UV Filter Instability: Consequences for the Human Lens LISA M. TAYLOR a , J. ANDREW AQUILINA a , JOANNE F. JAMIE b AND ROGER J. W. TRUSCOTT a * a Australian Cataract Research Foundation, University of Wollongong, Wollongong, N.S.W. 2522, Australia and b Department of Chemistry, Division of Environmental and Life Sciences, Macquarie University, N.S.W. 2109, Australia (Received 22 January 2002 and accepted in revised form 02 April 2002) Human lenses appear to become coloured with age primarily due to the covalent binding of UV ®lter compounds to lens proteins. These crystallin modi®cations result from the inherent instability of the kynurenine UV ®lters. Here we investigate this decomposition, the role this may have in the formation of other primate UV ®lters, and the interaction of the intermediates (a,b-ketoalkenes) with lens components. The UV ®lters kynurenine, 3-hydroxykynurenine and 3-hydroxykynurenine glucoside were incubated at neutral pH in the presence or absence of NADH or NADPH. The three UV ®lters underwent spontaneous deamination, such that at pH 7 less than half of the starting materials (kynurenine (42%), 3-hydroxykynurenine glucoside (30%) and 3-hydroxykynurenine (21%)) remained after 7 days. In the presence of NAD(P)H, the double bond of the UV ®lter-derived deamination compounds, were reduced. Deamination of 3-hydroxykynurenine glucoside, followed by reduction with NAD(P)H, could thus account for the formation of the major lens UV ®lter 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside. b-Benzoylacrylic acid, which possesses the same a,b-ketoalkene sidechain as the deaminated kynurenine UV ®lters, underwent Michael addition with glutathione, was reduced (hydrogenated) by NAD(P)H, however was unreactive with ascorbate. Surprisingly, at pH 7 the UV ®lter-derived a,b-ketoalkene intermediates, do 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 glutathione. On the basis of these data it is likely that glutathione and NAD(P)H, but not ascorbate, protect proteins in the lens from modi®cation by UV ®lters. # 2002 Elsevier Science Ltd. Key words: protein modi®cation; aging; cataract; glutathione; NAD(P)H; ascorbate. 1. Introduction The human lens absorbs most of the UV radiation transmitted by the cornea between 300 and 400 nm. This absorption is largely performed by a group of low molecular weight, ¯uorescent compounds (van Hey- ningen, 1973; Wood, 1993; Truscott et al., 1994; Wood and Truscott, 1994). These UV ®lter compounds are derived biosynthetically from tryptophan (Wood, 1993; Truscott et al., 1994; Wood et al., 1994; van Heyningen, 1973) and are thought to protect the lens and retina from UV-induced photodamage, aid visual acuity by eliminating chromatic aberration, and enhance the contrast sensitivity of the lens (Wood, 1993; Truscott et al., 1994; Wood et al., 1994; Zigman, 1988). The major UV ®lter compound found in the human lens is L-3-hydroxykynurenine O-b-D-glucoside (3OHKG), followed by 4-(2-amino-3-hydroxyphenyl)- 4-oxobutanoic acid O-b-D-glucoside (AHBG), L-kynur- enine (Kyn) and L-3-hydroxykynurenine (3OHKyn) (van Heyningen, 1971, 1973; Wood, 1993; Truscott et al., 1994; Wood et al., 1994). Model studies performed in our laboratory have provided evidence that AHBG is formed from 3OHKG. This is thought to occur via spontaneous deamination from the amino acid sidechain of 3OHKG to yield an a,b-ketoalkene intermediate, which then undergoes reduction of the double bond (Bova et al., 1999). Since Kyn and 3OHKyn also contain this amino acid sidechain, similar deamination and reduction reactions to form novel UV ®lters, analogous to AHBG, may account for some of the unidenti®ed UV ®lter compounds in the human lens. The aim of this study was to investigate the process of UV ®lter decomposition. Interaction of the UV ®lter intermediates (a,b-ketoalkenes) with lens components, such as NAD(P)H was examined in order to under- stand if these compounds in¯uenced the reaction pathway and if they may protect the crystallin proteins from covalent modi®cation by UV ®lters. The relative stability and reactivity of the three UV ®lters was compared by examining the decomposition products formed at pH 7 and pH 9, where deamination of the UV ®lter compounds is accelerated. 2. Materials and Methods Materials L-Kynurenine, L-3-hydroxykynurenine, tri¯uoroace- tic acid (TFA), b-nicotinamide adenine dinucleotide Exp. Eye Res. (2002) 75, 165±175 doi:10.1006/exer.2002.2012, available online at http://www.idealibrary.com on 0014-4835/02/$35.00/0 # 2002 Elsevier Science Ltd. * Address correspondence to: Roger J. W. Truscott, Australian Cataract Research Foundation, University of Wollongong, Wollon- gong, N.S.W. 2522, Australia. E-mail: roger_truscott@uow.edu.au