Summary of Study ST002179

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001387. The data can be accessed directly via it's Project DOI: 10.21228/M8FB0C This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002179
Study Title	Impact of nitisinone on the cerebrospinal fluid metabolome of a murine model of alkaptonuria
Study Summary	Background: Nitisinone induced hypertyrosinaemia is well documented in Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a decline in cognitive function and or mood by altering neurotransmitter metabolism. The aim of this work was to evaluate the impact of nitisinone on the cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to providing additional insight into metabolic changes that occur following treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c Hgd-/-mice (n=8, treated with nitisinone – 4 mg/L and n=9, no treatment). Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted feature extraction algorithm and an established in-house accurate mass retention time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3 minutes retention time window) were filtered based on their frequency and variability. Experimental groups were compared using a moderated t-test with Benjamini-Hochberg false-discovery rate adjustment. Results: Tyrosine, acetyl-tyrosine, γ-glutamyl-tyrosine, p-hydroxyphenylacetic acid and 3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold change 2.6-6.9, 3/5 were significant p<0.05) in the mice that received nitisinone. Several other metabolites of interest were matched but no significant differences were observed, including the aromatic amino acids phenylalanine and tryptophan, and monoamine metabolites adrenaline, 3-methoxy-4-hydroxyphenylglycol and octopamine. Conclusions: Evaluation of the CSF metabolome of a murine model of AKU showed a significant difference in the abundance of a limited number of metabolites. None of these have been reported in CSF from a murine model of AKU previously. Moreover this study confirms that some monoamine metabolites do not appear to be altered following nitisinone therapy.
Institute	University of Liverpool Institute of Life Course & Medical Sciences
Last Name	Davison
First Name	Andrew
Address	1. Department of Clinical Biochemistry and Metabolic Medicine, Liverpool Clinical Laboratories, Liverpool University Hospitals NHS Foundation Trust, Liverpool, UK; 2. Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK 3. School of Exercise Science, Liverpool John Moores University, Liverpool, UK
Email	andrew.davison@liverpoolft.nhs.uk
Phone	0151 706 4011
Submit Date	2022-05-13
Num Groups	2
Total Subjects	17
Raw Data Available	Yes
Raw Data File Type(s)	d, mzML
Analysis Type Detail	LC-MS
Release Date	2022-06-08
Release Version	1

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Project:

Project ID:	PR001387
Project DOI:	doi: 10.21228/M8FB0C
Project Title:	Impact of nitisinone on the cerebrospinal fluid metabolome of a murine model of alkaptonuria
Project Summary:	Background: Nitisinone induced hypertyrosinaemia is well documented in Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a decline in cognitive function and or mood by altering neurotransmitter metabolism. The aim of this work was to evaluate the impact of nitisinone on the cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to providing additional insight into metabolic changes that occur following treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c Hgd-/-mice (n=8, treated with nitisinone – 4 mg/L and n=9, no treatment). Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted feature extraction algorithm and an established in-house accurate mass retention time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3 minutes retention time window) were filtered based on their frequency and variability. Experimental groups were compared using a moderated t-test with Benjamini-Hochberg false-discovery rate adjustment. Results: Tyrosine, acetyl-tyrosine, γ-glutamyl-tyrosine, p-hydroxyphenylacetic acid and 3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold change 2.6-6.9, 3/5 were significant p<0.05) in the mice that received nitisinone. Several other metabolites of interest were matched but no significant differences were observed, including the aromatic amino acids phenylalanine and tryptophan, and monoamine metabolites adrenaline, 3-methoxy-4-hydroxyphenylglycol and octopamine. Conclusions: Evaluation of the CSF metabolome of a murine model of AKU showed a significant difference in the abundance of a limited number of metabolites. None of these have been reported in CSF from a murine model of AKU previously. Moreover this study confirms that some monoamine metabolites do not appear to be altered following nitisinone therapy.
Institute:	University of Liverpool Institute of Life Course & Medical Sciences
Last Name:	Davison
First Name:	Andrew
Address:	1. Department of Clinical Biochemistry and Metabolic Medicine, Liverpool Clinical Laboratories, Liverpool University Hospitals NHS Foundation Trust, Liverpool, UK; 2. Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK 3. School of Exercise Science, Liverpool John Moores University, Liverpool, UK
Email:	andrew.davison@liverpoolft.nhs.uk
Phone:	0151 706 4011
Contributors:	Davison AS1,2,*, Norman BP2, Sutherland H2,3, Milan AM1,2, Jarvis JC3, Gallagher JA2, Ranganath LR1,2

