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.     

The Relative UV Sensitizer Activity of Purified Advanced Glycation Endproducts

Abstract— The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L-Threose, the most active of these oxidation products, was reacted with N -acetyl lysine and six AGE peaks were isolated by RP-HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 μg/mL) generated5–10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100-fold less than the superoxide anion generated by kynurenic acid and 400-fold less than riboflavin.
Ultraviolet irradiation generated from 1.2 to 2.7 μmol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM-1 and Ac-FTP) and with kynurenine and 3-OH kynurenine. This considerable singlet oxygen formation, however, was still 40-fold less than that obtained with kynurenic acid and 100-fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated20–60-fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water-insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.     

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid-Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol . 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm² of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μ M as determined by the superoxide dismutase (SOD)-depen-dent increase in hydrogen peroxide formation and of 52 μ M by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.
Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N- dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H₂O₂ were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.     

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.     

THE GENERATION OF HYDROGEN PEROXIDE BY THE UVA IRRADIATION OF HUMAN LENS PROTEINS

Abstract— The water-insoluble proteins from aged human lens are known to contain protein-bound chromophores that act as UVA sensitizers. The irradiation of a sonication-solubilized, water-insoluble fraction from human lenses (55–75 years) with UVA light (1.5 kj/cm², λ > 338 nm) caused an oxygen-dependent photolysis of tryptophan, not seen when either α-crystallin or lysozyme were irradiated. The suggested requirement for active oxygen species was consistent with a linear increase in hydrogen peroxide formation, which was also observed. A final concentration of 55 µM H₂O₂ was attained, with no H₂0₂ being detected in either dark-incubated controls or in irradiated samples of native proteins. The UVA-dependent H₂O₂ formation was increased 50% by superoxide dismutase (SOD) and abolished by catalase, arguing for the initial generation of superoxide anion. A linear photolysis of histidine and tryptophan was also seen; however, the addition of SOD or SOD and catalase had no effect on the photolytic destruction of either amino acid. Superoxide dismutase increased the oxidation of protein SH groups implicating H₂O₂, but SOD and catalase caused a decrease in SH oxidation only at later time periods. The direct addition of H₂O₂ to a water-insoluble sonicate supernatant fraction caused only a slight oxidation of SH groups, but this was increased four- to eight-fold when the protein was denatured in 4.0 M guanidine hydrochloride. Overall, the data suggest a UVA-dependent oxidation of protein SH groups via H₂O₂ generated within the large protein aggregates of the water-insoluble fraction. These data also provide a mechanism for oxidation of the sulfur-containing amino acids in vivo—a process that is known to accompany the formation of age-onset cataracts.     

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm²= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm² of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm² of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA-irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz . 0.3 J/cm². Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.     

THE PHOTOREACTIONS OF 8-METHOXYPSORALEN WITH TRYPTOPHAN AND LENS PROTEINS*

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP) can be found in the lenses of rats injected (i.p.) with this drug, and that its presence can lead to a photosensitized enhancement of lenticular fluorescence. The cutaneous photosensitizing properties of psoralens are thought to be mediated via their excited triplet states, resulting in photoaddition cyclobutane products between pyri-midine bases and 8-MOP. We have now investigated the possibility that similar types of photoadducts could be generated between 8-MOP and the aromatic amino acid residues in lens proteins. Our experiments involved in vitro irradiation (at 360 nm) of aqueous solutions of 0.1 mM 8-MOP plus purified alpha, beta, or gamma crystallins from calf or normal human (under 20 years of age) lenses. UV absorption and fluorescence emission spectra were measured before and after radiation, and aliquots from all experiments were frozen and kept in the dark for subsequent phosphorescence and EPR spectroscopy. Similar experiments were performed with irradiated aqueous solutions of tryptophan or thymine plus 8-MOP. All controls consisted of solutions kept in the dark. NMR spectra demonstrated that the hydrogen atoms at the 3,4 and 4',5' positions of the 8-MOP molecule were lost following irradiation, suggesting that these two sites were involved in the photoproduct formed between tryptophan and 8-MOP. These studies strongly suggest that 8-MOP is capable of forming photoaddition products with tryptophan and with lens proteins as well as DNA in vivo, resulting in its permanent retention within the ocular lens.     

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.     

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.     

Helical peptide models for protein glycation: proximity effects in catalysis of the Amadori rearrangement.

INTRODUCTION: Non-enzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. The observation that only a fraction of all lysine residues undergo glycation indicates the role of the immediate chemical environment in the glycation reaction. Here we have constructed helical peptide models, which juxtapose lysine with potentially catalytic residues in order to probe their roles in the individual steps of the glycation reaction. RESULTS: The peptides investigated in this study are constrained to adopt helical conformations allowing residues in the i and i+4 positions to come into spatial proximity, while residues i and i+2 are far apart. The placing of aspartic acid and histidine residues at interacting positions with lysine modulates the steps involved in early peptide glycation (reversible Schiff base formation and its subsequent irreversible conversion to a ketoamine product, the Amadori rearrangement). Proximal positioning of aspartic acid or histidine with respect to the reactive lysine residue retards initial Schiff base formation. On the contrary, aspartic acid promotes catalysis of the Amadori rearrangement. Presence of the strongly basic residue arginine proximate to lysine favorably affects the pK(a) of both the lysine epsilon-amino group and the singly glycated lysine, aiding in the formation of doubly glycated species. The Amadori product also formed carboxymethyl lysine, an advanced glycation endproduct (AGE), in a time-dependent manner. CONCLUSIONS: Stereochemically defined peptide scaffolds are convenient tools for studying near neighbor effects on the reactivity of functional amino acid sidechains. The present study utilizes stereochemically defined peptide helices to effectively demonstrate that aspartic acid is an efficient catalytic residue in the Amadori arrangement. The results emphasize the structural determinants of Schiff base and Amadori product formation in the final accumulation of glycated peptides.     

