#METABOLOMICS WORKBENCH cadriano_20170802_143853_mwtab.txt DATATRACK_ID:1183 STUDY_ID:ST000881 ANALYSIS_ID:AN001437 PROJECT_ID:PR000611 VERSION 1 CREATED_ON October 29, 2017, 8:18 pm #PROJECT PR:PROJECT_TITLE Metabolomics analysis of leprosy patients with type 1 reaction PR:PROJECT_TYPE MS qualitative analysis and identification of polyunsaturated fatty acids in PR:PROJECT_TYPE retrospective serum samples of leprosy patients with type 1 reaction PR:PROJECT_SUMMARY Type 1 reaction (T1R) is an acute inflammatory episode that causes severe PR:PROJECT_SUMMARY neuronal damages in patients with leprosy. The factors that trigger this PR:PROJECT_SUMMARY pathology is still unknown, and further studies are needed to understand T1R and PR:PROJECT_SUMMARY to early prevent its start. It is well established that the host immune response PR:PROJECT_SUMMARY is linked to T1R and previous studies indicated that the metabolism of the host PR:PROJECT_SUMMARY also influences the adaptive immune response against M. leprae antigens. PR:PROJECT_SUMMARY Therefore, metabolomics-based analyses of sera from 7 patients with and 9 PR:PROJECT_SUMMARY without T1R were conducted via liquid chromatography–mass spectrometry. The PR:PROJECT_SUMMARY main goal of this project was to determine whether the metabolism of PR:PROJECT_SUMMARY polyunsaturated fatty acids (such as eicosanoids and omega-3 fatty acids) were PR:PROJECT_SUMMARY perturbed in leprosy patients with T1R. PR:INSTITUTE Colorado State University PR:DEPARTMENT Department of Microbiology, Immunology, and Pathology PR:LABORATORY Belisle PR:LAST_NAME Belisle PR:FIRST_NAME John PR:ADDRESS Rampart Road, 1682 Campus Delivery PR:EMAIL cadriano@rams.colostate.edu PR:PHONE 9704819160 PR:FUNDING_SOURCE This work was supported by the New York Community Trust (grant to J. T. B. as PR:FUNDING_SOURCE co–principle investigator [PI]), by the Heiser Foundation (grant to J. T. B. PR:FUNDING_SOURCE as co-PI), and by the Brazilian Coordination for the Improvement of Higher PR:FUNDING_SOURCE Education Personnel through the Science without Borders program (10546-13-8, for PR:FUNDING_SOURCE the postdoctoral scholarship to C. A. M. S.). PR:PROJECT_COMMENTS The institutional review boards of Colorado State University approved the use of PR:PROJECT_COMMENTS sera for the reported study. PR:PUBLICATIONS J Infect Dis. 2017 Feb 1;215(3):431-439. doi: 10.1093/infdis/jiw541. PR:CONTRIBUTORS Carlos A. M. Silva, Kristofor Webb, Barbara G. Andre, Maria Angela Marques, PR:CONTRIBUTORS Fernanda Marques Carvalho, Cristiana Santos de Macedo, Roberta Olmo Pinheiro, PR:CONTRIBUTORS Euzenir Nunes Sarno, Maria Cristina Vidal Pessolani, John T. Belisle. #STUDY ST:STUDY_TITLE Targeted metabolomics data of leprosy patients with type 1 reaction ST:STUDY_TYPE Targeted MS/MS analysis in retrospective serum samples of leprosy patients with ST:STUDY_TYPE type 1 reaction ST:STUDY_SUMMARY Targeted metabolomics-based analyses of pooled sera from 7 patients with T1R ST:STUDY_SUMMARY were conducted via LC-MS/MS. ST:INSTITUTE Colorado State University ST:DEPARTMENT Department of Microbiology, Immunology, and Pathology ST:LABORATORY Belisle ST:LAST_NAME Silva ST:FIRST_NAME Carlos ST:ADDRESS Rampart Road, 1682 Campus Delivery ST:EMAIL cadriano@rams.colostate.edu ST:PHONE 9702154962 ST:NUM_GROUPS 02 (first group was leprosy patients with type 1 reaction, N=9; and second group ST:NUM_GROUPS was leprosy patients without type 1 reaction, N=7) ST:TOTAL_SUBJECTS 16 ST:NUM_MALES 5 (3 were leprosy patients with type 1 reaction and 2 were leprosy patients ST:NUM_MALES without type 1 