PHOTOCHEMICAL AND PHOTOPHYSICAL STUDIES ON HUMAN LENS CONSTITUENTS

The young human lens contains species (3-hydroxy kynurenine; 3-HK and its glucoside; 3-HKG) which absorb most light between 300 and 400 nm. Photochemical studies have indicated that these compounds are relatively inefficient sensitizers of lens proteins. An investigation of the fluorescent properties of 3-HKG indicate that it contains a fast deactivation pathway (ps) which would be expected to have minimal photochemical effect on the integrity of the lens. Further phot physical studies on 3-HK indicates that it has an even faster fluorescent lifetime (less than 10 ps) with a much lower quantum yield of fluorescence (0.001 vs 0.03 for 3-HKG). With aging, the human lens proteins undergo numerous changes including a generalized yellowing. These chromophores exhibit a higher quantum yield of fluorescence, an increase in the fluorescent lifetime by 2 orders of magnitude and the formation of two long lived transient species (microsecond). These species might be expected to drastically increase the susceptibility of the human lens to ambient radiation. Based upon quantitative experimental comparisons with 3-HK this does not seem to be the case. Further time resolved studies on old lens proteins indicate that the two transient species are interconnected in that the first transient species is the precursor to the second. The implications of this mechanism on the integrity of the lens and origin of those chromophores is discussed.     

DETECTION OF PORPHYRIN EXCITED STATES IN THE INTACT BOVINE LENS

Previous steady state and time resolved spectroscopic studies on porphyrins have shown that the triplet lifetimes of those sensitizers that bind to lens proteins are lengthened by several orders of magnitude. Presented here is an extension of this experiment to measure these transients in an intact bovine lens. As demonstrated by steady state fluorescence spectroscopy and flash photolysis, mesotetra (p-sulfonatophenyl)porphyrin (TPPS) binds to lens proteins. In air-saturated aqueous solution, TPPS has a triplet lifetime of 2 microseconds. In an intact bovine lens the triplet state decayed via biexponential kinetics with lifetimes of 0.16 and 1.6 microseconds. In addition to a lengthening of the lifetime there was a red shift in the triplet transient spectra of 10-20 nm of the porphyrin in the intact lenses.     

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.     

FLUORESCENCE LIFETIMES OF OCULAR LENS CHROMOPHORES

Abstract— Fluorescence lifetimes are reported for intact human lenses in vitro. Two spectral regions were investigated: The first was excited at 296nm and detected at 332 or 370nm and corresponds to emission from tryptophan residues in the lens proteins. The second spectral region was excited at 359 nm and detected at 435 nm and corresponds to non-tryptophan 'fluorogen' fluorescence. The latter displayed a constant lifetime, 3.8 ns, independent of the anatomical part of the lens excited. This value was compared with measured lifetimes for some model fluorogens. The tryptophan fluorescence lifetime (332 nm detection) was found to vary from 1.8 to 2.8 ns depending on the anatomical part of the lens excited.     

The photochemical attachment of the O-glucoside of 3-hydroxykynurenine to alpha-crystallin: a model for lenticular aging

The young human lens contains a small metabolite from tryptophan called the O-glucoside of 3-hydroxykynurenine (3-HKG). Its function is to absorb most radiation between 295 and 400 nm, preventing it from reaching the retina. With age the concentration of this component decreases while the lens crystallins acquire covalently attached chromophores. This study investigates the photochemical attachment of 3-HKG to lens alpha-crystallin. Initial studies showed that alpha-crystallin photolyzed in the presence of 3-HKG developed a fluorescence (emission, 440 nm) and UV-visible spectrum similar to that found in aged human lens proteins. Extensive studies were then performed on the tryptic HPLC maps as monitored by photodiode array and fluorescent detection. Numerous photoproducts with either blue (emission, > 400 nm) or green (emission, > 500 nm) fluorescence were formed in addition to nonfluorescent compounds with absorption maxima above 300 nm. Comparisons were made between these model photoproducts and peptide maps from alpha-crystallin isolated from old human lenses. In terms of retention time and UV-visible spectra at least two of the peptides that appear in the model system are also present in the human samples. It is concluded that one of the aging processes in the human lens is the photochemically induced attachment of 3-HKG to lens proteins.     

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.     