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.     

ULTRAVIOLET ACTION SPECTRUM FOR FLUOROGEN PRODUCTION IN THE OCULAR LENS

Abstract— …Previous work has demonstrated that fluorescent material (360nm excitation, 440nm emission), whose concentration normally increases with age in human lenses, can be generated artificially by exposing cultured human or animal lenses to UV radiation. In the present paper we report measurements of the rate of production of this fluorescent material in rat lenses in vitro as a function of UV irradiation wavelength. A plot of the observed rate of fluorogen production normalized to constant photon flux vs irradiation wavelength shows little action at 360 or 320nm, increases sharply at 300nm, remains relatively constant in the range 300–280nm, and then exhibits a further gradual rise from 270–250nm. The results on rat lenses are compared with results reported elsewhere for tryptophan in aqueous solution. The action spectrum for photochemical destruction of tryptophan in solution closely parallels that for fluorogen production in rat lenses. This result and other evidence suggest that photochemical destruction of tryptophan might be the initial event in UV-induced fluorogen production in the ocular lens.     

Impairment of eye lens cell physiology and optics by broadband ultraviolet A-ultraviolet B radiation

The phototoxicity of ultraviolet A (UVA) alone and UVA plus ultraviolet B (UVB) combined on cultured porcine lenses was investigated by analyzing cellular function as measured with a fluorescence bioassay approach and optical integrity, in terms of sharpness of the lens focus as measured with a scanning laser system. The bioassay consisted of carboxyfluorescein diacetate-acetoxymethyl ester and alamarBlue fluorescent dyes. Aseptically dissected porcine lenses were maintained in modified medium 199 without phenol red supplemented with 1% penicillin-streptomycin and 4% porcine serum. At 1 week of preincubation, baseline measurements were obtained. Then the lenses were treated with single exposures of different UVA and UVB energy levels. The lenses treated with 86 J/cm2 UVA alone showed a significant (P < 0.05) decrease in cellular and optical integrity at 48 h after exposure, whereas those treated with 43 J/cm2 UVA alone did not show significant phototoxic effect. Lenses treated with 15.63 J/cm2 UVA plus 0.019 J/cm2 UVB combined showed significant adverse effects beginning from 48 h after exposure. Also, there was no recovery. These findings show that a high UVA dose alone and relatively low UVA in combination with low UVB radiant exposure can impair lens cellular and optical functions, respectively.     

Measurement of lens protein aggregation in vivo using dynamic light scattering in a guinea pig/UVA model for nuclear cataract

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.     

Glycation and insolubility of human lens protein.

To learn whether glycation plays a role in insolubilization or in senile cataractogenesis, the reactivity of lens protein from normal and senile cataractous lenses and individual crystallin prepared from human lens with various sugars [glucose, glucose-1-phosphate (G-1-P), glucose-6-phosphate (G-6-P) and fructose], and the insolubility of those proteins were determined. The reactivity of human lens protein to glucose was increased in a dose-dependent manner, and it was demonstrated that 17.9, 18.5 and 24 kDa proteins were susceptible to glycation with sugars. The study also showed that alpha-, beta-crystallins and high molecular weight (HMW) aggregate obtained from cataractous lens have some weak reactivity against sugars. It was demonstrated that the proteins obtained from normal lens of older age and from cataractous lenses have higher insolubilities to glucose than do normal younger ones. Measurement of glycosylated protein by affinity column chromatography revealed that cataractous lenses contained a larger amount of glycosylated protein than normal ones. These results suggest that there is an age-related increase of glycation in normal human lens protein, and that such glycation increases the amount of insolubilized protein with the effect of aging. The author also speculates that an abnormal acceleration of glycation in the human lens may induce senile cataract formation.     

LEVELS OF REDUCED PYRIDINE NUCLEOTIDES AND LENS PHOTODAMAGE

Since most of the known factors that are associated with cataract formation are oxidative in nature, one would expect that a highly reductive environment might arrest or retard the progress of cataract formation. Reduced nucleotides, both NADH and NADPH, are potent reductants with a large negative redox potential of -320 mV. Lenses of certain species contain high levels of these nucleotides, presumably due to the presence of taxon specific crystallins. We have utilized this situation to investigate whether the levels of reduced pyridine nucleotides modulate photo-oxidative damage to the lens. We have monitored the time dependent loss of tryptophan fluorescence upon photodamage for lenses from guinea pig, rabbit and frog (Rana) that contain high levels of pyridine nucleotides and compared with the lenses from rat, Xenopus and a mutant strain of guinea pig that contain significantly lower amounts of these nucleotides. About 75% and 90% of the initial fluorescence intensity is lost in the case of rat and Xenopus lenses, respectively, after a total of 35 min exposure. Rabbit, guinea pig and frog lenses, under identical conditions, show only about 35-40% loss of the initial fluorescence. It appears that the lenses that contain high levels of reduced nucleotides are less susceptible to photodamage. The observed anti-oxidative role of reduced nucleotides in the lenses indicates the possibility of testing reductants (NADPH, NADH and their functional analogues) as potential candidates to therapeutically intervene in the process of cataractogenesis.     

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.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

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.     

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.