Upon arrival at the imaging center, an MRI safe catheter was placed in participant’s left arm and samples were collected through antecubital venipuncture. Blood plasma samples were collected at baseline (before dinner) and at four time points following the dinner. Blood serum samples for ECS analysis were only collected at 7:30 pm while subjects were inside the MRI scanner, 90 min after the initiation of dinner (in between the 2nd and 3rd fMRI run). Prior to drawing each sample, 1.5–2 mL of blood were drawn off to remove potentially diluted blood from the dead space of the catheter. Samples were put on ice, centrifuged, aspirated, divided into aliquots, and stored at −80°C until assay.
Serum levels of 2-arachidonoylglycerol (2-AG) and 2-oleoylglycerol (2-OG) were extracted using the Bond Elut C18 solid-phase extraction columns (1 ml; Varian Inc, Lake Forest, CA). Serum samples were processed and the two compounds were quantified using chemical ionization liquid chromatography/mass spectrometry (LC-ESI-MS; Agilent LC-MSD 1100 series, Ramsey, MN), as previously described (Patel et al., 2005 (link)).
We used enzyme-linked immunosorbent assay (ELISA) kits for total plasma ghrelin (human ghrelin, Millipore), plasma leptin (human leptin, Millipore), and cobas e411 analyzer (Roche) for plasma insulin, and serum cortisol.
Serum levels of 2-arachidonoylglycerol (2-AG) and 2-oleoylglycerol (2-OG) were extracted using the Bond Elut C18 solid-phase extraction columns (1 ml; Varian Inc, Lake Forest, CA). Serum samples were processed and the two compounds were quantified using chemical ionization liquid chromatography/mass spectrometry (LC-ESI-MS; Agilent LC-MSD 1100 series, Ramsey, MN), as previously described (Patel et al., 2005 (link)).
We used enzyme-linked immunosorbent assay (ELISA) kits for total plasma ghrelin (human ghrelin, Millipore), plasma leptin (human leptin, Millipore), and cobas e411 analyzer (Roche) for plasma insulin, and serum cortisol.