Enzyme-linked this website immunosorbent assay (ELISA) was used to determine the IgA salivary levels, modified from the standard protocol used for measurement of IgA blood levels. IgA reacts with a specific antibody (anti-serum anti-IgA, Wiener Lab. 2000, Rosario, Argentina) forming insoluble

complexes. The turbidity formed by these complexes is proportional to the concentration of IgA in the sample and can be read at 340 nm in spectrophotometer. The calibration curve was obtained through calibrator proteins (Wiener Lab. 2000, Rosario, Argentina) diluted in saline solution at 1:10, 1:20, 1:40, 1:80 and 1:160 concentrations. The absorbance was read before (DO1) and after antiserum incubation for 30 min (DO2). The ΔA was 17-AAG concentration determined and IgA concentrations were expressed as μg/mL of saliva. For the analysis of ionized calcium concentrations, 80 μL of each saliva sample was used. To this sample, 16 μL of ionic strength adjuster for calcium (model ISA-932011,

Orion Research Inc., MA, USA) was added and then the calcium concentration ([Ca++]) was determined using a specific calcium electrode (model 9320BN, Orion) and a reference microelectrode (Analyzer) connected to a previously calibrated ion analyser (Orion 720A+). The analyses were expressed in mV and carried out in duplicates. The calibration curve was made with five different concentrations of calcium (10, 20, 40, 80 and 160 Ca++ μg/mL) obtained from the Thymidylate synthase standard solution of Ca++ (model 922006A, Orion Research

Inc.). Calcium ion concentration in the saliva of rats was calculated as Ca++ μg/mL of saliva. Calcium concentration was expressed by SFR as Ca++ μg/min/100 g. Salivary fluoride concentrations ([F−]) were determined by an ion-specific electrode (model 9409BN, Orion) and a reference microelectrode (Analyzer) connected to an ion analyser (Orion 720A+). The set was calibrated with standard fluoride concentrations at 0.15, 0.3, 0.6, 1.2 and 2.4 F− μg/mL, obtained by serial dilution, with pH adjustment solution (TISAB II, Orion). The readings were taken in mV and in duplicates. Fluoride ion concentration in the saliva of rats was calculated as F− μg/mL of saliva, and it was expressed by SFR as F− μg/min/100 g. The data were expressed as means ± standard error of the mean (SEM) and analysed by two-way ANOVA and Tukey post test. Some results were analysed by Student’s t test. Significance level within groups (normotensive or SHR) or across all groups was set at p < 0.05. Physiological parameters were compared between different ages (4 and 12 weeks old) into the same group and between Wistar and SHR groups in same age. At 12 weeks, SHR presented higher SBP mean values (161 ± 4 mmHg, n = 10) than Wistar rats (110 ± 4 mmHg, n = 10).