Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237277

Local Sample ID:	m321
Subject ID:	SU002458
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	wild type C57BL6/J
Age Or Age Range:	8 weeks
Weight Or Weight Range:	25-30g
Gender:	Male
Animal Animal Supplier:	Animal Resource Centre, Western Australai
Animal Housing:	standard
Animal Light Cycle:	12 h light dark cycle
Animal Feed:	normal chow
Animal Water:	autoclaved

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Sample Preparation:

Sampleprep ID:	SP002464
Sampleprep Summary:	Metabolites were extracted from 50 µl serum using 150 µl methanol and 50 µl chloroform, including 13C-sorbitol and 13C,15N-valine internal standards, and 30 µl of clarified sera was prepared for GC-MS analysis. Liver metabolites were extracted from 20-40 mg liver tissue (sampled from the same region of the left lateral lobe) in 600 µl methanol:MilliQ water (3:1), including 13C-sorbitol and 13C,15N-valine internal standards. The tissue was cryomilled at 4°C. 110 µl chloroform was added to 440 µg homogenate which was transferred to a new tube and the sample thermomixed for 20 minutes then centrifuged at 13,000 rpm for 15 minutes. 30 µl supernatant was dried in inserts for GC-MS analysis. Dried samples for targeted analysis were derivatised online using the Shimadzu AOC6000 autosampler robot. Derivatisation was achieved by the addition of 25 µL methoxyamine hydrochloride (30 mg/mL in pyridine, Merck) followed by shaking at 37°C for 2h. Samples were then derivatised with 25 µL of N,O-bis (trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA with 1% TMCS, Thermo Scientific) for 1h at 37°C. The sample was allowed to equilibrate at room temperature for 1 h before 1 µL was injected onto the GC column using a hot needle technique. Split (1:10) injections were performed for each sample.

Sampleprep ID:

SP002464

Sampleprep Summary:

Metabolites were extracted from 50 µl serum using 150 µl methanol and 50 µl chloroform, including 13C-sorbitol and 13C,15N-valine internal standards, and 30 µl of clarified sera was prepared for GC-MS analysis. Liver metabolites were extracted from 20-40 mg liver tissue (sampled from the same region of the left lateral lobe) in 600 µl methanol:MilliQ water (3:1), including 13C-sorbitol and 13C,15N-valine internal standards. The tissue was cryomilled at 4°C. 110 µl chloroform was added to 440 µg homogenate which was transferred to a new tube and the sample thermomixed for 20 minutes then centrifuged at 13,000 rpm for 15 minutes. 30 µl supernatant was dried in inserts for GC-MS analysis. Dried samples for targeted analysis were derivatised online using the Shimadzu AOC6000 autosampler robot. Derivatisation was achieved by the addition of 25 µL methoxyamine hydrochloride (30 mg/mL in pyridine, Merck) followed by shaking at 37°C for 2h. Samples were then derivatised with 25 µL of N,O-bis (trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA with 1% TMCS, Thermo Scientific) for 1h at 37°C. The sample was allowed to equilibrate at room temperature for 1 h before 1 µL was injected onto the GC column using a hot needle technique. Split (1:10) injections were performed for each sample.