#METABOLOMICS WORKBENCH cbeekman_20240621_055933 DATATRACK_ID:4947 STUDY_ID:ST003288 ANALYSIS_ID:AN005385 PROJECT_ID:PR002040
VERSION             	1
CREATED_ON             	July 2, 2024, 8:17 am
#PROJECT
PR:PROJECT_TITLE                 	Quantification of amoxicillin in intestinal contents of mice
PR:PROJECT_SUMMARY               	Antibiotics cause collateral damage to resident microbes that is associated with
PR:PROJECT_SUMMARY               	various health risks. To-date, studies have largely focused on impacts of
PR:PROJECT_SUMMARY               	antibiotics on large intestinal and fecal microbiota. Here, we employ a GI-wide
PR:PROJECT_SUMMARY               	integrated multiomic approach to reveal that amoxicillin (AMX) treatment reduces
PR:PROJECT_SUMMARY               	overall bacterial abundance, bile salt hydrolase activity and unconjugated bile
PR:PROJECT_SUMMARY               	acids in the small intestine (SI). An accompanying loss of fatty acids and
PR:PROJECT_SUMMARY               	increase in acyl-carnitines in the large intestine corresponded with
PR:PROJECT_SUMMARY               	spatially-distinct expansions of proteobacteria. Parasuterella excrementihominis
PR:PROJECT_SUMMARY               	utilized fatty acid biosynthesis, becoming dominant in the SI while multiple
PR:PROJECT_SUMMARY               	Klebsiella species employed fatty acid oxidation during expansion in the large
PR:PROJECT_SUMMARY               	intestine. We subsequently demonstrate that restoration of unconjugated bile
PR:PROJECT_SUMMARY               	acids can mitigate losses of commensals in the large intestine while also
PR:PROJECT_SUMMARY               	inhibiting the expansion of Proteobacteria during AMX treatment.
PR:INSTITUTE                     	Brown University
PR:LAST_NAME                     	Beekman
PR:FIRST_NAME                    	Chapman
PR:ADDRESS                       	BMC 613, 171 Meeting Street, Providence RI 02912
PR:EMAIL                         	Chapman_Beekman@brown.edu
PR:PHONE                         	4018635953
#STUDY
ST:STUDY_TITLE                   	Quantification of amoxicillin in mouse intestinal contents
ST:STUDY_SUMMARY                 	Conventionally housed mice were orally treated with amoxicillin for 24 hours
ST:STUDY_SUMMARY                 	with and without cholic acid (48h) and small intestinal and cecal contents were
ST:STUDY_SUMMARY                 	collected for targeted MS analysis. Samples were flash frozen and stored at -80°C
ST:STUDY_SUMMARY                 	prior to analysis. We found that amoxicillin levels in the small intestine were
ST:STUDY_SUMMARY                 	0-40 pmol/mg of contents and no significant difference in amoxicillin
ST:STUDY_SUMMARY                 	concentration was detected between small intestine and cecum or between mice
ST:STUDY_SUMMARY                 	receiving cholic acid vs amoxicillin alone.
ST:INSTITUTE                     	Brown University
ST:DEPARTMENT                    	Molecular Microbiology & Immunology
ST:LABORATORY                    	Molecular Microbiology & Immunology, Belenky Lab
ST:LAST_NAME                     	Beekman
ST:FIRST_NAME                    	Chapman
ST:ADDRESS                       	BMC 613, 171 Meeting Street, Providence RI 02912
ST:EMAIL                         	Chapman_Beekman@brown.edu
ST:PHONE                         	4018635953
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	Cec_05	Treatment:untreated + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_11.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_06	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_14.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_07	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_15.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_08	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_16.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_13	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_17.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_14	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_18.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_15	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_19.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_16	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_20.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_21	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_21.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_22	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_22.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_23	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_23.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_24	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_24.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_29	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_25.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_30	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_26.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_31	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_27.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_32	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_28.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_33	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_29.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_34	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_30.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_35	Treatment:AMX + std. diet | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_31.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_37	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_32.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_38	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_33.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_39	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_34.raw
SUBJECT_SAMPLE_FACTORS           	-	Cec_40	Treatment:AMX + CA | Sample source:mouse cecum	RAW_FILE_NAME(File)=Cec_35.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_05	Treatment:untreated + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_13.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_06	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_14.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_07	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_15.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_08	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_16.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_09	Treatment:untreated + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_17.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_10	Treatment:untreated + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_18.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_13	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_19.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_14	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_20.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_15	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_21.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_16	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_22.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_21	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_23.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_22	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_24.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_23	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_25.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_24	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_26.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_29	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_27.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_30	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_28.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_31	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_29.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_32	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_30.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_33	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_31.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_34	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_32.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_35	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_33.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_36	Treatment:AMX + std. diet | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_34.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_37	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_35.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_38	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_36.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_39	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_37.raw
SUBJECT_SAMPLE_FACTORS           	-	SI_40	Treatment:AMX + CA | Sample source:mouse small intestine	RAW_FILE_NAME(File)=SI_38.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	SI and cecal contents were homogenized at a concentration of 50-100 mg/mL in 70%
CO:COLLECTION_SUMMARY            	methanol (LCMS grade) containing 50 µM 13C6-labeled AMX as an internal standard
CO:COLLECTION_SUMMARY            	on the Bead Ruptor (Omni Inc.) without beads at a frequency setting of 15 for 5
CO:COLLECTION_SUMMARY            	minutes. Homogenized samples were then centrifuged at 16,000 X g for 10 minutes
CO:COLLECTION_SUMMARY            	and supernatants were used for analysis. AMX was quantitated with HPLC-MS on an
CO:COLLECTION_SUMMARY            	Ultimate 3000, Dionex coupled to Q Executive Classic (Thermo) with ESI
CO:COLLECTION_SUMMARY            	interface.
