#METABOLOMICS WORKBENCH jravilap_20250516_124353 DATATRACK_ID:5919 STUDY_ID:ST003931 ANALYSIS_ID:AN006456 PROJECT_ID:PR002462
VERSION             	1
CREATED_ON             	May 27, 2025, 9:52 pm
#PROJECT
PR:PROJECT_TITLE                 	Acylated putrescine therapeutic discovery for Inflammatory Bowel Diseases
PR:PROJECT_SUMMARY               	Gut microbial metabolites influence immune responses and play a key role in
PR:PROJECT_SUMMARY               	regulating host metabolism. Inflammatory bowel diseases (IBD) patients have
PR:PROJECT_SUMMARY               	altered gut microbial metabolic signatures driven by shifts in the microbiome.
PR:PROJECT_SUMMARY               	However, the microbial metabolites driving inflammation and remission and their
PR:PROJECT_SUMMARY               	immunomodulatory roles remain largely uncharacterized. We prioritized metabolic
PR:PROJECT_SUMMARY               	features from untargeted IBD fecal metabolomics and selected candidate features
PR:PROJECT_SUMMARY               	based on their microbial association for structural elucidation. We uncovered
PR:PROJECT_SUMMARY               	four acylated putrescines that are IBD-associated and microbially-dependent and
PR:PROJECT_SUMMARY               	investigated their bioactivity. One acylated putrescine, N-oleoylputrescine
PR:PROJECT_SUMMARY               	(NOP), has anti-inflammatory activity and suppressed type 1 immune responses.
PR:PROJECT_SUMMARY               	NOP ameliorated inflammation in several colitis models, modulating myeloid and
PR:PROJECT_SUMMARY               	lymphocyte cell populations and dampening proinflammatory responses, relevant
PR:PROJECT_SUMMARY               	for resolution of inflammation and maintenance of remission in IBD. We present
PR:PROJECT_SUMMARY               	an approach to identify clinically-relevant microbial metabolites from
PR:PROJECT_SUMMARY               	untargeted metabolomics useful for the discovery of bioactive molecules in IBD
PR:PROJECT_SUMMARY               	and other diseases involving host-microbial interactions.
PR:INSTITUTE                     	Broad Institute of MIT and Harvard
PR:LAST_NAME                     	Avila-Pacheco
PR:FIRST_NAME                    	Julian
PR:ADDRESS                       	415 Main Street, Cambridge, MA 02142 USA
PR:EMAIL                         	jravilap@broadinstitute.org
PR:PHONE                         	+1 (617) 714-1729
PR:CONTRIBUTORS                  	Sena Bae, Julian Avila-Pacheco, Slater L. Clay, Sunghee Bang, Monia Michaud,
PR:CONTRIBUTORS                  	Diogo Fonseca-Pereira, Eunyoung Chun, Y. Grace Cao, Yancong Zhang,
PR:CONTRIBUTORS                  	Amrisha Bhosle, Rosa M. Perez, Gleb Pishchany, Hera Vlamakis, Natalia
PR:CONTRIBUTORS                  	Andreeva, Geniver El Tekle, Xochitl C. Morgan, Jonathan N. Glickman, Ramnik
PR:CONTRIBUTORS                  	J. Xavier, Daniel B. Graham, Clary B. Clish, Jon Clardy, Eric A. Franzosa,
PR:CONTRIBUTORS                  	Curtis Huttenhower, and Wendy S. Garrett
#STUDY
ST:STUDY_TITLE                   	Acylated putrescine therapeutic discovery for Inflammatory Bowel Diseases:
ST:STUDY_TITLE                   	HILIC-neg, C8-pos and C18-neg profiling of mouse fecal samples
ST:STUDY_TYPE                    	Study 2: HILIC-neg, C8-pos and C18-neg profiling of mouse fecal samples.
ST:STUDY_SUMMARY                 	The data generated in this study using the HILIC-neg, C8-pos, and C18-neg
ST:STUDY_SUMMARY                 	profiling method were used to prioritize both disease-associated and
ST:STUDY_SUMMARY                 	microbe-associated metabolic features with potential bioactivity from untargeted
ST:STUDY_SUMMARY                 	metabolomics data. This mouse fecal microbiome data was used to develop a filter
ST:STUDY_SUMMARY                 	and prioritization tool: Metabolome Analysis and Combined Annotation Ranks to
ST:STUDY_SUMMARY                 	pRioritize Novel bioactives, that we call micro-MACARRoN. This workflow
ST:STUDY_SUMMARY                 	annotates the microbial association of gut metabolic features by comparing fecal
ST:STUDY_SUMMARY                 	metabolomics data among germ-free (GF), Altered Schaedler flora community (ASF),
ST:STUDY_SUMMARY                 	and specific pathogen free (SPF) mice. Application of this analysis pipeline to
ST:STUDY_SUMMARY                 	a publicly available dataset of 546 IBD fecal metabolomes from the Integrative
ST:STUDY_SUMMARY                 	Human Microbiome Project (HMP2/iHMP) cohort study enabled us to narrow our focus
ST:STUDY_SUMMARY                 	to metabolic features that are gut microbiome dependent. Using this tool we
ST:STUDY_SUMMARY                 	determined ~79% of gnotobiotic mouse-curated, IBD-associated HMP2 features are
ST:STUDY_SUMMARY                 	microbially associated (n=2,289 out of 2,883) indicating a substantial
ST:STUDY_SUMMARY                 	contribution of microbes to the gut metabolomes in IBD as would be expected.
