title: Transition metal-catalyzed oxidation of ascorbate in human cataract extracts: possible role of advanced glycation end products.
abstract: OBJECTIVE
  With age, human lens crystallins become more pigmented, oxidized, modified by ascorbate oxidation and advanced glycation end products (AGEs), and bind copper. The hypothesis was tested that the major AGE and ascorbylation product in the human lens, N(epsilon)-carboxymethyl-L-lysine (CML), has an EDTA-like structure, which may predispose it to bind redox active copper.
  
  
  METHODS
  Young, old, and cataractous human lens protein fractions were glycated with ascorbic acid and tested for their ability to bind Cu(II) by atomic absorption spectroscopy and oxidize (14C1)-ascorbate by radiometric thin-layer chromatography method. AGEs were assayed by high-performance liquid chromatography (HPLC). CML-rich proteins were immunoprecipitated from young, old, and cataractous crystallins using affinity-purified CML antibody and tested for their ability to oxidize ascorbate and generate hydroxyl radicals in the presence of H2O2 using 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) spin-trap and EPR spectroscopy.
  
  
  RESULTS
  Ascorbate oxidizing activity at 24 hours of native crystallins was significantly increased in both the water soluble (WS; P < 0.001) and insoluble (WIS; P < 0.05) fractions from cataractous and normal lenses. The chelator DTPA completely prevented oxidation up to 24 hours of incubation but less effectively thereafter. Mean endogenous Cu content in pooled young, old, and cataract fractions increased from 0.016 to 0.026 nmol/mg protein, respectively, in WS (P < 0.05) and WIS (P < 0.001) fractions, and Cu(II) binding was 20% to 30% increased in cataractous versus old and young lenses in WS (P < 0.01) and WIS (P < 0.001) fractions. Mean levels of the AGEs, CML, and pentosidine were markedly elevated in WS and WIS fractions from cataractous versus old or young crystallins (20% to severalfold, P < 0.05 to P < 0.001). In a separate experiment, protein-bound Fe was not elevated. Crystallins ascorbylated in vitro showed an increase in CML as well as Cu(II) binding. CML-rich proteins (immunoprecipitated from cataractous lenses) oxidized ascorbate approximately 4 times faster than similar proteins from young and old normal lenses (P < 0.01) and generated hydroxyl radicals in the presence of H2O2 and DMPO.
  
  
  CONCLUSIONS
  The association between CML formation, copper binding, and generation of free radicals by cataractous lens crystallins can be duplicated by ascorbylation in vitro. These effects are only in part attributable to CML itself, and other modifications (AGEs, conformational changes) may participate in the process. A vicious cycle between AGE formation, lipoxidation, and metal binding may exist in the aging lens, suggesting that chelation therapy could be beneficial in delaying cataractogenesis.
authors.affiliation: Institute of Pathology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: P
authors.initials: P
authors.lastname: Saxena
authors.forename: A K
authors.initials: AK
authors.lastname: Saxena
authors.forename: X L
authors.initials: XL
authors.lastname: Cui
authors.forename: M
authors.initials: M
authors.lastname: Obrenovich
authors.forename: K
authors.initials: K
authors.lastname: Gudipaty
authors.forename: V M
authors.initials: VM
authors.lastname: Monnier
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D002386:Cataract / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003300:Copper / Q000378:metabolism*; D003459:Crystallins / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D017127:Glycation End Products, Advanced / Q000502:physiology*; D006031:Glycosylation; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D010084:Oxidation-Reduction; D013054:Spectrophotometry, Atomic
chemical_list: D003459:Crystallins; D017127:Glycation End Products, Advanced; C067242:N(epsilon)-(carboxymethyl)hydroxylysine; D003300:Copper; D008239:Lysine; D001205:Ascorbic Acid