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X. Fan, F. J. Giblin, C. Strauch, I. Nemet, J. Zhang, X. Liu, V. M. Monnier; Metal Catalyzed Oxidation (MCO) in Rabbit Lens Crystallins after Hyperbaric Oxygen Treatment. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2539.
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Oxidative mechanisms during nuclear sclerosis are poorly understood. We previously reported that MCO products allysine (ALL) and 2-aminoadipic acid (2-AAA) increased with age, and that ALL but not 2-AAA was increased in diabetes. To clarify the mechanism, rabbit lenses were treated with hyp-O2, hypothesizing that glycation by methylglyoxal (MG) would increase if the Suyama(Eur J Biochem 269:2002) rather than the presumably MG-independent Stadtman pathway (FreeRadicBiolMed 9:1990) was involved.
Lenses (n=6 per group) were treated with 50 atm O2 in HEPES TC-199 buffer for 48 hours or normal atm. Nucleus (N) and cortex (C) were homogenized. Water soluble (WS) and insoluble (WI) proteins were delipidated and digested for LC-MS analysis. ALL, 2-AAA and the glycation products methylgloxal-(MG-H1) and glyoxal-(G-H1) hydroimidazolones, carboxyethyl-lysine(CEL), carboxymethyllysine (CML), glucosepane and fructoselysine(FL) were measured.
Lenses were swollen and nuclei were yellow after hyp-O2 which induced a 8- and 4-fold increase in ALL (p<0.0001) in the WS N and C fractions, respectively, vs. controls. A two-fold increase occurred in WI fractions (p<0.01). A dramatic increase in MG-H1 resulted from hyp-O2, with a six-fold elevation in C (p<0.05) and a 50% increase in N (p=NS). CEL, another MG-derived product, also increased in N in both WS (20%, p<0.05) and WI (100%, p<0.05) fractions. Surprisingly, no increase of 2-AAA occurred suggesting drastic conditions are needed to oxidize ALL into 2-AAA. Similarly, neither CML and G-H1 nor markers of lenticular glycemia (FL,glucospane) were elevated, excluding significant lipid peroxidation and glucose involvement.
The findings strongly implicate MCO, whereby hyp-O2 most likely first oxidizes ascorbic acid (ASA) to DHA and MG which binds to lysyl residues to make a Cu2+ complex leading to oxidative deamination of lysine. Paradoxically, the Suyama and Stadtman pathways may be identical since Stadtman often used ASA for MCO. An important translational conclusion is that chelating agents might help delay nuclear sclerosis.
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