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Xingjun Fan, Benlian Wang, David Sell, Daniel Wesson, Vincent M Monnier; Ascorbic acid oxidation contributes to methylglyoxal-derived hydroimidazolone formation in aged lens. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
Human lens crystallins become progressively pigmented and crosslinked with age, in part due to reactive carbonyl compounds producing advanced glycation end products (AGEs). The most prevalent AGE found in aged human lens crystallins, i.e. methylglyoxal (MG)-derived hydroimidazolones (MG-H1) is usually attributed to glucose-derived MG formation during glycolysis. Below, we hypothesized that ascorbic acid oxidation could be an important source for MG-H1 formation in the lens by in vitro and in vivo study.
In vitro: lens protein extract was incubated with glucose, ASA and ASA catabolic compounds at various concentration at 37oC for 7days. The MG-H1 formation was determined by both LC/MS and western-blot. The lens extract was also incubated with C113C213-ASA, C313-ASA and C513-ASA to determine the mechanisms of MG-H1 formation by ASA oxidation. In vivo: MG-H1 formation was determined in somatic and lens specific Vitamin C transporter 2(SVCT2) transgenic mouse tissues compared to WT. Universal C13 labeled ASA (U6-C13-ASA) was also delivered into glutathione biosynthesis KO and WT mouse brain via intra-ventricular injection, and the brain C13-MG-H1 was analyzed two weeks after injection.
In both anaerobic and aerobic conditions, ASA and most of its catabolic compounds can form MG-H1 after incubation with lens protein extract. The isotopic labeling experiments indicated that ASA was undergoing C3-C4 break during oxidative catabolism, and subsequent C4-6 backbone was involved in MG-H1 formation. There was five-fold elevation of lens protein bound MG-H1 (p<0.0001) and positive association (r2=0.732) with aging in the lens specific SVCT2 transgenic mouse compared to WT. Similar results were found in the brain cerebral cortex of somatic SVCT2 transgenic mouse at 12mos of age compared to age-matched WT. Direct evidence of MG-H1 formation from ASA oxidation was achieved by intra-ventricular injection of 10mM U6-C13-ASA in both WT and Gclm KO mouse. From this experiment, we were able to detect C13-labeled MG-H1 two weeks after injection with a three fold increase the C13-MG-H1 in KO brain compared to WT.
While much of MG-H1 formation is attributed to glucose degradation, the finding of elevated MG-H1 levels in tissues rich in ASA, as in the lens and brain, suggests that MG-H1 accumulation in these tissues reflects oxidative stress likely linked with impaired GSH homeostasis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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