#METABOLOMICS WORKBENCH juliehaines_20211122_115205 DATATRACK_ID:2938 STUDY_ID:ST001997 ANALYSIS_ID:AN003257 PROJECT_ID:000000
VERSION             	1
CREATED_ON             	November 22, 2021, 12:06 pm
PR:PROJECT_TITLE                 	Polyamine import and accumulation causes immunomodulation in macrophages
PR:PROJECT_TITLE                 	engulfing apoptotic cells
PR:PROJECT_SUMMARY               	Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue
PR:PROJECT_SUMMARY               	homeostasis and drives anti-inflammatory programming in engulfing macrophages.
PR:PROJECT_SUMMARY               	Here, we assess metabolites in naïve and inflammatory macrophages following
PR:PROJECT_SUMMARY               	engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to
PR:PROJECT_SUMMARY               	unique increases in the arginine-derived polyamines, spermidine and spermine, in
PR:PROJECT_SUMMARY               	vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does
PR:PROJECT_SUMMARY               	not arise from retention of apoptotic cell metabolites or de novo synthesis, but
PR:PROJECT_SUMMARY               	from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase.
PR:PROJECT_SUMMARY               	Blocking polyamine import prevents efferocytosis from suppressing macrophage
PR:PROJECT_SUMMARY               	IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine
PR:PROJECT_SUMMARY               	import and accumulation, and imported polyamines as mediators of
PR:PROJECT_SUMMARY               	efferocytosis-induced immune reprogramming.
PR:INSTITUTE                     	University of Colorado Denver
PR:LAST_NAME                     	Haines
PR:FIRST_NAME                    	Julie
PR:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
PR:EMAIL                         	julie.haines@cuanschutz.edu
PR:PHONE                         	3037243339
ST:STUDY_TITLE                   	Polyamine import and accumulation causes immunomodulation in macrophages
ST:STUDY_TITLE                   	engulfing apoptotic cells (Part 2)
ST:STUDY_SUMMARY                 	Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue
ST:STUDY_SUMMARY                 	homeostasis and drives anti-inflammatory programming in engulfing macrophages.
ST:STUDY_SUMMARY                 	Here, we assess metabolites in naïve and inflammatory macrophages following
ST:STUDY_SUMMARY                 	engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to
ST:STUDY_SUMMARY                 	unique increases in the arginine-derived polyamines, spermidine and spermine, in
ST:STUDY_SUMMARY                 	vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does
ST:STUDY_SUMMARY                 	not arise from retention of apoptotic cell metabolites or de novo synthesis, but
ST:STUDY_SUMMARY                 	from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase.
ST:STUDY_SUMMARY                 	Blocking polyamine import prevents efferocytosis from suppressing macrophage
ST:STUDY_SUMMARY                 	IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine
ST:STUDY_SUMMARY                 	import and accumulation, and imported polyamines as mediators of
ST:STUDY_SUMMARY                 	efferocytosis-induced immune reprogramming.
