Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA022591

Local Sample ID:	sample11
Subject ID:	SU000464
Subject Type:	Animal
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000464
Subject Type:	Animal
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
sample11	SA022591	FL005546	T1D good glycemic control	treatment

Collection:

Collection ID:	CO000458
Collection Summary:	At 5:00 AM after an overnight fast, baseline blood samples were collected from study participants. Plasma samples were stored at 80°C until analysis.
Sample Type:	Blood. Plasma was isolated for MS analysis.

Treatment:

Treatment ID:	TR000478
Treatment Summary:	Participants were admitted to the Clinical Research Unit at St Mary’s Hospital (Rochester, Minnesota) the evening before the study and spent overnight in the Clinical Research Unit. The participants were given a standard meal on the evening of the admission after which they fasted overnight. Participants with T1D were treated with insulin as per their usual individual programs. At 5:00 AM after an overnight fast, baseline blood samples were collected from study participants. Plasma samples were stored at 80°C until analysis.

Treatment ID:

TR000478

Treatment Summary:

Participants were admitted to the Clinical Research Unit at St Mary’s Hospital (Rochester, Minnesota) the evening before the study and spent overnight in the Clinical Research Unit. The participants were given a standard meal on the evening of the admission after which they fasted overnight. Participants with T1D were treated with insulin as per their usual individual programs. At 5:00 AM after an overnight fast, baseline blood samples were collected from study participants. Plasma samples were stored at 80°C until analysis.

Sample Preparation:

Sampleprep ID:	SP000471
Sampleprep Summary:	Plasma quality-control samples used in the study were prepared from pooled plasma spiked with a selection of metabolites to mimic elevated levels of metabolites during I− (insulin withdrawn) condition. Plasma was spiked with a standard mixture (3:1 ratio of plasma to spiking solution) containing 100 μg/mL niacin, hypoxanthine, leucine, isoleucine, phenylalanine, tryptophan, citric acid, glucose, hippuric acid, and taurocholic acid dissolved in 1:1 acetonitrile/water. All plasma samples (200 μL) were thawed on ice at 4°C followed by deproteinization with methanol (1:4 ratio of plasma to methanol) and vortexed for 10 s, followed by incubation at −20°C for 2 h. The samples were then centrifuged at 15,871g for 30 min at 4°C. The supernatants were lyophilized (Savant, Holbrook, NY) and stored at −20°C prior to analysis. The samples were reconstituted in 50% H2O/acetonitrile and passed through a Microcon YM3 filter (Millipore Corporation). The supernatants were transferred to analytical vials, stored in the autosampler at 4°C, and analyzed within 48 h of reconstitution in buffer.

Sampleprep ID:

SP000471

Sampleprep Summary:

Plasma quality-control samples used in the study were prepared from pooled plasma spiked with a selection of metabolites to mimic elevated levels of metabolites during I− (insulin withdrawn) condition. Plasma was spiked with a standard mixture (3:1 ratio of plasma to spiking solution) containing 100 μg/mL niacin, hypoxanthine, leucine, isoleucine, phenylalanine, tryptophan, citric acid, glucose, hippuric acid, and taurocholic acid dissolved in 1:1 acetonitrile/water. All plasma samples (200 μL) were thawed on ice at 4°C followed by deproteinization with methanol (1:4 ratio of plasma to methanol) and vortexed for 10 s, followed by incubation at −20°C for 2 h. The samples were then centrifuged at 15,871g for 30 min at 4°C. The supernatants were lyophilized (Savant, Holbrook, NY) and stored at −20°C prior to analysis. The samples were reconstituted in 50% H2O/acetonitrile and passed through a Microcon YM3 filter (Millipore Corporation). The supernatants were transferred to analytical vials, stored in the autosampler at 4°C, and analyzed within 48 h of reconstitution in buffer.

Combined analysis:

Analysis ID	AN000694
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 6890
Column	None
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5973
Ion Mode	POSITIVE
Units	micromolar

Chromatography:

Chromatography ID:	CH000502
Chromatography Summary:	The derivatized extracts were analyzed with an Agilent 6890 gas chromatograph coupled with an Agilent 5973 mass selective detector. The 1-μL aliquots of the extracts were injected into a DB5-MS capillary column (30 m × 250 μm i.d., 0.25-μm film thickness; J&W Scientific, Folson, CA) using PTV injection (Gerstel CIS4 injector) in the splitless mode. The temperature of the PTV was 70 °C during injection, and 0.6 min after injection, the temperature was raised to 300 °C at a rate of 2 °C/s and held at 300 °C for 20 min. The initial GC oven temperature was 70 °C, 5 min after injection the GC oven temperature was increased with 5 °C/min to 320 °C and held for 5 min at 320 °C. Helium was used as a carrier gas and pressure programmed such that the helium flow was kept constant at a flow rate of 1.7 mL/min. Detection was achieved using MS detection in electron impact mode and full scan monitoring mode (m/z 15−800). The temperature of the ion source was set at 250 °C and that of the quadrupole at 200 °C.
Instrument Name:	Agilent 6890
Column Name:	None
Chromatography Type:	GC

MS:

MS ID:	MS000616
Analysis ID:	AN000694
Instrument Name:	Agilent 5973
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE