#METABOLOMICS WORKBENCH abeerotb12_20231209_060007 DATATRACK_ID:4494 STUDY_ID:ST003171 ANALYSIS_ID:AN005204 PROJECT_ID:PR001971 VERSION 1 CREATED_ON April 15, 2024, 6:12 pm #PROJECT PR:PROJECT_TITLE Comberhincive Chemometric Metabolomic Profile for Maple Syrup Urine Disease Sick PR:PROJECT_TITLE Patients PR:PROJECT_TYPE Untargeted LCMS PR:PROJECT_SUMMARY Abstract: Background: A malfunction in the activity of the branched-chain PR:PROJECT_SUMMARY alpha-ketoacid dehydrogenase (BCKAD) complex results in maple syrup urine PR:PROJECT_SUMMARY disease (MSUD), a genetically inherited illness. Three amino acids—leucine, PR:PROJECT_SUMMARY isoleucine, and valine—are typically broken down by branched-chain alpha-keto PR:PROJECT_SUMMARY acid dehydrogenase complex. Abnormal activity in this process, therefore, can PR:PROJECT_SUMMARY affect vital body systems and result in metabolic dysregulation associated with PR:PROJECT_SUMMARY the consequences of this disease. The therapy and follow-up of ill MSUD patients PR:PROJECT_SUMMARY are greatly aided by many researched endogenous metabolites as well as PR:PROJECT_SUMMARY dysregulated biomarkers and pathways. Objectives: Our goal is to add to the PR:PROJECT_SUMMARY increasing knowledge of information about sick MSUD and the pathways that are PR:PROJECT_SUMMARY involved in improving patient outcomes by utilizing untargeted metabolomics to PR:PROJECT_SUMMARY examine the unique profile of MSUD in sick MSUD patients. Methods: This study PR:PROJECT_SUMMARY evaluated the metabolic changes in the dry blood spot (DBS) of 14 sick MSUD PR:PROJECT_SUMMARY patients and 14 healthy controls utilizing untargeted metabolomics studies PR:PROJECT_SUMMARY performed with liquid chromatography–mass spectrometry. Findings: Based on PR:PROJECT_SUMMARY metabolomics analysis,7754 metabolites were found to be highly dysregulated.Out PR:PROJECT_SUMMARY of them,3716 were up-regulated and 4038 were down-regulated.1557 of the PR:PROJECT_SUMMARY annotated metabolites were found to be endogenous metabolites. The research PR:PROJECT_SUMMARY found possible biomarkers for MSUD, including Glutathioselenol and dUDP, which PR:PROJECT_SUMMARY were elevated in sick MSUD relative to healthy controls and LysoPI downregulated PR:PROJECT_SUMMARY in sick MSUD. Moreover, the Sphingolipid metabolism, selenocompound metabolism PR:PROJECT_SUMMARY and porphyrin metabolism pathways were the most impacted in sick MSUD. In PR:PROJECT_SUMMARY summary, our findings shows that metabolomics is a noninvasive approach to PR:PROJECT_SUMMARY understanding the pathophysiology of the medical condition and a potentially PR:PROJECT_SUMMARY useful technique for assessing novel biomarkers in the early detection of sick PR:PROJECT_SUMMARY MSUD.Further research is required regarding the relationship of these PR:PROJECT_SUMMARY dysregulated metabolites to compromised pathways. PR:INSTITUTE King Saud University PR:DEPARTMENT Biochemistry PR:LABORATORY Clinical Biochemistry PR:LAST_NAME AlOtaibi PR:FIRST_NAME Abeer PR:ADDRESS King Fahad road,Riyadh 11211, Saudi Arabia PR:EMAIL 441203289@student.ksu.edu.sa PR:PHONE +966551933703 #STUDY ST:STUDY_TITLE Untargeted Metabolomics for Exploring Metabolomic Profile of