#METABOLOMICS WORKBENCH michaelsa93_20170809_154130_mwtab.txt DATATRACK_ID:1198 STUDY_ID:ST000847 ANALYSIS_ID:AN001371 PROJECT_ID:PR000600
VERSION             	1
CREATED_ON             	August 10, 2017, 3:01 pm
#PROJECT
PR:PROJECT_TITLE                 	Statin Immuno-Metabolomics in Asthma
PR:PROJECT_TYPE                  	Placebo-controled trial
PR:PROJECT_SUMMARY               	Innovative and novel therapies are urgently needed for the treatment of patients
PR:PROJECT_SUMMARY               	with severe asthma, especially those who are refractory to standard-of-care
PR:PROJECT_SUMMARY               	bronchodilators and inhaled corticosteroids. The Zeki lab is investigating the
PR:PROJECT_SUMMARY               	role of the mevalonate (MA) pathway, in the pathogenesis of airway inflammation
PR:PROJECT_SUMMARY               	and remodeling. Although statins all inhibit HMGCR in the same manner in terms
PR:PROJECT_SUMMARY               	of enzyme binding site, the statins’ varied physiochemical properties with
PR:PROJECT_SUMMARY               	respect to their polarity (i.e. lipophilicity) result in very different immune
PR:PROJECT_SUMMARY               	and lipid effects. The major significance of this work is to advance a new class
PR:PROJECT_SUMMARY               	of inhaler therapies for asthma; the statins which work by an entirely different
PR:PROJECT_SUMMARY               	mechanism than current ICS/LABA mainstays. Evidence suggests that statins may
PR:PROJECT_SUMMARY               	have an additive benefit to corticosteroids in asthma, thereby confirming a
PR:PROJECT_SUMMARY               	unique mechanism, namely via MVA pathway inhibition. This becomes particularly
PR:PROJECT_SUMMARY               	important in the severe asthma population which is highly
PR:PROJECT_SUMMARY               	corticosteroid-resistant, is poorly controlled with high exacerbation rates and
PR:PROJECT_SUMMARY               	hospitalizations, and has the highest healthcare costs of all asthma phenotypes.
PR:PROJECT_SUMMARY               	In essence, the potential public health impact of even an incremental
PR:PROJECT_SUMMARY               	improvement in asthma symptom control cannot be underestimated. Even the
PR:PROJECT_SUMMARY               	prevention of 1 asthma attack preserves lung function and reduces the adverse
PR:PROJECT_SUMMARY               	personal and financial impact. This study aimed to determine if statin polarity
PR:PROJECT_SUMMARY               	affects airway drug concentration and systemic drug absorption and to determine
PR:PROJECT_SUMMARY               	the effect of inhaled statins on naïve airway immune cell populations and
PR:PROJECT_SUMMARY               	alveolar-capillary membrane and epithelial barrier integrity in healthy rhesus
PR:PROJECT_SUMMARY               	monkeys. In this particular component of the study, we investigated the
PR:PROJECT_SUMMARY               	metabolic effects resulting from the use of statins in these healthy rhesus
PR:PROJECT_SUMMARY               	monkeys. Specifically, the Newman lab analyzed for lipid mediator (oxylipin,
PR:PROJECT_SUMMARY               	endocannabinoid, fatty acid, and nitro lipid) in lung and trachea tissue,
PR:PROJECT_SUMMARY               	plasma, and BAL and bile acid changes in the lung and trachea tissue and plasma.