Subject:

Subject ID:	SU002265
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment group
SA209243	Hgd81.3 + Nitisinone POS	Nitisinone
SA209244	Hgd81.2 + Nitisinone POS	Nitisinone
SA209245	Hgd81.1 + Nitisinone POS	Nitisinone
SA209246	Hgd82.1 + Nitisinone POS	Nitisinone
SA209247	Hgd82.2 + Nitisinone POS	Nitisinone
SA209248	Hgd83.2 - No treatment POS	Nitisinone
SA209249	Hgd83.1 + Nitisinone POS	Nitisinone
SA209250	Hgd81.1 + Nitisinone NEG	Nitisinone
SA209251	Hgd80.2 + Nitisinone POS	Nitisinone
SA209252	Hgd83.1 + Nitisinone NEG	Nitisinone
SA209253	Hgd82.1 + Nitisinone NEG	Nitisinone
SA209254	Hgd81.3 + Nitisinone NEG	Nitisinone
SA209255	Hgd81.2 + Nitisinone NEG	Nitisinone
SA209256	Hgd83.2 - No treatment NEG	Nitisinone
SA209257	Hgd82.2 + Nitisinone NEG	Nitisinone
SA209258	Hgd81.6 - No treatment POS	No Treatment (control)
SA209259	Hgd81.5 - No treatment POS	No Treatment (control)
SA209260	Hgd81.4 - No treatment POS	No Treatment (control)
SA209261	Hgd82.3 - No treatment POS	No Treatment (control)
SA209262	Hgd82.4 - No treatment POS	No Treatment (control)
SA209263	Hgd81.6 - No treatment NEG	No Treatment (control)
SA209264	Hgd83.4 - No treatment POS	No Treatment (control)
SA209265	Hgd83.3 - No treatment POS	No Treatment (control)
SA209266	Hgd80.4 - No treatment POS	No Treatment (control)
SA209267	Hgd80.3 - No treatment POS	No Treatment (control)
SA209268	Hgd83.4 - No treatment NEG	No Treatment (control)
SA209269	Hgd80.3 - No treatment NEG	No Treatment (control)
SA209270	Hgd80.4 - No treatment NEG	No Treatment (control)
SA209271	Hgd83.3 - No treatment NEG	No Treatment (control)
SA209272	Hgd81.5 - No treatment NEG	No Treatment (control)
SA209273	Hgd82.3 - No treatment NEG	No Treatment (control)
SA209274	Hgd82.4 - No treatment NEG	No Treatment (control)
SA209275	Hgd81.4 - No treatment NEG	No Treatment (control)

Showing results 1 to 33 of 33

Showing results 1 to 33 of 33

Collection:

Collection ID:	CO002258
Collection Summary:	A murine model of AKU was used for all experiments as described previously (Preston et al., 2014). Seventeen BALB/c Hgd-/- mice (Figure 5 for age and gender) were included in this study, 8 mice were administered nitisinone (4 mg/L) through their drinking water for 6 days, the remaining 9 mice received no nitisinone. All animals were housed in air conditioned rooms (with a 12 h dark/light cycle) at 20 °C and 53 % humidity, with access to food and water ad libitum at Liverpool John Moores University. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. All mice were culled by carbon dioxide asphyxiation and CSF was removed from the cisterna magna following the puncture technique of Liu et al. (56), with some modifications. The cisterna magna was exposed by dissecting skin and overlaying muscles. Cauterisation was used to dry up any bleeding from surrounding tissues. A pulled glass pipette with an internal diameter of ~0.4 mm was attached to silicone tubing and connected to a 1 mL syringe with a 19 g needle. The tip of the glass pipette was used to puncture the membrane and held still just below the membrane by 1 person. Through a double-headed microscope a second person then used the 1 mL syringe to apply gentle pressure to encourage the CSF to flow up the capillary tube. Only clear CSF samples (i.e. non blood stained) were collected. Once collected CSF samples were transferred to clear Eppendorf tubes and stored at -80 °C until analysis. Samples were not acidified. The same glass pipette was used to collect CSF from each animal, and was washed with pure water between each animal.
Sample Type:	CSF

Treatment:

Treatment ID:	TR002277
Treatment Summary:	9 mice control 8 mice received 4 mg/L nitisinone for 6 days in drinking water

