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Long-wavelength solar UV radiation is implicated in photodamage to the human eye. The human lens contains multiple tryptophan-derived compounds that have significant absorbance bands in the UVA region (λ 315-400 nm) that act as efficient physical filters for these wavelengths. The concentrations of many of these UV filter compounds decrease with increase in age, resulting in diminished protection, increased oxidative damage and the accumulation of modified proteins implicated in nuclear cataract formation. This damage may arise via the formation of α,β-unsaturated carbonyls from the UV filter compounds, adduction to lens proteins and subsequent action as photosensitizers, and/or via the reactions of redox-active transition metal ions that accumulate in aged human lenses. The latter may promote the oxidation of free, or protein-bound, o-aminophenols, such as the UV filter compounds 3-hydroxykynurenine (3OHKyn) and 3-hydroxyanthranilic acid (3OHAA). It is shown here that Cu(II), and to a lesser extent Fe(III), enhance oxidation of free 3OHKyn, 3OHAA and 3OHKyn bound to specific amino acids and lens proteins, with this resulting in increased cross-linking of lens proteins. These data indicate that elevated levels of transition metal ions in aging lenses can enhance the loss of protective UV filter compounds, and contribute to the formation of high-molecular-mass dysfunctional crystallin proteins in a light-independent manner. These reactions may contribute to the formation of lens cataracts in humans.

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.

Age-related nuclear (ARN) cataract is a major cause of world blindness. With the onset of ARN cataract, the normally transparent and colorless lens becomes opaque and can take on colors ranging from orange, brown, and even black. The molecular basis for this remarkable transformation is unknown. ARN cataract is also characterized by extensive oxidation, insolubilization, and cross-linking of polypeptides, particularly in the nucleus of the lens. It has been postulated that 3-hydroxykynurenine (3OHKyn) may be involved in these changes. This endogenous tryptophan metabolite is readily oxidized and is involved in the tanning of moth cocoons and the formation of pigments in the eyes of butterflies. 3OHKyn is a component of our primate-specific UV-filter pathway, and the brownish hue of ARN cataract lenses is also unique to humans. Because numerous colored compounds can be produced by autoxidation of 3OHKyn, this process could provide an explanation for the variety of lens colors and other changes seen in ARN cataract. For such a theory to be tenable, it needs to be demonstrated that 3OHKyn is bound to proteins in the human lens. Here, we show that all normal lenses older than 50 have 3OHKyn covalently attached to the nuclear proteins, most likely via cysteine residues. If indeed 3OHKyn is implicated in ARN cataract, a reduction in the levels that are bound in cataract, compared to normal lenses, would be expected. In agreement with this hypothesis, no bound 3OHKyn could be detected in proteins isolated from ARN cataract lenses.

The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: approximately 70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and approximately 30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -- with H2O2 as co-substrate -- indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.

Purpose: To determine the levels of free UV filters and selected amino acids in cataract lenses compared with normal lenses., Methods: Indian cataract lenses (n=39) and normal lenses (n=6) were examined by HPLC to quantify levels of UV filter compounds, the UV filter precursor amino acid tryptophan (Trp), as well as tyrosine (Tyr) and uric acid., Results: The levels of the two major primate UV filters, 3-hydroxykynurenine glucoside (3OHKG) and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG), in cataract lenses were markedly decreased compared with levels in normal lenses. By contrast, the levels of Trp were greatly increased. Mean Trp concentrations were an order of magnitude higher than in normal lenses, with 86% of dark-colored cataract lens nuclei having Trp concentrations greater than the mean level in the normal lenses. The concentrations of Tyr were also higher in cataract lenses. The levels of Kyn, however, were unchanged, and the uric acid levels were substantially lower., Conclusions: The levels of the free UV filter compounds 3OHKG and AHBG, and also of Trp, Tyr, and uric acid were different in cataract lenses compared to normal lenses. These data suggest that the metabolism of a large proportion of patients with cataract may be substantially different than in persons with normal lenses. Although the mechanism of such metabolic defects are unknown, the authors speculate that an amino acid transporter system may be upregulated in patients with cataract. Because kynurenine levels in cataract were not significantly different from those of normal lenses, there may be a defect in the lenticular UV filter pathway at one, or both, of the steps that convert kynurenine to 3OHKG.

