Tissue preparation and extraction

1. Frozen tissue was lyophilized for three days and homogenized.

2. 6.00 ± 6.05 mg of tissue was weighed into 1-dram glass vials for each sample.

3. We employed a biphasic extraction protocol, with non-polar metabolites partitioning into chloroform and polar metabolites partitioning into water.  

4. 1.5 mL chloroform with 10.0 µg/mL docosanol (non-polar internal standard) was added, and the vial vortexed and incubated for 1 hr at 50°C.

5. 1.5 mL of water containing 25 µg/mL ribitol (polar internal standard) was added and the biphasic system incubated for an additional hour at 50°C.

6. After cooling to room temperature, the vials were spun at 2900xg for 30 minutes to separate the two solvent layers.

7. 1.0 mL of each layer was collected and transferred to separate autosampler vials.

8. The polar layer was dried in a rotary evaporator and the non-polar layer dried under nitrogen. Samples were held at -80°C until processing.