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MB Sample ID: SA005013
| Local Sample ID: | 25 LH - T |
| Subject ID: | SU000110 |
| 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: | SU000110 |
| 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 |
|---|---|---|---|---|
| 25 LH - T | SA005013 | FL001315 | Insulin Treatment | Treatment |
Collection:
| Collection ID: | CO000093 |
| 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: | TR000111 |
| 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: | SP000106 |
| Sampleprep 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. |
Combined analysis:
| Analysis ID | AN000145 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| 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 | uM |
Chromatography:
| Chromatography ID: | CH000103 |
| Chromatography Summary: | High resolution separation was done using an Acquity UPLC system, injecting 1 l of derviatized solution, with a UPLC BEH C18 1.7 micron 2.1150 mm column from Waters. Column flow was set to 400 l/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 degree Celsius. |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 43 |
| Flow Gradient: | 99.9% A to 98% B |
| Flow Rate: | 401 µl/min |
| Solvent A: | 99% water/1% acetonitrile; 0.1% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000121 |
| Analysis ID: | AN000145 |
| Instrument Name: | Thermo Quantum Ultra |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | 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. |
| Ion Mode: | POSITIVE |
