UVA irradiation of riboflavin generates oxygen-dependent hydroxyl radicals

Objectives/methods: The aim of this study was to verify the formation of hydroxyl radicals (center dot OH) after ultraviolet A (UVA) irradiation of riboflavin (RF) by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and electron spin resonance spectroscopy. Results: We found that center dot OH were generated via hydrogen peroxide (H2O2) formation during UVA irradiation of RF. The . OH radicals were trapped with DMPO yielding 2-hydroxy-5,5-dimethyl-1-pyrroline-N-oxide (center dot DMPOOH). The formed radical adduct (center dot DMPO-OH) accumulated in the RF solution. Argon equilibration of the RF solution completely blocked the formation of the center dot DMPO-OH adduct whereas subsequent aeration restored radical adduct generation. The presence of catalase inhibited center dot DMPO-OH generation whereas BSA had no influence on center dot DMPO-OH formation. Stopping UVA irradiation led to decay of radical adducts. UVA irradiation of H2O2 in the presence of DMPO but without RF also induced the formation of center dot DMPO-OH adduct. When adding DMPO to an already irradiated RF solution significantly less center dot DMPO-OH was formed during further irradiation. Ultraviolet-visible spectroscopy and high-performance liquid chromatography analysis of RF indicated that RF decayed during UVA irradiation. Discussion: The formation of center dot OH during UVA irradiation of RF may be part of the oxygen-dependent mechanism involved in the cross-linking therapy of collagen in corneal stroma.