#METABOLOMICS WORKBENCH juliehaines_20241126_063750 DATATRACK_ID:5402 STUDY_ID:ST003607 ANALYSIS_ID:AN005928 PROJECT_ID:PR002231
VERSION             	1
CREATED_ON             	December 5, 2024, 7:47 pm
#PROJECT
PR:PROJECT_TITLE                 	Macrophages recycle phagocytosed bacteria to fuel immunometabolic responses
PR:PROJECT_SUMMARY               	Macrophages specialize in phagocytosis, a cellular process that eliminates
PR:PROJECT_SUMMARY               	extracellular matter, including microbes, through internalization and
PR:PROJECT_SUMMARY               	degradation. Despite the critical role of phagocytosis during bacterial
PR:PROJECT_SUMMARY               	infection, the fate of phagocytosed microbial cargo and its impact on host cell
PR:PROJECT_SUMMARY               	is poorly understood. Here, we reveal that ingested bacteria constitute an
PR:PROJECT_SUMMARY               	alternative nutrient source that skews immunometabolic host responses. Tracing
PR:PROJECT_SUMMARY               	stable isotope-labelled bacteria, we found that phagolysosomal degradation of
PR:PROJECT_SUMMARY               	bacteria provides carbon atoms and amino acids that are recycled into various
PR:PROJECT_SUMMARY               	metabolic pathways, including glutathione and itaconate biosynthesis, and
PR:PROJECT_SUMMARY               	satisfy macrophage bioenergetic needs. Metabolic recycling of
PR:PROJECT_SUMMARY               	microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and
PR:PROJECT_SUMMARY               	intricately tied to microbial viability. Dead bacteria, as opposed to live ones,
PR:PROJECT_SUMMARY               	are enriched in cyclic- adenosine monophosphate (AMP), sustain the cellular AMP
PR:PROJECT_SUMMARY               	pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1.
PR:PROJECT_SUMMARY               	Consequently, killed bacteria strongly fuel metabolic recycling and support
PR:PROJECT_SUMMARY               	macrophage survival, but elicit decreased reactive oxygen species (ROS)
PR:PROJECT_SUMMARY               	production and a reduced IL-1β secretion compared to viable bacteria. These
PR:PROJECT_SUMMARY               	results reveal a novel insight into the fate of engulfed microbes and highlights
PR:PROJECT_SUMMARY               	a microbial viability-associated metabolite that triggers host metabolic and
PR:PROJECT_SUMMARY               	immune responses. Our findings hold promise for shaping immunometabolic
PR:PROJECT_SUMMARY               	intervention in various immune-related pathologies.
PR:INSTITUTE                     	University of Colorado Anschutz Medical Campus
PR:LABORATORY                    	Lab of Angelo D'Alessandro in collaboration with lab of Johan Garaude (INSERM,
PR:LABORATORY                    	Fr)
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
#STUDY
ST:STUDY_TITLE                   	Global metabolomics and tracing of 13C-labeled metabolites of bone marrow
ST:STUDY_TITLE                   	derived macrophages following engulfment of dead 13C-labeled Citrobacter
ST:STUDY_TITLE                   	rodentium, Listeria innocua, Staphilococcus aureus, or Pseudomonas aeruginosa.
ST:STUDY_SUMMARY                 	Global metabolomics and 13C-tracing analysis of bone marrow derived macrophages
ST:STUDY_SUMMARY                 	exposed to heat-killed uniformly 13C-labelled Citrobacter rodentium, Listeria
ST:STUDY_SUMMARY                 	innocua, Staphilococcus aureus or Pseudomonas aeruginosa for 4h.
