Oxidative mechanisms during nuclear sclerosis of the lens are poorly understood, in particular metal-catalyzed oxidation. The lysyl oxidation product adipic semialdehyde (allysine, ALL) and its oxidized end-product 2-aminoadipic acid (2-AAA) were determined as a function of age and presence of diabetes. Surprisingly, whereas both ALL and 2-AAA increased with age and strongly correlated with cataract grade and protein absorbance at 350 nm, only ALL formation but not 2-AAA was increased by diabetes. To clarify the mechanism of oxidation, rabbit lenses were treated with hyperbaric oxygen (HBO) for 48 h, and proteins were analyzed by gas and liquid chromatography mass spectrometry for ALL, 2-AAA, and multiple glycation products. Upon exposure to HBO, rabbit lenses were swollen, and nuclei were yellow. Protein-bound ALL increased 8-fold in the nuclear protein fractions versus controls. A dramatic increase in methylglyoxal hydroimidazolone and carboxyethyl-lysine but no increase of 2-AAA occurred, suggesting more drastic conditions are needed to oxidize ALL into 2-AAA. Indeed the latter formed only upon depletion of glutathione and was catalyzed by H 2 O 2 . Neither carboxymethyl-lysine nor glyoxal hydroimidazolone, two markers of glyco-/lipoxidation, nor markers of lenticular glycemia (fructose-lysine, glucospane) were elevated by HBO, excluding significant lipid peroxidation and glucose involvement. The findings strongly implicate dicarbonyl/metal catalyzed oxidation of lysine to allysine, whereby low GSH combined with ascorbate-derived H 2 O 2 likely contributes toward 2-AAA formation, since virtually no 2-AAA formed in the presence of methylglyoxal instead of ascorbate. An important translational conclusion is that chelating agents might help delay nuclear sclerosis.The aging human lens accumulates a number of postsynthetic protein modifications that are thought to predispose lens crystallins toward aggregation and cataractogenesis. These include glycation by sugars, ascorbic acid, and oxoaldehydes, 3-hydroxy-kynurenination, oxidation, and photooxidation, deamidation, deamination, and protein truncation (reviewed in Ref. 1). Among the oxidative mechanisms, those involving formation of protein disulfides and mixed disulfides with glutathione or cysteine are well understood (2). Similarly, the presence of methionine sulfoxide in human lens crystallins has been known for several years (3, 4). In recent years, however, considerable interest has focused on the formation of so-called protein carbonyls as markers of oxidative stress in biology. Stadtman and co-workers (5) demonstrated that proteins incubated in the presence of redox active metals, such as Cu(II) and Fe(III), with hydrogen peroxide or ascorbic acid, underwent metal-catalyzed oxidation (MCO), 3 whereby histidine, proline, lysine, and arginine are preferentially oxidized. Lysine residues are oxidized into allysine (adipic semialdehyde) (Fig. 1), while proline and arginine residues into glutamic semialdehyde (6, 7). These "protein carbonyls" have been immuno...

The effects of anaerobic (lens) vs aerobic (skin) environment on carbonyl and oxidant stress are compared using de novo and existing data on advanced glycation and oxidation products in human crystallins and collagen. Almost all modifications increase with age. Methylglyoxal hydroimidazolones (MG-H1), carboxymethyl-lysine (CML), and carboxyethyl-lysine (CEL) are several folds higher in lens than skin, and markedly increase upon incubation of lens crystallins with 5 mM ascorbic acid. Vice-versa, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones (G-H1), metal catalyzed oxidation (allysine) and H 2 O 2 dependent modifications (2-aminoapidic acid and methionine sulfoxide) are markedly elevated in skin, but relatively suppressed in the aging lens. In both tissues ornithine is the dominant modification, implicating arginine residues as the principal target of the Maillard reaction in vivo. Diabetes (here mostly type 2 studied) increases significantly fructose-lysine and glucosepane in both tissues (P<0.001) but has surprisingly little effect on the absolute level of most other advanced glycation end products (AGEs) . However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide mediated modifications in the lens. Overall, the data suggest oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging non-cataractous lens, while aging skin collagen undergoes combined attack by non-oxidative glucose mediated modifications, as well as those from metal catalyzed oxidation and H 2 O 2 . Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptFree Radic Biol Med. Author manuscript; available in PMC 2011 September 1. Long lived proteins such as lens crystallins and collagen are prone to age-related accumulation of damage by glycation and oxidation which are thought to contribute to loss of protein solubility, impaired function, increased tissue stiffness and sclerosis during aging. The predilection for nucleophilic amino acids, in particular lysine and arginine residues as targets of glycation results in profound molecular changes characterized by increased anionic charge, formation of non-disulfide crossslinks [1,2], and increased binding of redox active catalytic metals [3], all of which impact both on the protein molecule itself and its interaction with ligands or cells [4,5].In the lens, crystallin modification can result in increased or decreased chaperone activity [6,7] and formation of light scattering aggregates that are observed ...