reaction) ST:NUM_FEMALES 11 (4 were leprosy patients with type 1 reaction and 7 were leprosy patients ST:NUM_FEMALES without type 1 reaction) ST:STUDY_COMMENTS For this study we used retrospective sera samples. ST:PUBLICATIONS J Infect Dis. 2017 Feb 1;215(3):431-439. doi: 10.1093/infdis/jiw541. #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 13 - 66 SU:GENDER Female and Male SU:HUMAN_ETHNICITY Brazillian SU:HUMAN_INCLUSION_CRITERIA Retrospective serum samples from leprosy patients diagnosed or not with type 1 SU:HUMAN_INCLUSION_CRITERIA reaction that were not receiving multidrug therapy or corticosteroid therapy at SU:HUMAN_INCLUSION_CRITERIA the time of blood collection. SU:HUMAN_EXCLUSION_CRITERIA Retrospective serum samples from leprosy patients that were under multidrug SU:HUMAN_EXCLUSION_CRITERIA therapy and/or corticosteroid therapy at the time of blood collection were SU:HUMAN_EXCLUSION_CRITERIA excluded from the study. SU:SUBJECT_COMMENTS The patients exhibited borderline tuberculoid, borderline borderline and SU:SUBJECT_COMMENTS borderline lepromatous forms of leprosy and was diagnosed or not with type 1 SU:SUBJECT_COMMENTS reaction. #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - S1 Type 1 reaction (T1R):Yes Gender=F; Bacillary index=-; Age=46; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S3 Type 1 reaction (T1R):Yes Gender=F; Bacillary index=0.5; Age=27; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BB SUBJECT_SAMPLE_FACTORS - S5 Type 1 reaction (T1R):Yes Gender=F; Bacillary index=0.5; Age=56; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BB SUBJECT_SAMPLE_FACTORS - S7 Type 1 reaction (T1R):Yes Gender=F; Bacillary index=-; Age=32; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S8 Type 1 reaction (T1R):Yes Gender=M; Bacillary index=-; Age=41; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S9 Type 1 reaction (T1R):Yes Gender=M; Bacillary index=2; Age=66; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BL SUBJECT_SAMPLE_FACTORS - S10 Type 1 reaction (T1R):Yes Gender=M; Bacillary index=1.5; Age=49; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BB SUBJECT_SAMPLE_FACTORS - S11 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=65; Serologic finding (+ or - for PGL-I)=-; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S12 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=46; Serologic finding (+ or - for PGL-I)=-; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S13 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=13; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S14 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=57; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S15 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=15; Serologic finding (+ or - for PGL-I)=-; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S16 Type 1 reaction (T1R):No Gender=M; Bacillary index=-; Age=34; Serologic finding (+ or - for PGL-I)=-; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S17 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=40; Serologic finding (+ or - for PGL-I)=+; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S18 Type 1 reaction (T1R):No Gender=M; Bacillary index=-; Age=55; Serologic finding (+ or - for PGL-I)=-; clinical form of leprosy=BT SUBJECT_SAMPLE_FACTORS - S19 Type 1 reaction (T1R):No Gender=F; Bacillary index=-; Age=64; Serologic finding (+ or - for PGL-I)=-; clinical form of leprosy=BT #COLLECTION CO:COLLECTION_SUMMARY The whole blood (10 mL) was collected in a Vacutainer tube from Becton Dickinson CO:COLLECTION_SUMMARY (BD). After