#METABOLOMICS WORKBENCH adavison_20220513_073938 DATATRACK_ID:3248 STUDY_ID:ST002179 ANALYSIS_ID:AN003568 PROJECT_ID:PR001387
VERSION             	1
CREATED_ON             	May 26, 2022, 5:52 pm
#PROJECT
PR:PROJECT_TITLE                 	Impact of nitisinone on the cerebrospinal fluid metabolome of a murine model of
PR:PROJECT_TITLE                 	alkaptonuria
PR:PROJECT_SUMMARY               	Background: Nitisinone induced hypertyrosinaemia is well documented in
PR:PROJECT_SUMMARY               	Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a
PR:PROJECT_SUMMARY               	decline in cognitive function and or mood by altering neurotransmitter
PR:PROJECT_SUMMARY               	metabolism. The aim of this work was to evaluate the impact of nitisinone on the
PR:PROJECT_SUMMARY               	cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to
PR:PROJECT_SUMMARY               	providing additional insight into metabolic changes that occur following
PR:PROJECT_SUMMARY               	treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c
PR:PROJECT_SUMMARY               	Hgd-/-mice (n=8, treated with nitisinone – 4 mg/L and n=9, no treatment).
PR:PROJECT_SUMMARY               	Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity
PR:PROJECT_SUMMARY               	II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass
PR:PROJECT_SUMMARY               	spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted
PR:PROJECT_SUMMARY               	feature extraction algorithm and an established in-house accurate mass retention
PR:PROJECT_SUMMARY               	time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3
PR:PROJECT_SUMMARY               	minutes retention time window) were filtered based on their frequency and
PR:PROJECT_SUMMARY               	variability. Experimental groups were compared using a moderated t-test with
PR:PROJECT_SUMMARY               	Benjamini-Hochberg false-discovery rate adjustment. Results: Tyrosine,
PR:PROJECT_SUMMARY               	acetyl-tyrosine, γ-glutamyl-tyrosine, p-hydroxyphenylacetic acid and
PR:PROJECT_SUMMARY               	3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold
PR:PROJECT_SUMMARY               	change 2.6-6.9, 3/5 were significant p<0.05) in the mice that received
PR:PROJECT_SUMMARY               	nitisinone. Several other metabolites of interest were matched but no
PR:PROJECT_SUMMARY               	significant differences were observed, including the aromatic amino acids
PR:PROJECT_SUMMARY               	phenylalanine and tryptophan, and monoamine metabolites adrenaline,
PR:PROJECT_SUMMARY               	3-methoxy-4-hydroxyphenylglycol and octopamine. Conclusions: Evaluation of the
PR:PROJECT_SUMMARY               	CSF metabolome of a murine model of AKU showed a significant difference in the
PR:PROJECT_SUMMARY               	abundance of a limited number of metabolites. None of these have been reported
PR:PROJECT_SUMMARY               	in CSF from a murine model of AKU previously. Moreover this study confirms that
PR:PROJECT_SUMMARY               	some monoamine metabolites do not appear to be altered following nitisinone
PR:PROJECT_SUMMARY               	therapy.
PR:INSTITUTE                     	University of Liverpool Institute of Life Course & Medical Sciences
PR:LAST_NAME                     	Davison
PR:FIRST_NAME                    	Andrew
PR:ADDRESS                       	1. Department of Clinical Biochemistry and Metabolic Medicine, Liverpool
PR:ADDRESS                       	Clinical Laboratories, Liverpool University Hospitals NHS Foundation Trust,
PR:ADDRESS                       	Liverpool, UK; 2. Department of Musculoskeletal and Ageing Science, Institute of
PR:ADDRESS                       	Life Course and Medical Sciences, University of Liverpool, Liverpool, UK 3.
PR:ADDRESS                       	School of Exercise Science, Liverpool John Moores University, Liverpool, UK
PR:EMAIL                         	andrew.davison@liverpoolft.nhs.uk
PR:PHONE                         	0151 706 4011
PR:CONTRIBUTORS                  	Davison AS1,2,*, Norman BP2, Sutherland H2,3, Milan AM1,2, Jarvis JC3, Gallagher
PR:CONTRIBUTORS                  	JA2, Ranganath LR1,2
#STUDY
ST:STUDY_TITLE                   	Impact of nitisinone on the cerebrospinal fluid metabolome of a murine model of
ST:STUDY_TITLE                   	alkaptonuria
ST:STUDY_SUMMARY                 	Background: Nitisinone induced hypertyrosinaemia is well documented in
ST:STUDY_SUMMARY                 	Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a
ST:STUDY_SUMMARY                 	decline in cognitive function and or mood by altering neurotransmitter
ST:STUDY_SUMMARY                 	metabolism. The aim of this work was to evaluate the impact of nitisinone on the
ST:STUDY_SUMMARY                 	cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to
ST:STUDY_SUMMARY                 	providing additional insight into metabolic changes that occur following
ST:STUDY_SUMMARY                 	treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c
ST:STUDY_SUMMARY                 	Hgd-/-mice (n=8, treated with nitisinone – 4 mg/L and n=9, no treatment).
ST:STUDY_SUMMARY                 	Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity
ST:STUDY_SUMMARY                 	II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass
ST:STUDY_SUMMARY                 	spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted
ST:STUDY_SUMMARY                 	feature extraction algorithm and an established in-house accurate mass retention
ST:STUDY_SUMMARY                 	time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3
ST:STUDY_SUMMARY                 	minutes retention time window) were filtered based on their frequency and
ST:STUDY_SUMMARY                 	variability. Experimental groups were compared using a moderated t-test with
ST:STUDY_SUMMARY                 	Benjamini-Hochberg false-discovery rate adjustment. Results: Tyrosine,
ST:STUDY_SUMMARY                 	acetyl-tyrosine, γ-glutamyl-tyrosine, p-hydroxyphenylacetic acid and
ST:STUDY_SUMMARY                 	3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold
ST:STUDY_SUMMARY                 	change 2.6-6.9, 3/5 were significant p<0.05) in the mice that received
ST:STUDY_SUMMARY                 	nitisinone. Several other metabolites of interest were matched but no
ST:STUDY_SUMMARY                 	significant differences were observed, including the aromatic amino acids
ST:STUDY_SUMMARY                 	phenylalanine and tryptophan, and monoamine metabolites adrenaline,
ST:STUDY_SUMMARY                 	3-methoxy-4-hydroxyphenylglycol and octopamine. Conclusions: Evaluation of the
ST:STUDY_SUMMARY                 	CSF metabolome of a murine model of AKU showed a significant difference in the
ST:STUDY_SUMMARY                 	abundance of a limited number of metabolites. None of these have been reported
ST:STUDY_SUMMARY                 	in CSF from a murine model of AKU previously. Moreover this study confirms that
ST:STUDY_SUMMARY                 	some monoamine metabolites do not appear to be altered following nitisinone
ST:STUDY_SUMMARY                 	therapy.
ST:INSTITUTE                     	University of Liverpool Institute of Life Course & Medical Sciences
ST:LAST_NAME                     	Davison
ST:FIRST_NAME                    	Andrew
ST:ADDRESS                       	1. Department of Clinical Biochemistry and Metabolic Medicine, Liverpool
ST:ADDRESS                       	Clinical Laboratories, Liverpool University Hospitals NHS Foundation Trust,
ST:ADDRESS                       	Liverpool, UK; 2. Department of Musculoskeletal and Ageing Science, Institute of
ST:ADDRESS                       	Life Course and Medical Sciences, University of Liverpool, Liverpool, UK 3.
