#METABOLOMICS WORKBENCH Mahamogren_20230810_093747 DATATRACK_ID:4216 STUDY_ID:ST002811 ANALYSIS_ID:AN004572 PROJECT_ID:PR001758 VERSION 1 CREATED_ON August 14, 2023, 9:28 am #PROJECT PR:PROJECT_TITLE Metabolomics panel associated with cystic fibrosis-related diabetes towards PR:PROJECT_TITLE biomarker discovery PR:PROJECT_SUMMARY Background: The most prevalent comorbidity among cystic fibrosis (CF) patients PR:PROJECT_SUMMARY is cystic fibrosis-related diabetes (CFRD). CFRD has been linked to one of the PR:PROJECT_SUMMARY worse clinical outcomes and higher mortality. Improved clinical results have PR:PROJECT_SUMMARY been related to earlier diagnosis and treatment of CFRD. Therefore, the present PR:PROJECT_SUMMARY study aimed to investigate the metabolome of human serum of patients with CFRD. PR:PROJECT_SUMMARY This might aid in identifying novel biomarkers linked with the pathophysiology PR:PROJECT_SUMMARY of CFRD and its diagnosis. Methods: The liquid chromatography–high-resolution PR:PROJECT_SUMMARY mass spectrometry (LC–HRMS) metabolomics approach was utilized for serum PR:PROJECT_SUMMARY samples from patients with CF (n= 36) and healthy control (n=36). Among the CF PR:PROJECT_SUMMARY group, nine patients were with CFRD and 27 were non-CFRD (nCFRD). Results: A PR:PROJECT_SUMMARY total of 2328 metabolites were significantly altered in CF compared to the PR:PROJECT_SUMMARY healthy control. Among those, 799 significantly dysregulated metabolites were PR:PROJECT_SUMMARY identified between CFRD and nCFRD. Arachidonic acid (AA), ascorbate, and PR:PROJECT_SUMMARY aldarate metabolism were the most common metabolic pathways dysregulated in CF. PR:PROJECT_SUMMARY L-homocysteic acid (L-HCA) levels were significantly reduced in CF and CFRD PR:PROJECT_SUMMARY compared to control and nCFRD, respectively. In addition, gamma-glutamylglycine PR:PROJECT_SUMMARY and L-5-hydroxytryptophan (5-HTP) had the highest discrimination between CFRD PR:PROJECT_SUMMARY and nCFRD with AUC (0.716 and 0.683, respectively). Conclusions: These PR:PROJECT_SUMMARY biomarkers might serve as diagnostic biomarkers and aid in understanding PR:PROJECT_SUMMARY potential metabolic changes linked to CF and CFRD. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:LAST_NAME Al Mogren PR:FIRST_NAME Maha PR:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia PR:EMAIL mmogren@kfshrc.edu.sa PR:PHONE 966541205332 #STUDY ST:STUDY_TITLE Metabolomics panel associated with cystic fibrosis-related diabetes towards ST:STUDY_TITLE biomarker discovery ST:STUDY_SUMMARY Background: The most prevalent comorbidity among cystic fibrosis (CF) patients ST:STUDY_SUMMARY is cystic fibrosis-related diabetes (CFRD). CFRD has been linked to one of the ST:STUDY_SUMMARY worse clinical outcomes and higher mortality. Improved