#METABOLOMICS WORKBENCH hormel101_20160729_093904_mwtab.txt DATATRACK_ID:690 STUDY_ID:ST000432 ANALYSIS_ID:AN000682 PROJECT_ID:PR000330 VERSION 1 CREATED_ON August 1, 2016, 10:11 am #PROJECT PR:PROJECT_TITLE Impact of Long-Term Poor and Good Glycemic Control on Metabolomics Alterations PR:PROJECT_TITLE in Type 1 Diabetic People. PR:PROJECT_TYPE Targeted LC-MS Metabolomics of Vitamin D PR:PROJECT_SUMMARY The objective of the study was to determine whether T1D with good glycemic PR:PROJECT_SUMMARY control have persistent abnormalities of metabolites and pathways that exist in PR:PROJECT_SUMMARY T1D with poor glycemic control. PR:INSTITUTE Mayo Clinic PR:DEPARTMENT Endocrinology PR:LABORATORY Mayo Clinic Metabolomics Resource Core PR:LAST_NAME Nair PR:FIRST_NAME Sreekumaran PR:ADDRESS 200 First Street SW, Rochester, MN 55905 PR:EMAIL Nair.K@mayo.edu PR:PHONE 507-285-2415 #STUDY ST:STUDY_TITLE Quantitative measurements of vitamin D in T1D poor control, good control, and ST:STUDY_TITLE controls. ST:STUDY_TYPE Quantitative measurements of vitamin D ST:STUDY_SUMMARY The objective of the study was to determine whether T1D with good glycemic ST:STUDY_SUMMARY control have persistent abnormalities of metabolites and pathways that exist in ST:STUDY_SUMMARY T1D with poor glycemic control. ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Endocrinology ST:LABORATORY Mayo Clinic Metabolomics Resource Core ST:LAST_NAME Nair ST:FIRST_NAME Sreekumaran ST:ADDRESS 200 First Street SW, Rochester, MN 55905 ST:EMAIL Nair.K@mayo.edu ST:PHONE 507-285-2415 #SUBJECT SU:SUBJECT_TYPE Animal 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 ms3332-19 sample11 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3332-23 sample15 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-19 sample58 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3333-6 sample35 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-21 sample13 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-16 sample56 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-16 sample22 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-16 sample26 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-1 sample1 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3332-8 sample8 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-22 sample14 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-15 sample21 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-1 sample45 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-5 sample5 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-21 sample46 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-11 sample17 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-5 sample55 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-13 sample19 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-20 sample38 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-7 sample47 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3333-18 sample57 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3333-11 sample50 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-3 sample3 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3332-20 sample12 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-20 sample16 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-25 sample24 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-3 sample54 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-14 sample20 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-14 sample41 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-13 sample52 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-36 sample36 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-9 sample49 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-10 sample10 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-12 sample43 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-24 sample23 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3332-12 sample18 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-6 sample6 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-15 sample53 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3333-22 sample48 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-7 sample7 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3332-2 