The Maillard reaction, the non-enzymatic reaction of sugars with proteins is hypothesized to play a significant role in the lens aging and cataractogenesis. In recent years, considerable interest has focused on the so-called advanced glycation, since crosslinks and fluorescent products derived at this stage are considered to accumulate and cause protein damage. In a series of preliminary studies, I was able to demonstrate with my colleagues that the two advanced Maillard products, LM-l and pentosidine are present in the aging human lens and can be synthesized from ascorbate oxidation products. These results raise a number of important questions concerning the' participation of ascorbate in lens senescence and cataractogenesis, which I plan to address in the coming 5 years. In the first specific aim, major crosslinks from ascorbate reacted with synthetic peptides will be isolated. The rationale is to use simple peptides instead of whole proteins to minimize the complexity of the reaction. The peptides (N-terminal protected) to be used will have a single amino group of lysine or guanido group of arginine residue to limit side reactions of ascorbate with other amino groups. Crosslinks (lysine- lysine or lysine-arginine) will be isolated by column chromatography and/or HPLC. The crosslinks will be synthesized with amino acids reacted with ascorbate to maximize the yield. The isolated crosslinks will be characterized using UV, NMR and mass spectroscopy. The second specific aim will focus on the detection and quantification of ascorbate-derived crosslinks in human lens. The major crosslinks will be quantified as a function of age and the degree of cataract (classified by CCRG method based on pigmentation and opacity) to establish their role in these processes. The third specific aim will focus on the ascorbate redox status and its Maillard reaction in experimental cataracts. In the first part, relationships between the crosslinks, redox status of ascorbate and, glutathione will be established in the lenses of rats with diabetes. In the second part, I will use naphthalene cataract in rats and rabbits as a model to study ascorbate degradation and Maillard reaction in vivo, since ascorbate in the eye is shown to undergo accelerated oxidation in this model. In these experiments ascorbate and its oxidation products will be quantitatively estimated by gas chromatography-mass spectroscopy (GC-MS).
| Ni, Jiaqian; Yuan, Xianglin; Gu, Jiayin et al. (2009) Plasma protein pentosidine and carboxymethyllysine, biomarkers for age-related macular degeneration. Mol Cell Proteomics 8:1921-33 |