PR:INSTITUTE                     	University of California, Davis
PR:DEPARTMENT                    	Internal Medicine
PR:LAST_NAME                     	Zeki
PR:FIRST_NAME                    	Amir
PR:ADDRESS                       	2825 J St. Suite 400 Sacramento, CA 95816
PR:EMAIL                         	aazeki@ucdavis.edu
PR:PHONE                         	(916) 734-8230
#STUDY
ST:STUDY_TITLE                   	Statin Immuno-Metabolomics in Asthma (part V)
ST:STUDY_TYPE                    	Placebo-controled trial
ST:STUDY_SUMMARY                 	Innovative and novel therapies are urgently needed for the treatment of patients
ST:STUDY_SUMMARY                 	with severe asthma, especially those who are refractory to standard-of-care
ST:STUDY_SUMMARY                 	bronchodilators and inhaled corticosteroids. The Zeki lab is investigating the
ST:STUDY_SUMMARY                 	role of the mevalonate (MA) pathway, in the pathogenesis of airway inflammation
ST:STUDY_SUMMARY                 	and remodeling. Although statins all inhibit HMGCR in the same manner in terms
ST:STUDY_SUMMARY                 	of enzyme binding site, the statins’ varied physiochemical properties with
ST:STUDY_SUMMARY                 	respect to their polarity (i.e. lipophilicity) result in very different immune
ST:STUDY_SUMMARY                 	and lipid effects. The major significance of this work is to advance a new class
ST:STUDY_SUMMARY                 	of inhaler therapies for asthma; the statins which work by an entirely different
ST:STUDY_SUMMARY                 	mechanism than current ICS/LABA mainstays. Evidence suggests that statins may
ST:STUDY_SUMMARY                 	have an additive benefit to corticosteroids in asthma, thereby confirming a
ST:STUDY_SUMMARY                 	unique mechanism, namely via MVA pathway inhibition. This becomes particularly
ST:STUDY_SUMMARY                 	important in the severe asthma population which is highly
ST:STUDY_SUMMARY                 	corticosteroid-resistant, is poorly controlled with high exacerbation rates and
ST:STUDY_SUMMARY                 	hospitalizations, and has the highest healthcare costs of all asthma phenotypes.
ST:STUDY_SUMMARY                 	In essence, the potential public health impact of even an incremental
ST:STUDY_SUMMARY                 	improvement in asthma symptom control cannot be underestimated. Even the
ST:STUDY_SUMMARY                 	prevention of 1 asthma attack preserves lung function and reduces the adverse
ST:STUDY_SUMMARY                 	personal and financial impact. This study aimed to determine if statin polarity
ST:STUDY_SUMMARY                 	affects airway drug concentration and systemic drug absorption and to determine
ST:STUDY_SUMMARY                 	the effect of inhaled statins on naïve airway immune cell populations and
ST:STUDY_SUMMARY                 	alveolar-capillary membrane and epithelial barrier integrity in healthy rhesus
ST:STUDY_SUMMARY                 	monkeys. In this particular component of the study, we investigated the
ST:STUDY_SUMMARY                 	metabolic effects resulting from the use of statins in these healthy rhesus
ST:STUDY_SUMMARY                 	monkeys. Specifically, the Newman lab analyzed for lipid mediator (oxylipin,
ST:STUDY_SUMMARY                 	endocannabinoid, fatty acid, and nitro lipid) in lung and trachea tissue,
ST:STUDY_SUMMARY                 	plasma, and BAL and bile acid changes in the lung and trachea tissue and plasma.
ST:INSTITUTE                     	USDA
ST:DEPARTMENT                    	Obesity and metabolism research unit
ST:LAST_NAME                     	Newman
ST:FIRST_NAME                    	John
ST:ADDRESS                       	430 West Health Sciences Dr. Davis, Ca, 95616
ST:EMAIL                         	John.Newman@ars.usda.gov
ST:PHONE                         	(530) 752-1009
#SUBJECT
SU:SUBJECT_TYPE                  	Animal
SU:SUBJECT_SPECIES               	Macaca mulatta
SU:TAXONOMY_ID                   	9544
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	Zeki R 74	Tissue:Right Lung | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki R 28 Repl Avg.	Tissue:Right Lung | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki R 48	Tissue:Right Lung | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki R 68	Tissue:Right Lung | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki R 40	Tissue:Right Lung | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki R 95	Tissue:Right Lung | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki L 74	Tissue:Left Lung | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki L 48	Tissue:Left Lung | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki L 28 Repl Avg.	Tissue:Left Lung | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki L 95	Tissue:Left Lung | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki L 40	Tissue:Left Lung | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki L 68	Tissue:Left Lung | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki T 74	Tissue:Trachea | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki T 48	Tissue:Trachea | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki T 28 Repl Avg.	Tissue:Trachea | Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	Zeki T 40	Tissue:Trachea | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki T 68	Tissue:Trachea | Treatment:Simvastatin	
SUBJECT_SAMPLE_FACTORS           	-	Zeki T 95	Tissue:Trachea | Treatment:Simvastatin	
#COLLECTION
CO:COLLECTION_SUMMARY            	Monkeys were treated with placebo or Provastatin for 12 days. Further, after the
CO:COLLECTION_SUMMARY            	wash out period animals were treated with Simvastatin for 12 days. Plasma was
CO:COLLECTION_SUMMARY            	collected at day 0, 8 and 12 of each treatment.