ST:INSTITUTE                     	University of Colorado Anschutz Medical Campus
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
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:GENDER                        	Not applicable
#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           	-	AA89-33_r67+	treatment:CTRL | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-33_r67
SUBJECT_SAMPLE_FACTORS           	-	AA89-34_r102+	treatment:CTRL | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-34_r102
SUBJECT_SAMPLE_FACTORS           	-	AA89-35_r39+	treatment:CTRL | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-35_r39
SUBJECT_SAMPLE_FACTORS           	-	AA89-36_r15+	treatment:CTRL | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-36_r15
SUBJECT_SAMPLE_FACTORS           	-	AA89-37_r55+	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-37_r55
SUBJECT_SAMPLE_FACTORS           	-	AA89-38_r8+	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-38_r8
SUBJECT_SAMPLE_FACTORS           	-	AA89-39_r42+	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-39_r42
SUBJECT_SAMPLE_FACTORS           	-	AA89-40_r32+	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-40_r32
SUBJECT_SAMPLE_FACTORS           	-	AA89-41_r53+	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-41_r53
SUBJECT_SAMPLE_FACTORS           	-	AA89-42_r38+	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-42_r38
SUBJECT_SAMPLE_FACTORS           	-	AA89-43_r65+	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-43_r65
SUBJECT_SAMPLE_FACTORS           	-	AA89-44_r43+	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-44_r43
SUBJECT_SAMPLE_FACTORS           	-	AA89-45_r105+	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-45_r105
SUBJECT_SAMPLE_FACTORS           	-	AA89-46_r70+	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-46_r70
SUBJECT_SAMPLE_FACTORS           	-	AA89-47_r81+	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-47_r81
SUBJECT_SAMPLE_FACTORS           	-	AA89-48_r80+	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-48_r80
SUBJECT_SAMPLE_FACTORS           	-	AA89-49_r108+	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-49_r108
SUBJECT_SAMPLE_FACTORS           	-	AA89-50_r36+	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-50_r36
SUBJECT_SAMPLE_FACTORS           	-	AA89-51_r33+	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-51_r33
SUBJECT_SAMPLE_FACTORS           	-	AA89-52_r12+	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	RAW_FILE_NAME(raw file name)=AA89-52_r12
#COLLECTION
CO:COLLECTION_SUMMARY            	BMDMs were seeded at 2.5E6 cell/well in a non-tissue treated 6-well plate (2
CO:COLLECTION_SUMMARY            	wells per conditions) the day before the experiment. For metabolite extraction,
CO:COLLECTION_SUMMARY            	BMDMs were washed with cold PBS and harvested with 5 mM EDTA in PBS, frozen as
CO:COLLECTION_SUMMARY            	dry cell pellet, and stored at -80˚C until processing.
CO:SAMPLE_TYPE                   	Macrophages
#TREATMENT
TR:TREATMENT_SUMMARY             	Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were
TR:TREATMENT_SUMMARY             	generated in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell
TR:TREATMENT_SUMMARY             	supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100
TR:TREATMENT_SUMMARY             	µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate
TR:TREATMENT_SUMMARY             	and 50 µM 2-mercaptoethanol (all from Gibco). BMDMs were used on day 5 to 7
TR:TREATMENT_SUMMARY             	after seeding. Period of differentiation of the cells, concentration of cells
TR:TREATMENT_SUMMARY             	when replating and time-lapse between replating and stimulation with bacteria
TR:TREATMENT_SUMMARY             	were important parameters to maintain metabolic backgrounds homogenous between
TR:TREATMENT_SUMMARY             	experiments. Preparation of viable and killed U-[13C]Bacteria: Citrobacter
TR:TREATMENT_SUMMARY             	rodentium, Staphylococus aureus, Listeria innocua, Pseudomonas aeruginosa were
TR:TREATMENT_SUMMARY             	grown overnight with shaking in LB, diluted 1/40, and grown until log-phase
TR:TREATMENT_SUMMARY             	[optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with
TR:TREATMENT_SUMMARY             	phosphate buffer saline (PBS) to remove LB salts before addition to cells. For
TR:TREATMENT_SUMMARY             	labeling, 10 µl of an overnight cultured of bacteria was added to 20 ml of a
TR:TREATMENT_SUMMARY             	filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM
TR:TREATMENT_SUMMARY             	thiamine, 1 mM MgSO4, 0.1 M CaCl2, and 0.5% U-13C glucose (Campro Scientific).
TR:TREATMENT_SUMMARY             	Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by
TR:TREATMENT_SUMMARY             	re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For
TR:TREATMENT_SUMMARY             	antibiotic killing, bacteria were incubated for 6h to 12h with streptomycin or
TR:TREATMENT_SUMMARY             	gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing
TR:TREATMENT_SUMMARY             	was confirmed by overnight plating on LB-agar plates. Treatment of macrophages:
TR:TREATMENT_SUMMARY             	2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated
TR:TREATMENT_SUMMARY             	6-well plate (BD Falcons). Cells were then stimulated with killed labelled
TR:TREATMENT_SUMMARY             	bacteria at MOI 25, centrifuged at 2000 rpm for 5 min. BMDMs were incubated for
TR:TREATMENT_SUMMARY             	5 min and washed with PBS to remove non-ingested bacteria and further incubated
TR:TREATMENT_SUMMARY             	for 4h.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites from frozen pellets were extracted at 4e6 cells per mL using ice
SP:SAMPLEPREP_SUMMARY            	cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4
SP:SAMPLEPREP_SUMMARY            	degrees C followed by centrifugation as described for 10 min at 18,000 g.
