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MB Sample ID: SA009039

Local Sample ID:7
Subject ID:SU000184
Subject Type:Animal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Genotype Strain:Sprague–Dawley
Gender:female
Species Group:Mammal

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Subject:

Subject ID:SU000184
Subject Type:Animal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Genotype Strain:Sprague–Dawley
Gender:female
Species Group:Mammal

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
7SA009039FL002135NoneMechanical loading hours/day
7SA009039FL002135ShamGroup

Collection:

Collection ID:CO000170
Collection Summary:The tibialis anterior (TA), extensor digitorum longus (EDL), plantaris, gastrocnemius and soleus muscles were dissected from the loaded left leg and the unloaded right leg immediately after death. One half of the soleus and EDL muscles together with TA and gastrocnemius were quickly frozen in liquid propane cooled by liquid nitrogen, and stored at ?160°C for further analyses. In the other halves of the soleus and EDL muscles, bundles of approximately 50 fibres were dissected from the muscles in relaxing solution at 4°C and tied to glass capillaries, stretched to about 110% of their resting slack length. The bundles were chemically skinned for 24 h in relaxing solution containing 50% (v/v) glycerol for 24 h at 4°C and were subsequently stored at ?20°C (Larsson & Moss, 1993). All the bundles were cryo-protected within 1 week after skinning by transferring the bundles every 30 min to relax solution containing increasing concentrations of sucrose, i.e. 0, 0.5, 1.0, 1.5 and 2.0 m, and subsequently frozen in liquid propane chilled with liquid nitrogen (Frontera & Larsson, 1997). The frozen bundles were stored at ?160°C pending use. One day before the experiments, a bundle was transferred to a 2.0 m sucrose solution for 30 min, subsequently incubated in solutions of decreasing sucrose concentration (1.5–0.5 m) and finally kept in a skinning solution at ?20°C.
Sample Type:Muscle

Treatment:

Treatment ID:TR000190
Treatment Summary:Mechanical stimulation|Sham operation
Treatment Protocol Comments:Sham operated controls and 46 anaesthetized and mechanically ventilated female Sprague–Dawley rats treated with ?-cobratoxin for durations varying from 6 h to 14 days were included in this study. The experimental model has previously been described in detail (Dworkin & Dworkin, 1990, 2004). For mechanical stimulation animals, left leg was mechanically stimulated and right leg was not. |Sham operated controls and 46 anaesthetized and mechanically ventilated female Sprague–Dawley rats treated with ?-cobratoxin for durations varying from 6 h to 14 days were included in this study. The experimental model has previously been described in detail (Dworkin & Dworkin, 1990, 2004). For sham operated controls, no stimulation was performed on either legs

Sample Preparation:

Sampleprep ID:SP000184
Sampleprep Summary:An i.v. bolus dose of [ring-13C6]phenylalanine (15 ?g g?1 body weight) was given 15 min prior to the animals being killed. Immediately after death, the gastrocnemius muscle was removed from the left and right hindlimb and split into a medial and lateral deep red and superficial white portion and frozen in liquid propane chilled by liquid nitrogen. Tissue fluid and mixed gastrocnemius muscle proteins were isolated according to the method of Ljungqvist et al. (1997). The mixed muscle protein precipitate from the isolation was hydrolysed by heating with 6 m HCl overnight at 110°C. Both tissue fluid and hydrolysed mixed gastrocnemius muscle amino acids were purified using a BioRad AG-50 × 8 ion exchange resin prior to mass spectrometry analysis.
Tissue fluid
The level of enrichment of [ring-13C6]phenylalanine in tissue fluid was analysed using ThermoFisher Quantum gas chromatography tandem mass spectrometry (GC/MS/MS) (San Jose, CA, USA). The heptafluorobutyryl isobutyl ester derivative was prepared as described by Ford et al. (1985) and the amino acids were measured under negative ion chemical ionisation conditions using isobutane as reactant gas. The [M-HF]? fragments reflecting the m0 and m+6 species were monitored (m/z transitions 397?377 and 403?383, respectively) and the enrichment of the label was measured against a calibration curve prepared from known amounts of labelled and unlabelled phenylalanine (range 0–30%).

Combined analysis:

Analysis ID AN000258
Analysis type MS
Chromatography type GC
Chromatography system
Column
MS Type IR MS
MS instrument type IR MS
MS instrument name Thermo DeltaPlus Isotope Ratio MS
Ion Mode NEGATIVE
Units

Chromatography:

Chromatography ID:CH000182
Chromatography Summary:The level of enrichment of [ring-13C6]phenylalanine derived from hydrolysed mixed muscle proteins were analysed using a ThermoFisher DeltaPlus Isotope Ratio mass spectrometer (IR/MS) (Bremen, Germany) fitted with an on-line gas chromatograph with oxidation and reduction furnaces as previously described (Balagopal et al. 1996). The amino acids were derivatized to their trimethyl acetyl, methyl esters according to the method of Metges et al. (1996). Any amino acid eluting from the gas chromatograph is converted to CO2 and N2 prior to entry into the IR/MS. The amino acids were derivatized to their trimethyl acetyl, methyl esters according to the method of Metges et al. (1996). Enrichment of the tracer was measured by monitoring the ratio of 13C to 12CO2 in the IR/MS and again referenced to a calibration curve (00.1%).
Chromatography Type:GC

MS:

MS ID:MS000208
Analysis ID:AN000258
Instrument Name:Thermo DeltaPlus Isotope Ratio MS
Instrument Type:IR MS
MS Type:IR MS
MS Comments:the amino acids were measured under negative ion chemical ionisation conditions using isobutane as reactant gas
Ion Mode:NEGATIVE
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