ST:INSTITUTE                     	Broad Institute of MIT and Harvard
ST:LAST_NAME                     	Avila-Pacheco
ST:FIRST_NAME                    	Julian
ST:ADDRESS                       	415 Main Street
ST:EMAIL                         	jravilap@broadinstitute.org
ST:PHONE                         	(617) 714-1729
ST:NUM_GROUPS                    	3
ST:TOTAL_SUBJECTS                	21
ST:NUM_MALES                     	10
ST:NUM_FEMALES                   	11
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:GENOTYPE_STRAIN               	C57BL/6 wild-type
SU:AGE_OR_AGE_RANGE              	6-9 weeks
SU:GENDER                        	Male and female
SU:ANIMAL_ANIMAL_SUPPLIER        	Jackson Laboratories
#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           	-	M1	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Male	Cage info=1; RAW_FILE_NAME(C18-NEG)=B0170_GAR_HMCs_2nd_CN-HMCs-2nd-61; RAW_FILE_NAME(C8-POS)=B0170_GAR_HMC_CP-HMCs-2nd-61; RAW_FILE_NAME(HILIC-NEG)=B0170_GAR_GMC_HN-HMCs-2nd-61
SUBJECT_SAMPLE_FACTORS           	-	M2	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Male	Cage info=1; RAW_FILE_NAME(C18-NEG)=B0190_GAR_HMCs_2nd_CN-HMCs-2nd-62; RAW_FILE_NAME(C8-POS)=B0190_GAR_HMC_CP-HMCs-2nd-62; RAW_FILE_NAME(HILIC-NEG)=B0190_GAR_GMC_HN-HMCs-2nd-62
SUBJECT_SAMPLE_FACTORS           	-	M3	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Male	Cage info=1; RAW_FILE_NAME(C18-NEG)=B0202_GAR_HMCs_2nd_CN-HMCs-2nd-63; RAW_FILE_NAME(C8-POS)=B0202_GAR_HMC_CP-HMCs-2nd-63; RAW_FILE_NAME(HILIC-NEG)=B0202_GAR_GMC_HN-HMCs-2nd-63
SUBJECT_SAMPLE_FACTORS           	-	M4	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Cage info=2; RAW_FILE_NAME(C18-NEG)=B0232_GAR_HMCs_2nd_CN-HMCs-2nd-64; RAW_FILE_NAME(C8-POS)=B0232_GAR_HMC_CP-HMCs-2nd-64; RAW_FILE_NAME(HILIC-NEG)=B0232_GAR_GMC_HN-HMCs-2nd-64
SUBJECT_SAMPLE_FACTORS           	-	M5	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Cage info=2; RAW_FILE_NAME(C18-NEG)=B0212_GAR_HMCs_2nd_CN-HMCs-2nd-65; RAW_FILE_NAME(C8-POS)=B0212_GAR_HMC_CP-HMCs-2nd-65; RAW_FILE_NAME(HILIC-NEG)=B0212_GAR_GMC_HN-HMCs-2nd-65
SUBJECT_SAMPLE_FACTORS           	-	M6	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Cage info=2; RAW_FILE_NAME(C18-NEG)=B0238_GAR_HMCs_2nd_CN-HMCs-2nd-66; RAW_FILE_NAME(C8-POS)=B0238_GAR_HMC_CP-HMCs-2nd-66; RAW_FILE_NAME(HILIC-NEG)=B0238_ri_GAR_GMC_HN-HMCs-2nd-66
SUBJECT_SAMPLE_FACTORS           	-	M7	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Cage info=2; RAW_FILE_NAME(C18-NEG)=B0214_GAR_HMCs_2nd_CN-HMCs-2nd-67; RAW_FILE_NAME(C8-POS)=B0214_GAR_HMC_CP-HMCs-2nd-67; RAW_FILE_NAME(HILIC-NEG)=B0214_GAR_GMC_HN-HMCs-2nd-67
SUBJECT_SAMPLE_FACTORS           	-	M8	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Cage info=3; RAW_FILE_NAME(C18-NEG)=B0162_GAR_HMCs_2nd_CN-HMCs-2nd-68; RAW_FILE_NAME(C8-POS)=B0162_GAR_HMC_CP-HMCs-2nd-68; RAW_FILE_NAME(HILIC-NEG)=B0162_GAR_GMC_HN-HMCs-2nd-68
SUBJECT_SAMPLE_FACTORS           	-	M9	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Cage info=3; RAW_FILE_NAME(C18-NEG)=B0173_GAR_HMCs_2nd_CN-HMCs-2nd-69; RAW_FILE_NAME(C8-POS)=B0173_GAR_HMC_CP-HMCs-2nd-69; RAW_FILE_NAME(HILIC-NEG)=B0173_GAR_GMC_HN-HMCs-2nd-69
SUBJECT_SAMPLE_FACTORS           	-	M10	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Cage info=3; RAW_FILE_NAME(C18-NEG)=B0210_GAR_HMCs_2nd_CN-HMCs-2nd-70; RAW_FILE_NAME(C8-POS)=B0210_GAR_HMC_CP-HMCs-2nd-70; RAW_FILE_NAME(HILIC-NEG)=B0210_GAR_GMC_HN-HMCs-2nd-70
SUBJECT_SAMPLE_FACTORS           	-	M11	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Cage info=3; RAW_FILE_NAME(C18-NEG)=B0207_GAR_HMCs_2nd_CN-HMCs-2nd-71; RAW_FILE_NAME(C8-POS)=B0207_GAR_HMC_CP-HMCs-2nd-71; RAW_FILE_NAME(HILIC-NEG)=B0207_GAR_GMC_HN-HMCs-2nd-71
SUBJECT_SAMPLE_FACTORS           	-	M12	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Cage info=4; RAW_FILE_NAME(C18-NEG)=B0204_GAR_HMCs_2nd_CN-HMCs-2nd-72; RAW_FILE_NAME(C8-POS)=B0204_GAR_HMC_CP-HMCs-2nd-72; RAW_FILE_NAME(HILIC-NEG)=B0204_GAR_GMC_HN-HMCs-2nd-72
SUBJECT_SAMPLE_FACTORS           	-	M13	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Cage info=4; RAW_FILE_NAME(C18-NEG)=B0220_GAR_HMCs_2nd_CN-HMCs-2nd-73; RAW_FILE_NAME(C8-POS)=B0220_GAR_HMC_CP-HMCs-2nd-73; RAW_FILE_NAME(HILIC-NEG)=B0220_ri_GAR_GMC_HN-HMCs-2nd-73