SP:SAMPLEPREP_SUMMARY            	Supernatants were maintained at 4°C until analysis that same day.
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Positive C18
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:FLOW_GRADIENT                 	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min
CH:FLOW_GRADIENT                 	95-5% B, 3-5 min hold at 5% B
CH:FLOW_RATE                     	450 uL/min
CH:COLUMN_TEMPERATURE            	45
CH:SAMPLE_INJECTION              	10 uL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Exploris 120
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975
MS:MS_COMMENTS                   	m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC)
MS:MS_COMMENTS                   	detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C,
MS:MS_COMMENTS                   	vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all
MS:MS_COMMENTS                   	nitrogen). Data converted to mzXML using RawConverter. Metabolites were
MS:MS_COMMENTS                   	annotated and integrated using Maven in conjunction with the KEGG database.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	AA89-33_r67+	AA89-34_r102+	AA89-35_r39+	AA89-36_r15+	AA89-37_r55+	AA89-38_r8+	AA89-39_r42+	AA89-40_r32+	AA89-41_r53+	AA89-42_r38+	AA89-43_r65+	AA89-44_r43+	AA89-45_r105+	AA89-46_r70+	AA89-47_r81+	AA89-48_r80+	AA89-49_r108+	AA89-50_r36+	AA89-51_r33+	AA89-52_r12+
Factors	treatment:CTRL | Sample source:murine bone marrow derived macrophages	treatment:CTRL | Sample source:murine bone marrow derived macrophages	treatment:CTRL | Sample source:murine bone marrow derived macrophages	treatment:CTRL | Sample source:murine bone marrow derived macrophages	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	treatment:Killed-Citro | Sample source:murine bone marrow derived macrophages	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	treatment:Killed-Listeria | Sample source:murine bone marrow derived macrophages	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	treatment:Killed_Staph | Sample source:murine bone marrow derived macrophages	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages	treatment:Killed-Pseudo | Sample source:murine bone marrow derived macrophages
Alanine	215653	354524	272542	184201	125448	177954	200139	280433	330788	363797	389461	269694	221473	244630	249905	197517	223605	278641	198796	261052
Arginine	2485193	3079702	2845060	2244313	1763949	1646419	1884363	2017153	1884219	2178053	2406837	2071309	1777563	1423755	2015685	1902686	1541960	1583675	1539819	1619473
Cysteine	50307	81892	71289	59912	61123	57953	91522	72095	119288	74578	119442	119300	16507	33742	29164	19238	42788	89115	86864	82178
Glutamate	721808	973313	686515	869991	2806637	2170096	2236222	2055006	1307007	1036483	910204	1176940	761626	757627	817536	804061	589027	487475	581011	530615
Glutamine	356643	502949	529743	407664	642058	472663	578190	463865	673694	613280	692325	599541	123774	161826	144141	95951	476935	558862	389771	351086
Histidine	237237	281403	235560	146737	204140	224470	193268	245773	253705	250329	301192	231123	116196	158533	99726	124852	157401	168547	157348	196791
Leucine/isoleucine	3167724	4368757	4981617	2456016	2326751	2565682	3201547	4293494	3943127	4487938	5109763	3611876	2449985	2652157	2507623	2043665	2755700	3058079	2715656	3200604
Lysine	4938863	6301252	4492699	3919210	4006349	3877910	4870409	5236058	4667769	5448142	6167599	4514664	3996175	3687879	3861719	3405873	4280902	4527765	3184774	4196638
