Tyrosine tyr

500 uL of plasma from each animal was precipitated with ammonium sulfate up to 60%, to remove the globulin, remaining over-night in the refrigerator. After centrifugation (3400 rpm, 5 min, eppendorf), precipitates were resuspended in phosphate buffer pH = 7 and dialyzed (Tubbing MW 10,000 daltons) for the removal of the salt used. Samples were filtered in millipore 0.25 um, and 20 uL was injected into HPLC. YL-9300 UV-coupled HPLC instrument 254 nm, C18 Luna 25 cm × 4.5 mm reverse phase column, temperature 27–30 °C. Separation was done in the following mobile phase: buffer sodium acetate 10 mM in MilliQ water (A) and acetonitrile (B): 0–1 min (20% B); 1.01–1.5 min (5% B); 1.51–8 min (4% B). The flow rate was kept constant at 1 mL/min and peaks were detected at 254 nm. All chemicals reagents used in the analysis, such as acetonitrile and acetate buffer we purchased from Sigma and Merck, (Rio de Janeiro Brazil), with high purity levels. The standards used were Kynerunin-Kyn (20 uM), Tryptophan-Trp (20 uM), Cysteine-Cys (10 mM), Arginine-Arg (10 mM), Metionine-Met (10 mM), Proline-Pro (10 mM), Valine-Val (10 mM), Alanine-Ala (10 mM), Phenilalanine-Phe (10 mM), Tyrosine-Tyr (10 mM) from Sigma and Merck. All were evaluated and separated by HPLC based on the peak at said retention time (Rt) and height peak in millivolt (mV) or concentration in uM or ug.