#METABOLOMICS WORKBENCH Karin_20241202_022726 DATATRACK_ID:5417 STUDY_ID:ST003766 ANALYSIS_ID:AN006182 PROJECT_ID:PR002349 VERSION 1 CREATED_ON March 3, 2025, 7:21 am #PROJECT PR:PROJECT_TITLE Metabolomics of Ndufs4 KO liver PR:PROJECT_TYPE Multi-platform metabolomics analysis PR:PROJECT_SUMMARY Mitochondrial diseases, including Leigh syndrome, frequently result from complex PR:PROJECT_SUMMARY I (CI) deficiencies and lack effective treatments. Investigating metabolic PR:PROJECT_SUMMARY alterations in affected tissues is essential to understanding disease PR:PROJECT_SUMMARY progression and identifying therapeutic targets. The aim of this project was to PR:PROJECT_SUMMARY characterize the metabolic profile of Ndufs4 knockout (KO) mouse livers, a model PR:PROJECT_SUMMARY of CI deficiency, using multi-platform metabolomics. Whole-liver extracts from PR:PROJECT_SUMMARY Ndufs4 KO and wild-type mice were analyzed via untargeted GC-TOFMS and PR:PROJECT_SUMMARY semi-targeted LC-MS/MS. Metabolic profiling revealed widespread disruptions, PR:PROJECT_SUMMARY including altered glucose, amino acid, and nucleotide metabolism, as well as TCA PR:PROJECT_SUMMARY cycle intermediates. Key findings included elevated levels of UDP-glucose and PR:PROJECT_SUMMARY UDP-N-acetylglucosamine, indicating shifts toward anabolic pathways such as PR:PROJECT_SUMMARY hexosamine biosynthesis and glycogen synthesis. These data highlight the PR:PROJECT_SUMMARY potential role of metabolic reprogramming in liver adaptation to mitochondrial PR:PROJECT_SUMMARY dysfunction and provide new directions for investigating interorgan metabolic PR:PROJECT_SUMMARY dynamics in mitochondrial diseases. PR:INSTITUTE North-West University PR:DEPARTMENT Biochemistry PR:LABORATORY Mitochondria Research Group PR:LAST_NAME Louw PR:FIRST_NAME Roan PR:ADDRESS Hoffman Street, Potchefstroom, North-West, 2520, South Africa PR:EMAIL Roan.Louw@nwu.ac.za PR:PHONE +27 18 299 4074 #STUDY ST:STUDY_TITLE Hepatic bioenergetics and metabolism in mitochondrial disease: Insights from the ST:STUDY_TITLE Ndufs4 KO mouse model ST:STUDY_TYPE Multi-platform metabolomics analysis ST:STUDY_SUMMARY Mitochondrial diseases, including Leigh syndrome, frequently result from complex ST:STUDY_SUMMARY I (CI) deficiencies and lack effective treatments. Investigating metabolic ST:STUDY_SUMMARY alterations in affected tissues is essential to understanding disease ST:STUDY_SUMMARY progression and identifying therapeutic targets. The aim of this project was to ST:STUDY_SUMMARY characterize the metabolic profile of Ndufs4 knockout (KO) mouse livers, a model ST:STUDY_SUMMARY of CI deficiency, using multi-platform metabolomics. Whole-liver extracts from ST:STUDY_SUMMARY Ndufs4 KO and wild-type mice were analyzed via untargeted GC-TOFMS and ST:STUDY_SUMMARY semi-targeted LC-MS/MS. Metabolic profiling revealed widespread disruptions, ST:STUDY_SUMMARY including altered glucose, amino acid, and nucleotide metabolism, as well as TCA ST:STUDY_SUMMARY cycle intermediates. Key findings included elevated levels of UDP-glucose and ST:STUDY_SUMMARY UDP-N-acetylglucosamine, indicating shifts toward anabolic pathways such as ST:STUDY_SUMMARY hexosamine biosynthesis and glycogen synthesis. These data highlight the ST:STUDY_SUMMARY potential role of metabolic reprogramming in liver adaptation to mitochondrial ST:STUDY_SUMMARY dysfunction and provide new directions for investigating interorgan metabolic ST:STUDY_SUMMARY dynamics in mitochondrial diseases. ST:INSTITUTE North-West University