Plasma concentrations of leptin were determined by a multi-species leptin RIA kit (XL-85K, Millipore, Molsheim, France). The limit of sensitivity of the assay was 2 ng·mL−1 and the inter- and intra-assay coefficients of variance were <9 and <4%, respectively.
Plasma concentrations of cortisol were determined by a Cortisol Express EIA Kit (AYN830, Cayman Chemical, Ann Arbor, MI, USA). The limit of sensitivity of the assay was 0.1 ng·mL−1, while the coefficients of variance for a dose of 5 ng were equal, respectively, to 11 and 6% for inter- and intra-assays. Plasma levels of insulin were assayed with Ultra Rat Insulin ELISA Kit (# 90060, Crystal Chem, Downers Grove, IL, USA) using hamster insulin standard (# 90330, Crystal Chem) following the instructions of the manufacturer. The limit of sensitivity of the assay was 0.1 ng·mL−1 and the coefficients of variance were <10% for both inter- and intra-assays.
Plasma concentrations of cortisol were determined by a Cortisol Express EIA Kit (AYN830, Cayman Chemical, Ann Arbor, MI, USA). The limit of sensitivity of the assay was 0.1 ng·mL−1, while the coefficients of variance for a dose of 5 ng were equal, respectively, to 11 and 6% for inter- and intra-assays. Plasma levels of insulin were assayed with Ultra Rat Insulin ELISA Kit (# 90060, Crystal Chem, Downers Grove, IL, USA) using hamster insulin standard (# 90330, Crystal Chem) following the instructions of the manufacturer. The limit of sensitivity of the assay was 0.1 ng·mL−1 and the coefficients of variance were <10% for both inter- and intra-assays.