#METABOLOMICS WORKBENCH cadriano_20170619_132232 DATATRACK_ID:1020 STUDY_ID:ST000882 ANALYSIS_ID:AN001438 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 negative 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                     	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               	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              	06/06/14
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                      	NEGATIVE
MS:CAPILLARY_VOLTAGE             	2.8kV
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_AN001438_Results.txt	UNITS:Peak area
#END