Summary of Study ST001665

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 PR001070. The data can be accessed directly via it's Project DOI: 10.21228/M8D12Q This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST001665
Study Title	Branched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the rat heart
Study Summary	Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine.
Institute	Duke University
Last Name	Walejko
First Name	Jacquelyn
Address	300 N Duke St
Email	jacquelyn.walejko@duke.edu
Phone	9194792304
Submit Date	2021-01-26
Analysis Type Detail	GC-MS
Release Date	2021-02-17
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP001748
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 and plasma 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:

SP001748

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 and plasma 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.