Preparation of Advanced Oxidation Protein Products

AOPPs were prepared according to the procedure described previously (Witko‐Sarsat et al., 1996). In brief, rat serum albumin (RSA, Sigma, St. Louis, MO, USA) solution (20 mg/ml) was incubated with 40 mm hypochlorous acid (Fluke, Buchs, Switzerland) in phosphate‐buffered saline (PBS, pH = 7.4) for 30 min at the room temperature. Prepared samples were dialyzed against PBS to remove free hypochlorous acid. To remove contaminated endotoxin, all samples were passed through a Detoxi‐Gel column (Pierce, Rockford, IL). Endotoxin levels in AOPP–RSA and unmodified RSA were then measured using a Limulus Amoebocyte Lysate kit (Sigma, St Louis, MO) and were found to be below 0.05 ng/mg protein. AOPP content in the sample was determined as described previously (Witko‐Sarsat et al., 1998).