Maple Syrup Urine ST:STUDY_TITLE Disease Sick Patients ST:STUDY_TYPE Untargeted LCMS ST:STUDY_SUMMARY Abstract non-newborn: Background: A malfunction in the activity of the ST:STUDY_SUMMARY branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex results in maple ST:STUDY_SUMMARY syrup urine disease (MSUD), a genetically inherited illness. Three amino ST:STUDY_SUMMARY acids—leucine, isoleucine, and valine—are typically broken down by this ST:STUDY_SUMMARY complex. Abnormal activity in this process, therefore, can affect vital body ST:STUDY_SUMMARY systems and result in metabolic dysregulation associated with the consequences ST:STUDY_SUMMARY of the disease. The therapy and follow-up of ill MSUD patients are greatly aided ST:STUDY_SUMMARY by many researched endogenous metabolites as well as dysregulated biomarkers and ST:STUDY_SUMMARY pathways. Objectives: Our goal is to add to the increasing knowledge of ST:STUDY_SUMMARY information about sick MSUD with relation to MSUD newborns and the pathways that ST:STUDY_SUMMARY are involved in improving patient outcomes by utilizing untargeted metabolomics ST:STUDY_SUMMARY to examine the unique profile of MSUD in sick MSUD patients. Methods: This study ST:STUDY_SUMMARY evaluated the metabolic changes in the dry blood spot (DBS) of 14 sick MSUD ST:STUDY_SUMMARY patients and 14 healthy controls utilizing untargeted metabolomics studies ST:STUDY_SUMMARY performed with liquid chromatography–mass spectrometry. Findings: Based on ST:STUDY_SUMMARY metabolomics analysis,7754 metabolites were found to be highly dysregulated.Out ST:STUDY_SUMMARY of them,3716 were up-regulated and 4038 were down-regulated.1557 of the ST:STUDY_SUMMARY annotated metabolites were found to be endogenous metabolites. The research ST:STUDY_SUMMARY found possible biomarkers for MSUD, including Glutathioselenol and dUDP, which ST:STUDY_SUMMARY were elevated in sick MSUD relative to healthy controls and LysoPI downregulated ST:STUDY_SUMMARY in sick MSUD. Moreover, the Sphingolipid metabolism, selenocompound metabolism ST:STUDY_SUMMARY and porphyrin metabolism pathways were the most impacted in MSUD newborns.This ST:STUDY_SUMMARY study shows 92 endogenous metabolites between newborn MSUD and sick MSUD. In ST:STUDY_SUMMARY summary, our findings shows that metabolomics is a noninvasive approach to ST:STUDY_SUMMARY understanding the pathophysiology of the medical condition and a potentially ST:STUDY_SUMMARY useful technique for assessing novel biomarkers in the early detection of sick ST:STUDY_SUMMARY MSUD.Further research is required regarding the relationship of these ST:STUDY_SUMMARY dysregulated metabolites to compromised pathways. ST:INSTITUTE King Saud University ST:DEPARTMENT Biochemistry ST:LABORATORY Clinical Biochemistry ST:LAST_NAME AlOtaibi ST:FIRST_NAME Abeer ST:ADDRESS 2808 ST:EMAIL 441203289@student.ksu.edu.sa ST:PHONE +966551933703 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 28 ST:NUM_MALES 7 ST:NUM_FEMALES 7 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE >14 days, SU:GENDER Male and female SU:HUMAN_INCLUSION_CRITERIA >14 days, MSUD sick patients DBS SU:HUMAN_EXCLUSION_CRITERIA </=14 days, any IEM's sick other than MSUD, unknown gender #FACTORS #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 - 20820068 Sample source:Whole blood | Sex:M | sample_type:Control age_years=18; RAW_FILE_NAME(Raw file name)=20820068_nNC_p; RAW_FILE_NAME(Raw file name)=20820068_nNC_N SUBJECT_SAMPLE_FACTORS - 20830764 Sample source:Whole blood | Sex:M | sample_type:Control age_years=14; RAW_FILE_NAME(Raw file name)=20830764_nNC_p; RAW_FILE_NAME(Raw file name)=20830764_nNC_N SUBJECT_SAMPLE_FACTORS - 20830816 Sample source:Whole blood | Sex:M | sample_type:Control age_years=10; RAW_FILE_NAME(Raw file name)=20830816_nNC_p; RAW_FILE_NAME(Raw file name)=20830816_nNC_N SUBJECT_SAMPLE_FACTORS - 20839516 Sample source:Whole blood | Sex:F | sample_type:Control age_years=11; RAW_FILE_NAME(Raw file name)=20839516_nNC_p; RAW_FILE_NAME(Raw file name)=20839516_nNC_N SUBJECT_SAMPLE_FACTORS - 21273683 Sample source:Whole blood | Sex:F | sample_type:Control age_years=11; RAW_FILE_NAME(Raw file name)=21273683_nNC_p; RAW_FILE_NAME(Raw file name)=21273683_nNC_N SUBJECT_SAMPLE_FACTORS - 21489196 Sample source:Whole blood | Sex:F | sample_type:Control age_years=11; RAW_FILE_NAME(Raw file name)=21489196_nNC_p; RAW_FILE_NAME(Raw file name)=21489196_nNC_N SUBJECT_SAMPLE_FACTORS - 21489309 Sample source:Whole blood | Sex:F | sample_type:Control age_years=13; RAW_FILE_NAME(Raw file name)=21489309_nNC_p; RAW_FILE_NAME(Raw file name)=21489309_nNC_N SUBJECT_SAMPLE_FACTORS - 21489947 Sample source:Whole blood | Sex:M | sample_type:Control age_years=10; RAW_FILE_NAME(Raw file name)=21489947_nNC_p; RAW_FILE_NAME(Raw file name)=21489947_nNC_N SUBJECT_SAMPLE_FACTORS - 21532106 Sample source:Whole blood | Sex:M | sample_type:Control age_years=11; RAW_FILE_NAME(Raw file name)=21532106_nNC_p; RAW_FILE_NAME(Raw file name)=21532106_nNC_N SUBJECT_SAMPLE_FACTORS - 21581573 Sample source:Whole blood | Sex:M | sample_type:Control age_years=8; RAW_FILE_NAME(Raw file name)=21581573_nNC_p; RAW_FILE_NAME(Raw file name)=21581573_nNC_N SUBJECT_SAMPLE_FACTORS - 21695964 Sample source:Whole blood | Sex:F | sample_type:Control age_years=10; RAW_FILE_NAME(Raw file name)=21695964_nNC_p; RAW_FILE_NAME(Raw file name)=21695964_nNC_N SUBJECT_SAMPLE_FACTORS - 21796397 Sample source:Whole blood | Sex:F | sample_type:Control age_years=12; RAW_FILE_NAME(Raw file name)=21796397_nNC_p; RAW_FILE_NAME(Raw file name)=21796397_nNC_N SUBJECT_SAMPLE_FACTORS - 21799570 Sample source:Whole blood | Sex:F | sample_type:Control age_years=17; RAW_FILE_NAME(Raw file name)=21799570_nNC_p; RAW_FILE_NAME(Raw file name)=21799570_nNC_N SUBJECT_SAMPLE_FACTORS - 21800326 Sample source:Whole blood | Sex:F | sample_type:Control age_years=4; RAW_FILE_NAME(Raw file name)=21800326_nNC_p; RAW_FILE_NAME(Raw file name)=21800326_nNC_N SUBJECT_SAMPLE_FACTORS - 148705 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=10; RAW_FILE_NAME(Raw file name)=148705_nNM_P; RAW_FILE_NAME(Raw file name)=148705_nNM_N SUBJECT_SAMPLE_FACTORS - 15383554 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=15; RAW_FILE_NAME(Raw file name)=15383554_nNM_P; RAW_FILE_NAME(Raw file name)=15383554_nNM_N SUBJECT_SAMPLE_FACTORS - 20613149 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=1; RAW_FILE_NAME(Raw file name)=20613149_nNM_P; RAW_FILE_NAME(Raw