PBS

The preparation of the brain extracts was based on the previous reference [1]. The frozen tissue was weighed and ground using an electric homogenizer with ice-cold methanol (4 mL/g) and distilled water (0.85 mL/g) at 4°C and the mixture was vortexed. Chloroform (2 mL/g) and ... distilled water (2 mL/g) was added and mixed again. After keeping on ice for 15 min, the homogenate was centrifuged at 1,000 g for 15 min at 4°C. The supernatant was extracted and lyophilized for about 24h. The metabolite mixture obtained was then weighed and dissolved in 0.6 mL of 99.5% D2O for NMR spectroscopy. One-dimensional ZGPR pulse sequence was used, and the main parameters were set as follows: data points, 64 K; spectral width, 12,000 Hz; relaxation delay, 10 s. The spectral segments for each NMR spectrum were normalized to the total sum of the spectral intensity to partially compensate for differences in concentration of the many metabolites in the samples [2].
--------------References--------------
[1] Gao H, Xiang Y, Sun N, Zhu H, Wang Y, Liu M, Ma Y, Lei H: Metabolic changes in rat prefrontal cortex and hippocampus induced by chronic morphine treatment studied ex vivo by high resolution 1H NMR spectroscopy. Neurochem Int 2007, 50:386-394. http://doi.org/ds7tm7
[2] Coen M, Lenz EM, Nicholson JK, Wilson ID, Pognan F, Lindon JC: An integrated metabonomic investigation of acetaminophen toxicity in the mouse using NMR spectroscopy. Chem Res Toxicol 2003, 16:295-303. http://doi.org/bhqmgj