BDNF analysis

Both plasma and serum BDNF were measured, given that plasma represents the unbound, bioavailable pool and serum represents unbound and platelet-contained BDNF (30). For serum BDNF, blood was drawn in serum separator tubes and left to clot at room temperature for 1 h (31). Serum tubes were then centrifuged at 1500 g at 4°C for 15 min. For plasma BDNF, blood was drawn in EDTA tubes and centrifuged immediately at 1500 g at 4°C for 15 min. The resultant supernatant was aliquoted and centrifuged at 10,000 g at 4°C for 10 min to yield platelet-poor plasma. Both serum and plasma samples were aliquoted and stored at −80°C until analysis. A commercially available enzyme-linked immunoassay (BEK-2211-2P, Biosensis, SA, Australia) was used to measure BDNF. The intra- and inter-assay coefficients of variation are 1.0% and 5.0%, respectively, as reported by an independent third party (32). Serum BDNF was diluted 100-fold in accordance with the manufacturer's recommendation and confirmed by in-house assay optimization. All other steps were followed in accordance with the manufacturer's instructions.