UVA-ketoprofen-induced hemoglobin radicals detected by immuno-spin trapping

Ketoprofen (3-benzoyl-alpha-methylbenzeneacetic acid, KP) is a widely used nonsteroidal anti-inflammatory drug (NSAID) that causes both phototoxicity and photoallergy. Here, we investigated the formation of hemoglobin radicals, in both purified hemoglobin and red blood cells (RBC), induced by ultraviolet A (UVA)-KP by using "immuno-spin trapping," a novel approach that combines the specificity of spin trapping with the sensitivity of antigen-antibody interactions. The methemoglobin (metHb) radicals react covalently with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form nitroxyl radical adducts that are oxidized to the corresponding nitrone adducts, which in turn are specifically recognized by antiserum against DMPO nitrone. We found that the formation of nitrone adducts in metHb depended on the UVA dose, the KP concentration and the presence of DMPO, as determined by enzyme-linked immunosorbent assay and Western blotting. Adduct formation decreased when irradiation was carried out in the presence of catalase or nitrogen, suggesting that H2O2 plays a key role in KP-UVA-induced metHb radical formation. KP in the dark did not generate metHb radical-derived nitrone adducts, whereas UVA alone resulted in the formation of metHb radical-derived nitrone adducts that increased with UVA dose from 4 to 10 J/cm2. However, KP (25 and 200 microM) plus UVA (4 and 10 J/cm2) resulted in a significant increase in the formation of metHb radical-derived nitrone adducts as compared with UVA or KP alone, indicating that KP photosensitized the production of the metHb radicals in the presence of UVA. In contrast, no metHb radical-derived nitrone adduct was detected in the absence of DMPO, even though KP and UVA were present. We also detected the hemoglobin radical formation in RBC as well as in hemolysates. The endogenous antioxidants and exogenous reduced glutathione inhibited the protein radical formation. These studies have shown that the immuno-spin-trapping technique can be used to detect radical damage in proteins as a result of photosensitizing reactions. The successful detection of protein radical formation caused by KP photosensitization could help further understand the photoallergic effect of this NSAID.