Contribution of glycation to human lens coloration

To study the contribution of glycation or the Maillard reaction to the spontaneous coloration of human crystalline lens in aging, we determined 1-deoxyfructosyl adduct and the fluorescent material, which are produced in the early stage of glycation, in the proteins of normal and colored human lenses of different ages. The amount of both glycation products in the lens increased significantly in proportion to aging or the advance of lens coloration. The insolubility of lens protein also increased with the advance of glycation. In addition, the present study showed that glucose and glucose-6-phosphate have higher reactivities with human lens protein than fructose and glucose-1-phosphate. This paper demonstrates that the deeper colored or older aged lens contains larger amounts of glycation products, and that glycation between lens protein and various sugars in vivo may be a serious factor in human lens coloration or insolubilization of lens protein.     

Application of solid‐phase extraction for the concentration of chromophores,fluorophores, and photosensitizers from lens protein digests

Solid‐phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid‐phase extraction was capable of concentrating the advanced glycation end‐products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.     

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

-Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm² total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. The incubation of lens proteins with dehydroascorbic acid or l -threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity. The relative sensitizer activity of the 4 week glycated sample was quantitatively very similar to that of a water-insoluble fraction from aged human lenses. These data are consistent with the hypothesis that the protein-bound brunescence in the lens may be advanced glycation endproducts, which are formed in large part by the oxidation products of ascorbic acid, and that these compounds may contribute significantly to the UVA sensitizer activity present in aged human lenses.     

Novel human lens metabolites from normal and cataractous human lenses

4-(2-Aminophenyl)-4-oxobutanoic acid, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid and glutathionyl-kynurenine have been identified as novel metabolites in normal and cataractous human lenses following total synthesis and comparison with authentic human lens samples. Their structures are consistent with those derived from the major human lens UV filters kynurenine and 3-hydroxykynurenine, and it is proposed that these compounds also play a role as UV filters. These metabolites were isolated in pmol/mg levels (dry mass) in lenses. 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid and glutathionyl-kynurenine were found to be unstable at physiological pH. Other potential metabolites, glutathionyl-3-hydroxykynurenine, kynurenine yellow and 3-hydroxykynurenine yellow, were not detected in either normal or cataractous lenses.     

Quantitative photoacoustic spectroscopy of cataractous human lenses

Quantitative photoacoustic spectra of the nuclei of cataractous human lenses with various degrees of colouration and opacification were measured in the spectral range 250-600 nm. The lens nuclei were obtained from 20 cataractous patients through extracapsular cataract extraction. These measurements yield the light loss per unit path length in the nucleus of cataractous lenses.     

Focal length variability and protein leakage as tools for measuring photooxidative damage to the lens

Hypericin is the ingredient used to standardize the popular over-the-counter antidepressant medication St. John's Wort. Because hypericin readily produces singlet oxygen and other excited state intermediates, it is a very efficient phototoxic agent in the eye that can potentially induce the development of the cataract photooxidative mechanism. Hypericin absorbs in the UV and visible ranges, binds to the lens crystallins (alpha, beta and gamma) and damages these proteins through a photooxidative mechanism. Effects were measured previously using fluorescence, UV and mass spectrometry. We report here two additional methods to monitor lens damage: (1) measuring focal length variability using a ScanTox instrument and (2) measuring protein leakage from the damaged lens. Because nonenzymic glycation results in free radical production, we chose to use elevated glucose concentrations as a convenient model for studying oxidative stress. To compare and contrast photooxidative damage against oxidative damage to the lens, we also measured the focal length variability and protein leakage induced by the presence of elevated glucose concentrations. We found that the total accumulated protein leakage was positively correlated (r = 0.9) with variability in focal length. Lenses treated with hypericin and irradiated with UVB had an increase in focal length variability as compared with the lenses that were only UVB-irradiated. Lenses without UVB irradiation had much lower focal length variability than irradiated lenses. For non-hypericin-treated lenses, UVB-irradiated lenses had a larger variability (4.58 mm) than the unirradiated lenses (1.78 mm). The lenses incubated in elevated glucose concentrations had a focal length variability (3.23 mm) equivalent to that of the unirradiated hypericin-treated lenses (3.54 mm). We conclude that photooxidative damage by hypericin results in changes in the optical properties of the lens, protein leakage and finally cataract formation. In contrast to this, high concentrations of glucose induced protein leakage but not changes in optical properties or the opacity associated with a cataract. This work provides further evidence that people should protect their eyes from intense sunlight when taking St. John's Wort.     

