Summary of Study ST003557

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 PR002191. The data can be accessed directly via it's Project DOI: 10.21228/M8DN9K 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 IDST003557
Study TitleMetabolomics analysis of kidney, brain, liver, and plasma from Aldh7a1-/- mice administered PBS or N-methyl-arginine (80 mg/kg) via a single intraperitoneal injection.
Study SummaryMetabolite analysis of kidney, brain, liver, and plasma isolated from Aldh7a1-/- mice fed diets consisting of 0.9% w/w lysine and 18 ppm pyridoxine. 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.
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/M8DN9K
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002191
Project DOI:doi: 10.21228/M8DN9K
Project Title:N-methyl-arginine is taken up into the liver, brain, and kidney of mice.
Project Type:Manuscript
Project Summary:N-methyl-arginine was identified as a transported substrate through multiple cationic amino acid transporters, which facilitate the uptake of lysine, arginine, and other structurally related amino acids. In cell culture, N-methyl-arginine inhibits the uptake and metabolism of lysine. In this project, we aimed to determine if N-methyl-arginine administered to mice at 80 mg/kg was sufficient to inhibit tissue lysine uptake and metabolism. Following a single injection, N-methyl-arginine significantly accumulated in mouse plasma and tissues but failed to significantly inhibit the level of lysine metabolites (e.g., saccharopine). These results are in-line with in vitro results suggesting that N-methyl-arginine is a relatively low affinity transport inhibitor.
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:SU003686
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Male and female
Animal Feed:0.9% w/w lysine, 18 ppm pyridoxine (standard diets)

Factors:

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

mb_sample_id local_sample_id Genotype Treatment Sample source
SA3885654Aldh7a1-/- N-methyl-arginine (80 mg/kg) Brain
SA3885666Aldh7a1-/- N-methyl-arginine (80 mg/kg) Brain
SA3885678Aldh7a1-/- N-methyl-arginine (80 mg/kg) Brain
SA38856810Aldh7a1-/- N-methyl-arginine (80 mg/kg) Brain
SA3885692Aldh7a1-/- N-methyl-arginine (80 mg/kg) Brain
SA38857028Aldh7a1-/- N-methyl-arginine (80 mg/kg) Kidney
SA38857122Aldh7a1-/- N-methyl-arginine (80 mg/kg) Kidney
SA38857224Aldh7a1-/- N-methyl-arginine (80 mg/kg) Kidney
SA38857326Aldh7a1-/- N-methyl-arginine (80 mg/kg) Kidney
SA38857418Aldh7a1-/- N-methyl-arginine (80 mg/kg) Liver
SA38857516Aldh7a1-/- N-methyl-arginine (80 mg/kg) Liver
SA38857620Aldh7a1-/- N-methyl-arginine (80 mg/kg) Liver
SA38857714Aldh7a1-/- N-methyl-arginine (80 mg/kg) Liver
SA38857812Aldh7a1-/- N-methyl-arginine (80 mg/kg) Liver
SA38857934Aldh7a1-/- N-methyl-arginine (80 mg/kg) Plasma
SA38858036Aldh7a1-/- N-methyl-arginine (80 mg/kg) Plasma
SA38858138Aldh7a1-/- N-methyl-arginine (80 mg/kg) Plasma
SA38858240Aldh7a1-/- N-methyl-arginine (80 mg/kg) Plasma
SA38858332Aldh7a1-/- N-methyl-arginine (80 mg/kg) Plasma
SA3885841Aldh7a1-/- PBS Brain
SA3885853Aldh7a1-/- PBS Brain
SA3885865Aldh7a1-/- PBS Brain
SA3885877Aldh7a1-/- PBS Brain
SA3885889Aldh7a1-/- PBS Brain
SA38858921Aldh7a1-/- PBS Kidney
SA38859023Aldh7a1-/- PBS Kidney
SA38859125Aldh7a1-/- PBS Kidney
SA38859227Aldh7a1-/- PBS Kidney
SA38859329Aldh7a1-/- PBS Kidney
SA38859419Aldh7a1-/- PBS Liver
SA38859513Aldh7a1-/- PBS Liver
SA38859615Aldh7a1-/- PBS Liver
SA38859717Aldh7a1-/- PBS Liver
SA38859811Aldh7a1-/- PBS Liver
SA38859933Aldh7a1-/- PBS Plasma
SA38860031Aldh7a1-/- PBS Plasma
SA38860135Aldh7a1-/- PBS Plasma
SA38860237Aldh7a1-/- PBS Plasma
SA38860339Aldh7a1-/- PBS Plasma
Showing results 1 to 39 of 39

Collection:

Collection ID:CO003679
Collection Summary:Liver, brain, kidney, or plasma samples collected from mice treated with PBS or N-methyl-arginine (80 mg/kg) as a single intraperitoneal injection. Tissues collected 7-hours after injection.
Sample Type:Tissue or Plasma

Treatment:

Treatment ID:TR003695
Treatment Summary:Mice were administered a single intraperitoneal injection containing either saline or N-methyl-arginine (80 mg/kg). 7-hours after the injection, the mice were euthanized by cardiac puncture, and tissues were isolated for analysis by LCMS.
Treatment Compound:N-methyl-arginine
Treatment Route:Intraperitoneal
Treatment Dose:80 mg/kg
Treatment Vehicle:PBS

Sample Preparation:

Sampleprep ID:SP003693
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.

Combined analysis:

Analysis ID AN005847 AN005848
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Vanquish Thermo Vanquish
Column Merck SeQuant ZIC-pHILIC (150 x 2.1mm, 5µm) Merck SeQuant ZIC-pHILIC (150 x 2.1mm, 5µm)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Exploris 240 Thermo Exploris 240
Ion Mode POSITIVE NEGATIVE
Units Ion counts Ion counts

Chromatography:

Chromatography ID:CH004440
Instrument Name:Thermo Vanquish
Column Name:Merck SeQuant ZIC-pHILIC (150 x 2.1mm, 5µm)
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

MS:

MS ID:MS005567
Analysis ID:AN005847
Instrument Name:Thermo Exploris 240
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer operating in heated electrospray ionization mode (HESI) for analysis. The following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion transfer tube temperature 320°C, and vaporizer temperature 75°C. The global parameters included an expected peak width of 20 seconds, mild trapping, and a default charge state of 1. A 40-min polarity switching data-dependent Top 5 method was used for positive mode and a data-dependent Top 3 method was used for negative mode. Full MS scan parameters for both positive and negative modes were set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time set to automatic. ddMS2 for positive mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 30%. For both positive and negative ddMS2, we applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was also included for pipecolate and P6C/P2C, and the retention time ranges were based on elution of authentic standards (pipecolate) or from positive samples (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode.
Ion Mode:POSITIVE
  
MS ID:MS005568
Analysis ID:AN005848
Instrument Name:Thermo Exploris 240
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer operating in heated electrospray ionization mode (HESI) for analysis. The following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion transfer tube temperature 320°C, and vaporizer temperature 75°C. The global parameters included an expected peak width of 20 seconds, mild trapping, and a default charge state of 1. A 40-min polarity switching data-dependent Top 5 method was used for positive mode and a data-dependent Top 3 method was used for negative mode. Full MS scan parameters for both positive and negative modes were set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time set to automatic. ddMS2 for positive mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 30%. For both positive and negative ddMS2, we applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was also included for pipecolate and P6C/P2C, and the retention time ranges were based on elution of authentic standards (pipecolate) or from positive samples (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode.
Ion Mode:NEGATIVE
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