file name)=20613149_nNM_N SUBJECT_SAMPLE_FACTORS - 21100943 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=6; RAW_FILE_NAME(Raw file name)=21100943_nNM_P; RAW_FILE_NAME(Raw file name)=21100943_nNM_N SUBJECT_SAMPLE_FACTORS - 21219108 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=0.27; RAW_FILE_NAME(Raw file name)=21219108_nNM_P; RAW_FILE_NAME(Raw file name)=21219108_nNM_N SUBJECT_SAMPLE_FACTORS - 21770104 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=16; RAW_FILE_NAME(Raw file name)=21770104_nNM_P; RAW_FILE_NAME(Raw file name)=21770104_nNM_N SUBJECT_SAMPLE_FACTORS - 21776205 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=18; RAW_FILE_NAME(Raw file name)=21776205_nNM_P; RAW_FILE_NAME(Raw file name)=21776205_nNM_N SUBJECT_SAMPLE_FACTORS - 21796944 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=14; RAW_FILE_NAME(Raw file name)=21796944_nNM_P; RAW_FILE_NAME(Raw file name)=21796944_nNM_N SUBJECT_SAMPLE_FACTORS - 21799172 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=14; RAW_FILE_NAME(Raw file name)=21799172_nNM_P; RAW_FILE_NAME(Raw file name)=21799172_nNM_N SUBJECT_SAMPLE_FACTORS - 21805181 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=16; RAW_FILE_NAME(Raw file name)=21805181_nNM_P; RAW_FILE_NAME(Raw file name)=21805181_nNM_N SUBJECT_SAMPLE_FACTORS - 21833519 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=18; RAW_FILE_NAME(Raw file name)=21833519_nNM_P; RAW_FILE_NAME(Raw file name)=21833519_nNM_N SUBJECT_SAMPLE_FACTORS - 21914504 Sample source:Whole blood | Sex:M | sample_type:MSUD age_years=17; RAW_FILE_NAME(Raw file name)=21914504_nNM_P; RAW_FILE_NAME(Raw file name)=21914504_nNM_N SUBJECT_SAMPLE_FACTORS - 27339556 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=9; RAW_FILE_NAME(Raw file name)=27339556_nNM_P; RAW_FILE_NAME(Raw file name)=27339556_nNM_N SUBJECT_SAMPLE_FACTORS - 21460012 Sample source:Whole blood | Sex:F | sample_type:MSUD age_years=8; RAW_FILE_NAME(Raw file name)=21460012_nNM_P; RAW_FILE_NAME(Raw file name)=21460012_nNM_N #COLLECTION CO:COLLECTION_SUMMARY Twenty-eight DBS samples were collected from biochemically and genetically CO:COLLECTION_SUMMARY confirmed MSUD sick patients (n=14) at King Faisal Specialist Hospital and CO:COLLECTION_SUMMARY Research Center (KFSHRC) and healthy controls (n=14). These healthy individuals CO:COLLECTION_SUMMARY were almost age-sex matched with MSUD's group (Female 50%). Samples from newborn CO:COLLECTION_SUMMARY patients and controls less than 14 days were excluded from this study, as well CO:COLLECTION_SUMMARY as any IEM other than MSUD excluded. CO:SAMPLE_TYPE Blood (whole) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment used. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY One punch, of size 3.3mm, DBS from MSUD newborn and healthy controls were SP:SAMPLEPREP_SUMMARY distributed in 96 V-shaped plate wells, then immersed with 250 μL of extraction SP:SAMPLEPREP_SUMMARY solvent composed of 20% Water: 20% MeOH:40% ACN. The samples were vortexed in SP:SAMPLEPREP_SUMMARY thermomixer (Eppendrof, Germany) at 600 rpm, 25˚C, for 2hrs. The samples were SP:SAMPLEPREP_SUMMARY spun down at 16.000 rpm, 4˚C, for 10 min. The supernatants were