ST:ADDRESS                       	School of Exercise Science, Liverpool John Moores University, Liverpool, UK
ST:EMAIL                         	andrew.davison@liverpoolft.nhs.uk
ST:PHONE                         	0151 706 4011
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	17
#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           	-	Hgd81.1 + Nitisinone NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_81.1_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.2 + Nitisinone NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_81.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.3 + Nitisinone NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_81.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.1 + Nitisinone NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_82.1_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.2 + Nitisinone NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_82.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.1 + Nitisinone NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_83.1_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.2 - No treatment NEG	Treatment group:Nitisinone	RAW_FILE_NAME=NEG_AKU_+ NTBC_83.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd80.3 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_80.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd80.4 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_80.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.4 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_81.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.5 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_81.5_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.6 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_81.6_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.3 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_82.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.4 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_82.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.3 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_83.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.4 - No treatment NEG	Treatment group:No Treatment (control)	RAW_FILE_NAME=NEG_AKU_83.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd80.2 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_80.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.1 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_81.1_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.2 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_81.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.3 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_81.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.1 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_82.1_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.2 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_82.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.1 + Nitisinone POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_83.1_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.2 - No treatment POS	Treatment group:Nitisinone	RAW_FILE_NAME=POS_AKU_+ NTBC_83.2_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd80.3 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_80.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd80.4 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_80.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.4 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_81.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.5 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_81.5_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd81.6 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_81.6_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.3 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_82.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd82.4 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_82.4_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.3 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_83.3_CSF
SUBJECT_SAMPLE_FACTORS           	-	Hgd83.4 - No treatment POS	Treatment group:No Treatment (control)	RAW_FILE_NAME=POS_AKU_83.4_CSF
#COLLECTION
CO:COLLECTION_SUMMARY            	A murine model of AKU was used for all experiments as described previously
CO:COLLECTION_SUMMARY            	(Preston et al., 2014). Seventeen BALB/c Hgd-/- mice (Figure 5 for age and
CO:COLLECTION_SUMMARY            	gender) were included in this study, 8 mice were administered nitisinone (4
CO:COLLECTION_SUMMARY            	mg/L) through their drinking water for 6 days, the remaining 9 mice received no
CO:COLLECTION_SUMMARY            	nitisinone. All animals were housed in air conditioned rooms (with a 12 h
CO:COLLECTION_SUMMARY            	dark/light cycle) at 20 °C and 53 % humidity, with access to food and water ad
CO:COLLECTION_SUMMARY            	libitum at Liverpool John Moores University. All animal experiments complied
CO:COLLECTION_SUMMARY            	with the ARRIVE guidelines and were carried out in accordance with the U.K.
CO:COLLECTION_SUMMARY            	Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU
CO:COLLECTION_SUMMARY            	Directive 2010/63/EU for animal experiments. All mice were culled by carbon
CO:COLLECTION_SUMMARY            	dioxide asphyxiation and CSF was removed from the cisterna magna following the
CO:COLLECTION_SUMMARY            	puncture technique of Liu et al. (56), with some modifications. The cisterna
CO:COLLECTION_SUMMARY            	magna was exposed by dissecting skin and overlaying muscles. Cauterisation was
CO:COLLECTION_SUMMARY            	used to dry up any bleeding from surrounding tissues. A pulled glass pipette
CO:COLLECTION_SUMMARY            	with an internal diameter of ~0.4 mm was attached to silicone tubing and
CO:COLLECTION_SUMMARY            	connected to a 1 mL syringe with a 19 g needle. The tip of the glass pipette was
CO:COLLECTION_SUMMARY            	used to puncture the membrane and held still just below the membrane by 1
CO:COLLECTION_SUMMARY            	person. Through a double-headed microscope a second person then used the 1 mL
CO:COLLECTION_SUMMARY            	syringe to apply gentle pressure to encourage the CSF to flow up the capillary
CO:COLLECTION_SUMMARY            	tube. Only clear CSF samples (i.e. non blood stained) were collected. Once
CO:COLLECTION_SUMMARY            	collected CSF samples were transferred to clear Eppendorf tubes and stored at