Role of xanthurenic acid 8-O-beta-D-glucoside,a novel fluorophore that accumulates in the brunescent human eye lens

We have been able to identify a blue fluorophore from the low-molecular weight soluble fraction of human adult nondiabetic brunescent cataract lenses as xanthurenic acid 8-O-beta-D-glucoside (XA8OG) (excitation = 338 nm and emission = 440 nm). To determine the role of this fluorophore in the lens, we have examined its photophysical and photodynamic properties. We found XA8OG to have a fluorescence quantum yield (phi) of 0.22 and a major emission lifetime of 12 ns. We found it to be a UVA-region sensitizer, capable of efficiently generating singlet oxygen species but little of superoxide. We also demonstrated that XA8OG oxidizes proteins when irradiated with UVA light, causing photodynamic covalent chemical damage to proteins. Its accumulation in the aging human lens (and the attendant decrease of its precursor O-beta-D-glucoside of 3-hydroxykynurenine) can, thus, add to the oxidative burden on the system. XA8OG, thus, appears to be an endogenous chromophore in the lens, which can act as a cataractogenic agent.     

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.     

AGE-RELATED CHANGES IN THE HUMAN LENS AS MONITORED BY DETECTION OF PORPHYRIN EXCITED STATES

Abstract— Previous studies have shown that the triplet state lifetimes of various porphyrins are increased by several orders of magnitude when they are bound to lens proteins. Flash photolysis studies of me-sotetra ( p -sulfonatophenyl)porphyrin (TPPS) on intact bovine lenses indicated a biexponential decay of the triplet state with lifetimes of 160 μs and 1.6 ms. Here we extend those measurements to TPPS associated with intact human lenses. Steady-state fluorescence measurements indicate that TPPS binds to both young and old human lenses. In an intact young human lens, the TPPS triplet state is observed to decay biexponentially with lifetimes of 50 and 680 μs. As the age of the lens increases, the lifetime of the shorter-lived component lengthens while that of the longer-lived component decreases slightly. In older human lenses, the two lifetimes coalesce and the triplet decay exhibits purely monoexponential behavior. These photophysical characteristics apparently are due to age-related modification(s) of the protein in the human lens resulting in an increasingly more homogeneous environment around the porphyrin.     

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.     

Transmission of light to the young primate retina: possible implications for the formation of lipofuscin

The purpose of this study was to determine the transmission properties of the anterior segment of young primate eyes and potentially relate those changes to photochemical processes in the retina that lead to the early, rapid formation of lipofuscin. A simple method has been developed to determine the optical properties of the anterior segment of the intact eye. Using this technique, the transmission/absorption properties of primate cadaver eyes were determined. A young primate anterior segment has a maximum absorption at 365nm due to the presence of the O-β-glucoside of 3-hydroxykynurenine in the lens. This is synthesized in the last trimester of gestation. Although this compound filters out most of the UV light from reaching the retina, there is a small window of transmission centered on an absorption minimum at 320nm. This closes by the second decade of life. The window of transmission of UV light to the primate retina may explain the initial accelerated formation of lipofuscin in the young human retina by a photochemical process. This would be exacerbated by any decrease in the ozone layer with concomitant increase in UV-B reaching the earth's surface.     

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.     

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.     

Sunlight induces N epsilon-(carboxymethyl)lysine formation from glycated polylysine-iron(III) complex

Sunlight was found to strongly induce the formation of N epsilon-(carboxymethyl)lysine (CML) from glycated polylysine in the presence of Fe(III) ion. The initial step of this Fe(III)-catalyzed CML formation was noted to be similar to that of blueprint photography as was confirmed by the production of Turnbull's blue in sunlight-exposed glycated human serum albumin ferricyanide solution in the presence of Fe(III). Based on this, photoinduced oxidative C-C bond cleavage of the Amadori compound was assumed to be initiated by photochemical single electron transfer front ligand to Fe(III) in the Fe(III)-Amadori compound complex affording the Fe(II)-Amadori compound radical intermediate, which eventually yields either CML or active oxygen species. CML is thus a useful oxidative stress marker. The mechanism proposed here would explain the high accumulation of CML in lens protein and skin actinic elastosis.     

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.     