Photosensitizing activity of advanced glycation endproducts on tryptophan, glucose 6-phosphate dehydrogenase, human serum albumin and ascorbic acid evaluated at low oxygen pressure

A comparative study of the photosensitizing activity of advanced glycation endproducts (AGEs) prepared by incubating glucose (Glc), threose (Threo) and ascorbate (AH-) in the presence of lysine (Lys) was performed. Photochemical activity was evaluated under low oxygen pressure with the aim to simulate the conditions of the eye lens. AGE-sensitized tryptophan and AH- photodecomposition and glucose 6-phosphate dehydrogenase inactivation were studied. In all systems, glucose-derived AGEs showed the highest photosensitizing efficiency, followed by ascorbate and threose. The presence of different sensitizers in glycation products mixtures was investigated. For this purpose, Trp decomposition quantum yields were determined at 344 and 367 nm. The values obtained at 344 nm are between three and six times higher than those observed at 367 nm, confirming the presence of at least two compounds with different photosensitizing activities in the mixtures. The chemiluminescence associated with the AGE-mediated oxidation of free Trp and Trp residues in human serum albumin was also studied, and a good correlation between the emission of light and the extent of Trp decomposition was found. In conclusion, it is demonstrated that glucose derived AGEs, which can be formed in vivo in the eye lens of diabetic patients and are accumulated in elderly lenses, have a higher photosensitizing efficiency, at low oxygen pressure, than those arising from ascorbate and threose. This high efficiency is especially significant when proteins are employed as photochemical targets, indicating that protein-sensitizer interaction and the local environment around the sensitizers play an important role.     

Mutation of two active-site residues converts a phosphatidylinositol-specific phospholipase C to a glucose phosphatase

Two mutations, R69D and K115E, converted a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) to a phosphatase with much higher specific activity toward glucose-6-phosphate than inositol-1-phosphate. PI-PLC single mutations R69D and K115E can cleave PI but lack any demonstrable phosphatase activity. The bacterial PI-PLC has no sequence homology with known glucose-6-phosphatase enzymes, which need His, Arg, and negatively charged residues (Asp or Glu) at the active site. The change in chemical reaction and substrate specificity can be rationalized by energy minimization of the mutant with I-1-P or G-6-P bound.     

Enhancement of the non-tryptophan fluorescence of human lens proteins after near-UV light exposure

Abstract. In this work, the non-tryptophan fluorescence (360 nm excited; 440 nm emitted) of human lens proteins was found to be intensified by exposing whole lens homogenates to near-UV light in the presence of tryptophan photoproducts. The induced fluorescence accumulates mainly in the soluble phase proteins, whereas in aging and brown cataractous lenses, the major fluorescence is found in the insoluble proteins. Using SDS-polyacrylamide gel electrophoresis with densitometric and fluorescence scanning techniques, the polypeptide chains of the three major protein fractions were analyzed for their specific non-tryptophan fluorescences. The same chains were found in all fractions. Two chains (11,000 and 45,000 daltons) were found to accumulate most of the induced fluorescence. These also contained the greatest intrinsic fluorescence initially. The data indicates that specific polypeptide chains in the lens proteins are most sensitive to modifications due to their exposure to near-UV light in the presence of tryptophan photoproducts.     

STUDIES ON HUMAN LENSES: II. DISTRIBUTION AND SOLUBILITY OF FLUORESCENT PIGMENTS IN CATARACTOUS AND NON-CATARACTOUS LENSES OF INDIAN ORIGIN

Abstract— Fluorometric studies of cataractous and non-cataractous human lenses were carried out to study the emission characteristics and the distribution and solubility of lenticular pigments. Most of the detected fluorophores were well distributed over the cortical and nuclear portion of the lens. The decrease in solubility of proteins with aging and cataract formation is concomitant with increasing photolysis of tryptophan. However, this is likely a phenomenon independent of the photochemical transformations of the lens proteins. The number of emitting species in the diseased lenses are higher than in the normal mature lenses. A species emitting around 375 or 388 nm is of particular interest (λ_cx, 330 nm) in that the emission characteristics of this fluorophore resemble kynurenic acid which has a high photosensitizing efficiency. The concentration of fluorescent pigments in the lenses of Indian origin is significantly high. The intense pigmentation could be attributed largely to the formation of photoproducts in the absence of normal endogenous antioxidant accumulation that is dependent on nutrition standard. If, indeed, any of these fluorescent pigments, because of their photosensitizing ability, are responsible for lenticular opacity, it is not the abundance of sunlight alone but also malnutrition that could account for the high incidence of cataract in India.     

