#METABOLOMICS WORKBENCH siddia_20230104_154319 DATATRACK_ID:3681 STUDY_ID:ST002432 ANALYSIS_ID:AN003960 PROJECT_ID:PR001565 VERSION 1 CREATED_ON January 5, 2023, 10:27 am #PROJECT PR:PROJECT_TITLE Metabolic impacts of metformin to seasonal influenza vaccination: a pilot study PR:PROJECT_TITLE of drug interaction with the immune response PR:PROJECT_SUMMARY We report here a double-blinded pilot study of seasonal influenza vaccination, PR:PROJECT_SUMMARY where half of the participants received daily metformin administration. Global PR:PROJECT_SUMMARY metabolomics was measured in the plasma samples at six timepoints. Metformin PR:PROJECT_SUMMARY signatures were successfully identified in the metabolomics data. Statistically PR:PROJECT_SUMMARY significant metabolite features were found both for the vaccination effect and PR:PROJECT_SUMMARY for the drug-vaccine interactions. PR:INSTITUTE The Jackson Laboratory For Genomic Medicine PR:LABORATORY Shuzhao Li Lab PR:LAST_NAME Siddiqa PR:FIRST_NAME Amnah PR:ADDRESS 10 Discovery Drive , Farmington, CT 06032 PR:EMAIL amnah.siddiqa@jax.org PR:PHONE 4049187223 #STUDY ST:STUDY_TITLE Metabolic impacts of metformin to seasonal influenza vaccination: a pilot study ST:STUDY_TITLE of drug interaction with the immune response ST:STUDY_SUMMARY We report here a double-blinded pilot study of seasonal influenza vaccination, ST:STUDY_SUMMARY where half of the participants received daily metformin administration. Global ST:STUDY_SUMMARY metabolomics was measured in the plasma samples at six timepoints. Metformin ST:STUDY_SUMMARY signatures were successfully identified in the metabolomics data. Statistically ST:STUDY_SUMMARY significant metabolite features were found both for the vaccination effect and ST:STUDY_SUMMARY for the drug-vaccine interactions. ST:INSTITUTE The Jackson Laboratory For Genomic Medicine ST:LABORATORY Shuzhao Li Lab ST:LAST_NAME Siddiqa ST:FIRST_NAME Amnah ST:ADDRESS 10 Discovery dr, Farmington, CT 06032 ST:EMAIL amnah.siddiqa@jax.org ST:PHONE 4049187223 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - "YW_20201210_033.mzML" "Visit":"V5" | "Treatment":"Placebo" RAW_FILE_NAME="YW_20201210_033.mzML" SUBJECT_SAMPLE_FACTORS - "YW_20201210_035.mzML" "Visit":"V4" | "Treatment":"Placebo" RAW_FILE_NAME="YW_20201210_035.mzML" SUBJECT_SAMPLE_FACTORS - "YW_20201210_037.mzML" "Visit":"V1" | "Treatment":"Placebo" RAW_FILE_NAME="YW_20201210_037.mzML" SUBJECT_SAMPLE_FACTORS - "YW_20201210_039.mzML" "Visit":"V3" | "Treatment":"Placebo" RAW_FILE_NAME="YW_20201210_039.mzML" SUBJECT_SAMPLE_FACTORS - "YW_20201210_041.mzML" "Visit":"V6" | "Treatment":"Placebo" RAW_FILE_NAME="YW_20201210_041.mzML" SUBJECT_SAMPLE_FACTORS - 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"YW_20201210_148.mzML" "Visit":"V6" | "Treatment":"Placebo" RAW_FILE_NAME="YW_20201210_148.mzML" #COLLECTION CO:COLLECTION_SUMMARY EDTA-treated whole blood was immediately centrifuged and the resultant plasma CO:COLLECTION_SUMMARY was stored at -80˚C until analyses. Plasma metabolites extraction was carried CO:COLLECTION_SUMMARY out by protein precipitation technique using extraction solvent, CO:COLLECTION_SUMMARY acetonitrile:methanol (8:1, v/v) containing 0.1% formic acid. All samples were CO:COLLECTION_SUMMARY vortexed and incubated with shaking at 1000 rpm for 10 min at 4˚C followed by CO:COLLECTION_SUMMARY centrifugation at 4˚ C for 15 min at 15,000 rpm. The supernatant was CO:COLLECTION_SUMMARY transferred into mass spec vials and 2 micro liter injected into UHPLC-MS. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY This pilot study is a double-blinded placebo-controlled trial in men and women TR:TREATMENT_SUMMARY over the age of 65 years. Subjects were screened rigorously for eligibility. TR:TREATMENT_SUMMARY Study exclusion criteria included the following: any unstable medical conditions TR:TREATMENT_SUMMARY or severe co-morbidities (severe COPD, severe congestive heart failure, advanced TR:TREATMENT_SUMMARY neurological disorders, etc), contraindications for metformin (severe renal or TR:TREATMENT_SUMMARY liver impairment), contraindication for flu vaccine (history of Guillain-Barre TR:TREATMENT_SUMMARY syndrome post vaccination or allergic to component of vaccine), TR:TREATMENT_SUMMARY immunosuppressive disorders, immunosuppressive medications, and active cancer. TR:TREATMENT_SUMMARY Importantly, participants were excluded if they were prediabetic or diabetic TR:TREATMENT_SUMMARY (HbA1c ≥ 5.7%) to avoid any confounding impact of metformin on diabetes TR:TREATMENT_SUMMARY status. Eligible participants were randomized to metformin (final dose 1500mg TR:TREATMENT_SUMMARY extended release (ER)/day) or placebo treatment. To limit gastrointestinal TR:TREATMENT_SUMMARY issues per current metformin label recommendations, participants started with 1 TR:TREATMENT_SUMMARY tablet a day for week 1 (500mg metformin ER/day or placebo), then 2 tablets a TR:TREATMENT_SUMMARY day for week 2 (1000mg metformin ER or placebo), and finally 3 tablets a day for TR:TREATMENT_SUMMARY week 3 until the completion of the study (1500mg metformin ER or placebo). TR:TREATMENT_SUMMARY Fifteen subjects (n=8 metformin, n=7 placebo) were randomized and completed the TR:TREATMENT_SUMMARY study on treatment with no differences in basic characteristics at baseline TR:TREATMENT_SUMMARY (placebo: 74.71±2.45 years old, 3 males, BMI: 27.31±1.68; metformin: TR:TREATMENT_SUMMARY 74.13±2.42 years old, 5 males, BMI: 26.43±1.47). All participants were TR:TREATMENT_SUMMARY vaccinated with Fluzone high-dose trivalent flu vaccine (Sanofi Pasteur Inc., TR:TREATMENT_SUMMARY Swiftwater, PA) after ~70 days of treatment. Blood was drawn via standard TR:TREATMENT_SUMMARY venipuncture into EDTA-treated vacutainers prior to treatment (Day 0), prior to TR:TREATMENT_SUMMARY vaccination (~day 35 and ~day 70), and 7, ~35, and ~70 days post vaccination. TR:TREATMENT_SUMMARY The study protocol was approved by the Institutional Review Board at the TR:TREATMENT_SUMMARY University of Connecticut Health Center (UCHC) and registered at TR:TREATMENT_SUMMARY ClinicalTrials.gov (NCT03996538). All study participants provided written TR:TREATMENT_SUMMARY informed consent to participate in the study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The plasma samples were thawed in ice. Metabolites extraction were carried out SP:SAMPLEPREP_SUMMARY by protein precipitation using extraction solvent, acetonitrile:methanol (8:1, SP:SAMPLEPREP_SUMMARY v/v) containing 0.1% formic acid and isotope labelled Trimethyl-13C3]-caffeine, SP:SAMPLEPREP_SUMMARY [13C5]-L-glutamic acid, [15N2]-Uracil, [15N,13C5]-L-methionine, [13C6]-D-glucose SP:SAMPLEPREP_SUMMARY and [15N]-L-tyrosin as spike-in controls. 30 μl of plasma was taken and 60 μl SP:SAMPLEPREP_SUMMARY of extraction solvent was added. All samples were vortexed and incubated with SP:SAMPLEPREP_SUMMARY shaking at 1000 rpm for 10 min at 4°C followed by centrifugation at 4°C for 15 SP:SAMPLEPREP_SUMMARY min at 15,000 rpm. The supernatant was transferred into mass spec vials and 2 SP:SAMPLEPREP_SUMMARY μl injected into UHPLC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo ScientificTM TranscendTM Duo LX-2 UHPLC CH:COLUMN_NAME HILIC column, AccucoreTM-150-Amide (50 x 2.1mm,2.6um) CH:SOLVENT_A 95% acetonitrile/5% water; 0.1% acetic acid; 10 mM ammonium acetate CH:SOLVENT_B 50% acetonitrile/50% water; 0.1% acetic acid; 10 mM ammonium acetate CH:FLOW_GRADIENT 0.55 ml/min: 0-0.1 min: 0% B, 0.10-5.0 min: 98% B, and 5 min for cleaning and CH:FLOW_GRADIENT equilibration of column. CH:FLOW_RATE 0.55 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Asari was used for data processing MS:MS_RESULTS_FILE ST002432_AN003960_Results.txt UNITS:m/z Has m/z:Yes Has RT:Yes RT units:Seconds #END