Methionine	413774	751530	535613	393278	376103	541722	542223	1010365	714788	758475	956882	471157	204492	243185	261731	221431	358380	610498	382920	830333
Phenylalanine	763575	880376	880647	612789	861725	796597	753183	1098630	813858	925350	897369	723004	420412	390926	392025	424692	671657	648336	560867	713810
Proline	4607837	5358200	4167351	3132934	4689746	3760509	6082733	5922532	4868121	6545808	6939244	4710990	7115878	8729704	8964096	7990548	4491553	5218076	3306410	4184780
Threonine	173775	232577	175624	151515	164805	192137	206936	192997	216620	226809	320900	207121	154431	171237	149248	149488	143310	173121	142659	177557
Tryptophan	535644	740631	848590	463506	835109	854328	1110997	1072396	837857	671254	777248	680601	473778	513423	468448	405617	551390	496585	381167	363664
Valine	1742720	2281535	1968391	1365703	17022846	15313103	20258878	18184092	2017352	2486474	2309651	2061116	6996379	8793343	9231545	9170227	1609982	1597845	1293258	1284300
Guanosine	12441	25694	22937	19831	37309	49226	76403	2887	51617	12250	19425	28204	2792	5424	0	13026	42268	4736	31837	19157
Guanine	31446	36683	37411	35432	56947	38189	65114	29885	12856	34608	19421	38284	20209	32604	16710	27814	6784	9048	20797	21250
Cytosine	766398	780180	670221	542312	33694	39723	41942	25209	699694	505691	389182	556820	13777	0	17081	11027	389041	256180	271942	272570
Pyridoxal	356438	520632	382935	370152	434870	374632	522257	463060	303563	308383	362370	414732	256740	234932	265618	218930	254579	104863	219405	225233
Nicotinamide	189281	235598	242485	183630	188132	145138	215318	197266	256441	273936	326602	261515	126972	125430	126685	121309	330217	222379	231126	225441
NAD+	239475	293640	235740	210725	424823	350974	377771	358153	256853	176656	124125	227074	69032	47446	42094	67680	86215	37725	82072	100755
gamma-L-Glutamyl-L-cysteine	13690	46404	25359	15813	47791	41083	144547	73963	110533	96933	95016	77076	0	0	13438	7151	12095	57717	40583	32285
gamma-L-Glutamyl-D-alanine	214488	337591	213982	197533	168143	174993	293970	258889	328460	421928	487129	319466	178653	217728	197623	116086	254372	404046	252769	317419
gamma-Glutamyl-gamma-aminobutyrate	343774	495215	229141	246368	256257	238408	429155	477755	458364	738077	625170	366269	358215	593672	536024	487227	436322	342937	315879	311521
gamma-L-Glutamylputrescine	1698632	2268219	1480699	1294787	1233115	1390637	1759698	2241432	1772046	2278625	2615052	1598724	1713361	1745984	1653594	1445366	1520988	1564055	974638	1669916
(5-L-Glutamyl)-L-glutamine	81715	119447	110790	80749	76548	53766	98850	103604	111142	142675	204753	94751	84111	130347	97691	70802	92606	163741	97283	141103
Cystathionine	13937	33977	12620	15090	20685	11434	11476	4443	48722	17639	58732	29154	32577	13963	20983	2594	81974	57140	30096	26309
Dimethylglycine	147332	262029	115061	80102	81950	69003	201611	187214	276907	274287	362968	173909	395909	384187	429861	353779	194655	222712	178860	170222
S-Adenosyl-L-methionine	0	5451	8808	7868	48715	55679	42101	44059	9682	12886	7283	11284	16086	25517	32843	14906	6902	6418	6304	7566
Ornithine	42843	34941	66115	25445	31024	26587	30233	11291	3852	59163	60950	38519	41746	120074	62654	58098	25031	18737	10231	12995
L-Citrulline	0	0	6673	0	8544	23061	29489	30982	67524	49630	88787	68928	169945	282165	271674	302566	36927	23766	24261	26419
Cadaverine	0	7142	0	0	6747	13469	16176	0	0	0	0	0	106651	88944	67343	133589	0	0	0	0
Putrescine	24198	56843	27570	22197	537502	466790	604373	503113	63655	57347	50738	69299	359238	450573	419164	465705	53639	42123	49919	15873