SUBJECT_SAMPLE_FACTORS           	-	M14	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Cage info=4; RAW_FILE_NAME(C18-NEG)=B0183_GAR_HMCs_2nd_CN-HMCs-2nd-74; RAW_FILE_NAME(C8-POS)=B0183_GAR_HMC_CP-HMCs-2nd-74; RAW_FILE_NAME(HILIC-NEG)=B0183_GAR_GMC_HN-HMCs-2nd-74
SUBJECT_SAMPLE_FACTORS           	-	M15	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Cage info=4; RAW_FILE_NAME(C18-NEG)=B0208_GAR_HMCs_2nd_CN-HMCs-2nd-75; RAW_FILE_NAME(C8-POS)=B0208_GAR_HMC_CP-HMCs-2nd-75; RAW_FILE_NAME(HILIC-NEG)=B0208_GAR_GMC_HN-HMCs-2nd-75
SUBJECT_SAMPLE_FACTORS           	-	M16	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Male	Cage info=5; RAW_FILE_NAME(C18-NEG)=B0153_GAR_HMCs_2nd_CN-HMCs-2nd-76; RAW_FILE_NAME(C8-POS)=B0153_GAR_HMC_CP-HMCs-2nd-76; RAW_FILE_NAME(HILIC-NEG)=B0153_GAR_GMC_HN-HMCs-2nd-76
SUBJECT_SAMPLE_FACTORS           	-	M17	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Male	Cage info=5; RAW_FILE_NAME(C18-NEG)=B0230_GAR_HMCs_2nd_CN-HMCs-2nd-77; RAW_FILE_NAME(C8-POS)=B0230_GAR_HMC_CP-HMCs-2nd-77; RAW_FILE_NAME(HILIC-NEG)=B0230_GAR_GMC_HN-HMCs-2nd-77
SUBJECT_SAMPLE_FACTORS           	-	M18	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Male	Cage info=5; RAW_FILE_NAME(C18-NEG)=B0219_GAR_HMCs_2nd_CN-HMCs-2nd-78; RAW_FILE_NAME(C8-POS)=B0219_GAR_HMC_CP-HMCs-2nd-78; RAW_FILE_NAME(HILIC-NEG)=B0219_ri_GAR_GMC_HN-HMCs-2nd-78
SUBJECT_SAMPLE_FACTORS           	-	M19	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Female	Cage info=6; RAW_FILE_NAME(C18-NEG)=B0221_GAR_HMCs_2nd_CN-HMCs-2nd-79; RAW_FILE_NAME(C8-POS)=B0221_GAR_HMC_CP-HMCs-2nd-79; RAW_FILE_NAME(HILIC-NEG)=B0221_ri_GAR_GMC_HN-HMCs-2nd-79
SUBJECT_SAMPLE_FACTORS           	-	M20	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Female	Cage info=6; RAW_FILE_NAME(C18-NEG)=B0157_GAR_HMCs_2nd_CN-HMCs-2nd-80; RAW_FILE_NAME(C8-POS)=B0157_GAR_HMC_CP-HMCs-2nd-80; RAW_FILE_NAME(HILIC-NEG)=B0157_GAR_GMC_HN-HMCs-2nd-80
SUBJECT_SAMPLE_FACTORS           	-	M21	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Female	Cage info=6; RAW_FILE_NAME(C18-NEG)=B0196_GAR_HMCs_2nd_CN-HMCs-2nd-81; RAW_FILE_NAME(C8-POS)=B0196_GAR_HMC_CP-HMCs-2nd-81; RAW_FILE_NAME(HILIC-NEG)=B0196_GAR_GMC_HN-HMCs-2nd-81
#COLLECTION
CO:COLLECTION_SUMMARY            	Stool pellets were collected from germ-free (GF), Altered Schaedler Flora (ASF)
CO:COLLECTION_SUMMARY            	and specified-pathogen free (SPF) C57BL/6 mice (6-9wks), snap-frozen
CO:COLLECTION_SUMMARY            	immediately, and stored at -80°C until untargeted metabolomics sample
CO:COLLECTION_SUMMARY            	preparation
CO:SAMPLE_TYPE                   	Feces
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatments were applied in this section of the study
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Snap-frozen stool samples were thawed on ice, and then aqueous homogenates were
SP:SAMPLEPREP_SUMMARY            	generated by homogenizing samples in 10 volumes of water (mg: µL) using a
SP:SAMPLEPREP_SUMMARY            	TissueLyser II (QIAGEN) bead mill set to two 2 min intervals at 20Hz. The
SP:SAMPLEPREP_SUMMARY            	homogenate for each sample was divided into two 10 µL and two 30 µL aliquots
SP:SAMPLEPREP_SUMMARY            	in 1.5mL centrifuge tubes for LC-MS sample preparation. Metabolites were
SP:SAMPLEPREP_SUMMARY            	extracted from these homogenates for each method as follows; C8-pos: Bacterial
SP:SAMPLEPREP_SUMMARY            	cultures (10 μL) using 190 μL isopropanol containing
SP:SAMPLEPREP_SUMMARY            	1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard
SP:SAMPLEPREP_SUMMARY            	(Avanti Polar Lipids; Alabaster, AL). After centrifugation (10 min, 9,000g,
SP:SAMPLEPREP_SUMMARY            	ambient temperature), supernatants (2 μL) were injected directly onto column. -
SP:SAMPLEPREP_SUMMARY            	C18-neg: Bacterial cultures (30 μL) were extracted using 90 μl methanol
SP:SAMPLEPREP_SUMMARY            	containing 15R-15-methyl Prostaglandin A2,15R-15-methyl Prostaglandin F2α,
SP:SAMPLEPREP_SUMMARY            	15S-15-methyl Prostaglandin D2, 15S-15-methyl Prostaglandin E1, and
SP:SAMPLEPREP_SUMMARY            	15S-15-methyl Prostaglandin E2 as internal standards (Cayman Chemical Co.) and
SP:SAMPLEPREP_SUMMARY            	centrifuged (10 min, 9,000g, 4°C). The supernatants (10 μL) were injected onto
SP:SAMPLEPREP_SUMMARY            	column. - HILIC-neg: Bacterial cultures (30 μL) were extracted with the