ST:INSTITUTE                     	University of Colorado Denver
ST:LAST_NAME                     	Haines
ST:FIRST_NAME                    	Julie
ST:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
ST:EMAIL                         	julie.haines@cuanschutz.edu
ST:PHONE                         	3037243339
SU:SUBJECT_TYPE                  	Cultured cells
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           	-	4h 1	category:4h unexposed	RAW_FILE_NAME=37
SUBJECT_SAMPLE_FACTORS           	-	4h 2	category:4h unexposed	RAW_FILE_NAME=40
SUBJECT_SAMPLE_FACTORS           	-	4h 3	category:4h unexposed	RAW_FILE_NAME=43
SUBJECT_SAMPLE_FACTORS           	-	4h 1-	category:4h non-engulfing	RAW_FILE_NAME=38
SUBJECT_SAMPLE_FACTORS           	-	4h 2-	category:4h non-engulfing	RAW_FILE_NAME=41
SUBJECT_SAMPLE_FACTORS           	-	4h 3-	category:4h non-engulfing	RAW_FILE_NAME=44
SUBJECT_SAMPLE_FACTORS           	-	4h 1+	category:4h engulfing	RAW_FILE_NAME=39
SUBJECT_SAMPLE_FACTORS           	-	4h 2+	category:4h engulfing	RAW_FILE_NAME=42
SUBJECT_SAMPLE_FACTORS           	-	4h 3+	category:4h engulfing	RAW_FILE_NAME=45
SUBJECT_SAMPLE_FACTORS           	-	8h 1	category:8h unexposed	RAW_FILE_NAME=46
SUBJECT_SAMPLE_FACTORS           	-	8h 2	category:8h unexposed	RAW_FILE_NAME=49
SUBJECT_SAMPLE_FACTORS           	-	8h 3	category:8h unexposed	RAW_FILE_NAME=52
SUBJECT_SAMPLE_FACTORS           	-	8h 1-	category:8h non-engulfing	RAW_FILE_NAME=47
SUBJECT_SAMPLE_FACTORS           	-	8h 2-	category:8h non-engulfing	RAW_FILE_NAME=50
SUBJECT_SAMPLE_FACTORS           	-	8h 3-	category:8h non-engulfing	RAW_FILE_NAME=53
SUBJECT_SAMPLE_FACTORS           	-	8h 1+	category:8h engulfing	RAW_FILE_NAME=48
SUBJECT_SAMPLE_FACTORS           	-	8h 2+	category:8h engulfing	RAW_FILE_NAME=51
SUBJECT_SAMPLE_FACTORS           	-	8h 3+	category:8h engulfing	RAW_FILE_NAME=54
CO:COLLECTION_SUMMARY            	Experimental animals This study was approved and performed in accordance with
CO:COLLECTION_SUMMARY            	the ethical guidelines of the Institutional Animal Care and Use Committee at
CO:COLLECTION_SUMMARY            	National Jewish Health. C57BL/6J (000664), B6-CD45.1 (002014) and CCR2KO
CO:COLLECTION_SUMMARY            	(004999) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA).
CO:COLLECTION_SUMMARY            	DsRed.T3 mice (available as 006051 at Jackson Labs) were generously shared by
CO:COLLECTION_SUMMARY            	the Nagy laboratory (Vintersten et al., 2004). CCR2KO mice were crossed with
CO:COLLECTION_SUMMARY            	DsRed mice until double homozygous. Mice used in experiments were between 8-16
CO:COLLECTION_SUMMARY            	weeks of age. Both male and female sex were used in experiments except bone
CO:COLLECTION_SUMMARY            	marrow chimera experiments, which used only male mice. Human tissue This study
CO:COLLECTION_SUMMARY            	used deidentified donor peripheral blood mononuclear cells (PBMC) collected and
CO:COLLECTION_SUMMARY            	isolated (Young et al., 2011) by the National Jewish Blood Prep Core (IRB No.
CO:COLLECTION_SUMMARY            	HS-1285). Age and sex of deidentified blood donors in this study is not known.
CO:COLLECTION_SUMMARY            	Cell Lines Jurkat (human T lymphocytes, clone E6-1) were obtained from ATCC and
CO:COLLECTION_SUMMARY            	cultured in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal
CO:COLLECTION_SUMMARY            	bovine serum (v/v) (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium
CO:COLLECTION_SUMMARY            	Pyruvate. Cells were cultured in a humidified CO2 incubator at 37C. The Jurkat
CO:COLLECTION_SUMMARY            	cell line was established from peripheral blood of a 14-year-old male diagnosed
CO:COLLECTION_SUMMARY            	with T cell leukemia. Murine primary cell cultures Peritoneal cells were
CO:COLLECTION_SUMMARY            	isolated from naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA.
