Before taste tests, unstimulated whole saliva was collected using the drooling technique. Each of the children rinsed their mouth with water and was asked to swallow to remove water and saliva from the mouth. Each of the children received instructions to drain all the saliva produced during 4 minutes to a tube maintained on ice. Saliva obtained was frozen at −20°C. After that, samples were thawed on ice and centrifuged at 13,000g for 30 min, at 4°C, to remove mucinous and insoluble material. Supernatant was divided into aliquots and frozen at −20°C until analysis.
For salivary leptin measurement, by enzyme-linked immunosorbent assay (ELISA), samples were thawed at room temperature. A commercial ELISA kit for human leptin (RayBio®, ELH-Leptin-1) was used and the assay was conducted according to the manufacturer's instructions: intra-assay variation was 1.4–8.6%. Each sample from each individual was run in duplicate. Samples were analyzed without dilution.
For salivary leptin measurement, by enzyme-linked immunosorbent assay (ELISA), samples were thawed at room temperature. A commercial ELISA kit for human leptin (RayBio®, ELH-Leptin-1) was used and the assay was conducted according to the manufacturer's instructions: intra-assay variation was 1.4–8.6%. Each sample from each individual was run in duplicate. Samples were analyzed without dilution.