#METABOLOMICS WORKBENCH wpathmasiri_20210809_053451 DATATRACK_ID:2790 STUDY_ID:ST001913 ANALYSIS_ID:AN003111 VERSION 1 CREATED_ON 02-02-2023 #PROJECT PR:PROJECT_TITLE Metabolomics Changes in the feces of infants exposed to arsenic PR:PROJECT_SUMMARY Exposure to arsenic (As) during the vulnerable window of fetal development and PR:PROJECT_SUMMARY early childhood has shown significant clinical effects. In highly exposed PR:PROJECT_SUMMARY populations, altered immunity is one of the most affected pathways and can lead PR:PROJECT_SUMMARY to an elevated risk of infection and a pre-disposition to allergy/atopy. Since PR:PROJECT_SUMMARY well water in the New Hampshire region has been determined to be a potential PR:PROJECT_SUMMARY source of As exposure, this study includes mother-infant dyads living in the PR:PROJECT_SUMMARY area, who obtain household water from private wells. This study seeks to PR:PROJECT_SUMMARY determine if in utero and early life As exposure is related to increase PR:PROJECT_SUMMARY occurrence of childhood: infections, allergy and atopy, and diminished vaccine PR:PROJECT_SUMMARY response. It has been observed that the microbiome is an important mediator of PR:PROJECT_SUMMARY immune impairment due to As exposure. Therefore, we will further investigate the PR:PROJECT_SUMMARY relation between in utero and early life As exposure on the development of the PR:PROJECT_SUMMARY infant intestinal microbiome in the first year of life. For this study fecal PR:PROJECT_SUMMARY samples, from infants at risk of As exposure, were collected at 6 weeks and 12 PR:PROJECT_SUMMARY months of age. Metabolomics data will allow for a more complete picture of the PR:PROJECT_SUMMARY relationships between the microbiome, As exposure and immune function. PR:INSTITUTE Dartmouth College PR:DEPARTMENT Department of Epidemiology PR:LAST_NAME Margaret PR:FIRST_NAME Karagas PR:ADDRESS One Medical Center Road, 7927 HB, Rubin Building, Lebanon, NH 03756 PR:EMAIL Margaret.Karagas@Dartmouth.edu PR:PHONE 603-653-9010 PR:DOI http://dx.doi.org/10.21228/M8H029 #STUDY ST:STUDY_TITLE Stool metabolomics in the New Hampshire Birth Cohort Study ST:STUDY_TYPE Untargeted and semi-targeted metabolomics analysis ST:STUDY_SUMMARY This is a study of data collected from fecal samples from larger New Hampshire ST:STUDY_SUMMARY Birth Cohort Study (NHBCS). 1H NMR metabolomic profiling of 524 infant stool ST:STUDY_SUMMARY samples collected at 6 week - 3 year age was performed and spectra were binned ST:STUDY_SUMMARY (untargeted metabolomics). A set of host-microbiome co-metabolites were library ST:STUDY_SUMMARY matched in individual sample spectra and their relative concentrations were ST:STUDY_SUMMARY determined. This study investigated associations of the functional metabolic ST:STUDY_SUMMARY response of the microbial milieu of the infant gut with environmental and other ST:STUDY_SUMMARY factors. ST:INSTITUTE University of North Carolina at Chapel Hill ST:DEPARTMENT Nutrition ST:LABORATORY Metabolomics and Exposome Laboratory, Nutrition Research Institute, UNC Chapel ST:LABORATORY Hill ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 500 Laureate Way, Kannapolis, NC 28081 ST:EMAIL susan_sumner@unc.edu