July 2003
Volume 44, Issue 7
Free
Lens  |   July 2003
The Effect of UVA Light on the Anaerobic Oxidation of Ascorbic Acid and the Glycation of Lens Proteins
Author Affiliations
  • Beryl J. Ortwerth
    From the Mason Eye Institute and the
  • Vitaliy Chemoganskiy
    From the Mason Eye Institute and the
  • Valeri V. Mossine
    Department of Biochemistry, University of Missouri, Columbia, Missouri.
  • Paul R. Olesen
    From the Mason Eye Institute and the
Investigative Ophthalmology & Visual Science July 2003, Vol.44, 3094-3102. doi:10.1167/iovs.02-0857
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Beryl J. Ortwerth, Vitaliy Chemoganskiy, Valeri V. Mossine, Paul R. Olesen; The Effect of UVA Light on the Anaerobic Oxidation of Ascorbic Acid and the Glycation of Lens Proteins. Invest. Ophthalmol. Vis. Sci. 2003;44(7):3094-3102. doi: 10.1167/iovs.02-0857.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. To determine whether UVA-excited human lens chromophores can cause the oxidation of ascorbic acid in the absence of oxygen, and whether these oxidation products are capable of glycating lens proteins.

methods. The oxidation of ascorbic acid, mediated by UVA irradiation in the presence of aged human lens proteins, was measured in the absence of oxygen by the decrease in absorbance at 265 nm in vitro. An action spectrum from 320 to 400 nm was determined for both ascorbate oxidation and the photobleaching of the lens yellow pigments at λ = 350 nm. The UVA-mediated oxidation products of [U-14C]ascorbate were quantified by HPLC. Glycation was assayed by the UVA-dependent incorporation of [U-14C]ascorbate into lens proteins with a water-insoluble (WI) fraction in vitro, with incubated whole human lenses, and with a WI fraction after a 5- to 7-day exposure to ambient sunlight. An enzymatic digest of [U-14C]ascorbate-labeled proteins was fractionated over HPLC columns and compared with the 330-nm absorbance profile of a proteolytic digest of aged human lens proteins.

results. Aged human lens WI proteins absorbed UVA light (86 J/h per square centimeter) and oxidized 33 to 45 nanomoles of ascorbate over 1 hour in the absence of oxygen. No ascorbate oxidation was detected, however, in the dark control. An action spectrum showed that ascorbate oxidation occurred throughout the UVA region, with λmax at 350 nm, which was similar to the action spectrum obtained for the photobleaching of the lens chromophores. Anaerobic UVA irradiation of aged human lens proteins for 2 hours with [U-14C]ascorbate resulted in a 40% loss of ascorbate with the accumulation of dehydroascorbic acid, diketogulonic acid, and oxalate. After subsequent incubation for 24 hours, the ascorbate oxidation products disappeared, with a corresponding incorporation of radioactivity into lens proteins. Chromatography of enzymatic digests of the labeled proteins produced peaks that coeluted with several of the 330-nm absorbing peaks in an aged human lens protein digest. Irradiation of whole human lenses for 2 hours caused a 33% loss of total lens ascorbate. UVA irradiation of aged human lenses for 2 hours resulted in the incorporation of ascorbate into lens proteins during the ensuing 24 hours in the dark. Exposure of aged human lens WI proteins to reflected ambient sunlight (1.1 J/h per square centimeter) for 5 to 7 days in the absence of oxygen also produced an increased incorporation of [14C]ascorbate into protein when compared with dark control samples.

conclusions. These data argue that UVA light can cause an oxidation of ascorbic acid in the absence of oxygen, due to the activation of the sensitizers present in aged human lens WI proteins. The oxidation products formed were the same as those seen in the presence of oxygen, and were rapidly incorporated into protein, apparently by Maillard-type chemistry. These data argue that ascorbate glycation can occur under the low oxygen levels thought to exist in the human lens nucleus in vivo.