CO:COLLECTION_SUMMARY            	-80 °C until analysis. Samples were not acidified. The same glass pipette was
CO:COLLECTION_SUMMARY            	used to collect CSF from each animal, and was washed with pure water between
CO:COLLECTION_SUMMARY            	each animal.
CO:SAMPLE_TYPE                   	CSF
#TREATMENT
TR:TREATMENT_SUMMARY             	9 mice control 8 mice received 4 mg/L nitisinone for 6 days in drinking water
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Murine CSF samples were prepared by diluting 2 µL CSF with 2 µL deionized
SP:SAMPLEPREP_SUMMARY            	water (DIRECT-Q 3UV Millipore water purification system). CSF samples were
SP:SAMPLEPREP_SUMMARY            	pipetted directly into a 150 µL glass insert with polymer feet, which sat in a
SP:SAMPLEPREP_SUMMARY            	2 mL amber screw vial (Agilent, Cheadle, UK). These vials were used to ensure
SP:SAMPLEPREP_SUMMARY            	maximal recovery of sample. Deionised water was also added directly to the glass
SP:SAMPLEPREP_SUMMARY            	insert, and mixed with the CSF sample using a pipette. The glass vials were
SP:SAMPLEPREP_SUMMARY            	centrifuged at 600 rpm for 10 min to ensure the diluted sample was at the bottom
SP:SAMPLEPREP_SUMMARY            	of the vial for sampling. 1 µL of diluted CSF was analysed in negative and
SP:SAMPLEPREP_SUMMARY            	positive polarities, respectively.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Liquid chromatography (LC) was performed on an Agilent 1290 Infinity II LC
CH:CHROMATOGRAPHY_SUMMARY        	system. An Atlantis dC18 column (3.0x100mm, 3µm, Waters, UK) was maintained at
CH:CHROMATOGRAPHY_SUMMARY        	60°C with a flow rate of 0.4mL/min. Mobile phases were (A) water and (B)
CH:CHROMATOGRAPHY_SUMMARY        	methanol both containing 5mmol/L ammonium formate and 0.1% formic acid. The
CH:CHROMATOGRAPHY_SUMMARY        	elution gradient started at 5% mobile phase B at 0-1 min increasing linearly to
CH:CHROMATOGRAPHY_SUMMARY        	100% B by 12 min, held at 100% B until 14 min, returning to 95% A for 5 min to
CH:CHROMATOGRAPHY_SUMMARY        	recondition the column. Injection volume was 1µL.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent Infinity II
CH:COLUMN_NAME                   	Atlantis dC18 column (3.0x100mm, 3µm, Waters, UK)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6550 QTOF
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Quadrupole time-of-flight mass spectrometry (QTOF-MS) conditions An Agilent 6550
MS:MS_COMMENTS                   	QTOF-MS equipped with a dual jet stream electrospray ionisation source was
MS:MS_COMMENTS                   	operated in 2GHz mode, over the mass range of 50-1700, in negative and positive
MS:MS_COMMENTS                   	polarities. A reference mass correction solution was continually infused at a
MS:MS_COMMENTS                   	flow rate of 0.5 mL/min via an external isocratic pump (Agilent, UK) for
MS:MS_COMMENTS                   	constant mass correction (see Preparation of reference mass correction
MS:MS_COMMENTS                   	solution). Capillary and fragmentor voltages were 4000 V and 380 V,
MS:MS_COMMENTS                   	respectively. Desolvation gas temperature was 200 °C with flow rate at 15
MS:MS_COMMENTS                   	L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min and
MS:MS_COMMENTS                   	nebulizer pressure was 40 psi and nozzle voltage 1000 V. Data acquisition rate
MS:MS_COMMENTS                   	was 3 spectra/s. Preparation of reference mass correction solution Reference
MS:MS_COMMENTS                   	mass correction solution was prepared in 95:5 methanol:water containing 5 mmol/L
MS:MS_COMMENTS                   	purine (CAS No. 120-73-0), 100 mmol/L trifluoroacetic acid ammonium salt (TFA,
MS:MS_COMMENTS                   	CAS No. 3336-58-1) and 2.5 mmol/L hexakis(1H, 1H,
MS:MS_COMMENTS                   	3H-tetrafluoropropoxy)phosphazine (HP-0921, CAS No. 58943-98-9) (Agilent,
MS:MS_COMMENTS                   	Cheadle, UK). Reference ions monitored were: purine (m/z 121.0509) and HP-0921
MS:MS_COMMENTS                   	(m/z 922.0098) (positive polarity) and TFA (m/z 112.9856), purine (m/z 119.0363)
MS:MS_COMMENTS                   	and HP-0921 (HP-0921 + formate adduct: m/z 966.0007) (negative polarity). Data
MS:MS_COMMENTS                   	acquisition and handling parameters Data were acquired using Acquisition (Build
MS:MS_COMMENTS                   	06.00, Agilent, Cheadle, UK). Quality checks and processing of raw data files
MS:MS_COMMENTS                   	(Agilent ‘.d’ files) were performed with Qualitative Analysis software
MS:MS_COMMENTS                   	(Build 07.00, Agilent, Cheadle, UK). Extracted ion chromatograms of reference
MS:MS_COMMENTS                   	masses were performed to check mass accuracy remained <5 ppm throughout the run
MS:MS_COMMENTS                   	and that the reference ion signal did not drop out during the chromatographic
MS:MS_COMMENTS                   	run. In addition, to check chromatographic reproducibility binary pump pressure
MS:MS_COMMENTS                   	curves for injections across each analytical sequence were overlaid. Mass
MS:MS_COMMENTS                   	accuracy and chromatographic reproducibility were acceptable for all experiments
MS:MS_COMMENTS                   	performed. Acquired profiling sample data were mined for signals against an
MS:MS_COMMENTS                   	established in-house AMRT database of compounds (contains theoretical accurate
MS:MS_COMMENTS                   	mass, measured retention time, and empirical formula for 469 intermediary
MS:MS_COMMENTS                   	metabolites, MW 72-785) using ‘targeted feature extraction’ with Profinder
MS:MS_COMMENTS                   	software (Build 08.00, Agilent, Cheadle, UK). Targeted feature extraction uses
MS:MS_COMMENTS                   	the molecular formulae from the AMRT database to extract and group spectral
MS:MS_COMMENTS                   	signals (i.e. adducts, isotopes and multimers) that correspond to individual
MS:MS_COMMENTS                   	database compounds. Feature extraction employed a window of theoretical accurate
MS:MS_COMMENTS                   	mass ±10 ppm and database retention time ±0.3 min. Allowed species were: H+,
MS:MS_COMMENTS                   	Na+ and NH4+ (positive polarity) and H- and CHO2- (negative polarity). Dimers
MS:MS_COMMENTS                   	were allowed for both polarities. Charge state range was 1-2. Data files were
MS:MS_COMMENTS                   	then exported from Profinder as ‘.CEF’ files as a whole batch for each
