#METABOLOMICS WORKBENCH sarmas_20210101_140802 DATATRACK_ID:2378 STUDY_ID:ST001649 ANALYSIS_ID:AN002695 PROJECT_ID:PR001055 VERSION 1 CREATED_ON January 13, 2021, 10:27 am #PROJECT PR:PROJECT_TITLE Urinary microbiota and metabolome in pediatric vesicoureteral reflux and PR:PROJECT_TITLE scarring PR:PROJECT_TYPE Observational/cross-sectional PR:PROJECT_SUMMARY Experiment to analyze the changes in the urinary microbiota and metabolome PR:PROJECT_SUMMARY related to pediatric veicoureteral reflux with special attention to the PR:PROJECT_SUMMARY differences related to renal scarring. PR:INSTITUTE University of Missouri-Columbia PR:DEPARTMENT Surgery PR:LAST_NAME Malm-Buatsi PR:FIRST_NAME Elizabeth PR:ADDRESS 1 Hospital Dr, MC 301, Columbia, MO 65212 PR:EMAIL malmbuatsie@health.missouri.edu PR:PHONE (573) 817-3311 #STUDY ST:STUDY_TITLE Urinary microbiota and metabolome in pediatric vesicoureteral reflux and ST:STUDY_TITLE scarring ST:STUDY_TYPE Observational/cross-sectional ST:STUDY_SUMMARY We enrolled girls and boys aged zero to nine years presenting to a pediatric ST:STUDY_SUMMARY urologist for recurrent urinary tract infection (UTI) or renal scarring ST:STUDY_SUMMARY (decreased uptake on a nuclear renal scan) or grade 3-5 vesico ureteral reflux ST:STUDY_SUMMARY (VUR). Exclusion criteria included other urogenital abnormalities, medical renal ST:STUDY_SUMMARY disease, immunodeficiency, syndromes associated with VUR, acute UTI, persistent ST:STUDY_SUMMARY UTI (ongoing positive urine culture 1-3 weeks after completing a treatment ST:STUDY_SUMMARY course), or global renal atrophy on imaging. At one patient visit, a urine ST:STUDY_SUMMARY specimen was collected for 16S and metabolomic analysis. ST:INSTITUTE University of Missouri-Columbia ST:DEPARTMENT MU Metabolomics Center ST:LAST_NAME Sarma ST:FIRST_NAME Saurav ST:ADDRESS 1201 Rollins street, Bond Life Science Center ST:EMAIL sarmas@missouri.edu ST:PHONE 5738825596 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 4.14 to 8.86 years SU:WEIGHT_OR_WEIGHT_RANGE 4.5 to 41.3 kg SU:HEIGHT_OR_HEIGHT_RANGE 56.0 to 143.1 inch SU:GENDER Male and female SU:HUMAN_RACE white #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 - A10 race:White | sex:F | category:No scarring age_at_collection(years)=5.115068493; weight(kg)=18.2; height(cm)=108; RAW_FILE_NAME=A-10.D SUBJECT_SAMPLE_FACTORS - B1 race:White | sex:F | category:No VUR age_at_collection(years)=4.520547945; weight(kg)=17.8; height(cm)=106; RAW_FILE_NAME=B-1.D SUBJECT_SAMPLE_FACTORS - B3 race:White | sex:M | category:No VUR age_at_collection(years)=0.243835616; weight(kg)=6.1; height(cm)=-; RAW_FILE_NAME=B-3.D SUBJECT_SAMPLE_FACTORS - B4 race:White | sex:F | category:No VUR age_at_collection(years)=1.287671233; weight(kg)=9.3; height(cm)=-; RAW_FILE_NAME=B-4.D SUBJECT_SAMPLE_FACTORS - B5 race:White | sex:F | category:No VUR age_at_collection(years)=4.975342466; weight(kg)=16.7; height(cm)=106.8; RAW_FILE_NAME=B-5.D SUBJECT_SAMPLE_FACTORS - B6 race:White | sex:F | category:No VUR age_at_collection(years)=4.868493151; weight(kg)=18.1; height(cm)=114.4; RAW_FILE_NAME=B-6.D