#METABOLOMICS WORKBENCH sdasari_20140321_9643551_mwtab.txt DATATRACK_ID:78 STUDY_ID:ST000045 ANALYSIS_ID:AN000074 PROJECT_ID:PR000044 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Concordance of changes in metabolic pathways based on plasma metabolomics and PR:PROJECT_TITLE muscle transcriptomics in type 1 diabetes. PR:PROJECT_TYPE Untargeted LC-MS Metabolomics PR:PROJECT_SUMMARY Insulin regulates many cellular processes, but the full impact of insulin PR:PROJECT_SUMMARY on cellular functions remains to be defined. Applying a mass spectrometry-based PR:PROJECT_SUMMARY metabolomics approach, we report here alterations of 330 plasma metabolites PR:PROJECT_SUMMARY 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic PR:PROJECT_SUMMARY These pathways included those known to be affected by insulin such as glucose, PR:PROJECT_SUMMARY acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto PR:PROJECT_SUMMARY to be altered including prostaglandin, arachidonic acid, leukotrienes, PR:PROJECT_SUMMARY nucleotides, and anti-inflammatory responses. A significant concordance of PR:PROJECT_SUMMARY and skeletal muscle transcriptome-based pathways supports an assumption that PR:PROJECT_SUMMARY metabolites are chemical fingerprints of cellular events. Although insulin PR:PROJECT_SUMMARY normalized plasma glucose and many other metabolites, there were 71 metabolites PR:PROJECT_SUMMARY 24 pathways that differed between nondiabetes and insulin-treated type 1 PR:PROJECT_SUMMARY Confirmation of many known pathways altered by insulin using a single blood PR:PROJECT_SUMMARY offers confidence in the current approach. Future research needs to be focused PR:PROJECT_SUMMARY newly discovered pathways affected by insulin deficiency and systemic insulin PR:PROJECT_SUMMARY to determine whether they contribute to the high morbidity and mortality in T1D PR:PROJECT_SUMMARY insulin treatment. PR:INSTITUTE Mayo Clinic PR:DEPARTMENT Endocrinology PR:LAST_NAME Nair PR:FIRST_NAME Sreekumaran PR:ADDRESS 200 First Street SW, Rochester, MN 55905 PR:EMAIL Dasari.Surendra@mayo.edu PR:PHONE 507-284-0513 PR:FUNDING_SOURCE R01 DK41973, UL1 RR024150-01 PR:PROJECT_COMMENTS 22415876 #STUDY ST:STUDY_TITLE Plasma metabolomics: Comparison of non-diabetic controls with T1D patients ST:STUDY_TYPE Drug effect study ST:STUDY_SUMMARY Non-diabetic controls whose metabolites were compared to T1D patients with and ST:STUDY_SUMMARY insulin. Seven C-peptide?negative T1D subjects were studied on two occasions: ST:STUDY_SUMMARY during insulin treatment and the other following withdrawal of insulin for 8 h ST:STUDY_SUMMARY compared with matched healthy ND participants ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Endocrinology ST:LAST_NAME Nair ST:FIRST_NAME Sree ST:ADDRESS - ST:EMAIL Dasari.Surendra@mayo.edu ST:PHONE - ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 7 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:SPECIES_GROUP Human #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS T1D_S01 Group1B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS T1D_S02 Group2B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS T1D_S03 Group3B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS T1D_S04 Group4B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS T1D_S05 Group5B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS T1D_S06 Group6B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS T1D_S07 Group7B Treatment:Insulin Withdrawal SUBJECT_SAMPLE_FACTORS ND_S01 Group1C Treatment:Saline Infusion SUBJECT_SAMPLE_FACTORS ND_S02 Group2C Treatment:Saline Infusion SUBJECT_SAMPLE_FACTORS ND_S03 Group3C Treatment:Saline Infusion SUBJECT_SAMPLE_FACTORS ND_S04 Group4C Treatment:Saline Infusion SUBJECT_SAMPLE_FACTORS ND_S05 Group5C Treatment:Saline Infusion SUBJECT_SAMPLE_FACTORS ND_S06 Group6C Treatment:Saline Infusion SUBJECT_SAMPLE_FACTORS ND_S07 Group7C Treatment:Saline Infusion #COLLECTION CO:COLLECTION_SUMMARY Arterialized venous blood was obtained from a catheterized hand vein maintained CO:COLLECTION_SUMMARY 60°C using a hot box for the duration of the study. Plasma samples were stored CO:COLLECTION_SUMMARY -80°C until analysis. CO:SAMPLE_TYPE Blood. Plasma was isolated for MS analysis. CO:COLLECTION_LOCATION Hand vein #TREATMENT TR:TREATMENT_SUMMARY Saline Infusion | Insulin Withdrawal | Insulin Infusion TR:TREATMENT_PROTOCOL_ID SI / IW / II TR:TREATMENT_PROTOCOL_COMMENTS ND participants were kept on a saline infusion from the evening following their TR:TREATMENT_PROTOCOL_COMMENTS | Insulin was discontinued for 8.6 +/- 0.6 h starting at 0400 h. | Insulin was TR:TREATMENT_PROTOCOL_COMMENTS into a forearm vein to maintain blood glucose between 4.44 and 5.56 mmol | L TR:TREATMENT_PROTOCOL_COMMENTS until 1200 h the next day. TR:TREATMENT_COMPOUND Saline | Insulin #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma quality-control samples used in the study were prepared from pooled SP:SAMPLEPREP_SUMMARY spiked with a selection of metabolites to mimic elevated levels of metabolites SP:SAMPLEPREP_SUMMARY I- (insulin withdrawn) condition. Plasma was spiked with a standard mixture SP:SAMPLEPREP_SUMMARY ratio of plasma to spiking solution) containing 100 µg/mL niacin, SP:SAMPLEPREP_SUMMARY leucine, isoleucine, phenylalanine, tryptophan, citric acid, glucose, hippuric SP:SAMPLEPREP_SUMMARY and taurocholic acid dissolved in 1:1 acetonitrile/water. All plasma samples SP:SAMPLEPREP_SUMMARY µL) were thawed on ice at 4°C followed by deproteinization with methanol (1:4 SP:SAMPLEPREP_SUMMARY of plasma to methanol) and vortexed for 10 s, followed by incubation at -20°C SP:SAMPLEPREP_SUMMARY 2 h. The samples were then centrifuged at 15,871g for 30 min at 4°C. The SP:SAMPLEPREP_SUMMARY were lyophilized (Savant, Holbrook, NY) and stored at -20°C prior to analysis. SP:SAMPLEPREP_SUMMARY samples were reconstituted in 50% H2O/acetonitrile and passed through a SP:SAMPLEPREP_SUMMARY YM3 filter (Millipore Corporation). The supernatants were transferred to SP:SAMPLEPREP_SUMMARY vials, stored in the autosampler at 4°C, and analyzed within 48 h of SP:SAMPLEPREP_SUMMARY in buffer. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME high-strength silica 2.1 150 mm, 1.8 m; Waters CH:CHROMATOGRAPHY_COMMENTS The liquid chromatography platform consisted of an Acquity UPLC system (Waters, CH:CHROMATOGRAPHY_COMMENTS MA). Plasma metabolite separation was achieved using both hydrophilic CH:CHROMATOGRAPHY_COMMENTS chromatography (ethylene-bridged hybrid 2.1 150 mm, 1.7 m; Waters) and CH:CHROMATOGRAPHY_COMMENTS liquid chromatography C18 (high-strength silica 2.1 150 mm, 1.8 m; Waters). For CH:CHROMATOGRAPHY_COMMENTS column, the run time was 20 min at a flow rate of 400 L/min. Reverse-phase CH:CHROMATOGRAPHY_COMMENTS was performed using 