Degree Name

Doctor of Philosophy


Department of Chemistry


Age-dependent human lens colouration and fluorescence may be explained by the covalent binding of U V filter compounds to lens crystallins. These crystallin modifications result from the inherent instability of the kynurenine (Kyn) UV filters at physiological pH. Thus, the process of UV filter decomposition and the interaction of the intermediates (α,β-ketoalkenes) with lens components clearly warrants further investigation.

Quantification of the U V filter compounds 3-hydroxykynurenine (30HKyn), Kyn and 3-hydroxykynurenine glucoside (30HKG) present in human lenses displayed a linear decrease with age, with slightly lower levels in the nucleus than the cortex. 4-(2- amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG) was found in higher levels in the nucleus than the cortex and decreased slowly in both regions with age. The glutathionyl-3-hydroxykynurenine glucoside (GSH-30HKG) adduct was present in higher concentrations in the nucleus, barely detectable in young lenses, but increased significantly after age 50. Reduced glutathione (GSH) levels were lower in the nucleus and decreased in both regions with age, while oxidized glutathione (GSSG) increased in the nucleus but remained constant in the cortex. These results are consistent with a predominantly nuclear origin for both AHBG and the GSH-3OHKG adduct. This is in accord with their proposed mechanism of formation, which involves an initial deamination of 30HKG . Reaction of the UV filter deamination products with lens crystallins therefore appears to be more likely in the nucleus.

Using the model compound, β-benzoylacrylic acid, it was demonstrated at neutral pH that NADH reduces (hydrogenates) the α,β-ketoalkene double bond, whilst GSH undergoes Michael addition to form an adduct and ascorbate is unreactive. The three UV filter compounds underwent spontaneous deamination, such that at pH7 less than half of the starting materials [Kyn (42%), 3OHKG (30%) and 3OHKyn (21%)] remained after 7 days. In the presence of NAD(P)H, the double bond of the UV filterderived deamination compounds, were reduced. Deamination of 30HKG , followed by reduction with NAD(P)H, could thus account for the formation of AHBG . The UV filter-derived α,β-ketoalkene intermediates, did not readily undergo intramolecular cyclization. This feature makes the double bond more available for reaction with protein nucleophilic residues and other lens components such as GSH . On the basis of these data it was proposed that GSH and NAD(P)H, but not ascorbate, protect proteins in the lens from modification by UV filter compounds.

To confirm this hypothesis, bovine crystallins were incubated with Kyn at pH7 in the presence and absence of GSH , ascorbate or NADH . Ascorbate, even at high (15 mM) levels, was not found to significantly retard the time-dependent covalent binding of Kyn to the proteins. The increase in protein UV absorbanoe and fluorescence was inhibited by GSH intercepting the reactive α,β-ketoalkene intermediate, to form a GS H-Kyn adduct. NADH seemed to protect by both reduction of the reactive α,β-ketoalkene intermediate and by competing with Kyn for presumably hydrophobic sites on the crystallins. This may indicate that the covalent attachment of aromatic Kyn molecules could be facilitated by initial hydrophobic interactions. Since GSH is present at far greater concentrations than N A D H , these results show that in primate lenses, GSH is most likely the key agent responsible for protecting the crystallins from UV filtermediated covalent modification.

Both the reduced 30HKG deamination product (AHBG) and the GSH-3OHKG adduct had been previously identified in human lenses. In this study, further investigations of both normal and cataractous lenses were performed in an attempt to identify additional UV filter decomposition products (eg. Kyn yellow, 3OHKyn yellow, 3OHKG yellow, GSH-Kyn, GSH-3OHKyn and the reduced Kyn and 30HKyn deamination products). These decomposition products were all detected in vitro, however, none of these compounds were detected in normal human lenses. This suggests that the conditions in the normal lens are such that other reactions are favoured. The reduced 30HKyn deamination product (AHB) was detected in a single type IV cataractous lens. Further analysis of cataractous lenses is necessary to confirm this result.

Finally, a new UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-diglucoside (AHBDG) , was identified in human lenses. The structure suggests that it is a further metabolic product of AHBG . Quantification studies on the new compound show that it decreases towards zero in both the nucleus and cortex as a function of age. The discovery of this novel disaccharide completes the identification of the major UV filter compounds present in the human lens.



Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.