Sample Preparation:

Sampleprep ID:	SP002271
Sampleprep Summary:	Murine CSF samples were prepared by diluting 2 µL CSF with 2 µL deionized water (DIRECT-Q 3UV Millipore water purification system). CSF samples were pipetted directly into a 150 µL glass insert with polymer feet, which sat in a 2 mL amber screw vial (Agilent, Cheadle, UK). These vials were used to ensure maximal recovery of sample. Deionised water was also added directly to the glass insert, and mixed with the CSF sample using a pipette. The glass vials were centrifuged at 600 rpm for 10 min to ensure the diluted sample was at the bottom of the vial for sampling. 1 µL of diluted CSF was analysed in negative and positive polarities, respectively.

Sampleprep ID:

SP002271

Sampleprep Summary:

Murine CSF samples were prepared by diluting 2 µL CSF with 2 µL deionized water (DIRECT-Q 3UV Millipore water purification system). CSF samples were pipetted directly into a 150 µL glass insert with polymer feet, which sat in a 2 mL amber screw vial (Agilent, Cheadle, UK). These vials were used to ensure maximal recovery of sample. Deionised water was also added directly to the glass insert, and mixed with the CSF sample using a pipette. The glass vials were centrifuged at 600 rpm for 10 min to ensure the diluted sample was at the bottom of the vial for sampling. 1 µL of diluted CSF was analysed in negative and positive polarities, respectively.

Combined analysis:

Analysis ID	AN003568	AN003569
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent Infinity II	Agilent Infinity II
Column	Atlantis dC18 (3.0x100mm,3m,Waters,UK)	Atlantis dC18 (3.0x100mm,3m,Waters,UK)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6550 QTOF	Agilent 6550 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	area	area

Chromatography:

Chromatography ID:	CH002639
Chromatography Summary:	Liquid chromatography (LC) was performed on an Agilent 1290 Infinity II LC system. An Atlantis dC18 column (3.0x100mm, 3µm, Waters, UK) was maintained at 60°C with a flow rate of 0.4mL/min. Mobile phases were (A) water and (B) methanol both containing 5mmol/L ammonium formate and 0.1% formic acid. The elution gradient started at 5% mobile phase B at 0-1 min increasing linearly to 100% B by 12 min, held at 100% B until 14 min, returning to 95% A for 5 min to recondition the column. Injection volume was 1µL.
Instrument Name:	Agilent Infinity II
Column Name:	Atlantis dC18 (3.0x100mm,3m,Waters,UK)
Column Temperature:	60
Flow Gradient:	The elution gradient started at 5% mobile phase B at 0-1 min increasing linearly to 100% B by 12 min, held at 100% B until 14 min, returning to 95% A for 5 min to recondition the column
Flow Rate:	0.4mL/min
Solvent A:	100% water; 0.1% formic acid; 5 mM ammonium formate
Solvent B:	100% acetonitrile; 0.1% formic acid;5 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003325
Analysis ID:	AN003568
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Quadrupole time-of-flight mass spectrometry (QTOF-MS) conditions An Agilent 6550 QTOF-MS equipped with a dual jet stream electrospray ionisation source was operated in 2GHz mode, over the mass range of 50-1700, in negative and positive polarities. A reference mass correction solution was continually infused at a flow rate of 0.5 mL/min via an external isocratic pump (Agilent, UK) for constant mass correction (see Preparation of reference mass correction solution). Capillary and fragmentor voltages were 4000 V and 380 V, respectively. Desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min and nebulizer pressure was 40 psi and nozzle voltage 1000 V. Data acquisition rate was 3 spectra/s. Preparation of reference mass correction solution Reference mass correction solution was prepared in 95:5 methanol:water containing 5 mmol/L purine (CAS No. 120-73-0), 100 mmol/L trifluoroacetic acid ammonium salt (TFA, CAS No. 3336-58-1) and 2.5 mmol/L hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine (HP-0921, CAS No. 58943-98-9) (Agilent, Cheadle, UK). Reference ions monitored were: purine (m/z 121.0509) and HP-0921 (m/z 922.0098) (positive polarity) and TFA (m/z 112.9856), purine (m/z 119.0363) and HP-0921 (HP-0921 + formate adduct: m/z 966.0007) (negative polarity). Data acquisition and handling parameters Data were acquired using Acquisition (Build 06.00, Agilent, Cheadle, UK). Quality checks and processing of raw data files (Agilent ‘.d’ files) were performed with Qualitative Analysis software (Build 07.00, Agilent, Cheadle, UK). Extracted ion chromatograms of reference masses were performed to check mass accuracy remained <5 ppm throughout the run and that the reference ion signal did not drop out during the chromatographic run. In addition, to check chromatographic reproducibility binary pump pressure curves for injections across each analytical sequence were overlaid. Mass accuracy and chromatographic reproducibility were acceptable for all experiments performed. Acquired profiling sample data were mined for signals against an established in-house AMRT database of compounds (contains theoretical accurate mass, measured retention time, and empirical formula for 469 intermediary metabolites, MW 72-785) using ‘targeted feature extraction’ with Profinder software (Build 08.00, Agilent, Cheadle, UK). Targeted feature extraction uses the molecular formulae from the AMRT database to extract and group spectral signals (i.e. adducts, isotopes and multimers) that correspond to individual database compounds. Feature extraction employed a window of theoretical accurate mass ±10 ppm and database retention time ±0.3 min. Allowed species were: H+, Na+ and NH4+ (positive polarity) and H- and CHO2- (negative polarity). Dimers were allowed for both polarities. Charge state range was 1-2. Data files were then exported from Profinder as ‘.CEF’ files as a whole batch for each profiling experiment and imported to Mass Profiler Professional (MPP) software for statistical analysis (Build 14.5, Agilent, Cheadle, UK).
Ion Mode:	POSITIVE