MS:MS_COMMENTS                   	profiling experiment and imported to Mass Profiler Professional (MPP) software
MS:MS_COMMENTS                   	for statistical analysis (Build 14.5, Agilent, Cheadle, UK).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	area
MS_METABOLITE_DATA_START
Samples	Hgd80.2 + Nitisinone POS	Hgd81.1 + Nitisinone POS	Hgd81.2 + Nitisinone POS	Hgd81.3 + Nitisinone POS	Hgd82.1 + Nitisinone POS	Hgd82.2 + Nitisinone POS	Hgd83.1 + Nitisinone POS	Hgd83.2 - No treatment POS	Hgd80.3 - No treatment POS	Hgd80.4 - No treatment POS	Hgd81.4 - No treatment POS	Hgd81.5 - No treatment POS	Hgd81.6 - No treatment POS	Hgd82.3 - No treatment POS	Hgd82.4 - No treatment POS	Hgd83.3 - No treatment POS	Hgd83.4 - No treatment POS
Factors	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:Nitisinone	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)	Treatment group:No Treatment (control)
L-Histidine	78933	72314	64514	36100	55730	49207	62860	64484	47029	57276	46393	45511	73233	54586	55459	70186	64495
Inosine	3903310	4005959	3069856	3496044	4261473	3752630	4043093	4188349	3794904	3774884	3899201	3892178	3670710	3659707	3958599	4380851	4397641
Adipic acid	139999	71929	45803	351712	144891	76400	138106	688082	105986	80090	111599	150378	85783	246463	245567	118607	225067
Sarcosine	178538	163956	141587	160669	254977	180632	166993	188636	163012	162463	148506	178385	200702	162211	179038	185995	173257
6-Deoxy-L-galactose	889697	1251635	660939	687862	397994	533422	782823	683651	1177064	1094671	839623	978886	929904	1038672	970744	1280665	1149318
L-Pipecolic acid	49302	21919	20078	28054	106503	69113	19099	17758	38017	48733	35146	57446	34751	65513	69077	27954	26177
Ethylmalonic acid	8950	6971	6802	13787	12484	16061	7428	14577	7816	11368	9340	14080	7236	1	11472	10403	12474
D-(-)-Lyxose	124071	153992	120694	127395	65086	90135	144368	129393	205460	167131	138531	116688	117816	82946	153443	160433	162398
Theobromine	37255	17965	19980	10385	21625	10307	6134	11392	24414	11825	18292	28486	26422	6476	19602	12037	14662
Hippuric acid	39405	10633	6284	6883	20252	9899	3260	13762	18653	12992	4761	75308	20282	5232	16716	10301	13724
Oleic Acid	35404	21421	50788	10013	16049	10111	32735	29385	29136	65847	31300	35682	44761	56559	47787	37487	28105
3,4-Dihydroxybenzoic acid	80115	58721	107102	102591	112969	96432	50443	86773	63977	95823	107245	102728	122295	71642	83618	89812	87811
Kynurenine	2609	1	1	1	1	1	1	1	1	1	1	1	2058	1	1	1	3442
Methyl jasmonate	55384	50979	56450	44666	39592	38396	48517	57443	42147	24305	56308	47681	58666	61778	8805	57913	45889
Thiamine monophosphate	11372	7958	11377	10713	8087	9739	9490	10827	9349	13469	8916	8667	10083	8751	10767	11222	12363
Dulcitol	99543	121818	241108	193481	64188	127342	75775	46056	92218	70610	87297	594385	59929	325844	143663	68748	246722
myo-Inositol	950049	188581	169483	405522	169087	111661	134825	212506	1003364	485925	245433	450223	706585	356792	748828	250961	1132243
Hypoxanthine	2867512	1.09E+07	8283657	1.01E+07	1.16E+07	1.10E+07	1.06E+07	1.07E+07	1.20E+07	3124488	1.14E+07	3584142	1.16E+07	1.10E+07	1.31E+07	1.21E+07	395998
Phosphocreatine	119741	94986	105833	105489	94482	102559	92077	102162	138748	134942	118316	112004	92737	87285	101397	102959	136210
Thymidine	69676	82300	48232	61958	96034	77261	93238	90870	65673	58931	60172	69530	80214	93119	76923	75956	88750
1-methylhistidine	15546	16117	19151	11284	47715	34710	19153	12829	10002	21228	29245	22835	22725	40741	47230	22310	18332
DL-pipecolic acid	49302	21913	20112	28058	106503	69115	19102	17758	37929	48680	35146	57439	34751	65513	69077	27951	26177
4-acetamidobutanoate	18573	15623	3067	25754	38081	18542	10690	36670	22654	24477	15873	35583	12747	31201	21486	42080	52261
Theophylline	13836	2179	7743	3704	7269	1037	1	1	8371	3191	6104	13140	9370	861	7886	5095	4108
L-Tryptophan	484165	363105	241291	314349	327042	290644	296658	472479	442315	458497	315894	575480	434960	178406	417303	452340	625359
Niacinamide	3911972	5049253	2614194	4285072	1.00E+07	6882845	6205599	5537467	5262513	5205195	6050538	6394836	5054419	6649651	7078067	6629124	6037039
Cytidine	4510614	5107240	3415030	4057828	3049089	4130054	5285237	5161713	4598287	4140941	4336203	5031404	4511121	5112675	5081954	4822599	4760439
L-Threonine	124077	106973	197165	105458	66717	75092	121062	121120	110201	112946	112564	123370	92918	101837	89639	195037	171001
Malic acid	13841	11967	20298	17293	36353	23538	13879	20476	9282	11781	22133	15343	28331	12785	21987	18406	18509
L-Isoleucine	648897	523715	457972	511396	755224	567192	478361	653580	661404	597771	492557	780631	592363	399523	635290	630352	729273
N-Acetyl-L-glutamic acid	24816	26799	21622	30367	30915	39825	36569	47558	41711	57776	48044	51853	63495	38967	70302	55714	61409
D-Mannitol	99460	121823	241108	193481	64188	127342	75775	46056	92228	70612	87297	594383	59927	325857	143663	68757	246722
L-Aspartic Acid	40690	43064	25709	26831	16201	29121	35989	40697	34298	38093	34426	33318	29628	40139	38218	43074	50055
Adenosine	1073354	1249020	9662401	215730	823376	896599	1331946	3244738	372430	284084	2251129	863852	9513782	68700	228664	589816	601080
5-Hydroxyindoleacetic acid	61991	45620	50676	9979	17919	13941	46445	40196	23893	13574	99686	68144	53576	24128	14469	74878	50863
Creatine	4.38E+07	4.14E+07	3.34E+07	4.01E+07	7.17E+07	4.89E+07	4.22E+07	4.78E+07	4.01E+07	4.13E+07	3.84E+07	4.80E+07	5.13E+07	4.10E+07	4.79E+07	4.72E+07	4.41E+07
2-Aminoisobutyric acid	9848	7422	19433	49971	31653	70764	21641	18670	19442	43558	17927	25601	28204	13607	48658	29985	26053
Acetyl-L-tyrosine	38506	34486	17714	20641	35271	18200	31330	39973	2523	2945	1574	3128	1899	2460	2984	3573	5565
p-Hydroxyphenylacetic acid	18927	11060	7490	6964	6206	4491	10471	11819	1	1	1	1	1	1	1	1	1
Quinic acid	11410	7543	7840	16447	1	11881	6471	4771	17581	14877	10319	10315	8672	9775	15567	8524	8843
Methyl N-(a-methylbutyryl)glycine	129548	70814	70924	803809	175604	102955	194797	1692926	129280	78299	118890	176404	104682	2091839	374433	153962	160638
3-Methyladenine	102093	121406	110355	157003	60323	62652	134773	121790	169751	169866	127049	74568	72724	63920	139495	101634	137192
Maleamic acid	18247	21090	1	13892	1	16745	18257	18253	1	1	1	20126	1	14430	16861	19234	37901
D-Aspartic acid	33921	34882	23402	23199	13752	25310	29649	33543	29307	33927	30440	27843	25733	34209	32642	36165	37642
12-Hydroxydodecanoic acid	19983	6859	19435	17832	3620	5502	13359	61441	3914	2853	9615	6114	5982	21797	4141	8467	8721
Histamine	33320	1	1	1	96482	40222	1	1	1	1	1	1	40055	1	1	1	1
Pantothenic Acid	4486677	3397242	2813678	3240376	2668242	2405047	4097222	4442885	4282374	4426220	4294624	4787631	3592439	2411664	2928244	4986370	5117808
