Quantification of another interesting target; retinol-binding protein (RBP) was performed using ELISA. Concentrations of RBP4 in serum samples of HC (n = 20), FC (n = 6), FM (n = 12) and VM (n = 12) patients were measured using AssayMax Human Retinol-Binding Protein-4 (RBP4) ELISA kit (Cat# ER3005-1) from AssayPro (USA) following the manufacturer's instructions. Briefly, quantitative sandwich enzyme assay was employed where RBP4 standard and serum samples (HC, FC, FM and VM) at a dilution of 1:100 were subjected to a microplate pre-coated with a polyclonal antibody specific for RBP4. Samples were sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for RBP4, which was recognized by a streptavidinperoxidase complex. Color development was performed through the addition of a peroxidase enzyme substrate and optical densities were measured at 450 nm and 570 nm using a SpectraMax M2 e (Molecular Devices, USA).