sample2 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-28 sample30 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-28 sample39 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-9 sample9 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3332-17 sample27 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-10 sample42 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-23 sample51 treatment:T1D poor glycemic control SUBJECT_SAMPLE_FACTORS ms3332-26 sample25 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-14 sample32 treatment:ND SUBJECT_SAMPLE_FACTORS ms3333-4 sample34 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-4 sample4 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-18 sample28 treatment:ND SUBJECT_SAMPLE_FACTORS ms3332-27 sample29 treatment:T1D good glycemic control SUBJECT_SAMPLE_FACTORS ms3333-8 sample40 treatment:ND #COLLECTION CO:COLLECTION_SUMMARY At 5:00 AM after an overnight fast, baseline blood samples were collected from CO:COLLECTION_SUMMARY study participants. Plasma samples were stored at 80°C until analysis. CO:SAMPLE_TYPE Blood. Plasma was isolated for MS analysis. #TREATMENT TR:TREATMENT_SUMMARY Participants were admitted to the Clinical Research Unit at St Mary’s Hospital TR:TREATMENT_SUMMARY (Rochester, Minnesota) the evening before the study and spent overnight in the TR:TREATMENT_SUMMARY Clinical Research Unit. The participants were given a standard meal on the TR:TREATMENT_SUMMARY evening of the admission after which they fasted overnight. Participants with TR:TREATMENT_SUMMARY T1D were treated with insulin as per their usual individual programs. At 5:00 AM TR:TREATMENT_SUMMARY after an overnight fast, baseline blood samples were collected from study TR:TREATMENT_SUMMARY participants. Plasma samples were stored at 80°C until analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma quality-control samples used in the study were prepared from pooled SP:SAMPLEPREP_SUMMARY plasma spiked with a selection of metabolites to mimic elevated levels of SP:SAMPLEPREP_SUMMARY metabolites during I− (insulin withdrawn) condition. Plasma was spiked with a SP:SAMPLEPREP_SUMMARY standard mixture (3:1 ratio of plasma to spiking solution) containing 100 μg/mL SP:SAMPLEPREP_SUMMARY niacin, hypoxanthine, leucine, isoleucine, phenylalanine, tryptophan, citric SP:SAMPLEPREP_SUMMARY acid, glucose, hippuric acid, and taurocholic acid dissolved in 1:1 SP:SAMPLEPREP_SUMMARY acetonitrile/water. All plasma samples (200 μL) were thawed on ice at 4°C SP:SAMPLEPREP_SUMMARY followed by deproteinization with methanol (1:4 ratio of plasma to methanol) and SP:SAMPLEPREP_SUMMARY vortexed for 10 s, followed by incubation at −20°C for 2 h. The samples were SP:SAMPLEPREP_SUMMARY then centrifuged at 15,871g for 30 min at 4°C. The supernatants were SP:SAMPLEPREP_SUMMARY lyophilized (Savant, Holbrook, NY) and stored at −20°C prior to analysis. The SP:SAMPLEPREP_SUMMARY samples were reconstituted in 50% H2O/acetonitrile and passed through a Microcon SP:SAMPLEPREP_SUMMARY YM3 filter (Millipore Corporation). The supernatants were transferred to SP:SAMPLEPREP_SUMMARY analytical vials, stored in the autosampler at 4°C, and analyzed within 48 h of SP:SAMPLEPREP_SUMMARY reconstitution in buffer. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Plasma samples and amino acid calibration standards were prepared with MassTrak CH:CHROMATOGRAPHY_SUMMARY Amino Acid Analysis Solution (AAA) kit from Waters according to instructions CH:CHROMATOGRAPHY_SUMMARY with slight modifications for detection on a mass spectrometer. A 10 point CH:CHROMATOGRAPHY_SUMMARY standard concentration curve was made from the calibration standard solution to CH:CHROMATOGRAPHY_SUMMARY calculate amino acid concentrations in plasma samples. A solution containing CH:CHROMATOGRAPHY_SUMMARY U-13C4-L-aspartic acid, U-13C3-L-alanine, U-13C4-L-threonine, U-13C5-L-proline, CH:CHROMATOGRAPHY_SUMMARY U-13C5-L-valine, U-13C6-leucine, U-13C6-phenylalanine all from Cambridge Isotope CH:CHROMATOGRAPHY_SUMMARY Laboratories, 13C6-tyrosine from Isotec, L-arginine (15N2, 2H2) from MassTrace, CH:CHROMATOGRAPHY_SUMMARY norvaline from Sigma dissolved in 0.01N HCl was used as the internal standard CH:CHROMATOGRAPHY_SUMMARY solution. Frozen plasma