Ethylene diamine tetraacetic acid (edta)

The concentrations of SZV 1287 and its four main metabolites (oxaprozin, L 2799, L 2805, and L 2811) were measured by both high-performance liquid chromatography-ultraviolet (HPLC-UV) and HPLC-fluorescence detector (FLD) analyses in the plasma and by HPLC-UV in the brain. The most likely route of formation of SZV 1287 metabolites is demonstrated in Figure S3. In principle, it is also possible that SZV 1287 is hydroxylated first and then the resulting hydroxy derivatives are oxidized to hydroxyoxaprozins (L 2799, L 2805, and L 2811). Blood samples were collected by cardiac puncture into ice-cold tubes containing 8 μL of 50 mg/mL EDTA at a pH of 7.5 (Reanal, Budapest, Hungary) immediately after the post-treatment nociceptive measurement. Then samples were centrifuged for 5 min at 1000 rpm and for 10 min at 10,000 rpm and stored at −80 °C until HPLC analyses. After collecting blood samples, brains were also removed and snap-frozen in liquid nitrogen. The samples were stored at −80 °C until HPLC analyses.

The L-phenylalanine ammonia-lyase (PAL) activity was determined by homogenization of the frozen material (200 mg FW each) in a 0.1 M Na-borate buffer (pH 8.8) containing 0.5 mM EDTA (Reanal, Budapest, Hungary) and 3 mM dithiothreitol (Reanal, Hungary) with the addition of insoluble polyvinylpyrrolidone (50% by weight of fresh tissue; Serva, Germany) [63 (link)]. The homogenate was filtered and centrifuged at 13,000× g for 40 min (centrifuge Minispin, Göttingen, Germany) and then the supernatant was used to determine the enzyme activity. These manipulations were carried out at a temperature of +4 °C. The PAL activity was determined by the change in optical density (wavelength 290 nm) in a reaction mixture of the following composition: supernatant, 0.1 M Na-borate buffer, 0.02 M L-phenylalanine in ratio 1:1:1 (Serva, Germany). The PAL activity was evaluated using the formation of trans-cinnamic acid from L-phenylalanine for 1 h. The PAL activity was expressed in U/mg protein [63 (link)]. The protein was analyzed according to Bradford [60 (link)].