transferred SP:SAMPLEPREP_SUMMARY into new 96 V-shaped plate and the punches discarded, and then the samples were SP:SAMPLEPREP_SUMMARY dried in a vacuum concentrator SpeedVac (Christ, City, Germany). Dry residue was SP:SAMPLEPREP_SUMMARY re-dissolved in 100 μL of methanol/water with a ratio (1:1) prior to LC-MS SP:SAMPLEPREP_SUMMARY analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME MSUD_Metabolites_Extraction.pdf SP:PROCESSING_STORAGE_CONDITIONS Room temperature SP:EXTRACT_STORAGE Room temperature #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Waters XSelect CSH C18 (100 x 2.1mm 2.5um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 50% MeOH/50% ACN; 0.1% formic acid CH:FLOW_GRADIENT 95–5% A [0–16 min], 5% A [16–19 min], 5–95% A [19–20 min], and CH:FLOW_GRADIENT 95–95% A [20–22 min]. CH:FLOW_RATE 300 μL/min CH:COLUMN_TEMPERATURE 55 CH:METHODS_FILENAME MSUD_LC_MS_Metabolomics.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Metabolomics_Pos_and_Neg.pdf #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The DIA data were gathered with a Masslynx™ V4.1 Software (Waters Inc., MS:MS_COMMENTS Milford, MA, USA) in continuum mode. Quality control samples (QCs) were made MS:MS_COMMENTS with aliquots from all samples and introduced to the instrument after the MS:MS_COMMENTS randomization of each group, after 10 samples to validate the stability of the MS:MS_COMMENTS system (Aldubayan, Rodan, Berry, & Levy, 2017). Data and Statistical Analyses: MS:MS_COMMENTS The raw MS data were processed using a standard pipeline, beginning from an MS:MS_COMMENTS alignment depending on the mass to charge ratio (m/s) and the retention time MS:MS_COMMENTS (RT) of ion signals’, picking the best peak, followed by the filtering of MS:MS_COMMENTS signal depending on the quality of peak by utilizing the Progenesis QI (v.3.0) MS:MS_COMMENTS software (Waters Technologies, Milford, MA, USA). A multivariate statistics was MS:MS_COMMENTS applied by using MetaboAnalyst (v.5.0) (McGill University, Montreal, QB, Canada) MS:MS_COMMENTS (http://www.metaboanalyst.ca) (Pang et al., 2021). All the imported data-groups MS:MS_COMMENTS (compounds’ names also their raw abundances information) were Pareto scaled, MS:MS_COMMENTS log transformed and applied for creating partial least squares discriminant MS:MS_COMMENTS analysis (PLS-DA) and orthogonal partial least squares discriminant analysis MS:MS_COMMENTS (OPLS-DA) models. The generated OPLS-DA model was measured through R2Y and Q2 MS:MS_COMMENTS values, that represents the fitness of the model and predictive ability, MS:MS_COMMENTS respectively (Worley & Powers, 2013). A univariate analysis was applied through MS:MS_COMMENTS Mass Profiler Professional (MPP) (v. 15.0) software (Agilent, Santa Clara, CA, MS:MS_COMMENTS USA). A volcano plot was applied to uncover significantly changed mass features MS:MS_COMMENTS based on a Moderated T-test, cut-off: no correction, p <0.05, FC 1.5. Heatmap MS:MS_COMMENTS analysis for altered features was performed using the Pearson distance measure MS:MS_COMMENTS according to the Pearson similarity test (Gu et al., 2020). MS:MS_RESULTS_FILE ST003171_AN005204_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END