7L2LoxP/LoxP) mice in hepatic fibrosis. PR:INSTITUTE UT Health San Antonio PR:LAST_NAME Norton PR:FIRST_NAME Luke PR:ADDRESS 7703 Floyd Curl Drive, San Antonio, TX 78229 PR:EMAIL nortonl@uthscsa.edu PR:PHONE (210)-567-0739 PR:DOI http://dx.doi.org/10.21228/M8NV72 #STUDY ST:STUDY_TITLE The Spatial Transcriptional Activity of Hepatic TCF7L2 Regulates Zonated ST:STUDY_TITLE Metabolic Pathways that Contribute to Liver Fibrosis ST:STUDY_SUMMARY Background and Aims: The molecular mechanisms regulating the zonal distribution ST:STUDY_SUMMARY of metabolism in liver are incompletely understood. Here we used single nuclei ST:STUDY_SUMMARY genomics techniques to examine the spatial transcriptional function of the ST:STUDY_SUMMARY transcription factor 7-like 2 (TCF7L2) in rodent liver. We also determined the ST:STUDY_SUMMARY consequences of TCF7L2 transcriptional inactivation on the metabolic ST:STUDY_SUMMARY architecture of the liver, and on the function of key zonated metabolic pathways ST:STUDY_SUMMARY that influence the development of fibrotic liver diseases. Methods: Multimodal ST:STUDY_SUMMARY 10X single nuclei RNA- and ATAC-Seq were used to dissect the cell-type specific ST:STUDY_SUMMARY expression and DNA binding activity of TCF7L2 and transcriptional co-regulators ST:STUDY_SUMMARY across the liver lobule. The transcriptional activity of TCF7L2 was targeted by ST:STUDY_SUMMARY excising exon 11 of Tcf7l2, which encodes part of the DNA binding domain (DBD). ST:STUDY_SUMMARY The consequences of TCF7L2 inactivation on zonation was investigated with a ST:STUDY_SUMMARY focus on the disruption to zonated metabolic pathways, including those linked to ST:STUDY_SUMMARY ammonia detoxification and glutamine metabolism, cholesterol homeostasis and ST:STUDY_SUMMARY bile acid synthesis. The susceptibility of Hep-TCF7L2ÎDBD mice to hepatic ST:STUDY_SUMMARY fibrosis under dietary stress was investigated using the Gubra Amylin Nash (GAN) ST:STUDY_SUMMARY and choline-deficient amino acid-defined high fat (CDAHFD) diets. Results: ST:STUDY_SUMMARY Tcf7l2 mRNA expression was ubiquitous across the liver lobule, but accessibility ST:STUDY_SUMMARY of the consensus TCF/LEF DNA binding motif was restricted to pericentral (PC) ST:STUDY_SUMMARY hepatocytes in zone 3. PC hepatocyte-specific gene expression was lost in ST:STUDY_SUMMARY Hep-TCF7L2ÎDBD mice, which we link to alterations in the transcriptional ST:STUDY_SUMMARY activity of zonal repressors Tbx3 and Tcf7l1. The absence of a classic zone 3 ST:STUDY_SUMMARY gene expression program in TCF7L2 mutant mice led to hepatic cholesterol ST:STUDY_SUMMARY accumulation, impaired bile acid synthesis, and significant disruptions to ST:STUDY_SUMMARY glutamine and glutamate homeostasis. In human liver biopsy tissue, TCF7L2 ST:STUDY_SUMMARY expression declined as the severity of fibrosis progressed, and Hep-TCF7L2ÎDBD ST:STUDY_SUMMARY mice developed more severe hepatic fibrosis when fed the CDAHFD. Conclusion: ST:STUDY_SUMMARY TCF7L2 is key transcriptional regulator of the PC hepatocyte gene expression ST:STUDY_SUMMARY program in zone 3, and regulates multiple zonated metabolic pathways that may ST:STUDY_SUMMARY contribute to the development of fibrotic liver diseases. ST:INSTITUTE UT Health San Antonio ST:LAST_NAME Norton ST:FIRST_NAME Luke ST:ADDRESS 7703 Floyd Curl Drive, San Antonio, TX 78229 ST:EMAIL nortonl@uthscsa.edu ST:PHONE (210)-567-0739 ST:SUBMIT_DATE 2024-12-23 #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]Additional sample data SUBJECT_SAMPLE_FACTORS 6697 10_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-10.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8005 11_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-11.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8015 12_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-12.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8152 13_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-13.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8156 14_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-14.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8160 15_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-15.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8164 16_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-16.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6682 9_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-9.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8006 1_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-1.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8402 2_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-2_20220425171539.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8124 3_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-3.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8125 4_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-4.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8153 5_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-5.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8154 6_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-6.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8155 7_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-7.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8172 8_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-8.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6697 10_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-10_20220411222209.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8005 11_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-11.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8015 12_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-12.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8152 13_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-13.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8156 14_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-14.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8160 15_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-15.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8164 16_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-16.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6682 9_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-09.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8006 1_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-01.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8402 2_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-02.