99% solvent A (5 mmol/L NH4 acetate, 0.1% formic acid, and CH:CHROMATOGRAPHY_COMMENTS acetonitrile) to 100% solvent B (95% acetonitrile with 0.1% formic acid). The CH:CHROMATOGRAPHY_COMMENTS was 0 min, 0% B; 1 min, 0% B; 3 min, 5% B; 13.0 min, 100% B; 16 min, 100% B; CH:CHROMATOGRAPHY_COMMENTS min, 0% B; and 20 min, 0% B. Other LC parameters were injection volume 5 L and CH:CHROMATOGRAPHY_COMMENTS temperature 50C. Each sample was injected in triplicate with blank injections CH:CHROMATOGRAPHY_COMMENTS each sample. Quality controls and standards were run at the beginning and end CH:CHROMATOGRAPHY_COMMENTS the sequence. #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6220 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Negative-ion mode/C18: A 6220 ToF MS (Agilent Technologies) was operated in MS:MS_COMMENTS positive and negative electrospray ionization modes using a scan range of MS:MS_COMMENTS m/z. The mass accuracy and mass resolution were <5 parts per million (ppm) and MS:MS_COMMENTS respectively. The instrument settings were as follows: nebulizer gas MS:MS_COMMENTS 325°C, capillary voltage 3.5 kV, capillary temperature 300°C, fragmentor MS:MS_COMMENTS 150 V, skimmer voltage 58 V, octapole voltage 250 V, cycle time 0.5 s, and run MS:MS_COMMENTS 15.0 min. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Raw MS Intensities MS_METABOLITE_DATA_START Samples Group1B Group2B Group3B Group4B Group5B Group6B Group7B Group1C Group2C Group3C Group4C Group5C Group6C Group7C Factors Treatment:Insulin Withdrawal Treatment:Insulin Withdrawal Treatment:Insulin Withdrawal Treatment:Insulin Withdrawal Treatment:Insulin Withdrawal Treatment:Insulin Withdrawal Treatment:Insulin Withdrawal Treatment:Saline Infusion Treatment:Saline Infusion Treatment:Saline Infusion Treatment:Saline Infusion Treatment:Saline Infusion Treatment:Saline Infusion Treatment:Saline Infusion 2-Butanone_4-[6-(sulfooxy)-2-naphthalenyl]- 155103.6667 276712.5000 250082.0000 338701.8333 222400.6667 261165.3333 314010.5000 220828.0000 165558.0000 301389.0000 239306.6667 301983.0000 273185.6667 2-Methoxyestrone3-glucuronide 72286.0000 63813.3333 117560.5000 39446.8333 58604.3333 29655.1667 51748.6667 33056.0000 40428.0000 25838.6667 32902.0000 63035.3333 68708.6667 50633.6667 2-Oxo-3-hydroxy-4-phosphobutanoate 4079481.3333 4428458.6667 1867693.0000 2276804.1667 2404574.6667 2739638.5000 5078547.5000 3035323.6667 8061903.3333 3092924.6667 2922559.6667 2813365.3333 5444678.6667 3795538.6667 2-oxoisocaproicacid 108390.5000 72992.8333 71886.8333 70315.8333 109658.1667 66437.1667 67753.0000 86543.6667 26415.3333 60830.6667 52972.3333 73860.3333 81458.0000 70861.6667 5-Aminopentanoicacid 378741.1667 277000.8333 180528.8333 215675.5000 343450.6667 214697.8333 244059.6667 388493.6667 96774.3333 207206.3333 212260.3333 239534.6667 225296.6667 306554.3333 5-Aminopentanoicacid-1.419143 48830.3333 19270.0000 19807.6667 19616.3333 29182.2500 35290.0000 25148.3333 14144.0000 14535.6667 6445.0000 Acetoaceticacid 319470.3333 313814.0000 304386.0000 275592.5000 318816.0000 263028.3333 283763.0000 157889.3333 122543.3333 138783.3333 151722.0000 148849.6667 149898.6667 160766.0000 a-hydroxybutyrate 1129218.7500 1055030.2500 510198.7500 309931.5000 900072.1667 958829.6000 238997.3333 99244.6667 333865.6667 165628.0000 140181.0000 138971.0000 311694.0000 102848.0000 beta-D-Fucose 6562.5000 12188.0000 