3-Ureidopropionic acid	56364	46945	37348	61562	21276	27813	63739	42959	139168	65260	82795	54495	43458	51045	34343	54205	99439
L-(-)-Arabitol	209690	194341	182293	173086	43195	88843	211035	155810	274643	201029	245813	152948	143206	152253	143710	188886	225755
Glutamine	9191101	9583462	4541594	4359480	4609960	4776238	7452358	7229892	7814806	7257660	6541876	7207384	6683188	9410353	6449269	8370120	6830842
2-Deoxy-D-Ribose	96830	71409	80198	99809	53841	98847	158334	83671	97319	64716	45314	54115	86225	49845	63008	70072	103871
salsolinol	15907	23488	8045	37025	58533	51407	8561	6863	134985	144296	17127	51062	2831	33083	76735	12970	13788
Creatinine	1136294	988610	929513	906217	566178	882648	849493	834809	990463	969407	894700	1036602	890863	895321	702092	926307	913870
Cytosine	2090936	2340845	1580444	1843658	1420428	1911003	2433749	2400016	2085904	1867587	1924390	2296859	2092098	2439508	2325851	2197861	2162801
Adenosine5'-monophosphate	262503	74535	80684	384935	3375091	513435	103799	90427	340015	887837	492611	292868	165791	206404	1257455	80601	158902
D-Mannose 6-phosphate	27883	20768	37869	37592	61973	28845	25591	33211	26223	39443	36361	27718	20945	36053	33442	22877	32939
a-N-Acetylglucosamine	360434	442099	291868	315003	140830	258389	371571	356361	374164	347179	374016	370484	348628	398491	301852	387635	362560
L-Alanine	186277	171963	149598	169553	263062	189912	176276	197514	171298	171559	156739	186586	208527	170640	187096	195756	182546
N-Acetyl-DL-serine	106010	121452	110214	108350	77638	98045	141117	115061	106832	95872	128409	97813	107605	140841	94679	99086	118928
N-Formylmethionine	5631	8094	4480	5299	10433	6438	6734	7823	8667	9105	8057	8630	6665	7386	7274	7108	9335
N-Acetyl-DL-methionine	61991	45620	50676	9979	17919	13941	46445	40196	23893	13574	99686	68144	53576	24128	14469	74878	50863
Diacetyl	58942	47797	62348	48928	45666	36127	53042	50207	43567	48787	48438	49913	63045	59772	37607	51168	42345
1-Methyladenosine	7826	9147	5762	3148	6004	5176	6866	7715	4838	4219	6356	7656	9653	8447	2703	7528	6250
Xanthurenic acid	3108	1	1	20488	40958	1	1	1	63402	100819	1	150	7223	9315	59007	1	1
L-Anserine	92778	88412	52555	60096	108108	69322	67889	76022	78176	78745	78115	77995	81264	87667	74114	84593	80558
D-Fructose 6-phosphate	27883	20768	37869	37592	61973	28845	25591	33211	26223	39443	36361	27718	20945	36053	33442	22877	32939
Diethanolamine	10937	35086	45274	24161	25767	64724	13791	7754	25172	15625	21538	122989	15579	17153	11163	18703	45891
Pimelic acid	25341	11678	5166	86281	28711	12932	25262	185237	16399	16464	18423	30127	16288	59591	46389	24314	45471
Suberic acid	69324	33370	24379	283325	68594	39843	74926	580392	59643	48476	52982	81658	51760	186128	120942	77105	94969
D-MANNOSAMINE	418732	379238	192566	199201	301297	764294	310352	312291	337509	414061	424808	294307	302410	544740	303492	304609	825685
D-Ribose	124562	153992	120675	127395	65086	90135	144368	129393	205460	167131	138531	116688	117816	82894	153443	160433	163217
Deoxycytidine	72472	99358	50588	65084	60406	77961	96248	97541	80813	69173	83816	87404	80269	145513	97991	105799	96236
N-Methyl-D-aspartic acid	3652	1	1	78716	120038	90951	1	14485	1	111442	1	13057	54143	1	83124	3447	5516
?-D-Glucose 1-phosphate	27883	20768	37869	37592	61973	28845	25591	33211	26223	39443	36361	27718	20945	36053	33442	22877	32939
S-(5-ADENOSYL)-L-METHIONINE	5534	5335	4735	5263	9198	5155	4927	5621	4900	7085	2160	5095	5083	5286	5755	5417	6161
Xanthine	202526	217944	136721	217129	254797	230629	200330	230096	210971	214055	189332	242014	212343	173839	277372	219403	213872
Glyceryl phosphate	99089	93451	114184	170614	122422	152552	101401	105599	141639	139771	113967	121356	126457	68517	141176	115957	106775
L-Norleucine	648897	523654	457726	512158	755224	569499	478361	653580	661404	598548	496412	780658	598324	398440	635290	635441	728542
L-Serine	23944	22999	48429	26925	26215	25360	29292	31457	28177	18045	25143	24844	36674	16747	21955	40606	30192
Xanthosine	12644	16113	11118	18867	31140	22400	16131	26757	18039	17507	13671	27158	19066	15296	29709	19130	20198
D-Ornithine	27998	14961	16689	9022	14641	9534	9165	12453	17512	12262	10167	16369	15772	10956	13076	22915	16352
D-(+)-Xylose	124071	153992	120714	127395	65086	90135	144368	129393	205460	167131	138531	116688	117816	82946	153443	160433	162398
Corticosterone	19516	1	17138	6597	1	7783	1	29747	6612	1	3428	13606	3251	1	1	14757	8504
N-Acetyl-?-D-mannosamine	360434	442099	291868	315003	140830	258389	371571	356361	374164	347179	374016	370484	348628	398491	301852	387635	362560
DL-Homoserine	124077	106973	197165	105458	66717	75092	121062	121120	110201	112946	112564	123370	92918	101837	89639	195037	171001
D-(+)-Malic acid	13841	7961	13442	17886	36727	25043	13879	24917	9355	11781	11285	16400	30880	13277	22027	20153	20823
4-Pyridoxic acid	13519	8247	6364	9684	8692	10181	10568	16328	8779	9367	9053	14013	1	4471	9582	14808	11138
Dihydroxyphenylacetic acid	133388	75825	53846	322258	139071	81742	134534	589007	106251	87973	116340	138867	86300	226977	222760	119535	202991
D-Glucosamine 6-phosphate	74047	95119	48395	54645	35172	49186	70260	71551	69842	64374	64737	74484	85423	90107	56684	79311	67953
N-Methylhistamine	4972	13641	14505	20310	13951	20191	12067	18105	11004	10142	18029	15938	21357	3614	72487	13711	17793
Ketoleucine	18251	11319	11653	14560	36997	33387	12453	21757	26723	12127	14267	8441	20268	21683	19576	16200	25149
AHBA	59162	65257	52769	48652	59456	48262	63940	55720	53240	59727	48355	60197	63497	58266	49755	59854	56817
L-Phenylalanine	1459871	1275819	737686	952560	1530803	1044089	1188752	1532597	1397008	1350516	993322	1696131	1281485	1006935	1449457	1402069	1644901
3-hydroxy-3-methyl-Glutaric acid	35052	27115	23092	33466	27977	29898	32057	39967	30836	37427	22783	38306	40219	25824	29072	43206	50802
L-Methionine	1930000	1458323	1484296	1548535	1974145	1904929	1424697	1848920	1668596	1796335	1431958	2234768	2403323	1561466	1569687	1566663	1503206
N-Acetyl-D-galactosamine	360434	442099	291868	315003	140830	258389	371571	356361	374164	347179	374016	370484	348628	398491	301852	387635	362560
N-Acetyl-L-alanine	145999	172313	113381	117602	159316	120155	153151	163606	138405	148652	150809	157931	138908	156697	154410	154707	172679
3-Methoxy-4-hydroxyphenylethylene glycol	25723	29847	28936	21719	16235	20595	22030	21381	17080	23535	26409	19573	24639	22267	18675	19674	19468
Ribitol	209690	194341	182293	173086	43195	88843	211035	155810	274643	201029	245813	152948	143206	152253	143710	188886	225755
?-Aminobutryic acid	6317	7422	19430	49968	31653	70680	21669	18670	19582	43558	17843	25601	27997	13607	48739	29985	25493
Cortexolone	19558	1	17344	6586	1	7739	1	29760	7597	1	3428	13522	3203	1	1	14735	8448