CO:SAMPLE_TYPE                   	Tissue
CO:TISSUE_CELL_IDENTIFICATION    	Trachea and lung tissue
#TREATMENT
TR:TREATMENT_SUMMARY             	Monkeys were treated (by inhalation) with placebo or Provastatin for 12 days.
TR:TREATMENT_SUMMARY             	Further, after the wash out period animals were treated with Simvastatin for 12
TR:TREATMENT_SUMMARY             	days. Plasma was collected at day 0, 8 and 12 of each treatment.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Oxylipins, endocannabinoids, bile acids and fatty acids were isolated using a
SP:SAMPLEPREP_SUMMARY            	Waters Ostro Sample Preparation Plate (Milford, MA). Lung and trachea samples
SP:SAMPLEPREP_SUMMARY            	were aliquoted (~40-60mg) into 2mL polypropylene tubes and spiked with a 5 µL
SP:SAMPLEPREP_SUMMARY            	anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL
SP:SAMPLEPREP_SUMMARY            	1000nM analytical deuterated surrogates. A total of 50 µL of methanol, 550µL
SP:SAMPLEPREP_SUMMARY            	isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water were
SP:SAMPLEPREP_SUMMARY            	added and the tube was placed in a Geno/Grinder for 30 sec before being
SP:SAMPLEPREP_SUMMARY            	centrifuged at 10,000g for 5 min at room temp. The supernate was then
SP:SAMPLEPREP_SUMMARY            	transferred into the plate wells and samples were eluted into glass inserts
SP:SAMPLEPREP_SUMMARY            	containing 10 μL 20% glycerol by applying a vacuum at 15 Hg for 10 min. Eluent
SP:SAMPLEPREP_SUMMARY            	was dried by speed vacuum for 35 min at the medium BP setting, before switching
SP:SAMPLEPREP_SUMMARY            	to an aqueous setting for an additional 35 min. Once dry, samples were
SP:SAMPLEPREP_SUMMARY            	re-constituted with the internal standard 1-cyclohexyl ureido, 3-dodecanoic acid
SP:SAMPLEPREP_SUMMARY            	(CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN),
SP:SAMPLEPREP_SUMMARY            	vortexed 1 min, transferred to a spin filter (0.1 µm, Millipore, Billerica,
SP:SAMPLEPREP_SUMMARY            	MA), centrifuged for 3 min at 6ºC at <4500g (rcf), before being transferred to
SP:SAMPLEPREP_SUMMARY            	2 mL LC-MS amber vials. Extracts were stored at -20ºC until analysis by
SP:SAMPLEPREP_SUMMARY            	UPLC-MS/MS. The internal standard was used to quantify the recovery of surrogate
SP:SAMPLEPREP_SUMMARY            	standards.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Aquity C18 BEH 1.7µm 100mm x 2.1mm column
CH:FLOW_GRADIENT                 	See protocol/methods file
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	60 °C
CH:SOLVENT_A                     	0.1% Formic Acid
CH:SOLVENT_B                     	0.1% Formic Acid in Acetonitrile
CH:INTERNAL_STANDARD             	See protocol/methods file
CH:RETENTION_TIME                	See protocol/methods file
CH:SAMPLE_INJECTION              	5 µL
CH:ANALYTICAL_TIME               	16 min
CH:WEAK_WASH_SOLVENT_NAME        	20% methanol, 10% isopropanol
CH:WEAK_WASH_VOLUME              	600 µL
CH:STRONG_WASH_SOLVENT_NAME      	50:50 Acetonitrile:Methanol
CH:STRONG_WASH_VOLUME            	600 µL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Newman
AN:OPERATOR_NAME                 	Kamil Borkowski
AN:DETECTOR_TYPE                 	API 6500 QTrap (AB Sciex, Framingham, MA, USA)
AN:SOFTWARE_VERSION              	AB Sciex MultiQuant version 3.0.2
AN:DATA_FORMAT                   	.mzML
#MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	ABI Sciex 6500 QTrap
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	Concentration (nM)
MS_METABOLITE_DATA_START
Samples	Zeki R 74	Zeki R 28 Repl Avg.	Zeki R 48	Zeki R 68	Zeki R 40	Zeki R 95	Zeki L 74	Zeki L 48	Zeki L 28 Repl Avg.	Zeki L 95	Zeki L 40	Zeki L 68	Zeki T 74	Zeki T 48	Zeki T 28 Repl Avg.	Zeki T 40	Zeki T 68	Zeki T 95
Factors	Tissue:Right Lung | Treatment:Control	Tissue:Right Lung | Treatment:Control	Tissue:Right Lung | Treatment:Control	Tissue:Right Lung | Treatment:Simvastatin	Tissue:Right Lung | Treatment:Simvastatin	Tissue:Right Lung | Treatment:Simvastatin	Tissue:Left Lung | Treatment:Control	Tissue:Left Lung | Treatment:Control	Tissue:Left Lung | Treatment:Control	Tissue:Left Lung | Treatment:Simvastatin	Tissue:Left Lung | Treatment:Simvastatin	Tissue:Left Lung | Treatment:Simvastatin	Tissue:Trachea | Treatment:Control	Tissue:Trachea | Treatment:Control	Tissue:Trachea | Treatment:Control	Tissue:Trachea | Treatment:Simvastatin	Tissue:Trachea | Treatment:Simvastatin	Tissue:Trachea | Treatment:Simvastatin
CA	11.3	10.55	12.5	35.6	10.8	468	423	6.58	34.1	16	31.5	27.3	46.9	21.1	35.3	86	14.8	455
CDCA	3.42	8.87	12.3	118	4.9	494	441	9.16	64.5	21.5	77.7	19.8	398	144	302	69.7	8.3	453
TCA	58.9	19.14	96	17	49.9	13.5	11.6	39.6	16.3	5.5	33.1	14.1	171	249	5.47	272	23.2	12.6
TCDCA	66	69.34	64.8	60.2	58.5	52.4	49.9	80.7	63.75	22.7	99.5	62.8	279	1160	53.2	409	139	45.4
GCA	213	92.6	200	106	200	89.9	80.1	163	174	26.3	257	202	618	399	92.6	2110	94.3	80.4
GCDCA	568	456	282	625	559	465	371	589	1076	305	1010	1190	1220	2720	1300	4310	890	410
DCA	15.3		41.7	365	24	683	642	79.5	288	77.3	294	126	588	72.5	< 0	560	55.7	687
LCA	4.83	2.86	8.05	10.3	2.85	4.04	9.21	0.78	3.18	1.8	4.75	7.26	5.69	5.53	4.8	5.54	3.31	7.59
TDCA	35.9	21.28	34.7	30.7	30.8	44.6	33.7	84.8	39.1	11.2	49.5	40	148	138	< 0	136	51.1	37.7
TLCA	1.93	3.67	1.13	2.91	1.69	1.61	1.4	4.14	3.21	1.4	3.67	2.85	8.05	6.22	0.66	4.36	3.26	1.51
GDCA	190	100	108	273	191	280	223	433	543	117	395	592	580	271	0.7	1510	227	228
GLCA	9.29	69.1		41.7	7.07	11.6	8.41	59.2	69.75	2.24	127	64.7	124	24.1	< 0	170	32.4	10.4
GHDCA	206	170	117	224	203	174	145	213	360	124	342	394	406	838	430	1260	305	157
UDCA	1.95	3.24	5.86	29.1	2.96	61.3	49.6	4.34	23.35	12.3	63.9	10.2	50	69	23.8	60	1.3	56.6
TUDCA	1.87	1.35	3.23	2.25	2.7	3.05	2.57	1.79	3.47	1.94	9.16	4.16	11.4	81.6	2.09	20.2	3.67	3.32
GUDCA	15.5	6.83	17.2	22.8	11.2	34.2	35.7	10.2	70.45	30.8	150	97.6	80.7	296	54.9	392	34.5	30.4
α-MCA																		
β-MCA																		
HCA screen																		
T-ω-MCA																		
T-α-MCA																		
T-β-MCA																		
THCA screen																		
GHCA screen																		
ω-MCA																		
THDCA screen																		
DHCA screen																		
TDHCA																		
GDHCA screen																		
MCA screen																		
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
CA
CDCA
TCA
TCDCA
GCA
GCDCA
DCA
LCA
TDCA
TLCA
GDCA
GLCA
GHDCA
UDCA
TUDCA
GUDCA
α-MCA
β-MCA
HCA screen
T-ω-MCA
T-α-MCA
T-β-MCA
THCA screen
GHCA screen
ω-MCA
THDCA screen
DHCA screen
TDHCA
GDHCA screen
MCA screen
METABOLITES_END
#END