Summary of Study ST003548

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 PR002184. The data can be accessed directly via it's Project DOI: 10.21228/M89V56 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST003548
Study TitleMetabolomics of Aldh7a1-/- mice treated with lysine alpha-oxidase from Trichoderma viride
Study SummaryMetabolite analysis of brain, liver, and plasma isolated from Aldh7a1-/- (n=3/each) fed diets consisting of 0.9% w/w lysine and 18 ppm pyridoxine administered with PBS or 150 U/kg lysine alpha-oxidase (LOX) from Trichoderma viride. Tissues were cryo-homogenized using a liquid nitrogen cooled mortar and pestle into a fine powder and approximately 25-50 mg of each tissue was transferred to a pre-weighed homogenization tube to which ~20 2.3 mm zirconia beads and 1 mL of 80% ice cold methanol were added. Tissues were homogenized using a bead mill and cleared by centrifugation at 4C, where each of the 3-4 cycles consisted of a 60 second homogenization at 6 m/s followed by a 30 second pause. A volume equivalent to 25 mg of tissue extract was transferred to a new tube, and 250 µL of 80% methanol containing 1 µL of stable isotope-labeled internal amino acid standard mix was added. Metabolites were concentrated using a SpeedVac until dry. For plasma metabolite analysis, 10 µL of plasma was added to 250 µL of 80% methanol containing 1 µL of stable isotope-labeled internal amino acid standard mix, vortexed at 4C for 10 minutes, and centrifuged. 0.9 ml of supernatant was transferred to a new tube and concentrated using a SpeedVac until dry. Metabolites were reconstituted into 25-50 µL of water, vortexed, centrifuged, and transferred to vials for analysis by LCMS. LCMS was performed using a ZIC-pHILIC LC column coupled to a Vanquish LC and a flow gradient consisting of 10 mM ammonium carbonate in water and pure acetonitrile. The LC was coupled to an Exploris 240 mass spectrometer operated in a polarity switching data-dependent Top 5 mode. Full MS scan parameters for both positive and negative mode were set to 67-1000 m/z at a resolution of 120k and ddMS2 were collected at a resolution of 30k. Data files are labeled with brain, liver, and plasma followed by a number. The number represents a mouse. Samples 1-3 are from PBS-treated mice and samples 4-6 are from LOX-treated mice.
Institute
University of British Columbia
DepartmentBiochemistry & Molecular Biology
LaboratoryParker laboratory
Last NameParker
First NameSeth
Address950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada
Emailseth.parker@bcchr.ca
Phone6048753121
Submit Date2024-11-01
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-11-15
Release Version1
Seth Parker Seth Parker
https://dx.doi.org/10.21228/M89V56
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002184
Project DOI:doi: 10.21228/M89V56
Project Title:Restricting lysine normalizes toxic catabolites associated with ALDH7A1-deficiency in cells and mice
Project Type:Manuscript
Project Summary:Lysine metabolism converges at α-aminoadipic semialdehyde dehydrogenase (ALDH7A1). Rare loss-of-function mutations in ALDH7A1 cause a toxic accumulation of lysine catabolites, including piperideine 6-carboxylate (P6C), that are thought to cause fatal seizures in children unless strictly managed with dietary lysine reduction. In this study, we perform metabolomics and expression analysis of tissues from Aldh7a1-deficient mice, which reveal tissue-specific differences in lysine metabolism and other metabolic pathways. We also develop a fluorescent biosensor to characterize lysine transporter activity and identify competitive substrates that reduce the accumulation of lysine catabolites in ALDH7A1-deficient HEK293 cells. Lastly, we show that intravenous administration of lysine α-oxidase from Trichoderma viride reduces lysine and P6C levels by >80% in mice. Our results improve our understanding of lysine metabolism and make inroads towards improving therapeutic strategies for lysine catabolic disorders.
Institute:University of British Columbia
Department:Biochemistry & Molecular Biology
Laboratory:Parker laboratory
Last Name:Parker
First Name:Seth
Address:950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada
Email:seth.parker@bcchr.ca
Phone:6048753121

Subject:

Subject ID:SU003677
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Age Or Age Range:10-17 week
Gender:Female
Animal Feed:0.9% w/w lysine, 18 ppm pyridoxine (standard diets)
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Genotype Treatment Sample source
SA387786Brain_LOX_2Aldh7a1-/- LOX (150 U/kg, intravenous) Brain
SA387787Brain_LOX_3Aldh7a1-/- LOX (150 U/kg, intravenous) Brain
SA387788Brain_LOX_1Aldh7a1-/- LOX (150 U/kg, intravenous) Brain
SA387789Liver_LOX_2Aldh7a1-/- LOX (150 U/kg, intravenous) Liver
SA387790Liver_LOX_1Aldh7a1-/- LOX (150 U/kg, intravenous) Liver
SA387791Liver_LOX_3Aldh7a1-/- LOX (150 U/kg, intravenous) Liver
SA387792Plasma_LOX_2Aldh7a1-/- LOX (150 U/kg, intravenous) Plasma
SA387793Plasma_LOX_3Aldh7a1-/- LOX (150 U/kg, intravenous) Plasma
SA387794Plasma_LOX_1Aldh7a1-/- LOX (150 U/kg, intravenous) Plasma
SA387795Brain_PBS_1Aldh7a1-/- PBS Brain
SA387796Brain_PBS_2Aldh7a1-/- PBS Brain
SA387797Brain_PBS_3Aldh7a1-/- PBS Brain
SA387798Liver_PBS_1Aldh7a1-/- PBS Liver
SA387799Liver_PBS_2Aldh7a1-/- PBS Liver
SA387800Liver_PBS_3Aldh7a1-/- PBS Liver
SA387801Plasma_PBS_2Aldh7a1-/- PBS Plasma
SA387802Plasma_PBS_3Aldh7a1-/- PBS Plasma
SA387803Plasma_PBS_1Aldh7a1-/- PBS Plasma
Showing results 1 to 18 of 18

Collection:

Collection ID:CO003670
Collection Summary:Liver, brain, or plasma samples collected from mice treated with PBS or Lysine alpha-oxidase (150 U/kg) as a single intravenous injection. Tissues collected 7-hours after injection.
Sample Type:Tissue or Plasma

Treatment:

Treatment ID:TR003686
Treatment Summary:PBS or Lysine alpha-oxidase (150 U/kg)
Treatment Route:intravenous
Treatment Vehicle:PBS

Sample Preparation:

Sampleprep ID:SP003684
Sampleprep Summary:Dried samples were reconstituted in 50 µL of HPLC-grade water. Samples were vortexed for ~10 minutes, then centrifuged at 21,000 x g for 15 min at 4°C. 40 microliters were transferred to LC vials containing glass inserts for analysis.

Chromatography:

Chromatography ID:CH004431
Instrument Name:Thermo Vanquish
Column Name:Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:25°C
Flow Gradient:80-20%B (0-30 min), 20-20%B (30-40 minute), and 20-80%B (40-40.5 minute); the LC column was re-equilibrated using 80-80%B from 40.5-52 minute before subsequent injections
Flow Rate:100 µL/min
Solvent A:100% Water; 10 mM Ammonium Carbonate, pH 9.0
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

Analysis:

Analysis ID:AN005831
Analysis Type:MS
Chromatography ID:CH004431
Num Factors:6
Num Metabolites:47
Rt Units:Minutes
Units:Ion counts
  
Analysis ID:AN005832
Analysis Type:MS
Chromatography ID:CH004431
Num Factors:6
Num Metabolites:47
Rt Units:Minutes
Units:Ion counts
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