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MB Sample ID: SA018731
| Local Sample ID: | 061215byusa1067_1 |
| Subject ID: | SU000414 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6J |
| Age Or Age Range: | 4 months |
| Gender: | Female |
| Human Smoking Status: | Former/Current |
| Animal Animal Supplier: | Jackson Laboratories |
| Animal Housing: | housed in a facility at 23°C |
| Animal Light Cycle: | 12∶12 hr light cycle, lights on 0700 |
| Animal Feed: | with free access to chow (4.5% fat/weight), for at least 1 week before being studied |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000414 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6J |
| Age Or Age Range: | 4 months |
| Gender: | Female |
| Human Smoking Status: | Former/Current |
| Animal Animal Supplier: | Jackson Laboratories |
| Animal Housing: | housed in a facility at 23°C |
| Animal Light Cycle: | 12∶12 hr light cycle, lights on 0700 |
| Animal Feed: | with free access to chow (4.5% fat/weight), for at least 1 week before being studied |
| Species Group: | Mammal |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 061215byusa1067_1 | SA018731 | FL004338 | mitochondria | Organ |
| 061215byusa1067_1 | SA018731 | FL004338 | 20 min | Time |
| 061215byusa1067_1 | SA018731 | FL004338 | palmitic acid 2.4 uM | Dose |
Collection:
| Collection ID: | CO000408 |
| Collection Summary: | Skeletal muscle mitochondria were isolated essentially according to Chappell and Perry [Chappell JB, Perry SV (1954) Biochemical and osmotic properties of skeletal muscle mitochondria. Nature 173: 1094–1095.]. All media were ice-cold, and procedures done on ice or at 4°C. Briefly, pectoral, forelimb and hindlimb muscles were rapidly dissected and placed in basic medium [BM (mM): KCl (140), HEPES (20), MgCl2 (5), EGTA (2); pH 7.0]. Together, these muscle groups comprise a mixed population of mainly type II oxidative and glycolytic fibers. Muscle was cleaned of connective tissue and fat, minced and placed in 15 vol of homogenizing medium (HM: BM with 1 mM ATP and 1% BSA (w/v)) containing one unit of protease (Subtilisin A) per g muscle wet weight. |
| Collection Protocol Filename: | Long-Chain_Fatty_Acid_Metabolite_Profiles_in_Skeletal_Muscle_Mitochondria.PDF |
| Sample Type: | Mitochondria |
Treatment:
| Treatment ID: | TR000428 |
| Treatment Summary: | Mitochondria (0.6 mg/ml) were supplied with three concentrations of palmitate corresponding to rates of β-oxidation: 1. low (2 µM) 2. medium (9 µM) 3. high (19 µM) Three ml aliquots of incubation medium [IM, (mM): KCl (120), HEPES (5), KH2PO4 (5), MgCl2 (5) and EGTA (1); pH 7.4] were supplemented with (mM) ATP (1), malate (0.05), coenzyme A (0.025), and carnitine (0.5) and added to 20-ml glass reaction vials. Solutions of low, medium and high palmitate concentrations were added to vials in a 6∶1 FA:BSA complex. Two additional incubations were performed as controls: 1. 0 µM palmitate 2. 9 µM palmitate + inhibitors |
| Treatment Protocol Filename: | Long-Chain_Fatty_Acid_Metabolite_Profiles_in_Skeletal_Muscle_Mitochondria.PDF |
| Treatment Protocol Comments: | The first control condition evaluated the metabolic profile of mitochondria oxidizing only malate, and included ATP, carnitine and CoA and ethanol (0.5%). The second control condition assessed effects of FA in the absence of complete oxidative catabolism, and consisted of malate, 9 µM palmitate, ATP, carnitine and CoA, and supplemented with the TCA cycle inhibitor malonate (10 mM) and the electron transport chain complex I inhibitor rotenone (5 µM) |
Sample Preparation:
| Sampleprep ID: | SP000421 |
| Sampleprep Summary: | Tissue was homogenized using a glass/Teflon Potter-Elvehjem tissue grinder (240 rpm) and fractionated by centrifugation at 800 g (10 min), and the supernatant collected and spun at 12000 g (9 min). The pellet was resuspended in 20 ml BM and incubated on ice for 5 min (myofibrillar repolymerization). Samples were spun at 800 g (8 min) to pellet actin-myosin polymers. The supernatant was then spun at 12000 g (9 min). The final pellet was resuspended in 220 µl of BM. |
| Sampleprep Protocol Filename: | Long-Chain_Fatty_Acid_Metabolite_Profiles_in_Skeletal_Muscle_Mitochondria.PDF |
| Sampleprep Protocol Comments: | This isolation procedure yields mitochondria with high respiratory control ratios (state 3/state 4; ∼8–10 when supplied with 10 mM pyruvate/5 mM malate), and which are capable of activating palmitate [29], a process dependent on the integrity of enzymes on the mitochondrial outer membrane. Protein concentration was determined by a modified Lowry method with BSA as standard. |
| Processing Method: | Homogenized |
Combined analysis:
| Analysis ID | AN000629 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Leco Pegasus III GC |
| Column | Restek Corporation Rtx-5Sil MS |
| MS Type | EI |
| MS instrument type | GC-TOF |
| MS instrument name | Leco Pegasus III GC TOF |
| Ion Mode | POSITIVE |
| Units | counts |
Chromatography:
| Chromatography ID: | CH000454 |
| Instrument Name: | Leco Pegasus III GC |
| Column Name: | Restek Corporation Rtx-5Sil MS |
| Column Pressure: | 7.7 PSI |
| Column Temperature: | 50-330C |
| Flow Rate: | 1 ml/min |
| Injection Temperature: | 50 C ramped to 250 C by 12 C/s |
| Sample Injection: | 0.5 uL |
| Transferline Temperature: | 230C |
| Washing Buffer: | Ethyl Acetate |
| Sample Loop Size: | 30 m length x 0.25 mm internal diameter |
| Randomization Order: | Excel generated |
| Chromatography Type: | GC |
MS:
| MS ID: | MS000562 |
| Analysis ID: | AN000629 |
| Instrument Name: | Leco Pegasus III GC TOF |
| Instrument Type: | GC-TOF |
| MS Type: | EI |
| Ion Mode: | POSITIVE |
| Ion Source Temperature: | 250 C |
| Ionization: | Pos |
| Ionization Energy: | 70 eV |
| Mass Accuracy: | Nominal |
| Source Temperature: | 250 C |
| Scan Range Moverz: | 85-500 Da |
| Scanning Cycle: | 17 Hz |
| Scanning Range: | 85-500 Da |
| Skimmer Voltage: | 1850 V |
