#METABOLOMICS WORKBENCH ebrown427_20150608_9947521_mwtab.txt DATATRACK_ID:292 STUDY_ID:ST000224 ANALYSIS_ID:AN000333 PROJECT_ID:PR000182 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Metabolomic analysis of the small intestinal content of malnourished mice PR:PROJECT_SUMMARY - PR:INSTITUTE University of British Columbia PR:DEPARTMENT Microbiology and Immunology PR:LABORATORY Dr. B. Brett Finlay PR:LAST_NAME Finlay PR:FIRST_NAME Brett PR:ADDRESS 2185 East Mall, Vancouver, BC, Canada V6T 1Z4 PR:EMAIL bfinlay@msl.ubc.ca PR:PHONE - PR:FUNDING_SOURCE Canadian Institutes for Health Research #STUDY ST:STUDY_TITLE Vitamin targeted metabolomics of the small intestine in malnourished and ST:STUDY_TITLE mice ST:STUDY_TYPE Targeted metabolomics ST:STUDY_SUMMARY A total of 8 samples from 6 week old, female C57BL/6 mice, treated for 3 weeks ST:STUDY_SUMMARY a malnourished diet or a control-fed isocaloric diet. Samples were taken from ST:STUDY_SUMMARY small intestinal fecal content at the terminus of the ileum for targeted ST:STUDY_SUMMARY of vitamin concentrations. ST:INSTITUTE University of Victoria ST:DEPARTMENT The Uvic Proteomics and Metabolomics Innovation Centre ST:LAST_NAME Borchers ST:FIRST_NAME Christoph ST:ADDRESS #3101-4464 Markham St Victoria, BC Canada, V8Z 7X8 ST:EMAIL christoph@proteincentre.com ST:PHONE - ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 8 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:AGE_OR_AGE_RANGE 6 weeks at collection SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs SU:ANIMAL_HOUSING Conventional SU:ANIMAL_FEED Low Protein, Low Fat Malnourished Diet/Isocaloric Control Diet SU:CELL_PRIMARY_IMMORTALIZED Malnourished/Control #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Control_SI_1 CON1 Diet:control SUBJECT_SAMPLE_FACTORS Control_SI_2 CON2 Diet:control SUBJECT_SAMPLE_FACTORS Control_SI_3 CON3 Diet:control SUBJECT_SAMPLE_FACTORS Control_SI_4 CON4 Diet:control SUBJECT_SAMPLE_FACTORS Malnourished_SI_1 MAL1 Diet:malnourished SUBJECT_SAMPLE_FACTORS Malnourished_SI_2 MAL2 Diet:malnourished SUBJECT_SAMPLE_FACTORS Malnourished_SI_3 MAL3 Diet:malnourished SUBJECT_SAMPLE_FACTORS Malnourished_SI_4 MAL4 Diet:malnourished #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE Small intestinal fecal contents CO:COLLECTION_TIME 6 weeks of age CO:STORAGE_CONDITIONS -80 degrees CO:TISSUE_CELL_QUANTITY_TAKEN 15-40mg #TREATMENT TR:TREATMENT_SUMMARY 4 mice were given a malnourished diet and 4 mice were untreated, fed a control #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Vitamin-targeted metabolomics. The samples were analyzed by UPLC-MRM/MS on a SP:SAMPLEPREP_SUMMARY UltiMate 3400 RSLC system coupled to an AB Sciex 4000 QTRAP triple-quadrupole SP:SAMPLEPREP_SUMMARY spectrometer equipped with an electrospray ionization source. The standard SP:SAMPLEPREP_SUMMARY of vitamin A (retinal, retinol, retinoic acid), B1 (thiamine), B2 (riboflavin), SP:SAMPLEPREP_SUMMARY (niacinamide), B6 (pydidoximine, pyridoxine, pyridoxal, SP:SAMPLEPREP_SUMMARY B7 (biotin), B9 (folic acid), D2, D3, E (?-tocopherol, ?-tocopherol, and SP:SAMPLEPREP_SUMMARY K1 and K2, were purchased either from Sigma-Aldrich or from Cayman Chemicals SP:SAMPLEPREP_SUMMARY The MRM transitions of individual analytes were optimized by direct infusion of SP:SAMPLEPREP_SUMMARY standard solution of each compound into the MS instrument. Each sample was SP:SAMPLEPREP_SUMMARY with a methanolic BHT (2 mg/mL) solution at a ratio of 15 ?L per mg of the SP:SAMPLEPREP_SUMMARY intestine digestate. Vitamins were extracted by homogenizing the samples at a SP:SAMPLEPREP_SUMMARY frequency of 30 Hz for 1 min twice using a Retsch MM400 mixer mill and with the SP:SAMPLEPREP_SUMMARY of two 3-mm stainless steel metal balls, followed by 5-min sonication in an icy SP:SAMPLEPREP_SUMMARY bath. The samples were then centrifuged in a micro-centrifuge at 12,500 rpm and SP:SAMPLEPREP_SUMMARY for 10 min. A 300-?L aliquot of the supernatant was transferred