Return to study ST000149 main page
MB Sample ID: SA008168
Local Sample ID: | 11D |
Subject ID: | SU000168 |
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: | SU000168 |
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 |
---|---|---|---|---|
11D | SA008168 | FL001970 | Low Ins +EAA | Sample Group |
Collection:
Collection ID: | CO000154 |
Collection Summary: | Percutaneous muscle biopsy specimens of the vastus lateralis (?300 mg) were performed at 180 and 480 minutes under local anesthesia (2% lidocaine). Muscle samples were blotted, were dissected free of fat and connective tissue, and then were frozen. Biopsy specimens were collected from the opposite legs on a single visit. Indirect calorimetry was performed at 11:00 am for 20 minutes using a ventilated hood (Parvo Medics). The final 10 minutes of steady-state oxygen consumption (Vo2) and carbon dioxide production (Vco2) determined the respiratory exchange ratio (RER = Vco2/Vo2). Urine was collected throughout the clamp and analyzed for urea content at a core laboratory. |
Sample Type: | Muscle |
Treatment:
Treatment ID: | TR000173 |
Treatment Summary: | Low Insulin | High Insulin | Low Ins+ Saline | Low Ins +EAA | High Insulin+Saline | High Insulin + EAA |
Sample Preparation:
Sampleprep ID: | SP000168 |
Sampleprep Summary: | MMP and muscle fractions were isolated from frozen samples using differential centrifugation (18, 19). Biopsy samples were homogenized with protease and phosphatase inhibitors (Halt; Thermo Fisher Scientific) and centrifuged to pellet myofibrillar (MYO) proteins. The supernatant was centrifuged to pellet mitochondrial (MITO) proteins, and the final supernatant was deproteinated with cold ethanol (1:9, v/v) and then centrifuged to pellet sarcoplasmic (SARC) proteins. Aliquots from MMP, MYO, SARC, and MITO were acid hydrolyzed, and free amino acids were purified using cation exchange columns and then were dried. Plasma phenylalanine enrichment was determined using gas chromatography (GC) and mass spectrometry (MS) as described previously (19). Samples were derivatized to a heptafluorobutyryl isobutyl ester and identified with a Micromass Quattro Micro triple quadrupole GC-MS system (Waters) operating under negative ion chemical ionization using isobutane as the reactant gas. Selected ion monitoring of m/z 399.2 and 403.2 M + 2 and M + 6 fragments of phenylalanine and the l-[ring-13C6]phenylalanine, respectively, was performed. |
Combined analysis:
Analysis ID | AN000236 |
---|---|
Analysis type | MS |
Chromatography type | |
Chromatography system | |
Column | |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | ABI Sciex API 5000 QQQ |
Ion Mode | POSITIVE |
Units | uM |
Chromatography:
Chromatography ID: | CH000165 |
MS:
MS ID: | MS000187 |
Analysis ID: | AN000236 |
Instrument Name: | ABI Sciex API 5000 QQQ |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Dried amino acids were prepared as isobutyl ester derivatives, and the mass was determined by an API 5000 triple quadrupole mass spectrometer with a TurboIonSpray source (Applied Biosystems). |
Ion Mode: | POSITIVE |