Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA152227

Local Sample ID:	212
Subject ID:	SU001742
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Age Or Age Range:	10 weeks
Gender:	Male

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU001742
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Age Or Age Range:	10 weeks
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
212	SA152227	FL017903	LY	Treatment

Collection:

Collection ID:	CO001735
Collection Summary:	Fed male Wistar rats 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:	TR001755
Treatment Summary:	All heart perfusions underwent an initial 15-minute equilibration period with Krebs Ringer bicarbonate buffer containing 119 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose, and 0.05 mM L-carnitine at a flow rate of 12 ml/minute. After the equilibrium period, hearts were perfused for 30 minutes with 3% BSA, 100 µU/mL insulin, 0.4 mM palmitate, physiologic concentrations of amino acids, and either DMSO (Veh), BT2, or LY3351337.

Treatment ID:

TR001755

Treatment Summary:

All heart perfusions underwent an initial 15-minute equilibration period with Krebs Ringer bicarbonate buffer containing 119 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose, and 0.05 mM L-carnitine at a flow rate of 12 ml/minute. After the equilibrium period, hearts were perfused for 30 minutes with 3% BSA, 100 µU/mL insulin, 0.4 mM palmitate, physiologic concentrations of amino acids, and either DMSO (Veh), BT2, or LY3351337.

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.

Combined analysis:

Analysis ID	AN002717
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:	CH002005
Instrument Name:	Agilent 7890B
Column Name:	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Chromatography Type:	GC

MS:

MS ID:	MS002514
Analysis ID:	AN002717
Instrument Name:	Agilent 5977A
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	Masshunter used for data acquisition and processing.
Ion Mode:	UNSPECIFIED