CO:COLLECTION_SUMMARY            	Lavage was estimated to contain 50% macrophages. Peritoneal macrophages were
CO:COLLECTION_SUMMARY            	isolated by adhesion purification of lavage cells in RPMI-10 (RPMI-1640
CO:COLLECTION_SUMMARY            	containing 10% heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM
CO:COLLECTION_SUMMARY            	HEPES, and 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well
CO:COLLECTION_SUMMARY            	plates for FACS sorting or 4x106 macrophage/well in 24-well plates for flow
CO:COLLECTION_SUMMARY            	cytometry. Non-adherent cells were removed by washing after 3-5 hours, and
CO:COLLECTION_SUMMARY            	macrophages were cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli
CO:COLLECTION_SUMMARY            	O111:B4 LPS (List Biological Laboratories Inc.) was added to generate
CO:COLLECTION_SUMMARY            	inflammatory macrophages 12h before the addition of target cells. Bone
CO:COLLECTION_SUMMARY            	marrow-derived macrophages were generated by 8-day culture of murine bone marrow
CO:COLLECTION_SUMMARY            	cells grown in media containing M-CSF (High glucose DMEM containing 10% v/v FBS,
CO:COLLECTION_SUMMARY            	20% v/v L929-conditioned media, 1X Pen/Strep/Glut, 100M Sodium Pyruvate,
CO:COLLECTION_SUMMARY            	55M beta-Mercaptoethanol). All macrophages were removed from culture dishes
CO:COLLECTION_SUMMARY            	by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping.
CO:SAMPLE_TYPE                   	Macrophages
TR:TREATMENT_SUMMARY             	LPS-primed murine peritoneal macrophages were fed fluorescently-tagged
TR:TREATMENT_SUMMARY             	IgG-opsonized live Jurkat cells for 1h. Unengulfed targets were washed off and
TR:TREATMENT_SUMMARY             	macrophages were left to degrade targets for a further 3 or 7 hours. Macrophages
TR:TREATMENT_SUMMARY             	were then collected and sorted into non-engulfing or engulfing populations,
TR:TREATMENT_SUMMARY             	alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and
TR:TREATMENT_SUMMARY             	dry pellets were snap frozen and stored at -80C until metabolite extraction.
SP:SAMPLEPREP_SUMMARY            	To process cells for assessment of intracellular metabolites, cells were
SP:SAMPLEPREP_SUMMARY            	pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and
SP:SAMPLEPREP_SUMMARY            	discarded; residual liquid was carefully wicked away from the pellet with a
SP:SAMPLEPREP_SUMMARY            	kimwipe. Dry pellets were immediately snap frozen and stored at -80C until
SP:SAMPLEPREP_SUMMARY            	processing. To process culture supernatants for assessment of metabolites,
SP:SAMPLEPREP_SUMMARY            	supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant
SP:SAMPLEPREP_SUMMARY            	was then transferred to a fresh tube, snap frozen, and stored at -80C until
SP:SAMPLEPREP_SUMMARY            	processing.
CH:CHROMATOGRAPHY_SUMMARY        	Isocratic flow of 95% phase A (0.1% formic acid in water) and 5% phase B (0.1%
CH:CHROMATOGRAPHY_SUMMARY        	formic acid in acetonitrile) at 250 uL/min.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
CH:FLOW_GRADIENT                 	isocratic
CH:FLOW_RATE                     	250 ul/min
AN:ANALYSIS_TYPE                 	MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Global metabolomics analyses were performed using a 3 min isocratic run in
MS:MS_COMMENTS                   	positive and negative ion modes (separate runs) as described previously (Nemkov
MS:MS_COMMENTS                   	et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed
MS:MS_COMMENTS                   	using a 5 min C18 gradient in positive and negative ion modes (separate runs) as
MS:MS_COMMENTS                   	described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS
MS:MS_COMMENTS                   	scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in
MS:MS_COMMENTS                   	conjunction with the KEGG database), integrated, and quality control performed
MS:MS_COMMENTS                   	using Maven (Princeton University) as described. Stable isotope tracing results
MS:MS_COMMENTS                   	were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and
MS:MS_COMMENTS                   	15N2 (Nemkov et al., 2017).