SP:SAMPLEPREP_SUMMARY            	addition of four volumes of 80% methanol containing inosine-15N4, thymine-d4 and
SP:SAMPLEPREP_SUMMARY            	glycocholate-d4 internal standards (Cambridge Isotope Laboratories). The samples
SP:SAMPLEPREP_SUMMARY            	were centrifuged (10 min, 9,000g, 4°C) and the supernatants 10 μL) were
SP:SAMPLEPREP_SUMMARY            	injected directly onto column.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Shimadzu Nexera X2
CH:COLUMN_NAME                   	Phenomenex Luna NH2 (150 x 2mm, 5um)
CH:SOLVENT_A                     	100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide
CH:SOLVENT_B                     	75% acetonitrile/25% methanol; 10 mM ammonium hydroxide
CH:FLOW_GRADIENT                 	The column was eluted with initial conditions of 10% mobile phase A and 90%
CH:FLOW_GRADIENT                 	mobile phase B followed by a 10 min linear gradient to 100% mobile phase A.
CH:FLOW_RATE                     	400 µL/min
CH:COLUMN_TEMPERATURE            	30°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Plus Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher
MS:MS_COMMENTS                   	Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon
MS:MS_COMMENTS                   	Tyne, UK). Metabolite identities were confirmed using authentic reference
MS:MS_COMMENTS                   	standards or reference samples.
MS:MS_RESULTS_FILE               	ST003931_AN006456_Results.txt	UNITS:Abudances	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Abudances
MS_METABOLITE_DATA_START
Samples	M1	M2	M3	M4	M5	M6	M7	M8	M9	M10	M11	M12	M13	M14	M15	M16	M17	M18	M19	M20	M21
Factors	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Male	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Male	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Male	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Sample source:Fecal Samples | Type:germ-free (GF) | Sex:Female	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Male	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Sample source:Fecal Samples | Type:Altered Schaedler Flora (ASF) | Sex:Female	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Male	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Male	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Male	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Female	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Female	Sample source:Fecal Samples | Type:specified-pathogen free (SPF) | Sex:Female
Thymine-d4 [iSTD]	361551889	525558113	503632899	318396926	465606774	381654526	414531170	267486372	249236495	277036408	219922574	347282392	245614554	299320804	327287229	272095925	285434827	352898044	288491331	271359084	293952411
Inosine-15N4 [iSTD]	25512474	38148091	33559916	23783648	33994402	27416227	28811918	21045998	21135824	22448715	22857527	26767559	18592766	23310490	24945136	20573929	22968251	25816200	21070267	20303723	23084702
Glycocholate-d4 [iSTD]	109328007	186768122	151738436	107350865	157942767	121029776	122109327	88814442	81616560	90011416	106381150	124178377	76993432	94708070	101395407	101520722	98709283	117579071	98279635	92470272	104256661
Tetrahydroharman-3-carboxylic acid	3470705	2468147	3302401	4799214	3615817	4039939	4290694	2783780	3100522	2935559	2664018	3235701	3562192	2992262	3146742	3121397	3758235	2796649	2774884	3325154	3397491
4-Hydroxybenzeneacetonitrile	407	359	71	520	331		291	113	91	206		72	144	30		1240	2608	2388	1552	386	2622
Gentisic acid	388298	354802	241983	316169	413917	1505407	279198	9639049	4858837	7827221	16135166	13930014	7121081	4958705	5791154	21116804	14186186	22374247	20532738	8238370	26827933
4-Hydroxymandelic acid	1181535	1105595	1111591	1022399	1266297	1049358	1156691	3816521	3574015	4226911	11145399	3792011	4439335	4041337	3825660	8104754	5432560	5398032	6185402	4133473	6769054
Homovanillic acid	106254	78142	92508	95403	70848	78681	86573	459864	368066	609134	788742	337822	374029	305905	452123	89047	81457	100723	34728	36698	76832
Vanillylmandelic acid	2683815	2438573	2548121	3118391	2151536	2901648	2738590	3951208	4158600	5253916	4837728	4280300	4578319	4486371	5127166	1855051		1298130			
Dihexose	1881070	1807672	2052763	2329186	1960981	2431473	2128296	271990	283232	288941	316969	345946	358194	272026	319547	290675	314425	346255	498763	253364	419898
