O tyr

Standards of o-Tyr, p-Tyr, Phe, 3NO₂-Tyr, 3Cl-Tyr, 8OHdG and 2dG (>96% w/w purity) were from Sigma-Aldrich (St. Louis, MO, USA). Internal standards (ISs) p-Tyr-D₂, 2dG-¹³C¹⁵N₂, and 8OHdG-¹³C¹⁵N₂ were acquired from Cambridge Isotope Laboratories and Phe-D₅ from CDN Isotopes (Pointe-Claire, Canada).
Individual stock solutions of o-Tyr (2 mM), 3NO₂-Tyr (2 mM), 3Cl-Tyr (2 mM), 8OHdG (2 mM), 2dG (2 mM), 2dG-¹³C¹⁵N₂ (5 mM), 8OHdG-¹³C¹⁵N₂ (5 mM), Phe-D₅ (10 mM) and Phe (75 mM) were prepared and dissolved as previously described [21 (link)]. Once prepared, solutions were stored at −20 °C. Multi-component working solutions were prepared and kept at −20 °C. Standard solutions were prepared by serial dilution of the working solutions. The preparation of the standards and reagents that we used were described in detail by Cascant-Vilaplana and colleagues [21 (link)].

Serum was prepared as described in refs. ^{6 (link),35 (link)}. Serum was then subjected to dialysis using a dialysis membrane with a 1 KD cutoff (width 2.5 cm) for 48 h. The dialyzable fractions were lyophilized and resuspended in a mixture of water:methanol:trifluoroacetic (87:12:1) for tyrosine determination. p-tyrosine (p-tyr), m-Tyrosine (m-tyr), and o-tyrosine (o-tyr) determinations in the dialyzable fractions from normal serum as well as serum from mice bearing PC3 or Calu6 tumors were carried out using an HPLC. Methodology is previously described in ref. ^{10 (link)}. Briefly p-Tyr, m-Tyr, and o-Tyr separation was performed in a C-18 Zorbax column (Agilent Santa Clara, CA, 7.5 mm × 4.6 mm i.d., 5 μm particle size). The mobile phase consisted in a mixture of water:methanol:trifluoroacetic (87:12:1), the flow rate was set at 0.5 ml/min, and the UV detection was performed at 280 nm. The retention times for p-Tyr, m-Tyr, and o-Tyr were: 2.12 min; 2.55 min; 3.34 min, respectively. Standards for p-Tyr, m-Tyr, and o-Tyr purchased from Sigma, Argentina, were used.