Samples	4h 1	4h 2	4h 3	4h 1-	4h 2-	4h 3-	4h 1+	4h 2+	4h 3+	8h 1	8h 2	8h 3	8h 1-	8h 2-	8h 3-	8h 1+	8h 2+	8h 3+
Factors	category:4h unexposed	category:4h unexposed	category:4h unexposed	category:4h non-engulfing	category:4h non-engulfing	category:4h non-engulfing	category:4h engulfing	category:4h engulfing	category:4h engulfing	category:8h unexposed	category:8h unexposed	category:8h unexposed	category:8h non-engulfing	category:8h non-engulfing	category:8h non-engulfing	category:8h engulfing	category:8h engulfing	category:8h engulfing
L-arginine	1.15E+04	3.79E+04	2.33E+04	2.04E+04	3.04E+04	3.68E+04	5.43E+05	4.23E+05	4.42E+05	2.69E+04	3.87E+04	3.32E+04	3.23E+04	2.10E+04	3.61E+04	2.61E+05	2.31E+05	2.70E+05
L-cysteine	6.20E+04	4.66E+04	4.19E+04	3.45E+04	3.45E+04	6.42E+04	1.59E+05	1.94E+05	2.40E+05	4.36E+04	8.09E+04	4.90E+04	4.81E+04	7.27E+04	4.35E+04	1.88E+05	1.53E+05	1.52E+05
L-glutamate	3.69E+05	4.36E+05	2.59E+05	2.93E+05	2.98E+05	5.50E+05	6.12E+05	4.85E+05	5.19E+05	4.14E+05	5.77E+05	4.06E+05	4.08E+05	5.36E+05	4.85E+05	7.93E+05	6.81E+05	5.74E+05
L-glutamine	1.60E+05	1.85E+05	1.28E+05	1.03E+05	1.26E+05	1.93E+05	4.35E+05	3.99E+05	4.83E+05	1.18E+05	1.58E+05	1.42E+05	1.51E+05	1.21E+05	1.87E+05	5.47E+05	4.11E+05	4.61E+05
glycine	1.41E+06	1.24E+06	1.42E+06	1.35E+06	1.29E+06	1.35E+06	1.59E+06	1.29E+06	1.28E+06	1.41E+06	1.29E+06	1.37E+06	1.30E+06	1.36E+06	1.17E+06	1.31E+06	1.46E+06	1.50E+06
L-leucine	5.39E+05	1.72E+06	5.41E+05	4.61E+05	5.93E+05	8.80E+05	2.25E+06	2.44E+06	3.34E+06	5.86E+05	5.93E+05	5.44E+05	6.40E+05	5.37E+05	7.73E+05	1.99E+06	2.02E+06	2.42E+06
L-lysine	4.36E+04	6.37E+04	5.81E+04	5.96E+04	4.62E+04	6.87E+04	1.28E+06	8.60E+05	1.06E+06	4.56E+04	7.18E+04	4.37E+04	6.90E+04	6.33E+04	5.63E+04	6.91E+05	5.79E+05	7.75E+05
L-methionine	1.22E+04	1.34E+05	2.05E+04	1.41E+04	2.23E+04	2.60E+04	1.44E+05	1.21E+05	1.65E+05	2.01E+04	1.93E+04	1.63E+04	2.68E+04	1.43E+04	2.54E+04	1.43E+05	1.02E+05	1.52E+05
L-phenylalanine	8.89E+04	2.98E+05	8.77E+04	9.70E+04	1.16E+05	1.69E+05	2.34E+05	2.63E+05	3.42E+05	1.14E+05	1.23E+05	1.14E+05	1.16E+05	8.65E+04	1.29E+05	3.13E+05	2.47E+05	3.00E+05
L-proline	6.42E+05	8.51E+05	4.67E+05	5.09E+05	5.85E+05	8.45E+05	1.34E+06	1.19E+06	1.67E+06	5.57E+05	6.61E+05	4.62E+05	7.20E+05	6.75E+05	7.05E+05	1.73E+06	1.52E+06	1.42E+06
