May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
Covalent Modifications of Glutathione by Glucose
Author Affiliations & Notes
  • M.D. Linetsky
    Mason Eye Institute, University of Missouri, Columbia, MO
  • E.V. Shipova
    Mason Eye Institute, University of Missouri, Columbia, MO
  • R.D. LeGrand
    Mason Eye Institute, University of Missouri, Columbia, MO
  • O.K. Argirov
    Mason Eye Institute, University of Missouri, Columbia, MO
  • Footnotes
    Commercial Relationships  M.D. Linetsky, None; E.V. Shipova, None; R.D. LeGrand, None; O.K. Argirov, None.
  • Footnotes
    Support  NIH Grant EY13244
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 2889. doi:
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      M.D. Linetsky, E.V. Shipova, R.D. LeGrand, O.K. Argirov; Covalent Modifications of Glutathione by Glucose . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2889.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose:We explored a possibility that glucose, which is present in diabetic lens in mM concentration could react non–enzymatically with reduced and oxidized forms of glutathione, resulting in the formation of the correspondent Amadori compounds. Methods: Sterile solutions of reduced (GSH) and oxidized (GSSG) forms of glutathione were incubated with glucose in rations 1:5 through 1:200 in 50 mM phosphate buffer (pH 7.0), containing 1 mM DTPA under argon for the duration of 48 hours at 37oC in dark. Aliquots of these reaction mixtures were acidified and analyzed by a gradient RP–HPLC equipped with mass spectrometric detector. Normal human aged lenses (55–75 years old, 10 lenses) and cataractous lenses of Indian origin (55–75 years old, 10 lenses) were homogenized in de–gassed and ice–cold 10% TCA and centrifuged. After ether extraction of TCA from the lens supernatants, these low–molecular weight fractions were analyzed by RP–HPLC–MS for the presence of Amadori compounds of glutathione Results: Under the chromatographic conditions used in these studies we observed time– and concentration–dependent formation of N–1–Deoxy–fructosyl GSH (FGH) as the major glycation product of GSH with glucose. FGH had a characteristic positively charged ion with m/z=470 Th in its LC–MS spectra. Mixtures of GSSG and glucose generated two compounds, N–1–Deoxy–fructosyl GSSG (FGG; m/z=775 Th) as major adduct and bis di–N, N’–1–Deoxy–fructosyl GSSG (FGGF; m/z=937 Th) as the minor one. All three compounds showed a resonance signal at 55.2 ppm in the 13C–NMR spectra as C(1) methylene group of deoxyfructosyl, which represents a direct evidence that they are Amadori compound. All three compounds also showed LC–MS/MS fragmentation patterns identical to the synthetically synthesized FGH, FGG and FGGF. FGH was shown to be a poor substrate for the lenticular GPx (6.7% of the enzyme’s original specific activity, SP), G–S–T (25.7% of the original enzyme’s SP) and glyoxylase I ( 32% of the enzyme’s SP). GR failed to recycle the disulfide bond within the structure of di–substituted FGGF, showing only 1% of the original enzyme’s SP, but retained its ability to recycle FGG by 57% of its original SP. In addition, the presence of FGH was found in the low molecular fractions from aged human lenses and in higher amounts in brunescent cataractous lenses, as was demonstrated by LC–MS analysis. Conclusions: The evidence of the presence of FGH in aged human normal and cataractous lenses argues that the reaction between GSH and glucose, which leads to the formation of FGH, can be responsible, at least in part, for the depletion of levels GSH in human lens with aging and in diabetic cataract development

Keywords: cataract • diabetes • protein modifications-post translational 

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