Serum leptin

Blood samples were collected in EDTA-containing tubes, centrifuged, placed in
aliquot tubes and stored at −70 to −80° C until analysis. All assays
were run after both study days were complete for each subject. For GLP-1, 30ɥl of
dipeptidyl peptidase IV inhibitor was added to the 4ml EDTA tube prior to collection.
GLP-1 assays were performed with Alpco Diagnostics ELISA (43-GPTHU-E01). Insulin
concentrations were measured using competitive radioimmunoassay (Millipore).
Radioimmunoassay was used to analyze serum leptin (Millipore), serum PYY concentrations
(Millipore Cat. #PYYT-66HK) and total serum ghrelin concentrations (Millipore Cat.
#GHRT-89HK). All radioimmunoassays were performed with a Perkin Elmer Wallac Gamma counter
using Maciel RIA-AID data reduction software. Assays for glucose, TG and FFA were
performed on the Olympus AU400e Chemistry Analyzer (Beckman). Reagents were purchased from
Beckman Coulter for glucose and TG and from WACO for FFA.

Study visits occurred at baseline, 3, 6, 12, and 18 months. Body composition by dual-energy X-ray absorptiometry (DXA), serum estradiol (high-performance liquid chromatography tandem mass spectrometry, Esoterix, Calabasas Hills, CA), and serum cortisol (electrochemiluminescent immunoassay, Roche Cobas, Mannheim, Germany) were obtained at baseline and at every 6 months. Serum leptin (radioimmunoassay, Millipore, St. Charles, MO) was measured at baseline (n = 11) and at 18 months (n = 10). The Beck Depression Inventory-I [7 (link)] and 26-item Eating Attitudes Test [8 (link)] were completed at baseline, 6–, 12–, and 18– month visits.