Spermidine	1210206	1218542	2448560	2402046	863495	697172	870676	748455	2184518	1245089	819415	1610780	187112	260093	167248	136377	1220039	606251	325393	511800
Spermine	65439	55796	142364	171511	16072	11557	25503	44912	151809	78090	50583	144007	5042	10295	3019	0	188958	111375	71934	126548
Carnosine	53343	89797	66836	41315	11147	30358	44109	53742	89465	64136	75138	54037	17496	32183	36489	21677	25307	69607	58874	52057
Creatine	5170484	6532323	6852102	4052879	2171938	1714102	2949155	2871167	8951659	6963896	6035974	6917322	2022230	2021062	1751653	1906597	5465068	5153052	4536600	3780977
Creatinine	163426	263399	396484	151335	40428	41443	85578	66423	377864	273072	299932	306706	92295	169702	156926	115990	211639	184759	144719	130128
N-Acetyl-L-citrulline	190799	261928	247817	109861	114232	133379	206870	221416	276749	443882	474190	326971	185750	321149	224670	192360	369918	392939	270658	321299
N-Acetylornithine	749948	1216745	747155	523230	441307	556214	771570	1003932	1403304	1151273	1769793	1094506	583903	779167	695500	476442	1038620	1120214	654124	931469
Guanidinoacetate	84926	108478	81474	77560	16671	12082	19133	22457	69503	39211	28439	55390	5372	16561	16702	12545	31307	7220	22139	23254
trans-4-Hydroxy-L-proline	124872	156881	169225	118037	136465	98935	137126	161173	203275	166521	166581	202026	195940	184618	190601	197494	161960	183631	166083	109405
L-Methionine S-oxide	277980	387043	259574	53327	212412	28940	136007	206092	298952	234280	411612	191264	338237	343153	320481	224150	382607	232242	114090	48972
Indole-3-acetate	95006	106845	416356	50298	0	5508	4132	0	68794	59835	55780	67816	28148	39778	42740	67456	58103	55163	47616	21023
Sphingosine	28397226	33532542	25378016	27404674	19165274	24348122	25659486	26857286	28727238	27316622	32123840	29643368	27446394	30566082	30874384	29464058	26490962	24596778	22003344	21693472
Choline	11549636	14559995	12607264	11647099	8264383	8094154	9942777	7601623	12578047	6679103	5372699	10954965	6495460	6053230	6874061	10043675	14107781	9214077	13673187	6714962
Acetylcholine	793997	1208583	823528	859384	680927	784697	1047507	1024014	969375	726993	608277	811011	385468	393200	440810	399235	545665	495694	419201	404207
L-Carnitine	3452535	5190419	5290727	4417653	12235643	11789857	11738395	12569387	4266542	3904166	3941868	3799415	1549409	1913419	1582915	1504416	3187214	2797170	2586355	2445068
acetyl-carnitine	169917	261188	291646	221780	161237	169151	242297	208442	249347	175257	158569	190995	107025	104831	71725	46155	227942	206365	190667	109547
propionyl-carnitine	184415	238855	208847	246225	374880	533255	634943	840974	1886986	1520712	1055055	1371774	71770	27945	33384	27847	600280	522045	432401	464612
acyl-C5	21870	28984	28518	28737	45957	71219	79757	64275	89135	67131	44031	67844	16825	16918	7793	6192	13846	35190	34585	54975
AC(5-OH)	40953	65536	56572	59464	43085	41170	24101	53089	76547	56629	47545	104126	37649	0	0	0	39886	6838	74137	55187
O-tetradecanoyl-L-carnitine	224909	287951	333088	302963	87197	95443	130608	131540	318782	215872	158509	251224	78687	70598	74998	51879	177866	138926	114317	84809
L-Palmitoylcarnitine	4015781	5528186	4935362	5020559	1124359	1591012	1745114	2096144	4408273	3461127	2581390	3435889	1175640	1183386	953953	1077730	2668346	2005668	2238031	1976186
Hexadecenoyl-carnitine	234938	270499	251441	281446	107915	145499	174966	183238	392430	246901	251414	331162	72396	81615	63153	77117	162414	134802	147152	116800