NEAR UV LIGHT AND CATARACTS*

Abstract. Sunlight and many types of artificial lighting contain near-UV light (300–400 nm). These wavelengths can enter the eye and are maximally absorbed in the lens due to its chemical composition. The lenses of certain animals develop cataracts from exposure to this light, and changes similar to those that occur in human lenses with certain types of cataracts and with aging are inducible in isolated human lenses. These changes seem to be associated with chemical alterations in the essential amino acid tryptophan either as a part of proteins or in free form. Such changes in tryptophan would result in lens cell toxicity, in increased pigmentation of the lens, and in large aggregates of proteins. The latter two changes would result in losses in the ability of the lens to transmit visible light needed for vision, and the abnormal state called cataract would result. Much more work is needed to prove that near-UV light can accelerate cataractous changes in the lenses of living humans. Studies at the basic chemical level are needed, but population studies would be most essential for the final proof. Many preventive measures could become available, including the use of special types of spectacles and dietary additives.     

STUDIES ON HUMAN LENSES: II. DISTRIBUTION AND SOLUBILITY OF FLUORESCENT PIGMENTS IN CATARACTOUS AND NON-CATARACTOUS LENSES OF INDIAN ORIGIN

Fluorometric studies of cataractous and non-cataractous human lenses were carried out to study the emission characteristics and the distribution and solubility of lenticular pigments. Most of the detected fluorophores were well distributed over the cortical and nuclear portion of the lens. The decrease in solubility of proteins with aging and cataract formation is concomitant with increasing photolysis of tryptophan. However, this is likely a phenomenon independent of the photochemical transformations of the lens proteins. The number of emitting species in the diseased lenses are higher than in the normal mature lenses. A species emitting around 375 or 388 nm is of particular interest (lambda cx 330 nm) in that the emission characteristics of this fluorophore resemble kynurenic acid which has a high photosensitizing efficiency. The concentration of fluorescent pigments in the lenses of Indian origin is significantly high. The intense pigmentation could be attributed largely to the formation of photoproducts in the absence of normal endogenous antioxidant accumulation that is dependent on nutrition standard. If, indeed, any of these fluorescent pigments, because of their photosensitizing ability, are responsible for lenticular opacity, it is not the abundance of sunlight alone but also malnutrition that could account for the high incidence of cataract in India.     

UVA PHOTOLYSIS USING THE PROTEIN-BOUND SENSITIZERS PRESENT IN HUMAN LENS

Abstract —This research was undertaken to demonstrate that the protein-bound chromophores in aged human lens can act as sensitizers for protein damage by UVA light. The water-insoluble (WI) proteins from pooled human and bovine lenses were solubilized by sonication in water and illuminated with UV light similar in output to that transmitted by the cornea. Analysis of the irradiated proteins showed a linear decrease in sulfiydryl groups with a 30% loss after 2 h. No loss was seen when native a-crystallin was irradiated under the same conditions. A 25% loss of histidine residues was also observed with the human lens WI fraction, and sodium dodecyl sulfate polyacrylamide gels indicated considerable protein cross-linking. Similar photodamage was seen with a WI fraction from old bovine lenses. While the data show the presence of UVA sensitizers, some histidine destruction and protein cross-linking were also obtained with a-crystallin and with lysozyme, which argue that part of the histidine loss in the human WISS was likely due to tryptophan acting as a sensitizer.
A preparation of human WI proteins was irradiated with a total of 200 J/cm² of absorbed light at 10 nm intervals from 290 to 400 nm. Photodamage of cysteine SH groups (35%) and methionine (28Y0) was maximum at 330 nm and diminished linearly at longer wavelengths. The major loss of tryptophan (80%) occurred at 290 nm, but destruction was observed throughout the UVA range. Tyrosine was 35% destroyed at 290 nm but decreased sharply to only 50 at 330 nm. A constant loss of histidine (20%) was seen at all wavelengths from 290 to 360 nm, with some loss (7–8%) even at 400 nm. These action spectra show that the human lens WI fraction contains a collection of protein-bound UVA sensitizers that can cause protein photodamage similar to that seen in cataractous lenses.     

Investigations on inorganic elements in human lenses of normal and senile cataractous character

Some essential trace elements as copper, zinc, iron, cobalt, scandium and rubidium have been measured by neutron activation analysis in normal and different types of senile cataractous lenses.     

Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo

The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor- kappaB (NF-κB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-oneweek- old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-κB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal- treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-κB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-κB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-κB-dependent and pro-apoptotic.     