Dynamics of proteins: light scattering study of dilute and dense colloidal suspensions of eye lens homogenates

We report a dynamic light scattering study on protein suspensions of bovine lens homogenates at conditions (pH and ionic strength) similar to the physiological ones. Light scattering data were collected at two temperatures, 20 and 37 degrees C, over a wide range of concentrations from the very dilute limit up to the dense regime approaching the physiological lens concentration. A comparison with experimental data from intact bovine lenses was advanced, revealing differences between dispersions and lenses at similar concentrations. In the dilute regime, two scattering entities were detected and identified with the long-time self-diffusion modes of alpha-crystallins and their aggregates, which naturally exist in lens nucleus. Upon increasing protein concentration, significant changes in time correlation function were observed starting at approximately 75 mg ml(-1), where a new mode originating from collective diffusive motions becomes visible. Self-diffusion coefficients are temperature insensitive, whereas the collective diffusion coefficient depends strongly on temperature revealing a reduction of the net repulsive interparticle forces with decreasing temperature. While there are no rigorous theoretical approaches on particle diffusion properties for multicomponent, nonideal hard sphere polydispersed systems, as the suspensions studied here, a discussion of the volume fraction dependence of the long-time self-diffusion coefficient in the context of existing theoretical approaches was undertaken. This study is purported to provide some insight into the complex light scattering pattern of intact lenses and the interactions between the constituent proteins that are responsible for lens transparency. This would lead to understand basic mechanisms of specific protein interactions that lead to lens opacification (cataract) under pathological conditions.     

Early Diagnosis of Diabetes through the Eye

Diabetes mellitus is a metabolic disorder characterized by high blood sugar levels which give rise to complications in the eye, kidneys and the brain. Diabetes triggers the development of ocular diseases like diabetic retinopathy and cataracts which are the leading cause of blindness around the world. The most common method for the diagnosis of diabetes involves measuring the blood sugar levels in the body. One major disadvantage of this method is the fluctuating blood sugar levels which contribute to false negative results. This leads to delay in treatment, eventually causing permanent damage to the organs. Therefore, diagnosis of diabetes at an early stage is very crucial. One biomarker for diabetes related diseases is the formation of Advanced Glycation End‐products (AGEs) that result from the Maillard reaction of proteins with glucose. α‐crystallin in the ocular lens is a small heat shock protein with no protein turnover and hence acts as a record for post‐translational modifications especially glycation which forms AGEs. We have used steady state and time resolved fluorescence measurements to study the spectroscopic changes in α‐crystallin with increase in time of glycation and the intact lenses from diabetic and nondiabetic donors. Overall, this study was focused on developing a noninvasive diagnostic tool for early detection of diabetes mellitus.     

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.     

Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescence

High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria. We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (< 10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence. SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells. Remarkably high efficiency (> 1,200,000 theoretical plates/m) was achieved with this rapid and efficient CE method. It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis. Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5. The limit of detection (LOD) and recovery were found to be 4.2 x 10(4) cells/mL and 70.0%, respectively. This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings.     

MONITORING LIGHT-INDUCED CHANGES IN ISOLATED, INTACT EYE LENSES

Fluorescence spectral changes occurring upon irradiation with 300 nm light have been monitored in situ in isolated, intact, whole lenses from the eyes of several species. The findings corroborate observations on other individual constituent protein molecules in the solution state, and also reveal features attributable to the supramolecular protein assembly that exists in the whole lens. Irradiation of the lens with 300 nm light causes red shifts in the tryptophan emission spectrum, suggesting alterations in the protein packing in the lens. Intermolecular energy transfer from tryptophan to one of the photoproducts, presumably N-formylkynurenine (N-FK), occurs in the condensed-phase sample. The N-FK formed is photodegraded efficiently in the lens, indicating that the photodynamic effects of endogenous N-FK might not be as severe as has been thought. Species variation in the photoevents are evident, particularly in avian lenses that contain the variant δ-crystallin as the core protein. The photoinduced changes in the near-UV circular dichroism of δ-crystallin (which is α-helical, as opposed to the β-sheet structure of α-, β-, and -γ-crystallins), isolated from chicken lenses, are remarkably different from other crystallins. Irradiation of δ-crystallin leads to a drastic reduction of circular dichroism intensity in the 250–300 nm region, whereas no changes are seen in the peptide absorption band.