18411.5000 12263.6667 22614.6667 29585.6667 21402.0000 26264.5000 Dihydroxyacetone(glycerone) 3534286.5000 2530330.6667 3548558.5000 2975987.1667 3186961.3333 2958080.8333 2247866.1667 703817.6667 1557802.0000 1952608.5000 1514704.5000 1873186.0000 2513913.0000 Histidine 25547.6000 26767.5000 29624.3333 30856.6667 30918.5000 27438.6000 23787.5000 44931.6667 21077.3333 33860.6667 38758.0000 35270.0000 38355.0000 41025.3333 Leucine 225531.6667 178984.8333 123778.0000 140035.8333 237999.3333 144332.8333 169941.5000 174531.3333 43227.3333 123953.3333 115283.0000 135799.6667 158499.0000 140522.3333 Leucine-2.2727776 332266.8333 287586.1667 208793.1667 282313.5000 492962.1667 235868.6667 272438.5000 383702.3333 96762.6667 256262.0000 240917.6667 267549.3333 248818.3333 326280.6667 Pyroglutamicacid 198636.5000 114987.6667 90853.3333 147322.8333 143489.6667 107966.3333 105326.1667 149750.0000 99442.3333 225820.6667 113553.6667 98383.0000 79593.3333 305836.3333 sebacicacid 17087.6667 12664.0000 4657.0000 12208.0000 25886.0000 Uricacid 764245.1667 465643.6667 704712.0000 704125.1667 770225.1667 652526.3333 833009.3333 380947.3333 337591.3333 481943.0000 664879.3333 975895.3333 594321.6667 746442.0000 C5 H5 Cl N2 S 1 34100 38328 34949 1 34176 33065 32062 28061 35633 37153 31878 48840 41144 C8 H6 N4 O4 S3 15225 14350 1 20591 19687 20331 1 17589 1 18601 20517 20725 17546 15917 C8 H Cl N2 O3 S 59375 1 59060 57480 49935 56617 60050 1 50760 60516 1 60148 1 47796 C4 H Cl3 N2 S3 54295 116624 141456 101240 56202 53111 50530 54179 43003 1 55010 54945 1 149483 C12 H24 O12 10619 1 1 11518 14643 9112 12879 11577 1 10574 10749 10338 1 1 C3 H4 O2 16001 18389 23112 18483 24856 19276 23120 8058 17289 17028 1 20204 25628 18026 C5 H16 N4 O S 1 1 1 17688 1 16408 36096 16758 1 1 19094 1 1 1 C4 H6 O2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C5 H6 O3 86832 146747 1 1 113990 1 116406 94330 1 1 1 1 147068 124215 Dihydroxyacetone (glycerone) 1741365 77244 2255401 1829768 1731917 1953802 2070933 40524 1 1532736 1654965 1518705 1875522 1 Acetoacetic acid 140128 202550 172317 158550 173575 160751 151008 157989 124957 135202 144443 147904 164941 161209 C8 H20 N6 O 1 14614 14978 1 8766 1 1 1 1 1 1 1 1 1 C6 H13 N O3 15066 13464 10215 13331 12398 8739 14626 18507 8777 12087 17360 17566 9927 14803 C10 H14 N4 O6 1 7586 1 1 6629 1 1 5640 1 6960 1 6327 1 1 2-Butanone, 4-[6-(sulfooxy)-2-naphthalenyl]- 160983 315232 278344 352213 276054 168198 297564 229133 1 229718 311195 236534 317551 278550 C12 H25 N O3 S2 4735 16419 6621 8091 6966 6060 6505 8334 6590 1 7604 5275 5488 8908 5-Aminopentanoic acid 264448 254525 166549 170914 275824 171064 196063 362194 97325 206043 210940 227307 236150 296717 C4 H3 N3 O2 31476 18858 38123 36887 32534 29435 39247 1 17548 25397 34630 46178 32931 41397 Uric acid 586827 371016 760710 721957 641719 579473 716740 370900 349460 501398 662197 956766 629017 746636 C11 H21 N O7 21921 12408 10190 17680 21861 13635 16571 47910 14521 12217 44654 19889 1 17331 5-Aminopentanoic acid - 1.419143 1 1 1 1 1 1 1 1 1 1 15730 1 1 1 Pyroglutamic acid 224326 151800 119703 159144 161197 144473 146227 140942 101793 242121 111902 116162 81111 292883 C17 H4 O2 1 25367 1 1 1 1 18854 1 1 1 1 1 1 1 C6 H10 N6 O4 1 26999 1 1 1 1 1 1 1 1 1 1 1 1 C10 H16 O3 S 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C15 H8 N6 O5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C9 H16 O3 