#METABOLOMICS WORKBENCH ebrown427_20150608_9947521_mwtab.txt DATATRACK_ID:292 STUDY_ID:ST000224 ANALYSIS_ID:AN000334 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 POSITIVE #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 A_Retinal 0.0075 0.0082 0.0074 0.0079 0.0007 0.0008 0.0009 0.0009 Vitamin A_Retinoic acid Vitamin A_Retinol 0.3736 0.3488 0.2424 0.3145 0.1774 0.1114 0.1457 0.1798 Vitamin D2 0.1831 0.1545 0.1709 0.1407 0.0854 0.1058 0.1002 0.0703 Vitamin D3 0.5489 0.5749 0.4471 0.6929 1.5762 3.0405 1.5821 1.0678 Vitamin E_a-tocopherol 13.2240 9.4649 7.1510 12.1395 1.8691 3.5904 2.6829 1.1860 Vitamin E_d-tocopherol 0.0524 0.0421 0.0398 0.0592 0.0057 0.0297 0.0090 0.0033 Vitamin E_d-tocotrienol 0.2240 0.2170 0.2200 0.2820 0.0640 0.1610 0.0900 0.0600 Vitamin K1 Vitamin K2 0.0036 0.0043 0.0034 0.0035 0.0051 0.0036 0.0034 0.0035 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 A_Retinal Uvictoria_ID Vitamin A_Retinoic acid Uvictoria_ID Vitamin A_Retinol Uvictoria_ID Vitamin D2 Uvictoria_ID Vitamin D3 Uvictoria_ID Vitamin E_a-tocopherol Uvictoria_ID Vitamin E_d-tocopherol Uvictoria_ID Vitamin E_d-tocotrienol Uvictoria_ID Vitamin K1 Uvictoria_ID Vitamin K2 Uvictoria_ID METABOLITES_END #END