Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with a-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.

Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long-term preservation of lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (Q O 2 ) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors onQ O 2 and partial pressure of oxygen (P O 2 ). The distribution of mitochondria was mapped in living lenses by 2-photon microscopy. We found that a steep gradient of P O 2 was maintained within the tissue, leading to P O 2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens; however, P O 2 gradients extended beyond the boundaries of the mitochondria-containing cell layer, indicating the presence of non-mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion-consumption model. Typical values were 3 × 10 −5 cm 2 s −1 for the effective diffusion coefficient and a 5 min time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibres (DF) that contained mitochondria and mature fibres (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease inQ O 2 and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen-rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of postsurgical nuclear cataract.

The degradation of L-ascorbate (AsA) and its primary oxidation products, L-dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis. The molar yield of ERU from 2,3-DKG at pH 7.0 37 degrees C and limiting O(2)97%. This novel ketose product of AsA degradation, was additionally qualitatively identified by gas-liquid chromatography, and by thin layer chromatography. ERU is an extremely reactive ketose, which rapidly glycates and crosslinks proteins, and therefore may mediate the AsA-dependent modification of protein (ascorbylation) seen in vitro, and also proposed to occur in vivo in human lens during diabetic and age-onset cataract formation.

Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5-to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products.advanced glycation end products ͉ ascorbic acid ͉ cataract ͉ diabetes ͉ oxidation

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.