Spermidine	297786	220242	104279	473988	1853405	819257	234826	253074	815999	1198652	570641	691812	663008	525840	1237556	163881	325102
R-(-)-Mandelic acid	12704	6881	21114	23842	25815	11298	42351	23880	19897	17387	22326	9224	9356	12424	27767	11103	26204
4-Hydroxy-L-phenylglycine	19734	15952	21026	24975	2338	21962	23519	24675	25212	13544	25446	24689	20433	35997	23621	19788	23485
Inosine 5'-monophosphate (IMP)	5374	1	1	9396	588420	15836	1	1	7198	7113	6456	7222	18711	16682	34414	1	1
Pyridoxamine	341228	381887	355368	398975	396262	373127	428085	450476	422605	358672	415862	420577	385567	302481	324379	501424	1
Taurine	164893	160782	210653	312008	1459491	590531	217879	298694	171914	199491	215705	264472	347092	137394	367011	256747	191605
N?,N?,N?-Trimethyllysine	27959	27464	29481	35007	27948	28838	27426	26282	33538	36084	33627	29657	27522	26538	45535	27154	28558
L-Ornithine	27998	14961	16689	9022	14641	9534	9165	12453	17512	12262	10167	16369	15772	10956	13102	22915	16352
L-Asparagine	9847	8104	10477	13717	11311	13744	9944	12456	2281	10503	7832	10693	10346	3048	10756	12408	11358
Urocanic acid	2589305	1048339	1526418	405762	1204334	339045	66451	468377	1481460	672216	1037673	1489301	1467676	406775	961315	2587575	1830999
D-Glucose 6-phosphate	27883	20768	37869	37592	61973	28845	25591	33211	26223	39443	36361	27718	20945	36053	33442	22877	32939
L-(-)Sorbose	950049	188581	169483	405522	179934	124673	134825	212506	1003364	485925	245433	450223	706585	1868784	748828	250601	1132243
3-Amino-4-hydroxybenzoic acid	59162	65257	52769	48652	59456	48262	63940	55720	53240	59727	48355	60197	63497	58266	49755	59854	56817
Guanine	79136	87519	795332	76397	77938	98133	140575	569368	78222	74653	109781	138459	103683	69264	68424	494307	95212
L-Lysine	204031	224923	212428	231501	218049	207200	239437	246269	229437	201940	241037	220877	225797	177986	169687	282753	298398
Allothreonine	124077	106964	197165	105458	66717	75092	121062	121120	110201	112946	112564	123370	92918	101837	89639	195037	171001
Citrulline	127289	122464	69096	44672	49108	35276	88040	90149	111857	92458	90098	104314	102615	115537	65372	112176	272903
D-Tryptophan	484165	363105	241291	314349	327042	290644	296658	472464	442315	458497	315894	575480	434960	178406	417303	452340	625393
D-Alanine	178538	163956	141587	160669	254977	180632	167830	189538	163012	162463	148506	178385	200702	163082	179795	186842	174153
(-)-Epinephrine	27232	23671	32336	51600	81158	61850	34275	37750	36012	44985	46892	29703	39013	25349	51395	33313	46004
O-Acetylserine	118789	133630	122382	123589	95443	103531	151203	127352	111071	102356	151496	110498	134376	151545	115299	112773	136068
Petroselinic acid	35404	21421	50788	10013	16049	10111	32735	29385	29136	65847	31300	35682	44761	56559	47787	37487	28311
L-Proline	1240560	787265	786858	888097	1408531	969542	672960	965627	1135148	1203924	808566	1135429	1058275	1881844	1423480	1080267	1595876
Isocitrate	289730	120459	239619	178658	155482	303215	168695	297665	137163	147273	163779	248030	290403	571501	320236	218178	227790
RAC-GLYCEROL 1-MYRISTATE	6326	1	4189	5997	6658	4264	6997	6266	7702	3859	5598	1	6079	1	1	6289	5302
Glucosamine	418728	379238	192566	199201	301297	764079	310352	312291	337509	414061	424808	294307	302410	544740	303492	304609	825528
Deoxyuridine monophosphate (dUMP)	13409	15836	9764	13130	14160	10301	13724	13589	14078	12703	11049	15439	12618	11672	10720	13795	14174
Glycerol 2-phosphate	99089	93451	114184	170614	122422	152480	101401	105599	141639	139771	113967	121356	126457	68517	141176	115957	106775
D-Sorbitol	99461	121823	241108	193481	64188	127342	75775	46056	92228	70614	87297	594383	59927	325857	143663	68757	246722
METHYL BETA-D-GALACTOSIDE	33955	53491	60288	78831	16656	44700	67635	59832	30337	26508	51807	25166	52199	42804	43891	80729	73374
?-D-Galactose 1-phosphate	27883	20768	37869	37592	61973	28845	25591	33211	26223	39443	36361	27718	20945	36053	33442	22877	32939
D-Ribose 5-phosphate	6170	3431	1	17875	38367	15208	1530	350	20345	26713	3689	592	1	5521	26871	547	2216
3-Hydroxyphenylacetic acid	19006	11295	7511	6964	6206	4491	10062	11819	1	1	1	1	1	1	1	1	1
Diaminopimelic acid	13068	15167	1	10680	4793	9257	11929	11515	19586	19058	14431	12849	1	23205	13172	1	17298
Uridine	445955	587302	394270	474033	619842	523319	666208	695903	580585	562245	625050	631342	603576	617596	681461	672543	682061
Caffeine	86507	53476	80540	51178	66229	27489	58987	74433	72191	56439	46725	85472	255426	22144	47152	50774	49899
O-Phosphorylethanolamine	9800	10276	15846	21510	15280	28034	12889	15326	12489	22127	15206	15779	16703	9091	18577	12730	14160
L-Norvaline	780862	539568	471701	503084	605157	499515	544525	642219	657687	569530	547108	800941	715389	576995	1826	634339	635745
L-Leucine	1366785	1248563	865529	1034572	1745832	1182367	1083663	1458566	1417130	1277322	1036614	1607846	1331533	1057715	1420164	1431289	1568522
Trigonelline	237796	243967	169543	194245	252398	255196	246608	283848	294815	328373	269741	352744	311621	1231363	290950	333746	323527
Xylitol	209690	194341	182293	173086	43195	88843	211035	155810	274643	201029	245813	152948	143206	152253	143710	188886	225755
Glutamyl-Tyrosine	38763	27781	18637	24045	30095	24243	24128	34167	4199	3730	1	4792	3296	24847	4105	5901	7351
L-Arginine	719830	588361	583241	524708	425052	434875	594700	673097	736464	769029	633215	715437	663594	790716	640376	686799	832491
5?-Deoxy-5?-(methylthio)adenosine	23881	27924	15512	17123	41392	18269	23337	26903	25056	33963	21365	24386	32996	28486	25109	26037	24905
2-Deoxyglucose	855902	1251411	660488	688022	397994	533422	782611	684868	1177109	1094647	840758	978886	929892	1054803	970744	1280694	1149305
L-Tyrosine	6038927	3903738	3202107	3059504	3753154	3166419	3427897	4290325	509967	535885	446213	608297	490969	408761	610635	689503	751775
Deoxyguanosine	1073354	1249020	9592584	215730	823376	896599	1331946	3244738	372430	284084	2251129	863852	9513782	68700	228664	589816	601080
N-acetylaspartate	324547	321204	318871	535930	370255	558106	486829	835096	253002	809402	355373	451879	1096768	252417	529230	506435	420094
3-Methylglutaric acid	134864	71718	38464	351712	144693	76400	136284	685265	105942	80090	111483	150387	85466	246463	245567	118639	225025
N-Acetylneuraminic Acid	787798	946528	865256	1064271	541457	836566	977123	894853	1060679	1024457	1052720	925977	839118	928057	927295	1002385	968096
Octopamine (p-Hydroxyphenylethanolamine)	21107	18479	14887	13772	10988	12423	17565	17351	21066	20708	18728	16910	16372	20966	18410	20193	19729
S-Adenosylhomocysteine	10050	12918	7692	16697	61984	18291	13839	16889	17810	23463	15619	17101	28333	12292	47936	15631	17845
