Blood was drawn into EDTA (Ethylenediaminetetraacetic acid)-coated vacutainers, mixed with a relevant preservative and centrifuged. The plasma was collected and stored at −80 °C until being assayed. Plasma samples were ethanol-extracted [21 (link)] before being analyzed for GLP-1, CCK-8 and PYY3-36 using established RIA (radioimmunoassay) procedures [22 (link),23 (link)]. Ghrelin was also measured via radioimmunoassay but used unextracted blood samples. For all analyses, samples were run in duplicate and all samples from a given participant were analyzed within the same batch. All 125I-Tracers used were purchased from PerkinElmer (PerkinElmer, Waltham, MA, USA). Antibodies for Ghrelin, PYY3-36 and GLP-1 were purchased from Bachem (T-4747, T-4090 and T-4056, respectively), while CCK-8 antibody C2581 was purchased from Sigma Aldrich (St. Louis, MO, USA). Detection limits and coefficient of variations (CV) for each of the RIA measured hormones are as follows: Ghrelin: 50–3200 pg/mL, inter-assay CV 13%, intra-assay CV 10%; GLP-1: 7.5–1000 pg/mL, inter-assay CV 12%, intra-assay CV 9%; PYY3-36: 3.7–250 pg/mL, inter-assay CV 14%, intra-assay CV 8%; CCK-8: 0.62–80 pg/mL, inter-assay CV 13%, intra-assay CV 8%.
Breath samples were analyzed using the Quintron GaSampler System (Qunitron Instruments) for hydrogen content.
Breath samples were analyzed using the Quintron GaSampler System (Qunitron Instruments) for hydrogen content.