Human leptin

Leptin and IL-6 levels were determined using an ELISA with commercial kits (Human Leptin and IL-6 Quantikine ELISA, respectively, R&D Systems, Minneapolis, MD, USA). The assay diluent was added to all wells of the 96-well microplate provided by kit, and then 100 μL of samples were added per well. Also, a standard curve of seven-points diluted in calibration diluent, and a blank was performed in duplicate. The Human Leptin or IL-6 conjugate was added to each well and incubated for 1 h. The colorimetric reaction occurred after the substrate solution (3,3′,5,5′- tetramethylbenzidine) was added to the microplate. The optical density was determined at a wavelength of 450 nm by spectrophotometry, and the results were expressed as picograms per milliliter (pg/mL).

Blood was collected into tubes without an anticoagulant and with ethylenediaminetetraacetic acid immediately before a midweek dialysis session to obtain serum, whole blood and plasma. The samples were processed within 2 h of collection; aliquots of plasma and serum were prepared and stored at −80 °C until assayed.
The hemoglobin was evaluated by an automatic blood cell counter (Sysmex K1000; Sysmex, Hamburg, Germany). The serum levels of albumin, calcium and phosphorus were measured by routine procedures, using an auto-analyser (Cobas Integra 400 Plus, Roche Diagnostics, Basel, Switzerland). The serum concentrations of parathyroid hormone (PTH) were determined by chemiluminescence immunoassay (Advia Centaur XP, Siemens, Erlangen, Germany).
The circulating levels of adiponectin, leptin, NT-proBNP and GDF-15 were analyzed through commercial enzyme-linked immunosorbent assay (ELISA) kits (human total adiponectin and human leptin, R&D Systems Inc., Minneapolis, MN, USA; proBNP human ELISA kit and human GDF-15 ELISA Kit, Abcam, Cambridge, UK, respectively).