L-threonine	4.58E+04	3.77E+04	2.80E+04	1.49E+04	3.71E+04	5.18E+04	1.03E+05	8.97E+04	1.32E+05	2.74E+04	4.84E+04	2.37E+04	3.36E+04	3.93E+04	5.17E+04	1.32E+05	1.03E+05	1.16E+05
L-tryptophan	3.03E+05	4.17E+05	2.74E+05	2.30E+05	2.72E+05	4.99E+05	1.15E+06	1.32E+06	1.50E+06	3.64E+05	5.22E+05	3.43E+05	3.60E+05	2.62E+05	4.40E+05	1.48E+06	1.39E+06	1.65E+06
L-tyrosine	7.59E+04	2.58E+05	5.67E+04	5.99E+04	7.10E+04	1.11E+05	1.93E+05	1.95E+05	2.80E+05	7.30E+04	8.11E+04	7.30E+04	8.18E+04	5.09E+04	1.12E+05	2.53E+05	2.16E+05	2.76E+05
L-valine	9.92E+05	9.33E+05	8.32E+05	7.77E+05	8.03E+05	1.22E+06	1.00E+06	9.99E+05	1.38E+06	8.61E+05	9.98E+05	7.69E+05	7.80E+05	8.07E+05	1.01E+06	1.13E+06	7.98E+05	1.06E+06
Adenosine	1.23E+06	6.37E+06	1.24E+06	9.14E+05	1.19E+06	2.33E+06	7.25E+05	6.02E+05	1.29E+06	1.91E+06	8.46E+05	1.35E+06	1.99E+06	1.01E+06	2.42E+06	8.44E+05	1.64E+06	1.36E+06
Adenine	3.33E+04	2.48E+04	3.08E+04	3.28E+04	3.32E+04	4.43E+04	1.94E+04	3.28E+04	2.59E+04	2.94E+04	2.77E+04	5.08E+04	2.81E+04	3.17E+04	3.05E+04	2.08E+04	2.90E+04	4.07E+05
Guanosine	7.25E+03	7.01E+04	5.60E+03	0.00E+00	2.88E+03	2.16E+04	1.40E+04	9.41E+03	1.85E+04	1.12E+04	2.94E+03	4.15E+03	1.19E+04	2.00E+03	8.73E+03	2.02E+04	1.26E+04	2.72E+04
Guanine	1.90E+03	2.02E+04	2.31E+03	2.16E+03	1.89E+03	3.85E+03	1.09E+05	1.05E+05	1.11E+05	2.42E+03	2.04E+03	2.38E+03	4.37E+03	0.00E+00	2.55E+03	3.99E+04	3.51E+04	5.45E+04
Cytosine	5.19E+03	9.69E+03	2.38E+03	1.41E+04	7.55E+03	1.95E+04	4.61E+05	2.97E+05	5.78E+05	7.00E+03	9.83E+03	7.81E+03	1.32E+04	1.72E+04	1.25E+04	4.01E+05	2.31E+05	4.01E+05
Thymidine	5.98E+03	5.01E+04	1.07E+04	0.00E+00	4.17E+04	3.76E+04	2.36E+04	7.93E+03	8.99E+03	3.68E+03	1.37E+05	3.45E+03	2.14E+04	5.73E+03	7.91E+03	1.46E+04	2.34E+05	1.20E+04
Thymine	3.05E+04	3.05E+04	2.33E+04	2.66E+04	2.92E+04	3.28E+04	1.08E+05	9.65E+04	1.35E+05	2.72E+04	2.47E+04	2.69E+04	2.74E+04	3.46E+04	9.29E+04	6.59E+04	7.24E+04	7.25E+04
Uracil	1.68E+05	1.97E+05	8.41E+04	8.21E+04	1.30E+05	1.56E+05	1.67E+05	1.23E+05	1.28E+05	9.75E+04	1.13E+05	6.18E+04	1.93E+05	1.11E+05	5.76E+04	9.73E+04	4.82E+04	7.75E+04
Inosine	7.94E+03	1.03E+05	1.46E+04	2.15E+03	1.65E+04	9.38E+03	2.80E+04	3.62E+04	2.41E+04	1.68E+04	5.53E+03	4.16E+03	9.29E+03	2.51E+03	1.15E+04	3.13E+04	2.18E+04	3.30E+04