Tryptophan-derived UV filters (kynurenine and 3-hydroxylkynurenine glucoside) have recently been shown to bind to human lens proteins. These UV filter adducts increase in amount with age and appear to be mainly responsible for the yellowing of the lens in man. On the basis of research performed in other tissues, it has been assumed that indoleamine 2,3-dioxygenase (IDO) may be the first and probably rate-limiting enzyme in UV filter biosynthesis. In this study, 25 human lenses were examined by a reliable and sensitive assay method with a monoclonal antibody specific for IDO. IDO activity was detected in all lenses ranging from 26 to 80 years, and there was no clear relationship of IDO activity with age. The mean activity was 0.85 + 0.49 nmol of kynurenine/h/lens. The level in the iris/ciliary body was negligible (<0.05 nmol of kynurenine/h). The lens IDO activity is consistent with UV filter turnover values obtained previously. These findings indicate that IDO is the first enzyme in the UV filter pathway and that UV filter biosynthesis is active even in aged lenses. Yellowing of the aged lens may therefore be preventable by drug-induced suppression of IDO activity.

1. Cataract is the single major cause of blindness worldwide; however, the reasons for the development of this condition remain unknown. It has been suggested that the essential amino acid tryptophan may be implicated in the aetiology but definitive evidence has been lacking. 2. The serum levels of tryptophan and seven of its metabolites have been measured in both cataract patients and control subjects, after administration of tryptophan, in order to determine the typical response profile and to discover whether differences could be found in tryptophan metabolism in the two groups. 3. Tryptophan, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, 5-hydroxyanthranilic acid, 5-hydroxytryptophan and anthranilic acid were measured by HPLC with dual electrochemical and programmable wavelength fluorescence detection. Fasting cataract patients (n = 42) and control subjects (n = 37) were given an oral dose of L-tryptophan and sera were sampled at 0, 1, 2, 4 and 6 h. 4. Statistically significant differences in the distribution of data between the two groups were observed. The responses of kynurenine and 5-hydroxyanthranilic acid were higher in cataract patients, but those of kynurenic acid and total tryptophan were lower than in control subjects. No statistically significant differences in free tryptophan, anthranilic acid, 3-hydroxyanthranilic acid, xanthurenic acid or 5-hydroxytryptophan levels were noted. 5. We conclude that there is a major subgroup of age-related cataract patients with a dysfunction in the metabolism of tryptophan. This may be related to the onset of cataract. The mechanism remains to be established but may operate via the action of tryptophan metabolites, such as 5-hydroxyanthranilic acid, which become reactive towards protein upon oxidation.

Although free tryptophan was found in the lens of both the kissing gourami (Helostoma temmincki) and the 3-spot gourami (Trichogaster trichopterus), only the lens of the latter species contained 3-hydroxykynurenine. Radiolabel experiments carried out on 3-spot gourami lenses in culture demonstrated that, as in humans, lenticular tryptophan is converted to 3-hydroxykynurenine probably via the intermediate kynurenine.

Primate lenses are unique in that they convert tryptophan (trp) into 3-hydroxykynurenine glucoside (30HKG). This is the major short-wave absorbing pigment present in human lenses and it may play a role in protecting the eye from UV-induced photodamage. A study has been performed on aspects of this metabolic pathway in human lenses. A significant rate of synthesis could be observed in a 24-hr period using intact lenses to which radiolabelled tryptophan had been added. Label was found in kynurenine (Kyn), 3-hydroxykynurenine (30HKyn) and 30HKG, although always to the greatest extent in the latter metabolite. Considerable variation in the proportion of label incorporated into 30HKG was observed. Older lenses tended to accumulate a greater percentage into the glucoside; the data indicating a generally greater flux through the trp catabolic pathway in lenses above 60 years of age. Pulse-chase experiments on lens pairs suggested that there may be a significant loss or metabolism of 30HKG. Biosynthesis of 30HKG was found to take place in the lens epithelial cells. A linear rate of 30HKG efflux from organ cultured lenses was observed indicating that one pathway for removal of this compound involves diffusion through the lens capsule. That this pathway also occurs in vivo was confirmed by analysing samples of human vitreous humour. Based on efflux rates from cultured lenses (1.07 x 10(-3) +/- 0.293 x 10(-3) mumol hr-1, n = 5), half-life values for 30HKG in the lens ranging between 7 and 40 hr were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

An isocratic method for the separation of most of the tryptophan metabolites in the oxidative degradation pathway is described. The chromatographic analysis utilizes the combined selectivity and sensitivity of amperometric and fluorimetric detection. The effect of pH, ionic strength and operating potential on retention times and detector responses are evaluated. The use of dual electrochemical cells at two operating potentials together with a programmable fluorescence detector allows for improved selectivity for the detection of metabolites. The sensitivity achieved with this method allows for the detection of the metabolites in biological fluids at the picomole level. The method has been used to monitor serum samples obtained during a tryptophan load test.