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8124 3_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-03.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8125 4_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-04.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8153 5_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-05.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8154 6_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-06.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8155 7_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-07.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8172 8_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-08.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6697 10_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-10.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-10.mzXML SUBJECT_SAMPLE_FACTORS 8005 11_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-11.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-11.mzXML SUBJECT_SAMPLE_FACTORS 8015 12_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-12.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-12.mzXML SUBJECT_SAMPLE_FACTORS 8152 13_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-13.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-13.mzXML SUBJECT_SAMPLE_FACTORS 8156 14_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-14.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-14.mzXML SUBJECT_SAMPLE_FACTORS 8160 15_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-15.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-15.mzXML SUBJECT_SAMPLE_FACTORS 8164 16_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-16.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-16.mzXML SUBJECT_SAMPLE_FACTORS 6682 9_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-9.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-09.mzXML SUBJECT_SAMPLE_FACTORS 8006 1_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-1.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-01.mzXML SUBJECT_SAMPLE_FACTORS 8402 2_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-2.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-02.mzXML SUBJECT_SAMPLE_FACTORS 8124 3_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-3.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-03.mzXML SUBJECT_SAMPLE_FACTORS 8125 4_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-4.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-04.mzXML SUBJECT_SAMPLE_FACTORS 8153 5_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-5.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-05.mzXML SUBJECT_SAMPLE_FACTORS 8154 6_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-6.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-06.mzXML SUBJECT_SAMPLE_FACTORS 8155 7_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-7.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-07.mzXML SUBJECT_SAMPLE_FACTORS 8172 8_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-8.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-08.mzXML #COLLECTION CO:COLLECTION_SUMMARY Tissues were collected from control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 CO:COLLECTION_SUMMARY mutant mice (Hep-TCF7L2ÎDBD) then stored at -80âC until further analysis was CO:COLLECTION_SUMMARY performed. CO:SAMPLE_TYPE Liver, Intestine, Gallbladder #TREATMENT TR:TREATMENT_SUMMARY Wild-type control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice TR:TREATMENT_SUMMARY (Hep-TCF7L2ÎDBD) were fed methionine and choline deficient, 60% HFD diet TR:TREATMENT_SUMMARY (CDAHFD, Research Diets, Cat#: A06071302) for 8-weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Gallbladder, liver and intestine were isolated from Wild-type control SP:SAMPLEPREP_SUMMARY (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice (Hep-TCF7L2ÎDBD). Bile SP:SAMPLEPREP_SUMMARY acids were extracted from liver and intestine in Optima Methanol containing SP:SAMPLEPREP_SUMMARY recovery bile acid spikes (G-UDCA, G-CDCA) overnight at room temp. The following SP:SAMPLEPREP_SUMMARY day, samples were centrifuged at 4000rpm for 10min. Prior to analysis, bile was SP:SAMPLEPREP_SUMMARY diluted 1:1000 into Optima Methanol, then further diluted 1:2 into autosampler SP:SAMPLEPREP_SUMMARY vials. Liver supernatant was vortexed then filtered through a 0.45 µm SP:SAMPLEPREP_SUMMARY syringe-driven filter unit directly into autosampler vials. Intestine SP:SAMPLEPREP_SUMMARY supernatant was thoroughly vortexed, then an aliquot centrifuged at 15,000xg for SP:SAMPLEPREP_SUMMARY 5min. Intestine samples were then diluted 1:20 directly into autosampler vials. SP:SAMPLEPREP_SUMMARY For bile acid analysis, bile, intestine (2uL) and liver (5uL) were injected via SP:SAMPLEPREP_SUMMARY an Ultimate3000 autosampler module. For C4 analysis, 10uL of liver sample was SP:SAMPLEPREP_SUMMARY injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Detection and separation of 7α-hydroxy-4-cholesten-3-one (C4) using a UHPLC CH:CHROMATOGRAPHY_SUMMARY gradient method. Duration = 15min. Please see ETV-Lab-C4-Methods.pdf for CH:CHROMATOGRAPHY_SUMMARY details. CH:METHODS_FILENAME ETV-Lab-C4-Methods.pdf CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME ACE Excel SuperC18 (150 x 2.1mm, 2um, 90à ) CH:COLUMN_TEMPERATURE 50 CH:FLOW_GRADIENT 0.3 ml/min: 0min: 60% C, 40% A, 0.5min: 60% C, 40% A, 2min: 90% C, 10% A, 5min: CH:FLOW_GRADIENT 100% C, 9min: 100% C, 10-15min: 60% C, 40% A CH:FLOW_RATE 0.3 mL/min CH:INJECTION_TEMPERATURE 5 CH:SOLVENT_A Water 80%/20% methanol; 0.1% formic acid; CH:SOLVENT_B - CH:CHROMATOGRAPHY_TYPE Reversed phase CH:SOLVENT_C 100% Methanol #ANALYSIS AN:LABORATORY_NAME Tarling-Vallim Lab, University of California, Los Angeles AN:ANALYSIS_TYPE MS AN:OPERATOR_NAME Madelaine Sholto #MS MS:INSTRUMENT_NAME Thermo TSQ Quantiva MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS C4 levels (uM) were quantified in Skyline-daily software (MacCross Lab Software) MS:MS_COMMENTS from a standard curve generated using C4 purified standards. Total C4 levels MS:MS_COMMENTS were determined per whole liver weight. MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/g dry weight of liver MS_METABOLITE_DATA_START Samples 10_L 11_L 12_L 13_L 14_L 15_L 16_L 9_L 1_L 2_L 3_L 4_L 5_L 6_L 7_L 8_L Factors Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type C4 0.0525 0.0264 0.3393 0.3662 0.5520 1.1413 2.3434 0.4021 0.1575 0.5506 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant C4 123743 C05455                      401.38 METABOLITES_END #END