Optical effects of exposing intact human lenses to ultraviolet radiation and visible light

<p>Abstract</p> <p>Background</p> <p>The human lens is continuously exposed to high levels of light. Ultraviolet radiation is believed to play a causative role in the development of cataract. In vivo, however, the lens is mainly exposed to visible light and the ageing lens absorbs a great part of the short wavelength region of incoming visible light. The aim of the present study was to examine the optical effects on human lenses of short wavelength visible light and ultraviolet radiation.</p> <p>Methods</p> <p>Naturally aged human donor lenses were irradiated with UVA (355 nm), violet (400 and 405 nm) and green (532 nm) lasers. The effect of irradiation was evaluated qualitatively by photography and quantitatively by measuring the direct transmission before and after irradiation. Furthermore, the effect of pulsed and continuous laser systems was compared as was the effect of short, intermediate and prolonged exposures.</p> <p>Results</p> <p>Irradiation with high intensity lasers caused scattering lesions in the human lenses. These effects were more likely to be seen when using pulsed lasers because of the high pulse intensity. Prolonged irradiation with UVA led to photodarkening whereas no detrimental effects were observed after irradiation with visible light.</p> <p>Conclusions</p> <p>Irradiation with visible light does not seem to be harmful to the human lens except if the lens is exposed to laser irradiances that are high enough to warrant thermal protein denaturation that is more readily seen using pulsed laser systems.</p

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

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. © 2005 New York Academy of Sciences.link_to_subscribed_fulltex

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A330-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A330-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60. Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins. © 2002 Elsevier Science B.V. All rights reserved.link_to_subscribed_fulltex

Separation of the yellow chromophores in individual brunescent cataracts

Quantitative changes in the 330 nm absorbing chromophores and 350/450 nm fluorophores of water-soluble (WS) and water-insoluble (WI) proteins of individual human cataract lenses were characterized and compared with aged normal human lens. Twenty-five brunescent cataract lenses from India were selected from five different stages (types I-V) based upon the color of the lens. The WS and WI proteins from each lens were collected and subjected to an extensive enzymatic digestion procedure under argon. The lens protein digests were separated by Bio-Gel P-2 size-exclusion chromatography and individual peaks were analyzed further by reversed-phase HPLC. The total WI proteins increased and the total WS protein decreased with the development of cataract, especially in the late stages of cataract (III-V). The total 330 nm absorbance and 350/450 nm fluorescence of the WI fraction also increased, however, the A330 and fluorescence per mg lens protein were constant except for type V (black) lenses. Bio-Gel P-2 chromatography separated the chromophores and fluorophores into four fractions. The main fraction (designated as peak 2+3) from the cataract WI proteins was several times higher than that present in aged normal human lens WI proteins. A significant increase of this fraction was observed in WI proteins, but not in WS proteins with cataract development. Similarly, fractions 1 and 4 in the WI proteins also increased gradually but fraction 5 did not. Reversed-phase HPLC resolved fraction (2+3) of the water-insoluble sonicate supernatant proteins into four 330 nm absorbing peaks and eight fluorescent peaks. Among these peaks, a late-eluting peak (peak 8) increased 10 to 15-fold with the progress of cataract, and accounted for 80% of the total chromophores in type V lenses. This peak may represent limit digests of advanced glycation end-products (AGEs) derived protein cross-links. HPLC profiles of fraction 5 from both WS and WI proteins showed numerous new peaks which were not observed in either WS protein from cataract or WI proteins from aged normal human. The severe coloration and the higher levels of numerous novel chromophores and fluorophores in brunescent cataractous lenses reveal the possibility that a different chemistry occurs during cataract development. © 2003 Elsevier Ltd. All rights reserved.link_to_subscribed_fulltex

2-Ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) Is a Newly Identified Advanced Glycation End Product in Cataractous and Aged Human Lenses

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the non-proteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nα-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nα -t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.link_to_subscribed_fulltex

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 ε-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 ε-amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species. © 2003 Elsevier Science B.V. All rights reserved.link_to_subscribed_fulltex

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: Evidence for ascorbic acid glycation during cataract formation

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A330 units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar Rf values for the long wavelength-absorbing fluorophores. Glycation with [U-14C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A330-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A330-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A330-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo. © 2001 Elsevier Science B.V. All rights reserved.link_to_subscribed_fulltex