CO:SAMPLE_TYPE                   	Intestine
#TREATMENT
TR:TREATMENT_SUMMARY             	AMX treatment was administered in drinking water (166.7 µg/mL) for a 24-hour
TR:TREATMENT_SUMMARY             	period. This concentration is based on previous studies38,39 and determined to
TR:TREATMENT_SUMMARY             	achieve a dosage of approximately 25mg/kg/day. Untreated mice received
TR:TREATMENT_SUMMARY             	pH-matched water and both AMX and vehicle solutions were sterile filtered prior
TR:TREATMENT_SUMMARY             	to administration. For cholic acid supplementation experiments standard mouse
TR:TREATMENT_SUMMARY             	chow was first blended into a course powder using a blender (Vitamix) and 5mg of
TR:TREATMENT_SUMMARY             	cholic acid was added per gram of powdered chow. Cholic acid was mixed evenly by
TR:TREATMENT_SUMMARY             	re-blending and provided in the powdered chow for a 48-hour period and 24 hours
TR:TREATMENT_SUMMARY             	prior to AMX treatment.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	SI and cecal contents were homogenized at a concentration of 50-100 mg/mL in 70%
SP:SAMPLEPREP_SUMMARY            	methanol (LCMS grade) containing 50 µM 13C6-labeled AMX as an internal standard
SP:SAMPLEPREP_SUMMARY            	on the Bead Ruptor (Omni Inc.) without beads at a frequency setting of 15 for 5
SP:SAMPLEPREP_SUMMARY            	minutes. Homogenized samples were then centrifuged at 16,000 X g for 10 minutes
SP:SAMPLEPREP_SUMMARY            	and supernatants were used for analysis. AMX was quantitated with HPLC-MS on an
SP:SAMPLEPREP_SUMMARY            	Ultimate 3000, Dionex coupled to Q Executive Classic (Thermo) with ESI
SP:SAMPLEPREP_SUMMARY            	interface.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% Water; 0.2% Formic acid
CH:SOLVENT_B                     	100% Methanol; 0.2% Formic acid
CH:FLOW_GRADIENT                 	mobile phase A/B set to 90%/10% from 0.00 to 0.50 min and 90%/10% to 5%/95% from
CH:FLOW_GRADIENT                 	0.5 to 1.6 min and held at A/B ratio of 5%/95% for 0.5 min from 1.6 to 2.1 min (wash) 
CH:FLOW_GRADIENT                 	and set back to the starting composition A/B 90%/10% from 2.1 to 4.5 (for equilibration)
CH:FLOW_RATE                     	0.35 mL/min
CH:COLUMN_TEMPERATURE            	30°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The mass spectrometer was operated in the positive ion mode in Full MS; at
MS:MS_COMMENTS                   	resolution 70,000; AGT target 5 E5 and Scan range 200-500 m/z. Spray voltage and
MS:MS_COMMENTS                   	source temperature were set at 3,500 volts, and 320°C, respectively.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	nmol / mg sample
MS_METABOLITE_DATA_START
Samples	Cec_05	Cec_06	Cec_07	Cec_08	Cec_13	Cec_14	Cec_15	Cec_16	Cec_21	Cec_22	Cec_23	Cec_24	Cec_29	Cec_30	Cec_31	Cec_32	Cec_33	Cec_34	Cec_35	Cec_37	Cec_38	Cec_39	Cec_40	SI_05	SI_06	SI_07	SI_08	SI_09	SI_10	SI_13	SI_14	SI_15	SI_16	SI_21	SI_22	SI_23	SI_24	SI_29	SI_30	SI_31	SI_32	SI_33	SI_34	SI_35	SI_36	SI_37	SI_38	SI_39	SI_40
Factors	Treatment:untreated + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + std. diet | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:AMX + CA | Sample source:mouse cecum	Treatment:untreated + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:untreated + std. diet | Sample source:mouse small intestine	Treatment:untreated + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + std. diet | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine	Treatment:AMX + CA | Sample source:mouse small intestine
Amoxicillin	0.000478191	0.010556799	0.006758489	0.013696958	0.002754542	0.011972901	0.011024439	0.005953429	0.009236975	0.011760107	0.023027511	0.016327816	0.00826398	0.002921206	0.008463409	0.013412824	0.018213263	0.023218755	0.010759273	0.020816723	0.00463665	0.013139592	0.00260343	0	0.002334794	3.36128E-05	0.00020701	0	0	0.001905616	0.000887662	0.010664744	0.006486571	0.018839681	0.025742551	0.003630705	0.00555748	0.000134208	0.002879549	0.003637932	0.004152593	0.010777048	0.04371411	0.040085569	0.029802898	0.011073324	0.036184023	0.008376806	0.003273649
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	m/z	retention time
Amoxicillin	372.13	0.905
METABOLITES_END
#END