clinical results have ST:STUDY_SUMMARY been related to earlier diagnosis and treatment of CFRD. Therefore, the present ST:STUDY_SUMMARY study aimed to investigate the metabolome of human serum of patients with CFRD. ST:STUDY_SUMMARY This might aid in identifying novel biomarkers linked with the pathophysiology ST:STUDY_SUMMARY of CFRD and its diagnosis. Methods: The liquid chromatography–high-resolution ST:STUDY_SUMMARY mass spectrometry (LC–HRMS) metabolomics approach was utilized for serum ST:STUDY_SUMMARY samples from patients with CF (n= 36) and healthy control (n=36). Among the CF ST:STUDY_SUMMARY group, nine patients were with CFRD and 27 were non-CFRD (nCFRD). Results: A ST:STUDY_SUMMARY total of 2328 metabolites were significantly altered in CF compared to the ST:STUDY_SUMMARY healthy control. Among those, 799 significantly dysregulated metabolites were ST:STUDY_SUMMARY identified between CFRD and nCFRD. Arachidonic acid (AA), ascorbate, and ST:STUDY_SUMMARY aldarate metabolism were the most common metabolic pathways dysregulated in CF. ST:STUDY_SUMMARY L-homocysteic acid (L-HCA) levels were significantly reduced in CF and CFRD ST:STUDY_SUMMARY compared to control and nCFRD, respectively. In addition, gamma-glutamylglycine ST:STUDY_SUMMARY and L-5-hydroxytryptophan (5-HTP) had the highest discrimination between CFRD ST:STUDY_SUMMARY and nCFRD with AUC (0.716 and 0.683, respectively). Conclusions: These ST:STUDY_SUMMARY biomarkers might serve as diagnostic biomarkers and aid in understanding ST:STUDY_SUMMARY potential metabolic changes linked to CF and CFRD. ST:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) ST:LAST_NAME Al Mogren ST:FIRST_NAME Maha ST:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia ST:EMAIL mmogren@kfshrc.edu.sa ST:PHONE 966541205332 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #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 - Ctrl1 Factor:Control RAW_FILE_NAME=Ctrl1 SUBJECT_SAMPLE_FACTORS - Ctrl2 Factor:Control RAW_FILE_NAME=Ctrl2 SUBJECT_SAMPLE_FACTORS - Ctrl3 Factor:Control RAW_FILE_NAME=Ctrl3 SUBJECT_SAMPLE_FACTORS - Ctrl4 Factor:Control RAW_FILE_NAME=Ctrl4 SUBJECT_SAMPLE_FACTORS - Ctrl5 Factor:Control RAW_FILE_NAME=Ctrl5 SUBJECT_SAMPLE_FACTORS - Ctrl6 Factor:Control RAW_FILE_NAME=Ctrl6 SUBJECT_SAMPLE_FACTORS - Ctrl7 Factor:Control RAW_FILE_NAME=Ctrl7 SUBJECT_SAMPLE_FACTORS - Ctrl8 Factor:Control RAW_FILE_NAME=Ctrl8 SUBJECT_SAMPLE_FACTORS - Ctrl9 Factor:Control RAW_FILE_NAME=Ctrl9 SUBJECT_SAMPLE_FACTORS - Ctrl10 Factor:Control RAW_FILE_NAME=Ctrl10 SUBJECT_SAMPLE_FACTORS - Ctrl11 Factor:Control RAW_FILE_NAME=Ctrl11 SUBJECT_SAMPLE_FACTORS - Ctrl12 Factor:Control RAW_FILE_NAME=Ctrl12 SUBJECT_SAMPLE_FACTORS - Ctrl13 Factor:Control RAW_FILE_NAME=Ctrl13 SUBJECT_SAMPLE_FACTORS - Ctrl14 Factor:Control