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MB Sample ID: SA332490

Local Sample ID:Muscle_505
Subject ID:SU003191
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6
Gender:Male

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Subject:

Subject ID:SU003191
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6
Gender:Male

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Muscle_505SA332490FL039354metforminTreatment

Collection:

Collection ID:CO003184
Collection Summary:C57BL/6 mice were housed North Carolina State University Biological Resources Facility with ad libitum access to food (Laboratory Rodent Diet 5001) and water on a 12-hour light/dark cycle. At the age of 8 weeks, mice were administrated metformin in their drinking water (1mg/ml) for 12 days. Before tissue and blood collection, mice were fasted for 5 hours (6am-11am). Mice were then anesthetized with isoflurane and sacrificed via cervical dislocation. Blood was collected into tubes containing EDTA anticoagulant via cardiac puncture. Plasma was obtained by centrifugation of whole blood at 1,500 g for 10 min at 4 °C and stored at -80 °C freezer. Tissues were immediately snap-frozen in liquid nitrogen, except that small intestines were rinsed with PBS and then the jejunum portion was stored in -80 °C freezer until further analysis.
Sample Type:plasma, adipose, liver, muscle, heart, kidney, intestine, adipose

Treatment:

Treatment ID:TR003200
Treatment Summary:C57BL/6 mice were housed North Carolina State University Biological Resources Facility with ad libitum access to food (Laboratory Rodent Diet 5001) and water on a 12-hour light/dark cycle. At the age of 8 weeks, mice were administrated metformin in their drinking water (1mg/ml) for 12 days.

Sample Preparation:

Sampleprep ID:SP003197
Sampleprep Summary:All tissue sample was first homogenized in liquid nitrogen and then 10-20 mg tissues were weighed into a new 1.7 ml Eppendorf tube. Ice cold extraction solvent (400 l 80% methanol/water) and 10 l metformin-d6 (50 ng/l in water) was added to each sample. Geno/Grinder homogenizer was used (1500 rpm, 1 to 2 min) to further break down the tissue chunk and form an even suspension. 200 ul supernatant containing polar metabolites was removed after centrifugation at 20,000 g at 4 °C for 10 min and transferred to LC vial for polar metabolite analysis using LC-HRMS. To extract metabolites and lipids from mouse plasma, 10 l plasma was mixed with 10 l water containing internal standards (50 ng/l metformin-d6, 5 mM [U-13C]-glucose and [U-13C]-lactate, and 80 l ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 20 l was transferred directly to LC vial without solvent evaporation, followed by LC-HRMS analysis (injection volume, 3 l) of polar metabolites such as glucose and metformin.

Combined analysis:

Analysis ID AN005034
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000
Column Waters Xbridge Amide (100 × 2.1 mm, 3.5 um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode UNSPECIFIED
Units arbitrary unit

Chromatography:

Chromatography ID:CH003805
Chromatography Summary:A hydrophilic interaction chromatography method (HILIC) with an Xbridge amide column (100 x 2.1 mm i.d., 3.5 m; Waters) was used for compound separation at 25 °C. Mobile phase A: water with 5 mM ammonium acetate (pH 6.8), and mobile phase B: 100% acetonitrile. Linear gradient is: 0 min, 85% B; 1.5 min, 85% B; 5.5 min, 35% B; 6.9 min, 35% B; 10.5 min, 35% B; 10.6 min, 10% B; 12.5 min, 10% B; 13.5 min, 85% B; 17.9 min, 85% B; 18 min, 85% B; 20 min, 85% B. For Ultimate 3000 UHPLC, the flow rate is: 0-5.5 min, 0.15 ml/min; 6.9-10.5 min, 0.17 ml/min; 10.6-17.9 min, 0.3 ml/min; 18-20 min, 0.15 ml/min.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters Xbridge Amide (100 × 2.1 mm, 3.5 um)
Column Temperature:25 °C
Flow Gradient:Linear gradient is: 0 min, 85% B; 1.5 min, 85% B; 5.5 min, 35% B; 6.9 min, 35% B; 10.5 min, 35% B; 10.6 min, 10% B; 12.5 min, 10% B; 13.5 min, 85% B; 17.9 min, 85% B; 18 min, 85% B; 20 min, 85% B.
Flow Rate:0.15 ml/min
Solvent A:100% water; 5 mM ammonium acetate
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS004773
Analysis ID:AN005034
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:When Q Exactive Plus mass spectrometer was used, the relevant parameters are as listed: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.6 kV for positive mode and 2.5 kV for negative mode; capillary temperature, 320°C; S-lens, 55. The resolution was set at 70,000 (at m/z 200). Maximum injection time (max IT) was set at 200 ms and automatic gain control (AGC) was set at 3 × 106. FA species were detected using amide column and the identification was based on MS1 and the retention time determined by standard compounds when available. Integrated peak area of analytes was used to calculate the fold change of analytes in different samples. Metabolite and free fatty acid analysis was performed using Sieve (Thermo Fisher Scientific) based on theoretical m/z and retention time determined based on standard compounds.
Ion Mode:UNSPECIFIED
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