TITLE: In Vivo Metabolomics
AUTHORS: Yoshitaka J Sei, Szu-Chi Liao, Johanna ten Hoeve-Scott, Ken Nakamura

[DESCRIPTION]
This protocol describes steps for targeting metabolites with [U-13C]glucose in mice for metabolomics analysis.

[STEPS]
SECTION: Tail vein infusion and tissue harvest
1. Materials
Surgical tools – scissors, tweezers, forceps, spatula, razor
Heating pad (K&H Thermo-Peep Heated Pad)
Tail illuminator (Braintree Scientific: MTI STD)
Glue (Loctite 1699233)
30g needles (BD: 305106)
PE tubing (BD: PE#10)
Syringe pump (WPI: SP220I)
1mL syringes (BD: 309628)
Vapor splitter (VetEquip: CX-IM 5 place manifold)
Dry ice
Sterile 1X PBS or saline
[U-13C]glucose solutions (Cambridge Isotope Laboratories: CLM-1396-1)
Glucometer (Zoetis: AlphaTrak 2)
Luer adapter

SECTION: Tail vein infusion and tissue harvest
3. Day of Procedure
SECTION: Metabolite Extraction
4. Protocol adapted from UCLA Metabolomics Center.

Materials
Aluminum foil
Metal Pad
Dry ice
Hammer
Microcentrifuge tubes
80% methanol
Vortex mixer
Ice
Centrifuge
Screw-cap glass vials
SECTION: Tail vein infusion and tissue harvest
2. Day Prior to Procedure
SECTION: Tail vein infusion and tissue harvest
2.1. Weigh mice.
SECTION: Tail vein infusion and tissue harvest
2.2. Fast mice overnight (14-16 hours).
SECTION: Tail vein infusion and tissue harvest
3.1. Make [U-13C]glucose solutions with PBS/saline.
Bolus: 200 µL of 0.4 mg/g.
Infusion: 75 µL of 0.72 mg/g.
SECTION: Tail vein infusion and tissue harvest
3.2. Anesthetize mice in an induction chamber with 2% isoflurane with flowrate set to 1.2 L/min.
SECTION: Tail vein infusion and tissue harvest
3.3. Move a mouse to the tail illuminator, cannulate the tail vein and glue needle in place.
SECTION: Tail vein infusion and tissue harvest
3.4. Test that the catheter works by infusing 0.1 mL of saline – the saline should flow up the tail vein if it was cannulated correctly.
SECTION: Tail vein infusion and tissue harvest
3.5. Gently move mouse and catheter to the isoflurane-splitter.
SECTION: Tail vein infusion and tissue harvest
3.6. Repeat steps 3-4 to fill up the remaining spaces on the isoflurane-splitter (up to 5 mice).
SECTION: Tail vein infusion and tissue harvest
3.7. Prick the paw of each mouse with a needle (18G) to measure the pre-infusion blood glucose level.
SECTION: Tail vein infusion and tissue harvest
3.8. Inject the bolus glucose solution into each mouse and transfer the luer adapter to the infusion syringes.
SECTION: Tail vein infusion and tissue harvest
3.9. Start infusion at a flow rate of 150 µL/h for 75 µL (30 min).
SECTION: Tail vein infusion and tissue harvest
3.10. After infusion is done, euthanize mice by cervical dislocation.
SECTION: Tail vein infusion and tissue harvest
3.11. Quickly dissect out the brain and flash freeze in isopentane cooled to -80C or liquid nitrogen.
SECTION: Tail vein infusion and tissue harvest
3.12. Store samples in -80°C until further processing.
SECTION: Metabolite Extraction
5. Wrap frozen brains in pre-cooled aluminum foil and place on a metal pad on ground dry ice.
SECTION: Metabolite Extraction
6. Pulverize tissue with a hammer.
SECTION: Metabolite Extraction
7. Transfer the ground tissue into a pre-cooled microcentrifuge tube on dry ice.
SECTION: Metabolite Extraction
8. Add 1mL 80% MeOH at -80°C and vortex vigorously for 30 s.
SECTION: Metabolite Extraction
9. Incubate samples at -80°C for an hour.
SECTION: Metabolite Extraction
10. Allow samples to warm up a bit on ice.
SECTION: Metabolite Extraction
11. Vortex samples again for 30 s.
SECTION: Metabolite Extraction
12. Centrifuge at top speed (16,000 g) for 15 min at 4°C.
SECTION: Metabolite Extraction
13. Transfer the supernatant to a screw-cap glass vial and store at -80°C until shipping to UCLA Metabolomics Core.
SECTION: Metabolomics Analysis
14. Protocol from UCLA Metabolomics Center.

Materials
Q Exactive mass spectrometer (Thermo Scientific)
Vanquish Flex UHPLC (Thermo Scientific)
Ion Chromatography System ICS-5000+
Luna NH2 3um 100A (150 × 2.0 mm) column (Phenomenex) 
LC-MS grade acetonitrile (ACN)
LC-MS grade water
5 M ammonium acetate
ammonium hydroxide
SECTION: Metabolomics Analysis
15. Dilute the MeOH extract with 50% ACN to 50 mg tissue/ml concentration (1:5-1:10).
SECTION: Metabolomics Analysis
16. Spin 10 min at full speed at 4C to clarify the samples.
SECTION: Metabolomics Analysis
17. Transfer the supernatant to glass vials with glass insert.
SECTION: Metabolomics Analysis
18. Combine 10 ul of each sample to make a pool sample used to condition the column prior loading the individual samples.
SECTION: Metabolomics Analysis
19. Place samples in autosampler 4C.
SECTION: Metabolomics Analysis
20. Load 5 ul onto a Luna NH2 column using a Vanquish Flex.
SECTION: Metabolomics Analysis
21. Separate the metabolites at a flow rate of 200 μl/min with mobile phases A (5 mM NH4AcO pH 9.9) and B (100% ACN) using a linear gradient from 15% A to 95% A over 18 min followed by a 7 min isocratic wash at 95% A.
SECTION: Metabolomics Analysis
22. Re-equilibrate the column to 15% A for 15 min prior to loading the next sample.
SECTION: Metabolomics Analysis
23. Acquire data with a Q Exactive mass spectrometer using polarity switching in full scan mode using a range of 70-975 m/z and 70.000 resolution.
SECTION: Metabolomics Analysis
24. Convert the RAW files to mzXML format using the msConverter (part of open source ProteoWizard).
SECTION: Metabolomics Analysis
25. Extract metabolite intensities with Maven (v 8.1.27.11) using a targeted list of polar metabolites of central carbon metabolism with expected retention time and accurate mass measurements (< 5 ppm) for identification.
SECTION: Metabolomics Analysis
26. Perform data analysis using R scripts functions (or other tools). Correct isotopologue data for the natural abundance of C13 using AccuCor.