SUBJECT_SAMPLE_FACTORS - B7 race:White | sex:F | category:No VUR age_at_collection(years)=4.797260274; weight(kg)=15.6; height(cm)=103; RAW_FILE_NAME=B-7.D SUBJECT_SAMPLE_FACTORS - B9 race:White | sex:F | category:No VUR age_at_collection(years)=8.657534247; weight(kg)=39.3; height(cm)=134.5; RAW_FILE_NAME=B-9.D SUBJECT_SAMPLE_FACTORS - B12 race:White | sex:F | category:No VUR age_at_collection(years)=1.112328767; weight(kg)=11.6; height(cm)=76.2; RAW_FILE_NAME=B-12.D SUBJECT_SAMPLE_FACTORS - B13 race:White | sex:F | category:No VUR age_at_collection(years)=2.37260274; weight(kg)=15; height(cm)=-; RAW_FILE_NAME=B-13.D SUBJECT_SAMPLE_FACTORS - B15 race:White | sex:F | category:No VUR age_at_collection(years)=4.942465753; weight(kg)=22.2; height(cm)=114.4; RAW_FILE_NAME=B-15.D SUBJECT_SAMPLE_FACTORS - B16 race:White | sex:F | category:No VUR age_at_collection(years)=4.575342466; weight(kg)=25.4; height(cm)=115; RAW_FILE_NAME=B-16.D SUBJECT_SAMPLE_FACTORS - A1 race:White | sex:F | category:No VUR age_at_collection(years)=4.139726027; weight(kg)=17.2; height(cm)=-; RAW_FILE_NAME=A-1.D SUBJECT_SAMPLE_FACTORS - A2 race:White | sex:F | category:Scarring age_at_collection(years)=4.364383562; weight(kg)=23.2; height(cm)=-; RAW_FILE_NAME=A-2.D SUBJECT_SAMPLE_FACTORS - A3 race:White | sex:F | category:No scarring age_at_collection(years)=0.498630137; weight(kg)=7.8; height(cm)=-; RAW_FILE_NAME=A-3.D SUBJECT_SAMPLE_FACTORS - A4 race:White | sex:F | category:Scarring age_at_collection(years)=4.709589041; weight(kg)=16.3; height(cm)=-; RAW_FILE_NAME=A-4.D SUBJECT_SAMPLE_FACTORS - A5 race:White | sex:F | category:No scarring age_at_collection(years)=0.295890411; weight(kg)=4.7; height(cm)=-; RAW_FILE_NAME=A-5.D SUBJECT_SAMPLE_FACTORS - A6 race:White | sex:F | category:Scarring age_at_collection(years)=2.720547945; weight(kg)=13.76; height(cm)=91; RAW_FILE_NAME=A-6.D SUBJECT_SAMPLE_FACTORS - A8 race:White | sex:F | category:Scarring age_at_collection(years)=4.928767123; weight(kg)=18.5; height(cm)=-; RAW_FILE_NAME=A-8.D SUBJECT_SAMPLE_FACTORS - A7 race:White | sex:F | category:Scarring age_at_collection(years)=7.082191781; weight(kg)=24.5; height(cm)=120.7; RAW_FILE_NAME=A-7.D SUBJECT_SAMPLE_FACTORS - A9 race:White | sex:F | category:No scarring age_at_collection(years)=6.38630137; weight(kg)=25.3; height(cm)=121.5; RAW_FILE_NAME=A-9.D SUBJECT_SAMPLE_FACTORS - B8 race:White | sex:F | category:No scarring age_at_collection(years)=0.306849315; weight(kg)=6.7; height(cm)=61; RAW_FILE_NAME=B-8.D SUBJECT_SAMPLE_FACTORS - A11 race:White | sex:F | category:No scarring age_at_collection(years)=0.079452055; weight(kg)=4.5; height(cm)=58; RAW_FILE_NAME=A-11.D SUBJECT_SAMPLE_FACTORS - A12 race:Black | sex:F | category:No scarring age_at_collection(years)=5.057534247; weight(kg)=20.4; height(cm)=109.6; RAW_FILE_NAME=A-12.D SUBJECT_SAMPLE_FACTORS - A13 race:White | sex:F | category:No scarring age_at_collection(years)=4.402739726; weight(kg)=14.3; height(cm)=102.8; RAW_FILE_NAME=A-13.D SUBJECT_SAMPLE_FACTORS - A14 race:White | sex:F | category:No scarring