RAW_FILE_NAME=Ctrl14 SUBJECT_SAMPLE_FACTORS - Ctrl15 Factor:Control RAW_FILE_NAME=Ctrl15 SUBJECT_SAMPLE_FACTORS - Ctrl16 Factor:Control RAW_FILE_NAME=Ctrl16 SUBJECT_SAMPLE_FACTORS - Ctrl17 Factor:Control RAW_FILE_NAME=Ctrl17 SUBJECT_SAMPLE_FACTORS - Ctrl18 Factor:Control RAW_FILE_NAME=Ctrl18 SUBJECT_SAMPLE_FACTORS - Ctrl19 Factor:Control RAW_FILE_NAME=Ctrl19 SUBJECT_SAMPLE_FACTORS - Ctrl20 Factor:Control RAW_FILE_NAME=Ctrl20 SUBJECT_SAMPLE_FACTORS - Ctrl21 Factor:Control RAW_FILE_NAME=Ctrl21 SUBJECT_SAMPLE_FACTORS - Ctrl22 Factor:Control RAW_FILE_NAME=Ctrl22 SUBJECT_SAMPLE_FACTORS - Ctrl23 Factor:Control RAW_FILE_NAME=Ctrl23 SUBJECT_SAMPLE_FACTORS - Ctrl24 Factor:Control RAW_FILE_NAME=Ctrl24 SUBJECT_SAMPLE_FACTORS - Ctrl25 Factor:Control RAW_FILE_NAME=Ctrl25 SUBJECT_SAMPLE_FACTORS - Ctrl26 Factor:Control RAW_FILE_NAME=Ctrl26 SUBJECT_SAMPLE_FACTORS - Ctrl27 Factor:Control RAW_FILE_NAME=Ctrl27 SUBJECT_SAMPLE_FACTORS - Ctrl28 Factor:Control RAW_FILE_NAME=Ctrl28 SUBJECT_SAMPLE_FACTORS - Ctrl29 Factor:Control RAW_FILE_NAME=Ctrl29 SUBJECT_SAMPLE_FACTORS - Ctrl30 Factor:Control RAW_FILE_NAME=Ctrl30 SUBJECT_SAMPLE_FACTORS - Ctrl31 Factor:Control RAW_FILE_NAME=Ctrl31 SUBJECT_SAMPLE_FACTORS - Ctrl32 Factor:Control RAW_FILE_NAME=Ctrl32 SUBJECT_SAMPLE_FACTORS - Ctrl33 Factor:Control RAW_FILE_NAME=Ctrl33 SUBJECT_SAMPLE_FACTORS - Ctrl34 Factor:Control RAW_FILE_NAME=Ctrl34 SUBJECT_SAMPLE_FACTORS - Ctrl35 Factor:Control RAW_FILE_NAME=Ctrl35 SUBJECT_SAMPLE_FACTORS - Ctrl36 Factor:Control RAW_FILE_NAME=Ctrl36 SUBJECT_SAMPLE_FACTORS - CF1 Factor:Patient RAW_FILE_NAME=CF1 SUBJECT_SAMPLE_FACTORS - CF2 Factor:Patient RAW_FILE_NAME=CF2 SUBJECT_SAMPLE_FACTORS - CF3 Factor:Patient RAW_FILE_NAME=CF3 SUBJECT_SAMPLE_FACTORS - CF4 Factor:Patient RAW_FILE_NAME=CF4 SUBJECT_SAMPLE_FACTORS - CF5 Factor:Patient RAW_FILE_NAME=CF5 SUBJECT_SAMPLE_FACTORS - CF6 Factor:Patient RAW_FILE_NAME=CF6 SUBJECT_SAMPLE_FACTORS - CF7 Factor:Patient RAW_FILE_NAME=CF7 SUBJECT_SAMPLE_FACTORS - CF8 Factor:Patient RAW_FILE_NAME=CF8 SUBJECT_SAMPLE_FACTORS - CF9 Factor:Patient RAW_FILE_NAME=CF9 SUBJECT_SAMPLE_FACTORS - CF10 Factor:Patient RAW_FILE_NAME=CF10 SUBJECT_SAMPLE_FACTORS - CF11 Factor:Patient RAW_FILE_NAME=CF11 SUBJECT_SAMPLE_FACTORS - CF12 Factor:Patient RAW_FILE_NAME=CF12 SUBJECT_SAMPLE_FACTORS - CF13 Factor:Patient RAW_FILE_NAME=CF13 SUBJECT_SAMPLE_FACTORS - CF14 Factor:Patient RAW_FILE_NAME=CF14 SUBJECT_SAMPLE_FACTORS - CF17 Factor:Patient RAW_FILE_NAME=CF17 SUBJECT_SAMPLE_FACTORS - CF18 Factor:Patient RAW_FILE_NAME=CF18 SUBJECT_SAMPLE_FACTORS - CF19 Factor:Patient RAW_FILE_NAME=CF19 SUBJECT_SAMPLE_FACTORS - CF20 Factor:Patient RAW_FILE_NAME=CF20 SUBJECT_SAMPLE_FACTORS - CF21 Factor:Patient RAW_FILE_NAME=CF21 SUBJECT_SAMPLE_FACTORS - CF25 Factor:Patient RAW_FILE_NAME=CF25 SUBJECT_SAMPLE_FACTORS - CF26 Factor:Patient RAW_FILE_NAME=CF26 SUBJECT_SAMPLE_FACTORS - CF27 Factor:Patient RAW_FILE_NAME=CF27 SUBJECT_SAMPLE_FACTORS - CF28 Factor:Patient RAW_FILE_NAME=CF28 SUBJECT_SAMPLE_FACTORS - CF29 Factor:Patient RAW_FILE_NAME=CF29 SUBJECT_SAMPLE_FACTORS - CF30 Factor:Patient RAW_FILE_NAME=CF30 SUBJECT_SAMPLE_FACTORS - CF31 Factor:Patient RAW_FILE_NAME=CF31 SUBJECT_SAMPLE_FACTORS - CF33 Factor:Patient RAW_FILE_NAME=CF33 SUBJECT_SAMPLE_FACTORS - CF34 Factor:Patient RAW_FILE_NAME=CF34 SUBJECT_SAMPLE_FACTORS - CF35 Factor:Patient RAW_FILE_NAME=CF35 SUBJECT_SAMPLE_FACTORS - CF36 Factor:Patient RAW_FILE_NAME=CF36 SUBJECT_SAMPLE_FACTORS - CF37 Factor:Patient RAW_FILE_NAME=CF37 SUBJECT_SAMPLE_FACTORS - CF38 Factor:Patient RAW_FILE_NAME=CF38 SUBJECT_SAMPLE_FACTORS - CF39 Factor:Patient RAW_FILE_NAME=CF39 SUBJECT_SAMPLE_FACTORS - CF40 Factor:Patient RAW_FILE_NAME=CF40 SUBJECT_SAMPLE_FACTORS - CF41 Factor:Patient RAW_FILE_NAME=CF41 SUBJECT_SAMPLE_FACTORS - CF42 Factor:Patient RAW_FILE_NAME=CF42 #COLLECTION CO:COLLECTION_SUMMARY CF patients (n=36) and age- and gender-matched healthy controls (n=36) were CO:COLLECTION_SUMMARY enrolled in this study. Among 36 patients with CF, nine patients had CFRD. The CO:COLLECTION_SUMMARY institutional review board at King Faisal Specialist Hospital and Research CO:COLLECTION_SUMMARY Center (KFSHRC) approved this study (RAC# 2160 031). The exclusion criteria were CO:COLLECTION_SUMMARY patients who participated in other clinical studies in the last 30 days and were CO:COLLECTION_SUMMARY unable or unwilling to provide informed consent. The samples were collected as CO:COLLECTION_SUMMARY previously described [17]. In brief, blood samples were taken from adult CF CO:COLLECTION_SUMMARY patients who visited the adult CF-Pulmonology clinic at the King Faisal CO:COLLECTION_SUMMARY Specialist Hospital and Research Center (KFSHRC) in Riyadh, Saudi Arabia. The CO:COLLECTION_SUMMARY samples were centrifugated to separate the serum and frozen at −80 ◦C for CO:COLLECTION_SUMMARY further analysis. CO:COLLECTION_PROTOCOL_FILENAME CF_Sample Collection CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY None #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A standard procedure was used for metabolite extraction [18]. Briefly, SP:SAMPLEPREP_SUMMARY metabolites were extracted from plasma samples by adding an extraction solvent SP:SAMPLEPREP_SUMMARY of ACN: MeOH (1:1) followed by vortexing at 600 rpm, 4 °C for 1 hour in SP:SAMPLEPREP_SUMMARY Thermomixer (Eppendorf, Germany). The samples were then centrifugated at 16000 SP:SAMPLEPREP_SUMMARY rpm for 10 min at 4°C. Subsequently, the supernatant was dried using SpeedVac SP:SAMPLEPREP_SUMMARY (Christ, Germany) and resuspended in 100 ul of 50% A: B mobile phase before SP:SAMPLEPREP_SUMMARY