Lipid fatty acid analysis

100 µl of plasma was thawed and decanted into a screw top test tube. 100 μg of free nonadecanoic acid (19:0) was added as an internal fatty acid standard (odd chain fatty acids are found in low abundance in non-rumen animals). Plasma lipids were then converted to fatty acid methyl esters with the addition of sulfuric acid in methanol and heated for 60 min at 100 °C⁶⁷. Fatty acid methyl esters were extracted from sample tubes with the addition of water and distilled petroleum ether (mixed hexanes). The resulting non-polar ether phase was decanted and dried down under a gentle stream of nitrogen at ambient temperature.

Dried samples were immediately reconstituted with 100 µl of petroleum ether and decanted to a sample vial. Fatty acid methyl esters were then analyzed with a Shimadzu gas chromatograph (GC) model 2010. Samples were injected in split mode into a Restek (Bellefonte, PA) FAMEWAX 30 m column. The GC was programmed from 160 to 220 °C and detection conducted with flame ionization detection (FID). White adipose tissues were processed as described above with additional vortexing. For chow analysis, chow was homogenized with a standard coffee grinder into a fine particulate. The fatty acid and fat content of chow were determined as detailed above. Chromatograms and data were reviewed and calculated with Shimadzu Class VP software. Data are expressed as µg fatty acid per ml of plasma or mg of tissue (wet weight).