Hypoxanthine	5.58E+04	1.84E+05	6.57E+04	6.31E+04	6.90E+04	6.92E+04	4.38E+05	4.69E+05	4.52E+05	5.35E+04	6.35E+04	5.83E+04	6.43E+04	7.27E+04	7.09E+04	2.29E+05	2.09E+05	2.85E+05
Xanthine	7.95E+03	5.10E+04	1.10E+04	5.71E+03	9.38E+03	1.02E+04	2.75E+04	3.79E+04	4.06E+04	1.31E+04	7.10E+03	5.36E+03	8.29E+03	4.52E+03	1.22E+04	2.87E+04	3.82E+04	3.12E+04
3',5'-Cyclic IMP	1.23E+05	1.43E+05	1.49E+05	1.32E+05	1.38E+05	1.05E+05	1.24E+05	1.25E+05	1.12E+05	1.00E+05	7.97E+04	1.13E+05	1.12E+05	8.85E+04	1.08E+05	9.10E+04	8.64E+04	9.96E+04
Nicotinamide	3.76E+06	1.28E+07	3.80E+06	3.63E+06	3.65E+06	4.43E+06	4.90E+06	4.15E+06	5.19E+06	7.04E+06	4.51E+06	4.14E+06	5.80E+06	4.63E+06	5.02E+06	5.36E+06	5.76E+06	5.31E+06
Glutathione disulfide	3.21E+04	2.77E+05	3.92E+04	3.76E+04	5.07E+04	4.18E+04	6.90E+04	4.55E+04	5.82E+04	8.86E+04	5.49E+04	6.00E+04	8.92E+04	6.15E+04	6.14E+04	8.91E+04	7.15E+04	7.69E+04
5-Oxoproline	1.52E+05	2.40E+05	1.50E+05	1.28E+05	1.44E+05	2.03E+05	2.81E+05	2.35E+05	2.87E+05	1.52E+05	1.76E+05	1.69E+05	1.83E+05	1.61E+05	2.55E+05	2.67E+05	2.48E+05	2.42E+05
Phosphoserine	2.27E+05	3.64E+05	2.59E+05	2.09E+05	2.41E+05	2.92E+05	2.47E+05	2.92E+05	2.97E+05	2.33E+05	3.49E+05	1.56E+05	2.56E+05	2.20E+05	2.48E+05	2.39E+05	1.40E+05	1.69E+05
3-Phosphonooxypyruvate	3.55E+07	3.21E+07	3.99E+07	2.93E+07	2.87E+07	4.26E+07	3.58E+07	3.34E+07	3.20E+07	3.14E+07	2.94E+07	3.54E+07	3.20E+07	2.78E+07	3.29E+07	3.47E+07	3.36E+07	3.20E+07
Ornithine	4.14E+04	4.67E+04	3.52E+04	2.55E+04	3.60E+04	5.29E+04	1.96E+05	1.67E+05	1.24E+05	2.96E+04	4.40E+04	2.66E+04	3.47E+04	2.72E+04	3.93E+04	1.67E+05	1.64E+05	1.48E+05
L-Citrulline	2.37E+05	2.20E+05	2.02E+05	1.96E+05	1.89E+05	4.64E+05	4.03E+05	2.61E+05	4.06E+05	7.62E+04	1.36E+05	1.47E+05	1.23E+05	1.02E+05	1.96E+05	2.38E+05	1.61E+05	2.37E+05
Putrescine	1.18E+06	1.30E+06	9.59E+05	1.08E+06	8.85E+05	1.12E+06	1.07E+06	9.16E+05	9.23E+05	8.85E+05	8.98E+05	7.96E+05	8.65E+05	9.21E+05	6.82E+05	1.22E+06	1.11E+06	8.39E+05
Spermidine	8.33E+06	7.04E+06	9.05E+06	1.45E+07	7.97E+06	6.71E+06	8.53E+06	8.96E+06	1.00E+07	8.95E+06	8.52E+06	7.74E+06	1.20E+07	8.67E+06	6.42E+06	1.04E+07	1.47E+07	1.17E+07
Spermine	6.50E+05	6.07E+05	9.01E+05	1.29E+06	7.61E+05	5.99E+05	9.59E+05	1.17E+06	1.32E+06	6.09E+05	6.55E+05	5.64E+05	7.53E+05	5.80E+05	3.99E+05	1.24E+06	1.32E+06	2.24E+06
