title: UVA irradiation of riboflavin generates oxygen-dependent hydroxyl radicals.
abstract: OBJECTIVE
  The aim of this study was to verify the formation of hydroxyl radicals (·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 ·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 (·DMPO-OH). The formed radical adduct (·DMPO-OH) accumulated in the RF solution. Argon equilibration of the RF solution completely blocked the formation of the ·DMPO-OH adduct whereas subsequent aeration restored radical adduct generation. The presence of catalase inhibited ·DMPO-OH generation whereas BSA had no influence on ·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 ·DMPO-OH adduct. When adding DMPO to an already irradiated RF solution significantly less ·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.
  
  
  CONCLUSIONS
  The formation of ·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.
authors.forename: Saadettin
authors.initials: S
authors.lastname: Sel
authors.forename: Norbert
authors.initials: N
authors.lastname: Nass
authors.forename: Sandy
authors.initials: S
authors.lastname: Pötzsch
authors.forename: Stefanie
authors.initials: S
authors.lastname: Trau
authors.forename: Andreas
authors.initials: A
authors.lastname: Simm
authors.forename: Thomas
authors.initials: T
authors.lastname: Kalinski
authors.forename: Gernot Iw
authors.initials: GI
authors.lastname: Duncker
authors.forename: Friedrich E
authors.initials: FE
authors.lastname: Kruse
authors.forename: Gerd U
authors.initials: GU
authors.lastname: Auffarth
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Brömme
mesh_terms: D002851:Chromatography, High Pressure Liquid; D017665:Hydroxyl Radical / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D012256:Riboflavin / Q000737:chemistry* / Q000528:radiation effects*; D014466:Ultraviolet Rays
chemical_list: D017665:Hydroxyl Radical; D010100:Oxygen; D012256:Riboflavin
keywords: 5; 5-Dimethyl-1-pyrroline N-oxide; Catalase; Electron spin resonance; High-performance liquid chromatography; Hydrogen peroxide; Hydroxyl radical; Riboflavin; Spin trapping; UV-VIS spectroscopy; UVA