Xylose	80320	76099	84605	112408	95556	120242	88307	40493	46473	39225	43693	48256	56360	42111	42066	57495	132970	81054	166585	56586	137679
Hexose	125533473	122402622	129967926	169151269	139499381	159815131	150647439	11373576	8346577	10030494	9190894	11311472	16098136	11998342	9627405	31243192	49622106	53570232	69409808	27067656	41685666
Glyceric acid	40062572	38330241	31357507	32174149	35401446	36058222	25972241	45981031	35698781	46703901	57851462	55911222	83351310	36420622	27166952	63578272	62934871	74840800	129015437	29330846	94318474
Erythronic and or Threonic acid	885600	1217812	686398	696383	737694	757816	667093	1217184	1123287	1323400	2778926	1180984	1321272	1026414	1092239	274417	101909	181100	128056	91756	183267
Tartaric acid	2726	3085	1270	2069	1527	1800	1522	953	1520	522	1165	813	780	2244	1382	1216	1661	1395	372	380	1036
Glucuronic acid	5646488	5824629	6137109	6738332	5996797	5878942	6869983	236274	253331	255955	1491128	336303	234504	261580	269547	209037	388704	303619	670234	273403	515015
Ribitol	4043363	4008460	4346895	4474539	4270540	4438045	4375000	2519201	2516849	2408892	3262168	2429709	2676886	2593214	2523411	86078	63165	58992	197330	81348	121061
Sorbitol	39266825	39337112	42441162	47316483	44123913	47195050	45030458	3692837	3673664	2854832	4244950	3289283	4504304	3358196	3243287	218947	234613	180715	485231	342393	294171
2-Aminobutyric acid								3361776	1970787	4994943	2679193	6262752	6046682	1266642	2249937	3964503	2410461	5092685	3807908	3512856	2321278
Isovaleric and or Valeric and or Methylbutyric acid	574596	444156	397208	144366	150725	258999	359972	10394182	8222268	13491337	13250952	15329930	9181395	7190588	12267592	47456337	17497101	26793722	40938109	18703455	36656785
Ketoisovaleric acid	14860	11609	9785	8475	8561	9845	8470	41175	29475	36774	69792	40673	97583	35211	37087	25023	29906	24805	196380	22767	40169
Oxalic acid	17461	29402	15795	12639	16801	12677	12336	14165	16322	14028	20664	23351	8023	11568	12273	13639	22147	14001	17916	16537	20938
Malonic acid	3085077	2702240	2184599	2168550	2436068	2338046	2354988	5298430	4762389	4633084	5710388	6218945	4882703	4724431	4375721	1651148	1194389	1325490	2278567	1003698	1868000
3E-Methylglutaconic acid	4833570	6378775	2343155	3025477	2769112	4064292	3853740	3548191	3584090	2347065	11970844	2140647	3192702	3091589	2370932	2703160	1751024	2241849	2374925	2042098	2675178
Adipic and or Methylglutaric acid	5237801	6217949	3506644	3332633	3262897	3340015	3292051	2918986	3944947	2665111	8915884	3300968	3154838	3620350	3096500	3546033	2969642	3325271	3416870	3259993	4165425
3-Methyladipic and or Pimelic acid	66248	56779	22141	22910	30342	25180	29726	38605	53073	24511	80191	28277	31978	41386	28583	57895	46075	46976	63764	50455	87449
Suberic acid	4462646	4904893	4088544	3681641	3855339	3579709	3275023	3795114	4506529	3856534	5759409	4005382	5188509	3615863	3225780	6387127	6824371	6149092	7624474	5638098	9322793
Sebacic acid	6760272	6899146	4847794	3954706	4254878	3939367	4088521	4079113	5768200	5852883	21606672	2732508	3125481	3151490	2615819	14542770	13351158	13108971	17686766	12788980	19529428
3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid	6532	5755	7096	6527	7082	5356	6900	86194	86126	79979	113158	87756	106681	91889	84235	18128	25577	17840	14512	14365	16769
Hydroxybutyric and or Hydroxyisobutyric acid	4397786	5743995	3548805	1916069	3155845	3061945	3056288	12404600	9306389	12992281	15594714	16101018	10550604	8947784	7436504	7315058	3479989	6365252	3432944	3494904	5084466
2-Hydroxyvaleric and or 2-Hydroxyisovaleric acid	22549	37257	33018	26745	36481	36509	34762	200324	158631	182610	247046	214715	199429	139911	135166	50031	33689	50895	40809	32926	39459
2-Hydroxycaproic and or 2-Hydroxyisocaproic acid	18924	25302	16895	14843	17046	14130	16101	260424	122477	280929	531334	338867	157260	107091	140657	56163	36341	57418	47116	50101	71253
2-Hydroxyglutaric acid	965217	632599	326183	354628	372271	486781	493911	407619	480053	328190	1109006	324226	337491	349341	315514	992424	284667	553294	479147	430353	580495
2-Isopropylmalic acid	16994	14967	5780	2359	5063	3569	5589	24096	21480	12537	99163	22111	71037	14350	10499	24509	15354	17200	36498	9382	30923
Butyric acid		2467894		1857408				9875445	6700245	7297363	7287183	13308828	4959353	5533643	7827681	65254993	19332120	33076483	59366353	18954223	44758091