D-Glucosamine	4.81E+04	1.44E+05	6.85E+04	8.07E+04	8.65E+04	7.48E+04	5.85E+04	7.69E+04	4.54E+04	4.52E+04	3.86E+04	6.80E+04	5.99E+04	4.90E+04	5.36E+04	6.06E+04	7.15E+04	8.82E+04
Creatine	1.05E+06	1.36E+06	1.01E+06	8.20E+05	7.24E+05	1.72E+06	8.62E+05	9.31E+05	1.28E+06	2.57E+06	2.70E+06	2.80E+06	2.58E+06	2.75E+06	3.31E+06	3.46E+06	3.41E+06	3.55E+06
4-Acetamidobutanoate	9.62E+04	1.51E+05	9.00E+04	9.76E+04	9.51E+04	1.26E+05	1.91E+05	1.01E+05	1.84E+05	8.60E+04	9.83E+04	1.04E+05	1.34E+05	1.03E+05	1.31E+05	1.61E+05	1.72E+05	2.41E+05
trans-4-Hydroxy-L-proline	1.38E+05	1.22E+05	9.88E+04	9.45E+04	1.03E+05	1.37E+05	1.22E+05	1.12E+05	9.43E+04	1.15E+05	1.30E+05	1.04E+05	1.28E+05	1.21E+05	1.07E+05	1.26E+05	1.13E+05	1.22E+05
Pantothenol	3.14E+04	8.65E+04	2.39E+04	2.75E+04	2.64E+04	2.65E+04	3.41E+04	2.93E+04	3.48E+04	3.19E+04	3.27E+04	2.92E+04	3.82E+04	3.48E+04	3.45E+04	6.00E+04	4.72E+04	4.69E+04
L-Methionine S-oxide	4.07E+03	4.71E+04	8.80E+03	1.14E+04	1.39E+04	1.52E+04	5.13E+04	7.47E+04	8.82E+04	1.43E+04	6.77E+03	9.78E+03	1.07E+04	1.24E+04	1.27E+04	4.92E+04	5.39E+04	5.48E+04
Picolinic acid	6.52E+04	1.59E+05	5.17E+04	4.41E+04	1.22E+05	5.32E+04	5.34E+04	6.73E+04	5.45E+04	6.27E+04	7.01E+04	6.40E+04	5.60E+04	5.45E+04	6.24E+04	6.00E+04	6.80E+04	1.61E+05
Sphingosine	1.43E+04	1.06E+04	4.31E+04	1.03E+04	3.06E+04	3.40E+04	8.11E+03	1.49E+04	3.37E+04	2.33E+04	2.98E+04	1.34E+04	1.71E+04	1.26E+04	1.96E+04	2.06E+04	1.27E+04	2.69E+04
Acetylcholine	5.47E+05	4.40E+05	3.79E+05	4.11E+05	3.81E+05	4.83E+05	4.47E+05	4.05E+05	4.82E+05	3.21E+05	4.52E+05	3.34E+05	4.84E+05	5.64E+05	4.13E+05	6.41E+05	4.49E+05	3.85E+05
Choline	6.76E+06	7.43E+06	6.68E+06	6.09E+06	5.05E+06	8.95E+06	8.38E+06	6.48E+06	7.88E+06	1.03E+07	1.12E+07	1.03E+07	1.23E+07	1.13E+07	1.09E+07	1.24E+07	1.15E+07	1.14E+07
Triacanthine	9.09E+05	6.46E+06	8.55E+05	9.68E+05	9.77E+05	1.17E+06	1.20E+06	7.96E+05	9.43E+05	9.10E+05	6.91E+05	8.14E+05	1.12E+06	1.03E+06	9.48E+05	8.49E+05	9.54E+05	6.73E+05
6-Lactoyl-5-6-7-8-tetrahydropterin	3.33E+04	8.48E+04	3.01E+04	4.48E+04	4.17E+04	5.05E+04	5.06E+04	4.96E+04	8.08E+04	4.56E+04	3.18E+04	2.47E+04	4.26E+04	4.40E+04	3.51E+04	5.58E+04	5.71E+04	5.50E+04