Riboflavin (Vitamin B2)	8899	8239	3990	5663	3290	6002	7891	8766	6959	6801	5709	8622	9090	6557	4805	8490	8746
4-Hydroxyphenylpyruvic acid	11499	1	1	1	15602	1	1	1	1	1	1	1	9326	1	1	7837	17085
2-Methylglutaric acid	134864	71802	41831	351712	144693	76825	136343	685276	105935	84112	116688	150378	85646	246298	245629	118716	224994
Adenine	4685311	4213022	3256026	3436828	3731929	3976501	4443544	5147027	3781000	3718905	3878207	5033695	4386665	4120257	4152212	5207635	4734669
Pyroglutamic acid	1248581	953252	951719	672756	1182774	817781	695407	904925	1073549	777653	968436	942993	1199085	777922	1012899	1242077	808480
Uracil	49645	69196	44440	54237	65129	77469	62309	71676	57868	39023	53911	75507	60674	38797	47372	61007	55904
Carnitine	2151584	2106837	1380844	2755657	1.01E+07	4155693	2283134	2861165	2306673	2841788	2478362	2698669	2891915	2282963	3499471	2838670	2591990
L-Valine	780862	539568	468347	503854	605157	499515	544525	577092	657687	586462	547552	800941	722525	576995	576825	634339	635745
1-Aminocyclopropane-1-carboxylic acid	20321	18524	16502	9583	11444	9490	17915	16768	15922	13925	12281	15579	16363	18628	12248	19172	16363
10-hydroxy capric acid	32460	23545	34322	58148	34162	33610	34777	114090	28563	54431	27225	27185	27806	52722	70907	25491	25076
Erythritol	135207	172514	257941	193322	62063	122851	160253	114516	234040	43662	273850	40546	139093	69984	35150	141541	42384
Chenodeoxycholic Acid	325025	330100	310924	289885	245478	297262	300940	263860	307355	315898	263790	293772	300664	305759	354653	295998	258040
DL-3-amino-isobutanoic acid	6317	7422	19427	50217	31653	70764	21657	18670	20266	43558	17927	25601	28207	13607	48739	29985	25522
N-ALPHA-ACETYL-L-ASPARAGINE	224029	253772	141238	206707	168838	207093	226978	235929	245821	208458	221492	250622	193959	270835	280103	247126	379049
Monomethyl glutaric acid	1	1	1574	16807	4422	1	6498	38293	2197	1	5120	2617	4475	9385	10470	1	1
Succinylacetone HT-1	1	1	1	8439	1	1	1	20494	1	1	1	1	1	10792	6509	1	1
Glyceraldehyde	1	18338	1	17164	1	1	20391	1	1	1	20755	1	1	1	1	22172	1
Uridine monophosphate (UMP)	1	1	1	1	20919	3985	1	1	2494	5098	1	1	1	1	6682	1	1
Dihydrouracil	1	1	1	1	1	13308	1	1	1	1	1	1	1	1	1	1	1
5-Methylcytosine	1	1	1	1	3739	1	1	1	1	1	1	1	1	1	1	1	1
Pyridoxal Phosphate	1	3117	1	1	1	1	1	1	1	6298	1	1	1	1	1	1	1
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Mass	Retention Time	Ion Species	CAS Number
L-Histidine	155.0697	1.257	[M+H]+ [M+K]+ [M+Na]+	71-00-1
Inosine	268.0809	2.9869998	[M+H]+ [M+K]+ [M+Na]+	58-63-9
Adipic acid	146.058	4.713	[M+H]+ [M+K]+ [M+Na]+	124-04-9
Sarcosine	89.048	1.3640002	[M+H]+ [M+Na]+	107-97-1
6-Deoxy-L-galactose	164.0686	1.4799998	[M+H]+ [M+K]+ [M+Na]+	2438-80-4
L-Pipecolic acid	129.079	1.719	[M+H]+ [M+Na]+	3105-95-1
Ethylmalonic acid	132.0422	3.9780004	[M+H]+ [M+K]+ [M+Na]+	601-75-2
D-(-)-Lyxose	150.0529	1.434	[M+H]+ [M+K]+ [M+Na]+	1114-34-7
Theobromine	180.0648	4.611	[M+H]+ [M+K]+ [M+Na]+	83-67-0
Hippuric acid	179.0586	5.629	[M+H]+ [M+K]+ [M+Na]+	495-69-2
Oleic Acid	282.256	13.3150015	[M+H]+ [M+Na]+	112-80-1
3,4-Dihydroxybenzoic acid	154.0244	4.1679997	[M+H]+ [M+K]+ [M+Na]+	99-50-3
Kynurenine	208.0849	3.51	[M+H]+	343-65-7
Methyl jasmonate	224.1411	10.373	[M+H]+ [M+K]+ [M+Na]+	39924-52-2
Thiamine monophosphate	344.0703	1.2840002	[M+H]+ [M+K]+ [M+Na]+	532-40-1
Dulcitol	182.079	1.3500003	[M+H]+ [M+K]+ [M+Na]+	608-66-2
myo-Inositol	180.0637	1.4160002	[M+H]+ [M+K]+ [M+Na]+	87-9-8
Hypoxanthine	136.0393	2.1650004	[M+H]+ [M+K]+ [M+Na]+	68-94-0
Phosphocreatine	211.0358	1.419	[M+H]+ [M+Na]+	67-07-2
Thymidine	242.0902	3.9780004	[M+H]+ [M+K]+ [M+Na]+	50-89-5
1-methylhistidine	169.0853	1.2749999	[M+H]+ [M+K]+ [M+Na]+	332-80-9
DL-pipecolic acid	129.079	1.719	[M+H]+ [M+Na]+	535-75-1
4-acetamidobutanoate	145.0742	3.2809992	[M+H]+ [M+K]+ [M+Na]+	3025-96-5
Theophylline	180.0648	5.1080008	[M+H]+ [M+K]+ [M+Na]+	58-55-9
L-Tryptophan	204.0899	4.611	[M+H]+ [M+K]+ [M+Na]+	73-22-3
Niacinamide	122.0485	2.4039998	[M+H]+ [M+K]+ [M+Na]+	98-92-0
Cytidine	243.0862	1.5179998	[M+H]+ [M+K]+ [M+Na]+	65-46-3
L-Threonine	119.0586	1.324	[M+H]+ [M+K]+ [M+Na]+	72-19-5
Malic acid	134.0215	1.625	[M+H]+ [M+K]+ [M+Na]+	6915-15-7
L-Isoleucine	131.095	2.4900002	[M+H]+ [M+K]+ [M+Na]+	73-32-5
N-Acetyl-L-glutamic acid	189.0636	2.2970004	[M+H]+ [M+K]+ [M+Na]+	1188-37-0
D-Mannitol	182.079	1.3500003	[M+H]+ [M+K]+ [M+Na]+	69-65-8
L-Aspartic Acid	133.0375	1.462	[M+H]+ [M+K]+ [M+Na]+	56-84-8
Adenosine	267.097	3.1959996	[M+H]+ [M+K]+ [M+Na]+	58-61-7
5-Hydroxyindoleacetic acid	191.0593	5.0950003	[M+H]+ [M+K]+ [M+Na]+	54-16-0
Creatine	131.0702	1.367	[M+H]+ [M+K]+ [M+Na]+	57-00-1
2-Aminoisobutyric acid	103.0636	1.279	[M+H]+ [M+K]+ [M+Na]+	62-57-7
Acetyl-L-tyrosine	223.0842	5.0619993	[M+H]+ [M+K]+ [M+Na]+	537-55-3
p-Hydroxyphenylacetic acid	152.047	5.493	[M+H]+ [M+K]+ [M+Na]+	156-38-7
Quinic acid	192.0643	1.433	[M+H]+ [M+K]+ [M+Na]+	77-95-2
Methyl N-(a-methylbutyryl)glycine	188.1052	8.21	[M+H]+ [M+K]+ [M+Na]+	123-99-9
3-Methyladenine	149.0694	1.4379998	[M+H]+ [M+K]+ [M+Na]+	5142-23-4
Maleamic acid	115.0269	1.571	[M+H]+	557-24-4
D-Aspartic acid	133.0375	1.4599998	[M+H]+ [M+K]+ [M+Na]+	1783-96-6
12-Hydroxydodecanoic acid	216.1725	10.639002	[M+H]+ [M+K]+ [M+Na]+	505-95-3
Histamine	111.0801	1.06	[M+H]+	51-45-6
Pantothenic Acid	219.1112	4.364999	[M+H]+ [M+K]+ [M+Na]+	137-08-6
3-Ureidopropionic acid	132.0537	1.7450004	[M+H]+ [M+K]+ [M+Na]+	462-88-4
L-(-)-Arabitol	152.0686	1.4109999	[M+H]+ [M+K]+ [M+Na]+	7643-75-6
Glutamine	146.0697	1.325	[M+H]+ [M+K]+ [M+Na]+	56-85-9
2-Deoxy-D-Ribose	134.0602	8.989999	[M+H]+ [M+Na]+	533-67-5
salsolinol	179.094	2.0989995	[M+H]+ [M+Na]+	27740-96-1
Creatinine	113.0594	1.309	[M+H]+ [M+K]+ [M+Na]+	60-27-5
Cytosine	111.044	1.5160002	[M+H]+	71-30-7
Adenosine5'-monophosphate	347.0626	1.7999997	[M+H]+ [M+K]+ [M+Na]+	149022-20-8
D-Mannose 6-phosphate	260.0298	1.3419998	[M+H]+ [M+K]+ [M+Na]+	3672-15-9
a-N-Acetylglucosamine	221.0901	1.465	[M+H]+ [M+K]+	7512-17-6
L-Alanine	89.048	1.3640002	[M+H]+ [M+Na]+	56-41-7
N-Acetyl-DL-serine	147.0533	1.6879998	[M+H]+ [M+K]+ [M+Na]+	94-14-3
N-Formylmethionine	177.046	4.602	[M+H]+ [M+K]+ [M+Na]+	4289-98-9
N-Acetyl-DL-methionine	191.0593	5.0950003	[M+H]+ [M+K]+ [M+Na]+	1115-47-5
Diacetyl	86.0369	9.615999	[M+H]+	431-03-8
1-Methyladenosine	281.1123	4.383	[M+H]+ [M+K]+	15763-06-1
Xanthurenic acid	205.0373	5.204	[M+H]+ [M+K]+ [M+Na]+	59-00-7
L-Anserine	240.1222	1.265	[M+H]+ [M+K]+ [M+Na]+	10030-52-1
D-Fructose 6-phosphate	260.0298	1.3419998	[M+H]+ [M+K]+ [M+Na]+	643-13-0
Diethanolamine	105.0792	1.273	[M+H]+ [M+K]+ [M+Na]+	111-42-2
Pimelic acid	160.0737	5.997001	[M+H]+ [M+K]+ [M+Na]+	111-16-0
Suberic acid	174.0895	7.1869984	[M+H]+ [M+K]+ [M+Na]+	505-48-6
D-MANNOSAMINE	179.0796	1.3460001	[M+H]+ [M+K]+ [M+Na]+	5505-63-5
