#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