title: The effect of UVA light on the anaerobic oxidation of ascorbic acid and the glycation of lens proteins.
abstract: OBJECTIVE
  To determine whether UVA-excited human lens chromophores can cause the oxidation of ascorbic acid in the absence of oxygen, and whether these oxidation products are capable of glycating lens proteins.
  
  
  METHODS
  The oxidation of ascorbic acid, mediated by UVA irradiation in the presence of aged human lens proteins, was measured in the absence of oxygen by the decrease in absorbance at 265 nm in vitro. An action spectrum from 320 to 400 nm was determined for both ascorbate oxidation and the photobleaching of the lens yellow pigments at lambda = 350 nm. The UVA-mediated oxidation products of [U-(14)C]ascorbate were quantified by HPLC. Glycation was assayed by the UVA-dependent incorporation of [U-(14)C]ascorbate into lens proteins with a water-insoluble (WI) fraction in vitro, with incubated whole human lenses, and with a WI fraction after a 5- to 7-day exposure to ambient sunlight. An enzymatic digest of [U-(14)C]ascorbate-labeled proteins was fractionated over HPLC columns and compared with the 330-nm absorbance profile of a proteolytic digest of aged human lens proteins.
  
  
  RESULTS
  Aged human lens WI proteins absorbed UVA light (86 J/h per square centimeter) and oxidized 33 to 45 nanomoles of ascorbate over 1 hour in the absence of oxygen. No ascorbate oxidation was detected, however, in the dark control. An action spectrum showed that ascorbate oxidation occurred throughout the UVA region, with lambda(max) at 350 nm, which was similar to the action spectrum obtained for the photobleaching of the lens chromophores. Anaerobic UVA irradiation of aged human lens proteins for 2 hours with [U-(14)C]ascorbate resulted in a 40% loss of ascorbate with the accumulation of dehydroascorbic acid, diketogulonic acid, and oxalate. After subsequent incubation for 24 hours, the ascorbate oxidation products disappeared, with a corresponding incorporation of radioactivity into lens proteins. Chromatography of enzymatic digests of the labeled proteins produced peaks that coeluted with several of the 330-nm absorbing peaks in an aged human lens protein digest. Irradiation of whole human lenses for 2 hours caused a 33% loss of total lens ascorbate. UVA irradiation of aged human lenses for 2 hours resulted in the incorporation of ascorbate into lens proteins during the ensuing 24 hours in the dark. Exposure of aged human lens WI proteins to reflected ambient sunlight (1.1 J/h per square centimeter) for 5 to 7 days in the absence of oxygen also produced an increased incorporation of [(14)C]ascorbate into protein when compared with dark control samples.
  
  
  CONCLUSIONS
  These data argue that UVA light can cause an oxidation of ascorbic acid in the absence of oxygen, due to the activation of the sensitizers present in aged human lens WI proteins. The oxidation products formed were the same as those seen in the presence of oxygen, and were rapidly incorporated into protein, apparently by Maillard-type chemistry. These data argue that ascorbate glycation can occur under the low oxygen levels thought to exist in the human lens nucleus in vivo.
authors.affiliation: Mason Eye Institute and the. Department of Biochemistry, University of Missouri, Columbia, Missouri 65201, USA. ortwerthb@health.missouri.edu
authors.forename: Beryl J
authors.initials: BJ
authors.lastname: Ortwerth
authors.forename: Vitaliy
authors.initials: V
authors.lastname: Chemoganskiy
authors.forename: Valeri V
authors.initials: VV
authors.lastname: Mossine
authors.forename: Paul R
authors.initials: PR
authors.lastname: Olesen
mesh_terms: D000368:Aged; D000693:Anaerobiosis; D001205:Ascorbic Acid / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000378:metabolism / Q000528:radiation effects*; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism / Q000528:radiation effects*; D010084:Oxidation-Reduction; D014466:Ultraviolet Rays
chemical_list: D003459:Crystallins; D005947:Glucose; D001205:Ascorbic Acid