Octadecanoyl-L-carnitine	1535019	2182915	1947386	1453066	649952	1344138	879432	825427	1601574	1194739	998404	1114425	670795	1759067	1187115	845939	1341324	977596	902359	687026
O-octadecenoyl-L-carnitine	2380553	2975683	2899062	2596691	758291	1136317	1488987	1400575	3422501	2884948	2157052	2618437	529160	660843	568875	582376	1489127	1180986	1095860	1036384
acyl-C18:2	75130	83369	88475	82268	35170	48785	60198	50250	155336	116669	110659	124691	21737	11037	5316	11407	60998	39251	38156	31570
Arginine 13C4	45643	54493	41144	31797	53624	105264	24372	125708	104131	95859	119731	80682	16382	9665	67486	47684	69795	66438	55083	78000
Arginine 13C5	19729	15099	11150	4971	9744	38810	33358	49945	28576	17828	56368	12052	32297	10836	5232	6655	11838	11461	122139	17932
Arginine 13C6	0	0	0	0	54713	34241	40992	44069	0	0	0	0	64615	31241	52311	43889	0	0	7097	5536
Glutamate 13C2	78	2146	0	1226	29542	24531	11350	25793	14448	14393	0	4677	3	4409	6519	2548	0	0	0	5336
Glutamate 13C3	1985	0	0	2608	3840	0	11647	3766	3415	0	0	0	0	0	2156	5982	7282	0	0	0
Glutamate 13C4	38047	10389	35992	37130	46491	67181	56221	58246	40429	69611	19742	15362	31939	19971	44062	11661	49772	42695	45184	36790
Glutamate 13C5	0	0	0	0	12447	7550	13034	20588	0	2829	11600	0	12118	8929	6209	15575	0	2587	0	5049
Methionine 13C2	16571	18013	26715	33810	8774	12151	10340	20690	17250	28370	40626	22360	21485	18449	10117	11513	34327	47976	24607	19205
Methionine 13C5	13161	10433	7962	12788	25498	19119	30323	28681	12137	11180	9577	16316	10482	10503	21894	24515	13067	11471	6756	18088
Glutathione 13C2	0	0	0	0	11431	5039	20336	1600	15173	3467	0	16655	0	7568	25369	1270	1100	10688	16728	0
Glutathione 13C3	0	0	0	2244	344932	159839	308526	183809	0	0	1893	13870	0	3371	8914	5309	0	0	5715	9845
Glutathione 13C4	2879	0	1446	12408	288653	173903	230915	230419	0	0	0	0	34571	27344	29743	43796	0	0	2452	0
Glutathione 13C5	0	5115	4847	0	7057	2805	9869	3632	4756	12535	0	0	4911	3375	0	0	0	18999	0	0
Glutathione 13C6	93714	129163	80157	134814	92985	68066	100132	77095	106471	115033	76164	83034	27634	27526	29794	25520	145270	146249	119997	83725
Glutathione 13C8	9777	2316	2290	3999	21388	39258	76838	18245	11003	6715	33851	15886	13895	0	21443	1540	17416	25428	14371	20129
Glutathione disulfide 13C2	82016	111796	63071	81661	61366	44949	36558	60818	63156	104150	56813	91609	8002	0	27372	17495	14350	38944	33329	33131
Glutathione disulfide 13C3	11366	0	0	19840	0	35361	0	7449	0	0	8342	17308	0	0	0	0	13488	1768	1488	3045
Cystathionine 13C7	20854	0	9369	9055	60984	199779	202051	340482	46013	92294	27576	9254	0	0	0	0	40008	14305	30270	9664
Putrescine 13C4	0	0	0	0	161701	116846	176865	126484	10501	16963	16701	20476	70999	55325	80619	56311	37178	0	15386	17782
Spermidine 13C2	0	0	0	0	0	0	0	0	71291	20914	7876	44683	0	5981	4029	0	50507	17400	4227	10714
Spermidine 13C3	0	0	0	0	8336	18686	25377	8538	154926	63026	51194	110484	14202	21821	9398	5813	132188	34993	19202	40116
Spermidine 13C4	0	0	0	0	119585	60496	94994	74814	247429	115541	63278	159974	28100	39785	15355	26598	206459	123018	57163	63493
Spermidine 13C5	0	0	0	0	1665	0	9131	0	290169	172887	127729	253993	8396	23222	21476	5282	313787	168301	83485	165958
Spermidine 13C6	0	0	0	0	55508	37834	60672	38051	366730	215709	126580	266418	11578	18792	10874	5459	336493	189490	119529	173434
Spermidine 13C7	4513	0	0	0	464411	382513	517506	449439	384611	222522	130335	284413	80566	81528	59599	66054	552837	260859	164769	238619
Ornithine 13C3	0	0	2031	0	6934	4649	5824	6494	0	1616	0	8145	4754	19334	6861	11085	2927	2300	2517	0
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	m/z	r.t.