age_at_collection(years)=4.865753425; weight(kg)=16.4; height(cm)=107.3; RAW_FILE_NAME=A-14.D SUBJECT_SAMPLE_FACTORS - B10 race:White | sex:F | category:No VUR age_at_collection(years)=8.860273973; weight(kg)=38.05; height(cm)=143.1; RAW_FILE_NAME=B-10.D SUBJECT_SAMPLE_FACTORS - A15 race:White | sex:F | category:No scarring age_at_collection(years)=2.539726027; weight(kg)=12.1; height(cm)=89.5; RAW_FILE_NAME=A-15.D SUBJECT_SAMPLE_FACTORS - B11 race:White | sex:M | category:No scarring age_at_collection(years)=0.156164384; weight(kg)=5.1; height(cm)=56; RAW_FILE_NAME=B-11.D SUBJECT_SAMPLE_FACTORS - A16 race:White | sex:F | category:No VUR age_at_collection(years)=3.520547945; weight(kg)=14.3; height(cm)=97.5; RAW_FILE_NAME=A-16.D SUBJECT_SAMPLE_FACTORS - A17 race:Other | sex:F | category:VUR age_at_collection(years)=0.849315068; weight(kg)=10.8; height(cm)=77.6; RAW_FILE_NAME=A-17.D SUBJECT_SAMPLE_FACTORS - A18 race:White | sex:M | category:Scarring age_at_collection(years)=2.205479452; weight(kg)=12.9; height(cm)=-; RAW_FILE_NAME=A-18.D SUBJECT_SAMPLE_FACTORS - B14 race:White | sex:F | category:No VUR age_at_collection(years)=8.575342466; weight(kg)=41.3; height(cm)=133.2; RAW_FILE_NAME=B-14.D SUBJECT_SAMPLE_FACTORS - A20 race:White | sex:F | category:Scarring age_at_collection(years)=4.197260274; weight(kg)=16; height(cm)=102; RAW_FILE_NAME=A-20.D SUBJECT_SAMPLE_FACTORS - A22 race:White | sex:F | category:No scarring age_at_collection(years)=7.553424658; weight(kg)=20.5; height(cm)=121.5; RAW_FILE_NAME=A-22.D SUBJECT_SAMPLE_FACTORS - B17 race:White | sex:F | category:No VUR age_at_collection(years)=5.317808219; weight(kg)=18.3; height(cm)=110.2; RAW_FILE_NAME=B-17.D SUBJECT_SAMPLE_FACTORS - A23 race:White | sex:F | category:No scarring age_at_collection(years)=5.043835616; weight(kg)=18.6; height(cm)=103.5; RAW_FILE_NAME=A-23.D SUBJECT_SAMPLE_FACTORS - A24 race:White | sex:F | category:No scarring age_at_collection(years)=0.350684932; weight(kg)=5.65; height(cm)=59.5; RAW_FILE_NAME=A-24.D SUBJECT_SAMPLE_FACTORS - B18 race:White | sex:F | category:No scarring age_at_collection(years)=2.117808219; weight(kg)=14.6; height(cm)=89; RAW_FILE_NAME=B-18.D SUBJECT_SAMPLE_FACTORS - A25 race:White | sex:F | category:VUR age_at_collection(years)=4.824657534; weight(kg)=17.4; height(cm)=106; RAW_FILE_NAME=A-25.D SUBJECT_SAMPLE_FACTORS - A26 race:White | sex:F | category:No scarring age_at_collection(years)=4.073972603; weight(kg)=15.9; height(cm)=100; RAW_FILE_NAME=A-26.D SUBJECT_SAMPLE_FACTORS - A27 race:White | sex:F | category:No scarring age_at_collection(years)=4.375342466; weight(kg)=18.3; height(cm)=106; RAW_FILE_NAME=A-27.D #COLLECTION CO:COLLECTION_SUMMARY Urine was collected from eligible children at clinic visits to a pediatric CO:COLLECTION_SUMMARY urologist CO:SAMPLE_TYPE Urine CO:COLLECTION_METHOD Clean catch or catheterized CO:COLLECTION_LOCATION Pediatric urology clinic CO:COLLECTION_FREQUENCY Once CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatments #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100 µL of freshly prepared 1 