DSC examinations on cataractous lens materials obtained by phacoemulsification

Modifications of lens proteins play a crucial role in the formation of cataract, which is among the leading causes of world blindness in the ageing population. Although modern cataract surgery by phacoemulsification is a very successful and safe procedure, the prevention of cataract formation would be a real breakthrough in this field of ophthalmology. The aim of our study was to analyse the thermal denaturation of the cataractous lens proteins by the method of differential scanning calorimetry (DSC), to understand cataract formation and to work on its prevention possibilities. Samples were obtained from cataract patients of different age, sex, patients with and without diabetes mellitus. Samples were obtained from lenses of mature degree and progredient degree of cataract as well. Previous DSC examinations were performed on manually extracted human cataractous lens materials, however to the best of our knowledge, this is the first study, in which DSC examinations were performed on lens materials obtained by the phacoemulsification technique, which gained acceptance world-wide in the last decade. The online version of the original article can be found at     

Development of Monoclonal Antibodies Against a Riboflavin-Tryptophan Photoinduced Adduct: Reactivity to Eye Lens Proteins*

We describe here the development of monoclonal antibodies to the hapten tryptophan-riboflavin, generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. The specificity of the three obtained monoclonal antibodies, named lE6, 5H5, 5AS all belonging to the IgGl isotype, was assessed by a competitive enzyme-linked immunosorbent assay in the presence of an increasing concentration of the tryptophan-riboflavin adduct, obtained from an irradiated riboflavin-sensitized tryptophan solution. It was demonstrated that the tryptophan-riboflavin antibodies react with the soluble proteins of the eye lens; this reaction was more intense in the old rat lenses as compared to the young ones, and a maximum binding of the antibodies was obtained with the soluble protein fraction from the human catar-actous lens. By indirect immunofluorescence, a reactivity associated with the protein matrix, localized in the lens central zone, was observed. In the peripheral zone of the lens, where the younger cells are found, a marked im-munofluorescent emission was observed on structures preferentially localized in the nuclei.     

PHOTOSENSITIZED OXIDATION IN THE OCULAR LENS: EVIDENCE FOR PHOTOSENSITIZERS ENDOGENOUS TO THE HUMAN LENS

Abstract— Numerous investigators have attempted to associate near UV light exposure with various changes which occur to lens crystallins during aging and cataractogenesis. Recently we have shown in vitro that singlet oxygen mediated oxidation of lens crystallins produces effects very similar to those documented for crystallins from old or cataractous lenses and suggested that near UV photodynamic effects may play a major role in vivo in aging in the human lens. In the present work we demonstrate that certain oxidation products of tryptophan which have been identified in human lens can act as near UV photosensitizers, producing singlet oxygen. The insoluble protein fraction from human cataracts is shown to have the capacity to act as a photosensitizer. An age-related increase in photosensitizing capacity is also demonstrated in the soluble crystallins from human lens. These findings are discussed with respect to development of pigmented nuclear cataracts.     

Photooxidation of lens proteins with xanthurenic acid: a putative chromophore for cataractogenesis.

The tryptophan metabolite, xanthurenic acid (Xan), is produced through a transamination reaction in high concentrations in human lenses with age and has been isolated from aged human cataractous lenses. It has appreciable absorption between 300 and 400 nm (lambda max = 334 nm), the range absorbed by the human lens. Our recent studies have shown that unlike most tryptophan metabolites in the eye, Xan is photochemically active, producing both superoxide and singlet oxygen. To determine if Xan could act as a photosensitizer and photooxidize cytosolic lens proteins, alpha-, beta- and gamma-crystallins were irradiated (lambda > 300 nm, 12 mW/cm2) in the presence and absence of Xan. Upon irradiation and in the presence of Xan, lens proteins polymerized in the order alpha > beta > gamma as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Further analysis of the photolyzed alpha-crystallin by mass spectrometry indicated that histidine, tryptophan and methionine residues were oxidized at specific positions in a dose-dependent (irradiation time) manner. In alpha A-crystallin two forms of oxidized histidine 154 were observed, 2-imidazolone and 2-oxohistidine. Our results suggest that naturally occurring Xan is a chromophore capable of photosensitization and photooxidation of lens proteins. Furthermore, this compound could play a role in age-related cataractogenesis.     

Glycation pattern of peptides condensed with maltose,lactose and glucose determined by ultraviolet matrix‐assisted laser desorption/ionization tandem mass spectrometry

Protein glycation is the non‐enzymatic condensation of sugars with proteins. Although commonly occurring in both the therapeutic and food/beverage industries, protein glycation has not been the focus of many proteomic investigations. This study aims to establish a reliable mass spectrometric method for screening large tandem mass spectrometric (MSMS) datasets for protein glycation with glucose, lactose and maltose. Control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in MSMS analysis for glucose, lactose and maltose condensed with peptides. Valid in both tandem time‐of‐flight (TOFTOF) and quadrupole ion trap time‐of‐flight matrix‐assisted laser desorption/ionization (QIT TOF MALDI) mass spectrometers, these neutral loss signatures were then applied to elucidation of modified peptides from a complex human serum albumin (HSA) digest glycated with each of the proposed sugars. Screening of these large datasets was made possible by specifically designed software solutions that enable the input of detailed user‐defined post‐translational modifications that are not included in the universally available databases such as Unimod. Copyright © 2010 John Wiley & Sons, Ltd.