Blood collection
Fasted blood samples were collected using an intravenous (IV) catheter to assess glycerol and free fatty acids. Vials of blood were prepared, labeled, and collected using standard phlebotomy techniques according to the requirements of each assay. A Serum Separator Tube (SST) and a dipotassium ethylenediaminetetraacetic acid tube (KSerum samples collected in SST were sent to Quest Diagnostics (Irving, TX) to be assayed for free fatty acids, and serum samples in the microtubes for glycerol were stored at -80°F until they were sent off for analysis. Duplicate undiluted serum samples were analyzed using a commercially available fluorometric-free glycerol assay kit (Cell Biolabs; San Diego, CA). After following standard assay procedures, standards, controls, and samples were analyzed using a fluorometric plate reader (Biotek Synergy 2; Santa Clara, CA) with an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Unknown sample concentrations were calculated from the standard curve. All samples were analyzed on the same day to minimize variation, and the intraassay coefficient of variability (CV) was tested as <8%.