Adenine	142987	158225	132657	142980	171238	145663	132774	526785	441598	781464	416721	1404357	527243	1081009	1139342	476423	305543	257163	332392	679209	633675
Hypoxanthine	34662	104278	59533	57506	164625	84623	36597	218531	139605	133793	143893	326042	818814	191077	210901	128071	340772	206015	1303393	441047	556458
Guanine								462321	184661	873725	2795871		114770	334840	709255	3074887	5861795	4852647	11169252	5593660	5732598
Inosine	443628	824516	695825	700634	880731	673613	664830	16233	37206	6293	19260	11714	27393	10535	6955	706181	251786	541340	1091632	377170	383672
Guanosine	359269	267398	380651	733228	339206	446301	614419									167654	227433	269747	234040	236884	118914
Succinyladenosine	22437664	25881394	23494274	22406111	26976078	23845296	23495801	15700211	15050328	10607650	12224466	15545835	11499058	19537839	16559812		107331	496680			605966
Xanthine	1272174	2526389	2223463	1565966	3507566	2254629	1525595	1871327	1039965	1386545	1322023	3496693	4227210	1670818	1779994	1471455	4143849	2084953	10572320	4208573	4807021
1-Methylxanthine								3217211	2435188	1885954	1789740	6500286	20766358	4740088	2665082	392619	224610	261682	191826	251486	213269
Uric acid	783734	946693	1059489	816890	1276299	821498	1048325	130011	77685	198248	2264453	246208	326298	96649	100287	153236	164677	180834	403007	128986	203743
1-Methyluric acid	2104063	1389939	2047895	1634561	1900946	1819967	2043134	42821567	48821996	39650246	44860517	30743488	10938281	35672611	37600129	10014057	2503863	8257129	2162448	3041666	2928473
Orotic acid	1346184	1125961	1499925	1417474	1118691	955624	715730	3136293	2960478	4879993	17908240	2700620	2685047	2575156	3599032		522160		2964564		929778
dTMP	67859	74988		102892	258722	182401	60278			140837	657979		104783	201353		3378759	4853695	4189722	7396860	7077321	4473160
Cytidine	254064	202788	211933	392156	233530	301208	410745	41143	42098	58891	56787	62083	56146	63023	54413	32569	171607	74096	186576	90068	130421
Uridine	8617471	11208847	11390835	12193258	13514566	11728676	12730440	2691363	2951106	3616234	4182086	3168288	3011070	3711560	3341041	8117546	7003025	8225694	7027342	11013248	5332167
Pseudouridine	61691766	61065491	68676416	91916024	83999094	86088134	99056235	5897127	5934480	28012063	413010313	13368181	6358882	8131785	11007889	17399906	16551726	19649294	161705405	41050602	43669204
Uracil	340111	510669	475205	334431	727491	525933	391391	699884	451897	426641	537964	842920	1255941	510380	641297	555958	899022	685357	3150439	1430363	1542624
Thymine	235233	249562	330275	436373	385588	431172	393157	122814	100743	169112	420597	162304	214280	153545	128299	156324	233697	172851	551505	303070	247150
Aspartic acid	511656	817956	1019980	991305	1397295	1300805	957935	821636	433393	643596	379664	1272015	2142492	632611	874755	1064075	1254612	1474718	2684744	755969	1326068
Glutamic acid	1494065	2056083	2521627	2720357	3292790	3185222	2754280	2057706	1266970	1864389	1346665	2912585	4564108	1797495	2382112	2100057	2445963	2562968	7259125	2886369	3778894
N-Acetylglutamic acid	29842	41137	29344	30765	52106	34006	34182	105079	85620	83458	486379	156017	287487	79017	102439	199722	200485	194428	1653805	288726	591323
Pantothenic acid	236233	455946	321013	326085	475281	315136	216967	3089438	2104549	2345767	8872555	3751795	3996965	2171112	2118971	1544246	1469831	1670410	4997163	2298013	2592284
N-Acetylcarnosine	10748	68384	25514	10726	57091	33282	8914	4267	2974	3208	8891	20085	8144	5590	3492	16797	3160	12987	18268	740	14894
Ketocaproic and or Ketoisocaproic acid	198360	164624	105836	100862	115799	111761	112807	257789	188556	200356	398537	255388	602289	199116	222134	256277	276547	242603	1507362	256930	486922
Lactic acid	699907	982317	769933	628345	837042	655208	628429	390031	522360	329145	554100	618314	716015	490408	387372	754423	407987	985057	531332	533445	798839
Fumaric and or Maleic acid	127234	400041	311913	132790	478891	300416	114391	377339	209117	195211	199000	949965	1298206	378342	405011	471613	327569	454831	745534	452351	742149
Succinic acid	115154	199712	117313	74414	97823	105066	102938	468687	334166	418137	759935	998015	515261	374238	463602	593170	476278	605434	1685298	712092	1011701
Malic acid	171056	702742	528055	169546	848226	508400	135582	663196	333245	309227	319399	1777666	2504558	670387	703894	820607	541600	777975	1312676	776979	1324042