mg/mL urase solution was added to 100 µL of each SP:SAMPLEPREP_SUMMARY of the urine samples then vortexed for 20 seconds and allowed to stand at -20 oC SP:SAMPLEPREP_SUMMARY for 1 h. Next, 20 µL of 1 mg/mL of ribitol in water was added to each sample SP:SAMPLEPREP_SUMMARY followed by addition of 1 mL of MeOH that was precooled to -20 ºC. The solution SP:SAMPLEPREP_SUMMARY was again vortexed for 20 seconds and allowed to stand at -20 ºC for another 2 SP:SAMPLEPREP_SUMMARY hours. The samples were centrifuged at 13000g for 15 minutes, and 1 ml of the SP:SAMPLEPREP_SUMMARY supernatant from each sample was transferred to an autosampler vial and dried SP:SAMPLEPREP_SUMMARY under gaseous nitrogen stream. Additionally, a pooled sample was prepared by SP:SAMPLEPREP_SUMMARY combining 200 µL of supernatants from each sample and dried under nitrogen. SP:SAMPLEPREP_SUMMARY Dried extracts were stored at -20 C until derivatization. SP:PROCESSING_STORAGE_CONDITIONS -20℃ SP:EXTRACTION_METHOD For GCMS-polar analysis samples were extracted with water, containing 1 mg/ml SP:EXTRACTION_METHOD ribitol, and 1 ml methanol. SP:EXTRACT_ENRICHMENT 1 mL each of supernatant was collected from each sample for GCMS-polar analysis. SP:EXTRACT_STORAGE -20℃ SP:SAMPLE_RESUSPENSION For GCMS analysis extracts dried under nitrogen flow and storred at -20C then SP:SAMPLE_RESUSPENSION resuspended in 25 μl of pyridine containing 15 mg/ml methoxyamine hydrochloride SP:SAMPLE_DERIVATIZATION GCMS samples were derivatized with 25 μl MSTFA SP:SAMPLE_DERIVATIZATION (N-methyl-N-(trimethyl-silyl)trifluoroacetamide) + 1% TMCS SP:SAMPLE_DERIVATIZATION (chlorotrimethylsilane) SP:SAMPLE_SPIKING Ribitol was used as internal standard for GCMS-polar analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Gas chromatography integrated to mass spectrometer CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 6890N CH:COLUMN_NAME DB-5MS (J&W Scientific) 60m CH:FLOW_RATE 1mL/Min of Helium gas CH:COLUMN_TEMPERATURE 80 0C for 2 minutes, then ramped at 5 0C /min to 315 ºC and held at 315 ºC for CH:COLUMN_TEMPERATURE 12 minutes CH:COLUMN_PRESSURE variable CH:INJECTION_TEMPERATURE 280C CH:INTERNAL_STANDARD Ribitol CH:RETENTION_TIME 9 to 61 min CH:ANALYTICAL_TIME 61 min CH:OVEN_TEMPERATURE 80C to 315C CH:CHROMATOGRAPHY_COMMENTS 1:1 split injection with 1uL injection volume #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME MU Metabolomics Center AN:OPERATOR_NAME Saurav Sarma AN:DETECTOR_TYPE Mass Spectrometer AN:DATA_FORMAT .d #MS MS:INSTRUMENT_NAME Agilent 5973 MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Agilent Chem Station software used for data acquisition, Enhanced Data Analysis MS:MS_COMMENTS software used for converting data to .cdf file, AMDIS was used for peak MS:MS_COMMENTS deconvolution and NIST17 GCMS library was used for compound identification. MS:HELIUM_FLOW 1mL/min MS:ION_SOURCE_TEMPERATURE 230C MS:IONIZATION_ENERGY 70ev MS:REAGENT_GAS Helium MS:SOURCE_TEMPERATURE 230C MS:DATAFORMAT .d MS:MS_RESULTS_FILE ST001649_AN002695_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END