collection of the whole blood, it was allowed the blood to clot by CO:COLLECTION_SUMMARY leaving it undisturbed at room temperature for 30 minutes. The clot was removed CO:COLLECTION_SUMMARY by centrifuging at 1,000–2,000 x g for 10 minutes in a refrigerated centrifuge CO:COLLECTION_SUMMARY (4 C). The resulting supernatant is designated serum. The serum samples were CO:COLLECTION_SUMMARY aliquoted in 100 microliters and stored in a serum bank at -80C. These samples CO:COLLECTION_SUMMARY were collected in the Leprosy Outpatient Unit (Oswaldo Cruz Foundation, Rio de CO:COLLECTION_SUMMARY Janeiro, Brazil) and then they were sent in dry ice to Colorado State CO:COLLECTION_SUMMARY University, where it was processed and analyzed for metabolomics. At Colorado CO:COLLECTION_SUMMARY State University the samples were stored at -80C until processing. CO:SAMPLE_TYPE Serum CO:COLLECTION_LOCATION Leprosy Outpatient Unit (Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) CO:COLLECTION_DURATION 30 minutes CO:STORAGE_CONDITIONS -80C CO:ADDITIVES no CO:BLOOD_SERUM_OR_PLASMA Serum #TREATMENT TR:TREATMENT_SUMMARY No treatment of the patients was performed #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum samples (7 serum samples from leprosy patients with T1R) were pooled prior SP:SAMPLEPREP_SUMMARY the extraction. These pooled serum samples were incubated with 3 volumes of cold SP:SAMPLEPREP_SUMMARY methanol (100%) for 1 hour at -20C. After this incubation, the samples were SP:SAMPLEPREP_SUMMARY centrifuged for 30 minutes at 18,000 xg at 4C to clear the supernatant. The SP:SAMPLEPREP_SUMMARY supernatant was then transferred and dried under vacuum. Samples were SP:SAMPLEPREP_SUMMARY re-suspended in 80 μl of 50% methanol. Prior to the injections on the LC-MS SP:SAMPLEPREP_SUMMARY instrument, the samples were centrifuged for 30 minutes at 18,000 xg at 4oC. SP:SAMPLEPREP_PROTOCOL_ID SP000100 SP:SAMPLEPREP_PROTOCOL_FILENAME 060613_Methanol_Serum_Extraction SP:SAMPLEPREP_PROTOCOL_COMMENTS Use always nitrile gloves. Do not use gloves with powder. Use pipette tips with SP:SAMPLEPREP_PROTOCOL_COMMENTS aerosol barrier. SP:PROCESSING_METHOD Homogenization and solvent removal with Speed Vac SP:PROCESSING_STORAGE_CONDITIONS on ice SP:EXTRACTION_METHOD Cold Methanol extraction (3:1 methanol/water) SP:EXTRACT_ENRICHMENT no enrichment was performed SP:EXTRACT_CLEANUP Centrifugation SP:EXTRACT_STORAGE -80C SP:SAMPLE_RESUSPENSION 50% methanol SP:SAMPLE_DERIVATIZATION no derivatization process was performed SP:SAMPLE_SPIKING Samples were spiked with the following commercial standards: arachidonic acid, SP:SAMPLE_SPIKING docosahexaenoic acid, eicosapentaenoic acid and leukotriene B4 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY This analysis was performed only on negative mode. The solvent gradient used for CH:CHROMATOGRAPHY_SUMMARY all chromatography was 2% solvent B for 3 min followed by a 15 min linear CH:CHROMATOGRAPHY_SUMMARY gradient of 2% to 98% solvent B, which was held for 0.3 min. Then returned to CH:CHROMATOGRAPHY_SUMMARY starting conditions over 1.2 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:INSTRUMENT_NAME quadrupole time-of-flight (Q-TOF) mass spectrometer. CH:COLUMN_NAME Waters Xbridge BEH C18 XP (100 x 2.1mm, 2.5um) CH:COLUMN_NAME Millford, MA, USA) CH:FLOW_GRADIENT 0.25 mL/min CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 50C CH:SOLVENT_A 89% water, 5% acetonitrile, 5% isopropanol and 1% 500 mM ammonium acetate CH:SOLVENT_B 49.5% acetonitrile, 49.5% isopropanol and 1% 500 mM ammonium acetate CH:INJECTION_TEMPERATURE 4C