title: Vitamin C metabolomic mapping in the lens with 6-deoxy-6-fluoro-ascorbic acid and high-resolution 19F-NMR spectroscopy.
abstract: OBJECTIVE
  Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C.
  
  
  METHODS
  Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy.
  
  
  RESULTS
  F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA.
  
  
  CONCLUSIONS
  These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.
authors.affiliation: Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Satake
authors.forename: Barbara
authors.initials: B
authors.lastname: Dmochowska
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nishikawa
authors.forename: Janusz
authors.initials: J
authors.lastname: Madaj
authors.forename: Jie
authors.initials: J
authors.lastname: Xue
authors.forename: Zhongwu
authors.initials: Z
authors.lastname: Guo
authors.forename: D Venkat
authors.initials: DV
authors.lastname: Reddy
authors.forename: Peter L
authors.initials: PL
authors.lastname: Rinaldi
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Monnier
mesh_terms: D015083:2,3-Diketogulonic Acid / Q000378:metabolism; D000328:Adult; D000368:Aged; D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D001692:Biological Transport; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism; D018929:Cell Culture Techniques; D003571:Cytochalasin B / Q000494:pharmacology; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D005462:Fluorine Radioisotopes; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D018384:Oxidative Stress; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004791:Enzyme Inhibitors; D005462:Fluorine Radioisotopes; C076401:6-deoxy-6-fluoroascorbic acid; D015083:2,3-Diketogulonic Acid; D003571:Cytochalasin B; D019328:Buthionine Sulfoximine; D005978:Glutathione; D005947:Glucose; D001205:Ascorbic Acid; D005690:Galactose; D003683:Dehydroascorbic Acid