samples were thawed, spiked with internal standard then CH:CHROMATOGRAPHY_SUMMARY deproteinized with cold MeOH followed by centrifugation at 10,000 g for 5 CH:CHROMATOGRAPHY_SUMMARY minutes prior to derivatization according to MassTrak instructions. The amino CH:CHROMATOGRAPHY_SUMMARY acid derivatizing reagent used was 6-aminoquinolyl-N-hydroxysuccinimidyl CH:CHROMATOGRAPHY_SUMMARY carbamate. High resolution separation was done using an Acquity UPLC system, CH:CHROMATOGRAPHY_SUMMARY injecting 1 µl of derviatized solution, with a UPLC BEH C18 1.7 micron 2.1×150 CH:CHROMATOGRAPHY_SUMMARY mm column from Waters. Column flow was set to 400 µl/min with a gradient from CH:CHROMATOGRAPHY_SUMMARY 99.9%A to 98%B where buffer A is 1% acetonitrile in 0.1% formic acid and buffer CH:CHROMATOGRAPHY_SUMMARY B is 100% acetonitrile. A column temp of 43 degrees Celsius and a sample tray CH:CHROMATOGRAPHY_SUMMARY temp of 6% Celsius. Mass detection was completed on a TSQ Ultra Quantum from CH:CHROMATOGRAPHY_SUMMARY Thermo Finnigan running in ESI positive mode. A scan width of 0.002, scan time CH:CHROMATOGRAPHY_SUMMARY of 0.04 seconds per transition mass, collision energy of 25, collision gas CH:CHROMATOGRAPHY_SUMMARY pressure of 1.5 mTorr, tube lens value set to 90, monitoring a signature ion of CH:CHROMATOGRAPHY_SUMMARY the derivitized amines at m/z 171.04 by selected reaction monitoring. CH:CHROMATOGRAPHY_TYPE UPLC CH:INSTRUMENT_NAME Thermo TSQ Quantum Ultra CH:COLUMN_NAME None #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Quantum Ultra MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/mL MS_METABOLITE_DATA_START Samples sample11 sample15 sample58 sample35 sample13 sample56 sample22 sample26 sample1 sample8 sample14 sample21 sample45 sample5 sample46 sample17 sample55 sample19 sample38 sample47 sample57 sample50 sample3 sample12 sample16 sample24 sample54 sample20 sample41 sample52 sample36 sample49 sample10 sample43 sample23 sample18 sample6 sample53 sample48 sample7 sample2 sample30 sample39 sample9 sample27 sample42 sample51 sample25 sample32 sample34 sample4 sample28 sample29 sample40 Factors treatment:T1D good glycemic control treatment:T1D good glycemic control treatment:T1D poor glycemic control treatment:ND treatment:T1D good glycemic control treatment:T1D poor glycemic control treatment:ND treatment:ND treatment:T1D good glycemic control treatment:ND treatment:ND treatment:T1D good glycemic control treatment:T1D poor glycemic control treatment:T1D good glycemic control treatment:T1D poor glycemic control treatment:T1D good glycemic control treatment:T1D poor glycemic control treatment:T1D good glycemic control treatment:ND treatment:T1D poor glycemic control treatment:T1D poor glycemic control treatment:T1D poor glycemic control treatment:T1D good glycemic control treatment:ND treatment:ND treatment:ND treatment:T1D poor glycemic control treatment:ND treatment:ND treatment:T1D poor glycemic control treatment:ND treatment:T1D poor glycemic control treatment:ND treatment:ND treatment:T1D good glycemic control treatment:ND treatment:ND treatment:T1D poor glycemic control treatment:T1D poor glycemic control treatment:T1D good glycemic control treatment:ND treatment:ND treatment:ND treatment:T1D good glycemic control treatment:T1D good glycemic control treatment:ND treatment:T1D poor glycemic control treatment:T1D good glycemic control treatment:ND treatment:ND treatment:ND treatment:ND treatment:T1D good glycemic control treatment:ND 24.25.DHVD.D2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NA 0 0 0 0 0 0 0 0 0 0 0 0 0.7 0 0 0 0 0 0 0 0 0 0.84 0 0 0 0 0 0 0 NA 0 24.25.DHVD.D3 4.91 1.98 1.18 6.12 1.14 4.24 0.69 0.69 2.1 1.3 3.27 4.43 2.12 2.67 0.4 3.25 2.14 3.11 0.69 1.4 1.77 NA 3.44 1.51 1.51 3.33 1.21 1.47 1.47 3.2 2.4 1.33 5.57 2.74 1.25 4.46 2.78 6.46 2.63 3.31 1.85 2.48 2.48 2.6 1.25 2.22 2.23 1.23 4.27 2.55 2.88 3.28 NA 0.65 Total.24.25.DHVD 4.91 1.98 1.18 6.12 1.14 4.24 0.69 0.69 2.1 1.3 3.27 4.43 2.12 2.67 0.4 3.25 2.14 3.11 0.69 1.4 1.77 NA 3.44 1.51 1.51 3.33 1.21 1.47 1.47 3.2 2.4 1.33 5.57 2.74 1.95 4.46 2.78 6.46 2.63 3.31 1.85 2.48 2.48 2.6 2.09 2.22 2.23 1.23 4.27 2.55 2.88 3.28 NA 0.65 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 24.25.DHVD.D2 24.25.DHVD.D3 Total.24.25.DHVD METABOLITES_END #END