Ognyan K. Argirov

Isolation and characterization of a new advanced glycation endproduct of dehydroascorbic acid and lysine

Proteins are subject of posttranslational modification by sugars and their degradation products in vivo. The process is often referred as glycation. L-Dehydroascorbic acid (DHA), an oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. A new product of modification of lysine epsilon -amino group by DHA was discovered as a result of the interaction between Boc-Lys and dehydroascorbic acid. The chromatographic and spectral analyses revealed that the structure of the product was 1-(5-ammonio-5-carboxypentyl)-3-oxido-4-(hydroxymethyl)pyridinium. The same compound was isolated from DHA modified calf lens protein after hydrolysis and chromatographic separation. The study confirmed that L-erythrulose is an important intermediate of modification of proteins by DHA. The structure of the reported product and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under conditions similar to physiological. The present study revealed that the modification of epsilon -amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species.

K2P—A Novel Cross-Link from Human Lens Protein

Abstract: We report here the isolation of a novel acid‐labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC‐MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One‐ and two‐dimensional NMR analyses elucidated the structure as being 1‐(5‐amino‐5‐carboxypentyl)‐4‐(5‐amino‐5‐carboxypentyl‐amino)‐3‐hydroxy‐2, 3‐dihydropyridinium, a cross‐link between the ε‐amino groups of two lysine residues and a five‐carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water‐soluble and water‐insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 ± 362 pmol/mg of water‐insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of water‐soluble [WS] protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross‐link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

Phototransformations of advanced glycation end products in the human eye lens due to ultraviolet A light irradiation.

Abstract: Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP‐lysine and argpyrimidine are two UVA‐absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP‐lysine, OP‐phenethylamine (a phenethylamine analogue of OP‐lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP‐lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP‐lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP‐lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP‐phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.

Region-specific pathophysiological alterations occurring in calf lenses in vitro during hyperglycemia.

Abstract.Background: The early onset of cataract during diabetes may come about via a variety of pathogenic pathways, but an uncertainty about the significance of each of them exists. Methods: Calf lenses cultured in a high glucose medium were investigated for regional variations in sorbitol accumulation, changes in lactate dehydrogenase activity, and formation of carbonyl groups in proteins. The results obtained were used to evaluate the contributions of various pathways to the alterations in the lens during hyperglycemia and to relate these findings to morphologically diverse lens substructures. Results: The highest sorbitol accumulation was found in both the anterior and posterior cortex of lenses incubated in hyperglycemic medium. Lactate dehydrogenase activity was strongly affected by high sugar concentration, but the alterations in the equatorial part of lenses were more moderate relative to other substructures. After incubation with glucose, the concentration of Amadori products did not increase significantly compared to non-incubated and incubated controls. Nuclear proteins exhibited the highest level of oxidation. Conclusion: The process of sorbitol accumulation is more evident than glycation in the initial stage of hyperglycemia. Lens cortex is affected faster by elevated glucose, while the nucleus is more susceptible to prolonged effects of oxidation, glycation, and glycoxidation.