#METABOLOMICS WORKBENCH cadriano_20170619_132232 DATATRACK_ID:1020 STUDY_ID:ST000882 ANALYSIS_ID:AN001439 PROJECT_ID:PR000611 VERSION 1 CREATED_ON October 29, 2017, 9:20 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 9702154962 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 Untargeted metabolomics data of leprosy patients with type 1 reaction ST:STUDY_TYPE Untargeted MS qualitative analysis in retrospective serum samples of leprosy ST:STUDY_TYPE patients with type 1 reaction ST:STUDY_SUMMARY Untargeted metabolomics-based analyses of sera from 7 patients with and 9 ST:STUDY_SUMMARY without T1R were conducted via liquid chromatography–mass spectrometry. 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_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:STORAGE_CONDITIONS -80C CO:BLOOD_SERUM_OR_PLASMA serum #TREATMENT TR:TREATMENT_SUMMARY No treatment of the patients was performed #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The retrospective serum samples (80 microliters[ul]) were incubated with 3 SP:SAMPLEPREP_SUMMARY volumes of cold methanol (100%) for 1 hour at -20C. After this incubation, the SP:SAMPLEPREP_SUMMARY samples were centrifuged for 30 minutes at 18,000 xg at 4C to clear the SP:SAMPLEPREP_SUMMARY supernatant. The supernatant was then transferred and dried under vacuum. SP:SAMPLEPREP_SUMMARY Samples were re-suspended in 80 μl of 50% methanol. Prior to the injections on SP:SAMPLEPREP_SUMMARY the LC-MS instrument, the samples were centrifuged for 30 minutes at 18,000 xg SP:SAMPLEPREP_SUMMARY 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_STORAGE_CONDITIONS -80C SP:EXTRACTION_METHOD Methanol extraction SP:EXTRACT_CLEANUP Centrifugation SP:EXTRACT_STORAGE -80C SP:SAMPLE_RESUSPENSION 50% methanol SP:SAMPLE_DERIVATIZATION no derivatization process was performed #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY This was used for positive mode. The solvent gradient used for all CH:CHROMATOGRAPHY_SUMMARY chromatography was 2% solvent B for 3 min followed by a 15 min linear gradient CH:CHROMATOGRAPHY_SUMMARY of 2% to 98% solvent B, which was held for 0.3 min. Then returned to starting CH:CHROMATOGRAPHY_SUMMARY conditions over 1.2 min. Triplicate LC-MS injections were made of each sample CH:CHROMATOGRAPHY_SUMMARY and the order of sample injection (including triplicates) was randomized. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:INSTRUMENT_NAME Accurate-Mass Time-of-Flight (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 5% acetonitrile with 0.1% formic acid CH:SOLVENT_B 95% acetonitrile with 0.1% formic acid CH:INJECTION_TEMPERATURE 4C CH:SAMPLE_INJECTION 20uL CH:CAPILLARY_VOLTAGE 4.0kV #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Belisle 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:ACQUISITION_DATE 06/022/14 AN:DATA_FORMAT .d #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6224 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_VOLTAGE 4.0kV MS:DRY_GAS_FLOW 5 L/min MS:DRY_GAS_TEMP 310C MS:FRAGMENT_VOLTAGE 120V MS:NEBULIZER 20 psig MS:SCANNING_RANGE 2 spectra per second MS:SKIMMER_VOLTAGE 50V MS:MS_RESULTS_FILE ST000882_AN001439_Results.txt UNITS:Peak area #END