ST:PHONE (919) 622-4456 ST:SUBMIT_DATE 2021-08-09 #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]Additional sample data SUBJECT_SAMPLE_FACTORS - BD1_P1 Sample type:Batch-QC RAW_FILE_NAME=BD1_P1 SUBJECT_SAMPLE_FACTORS - BD1_P2 Sample type:Batch-QC RAW_FILE_NAME=BD1_P2 SUBJECT_SAMPLE_FACTORS - BD1_P3 Sample type:Batch-QC RAW_FILE_NAME=BD1_P3 SUBJECT_SAMPLE_FACTORS - BD1_P4 Sample type:Batch-QC RAW_FILE_NAME=BD1_P4 SUBJECT_SAMPLE_FACTORS - BD2_P1 Sample type:Batch-QC RAW_FILE_NAME=BD2_P1 SUBJECT_SAMPLE_FACTORS - BD2_P2 Sample type:Batch-QC RAW_FILE_NAME=BD2_P2 SUBJECT_SAMPLE_FACTORS - 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S_551 Sample type:Sample RAW_FILE_NAME=S_551 SUBJECT_SAMPLE_FACTORS - S_552 Sample type:Sample RAW_FILE_NAME=S_552 SUBJECT_SAMPLE_FACTORS - S_56 Sample type:Sample RAW_FILE_NAME=S_56 SUBJECT_SAMPLE_FACTORS - S_57 Sample type:Sample RAW_FILE_NAME=S_57 SUBJECT_SAMPLE_FACTORS - S_58 Sample type:Sample RAW_FILE_NAME=S_58 SUBJECT_SAMPLE_FACTORS - S_59 Sample type:Sample RAW_FILE_NAME=S_59 SUBJECT_SAMPLE_FACTORS - S_6 Sample type:Sample RAW_FILE_NAME=S_6 SUBJECT_SAMPLE_FACTORS - S_60 Sample type:Sample RAW_FILE_NAME=S_60 SUBJECT_SAMPLE_FACTORS - S_62 Sample type:Sample RAW_FILE_NAME=S_62 SUBJECT_SAMPLE_FACTORS - S_63 Sample type:Sample RAW_FILE_NAME=S_63 SUBJECT_SAMPLE_FACTORS - S_64 Sample type:Sample RAW_FILE_NAME=S_64 SUBJECT_SAMPLE_FACTORS - S_65 Sample type:Sample RAW_FILE_NAME=S_65 SUBJECT_SAMPLE_FACTORS - S_66 Sample type:Sample RAW_FILE_NAME=S_66 SUBJECT_SAMPLE_FACTORS - S_67 Sample type:Sample RAW_FILE_NAME=S_67 SUBJECT_SAMPLE_FACTORS - S_68 Sample type:Sample RAW_FILE_NAME=S_68 SUBJECT_SAMPLE_FACTORS - S_69 Sample type:Sample RAW_FILE_NAME=S_69 SUBJECT_SAMPLE_FACTORS - S_7 Sample type:Sample RAW_FILE_NAME=S_7 SUBJECT_SAMPLE_FACTORS - S_70 Sample type:Sample RAW_FILE_NAME=S_70 SUBJECT_SAMPLE_FACTORS - S_71 Sample type:Sample RAW_FILE_NAME=S_71 SUBJECT_SAMPLE_FACTORS - S_72 Sample type:Sample RAW_FILE_NAME=S_72 SUBJECT_SAMPLE_FACTORS - S_74 Sample type:Sample RAW_FILE_NAME=S_74 SUBJECT_SAMPLE_FACTORS - S_75 Sample type:Sample RAW_FILE_NAME=S_75 SUBJECT_SAMPLE_FACTORS - S_76 Sample type:Sample RAW_FILE_NAME=S_76 SUBJECT_SAMPLE_FACTORS - S_77 Sample type:Sample RAW_FILE_NAME=S_77 SUBJECT_SAMPLE_FACTORS - S_78 Sample type:Sample RAW_FILE_NAME=S_78 SUBJECT_SAMPLE_FACTORS - S_79 Sample type:Sample RAW_FILE_NAME=S_79 SUBJECT_SAMPLE_FACTORS - S_8 Sample type:Sample RAW_FILE_NAME=S_8 SUBJECT_SAMPLE_FACTORS - S_80 Sample type:Sample RAW_FILE_NAME=S_80 SUBJECT_SAMPLE_FACTORS - S_81 Sample type:Sample RAW_FILE_NAME=S_81 SUBJECT_SAMPLE_FACTORS - S_82 Sample type:Sample RAW_FILE_NAME=S_82 SUBJECT_SAMPLE_FACTORS - S_83 Sample type:Sample RAW_FILE_NAME=S_83 SUBJECT_SAMPLE_FACTORS - S_84 Sample type:Sample RAW_FILE_NAME=S_84 SUBJECT_SAMPLE_FACTORS - S_85 Sample type:Sample RAW_FILE_NAME=S_85 SUBJECT_SAMPLE_FACTORS - S_86 Sample type:Sample RAW_FILE_NAME=S_86 SUBJECT_SAMPLE_FACTORS - S_87 Sample type:Sample RAW_FILE_NAME=S_87 SUBJECT_SAMPLE_FACTORS - S_88 Sample type:Sample RAW_FILE_NAME=S_88 SUBJECT_SAMPLE_FACTORS - S_89 Sample type:Sample RAW_FILE_NAME=S_89 SUBJECT_SAMPLE_FACTORS - S_9 Sample type:Sample RAW_FILE_NAME=S_9 