MS ID:	MS003326
Analysis ID:	AN003569
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Quadrupole time-of-flight mass spectrometry (QTOF-MS) conditions An Agilent 6550 QTOF-MS equipped with a dual jet stream electrospray ionisation source was operated in 2GHz mode, over the mass range of 50-1700, in negative and positive polarities. A reference mass correction solution was continually infused at a flow rate of 0.5 mL/min via an external isocratic pump (Agilent, UK) for constant mass correction (see Preparation of reference mass correction solution). Capillary and fragmentor voltages were 4000 V and 380 V, respectively. Desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min and nebulizer pressure was 40 psi and nozzle voltage 1000 V. Data acquisition rate was 3 spectra/s. Preparation of reference mass correction solution Reference mass correction solution was prepared in 95:5 methanol:water containing 5 mmol/L purine (CAS No. 120-73-0), 100 mmol/L trifluoroacetic acid ammonium salt (TFA, CAS No. 3336-58-1) and 2.5 mmol/L hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine (HP-0921, CAS No. 58943-98-9) (Agilent, Cheadle, UK). Reference ions monitored were: purine (m/z 121.0509) and HP-0921 (m/z 922.0098) (positive polarity) and TFA (m/z 112.9856), purine (m/z 119.0363) and HP-0921 (HP-0921 + formate adduct: m/z 966.0007) (negative polarity). Data acquisition and handling parameters Data were acquired using Acquisition (Build 06.00, Agilent, Cheadle, UK). Quality checks and processing of raw data files (Agilent ‘.d’ files) were performed with Qualitative Analysis software (Build 07.00, Agilent, Cheadle, UK). Extracted ion chromatograms of reference masses were performed to check mass accuracy remained <5 ppm throughout the run and that the reference ion signal did not drop out during the chromatographic run. In addition, to check chromatographic reproducibility binary pump pressure curves for injections across each analytical sequence were overlaid. Mass accuracy and chromatographic reproducibility were acceptable for all experiments performed. Acquired profiling sample data were mined for signals against an established in-house AMRT database of compounds (contains theoretical accurate mass, measured retention time, and empirical formula for 469 intermediary metabolites, MW 72-785) using ‘targeted feature extraction’ with Profinder software (Build 08.00, Agilent, Cheadle, UK). Targeted feature extraction uses the molecular formulae from the AMRT database to extract and group spectral signals (i.e. adducts, isotopes and multimers) that correspond to individual database compounds. Feature extraction employed a window of theoretical accurate mass ±10 ppm and database retention time ±0.3 min. Allowed species were: H+, Na+ and NH4+ (positive polarity) and H- and CHO2- (negative polarity). Dimers were allowed for both polarities. Charge state range was 1-2. Data files were then exported from Profinder as ‘.CEF’ files as a whole batch for each profiling experiment and imported to Mass Profiler Professional (MPP) software for statistical analysis (Build 14.5, Agilent, Cheadle, UK).
Ion Mode:	NEGATIVE