D-Ribose	150.0529	1.434	[M+H]+ [M+K]+ [M+Na]+	50-69-1
Deoxycytidine	227.0907	1.6369996	[M+H]+ [M+K]+ [M+Na]+	951-77-9
N-Methyl-D-aspartic acid	147.0535	1.3310001	[M+H]+ [M+K]+ [M+Na]+	6384-92-5
?-D-Glucose 1-phosphate	260.0298	1.3419998	[M+H]+ [M+K]+ [M+Na]+	59-56-3
S-(5-ADENOSYL)-L-METHIONINE	398.1365	1.2840002	[M+H]+ [M+Na]+	17176-17-9
Xanthine	152.0335	2.3740003	[M+H]+ [M+K]+ [M+Na]+	69-89-6
Glyceryl phosphate	172.0138	1.426	[M+H]+ [M+K]+ [M+Na]+	57-03-4
L-Norleucine	131.095	2.4900002	[M+H]+ [M+K]+ [M+Na]+	327-57-1
L-Serine	105.0428	1.2819998	[M+H]+ [M+K]+ [M+Na]+	56-45-1
Xanthosine	284.0751	3.5380006	[M+H]+ [M+K]+ [M+Na]+	146-80-5
D-Ornithine	132.0901	1.104	[M+H]+ [M+K]+ [M+Na]+	348-66-3
D-(+)-Xylose	150.0529	1.434	[M+H]+ [M+K]+ [M+Na]+	58-86-6
Corticosterone	346.2143	10.036001	[M+H]+ [M+K]+ [M+Na]+	50-22-6
N-Acetyl-?-D-mannosamine	221.0901	1.465	[M+H]+ [M+K]+	7772-94-3
DL-Homoserine	119.0586	1.324	[M+H]+ [M+K]+ [M+Na]+	1927-25-9
D-(+)-Malic acid	134.0215	1.6240001	[M+K]+ [M+Na]+	636-61-3
4-Pyridoxic acid	183.0533	3.271	[M+H]+	82-82-6
Dihydroxyphenylacetic acid	168.04	4.713	[M+H]+ [M+K]+ [M+Na]+	102-32-9
D-Glucosamine 6-phosphate	259.0459	1.479	[M+H]+ [M+Na]+	3616-42-0
N-Methylhistamine	125.0957	1.092	[M+H]+ [M+K]+	673-50-7
Ketoleucine	130.0631	4.985	[M+H]+ [M+K]+ [M+Na]+	816-66-0
AHBA	153.0404	2.616	[M+H]+ [M+Na]+	76045-71-1
L-Phenylalanine	165.0794	3.8189998	[M+H]+ [M+K]+ [M+Na]+	63-91-2
3-hydroxy-3-methyl-Glutaric acid	162.0527	2.7960002	[M+H]+ [M+K]+ [M+Na]+	503-49-1
L-Methionine	149.0516	1.868	[M+H]+ [M+K]+ [M+Na]+	63-68-3
N-Acetyl-D-galactosamine	221.0901	1.465	[M+H]+ [M+K]+	1811-31-0
N-Acetyl-L-alanine	131.0587	2.616	[M+H]+ [M+K]+ [M+Na]+	97-69-8
3-Methoxy-4-hydroxyphenylethylene glycol	184.0736	3.7809992	[M+H]+ [M+K]+ [M+Na]+	67423-45-4
Ribitol	152.0686	1.4109999	[M+H]+ [M+K]+ [M+Na]+	488-81-3
?-Aminobutryic acid	103.0636	1.279	[M+H]+ [M+K]+ [M+Na]+	56-12-2
Cortexolone	346.2143	10.036001	[M+H]+ [M+K]+ [M+Na]+	152-58-9
Spermidine	145.1583	0.97500014	[M+H]+	124-20-9
R-(-)-Mandelic acid	152.0451	4.801	[M+H]+ [M+Na]+	90-64-2
4-Hydroxy-L-phenylglycine	167.0582	1.4659997	[M+H]+ [M+Na]+	32462-30-9
Inosine 5'-monophosphate (IMP)	348.0471	1.9330001	[M+H]+ [M+K]+ [M+Na]+	131-99-7
Pyridoxamine	168.0875	1.12	[M+H]+ [M+K]+ [M+Na]+	85-87-0
Taurine	125.0149	1.2959998	[M+H]+ [M+K]+ [M+Na]+	107-35-7
N?,N?,N?-Trimethyllysine	189.1561	1.2679999	[M+H]+ [M+K]+ [M+Na]+	55528-53-5
L-Ornithine	132.0901	1.104	[M+H]+ [M+K]+ [M+Na]+	3184-13-2
L-Asparagine	132.0534	1.286	[M+H]+ [M+K]+ [M+Na]+	70-47-3
Urocanic acid	138.0435	1.6900004	[M+H]+ [M+K]+ [M+Na]+	104-98-3
D-Glucose 6-phosphate	260.0298	1.3419998	[M+H]+ [M+K]+ [M+Na]+	56-73-5
L-(-)Sorbose	180.0637	1.4169999	[M+H]+ [M+K]+ [M+Na]+	87-79-6
3-Amino-4-hydroxybenzoic acid	153.0404	2.616	[M+H]+ [M+Na]+	1571-72-8
Guanine	151.0484	1.861	[M+H]+ [M+K]+ [M+Na]+	73-40-5
L-Lysine	146.1057	1.1090001	[M+H]+ [M+K]+ [M+Na]+	56-87-1
Allothreonine	119.0586	1.324	[M+H]+ [M+K]+ [M+Na]+	144-98-9
Citrulline	175.096	1.3290002	[M+H]+ [M+K]+	372-75-8
D-Tryptophan	204.0899	4.611	[M+H]+ [M+K]+ [M+Na]+	153-94-6
D-Alanine	89.048	1.3640002	[M+H]+ [M+Na]+	338-69-2
(-)-Epinephrine	183.0872	1.2910001	[M+H]+	51-43-4
O-Acetylserine	147.0533	1.6879998	[M+H]+ [M+K]+ [M+Na]+	5147-00-2
Petroselinic acid	282.256	13.3150015	[M+H]+ [M+Na]+	593-39-5
L-Proline	115.0643	1.4470003	[M+H]+ [M+Na]+	147-85-3
Isocitrate	192.0269	1.9160001	[M+H]+ [M+K]+ [M+Na]+	320-77-4
RAC-GLYCEROL 1-MYRISTATE	302.245	13.242997	[M+H]+ [M+K]+ [M+Na]+	589-68-4
Glucosamine	179.0796	1.3460001	[M+H]+ [M+K]+ [M+Na]+	3416-24-8
Deoxyuridine monophosphate (dUMP)	308.0419	1.944	[M+H]+ [M+K]+ [M+Na]+	964-26-1
Glycerol 2-phosphate	172.0138	1.426	[M+H]+ [M+K]+ [M+Na]+	17181-54-3
D-Sorbitol	182.079	1.3500003	[M+H]+ [M+K]+ [M+Na]+	50-70-4
METHYL BETA-D-GALACTOSIDE	194.0788	1.6800002	[M+H]+ [M+K]+ [M+Na]+	1824-94-8
?-D-Galactose 1-phosphate	260.0298	1.3419998	[M+H]+ [M+K]+ [M+Na]+	2255-14-3
D-Ribose 5-phosphate	230.0193	1.4150002	[M+H]+ [M+K]+ [M+Na]+	4300-28-1
3-Hydroxyphenylacetic acid	152.047	5.493	[M+H]+ [M+K]+ [M+Na]+	621-37-4
Diaminopimelic acid	190.0961	1.411	[M+H]+ [M+K]+ [M+Na]+	583-93-7
Uridine	244.0695	2.2819998	[M+H]+ [M+K]+ [M+Na]+	58-96-8
Caffeine	194.0804	6.0569997	[M+H]+ [M+K]+ [M+Na]+	58-08-2
O-Phosphorylethanolamine	141.0197	1.2760003	[M+H]+ [M+K]+ [M+Na]+	1071-23-4
L-Norvaline	117.0794	1.6720004	[M+H]+ [M+K]+ [M+Na]+	6600-40-4
L-Leucine	131.0951	2.664	[M+H]+ [M+K]+ [M+Na]+	61-90-5
Trigonelline	137.0476	1.4500002	[M+H]+	535-83-1
Xylitol	152.0686	1.4109999	[M+H]+ [M+K]+ [M+Na]+	87-99-0
Glutamyl-Tyrosine	310.1162	4.007	[M+H]+ [M+K]+ [M+Na]+	9046-27-9
L-Arginine	174.1117	1.2590001	[M+H]+ [M+K]+ [M+Na]+	74-79-3
5?-Deoxy-5?-(methylthio)adenosine	297.0892	5.316001	[M+H]+ [M+Na]+	2457-80-9
2-Deoxyglucose	164.0686	1.4799998	[M+H]+ [M+K]+ [M+Na]+	154-17-6
L-Tyrosine	181.0743	2.229	[M+H]+ [M+K]+ [M+Na]+	60-18-4
Deoxyguanosine	267.097	3.1959996	[M+H]+ [M+K]+ [M+Na]+	961-07-9
N-acetylaspartate	175.0484	1.8940005	[M+H]+ [M+K]+ [M+Na]+	997-55-7
3-Methylglutaric acid	146.058	4.713	[M+H]+ [M+K]+ [M+Na]+	626-51-7
N-Acetylneuraminic Acid	309.1058	1.4610002	[M+H]+ [M+K]+ [M+Na]+	131-48-6
Octopamine (p-Hydroxyphenylethanolamine)	153.08	1.2900002	[M+K]+ [M+Na]+	104-14-3
S-Adenosylhomocysteine	384.1207	2.5130002	[M+H]+	979-92-0
Riboflavin (Vitamin B2)	376.1375	6.5299993	[M+H]+ [M+K]+ [M+Na]+	83-88-5
4-Hydroxyphenylpyruvic acid	180.0426	3.295	[M+Na]+	156-39-8
2-Methylglutaric acid	146.058	4.713	[M+H]+ [M+K]+ [M+Na]+	617-62-9
Adenine	135.0552	1.7239997	[M+H]+ [M+K]+ [M+Na]+	73-24-5
Pyroglutamic acid	129.0433	2.111	[M+H]+ [M+K]+ [M+Na]+	98-79-3
Uracil	112.0276	1.8589997	[M+H]+ [M+K]+ [M+Na]+	66-22-8
Carnitine	161.1059	1.3059999	[M+H]+ [M+K]+ [M+Na]+	541-15-1
L-Valine	117.0794	1.6720004	[M+H]+ [M+K]+ [M+Na]+	72-18-4
1-Aminocyclopropane-1-carboxylic acid	101.0482	1.325	[M+H]+ [M+Na]+	22059-21-8
10-hydroxy capric acid	188.1415	9.354998	[M+H]+ [M+K]+ [M+Na]+	1679-53-4
Erythritol	122.0603	1.4069997	[M+H]+ [M+Na]+	149-32-6
Chenodeoxycholic Acid	392.3002	12.431	[M+H]+ [M+K]+ [M+Na]+	474-25-9
DL-3-amino-isobutanoic acid	103.0636	1.279	[M+H]+ [M+K]+ [M+Na]+	144-90-1
N-ALPHA-ACETYL-L-ASPARAGINE	174.0642	1.6289998	[M+H]+ [M+K]+ [M+Na]+	4033-40-3
Monomethyl glutaric acid	146.0584	5.4770007	[M+H]+ [M+K]+ [M+Na]+	1501-27-5
Succinylacetone HT-1	158.0578	4.225	[M+H]+ [M+K]+ [M+Na]+	51568-18-4
Glyceraldehyde	90.0318	1.422	[M+H]+ [M+Na]+	56-82-6
Uridine monophosphate (UMP)	324.0353	1.7229999	[M+H]+ [M+Na]+	58-97-9
Dihydrouracil	114.043	1.483	[M+H]+	504-07-4
5-Methylcytosine	125.0588	1.616	[M+H]+	554-01-8
Pyridoxal Phosphate	247.0245	2.722	[M+H]+	54-47-7
METABOLITES_END
#END