Fe(III)dicitrate	1.29E+05	1.64E+05	1.49E+05	1.42E+05	1.47E+05	1.54E+05	1.52E+05	1.13E+05	1.47E+05	1.21E+05	1.55E+05	1.69E+05	1.46E+05	1.40E+05	1.55E+05	1.54E+05	1.75E+05	1.23E+05
Pyridoxamine	0.00E+00	7.54E+03	1.34E+04	1.10E+04	1.40E+04	3.63E+03	1.21E+05	1.24E+05	1.64E+05	1.50E+04	1.13E+04	1.72E+04	7.89E+03	1.64E+04	7.48E+03	1.01E+05	9.94E+04	5.27E+04
L-Noradrenaline	1.48E+04	1.89E+04	1.46E+04	1.67E+04	7.00E+03	1.32E+04	1.60E+04	1.61E+04	1.69E+04	1.32E+04	1.78E+04	1.80E+04	1.74E+04	1.40E+04	1.68E+04	1.09E+04	1.36E+04	2.49E+04
metabolite_name	KEGG ID	m/z	r.t.
L-arginine	C00062	175.1190	0.64
L-cysteine	C00097	122.0272	0.68
L-glutamate	C00025	148.0604	0.67
L-glutamine	C00064	147.0764	0.67
glycine	C00037	76.0402	1.87
L-leucine	C00123	132.1020	0.99
L-lysine	C00047	147.1128	0.61
L-methionine	C00073	150.0584	0.89
L-phenylalanine	C00079	166.0864	1.39
L-proline	C00148	116.0710	0.68
L-threonine	C00188	120.0658	0.67
L-tryptophan	C00078	205.0972	1.72
L-tyrosine	C00082	182.0813	0.92
L-valine	C00183	118.0866	0.68
Adenosine	C00212	268.1039	0.91
Adenine	C00147	136.0618	0.26
Guanosine	C00387	284.0988	0.94
Guanine	C00242	152.0567	0.97
Cytosine	C00380	112.0509	0.69
Thymidine	C00214	243.0991	1.74
Thymine	C00178	127.0504	1.27
Uracil	C00106	113.0361	0.59
Inosine	C00294	269.0879	0.95
Hypoxanthine	C00262	137.0459	1.05
Xanthine	C00385	153.0407	0.89
3',5'-Cyclic IMP	C00943	331.0447	0.97
Nicotinamide	C00153	123.0555	0.91
Glutathione disulfide	C00127	613.1587	0.89
5-Oxoproline	C01879	130.0500	0.69
Phosphoserine	C02532	186.0186	0.59
3-Phosphonooxypyruvate	C03232	184.9855	3.79
Ornithine	C01602	133.0972	0.61
L-Citrulline	C00327	176.1029	0.67
Putrescine	C00134	89.1080	0.61
Spermidine	C00315	146.1652	0.61
Spermine	C00750	203.2230	0.60
D-Glucosamine	C00329	180.0866	0.90
Creatine	C00300	132.0769	0.67
4-Acetamidobutanoate	C02946	146.0812	1.08
trans-4-Hydroxy-L-proline	C01157	132.0656	0.71
Pantothenol	C00864	220.1180	1.65
L-Methionine S-oxide	C02989	166.0533	0.88
Picolinic acid	C10164	124.0395	0.23
Sphingosine	C00319	300.2896	2.74
Acetylcholine	C01996	146.1175	0.72
Choline	C00114	104.1075	0.66
Triacanthine	C08435	204.1231	0.89
6-Lactoyl-5-6-7-8-tetrahydropterin	C04244	240.1090	0.89
Fe(III)dicitrate	C06229	245.9450	0.86
Pyridoxamine	C00534	169.0961	0.60
L-Noradrenaline	C00547	170.0811	0.95