LC/MS analysis. (A: 0.1% Formic acid in dH2O, B: 0.1% FA in 50% ACN: MeOH). SP:SAMPLEPREP_SUMMARY Pooled QC was prepared from all samples to check the instrument performance. SP:SAMPLEPREP_PROTOCOL_FILENAME Metabolites extraction #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Untargeted metabolomics analyses were performed using LCMS as previously CH:CHROMATOGRAPHY_SUMMARY reported [19]. Waters Acquity UPLC system coupled with a Xevo G2-S QTOF mass CH:CHROMATOGRAPHY_SUMMARY spectrometer equipped with an electrospray ionization source (ESI) was used. The CH:CHROMATOGRAPHY_SUMMARY samples were separated by ACQUITY UPLC and eluted through XSelect (100×2.1mm CH:CHROMATOGRAPHY_SUMMARY 2.5 mm) column (Waters Ltd., Elstree, UK). The composition of mobile phase CH:CHROMATOGRAPHY_SUMMARY solvent A was 0.1% formic acid in dH2O, and solvent B was 0.1% formic acid in CH:CHROMATOGRAPHY_SUMMARY 50% ACN: MeOH. The flow rate was at 0.300 µL/min. The gradient started with 95- CH:CHROMATOGRAPHY_SUMMARY 5% A for 16 minutes, followed by 4 min of 5% - 95% A, 1 min of 5-95% A, then 2 CH:CHROMATOGRAPHY_SUMMARY min of 95- 5% A. The total run time was 23 minutes with an injection volume of 5 CH:CHROMATOGRAPHY_SUMMARY μL. MS spectra were acquired under positive and negative electrospray CH:CHROMATOGRAPHY_SUMMARY ionization modes (ESI+, ESI−). MS conditions were as follows: the source and CH:CHROMATOGRAPHY_SUMMARY desolvation temperatures were set at 150°C and 500°C (ESI+) or 140 (ESI−), CH:CHROMATOGRAPHY_SUMMARY respectively. The capillary voltage was 3.20 kV (ESI+) or 3 kV (ESI−), the CH:CHROMATOGRAPHY_SUMMARY cone voltage was 40 V, the desolvation gas flow was 800.0 L/h, cone gas flow was CH:CHROMATOGRAPHY_SUMMARY 50 L/h. The collision energies of low and high functions were set off at 10 V CH:CHROMATOGRAPHY_SUMMARY and 50 V, respectively, in MSE mode. Data were collected in continuum mode with CH:CHROMATOGRAPHY_SUMMARY a MasslynxTM V4.1 workstation (Waters Inc., Milford, MA, USA). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Waters XSelect HSS C18 (100 × 2.1mm,2.5um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 50% methanol/50% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-16 min 95- 5% A, 16-19 min 5% A, 19-20 min 5-95% A, 20-22 min 95- 95% A CH:FLOW_RATE 300 µL/min CH:COLUMN_TEMPERATURE 55 CH:METHODS_FILENAME LC/MS Metabolomics #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE LC/MS Metabolomics #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 collected with a Masslynx™ V4.1 workstation in continuum MS:MS_COMMENTS mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a MS:MS_COMMENTS standard pipeline using the Progenesis QI v.3.0 software. MS:MS_RESULTS_FILE ST002811_AN004572_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END