into a 3-mL SP:SAMPLEPREP_SUMMARY glass test tube and mixed with 300 ?L of water and 900 ?L of hexane. After 1 SP:SAMPLEPREP_SUMMARY vortex mixing, the tubes were centrifuged at 4000 rpm and 10 oC in a Beckman SP:SAMPLEPREP_SUMMARY centrifuge to separate the supernatant organic phase from the lower aqueous SP:SAMPLEPREP_SUMMARY The supernatants were carefully pipetted out to another sets of 3-mL test SP:SAMPLEPREP_SUMMARY The fat-soluble vitamins were further extracted from the aqueous phase with 900 SP:SAMPLEPREP_SUMMARY of hexane two more times. After liquid-liquid extraction, the pooled organic SP:SAMPLEPREP_SUMMARY for each sample was dried down in a speed-vacuum concentrator at room SP:SAMPLEPREP_SUMMARY The dried residue was reconstituted in 100 ?L of ethanol. A 20-?L aliquot was SP:SAMPLEPREP_SUMMARY for quantitation of the fat-soluble vitamins by LC-(+)ESI-MRM/MS on Waters BEH SP:SAMPLEPREP_SUMMARY (2.1 x 50 mm, 1.7 ?m) UPLC column and with 0.1% formic acid in water and SP:SAMPLEPREP_SUMMARY as the mobile phase for binary solvent gradient elution. An efficient elution SP:SAMPLEPREP_SUMMARY was 50% to 100% B in 10 min. The column temperature was 50oC and the flow rate SP:SAMPLEPREP_SUMMARY 300 ?L/min. The aqueous phases were loaded onto reversed-phase polymeric HLB SP:SAMPLEPREP_SUMMARY (60 mg/1mL, Waters Inc.), which have been activated with 1 mL of methanol and SP:SAMPLEPREP_SUMMARY with 1 mL of 50% methanol before use. Under a 5-inch Hg vaccum, the flow-throw SP:SAMPLEPREP_SUMMARY were collected and the resins were washed with 1 mL of 50% methanol with the SP:SAMPLEPREP_SUMMARY fractions collected. The pooled flow-through fractions were dried in a nitrogen SP:SAMPLEPREP_SUMMARY at 30 oC. The residue from each sample was reconstituted in 100 ?L of 2% SP:SAMPLEPREP_SUMMARY A 20-?L aliquot was injected for quantitation of the water-soluble vitamins by SP:SAMPLEPREP_SUMMARY with (+) or (-) ESI and on a Waters BEH C18 (2.1 x 150 mm, 1.7 ?m) UPLC column SP:SAMPLEPREP_SUMMARY using 0.01% formic acid in water and methanol as the mobile phase for binary SP:SAMPLEPREP_SUMMARY gradient elution. The efficient elution gradient was 2% B for 0.5 min and 2% to SP:SAMPLEPREP_SUMMARY B in 8 min. The column temperature was 30 oC and the flow rate was 250 ?L/min. SP:SAMPLEPREP_SUMMARY concentrations of all the detected vitamins were calculated from the standard SP:SAMPLEPREP_SUMMARY curves of individual vitamins, which were prepared with the use of their SP:SAMPLEPREP_SUMMARY compounds. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex API 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/g MS_METABOLITE_DATA_START Samples CON1 CON2 CON3 CON4 MAL1 MAL2 MAL3 MAL4 Factors Diet:control Diet:control Diet:control Diet:control Diet:malnourished Diet:malnourished Diet:malnourished Diet:malnourished Vitamin B1 5.0100 5.9600 2.4000 7.4000 0.7600 0.7400 0.6900 0.5700 Vitamin B12 0.0024 0.0015 0.0017 0.0066 0.0000 0.0000 0.0000 0.0000 Vitamin B2 2.2500 0.3200 0.3000 0.0200 0.3900 0.2300 0.6300 1.0500 Vitamin B3_nicotinamide 1.1400 2.7500 2.9000 1.4600 2.5900 2.0200 9.0000 1.2300 Vitamin B5 1.1200 0.9000 0.6200 0.2900 2.8300 1.7100 3.7600 4.0300 Vitamin B6_Pyridoxal 0.0280 0.0370 0.0350 0.0250 0.0160 0.0100 0.0120 0.0120 Vitamin B6_Pyridoxal_Phosphate 0.0115 0.0097 0.0217 0.0046 0.0058 0.0021 0.0025 0.0078 Vitamin B6_Pyridoxamine 2.3300 0.5100 0.6600 0.3600 0.1500 0.1600 0.1700 0.3700 Vitamin B6_Pyridoxine 0.0630 0.0550 0.0590 0.0850 0.0110 0.0180 0.0190 0.0140 Vitamin B7 0.0780 0.0500 0.0750 0.0690 0.1310 0.1450 0.1340 0.1660 Vitamin B9 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type Vitamin B1 UVictoria_ID Vitamin B12 UVictoria_ID Vitamin B2 UVictoria_ID Vitamin B3_nicotinamide UVictoria_ID Vitamin B5 UVictoria_ID Vitamin B6_Pyridoxal UVictoria_ID Vitamin B6_Pyridoxal_Phosphate UVictoria_ID Vitamin B6_Pyridoxamine UVictoria_ID Vitamin B6_Pyridoxine UVictoria_ID Vitamin B7 UVictoria_ID Vitamin B9 UVictoria_ID METABOLITES_END #END