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MB Sample ID: SA001923
Local Sample ID: | Group7B |
Subject ID: | SU000063 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU000063 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Human |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Group7B | SA001923 | FL000619 | Insulin Withdrawal | Treatment |
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from pooled plasma spiked with a selection of metabolites to mimic elevated levels of metabolites during I- (insulin withdrawn) condition.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 |