ST:DEPARTMENT Biochemistry ST:LABORATORY Mitochondria Research Group ST:LAST_NAME Louw ST:FIRST_NAME Roan ST:ADDRESS Hoffman Street, Potchefstroom, North-West, 2520, South Africa ST:EMAIL Roan.Louw@nwu.ac.za ST:PHONE +27 18 299 4074 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN Ndufs4, https://www.jax.org/strain/027058 SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratory (ME, USA) SU:ANIMAL_LIGHT_CYCLE 12:12 h SU:ANIMAL_FEED Rodent Breeder, Cat. #RM1845, LabChef, Nutritionhub SU:ANIMAL_WATER ad libitum #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 - 2L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=2.d; RAW_FILE_NAME(GCTOF file name)=2.L_1.SMP SUBJECT_SAMPLE_FACTORS - 3L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=3.d; RAW_FILE_NAME(GCTOF file name)=3.L_1.SMP SUBJECT_SAMPLE_FACTORS - 7L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=7.d; RAW_FILE_NAME(GCTOF file name)=7.L_1.SMP SUBJECT_SAMPLE_FACTORS - 8L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=8.d; RAW_FILE_NAME(GCTOF file name)=8.L_1.SMP SUBJECT_SAMPLE_FACTORS - 9L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=9.d; RAW_FILE_NAME(GCTOF file name)=9.L_1.SMP SUBJECT_SAMPLE_FACTORS - 10L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=10.d; RAW_FILE_NAME(GCTOF file name)=10.L_1.SMP SUBJECT_SAMPLE_FACTORS - 11L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=11.d; RAW_FILE_NAME(GCTOF file name)=11.L_1.SMP SUBJECT_SAMPLE_FACTORS - 13L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=13.d; RAW_FILE_NAME(GCTOF file name)=13.L_1.SMP SUBJECT_SAMPLE_FACTORS - 17L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=17.d; RAW_FILE_NAME(GCTOF file name)=17.L_1.SMP SUBJECT_SAMPLE_FACTORS - 18L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=18.d; RAW_FILE_NAME(GCTOF file name)=18.L_1.SMP SUBJECT_SAMPLE_FACTORS - 20L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=20.d; RAW_FILE_NAME(GCTOF file name)=20.L_1.SMP SUBJECT_SAMPLE_FACTORS - 24L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=24.d; RAW_FILE_NAME(GCTOF file name)=24.L_1.SMP SUBJECT_SAMPLE_FACTORS - 25L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=25.d; RAW_FILE_NAME(GCTOF file name)=25.L_1.SMP SUBJECT_SAMPLE_FACTORS - 26L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=26.d; RAW_FILE_NAME(GCTOF file name)=26.L_1.SMP SUBJECT_SAMPLE_FACTORS - 28L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=28.d; RAW_FILE_NAME(GCTOF file name)=28.L_1.SMP SUBJECT_SAMPLE_FACTORS - 29L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=29.d; RAW_FILE_NAME(GCTOF file name)=29.L_1.SMP SUBJECT_SAMPLE_FACTORS - 32L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=32.d; RAW_FILE_NAME(GCTOF file name)=32.L_1.SMP SUBJECT_SAMPLE_FACTORS - 36L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=36.d; RAW_FILE_NAME(GCTOF file name)=36.L_1.SMP SUBJECT_SAMPLE_FACTORS - 37L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=37.d; RAW_FILE_NAME(GCTOF file name)=37.L_1.SMP SUBJECT_SAMPLE_FACTORS - 38L Genotype:WT | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=38.d; RAW_FILE_NAME(GCTOF file name)=38.L_1.SMP SUBJECT_SAMPLE_FACTORS - 1L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=1.d; RAW_FILE_NAME(GCTOF file name)=1.L_1.SMP SUBJECT_SAMPLE_FACTORS - 4L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=4.d; RAW_FILE_NAME(GCTOF file name)=4.L_1.SMP SUBJECT_SAMPLE_FACTORS - 5L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=5.d; RAW_FILE_NAME(GCTOF file name)=5.L_1.SMP