cis-Aconitic acid	2952576	2943032	2798797	2724839	2520626	2463867	2540716	2245290	2458237	4632302	82932796	3211489	3900018	2445576	2966699	431391	762906	408484	754686	411376	812416
Citric and or Isocitric acid	196652	260738	212254	188006	252607	161039	139912	171654	153326	434527	6150962	401685	592472	176640	228746	54348	79464	44892	103830	34348	89542
Threitol	832910	730116	731704	523191	599659	653486	691112	9990566	9192782	9164975	14285408	8284385	9119506	9094981	7746826	228529		175715		132358	
Quinic acid	2834957	2866284	3228481	3471195	3409728	3382276	3678901	2247643	2230442	2306919	1706462	2505581	2372411	2548394	2741504	346308	386449	363205	559650	454129	488337
4-Hydroxybenzaldehyde	69563	47006	46567	70620	64258	74066	52474	68287	65627	70480	145934	77955	156191	52828	72434	117053	56999	96776	280029	71414	103385
Indolelactic acid	376892							18958867	12242954	23697960	28876140	18497365	16945236	13438556	17454510	2452159	931287	1835287		985842	1162288
Glucosan	193892	131491	63200	62514	76856	68669	72916	312420	344487	316191	966944	466834	754373	312150	326589	124608	105533	90499	156136	87730	216924
Folic acid	134823	99260	155606	127526	192525	156437	125753	88326	100609	114218	142042	63495	97846	58630	64755	6482	9699	5519	13042	8038	11939
Nicotinic acid	5898	5719	5303	5295	4999	4747	5127	151326	104122	120430	99957	235580	264843	111707	135252	79566	88831	78245	375562	118115	164014
Quinolinic acid	1142499	1351424	1256979	1270057	1077808	1510317	1448628	605353	692407	835100	947620	888782	811350	945815	911802	2104386	1858424	2014031	2046805	1948497	2761189
4-Pyridoxic acid	451135	441159	451642	317848	329301	354205	345226	649482	589359	670978	1231842	541025	581798	327386	317872	1425418	896692	1110469	1009672	624218	1126214
Xanthurenic acid	19298	20384	20255	18081	15417	15819	18122	135119	148324	278414	2026826	78129	85944	72617	75410	160033	69560	87057	23911	21767	53016
Bilirubin	328	701	7232	5241			19218	2470	7393	48017	36343	28403		63892	21137	65591	88219	178721	140329	305629	154460
3-(3-Hydroxyphenyl)propanoic acid								2556648	559911	1898579	2346110	5414138	606777		306964	30480536	29852231	24736780	3036086	1208626	16638284
Lithocholic acid								301								1090468	1955762	1549319	3529115	898673	2621524
Deoxycholic acid	162	146	173	398	258	259	514	109074	71167	92135	46155	91567	89275	245703	440618	13873070	14045060	18118076	24420826	31806821	31723096
Cholic acid	296240	222671	212399	144091	170942	136845	156228	2030716	1231022	1291816	776219	1816632	828243	4293216	9134473	36012973	19456969	42704608	66156730	286267498	67218636
Glycocholic acid	587422	1368187	920105	625702	897511	685079	682820	436755	436784	526494	591163	959114	326712	1002668	1116351	1231451	476199	1446542	532150	1979898	558433
Taurdeoxycholic and or Taurochenodeoxycholic acid	2139972	570977	1383765	3796275	4884785	2296786	3206774	1109824	753167	893688	997969	2931933	1322413	7119973	4926082	264671	330485	402461	414377	1456301	738355
Taurocholic acid	72509990	16506716	22756305	80581801	59085109	56576546	51035168	52040602	35096922	36950012	38124868	123981645	42981966	295511901	270308340	2548313	2834869	4264991	4414521	48023370	11782345
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Study_Compound_ID	MZ	RT	HMDB_ID
Thymine-d4 [iSTD]	TF30	129.0608	1.37	internal standard
Inosine-15N4 [iSTD]	TF19	271.06180000000001	3.49	internal standard
Glycocholate-d4 [iSTD]	TF16	468.32709999999997	3.93	internal standard
Tetrahydroharman-3-carboxylic acid	TF28	229.0984	3.81	HMDB0037942
4-Hydroxybenzeneacetonitrile	QI01494	132.04557890000001	1.432833333	HMDB0029757
Gentisic acid	TF13	153.01939999999999	4.6100000000000003	HMDB0000152
4-Hydroxymandelic acid	TF06	167.0351	4.5599999999999996	HMDB0000822
Homovanillic acid	QI08219	181.05081970000001	4.4081666669999997	HMDB0000118
Vanillylmandelic acid	TF31	197.04570000000001	4.53	HMDB0000291
Dihexose	QI03659	341.1094435	2.5945999999999998	HMDB0000258
Xylose	QI02078	149.04564959999999	1.8545499999999999	HMDB0000098
Hexose	TF12	179.05619999999999	2.31	HMDB0000122
Glyceric acid	TF14	105.0193	4.22	HMDB0000139
Erythronic and or Threonic acid	QI07865	135.03000900000001	4.2461333330000004	HMDB0000613
Tartaric acid	QI11736	149.00926190000001	6.9972500000000002	HMDB0000956
Glucuronic acid	QI09018	193.03554500000001	4.9511666669999999	HMDB0000127
Ribitol	QI02096	151.06132289999999	1.8589166669999999	HMDB0000508
Sorbitol	QI03163	181.07195429999999	2.1479333330000001	HMDB0000247
