Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA152236

Local Sample ID:	35
Subject ID:	SU001743
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	8 weeks
Gender:	Male

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU001743
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	8 weeks
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
35	SA152236	FL017905	SLC25A44	Treatment

Collection:

Collection ID:	CO001736
Collection Summary:	Fed male C57BL/6J mice were anesthetized with 5% isoflurane, and isolated hearts were perfused in the Langendorff mode at 37°C with non-recirculating perfusate. The hearts were allowed to beat spontaneously throughout the perfusion. At the end of each perfusion, hearts were immediately freeze-clamped in liquid nitrogen using the Wollenberger technique and stored at -80 oC for further analysis.
Sample Type:	Heart

Treatment:

Treatment ID:	TR001756
Treatment Summary:	SLC25A44 and GFP expression plasmids with a CMV promoter containing ITRs were used to prepare recombinant AAV9 by the UMass Gene Therapy Core. Adult 8-week-old male mice were injected intravenously with 100 µl of 5.1 x 1011 vector genomes per mouse.

Sample Preparation:

Sampleprep ID:	SP001749
Sampleprep Summary:	Frozen tissues were pulverized under liquid nitrogen using a mortar and pestle. Metabolites were then extracted using sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, tissue lysates were prepared with a Tissue Lyser (Qiagen) for 60 seconds at 30Hz. Similarly, plasma metabolites (20 μL) were extracted by sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, samples were vortexed for 30 seconds. Tissue extracts were then centrifuged at 4°C and 14400 x g for 20 minutes and the clarified aqueous phase was transferred to a fresh Eppendorf and stored in -80°C until processing for GC-MS analysis. For GC-MS analysis, the extracted tissue was dried under N2 gas-flow at 37°C using an evaporator. Amino and organic acids were derivatized via methoximation and silylation. Briefly, metabolites were resuspended in 25 μL of methoxylamine hydrochloride (2% (w/v) in pyridine) and incubated at 40°C for 90 minutes on a heating block. After brief centrifugation, 35 μL of MTBSTFA + 1% TBDMS was added and the samples were incubated at 60°C for 30 minutes.

Sampleprep ID:

SP001749

Sampleprep Summary:

Frozen tissues were pulverized under liquid nitrogen using a mortar and pestle. Metabolites were then extracted using sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, tissue lysates were prepared with a Tissue Lyser (Qiagen) for 60 seconds at 30Hz. Similarly, plasma metabolites (20 μL) were extracted by sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, samples were vortexed for 30 seconds. Tissue extracts were then centrifuged at 4°C and 14400 x g for 20 minutes and the clarified aqueous phase was transferred to a fresh Eppendorf and stored in -80°C until processing for GC-MS analysis. For GC-MS analysis, the extracted tissue was dried under N2 gas-flow at 37°C using an evaporator. Amino and organic acids were derivatized via methoximation and silylation. Briefly, metabolites were resuspended in 25 μL of methoxylamine hydrochloride (2% (w/v) in pyridine) and incubated at 40°C for 90 minutes on a heating block. After brief centrifugation, 35 μL of MTBSTFA + 1% TBDMS was added and the samples were incubated at 60°C for 30 minutes.

Combined analysis:

Analysis ID	AN002718
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890B
Column	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5977A
Ion Mode	UNSPECIFIED
Units	Concentration of 13C (uM)

Chromatography:

Chromatography ID:	CH002006
Instrument Name:	Agilent 7890B
Column Name:	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Chromatography Type:	GC

MS:

MS ID:	MS002515
Analysis ID:	AN002718
Instrument Name:	Agilent 5977A
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	Masshunter used for data acquisition and processing.
Ion Mode:	UNSPECIFIED