Photochemical properties of UV Filter molecules of the human eye.

PURPOSE: To compare the photochemical properties of UV filter molecules present in the human lens (kynurenine, KN; 3-hydroxykynurenine, 3OHKN; 3-hydroxykynurenine O-ß-D-glucoside, 3OHKG; 4-(2-aminophenyl)-4-oxobutanoic acid, AHA; and glutathionyl-kynurenine, GSH-KN) with the use of the following parameters: excited singlet lifetime τ(S), fluorescence quantum yield Φ(fl), triplet quantum yield Φ(T), and photodecomposition quantum yield Φ(dec). METHODS: The excited singlet lifetimes were measured with the use of fluorescence upconversion (time resolution, 210 fs) and pump-probe transient absorption (time resolution, 200 fs) methods. The fluorescence quantum yields were determined relative to an aqueous solution of quinine bisulfate. The triplet quantum yields were measured with the use of nanosecond laser flash photolysis. The photodecomposition quantum yields were determined by steady state photolysis followed by the high-performance liquid chromatography analysis. RESULTS: The secondary UV filters--AHA and GSH-KN are better photosensitizers than the primary ones--KN, 3OHKN and 3OHKG: the singlet state lifetimes of the secondary UV filters are longer, and the quantum yields of fluorescence and triplet state formation are higher. CONCLUSIONS: With aging, the ratio primary/secondary UV filters in the human lens decreases from approximately 10:1 to 2:1. The obtained results demonstrate that the quality of the secondary UV filters is inferior compared to the primary ones, which may result in a higher susceptibility of old lenses to UV light. That might be an important factor for the development of the age-related cataract.

Age-related changes in the water-soluble lens protein composition of Wistar and accelerated-senescence OXYS rats.

PURPOSE: To determine the age-related and the cataract-specific changes in the crystallin composition in lenses of accelerated-senescence OXYS (cataract model) and Wistar (control) rats. METHODS: The water soluble (WS) and insoluble (WIS) fractions of the lens proteins were separated; the identity and relative abundance of each crystallin in WS fraction were determined with the use of two-dimensional electrophoresis (2-DE) and Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) mass spectrometry. All statistical calculations were performed using the software package Statistica 6.0 by factor dispersion analysis (ANOVA/MANOVA) and Newman-Keuls post-hoc test for comparison of group mean values. RESULTS: The WIS protein content increased significantly in the aged animal lenses; the WIS/WS ratio increases in approximately 8 times to the age of 62 weeks. The interstrain difference was insignificant in this experiment. 2-DE maps of the young rat lenses (3 weeks) showed single spots for each lens protein while in older lenses (12 and 62 weeks) each crystallin was presented by several spots. The abundance of γA-γF-crystallins in WS fraction significantly decreases with age. A significant increase in the percentage abundance was also found for α-crystallins and ßB2-crystallin from 3 to 12 weeks. The major differences between Wistar and OXYS lenses are the faster decay of the content of γA-γF-crystallins in OXYS lenses, and the significant decrease of unmodified αA-crystallin abundance in old OXYS lenses. CONCLUSIONS: The presented results demonstrate that the increase of the water-insoluble (WIS) protein fraction is rather age-specific than cataract-specific phenomenon. The major age-related changes in WS protein composition are the fast insolubilization of γ-crystallins, and the increase of αB- and ßB2-crystallin abundance. The main interstrain differences, which could be attributed to the cataract-specific processes, are the faster decay of the content of γ-crystallins and the significant decrease of unmodified αA-crystallin abundance in the OXYS lenses.

Photophysics and photochemistry of the UV filter kynurenine covalently attached to amino acids and to a model protein.

The photophysics and photochemistry of kynurenine (KN) covalently bound to the amino acids lysine, cysteine, and histidine, the antioxidant glutathione, and the protein lysozyme have been studied by optical spectroscopy with femto- and nanosecond time resolution. The fluorescence quantum yield of the adducts of KN to amino acids is approximately 2 times higher than that of the free KN in solution; KN attached to protein exhibits a 7-fold increase in the fluorescence quantum yield. The S(1) state dynamics of KN-modified lysozyme reveals a multiphasic decay with a broad dispersion of time constants from 1 ps to 2 ns. An increase of the triplet yield of KN bound to lysozyme is also observed; the triplet state undergoes fast intramolecular decay. The obtained results reveal an increase of the photochemical activity of KN after its covalent attachment to amino acids and proteins, which may contribute to the development of oxidative stress in the human lenses-the main causative factor for the cataract onset.