CH:SAMPLE_INJECTION 20uL CH:CAPILLARY_VOLTAGE 2.8 kV #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Mycobacteria Research Laboratories AN:OPERATOR_NAME Kristofor Webb and Carlos AM Silva AN:SOFTWARE_VERSION Agilent Mass Hunter Workstation Software version, Qualitative Analysis, Version AN:SOFTWARE_VERSION B.05.00 AN:DATA_FORMAT .d #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6510 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 2.8kV MS:COLLISION_ENERGY 20eV MS:COLLISION_GAS Nitrogen MS:DRY_GAS_FLOW 10 L/min MS:DRY_GAS_TEMP 310C MS:FRAGMENT_VOLTAGE 120V MS:NEBULIZER 45 psig MS:SCANNING_RANGE 2 spectra per second MS:SKIMMER_VOLTAGE 50V MS:MS_RESULTS_FILE ST000881_AN001437_Results.txt UNITS:arbitrary units #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS MS peak area MS_METABOLITE_DATA_START Samples S19 S19 S19 S18 S18 S18 S14 S14 S14 S12 S12 S12 S16 S16 S16 S11 S11 S11 S15 S15 S15 S17 S17 S17 S13 S13 S13 S10 S10 S10 S1 S1 S1 S3 S3 S3 S5 S5 S5 S7 S7 S7 S8 S8 S9 S9 S9 Factors Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):No Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes Type 1 reaction (T1R):Yes arachidonic acid 7299911.143 7865161.81 7936921.548 5562575.436 5537228.911 5559891.47 4680144.135 4835802.763 4816532.524 8357758.299 8862133.547 8343606.247 8418441.688 8342810.663 8312042.446 4136148.492 4025418.911 4260880.609 4233553.897 4346004.752 4203527.537 5292458.743 5121234.83 5188449.248 4479098.224 4528766.337 4419431.369 31708991.27 32862074.22 32171297.07 7718777.643 7429486.983 7396863.005 20866392.34 20688327.31 20764686.47 3481418.866 3320437.877 3410989.698 11618443.14 11451317.53 11438085.37 18047570.85 17777816.43 38371185 37472315.29 40211943.32 docosahexaenoic acid 2686477.402 2882809.909 2918531.495 2138632.78 2110494.349 2126677.293 1109918.974 1156759.048 1160184.902 1815184.813 1947984.61 1768356.609 2414648.318 2374502.446 2376769.736 1764796.054 1703935.757 1799836.869 2079716.644 2129107.69 2066720.114 1856020.532 1798867.908 1819937.346 1344961.234 1358905.61 1333346.88 10423706.86 11729078.18 11359754.35 3447777.931 3319237.391 3289928.37 6438512.579 6362206.05 6517555.083 1579765.474 1517989.64 1566183.678 3363245.455 3281245.194 3224927.67 6097781.515 5951942.001 10864616.55 11164997.78 11531546.11 eicosapentaenoic acid 648846.6632 727768.5616 744377.0623 923821.0842 895982.836 920976.3724 97437.54853 103557.6025 103139.337 870042.7675 946178.2206 832269.0022 897563.8611 897627.2845 893886.9039 1020601.157 950535.8871 1037429.957 337390.9904 327778.2676 330488.2954 381725.3189 371663.0527 371476.0546 354048.6915 359007.4749 339867.5464 12621337.18 13336485.57 12927592.16 1148844.315 1079039.972 1080763.992 2567602.737 2542341.054 2607795.945 809095.3878 756894.3451 792520.8231 1717837.072 1812159.074 1815303.276 2551800.149 2514850.163 5406129.443 5625927.864 5856024.135 leukotriene B4 94178.36922 103950.1958 NA NA 30424.8453 35897.19797 NA NA NA NA 72213.07777 64022.06455 NA 31280.61113 31468.15348 NA 23796.41231 NA NA NA 34383.10702 94209.2556 90014.89524 93400.19634 28021.7732 27601.6116 26461.06465 1393464.481 1521256.503 1481179.511 199025.8664 205680.5836 190631.8112 1553250.332 1551457.533 1525152.203 NA NA NA 1853208.387 NA 1914268.658 1861406.666 1765671.804 2281901.324 2499739.085 2580232.762 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID HMDB ID arachidonic acid 303.23 444899 C00219 HMDB01043 docosahexaenoic acid 327.23 445580 C06429 HMDB02183 eicosapentaenoic acid 301.22 446284 C06428 HMDB01999 leukotriene B4 335.22 5280492 C02165 HMDB01085 METABOLITES_END #END