Calibrator f a s solution

Saliva samples were stored at -30 °C after collection for later analyses. Before cortisol and sAA measurement, two freeze-thaw cycles were performed. Immediately before measurement, samples were centrifuged at 2,000 × g and 20 °C for 10 min. sAA was measured with an in-house enzyme kinetic assay using reagents using reagents from DiaSys Diagnostic Systems GmbH (Holzheim, Germany), as previously described (Bosch et al., Citation2003; Rohleder & Nater, Citation2009) . In brief, saliva was diluted at 1:625 with ultrapure water, and diluted saliva was incubated with substrate reagent (a-amylase CC FS; DiaSys Diagnostic Systems) at 37 °C for three minutes before a first absorbance reading was taken at 405 nm with a Tecan Infinite 200 PRO reader (Tecan, Crailsheim, Germany). A second reading was taken after 5 min incubation at 37 °C and increase in absorbance was transformed to sAA concentration (U/mL), using a standard curve prepared using "Calibrator f.a.s." solution (Roche Diagnostics, Mannheim, Germany). Salivary cortisol concentrations were determined in duplicate using chemiluminescence immunoassay (CLIA, IBL, Hamburg, Germany). Intra-and inter-assay coefficients of variation were below 10% for both sAA and cortisol.