Tryptophan and kynurenine levels in lenses of Wistar and accelerated-senescence OXYS rats.

PURPOSE: To determine the levels of kynurenine (KN) and its metabolic precursor tryptophan (Trp) in lenses of accelerated-senescence OXYS (cataract model) and Wistar (control) rats at ages from 1 day to 24 months. METHODS: Protein-free lens extracts were prepared from Wistar and senescent-accelerated OXYS rat lenses. The presence and levels of KN and Trp were determined using high performance liquid chromatography (HPLC) analysis and mass spectrometric measurements. All statistical calculations were made using the software package Statistica 6.0, using factor dispersion analysis and Newman-Keuls post-hoc test for comparison of group mean values. RESULTS: The levels of KN, which plays the role of a molecular Ultraviolet (UV) filter in the human lens, and its metabolic precursor Trp in the rat lens significantly depend on the rat strain and age. During the first 20 days after birth, before the first signs of cataract in OXYS rats, there is a strong difference in the content of both Trp and KN between Wistar and OXYS lenses. This difference becomes insignificant in lenses of 1 month and older. The levels of Trp and KN in young lenses are higher than that in lenses of 1 month and older for both strains. CONCLUSIONS: The presented results demonstrate that the KN pathway of Trp catabolism does not play a significant role in cataract development in the rat lens at the stages of cataract manifestation; however, in the first 3 weeks of postnatal development, the interstrain difference in KN and Trp levels is very strong. The obtained results show a correlation between the low level of KN and the high level of Trp at the stage of lens maturation and future cataractogenesis, and suggest an imbalance in the KN pathway of Trp catabolism in potentially cataractous lenses.

Kinetics and mechanism of thermal decomposition of kynurenines and biomolecular conjugates: ramifications for the modification of mammalian eye lens proteins.

Thermal degradation reactions of kynurenine (KN), 3-hydroxykynurenine (3OHKN), and several adducts of KN, to amino acids and reduced glutathione (GSH) have been studied at physiological temperature. These compounds are all implicated in age-related mammalian eye lens cataract formation at the molecular level. The main reaction pathway for both KN and 3OHKN is deamination via beta-elimination to carboxyketoalkenes CKA and 3OHCKA. These reactions show a weak pH dependence below pH values of approximately 8, and a strong pH dependence above this value. The 3OHKN structure deaminates at a faster rate than KN. A mechanism for the deamination reaction is proposed, involving an aryl carbonyl enol/enolate ion, that is strongly supported by the structural, kinetic, and pH data. The degradation of Lys, His, Cys and GSH adducts of the CKA moieties was also studied. The Lys adduct was found to be relatively stable over 200 h at 37 degrees C, while significant degradation was observed for the other adducts. The results are discussed in terms of known post-translational modification reactions of the lens proteins and compared to incubation studies involving KN and related compounds in the presence of proteins.

UV filter decomposition. A study of reactions of 4-(2-aminophenyl)-4-oxocrotonic acid with amino acids and antioxidants present in the human lens.

Deamination of UV filters, such as kynurenine (KN), in the human lens results in protein modification. Thermal reactions of the product of kynurenine deamination, 4-(2-aminophenyl)-4-oxocrotonic acid (CKA), with amino acids (histidine, lysine, methionine, tryptophan, tyrosine, cysteine) and antioxidants (ascorbate, NADH, glutathione reduced) were studied. The rate constants of the reactions under physiological conditions were measured. The rate constants of CKA addition to cysteine k(Cys)=36+/-4M(-1)s(-1) and to glutathione k(GSH)=2.1+/-0.2M(-1)s(-1) are 4-5 orders of magnitude higher than the rate constants of CKA reactions with the other amino acids and antioxidants. The Arrhenius parameters for k(Cys) and k(GSH) were determined: A(GSH)=(1.8+/-0.7)x10(5)M(-1)s(-1), E(GSH)=29.2+/-5.6kJmol(-1), A(Cys)=(2.7+/-0.9)x10(8)M(-1)s(-1), E(Cys)=40.4+/-5.7kJmol(-1). The large difference in frequency factors for k(Cys) and k(GSH) is attributed to steric hindrance, peculiar to the bulky GSH molecule.