Alanine	C00041	90.05513	0.614
Arginine	C00062	175.119049	0.586
Cysteine	C00097	122.027229	0.626
Glutamate	C00025	148.060516	0.615
Glutamine	C00064	147.076462	0.611
Histidine	C00135	156.076797	0.571
Leucine/isoleucine	C00123	132.102036	0.654
Lysine	C00047	147.112869	0.566
Methionine	C00073	150.058395	0.636
Phenylalanine	C00079	166.086227	0.726
Proline	C00148	116.070801	0.639
Threonine	C00188	120.065704	0.62
Tryptophan	C00078	205.09697	1.709
Valine	C00183	118.086418	0.63
Guanosine	C00387	284.098969	0.68
Guanine	C00242	152.056671	0.657
Cytosine	C00380	112.050766	0.636
Pyridoxal	C00250	168.06546	0.638
Nicotinamide	C00153	123.05555	0.66
NAD+	C00003	664.117004	0.629
gamma-L-Glutamyl-L-cysteine	C00669	251.069519	0.637
gamma-L-Glutamyl-D-alanine	C03738	219.097626	0.631
gamma-Glutamyl-gamma-aminobutyrate	C15767	233.113266	0.635
gamma-L-Glutamylputrescine	C15699	218.149857	0.561
(5-L-Glutamyl)-L-glutamine	C05283	276.119049	0.629
Cystathionine	C00542	223.074768	0.647
Dimethylglycine	C01026	104.070808	0.644
S-Adenosyl-L-methionine	C00019	399.144531	0.605
Ornithine	C01602	133.097244	0.569
L-Citrulline	C00327	176.102905	0.617
Cadaverine	C01672	103.123207	0.567
Putrescine	C00134	89.10746	0.577
Spermidine	C00315	146.165131	0.566
Spermine	C00750	203.222931	0.563
Carnosine	C00386	227.113724	0.57
Creatine	C00300	132.076797	0.626
Creatinine	C00791	114.066399	0.665
N-Acetyl-L-citrulline	C15532	218.113373	0.631
N-Acetylornithine	C00437	175.107666	0.636
Guanidinoacetate	C00581	118.061378	0.614
trans-4-Hydroxy-L-proline	C01157	132.065582	0.663
L-Methionine S-oxide	C02989	166.053253	0.638
Indole-3-acetate	C00954	176.070541	1.925
Sphingosine	C00319	300.289703	2.023
Choline	C00114	104.107285	0.611
Acetylcholine	C01996	146.117584	0.635
L-Carnitine	C00318	162.112381	0.616
acetyl-carnitine	C02571	204.123032	0.639
propionyl-carnitine	C03017	218.138672	0.64
acyl-C5	C20826	246.169952	1.746
AC(5-OH)	HMDB0062555	262.164948	0.637
O-tetradecanoyl-L-carnitine	HMDB05066	372.31076	2.033
L-Palmitoylcarnitine	C02990	400.342133	2.114
Hexadecenoyl-carnitine	HMDB06317	398.327209	2.046
Octadecanoyl-L-carnitine	HMDB00848	428.373199	2.229
O-octadecenoyl-L-carnitine	HMDB06351	426.35791	2.136
acyl-C18:2	HMDB01064	424.34198	2.073
Arginine 13C4	C00062	179.128723	0.628
Arginine 13C5	C00062	180.136261	0.618
Arginine 13C6	C00062	181.138931	0.591
Glutamate 13C2	C00025	150.066727	0.635
Glutamate 13C3	C00025	151.070984	0.671
Glutamate 13C4	C00025	152.070648	0.641
Glutamate 13C5	C00025	153.076691	0.665
Methionine 13C2	C00073	152.068008	0.614
Methionine 13C5	C00073	155.073517	0.654
Glutathione 13C2	C00051	310.097107	0.644
Glutathione 13C3	C00051	311.100952	0.654
Glutathione 13C4	C00051	312.1026	0.661
Glutathione 13C5	C00051	313.107147	0.642
Glutathione 13C6	C00051	314.105286	0.573
Glutathione 13C8	C00051	316.121887	0.651
Glutathione disulfide 13C2	C00127	615.157227	0.634
Glutathione disulfide 13C3	C00127	616.168091	0.66
Cystathionine 13C7	C00542	230.09758	0.626
Putrescine 13C4	C00134	93.120972	0.573
Spermidine 13C2	C00315	148.171646	0.567
Spermidine 13C3	C00315	149.175232	0.574
Spermidine 13C4	C00315	150.178665	0.56
Spermidine 13C5	C00315	151.182007	0.565
Spermidine 13C6	C00315	152.18541	0.566
Spermidine 13C7	C00315	153.188599	0.569
Ornithine 13C3	C001602	136.10611	0.627
METABOLITES_END
#END