SUBJECT_SAMPLE_FACTORS - 6L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=6.d; RAW_FILE_NAME(GCTOF file name)=6.L_1.SMP SUBJECT_SAMPLE_FACTORS - 12L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=12.d; RAW_FILE_NAME(GCTOF file name)=12.L_1.SMP SUBJECT_SAMPLE_FACTORS - 14L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=14.d; RAW_FILE_NAME(GCTOF file name)=14.L_1.SMP SUBJECT_SAMPLE_FACTORS - 15L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=15.d; RAW_FILE_NAME(GCTOF file name)=15.L_1.SMP SUBJECT_SAMPLE_FACTORS - 16L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=16.d; RAW_FILE_NAME(GCTOF file name)=16.L_1.SMP SUBJECT_SAMPLE_FACTORS - 19L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=19.d; RAW_FILE_NAME(GCTOF file name)=19.L_1.SMP SUBJECT_SAMPLE_FACTORS - 21L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=21.d; RAW_FILE_NAME(GCTOF file name)=21.L_1.SMP SUBJECT_SAMPLE_FACTORS - 22L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=22.d; RAW_FILE_NAME(GCTOF file name)=22.L_1.SMP SUBJECT_SAMPLE_FACTORS - 23L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=23.d; RAW_FILE_NAME(GCTOF file name)=23.L_1.SMP SUBJECT_SAMPLE_FACTORS - 27L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=27.d; RAW_FILE_NAME(GCTOF file name)=27.L_1.SMP SUBJECT_SAMPLE_FACTORS - 31L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=31.d; RAW_FILE_NAME(GCTOF file name)=31.L_1.SMP SUBJECT_SAMPLE_FACTORS - 33L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=33.d; RAW_FILE_NAME(GCTOF file name)=33.L_1.SMP SUBJECT_SAMPLE_FACTORS - 34L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=34.d; RAW_FILE_NAME(GCTOF file name)=34.L_1.SMP SUBJECT_SAMPLE_FACTORS - 35L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=35.d; RAW_FILE_NAME(GCTOF file name)=35.L_1.SMP SUBJECT_SAMPLE_FACTORS - 39L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=39.d; RAW_FILE_NAME(GCTOF file name)=39.L_1.SMP SUBJECT_SAMPLE_FACTORS - 40L Genotype:KO | Sample source:Mouse liver RAW_FILE_NAME(LC file name)=40.d; RAW_FILE_NAME(GCTOF file name)=40.L_1.SMP #COLLECTION CO:COLLECTION_SUMMARY Mice were euthanized between postnatal day (P) 45-50 via cervical dislocation at CO:COLLECTION_SUMMARY the same time of day (8:00-9:00 AM) after overnight (12-h) fasting. The liver CO:COLLECTION_SUMMARY was removed, snap-frozen in liquid nitrogen, and stored at −80°C until used. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The animals did not receive any treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction was achieved using a modified monophasic Bligh–Dyer SP:SAMPLEPREP_SUMMARY extraction method with a solvent ratio of 3:1:1 (methanol:water:chloroform). The SP:SAMPLEPREP_SUMMARY water used during extraction contained internal standards. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Restek Rxi-5MS (28.6m x 0.25mm,0.25um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE - CH:COLUMN_TEMPERATURE 70°C for 1 min, 7°C/min minute increase until 120°C, 10°C/min increase until CH:COLUMN_TEMPERATURE 230°C, 13°C/min increase until 300°C, held for 1 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Leco Pegasus HT TOF MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS After a solvent delay of 250 seconds, data were gathered at a rate of 20 spectra MS:MS_COMMENTS per second, employing a mass-to-charge (m/z) ratio scan range spanning from 50 MS:MS_COMMENTS to 950 amu. MS:MS_RESULTS_FILE ST003766_AN006182_Results.txt UNITS:Normalised peak areas Has m/z:Yes Has RT:Yes RT units:Seconds #END