2.5 High performance liquid chromatography analysis (HPLC)
Tissue samples were weighed after extraction and homocysteine as an internal standard was added. After that the samples were homogenized in 10 volumes of 5% trichloroacetic acid (TCA) and the homogenates were centrifuged at 180006g at 4uC for 30 min. To remove TCA, the supernatants were washed three times with water-saturated diethylether. The resultant samples were used for HPLC analysis. Amino acid enantiomers were separated by HPLC using a carbon 18 reverse-phase column (250 mm) (Knauer, Advanced scientific instruments, Germany) with fluorimetric detection after derivatization with N-isobutyryl-L-cysteine and O-phthalaldehyde (Tomassoni-Ardori et al.). The Nisobutyryl-L-cysteine/OPA derivatives were immediately applied to the HPLC system (Knauer, Advanced scientific instruments, Germany). Mobile phase was 8% MeCN in 0.1 M sodium acetate buffer (pH 6.0). Amino acids were separated isocratically for 37 minutes and then the column was eluted with 50% H2O/50% MeCN for 5 minutes. After that, the column was rebalanced for 15 minutes under the initial conditions. The flow-rate was 0.125 ml min-1. Fluorescence detection of each amino acid derivative was carried out at 443nm with excitation at 344nm. The absolute D-serine levels referring to the internal standard were calculated and data were related to the wet weight of the initial tissue samples.