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MB Sample ID: SA001935

Local Sample ID:Group5C
Subject ID:SU000063
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

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Subject:

Subject ID:SU000063
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Group5CSA001935FL000620Saline InfusionTreatment

Collection:

Collection ID:CO000046
Collection Summary:Arterialized venous blood was obtained from a catheterized hand vein maintained at 60°C using a hot box for the duration of the study. Plasma samples were stored at -80°C until analysis.
Sample Type:Blood. Plasma was isolated for MS analysis.
Collection Location:Hand vein

Treatment:

Treatment ID:TR000064
Treatment Summary:Saline Infusion | Insulin Withdrawal | Insulin Infusion
Treatment Protocol ID:SI / IW / II
Treatment Protocol Comments:ND participants were kept on a saline infusion from the evening following their meal. | Insulin was discontinued for 8.6 +/- 0.6 h starting at 0400 h. | Insulin was infused into a forearm vein to maintain blood glucose between 4.44 and 5.56 mmol | L overnight until 1200 h the next day.
Treatment Compound:Saline | Insulin

Sample Preparation:

Sampleprep ID:SP000059
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from pooled plasma spiked with a selection of metabolites to mimic elevated levels of metabolites during I- (insulin withdrawn) condition.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 AN000072 AN000073 AN000074 AN000075
Analysis type MS MS MS MS
Chromatography type Reversed phase HILIC Reversed phase HILIC
Chromatography system Waters Acquity Waters Acquity Waters Acquity Waters Acquity
Column Waters high-strength silica (150 x 2.1mm,1.8um) Waters ethylene-bridged hybrid (150 x 2.1mm,1.7um) Waters high-strength silica (150 x 2.1mm,1.8um) Waters ethylene-bridged hybrid (150 x 2.1mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type TOF TOF TOF TOF
MS instrument name Agilent 6220 TOF Agilent 6220 TOF Agilent 6220 TOF Agilent 6220 TOF
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units Raw MS Intensities Raw MS Intensities Raw MS Intensities Raw MS Intensities

Chromatography:

Chromatography ID:CH000047
Chromatography Summary:C18
Chromatography Comments:The liquid chromatography platform consisted of an Acquity UPLC system (Waters, Milford, MA). Plasma metabolite separation was achieved using both hydrophilic interaction chromatography (ethylene-bridged hybrid 2.1 150 mm, 1.7 m; Waters) and reversed-phase liquid chromatography C18 (high-strength silica 2.1 150 mm, 1.8 m; Waters). For each column, the run time was 20 min at a flow rate of 400 L/min. Reverse-phase chromatography was performed using 99% solvent A (5 mmol/L NH4 acetate, 0.1% formic acid, and 1% acetonitrile) to 100% solvent B (95% acetonitrile with 0.1% formic acid). The gradient was 0 min, 0% B; 1 min, 0% B; 3 min, 5% B; 13.0 min, 100% B; 16 min, 100% B; 16.5 min, 0% B; and 20 min, 0% B. Other LC parameters were injection volume 5 L and column temperature 50C. Each sample was injected in triplicate with blank injections between each sample. Quality controls and standards were run at the beginning and end of the sequence.
Instrument Name:Waters Acquity
Column Name:Waters high-strength silica (150 x 2.1mm,1.8um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH000048
Chromatography Summary:HILIC
Chromatography Comments:The liquid chromatography platform consisted of an Acquity UPLC system (Waters, Milford, MA). Plasma metabolite separation was achieved using both hydrophilic interaction chromatography (ethylene-bridged hybrid 2.1 150 mm, 1.7 m; Waters) and reversed-phase liquid chromatography C18 (high-strength silica 2.1 150 mm, 1.8 m; Waters). For each column, the run time was 20 min at a flow rate of 400 L/min. The hydrophilic interaction chromatography gradient was as follows: 0 min, 100% B; 1 min, 100% B; 5 min, 90% B; 13.0 min, 0% B; 16 min, 0% B; 16.5 min, 100% B; and 20 min, 100% B. Other LC parameters were injection volume 5 L and column temperature 50C. Each sample was injected in triplicate with blank injections between each sample. Quality controls and standards were run at the beginning and end of the sequence.
Instrument Name:Waters Acquity
Column Name:Waters ethylene-bridged hybrid (150 x 2.1mm,1.7um)
Chromatography Type:HILIC

MS:

MS ID:MS000053
Analysis ID:AN000072
Instrument Name:Agilent 6220 TOF
Instrument Type:TOF
MS Type:ESI
MS Comments:Positive-ion mode/C18: A 6220 ToF MS (Agilent Technologies) was operated in both positive and negative electrospray ionization modes using a scan range of 50?1,200 m/z. The mass accuracy and mass resolution were <5 parts per million (ppm) and ~20,000, respectively. The instrument settings were as follows: nebulizer gas temperature 325°C, capillary voltage 3.5 kV, capillary temperature 300°C, fragmentor voltage 150 V, skimmer voltage 58 V, octapole voltage 250 V, cycle time 0.5 s, and run time 15.0 min.
Ion Mode:POSITIVE
  
MS ID:MS000054
Analysis ID:AN000073
Instrument Name:Agilent 6220 TOF
Instrument Type:TOF
MS Type:ESI
MS Comments:Positive-ion mode/HILIC: A 6220 ToF MS (Agilent Technologies) was operated in both positive and negative electrospray ionization modes using a scan range of 50?1,200 m/z. The mass accuracy and mass resolution were <5 parts per million (ppm) and ~20,000, respectively. The instrument settings were as follows: nebulizer gas temperature 325°C, capillary voltage 3.5 kV, capillary temperature 300°C, fragmentor voltage 150 V, skimmer voltage 58 V, octapole voltage 250 V, cycle time 0.5 s, and run time 15.0 min.
Ion Mode:POSITIVE
  
MS ID:MS000055
Analysis ID:AN000074
Instrument Name:Agilent 6220 TOF
Instrument Type:TOF
MS Type:ESI
MS Comments:Negative-ion mode/C18: A 6220 ToF MS (Agilent Technologies) was operated in both positive and negative electrospray ionization modes using a scan range of 50?1,200 m/z. The mass accuracy and mass resolution were <5 parts per million (ppm) and ~20,000, respectively. The instrument settings were as follows: nebulizer gas temperature 325°C, capillary voltage 3.5 kV, capillary temperature 300°C, fragmentor voltage 150 V, skimmer voltage 58 V, octapole voltage 250 V, cycle time 0.5 s, and run time 15.0 min.
Ion Mode:NEGATIVE
  
MS ID:MS000056
Analysis ID:AN000075
Instrument Name:Agilent 6220 TOF
Instrument Type:TOF
MS Type:ESI
MS Comments:Negative-ion mode/HILIC: A 6220 ToF MS (Agilent Technologies) was operated in both positive and negative electrospray ionization modes using a scan range of 50?1,200 m/z. The mass accuracy and mass resolution were <5 parts per million (ppm) and ~20,000, respectively. The instrument settings were as follows: nebulizer gas temperature 325°C, capillary voltage 3.5 kV, capillary temperature 300°C, fragmentor voltage 150 V, skimmer voltage 58 V, octapole voltage 250 V, cycle time 0.5 s, and run time 15.0 min.
Ion Mode:NEGATIVE
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