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(Investigative Ophthalmology and Visual Science. 2007;48:1718-1723.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1134
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Protein-Bound UV Filters in Normal Human Lenses: The Concentration of Bound UV Filters Equals That of Free UV Filters in the Center of Older Lenses

Anastasia Korlimbinis,1,2 J. Andrew Aquilina,3 and Roger J. W. Truscott1,2

1From the Save Sight Institute, University of Sydney, NSW, Sydney, Australia; and the 2Australian Cataract Research Foundation, and the 3School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters.

METHODS. Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins.

RESULTS. The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total protein-bound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts.

CONCLUSIONS. The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people.

The primate lens is unique among both other primate tissues and the lenses of other species, in that it synthesizes 3-hydroxykynurenine glucoside (3OHKG) as a UV filter from the amino acid tryptophan (Trp).1 The immediate precursors kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) are metabolites found in all organs of the body.

All three of these Trp metabolites are unstable at physiological pH and undergo side chain deamination to yield {alpha},ß-unsaturated ketones that are prone to nucleophilic attack.2 In tissues that contain sufficient glutathione (GSH), it is likely that GSH will react with the deamination products before they bind to proteins3 and that the adducts thus formed will diffuse out of the lens. In the interior of the lens, once the barrier to diffusion forms at middle age,4 5 6 the nucleus becomes a partially uncoupled region in which metabolites spend a longer time than in the young lens. This factor leads to an environment in the older lens nucleus that favors greater decomposition of intrinsically unstable molecules. Coupled with a diminished flux of GSH from the cortex, this results in increased covalent binding of UV filters to nuclear proteins after middle age.7 8 9

3OHKG, Kyn, and 3OHKyn deaminate at slightly different rates,2 and little is known about the stabilities of the UV filter adducts once they are formed. Also, it is not known whether all three UV filters are protein bound in an older lens, whether the extent of binding varies significantly with age, and whether the amounts reflect the levels of the free UV filters.

In this study, we set out to examine the levels of all three bound UV filters in normal lenses using a novel assay system in which lens proteins were incubated with excess GSH at pH 9.5.9 Under these conditions, the UV filters that are attached to proteins are released, and the GSH adducts thus formed can be quantified by HPLC.

UV filters were found to be covalently bound to proteins from all lenses older than 50 years. Such posttranslational modifications may have important consequences in terms of the susceptibility of the proteins to photooxidation and, in the case of 3OHKyn, the sensitivity to an oxidative environment such as that in the nuclei of lenses with age-related nuclear cataract.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Purified water (purified to 18.2 M{Omega}/cm2; Milli-Q; Millipore, Bedford, MA) was used in the preparation of all solutions. All organic solvents were HPLC grade (Ajax, Auburn, NSW, Australia). 3OHKyn, reduced GSH, trifluoroacetic acid (TFA), and guanidine HCl were obtained from Sigma-Aldrich (St. Louis, MO).

Reversed-Phase HPLC
Reversed-phase (RP)-HPLC was performed on a Shimadzu system (Kyoto, Japan). For analytical scale separations, a column (Jupiter, 5 µm, C18, 300 Å, 250 x 4.6 mm; Phenomenex, Torrance, CA) was used with the following mobile phase conditions: solvent A (aqueous 0.1% vol/vol TFA) for 5 minutes followed by a linear gradient of 0% to 50% solvent B (80% vol/vol acetonitrile/H2O, 0.1% vol/vol TFA) over 20 minutes, followed by a linear gradient of 50% to 100% B over 15 minutes and re-equilibration in the aqueous phase for 15 minutes. The flow rate was 0.5 mL/min and with detection at 360 nm.

Human Lenses
Normal human aged lenses were obtained from the NSW Lions Eye Bank (Sydney, Australia). Human tissue was handled in accordance with the tenets of the Declaration of Helsinki, with ethics clearance from both Wollongong and Sydney Universities. All lenses were stored at –80°C. Each lens was separated into the nucleus and cortex using a cork borer (5 mm), and the ends (~1 mm) of the nuclear core were removed. Twenty-two normal lenses ranging in age from 17 to 83 years were used.

Extraction of Free UV Filters from Lenses
Each nucleus and cortex was extracted with 100% ethanol (300 µL) and centrifuged (10,000 g, 10°C, 20 minutes). The insoluble protein was further extracted with 80% ethanol (300 µL) and then centrifuged (10,000 g, 10°C, 20 minutes). The supernatant was collected and combined with that from the first extraction. The ethanol extracts for each of the nuclei and cortices were dried and resuspended in 0.1% (vol/vol) TFA and analyzed by HPLC for levels of free UV filters.

Quantification of Protein-Bound UV Filters
The insoluble protein from each extracted cortex and nucleus was then dissolved in 6 M guanidine HCl (pH 5.5; 2 mL) and dialyzed overnight against 0.1 M sodium acetate-acetic acid buffer (pH 4), with several changes to ensure the removal of all noncovalently bound material. The protein was freeze dried and then weighed. The protein and reduced GSH (100 mg) were dissolved in 6 M guanidine HCl (0.4 mL) and 50 mM Na2CO3/NaHCO3 buffer (pH 9.5; 1.1 mL). The pH was readjusted to 9.5 with 6 M NaOH, and the resultant solution was sealed and incubated for 4 hours at 37°C. After the pH was adjusted to less than 5 with acetic acid the solution was centrifuged (6000g, 4°C, 60 minutes) in a concentrator (10,000 MW cutoff; Vivaspin; Sartorius, Edgewood, NY) to separate the noncovalently bound material (filtrate) from the protein. The filtrate was examined by HPLC. Standard curves were used for quantification of UV filters. Values are expressed per milligram of protein on the basis of the weight of dried lens tissue after dialysis.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
The primary purpose of this study was to quantify the protein-bound UV filters in individual human normal lenses. In particular, we wished to compare the levels attached to the nuclear proteins with those from the lens cortex and to see whether there was any relationship with the levels of the free UV filters. Model studies have shown that UV filters covalently attach to Cys, His, and Lys residues of proteins.10 11 12 Acid hydrolysis has been used to quantify levels of Kyn bound to His and Lys in normal and cataractous lens proteins8 13 ; however, acid hydrolysis cannot be used for determining levels of protein-bound 3OHKG, because it will cleave the glucose moiety.

In this study, we used a novel method for determining the levels of protein-bound UV filters in both the lens cortex and nucleus.9 The method involves incubating the proteins with excess GSH at pH 9.5 for 4 hours. At this pH, protein-bound UV filters are hydrolyzed and the {alpha},ß-unsaturated ketones corresponding to each UV filter are released from the protein. In the presence of excess GSH, each UV filter product that is released, forms its corresponding glutathionyl UV filter adduct (Fig. 1) which can then be separated by HPLC. All three adducts are diastereoisomers and elute as doublet peaks.


Figure 1
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  		FIGURE 1. Basis of the assay for protein-bound UV filters, showing decomposition of UV filter amino acid adducts, and formation of GSH adducts. Free UV filters were removed from the lens protein, and the protein was incubated with excess GSH at pH 9.5 for 4 hours.

 
In a sample of lens nuclear protein treated in this way, the GSH adducts were well separated (Fig. 2A) . Typically, 3OHKG eluted at 29 minutes, 3OHKyn at 30 minutes, and Kyn at 33 minutes. Large peaks corresponding to Kyn and 3OHKG were observed in the protein samples from both lens cortex and nucleus (Fig. 2) . A clear doublet due to the GSH adduct of 3OHKyn was detected for the nuclear proteins; however, this peak was absent in the cortical protein sample (Fig. 2B) .


Figure 2
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  		FIGURE 2. HPLC chromatograms of GSH-conjugated UV filters released from human lens proteins. (A) Normal 79-year-old nucleus; (B) normal 79-year-old cortex.

 
To ensure that free UV filters in the lens did not interfere with the assay system used, a 73-year-old lens was halved and to one-half, Kyn was added at approximately five times the level expected in the lens. The filters were then extracted from the lenses as normal. The HPLC chromatograms of the ethanol extracts of the lens halves showed that all the excess Kyn was present in the chromatogram of the spiked sample. The HPLC chromatograms of the proteins did not show any changes in the level of the Kyn-GSH adduct.

Twenty-two normal human lenses ranging in age from 17 to 83 years were examined. The reproducibility of the HPLC assay over five separate incubations using calf lens protein modified by 3OHKyn was 57.5 ± 2.4 picomoles/mg protein.

3-Hydroxykynurenine Glucoside
Nucleus.
The levels of protein-bound 3OHKG in the center of normal lenses are shown in Figure 3Ai . The graph shows that, with age, the levels of protein-bound 3OHKG in the nucleus increase significantly after age 40 years, reaching a maximum value of 2415 picomoles/mg of protein in a 68-year-old lens. To facilitate a general comparison of the levels of 3OHKG and the other two UV filters in the different lenses and between lens regions, we decided to use an average value after age 50 in each case. The average amount of protein-bound 3OHKG in the normal lens nucleus over the age of 50 was 1307 picomoles/mg protein (Table 1) .


Figure 3
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  		FIGURE 3. Protein-bound UV filters in normal lenses. The concentration of UV filters covalently bound to the proteins of normal human lenses as a function of age. (A) Nucleus: (Ai) 3OHKG, (Aii) Kyn (Aiii) 3OHKyn. (B) Cortex: (Bi) 3OHKG and (Bii) Kyn.

 

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  		TABLE 1. Average Concentration of Protein-Bound and Free Levels of UV Filters in the Nucleus and Cortex of Normal Lenses More Than 50 Years of Age

 
Cortex.
The average amount of bound 3OHKG in the cortices of normal lenses (Fig. 3Bi) more than 50 years of age was 56 picomoles/mg protein (Table 1) . Again, there was a clear difference in the levels before and after 40 years; however, in the cortex, the concentration of bound 3OHKG appeared to increase steadily after this age unlike the nuclear values in the same lenses.

Kynurenine
Nucleus.
The levels of protein-bound Kyn in the normal lens nucleus are shown in Figure 3Aii . The average amount of bound Kyn in the nucleus of normal lenses older than 50 years was 37 picomoles/mg protein (Table 1) . The appearance of the points on the graph are similar to those for 3OHKG measured in the same lenses; however, the values for Kyn are more than an order of magnitude lower.

Cortex.
The average amount of bound Kyn in the cortices of normal lenses older than 50 years was 2 picomoles/mg protein (Table 1 ; Fig. 3Bii ). Again, as found for 3OHKG, this is more than an order of magnitude lower than the nuclear Kyn values from the same lenses.

3-Hydroxykynurenine
Nucleus.
3OHKyn was not detected in the nuclei of normal lenses younger than 50 years (Fig. 3Aiii) . The average amount of 3OHKyn bound to the nucleus of normal lenses older than 50 years, was 9 picomoles/mg protein (Table 1) and past middle age, there appeared to be no clear relationship with age.

Cortex.
The cortical proteins of the same lenses were analyzed, however 3OHKyn could not be detected in any of the samples.

Free UV Filters
The mean levels of free UV filters determined in this study are summarized in Table 1 . The concentrations observed in normal lenses in this study, are comparable to the levels reported previously by Bova et al.,14 and Streete et al.15

To determine whether there was any relationship between the concentration of free UV filters and the bound levels, these levels were plotted (Fig. 4) . The results for 3OHKG and Kyn are comparable, in that there was no clear relationship between the levels of free and corresponding protein-bound concentrations. This finding suggests that the environment of the lens—for example, the extent of the barrier, and the availability of reducing agents such as GSH, cysteine, and NADH, that can intercept the reactive intermediates—may be of greater importance than simply the amount of free UV filter available for deamination.


Figure 4
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  		FIGURE 4. Relationship of free and bound 3OHKG. (A) nucleus, (B) cortex.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
A dedicated metabolic pathway for the synthesis of UV filters from Trp is present in primate lenses.1 16 The end product is 3OHKG, a metabolite detected only in the lens, although the immediate precursors Kyn and 3OHKyn are found throughout the body. Kyn, 3OHKyn, and 3OHKG are unstable at pH 7 and lose ammonia from the amino acid side chains.2 Spontaneous deamination of 3OHKG leads to the formation of other major lenticular UV filters, such as AHBG,17 3OHKG-GSH,3 and 3OHKG-Cys,18 that are formed in the human lens from the {alpha},ß-unsaturated ketone by reduction and thiol addition, respectively (Fig. 1) . Because the unsaturated ketones that result from UV filter deamination are reactive, it is perhaps not surprising that, in the very high protein concentration in the lens, nucleophilic groups of proteins may also couple with these intermediates. This binding appears to be exacerbated when the lens barrier forms at middle age, because the increased time that these small molecules spend in the nucleus allows a greater percentage of the unstable molecules to decompose.6 The barrier also restricts the flow of GSH from the cortex.4 These two factors combine to cause an increase in modification of nuclear proteins after age 50. It is not simply a matter of the time of reaction of the crystallins with UV filters since, in an adult, before barrier formation, proteins in the lens center may have been in contact with UV filters for more than 40 years, with little or no covalent modification.

Since crystallins in the nucleus are present in the lens before birth and are not replaced, several posttranslational modifications have been reported in proteins extracted from adult lenses (Table 2) . UV filter modification is one such modification, and it has been suggested that the brown and black coloration that is found only in human age-related nuclear cataract lenses could be the result of oxidation of these primate-specific, protein-bound UV filters.7 9 25 26 27 If this were true, one would predict lower levels of bound UV filters in cataract lenses compared with normal lenses. These levels will be examined in a separate investigation.


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  		TABLE 2. Novel Post Translational Modifications in Human Lens Proteins

 
In this study, we examined protein-bound UV filters in normal lenses. For each of the UV filters, the overall pattern was qualitatively similar, in that very little or none of the three UV filters could be detected bound to protein before middle age. As noted earlier, this finding is consistent with the formation of the lens barrier. Before age 40 to 50, free UV filters are present at higher concentrations than in older lenses14 ; however, based on data for other small molecules such as H2O,5 and Cys,4 they spend on average shorter times in the lens nucleus, based on signal decay for H2O when placed in a D2O based medium,5 giving them less time to deaminate. In addition, nuclear GSH levels are higher in younger lenses,14 so that {alpha},ß-unsaturated ketones that do form, will likely be intercepted before they can bind to proteins. Consistent with this view, young lenses contained either low, or undetectable, levels of the three protein-bound UV filters (Fig. 3) . By contrast, every lens aged more than 50 years was found to have all three of the UV filters covalently attached to the nuclear proteins as Cys or Lys adducts.

Despite individual variation, after age 50, the concentrations of each of the UV filters were relatively constant in the nucleus (Fig. 3) . The mean values for normal lenses (>age 50) were: 3OHKG, 1307 picomoles/mg protein; Kyn 37 picomoles/mg protein, and 3OHKyn 9 picomoles/mg protein, corresponding to a ratio of protein-bound 3OHKG-Kyn-3OHKyn of 145:4:1. These ratios parallel the ratios of free UV filters (178:5:1): the mean values of free UV filters were 3OHKG, 534 picomoles/mg tissue; Kyn 16, picomoles/mg tissue; and 3OHKyn, 3 picomoles/mg tissue. The data suggest that the UV filters are approximately equally able to bind to proteins in the lens, and the adducts formed may have similar stabilities. To compare the total free with the total bound levels in whole individual normal lens nuclei, we calculated the overall amount of protein-bound UV filters, based on the protein content. In lens nuclei older than 50 years, there was 9,225 ± 2,100 picomoles (n = 5) of bound and 11,177 ± 4,280 picomoles (n = 5) of free 3OHKG, 304 ± 173 picomoles (n = 5) of bound and 311 ± 83 picomoles (n = 5) of free Kyn, and 59 ± 28 picomoles (n = 5) of bound and 50 ± 32 picomoles (n = 5) of free 3OHKyn. Thus, in the center of older normal human lenses, UV light would be absorbed equally by protein-bound and free UV filters. This is in marked contrast to the young lens.

The amounts attached to the proteins from the lens cortex were always markedly lower than those in the nucleus. The mean cortical values for lenses aged more than 50 years were 3OHKG, 56 picomoles/mg protein, and Kyn, 2 picomoles/mg protein, corresponding to levels that are 23- and 19-fold less, respectively, than those in the nucleus. 3OHKyn attached to the cortical lens proteins was below the limits of detection.

What are the chief conclusions to be drawn from these findings? First, every normal lens examined >50 years was found to have all three UV filters bound to the nuclear proteins. Second, from a comparison of bound and free UV filters in the center of individual older lenses, it is clear that significant absorption of UVA light is mediated via modified proteins. This effect will likely be exacerbated by other posttranslational modifications that are age-related (Table 2) . Since binding of Kyn to calf lens proteins causes them to become susceptible to photooxidation by the wavelengths of light that penetrate the cornea, and this is accompanied by the generation of reactive oxygen species,28 these UV filter modifications may make the nuclear lens proteins prone to photooxidation after middle age. 3OHKyn is also extremely reactive under oxidative conditions and the presence of such a UV filter bound to the nuclear proteins of older normal lenses may predispose them to oxidation and therefore the sorts of changes, such as cross-linking, insolubilization and oxidation that are characteristic of age-related nuclear cataract.

It should be noted that the assay system that we used allows quantification of UV filter adducts of Lys and Cys, but not of His, which are significantly more stable.9 According to the same assay, when the Lys, Cys, and His adducts of 3OHKyn were incubated with GSH, the yield of the GSH conjugates of Lys, Cys, and His were 73%, 61%, and 0.5%, respectively.9 Therefore, the amounts of bound UV filters quoted herein are likely to be an underestimate. Model studies indicate however that Cys adducts are the most prevalent when proteins are incubated with UV filters. Even allowing for this underestimation, the total levels of UV filters found in this study (~1350 picomoles/mg protein) are comparable with those of other major posttranslational modifications in the human lens (Table 2) .

In summary, proteins isolated from all normal lenses older than 50 years contained measurable levels of each of the three kynurenine UV filters. 3OHKG was always present in the largest amounts, with lower levels of Kyn and 3OHKyn, which reflects the concentrations of the free UV filter compounds in the lens. Despite this finding, there was no clear relationship in individual lenses between free and bound UV filter concentrations, which implies that the free UV filter concentration, although giving rise to a population of reactive intermediates, does not directly correspond to the level of protein-bound UV filters. This result is consistent with the fact that mechanisms such as reduction of reactive intermediates by NADH and GSH, as well as the permeability of the barrier to small molecule diffusion, are key variables in the transport and reactivity of UV filters. The attachment of UV filters to older lenses may predispose them to oxidative damage.


   Footnotes
 
Supported by Grants R01 EY013570-03 from the National Eye Institute and 307615 from the National Health and Medical Research Council (NH&MRC), Australia. RJWT is a Senior Research Fellow and JAA is an R. D. Wright Fellow of the NH&MRC.

Submitted for publication September 21, 2006; revised October 23, 2006; accepted January 10, 2007.

Disclosure: A. Korlimbinis, None; J.A. Aquilina, None; R.J.W. Truscott, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Roger J. W. Truscott, Save Sight Institute, University of Sydney, NSW 2001, Australia; rjwt{at}eye.usyd.edu.au.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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