Return to study ST000435 main page
MB Sample ID: SA021880
| Local Sample ID: | sample43 |
| Subject ID: | SU000456 |
| Subject Type: | Animal |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000456 |
| 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 |
|---|---|---|---|---|
| sample43 | SA021880 | FL005334 | ND | treatment |
Collection:
| Collection ID: | CO000450 |
| 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: | TR000470 |
| 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: | SP000463 |
| 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from pooled plasma spiked with a selection of metabolites to mimic elevated levels of metabolites during I− (insulin withdrawn) condition.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 | AN000685 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo TSQ Quantum Ultra |
| Column | Waters Acquity BEH C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Thermo Quantum Ultra |
| Ion Mode | POSITIVE |
| Units | micromolar |
Chromatography:
| Chromatography ID: | CH000497 |
| Chromatography Summary: | Plasma samples and amino acid calibration standards were prepared with MassTrak Amino Acid Analysis Solution (AAA) kit from Waters according to instructions with slight modifications for detection on a mass spectrometer. A 10 point standard concentration curve was made from the calibration standard solution to calculate amino acid concentrations in plasma samples. A solution containing U-13C4-L-aspartic acid, U-13C3-L-alanine, U-13C4-L-threonine, U-13C5-L-proline, U-13C5-L-valine, U-13C6-leucine, U-13C6-phenylalanine all from Cambridge Isotope Laboratories, 13C6-tyrosine from Isotec, L-arginine (15N2, 2H2) from MassTrace, norvaline from Sigma dissolved in 0.01N HCl was used as the internal standard solution. Frozen plasma samples were thawed, spiked with internal standard then deproteinized with cold MeOH followed by centrifugation at 10,000 g for 5 minutes prior to derivatization according to MassTrak instructions. The amino acid derivatizing reagent used was 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. High resolution separation was done using an Acquity UPLC system, injecting 1 µl of derviatized solution, with a UPLC BEH C18 1.7 micron 2.1×150 mm column from Waters. Column flow was set to 400 µl/min with a gradient from 99.9%A to 98%B where buffer A is 1% acetonitrile in 0.1% formic acid and buffer B is 100% acetonitrile. A column temp of 43 degrees Celsius and a sample tray temp of 6% Celsius. Mass detection was completed on a TSQ Ultra Quantum from Thermo Finnigan running in ESI positive mode. A scan width of 0.002, scan time of 0.04 seconds per transition mass, collision energy of 25, collision gas pressure of 1.5 mTorr, tube lens value set to 90, monitoring a signature ion of the derivitized amines at m/z 171.04 by selected reaction monitoring. |
| Instrument Name: | Thermo TSQ Quantum Ultra |
| Column Name: | Waters Acquity BEH C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 43 |
| Flow Gradient: | from 99.9%A to 98%B |
| Flow Rate: | 400 µl/min |
| Solvent A: | 99% water/1% acetonitrile; 0.1% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000611 |
| Analysis ID: | AN000685 |
| Instrument Name: | Thermo Quantum Ultra |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