2-Aminobutyric acid	TF03	102.056	3.12	HMDB0000452
Isovaleric and or Valeric and or Methylbutyric acid	TF20	101.0608	3.69	HMDB0000718
Ketoisovaleric acid	QI05254	115.0401107	3.4523666670000002	HMDB0000019
Oxalic acid	QI12227	88.988028610000001	7.3519666670000001	HMDB0002329
Malonic acid	TF21	103.00369999999999	7.07	HMDB0000691
3E-Methylglutaconic acid	TF05	143.0351	6.79	HMDB0000522
Adipic and or Methylglutaric acid	TF08	145.05070000000001	6.78	HMDB0000448
3-Methyladipic and or Pimelic acid	QI11246	159.0664031	6.7345166670000003	HMDB0000555
Suberic acid	TF26	173.08199999999999	6.77	HMDB0000893
Sebacic acid	TF25	201.11340000000001	6.7	HMDB0000792
3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid	QI11093	239.0926892	6.6556833329999998	HMDB0061112
Hydroxybutyric and or Hydroxyisobutyric acid	TF09	103.0401	3.78	HMDB0000008
2-Hydroxyvaleric and or 2-Hydroxyisovaleric acid	QI05910	117.0557862	3.632933333	HMDB0000407
2-Hydroxycaproic and or 2-Hydroxyisocaproic acid	QI05742	131.0714189	3.5760000000000001	HMDB0000317
2-Hydroxyglutaric acid	QI11613	147.03003369999999	6.9315666670000002	HMDB0059655
2-Isopropylmalic acid	QI11739	175.06125209999999	6.9972500000000002	HMDB0000402
Butyric acid	TF10	87.045100000000005	3.8	HMDB0000039
Adenine	QI03458	134.04732870000001	2.3887833330000001	HMDB0000034
Hypoxanthine	QI04886	135.03135180000001	3.3428833330000001	HMDB0000157
Guanine	TF17	150.04220000000001	2.98	HMDB0000132
Inosine	QI05425	267.07365850000002	3.4917666669999998	HMDB0000195
Guanosine	QI04566	282.08408470000001	3.1764833330000002	HMDB0000133
Succinyladenosine	TF27	382.10019999999997	7.29	HMDB0000912
Xanthine	QI09073	151.02624700000001	5.012466667	HMDB0000292
1-Methylxanthine	TF02	165.0419	4.07	HMDB0010738
Uric acid	QI09284	167.02120930000001	5.2095333330000004	HMDB0000289
1-Methyluric acid	TF01	181.0369	4.57	HMDB0003099
Orotic acid	TF22	155.00989999999999	4.53	HMDB0000226
dTMP	TF11	321.04899999999998	7.26	HMDB0001227
Cytidine	QI03332	242.07851439999999	2.2793166669999998	HMDB0000089
Uridine	QI02066	243.06238070000001	1.8370333329999999	HMDB0000296
Pseudouridine	TF23	243.06229999999999	2.4300000000000002	HMDB0000767
Uracil	QI01727	111.0200384	1.5830333329999999	HMDB0000300
Thymine	QI01424	125.0357259	1.392116667	HMDB0000262
Aspartic acid	QI09247	132.03027370000001	5.1745000000000001	HMDB0000191
Glutamic acid	QI09231	146.04594130000001	5.1569833330000003	HMDB0000148
N-Acetylglutamic acid	QI11089	188.056613	6.6644500000000004	HMDB0001138
Pantothenic acid	QI06964	218.10356179999999	3.9615	HMDB0000210
N-Acetylcarnosine	QI07147	267.11004989999998	4.0009166670000003	HMDB0012881
Ketocaproic and or Ketoisocaproic acid	QI05080	129.055823	3.4041833330000002	HMDB0000491
Lactic acid	QI06831	89.024413620000004	3.9045666670000001	HMDB0000190
Fumaric and or Maleic acid	QI11600	115.0037245	6.9228166670000002	HMDB0000134
Succinic acid	QI11572	117.0193696	6.8877833329999998	HMDB0000254
Malic acid	QI11602	133.01430569999999	6.9228166670000002	HMDB0000156
cis-Aconitic acid	TF07	173.00919999999999	8.7100000000000009	HMDB0000072
Citric and or Isocitric acid	QI12976	191.01994049999999	8.6984333330000005	HMDB0000094
Threitol	TF29	121.0506	1.67	HMDB0004136
Quinic acid	QI07924	191.05627380000001	4.2811666669999999	HMDB0003072
4-Hydroxybenzaldehyde	QI03646	121.0295594	2.5945999999999998	HMDB0011718
Indolelactic acid	TF18	204.0667	4.42	HMDB0000671
Glucosan	QI11300	161.045692	6.760783333	HMDB0000640
Folic acid	QI02272	440.13291609999999	10.18703333	HMDB0000121
Nicotinic acid	QI07408	122.02482550000001	4.0797333330000001	HMDB0001488
Quinolinic acid	TF24	166.0146	7.3	HMDB0000232
4-Pyridoxic acid	QI06274	182.04602410000001	3.7644500000000001	HMDB0000017
Xanthurenic acid	QI12810	204.03041379999999	8.3584833330000006	HMDB0000881
Bilirubin	QI02914	583.2572576	15.19708333	HMDB0000054
3-(3-Hydroxyphenyl)propanoic acid	TF04	165.0557	4.38	HMDB0000375
Lithocholic acid	QI06715	375.29108100000002	3.8695333330000001	HMDB0000761
Deoxycholic acid	QI06947	391.28590259999999	3.9527333329999998	HMDB0000626
Cholic acid	QI07818	407.28080999999997	4.2242499999999996	HMDB0000619
Glycocholic acid	TF15	464.3021	3.93	HMDB0000138
Taurdeoxycholic and or Taurochenodeoxycholic acid	QI05626	498.28994290000003	3.539933333	HMDB0000896
Taurocholic acid	QI06236	514.28445769999996	3.7381666670000002	HMDB0000036
METABOLITES_END
#END