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B. J. Ortwerth, J. Bhattacharyya, E. Shipova; Isolation and Identification of the Low Molecular Weight Sensitizers Responsible for the UVA Light-Mediated Oxidation of Ascorbic Acid. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2269.
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To determine the chemical nature and ascorbic acid oxidizing activity of the low molecular weight (LMW) UVA-absorbing sensitizers present in human lens tissue.
The protein and LMW components were isolated from both the WS and the sonicated WI fractions from human lenses by filtration through a 3 kD filter. Each fraction was assayed for the UVA light-dependent oxidation of ascorbic acid by measuring the loss of absorbance of ascorbate at 265nm over a 30 min irradiation. Both LMW fractions were subjected to RP-HPLC on a Prodigy HPLC column and monitored at 350 nm. The major 350 nm peaks were isolated and identified by their LC/MS/MS and absorption spectra. Each compound was assayed for ascorbic acid-oxidizing activity where a unit of activity was defined as 1.0 nmole of ascorbate oxidized during 10 min of UVA irradiation (100 mJ of absorbed light). Activites are expressed either as units/lens or units/mg BCA protein.
Adult human lenses contain LMW sensitizers in both the WS and WI fraction, which oxidize ascorbic acid in response to UVA irradiation. These decrease with age while the activity in the lens protein fractions increase. This oxidation does not require oxygen, and appears to be due to a direct reaction of ascorbate with the triplet state anion to form ascorbyl free radical. This reaction, therefore, could account for the oxidation of ascorbate in vivo under the anaerobic conditions present in the human lens, producing ascorbate oxidation products, which are very active glycating agents.Two LMW compounds that absorb in the UVA region were present in the WS and WI fraction. Each was isolated by RP-HPLC and subjected to mass spectrometry. The absorption and mass spectra were identical to the published spectra for 3-OH-kynurenine-O-beta-glucoside and alpha-deamino-3-OH-kynurenine-O-beta-glucoside, both of which are sensitizers for UVA-dependent ascorbate oxidation. These sensitizers account for most of the UVA -absorbing LMW compounds in the human lens but their ascorbate-oxidizing activity was equal to only 0.1% of the classic sensitizer, riboflavin.
Human lenses contain two UVA-absorbing LMW sensitizers, which exist both free and bound to the proteins in the WI fraction. These sensitizers, previously considered inactive filter compounds, absorb UVA light and oxidize ascorbate in the absence of oxygen. The oxidized ascorbate can then glycate lens proteins and produce the extensive protein modifications that have been shown by mass spectrometry to be identical to those present in aged human lenses and cataracts.
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