S 1 25720 1 1 1 1 1 1 1 1 1 1 1 1 C14 H8 N6 O5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C6 H6 O3 1 9934 1 1 1 1 1 1 1 1 1 1 1 1 C8 H15 N7 O3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Leucine - 2.2727776 222754 214688 172250 201689 382833 205040 213528 378794 94976 246574 246611 185497 146127 335502 C9 H16 O3 S - 2.6250772 14676 17633 1 1 1 1 1 13681 1 1 1 1 1 1 C16 H25 N O2 S2 1 1 1 1 1 1 1 11599 1 1 1 1 1 1 C12 H6 N6 S 17011 1 16637 1 1 1 12645 19761 1 13821 1 1 1 16090 C20 H10 O18 8736 8239 11056 1 14549 10075 11181 8635 1 11032 1 17496 19320 14162 2-oxoisocaproic acid 67721 69392 66148 44712 67552 44615 56210 84168 27749 61908 54025 74649 80389 69422 C27 H2 N2 O23 1 16842 1 1 24485 1 23298 23985 10395 18990 20184 21817 18562 23539 C18 H6 N2 O19 S 1 1 1 53437 1 1 55662 1 40868 50478 46466 55381 47597 1 C21 H6 N2 O25 240402 536805 307400 357830 376832 343354 457505 435533 888179 461755 441807 428919 703316 545923 C13 H6 O18 936156 1687548 1053469 999300 1023743 930194 1314257 1352285 2505539 1300824 1231294 1154744 1801145 1571656 sebacic acid 1 5485 1 1 1 1 1 1 1 1 1 1 1 1 2-Methoxyestrone 3-glucuronide 42694 48226 124441 35187 46443 14607 31118 29962 41484 25477 33399 61918 66891 48150 C23 H28 N4 O3 1 1 12802 1 1 1 1 1 1 1 1 5893 7119 1 C32 H20 O6 S3 7674 6422 6610 6928 6970 6916 6608 6937 7048 7887 6189 7196 7371 1 C12 H12 N2 O9 23170 12028 11852 8743 10030 1 20939 21182 1 9707 12230 12710 9356 10606 C6 H13 N5 O2 S 64776 52784 39953 46441 47750 34971 1 1 1 1 54353 48638 39258 52261 C6 H13 Cl O5 1 1 1 1 1 1 1 49161 41739 27242 33473 1 28727 74548 C4 H2 O4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C12 H2 O15 1 8394 11016 13883 1 1 11907 1 16215 11902 12136 1 1 1 C18 H21 N O4 S 1 1 1 1 7357 1 1 1 1 1 1 5731 1 1 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 2-Butanone,4-[6-(sulfooxy)-2-naphthalenyl]- 294.0563 0.9280167 Mayo_RI 2-Methoxyestrone3-glucuronide 476.2022 10.172761 Mayo_RI 443077 2-Oxo-3-hydroxy-4-phosphobutanoate 213.9867 7.591397 Mayo_RI 25245610 2-oxoisocaproicacid 130.063 4.9944606 Mayo_RI 70 5-Aminopentanoicacid 117.0789 1.1872064 Mayo_RI 5-Aminopentanoicacid-1.419143 117.079 1.419143 Mayo_RI Acetoaceticacid 102.0318 0.896349 Mayo_RI a-hydroxybutyrate 104.0474 2.2411954 Mayo_RI beta-D-Fucose 164.0686 0.9833889 Mayo_RI 439650 Dihydroxyacetone(glycerone) 90.0318 0.89933956 Mayo_RI Histidine 155.0695 0.83680314 Mayo_RI Leucine 131.0946 2.0676668 Mayo_RI Leucine-2.2727776 131.0946 2.2727776 Mayo_RI Pyroglutamicacid 129.0425 1.4762219 Mayo_RI sebacicacid 202.1206 9.9398 Mayo_RI 5192 Uricacid 168.0282 1.3378096 Mayo_RI C5 H5 Cl N2 S C8 H6 N4 O4 S3 C8 H Cl N2 O3 S C4 H Cl3 N2 S3 C12 H24 O12 C3 H4 O2 C5 H16 N4 O S C4 H6 O2 C5 H6 O3 Dihydroxyacetone (glycerone) Acetoacetic acid C8 H20 N6 O C6 H13 N O3 C10 H14 N4 O6 2-Butanone, 4-[6-(sulfooxy)-2-naphthalenyl]- C12 H25 N O3 S2 5-Aminopentanoic acid C4 H3 N3 O2 Uric acid C11 H21 N O7 5-Aminopentanoic acid - 1.419143 Pyroglutamic acid C17 H4 O2 C6 H10 N6 O4 C10 H16 O3 S C15 H8 N6 O5 C9 H16 O3 S C14 H8 N6 O5 C6 H6 O3 C8 H15 N7 O3 Leucine - 2.2727776 C9 H16 O3 S - 2.6250772 C16 H25 N O2 S2 C12 H6 N6 S C20 H10 O18 2-oxoisocaproic acid C27 H2 N2 O23 C18 H6 N2 O19 S C21 H6 N2 O25 C13 H6 O18 sebacic acid 2-Methoxyestrone 3-glucuronide C23 H28 N4 O3 C32 H20 O6 S3 C12 H12 N2 O9 C6 H13 N5 O2 S C6 H13 Cl O5 C4 H2 O4 C12 H2 O15 C18 H21 N O4 S METABOLITES_END #END