SUBJECT_SAMPLE_FACTORS - S_90 Sample type:Sample RAW_FILE_NAME=S_90 SUBJECT_SAMPLE_FACTORS - S_91 Sample type:Sample RAW_FILE_NAME=S_91 SUBJECT_SAMPLE_FACTORS - S_92 Sample type:Sample RAW_FILE_NAME=S_92 SUBJECT_SAMPLE_FACTORS - S_93 Sample type:Sample RAW_FILE_NAME=S_93 SUBJECT_SAMPLE_FACTORS - S_94 Sample type:Sample RAW_FILE_NAME=S_94 SUBJECT_SAMPLE_FACTORS - S_95 Sample type:Sample RAW_FILE_NAME=S_95 SUBJECT_SAMPLE_FACTORS - S_96 Sample type:Sample RAW_FILE_NAME=S_96 SUBJECT_SAMPLE_FACTORS - S_97 Sample type:Sample RAW_FILE_NAME=S_97 SUBJECT_SAMPLE_FACTORS - S_98 Sample type:Sample RAW_FILE_NAME=S_98 SUBJECT_SAMPLE_FACTORS - S_99 Sample type:Sample RAW_FILE_NAME=S_99 SUBJECT_SAMPLE_FACTORS - D1_SP1 Sample type:Study-QC RAW_FILE_NAME=D1_SP1 SUBJECT_SAMPLE_FACTORS - D1_SP2 Sample type:Study-QC RAW_FILE_NAME=D1_SP2 SUBJECT_SAMPLE_FACTORS - D1_SP3 Sample type:Study-QC RAW_FILE_NAME=D1_SP3 SUBJECT_SAMPLE_FACTORS - D1_SP4 Sample type:Study-QC RAW_FILE_NAME=D1_SP4 SUBJECT_SAMPLE_FACTORS - D2_SP1 Sample type:Study-QC RAW_FILE_NAME=D2_SP1 SUBJECT_SAMPLE_FACTORS - D2_SP2 Sample type:Study-QC RAW_FILE_NAME=D2_SP2 SUBJECT_SAMPLE_FACTORS - D2_SP3 Sample type:Study-QC RAW_FILE_NAME=D2_SP3 SUBJECT_SAMPLE_FACTORS - D2_SP4 Sample type:Study-QC RAW_FILE_NAME=D2_SP4 SUBJECT_SAMPLE_FACTORS - D3_SP1 Sample type:Study-QC RAW_FILE_NAME=D3_SP1 SUBJECT_SAMPLE_FACTORS - D3_SP2 Sample type:Study-QC RAW_FILE_NAME=D3_SP2 SUBJECT_SAMPLE_FACTORS - D3_SP3 Sample type:Study-QC RAW_FILE_NAME=D3_SP3 SUBJECT_SAMPLE_FACTORS - D3_SP4 Sample type:Study-QC RAW_FILE_NAME=D3_SP4 SUBJECT_SAMPLE_FACTORS - D4_SP1 Sample type:Study-QC RAW_FILE_NAME=D4_SP1 SUBJECT_SAMPLE_FACTORS - D4_SP2 Sample type:Study-QC RAW_FILE_NAME=D4_SP2 SUBJECT_SAMPLE_FACTORS - D4_SP3 Sample type:Study-QC RAW_FILE_NAME=D4_SP3 SUBJECT_SAMPLE_FACTORS - D4_SP4 Sample type:Study-QC RAW_FILE_NAME=D4_SP4 SUBJECT_SAMPLE_FACTORS - D5_SP1 Sample type:Study-QC RAW_FILE_NAME=D5_SP1 SUBJECT_SAMPLE_FACTORS - D5_SP2 Sample type:Study-QC RAW_FILE_NAME=D5_SP2 SUBJECT_SAMPLE_FACTORS - D5_SP3 Sample type:Study-QC RAW_FILE_NAME=D5_SP3 SUBJECT_SAMPLE_FACTORS - D5_SP4 Sample type:Study-QC RAW_FILE_NAME=D5_SP4 SUBJECT_SAMPLE_FACTORS - D6_SP1 Sample type:Study-QC RAW_FILE_NAME=D6_SP1 SUBJECT_SAMPLE_FACTORS - D6_SP2 Sample type:Study-QC RAW_FILE_NAME=D6_SP2 SUBJECT_SAMPLE_FACTORS - D6_SP3 Sample type:Study-QC RAW_FILE_NAME=D6_SP3 SUBJECT_SAMPLE_FACTORS - D6_SP4 Sample type:Study-QC RAW_FILE_NAME=D6_SP4 #COLLECTION CO:COLLECTION_SUMMARY Infant stool samples were collected at home using provided diapers, sealed in a CO:COLLECTION_SUMMARY separate polyethylene bag and frozen in a home freezer or kept chilled until CO:COLLECTION_SUMMARY transport. Samples were transported in a cooler with ice packs and brought to CO:COLLECTION_SUMMARY the routine 6-week post-partum visit within 24 hours of collection. Stool CO:COLLECTION_SUMMARY samples remained frozen until processing where they were thawed at 4 °C. CO:COLLECTION_SUMMARY Using sterile applicators, 0.5–1 g of stool was aliquoted into trace CO:COLLECTION_SUMMARY element-free cryovial tubes and then frozen at −80 °C in a biorepository CO:SAMPLE_TYPE Feces CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY NA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY De-identified stool aliquots were shipped on dry ice and immediately stored at SP:SAMPLEPREP_SUMMARY -80 °C for metabolomics analysis. Samples were randomized into batches. For SP:SAMPLEPREP_SUMMARY each batch, samples were thawed and ~150mg of stool was transferred to MagNA SP:SAMPLEPREP_SUMMARY Lyser tubes after recording the weight, Samples were then homogenized with 50% SP:SAMPLEPREP_SUMMARY acetonitrile in water by using the Omni Bead Disruptor (Omni International, GA, SP:SAMPLEPREP_SUMMARY USA). Homogenized samples were centrifuged at 16000 rcf and the supernatant was SP:SAMPLEPREP_SUMMARY separated into another tube. An aliquot (1000 uL, 100 mg equivalent of fecal SP:SAMPLEPREP_SUMMARY mass) was transferred into Eppendorf tube and lyophilized overnight. The dried SP:SAMPLEPREP_SUMMARY extract was reconstituted in 700 uL of NMR master mix (containing 0.2M SP:SAMPLEPREP_SUMMARY phosphate, 0.5 mM DSS-d6, and 0.2% sodium azide), vortexed on a multitube SP:SAMPLEPREP_SUMMARY vortexer at speed 5 for 2 min and centrifuged at 16000 rcf for 5 min. A 600 µl SP:SAMPLEPREP_SUMMARY aliquot of the supernatant was transferred into a pre-labeled 5mm NMR tube for SP:SAMPLEPREP_SUMMARY data acquisition on a 700 MHz spectrometer. Additionally, study pooled samples SP:SAMPLEPREP_SUMMARY (created from randomly selected study samples) and batch pooled quality control SP:SAMPLEPREP_SUMMARY (QC) samples were generated from supernatants of study samples and aliquots of SP:SAMPLEPREP_SUMMARY supernatants were dried and reconstituted similar to study samples described SP:SAMPLEPREP_SUMMARY above, and used for QC purposes. SP:SAMPLEPREP_PROTOCOL_FILENAME 1. Dartmouth-Fecal Metabolomics_NMR_Batch2 Procedures SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACTION_METHOD 50% Acetonitrile in Water SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION In phosphate buffer containing D2O #CHROMATOGRAPHY #ANALYSIS AN:LABORATORY_NAME Metabolomics and Exposome Lab at UNC Nutrition Research Institute AN:ANALYSIS_TYPE NMR AN:SOFTWARE_VERSION TopSpin 3.5 AN:OPERATOR_NAME Wimal Pathmasiri AN:DETECTOR_TYPE NMR AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Avance III 700 MHz NMR Spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK 3015 Hz, D2O NM:STANDARD_CONCENTRATION 0.5 mM NM:SPECTROMETER_FREQUENCY 700 MHz NM:NMR_PROBE CP QCI/HPCN NM:NMR_SOLVENT 90% H2O, 10% D2O NM:NMR_TUBE_SIZE 5 mm NM:SHIMMING_METHOD topshim NM:PULSE_SEQUENCE noesygppr1d NM:WATER_SUPPRESSION pre-saturation NM:PULSE_WIDTH 14.59 us NM:POWER_LEVEL 12.3 Watts NM:RECEIVER_GAIN 36 NM:OFFSET_FREQUENCY 3290.61 Hz NM:PRESATURATION_POWER_LEVEL 0.0000964 Watts NM:CHEMICAL_SHIFT_REF_CPD DSS-d6 NM:TEMPERATURE 25 C NM:NUMBER_OF_SCANS 64 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 3.9 seconds NM:RELAXATION_DELAY 2 seconds NM:SPECTRAL_WIDTH 12 ppm NM:NUM_DATA_POINTS_ACQUIRED 65536 NM:REAL_DATA_POINTS 32768 NM:LINE_BROADENING 0.5 Hz NM:ZERO_FILLING Yes NM:APODIZATION Lorentzian NM:BASELINE_CORRECTION_METHOD Polynomial NM:CHEMICAL_SHIFT_REF_STD DSS-d6 NM:BINNED_INCREMENT 0.04ppm NM:BINNED_DATA_EXCLUDED_RANGE 4.79 - 4.85 ppm (Water region) #END