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MB Sample ID: SA006130
| Local Sample ID: | D-25 |
| Subject ID: | SU000134 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 13-wk-old |
| Gender: | Male |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000134 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 13-wk-old |
| Gender: | Male |
| Species Group: | Mammal |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| D-25 | SA006130 | FL001580 | diabetic untreated | Treatment |
Collection:
| Collection ID: | CO000118 |
| Collection Summary: | Mitochondria were isolated from quadriceps muscle by differential centrifugation, as described previously (38). Briefly, quadriceps muscle samples were homogenized on ice using a motor-driven Potter-Elvehjem tissue grinder. After initial centrifugation, the supernatant containing the mitochondrial and sarcoplasmic fraction was transferred to a chilled microcentrifuge tube and centrifuged at 10,000 g for 2 min to pellet mitochondria. The supernatant containing sarcoplasmic fraction was frozen for further analysis. Mitochondrial pellet was washed twice by resuspending/centrifugation and finally suspended in a mitochondrial storage buffer. The levels of both the LCFa-CoA and sphingolipids in homogenates and various muscle fractions were normalized to total protein content, as measured by 660 nm Protein Assay (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL). |
| Sample Type: | Blood |
Treatment:
| Treatment ID: | TR000136 |
| Treatment Summary: | Control/Diabetic; insulin treated/Diabetic; insulin deprived |
| Treatment Compound: | blank/Insulin/Insulin |
| Treatment Route: | Skin implants |
| Animal Anesthesia: | phenobarbital |
| Animal Endp Euthanasia: | 5 weeks after treatment |
| Animal Endp Tissue Coll List: | plasma, muscle, liver and skin |
| Animal Endp Tissue Proc Method: | see attached reference publication PDF |
Sample Preparation:
| Sampleprep ID: | SP000131 |
| Sampleprep Summary: | Mitochondria were isolated from quadriceps muscle by differential centrifugation, as described previously (38). Briefly, quadriceps muscle samples were homogenized on ice using a motor-driven Potter-Elvehjem tissue grinder. After initial centrifugation, the supernatant containing the mitochondrial and sarcoplasmic fraction was transferred to a chilled microcentrifuge tube and centrifuged at 10,000 g for 2 min to pellet mitochondria. The supernatant containing sarcoplasmic fraction was frozen for further analysis. Mitochondrial pellet was washed twice by resuspending/centrifugation and finally suspended in a mitochondrial storage buffer. The levels of both the LCFa-CoA and sphingolipids in homogenates and various muscle fractions were normalized to total protein content, as measured by 660 nm Protein Assay (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL). / Plasma free fatty acid concentrations were measured by liquid chromatography/mass spectrometry (LC/MS), as described previously (51). Briefly, 50 ?l of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M ? H]? ions. plasma LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 × 150 mm, 1.7 ?m (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids. |
| Sampleprep Protocol Filename: | PMID-24368672-Zabielski-Nair-AJPEM-2014.pdf |
| Sampleprep Protocol Comments: | Pubmed ID: 24368672 |
Combined analysis:
| Analysis ID | AN000195 | AN000196 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | ||
| Chromatography system | ||
| Column | ||
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | ABI Sciex API 5000 QQQ | Thermo TSQ Quantum Ultra |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | uM | uM |
Chromatography:
| Chromatography ID: | CH000129 |
| Chromatography Summary: | Plasma free fatty acid concentrations were measured by liquid chromatography/mass spectrometry (LC/MS), as described previously (51). Briefly, 50 ?l of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M ? H]? ions. LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 150 mm, 1.7 ?m (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids. |
| Methods Filename: | PMID-24368672-Zabielski-Nair-AJPEM-2014.pdf |
| Chromatography Comments: | Pubmed ID: 24368672 |
MS:
| MS ID: | MS000158 |
| Analysis ID: | AN000195 |
| Instrument Name: | ABI Sciex API 5000 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Briefly, 50 ul of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M - H]- ions. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS000159 |
| Analysis ID: | AN000196 |
| Instrument Name: | Thermo TSQ Quantum Ultra |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 × 150 mm, 1.7 um (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids. |
| Ion Mode: | POSITIVE |
