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.