Human lens proteins accumulate yellow modifications during aging that may play a causative role in the formation of age-related cataract. Considerable evidence has accumulated suggesting that these yellow compounds are advanced glycation end products (AGEs) 1 formed when lens proteins react with the oxidation products of ascorbic acid. 2 3 4 Ascorbate may be continually oxidized in vivo, because it functions as the major water-soluble (WS) antioxidant in lens tissue, protecting against oxidative damage. 5 It is known that UVA irradiation of water insoluble (WI) proteins in aged human lens produces several reactive oxygen species, but singlet oxygen is by far the predominant species. 6 Singlet oxygen–mediated damage to Trp and His residues in lens proteins, however, has been reported only in the most advanced forms of brunescent cataract. 7 Also, extensive irradiation of aged human lenses showed no loss of either Trp or His residues in contrast to the irradiation of total lens homogenates in air. 8 These data suggest that there may be insufficient oxygen levels in the normal human lens nucleus to permit significant amounts of singlet oxygen to be formed. Low levels of oxygen in lens have been reported by several laboratories 9 10 (Liang J, Barazabetto I, Zheng L, Dillon J, ARVO Abstract 2380, 2002; McNulty R, Truscott RJW, Bassnett S, ARVO Abstracts 2374, 2002 and 3140, 2003). Although lens epithelial cells have sufficient oxygen to support oxidative metabolism in the mitochondria, Eaton 11 has argued that the lens fiber cells may exist in a completely anaerobic state. 
If the human lens is largely anaerobic, then it could be argued that ascorbate oxidation products would not be available to glycate lens proteins in vivo. Recent investigations have shown that UVA irradiation of whole lenses causes extensive bleaching of the lens yellow chromophores. 8 Preparations of the WI proteins from aged human lenses and from brunescent cataracts have shown a similar bleaching, which was markedly accelerated by the addition of ascorbic acid. 8 Based on these data, we argued that ascorbate may participate in a type I reaction with the triplet state of the yellow sensitizers, causing a reductive bleaching of the yellow chromophores and the concomitant formation of ascorbyl free radical, as shown in Figure 1 . Because ascorbyl radical can readily dismutate to one molecule of dehydroascorbate and one molecule of ascorbate, 12 this reaction may provide glycation-active compounds from ascorbate, even in the absence of oxygen. 
In this manner, ascorbate glycation could be triggered by UVA light, which has been shown to be completely absorbed within the aged human lens. 13 In this study, we confirmed that ascorbate is indeed oxidized by UVA light in the complete absence of oxygen and that the oxidation products formed are capable of covalently modifying lens proteins both in solution and in whole lenses. 
Materials and Methods
Human lenses were obtained from the Heartland Lions Eye Tissue Bank of Missouri after removal of the cornea. Procedures adhered to the provisions of the Declaration of Helsinki for research in human tissue. Newborn calf lenses were obtained from Pel-Freeze Biologicals (Rogers, AR), shipped in dry ice, and stored frozen at −70°C until used. A pool of 10 to 20 lenses were homogenized, and the dialyzed water-soluble (WS) proteins and the water-insoluble sonication-solubilized (WISS) proteins were prepared as described previously, 14 15 and also dialyzed before use. l-Ascorbic acid was obtained as an ultrapure reagent from Sigma-Aldrich (St. Louis, MO). l-Dehydroascorbic acid (DHA) and l-diketogulonic acid (DKG) were prepared from l-ascorbic acid, according to published procedures. 16 DHA was also purchased commercially from several suppliers. Solutions of this reagent were yellow, but one pass through a charcoal filter removed the colored impurities, and the nuclear magnetic resonance (NMR) spectrum showed only DHA resonances. l-Erythrulose was obtained from Sigma-Aldrich. The chromatography standard, [U-14C]oxalate (OA), was purchased from DuPont (Wilmington, DE), whereas [1-14C]erythrulose was synthesized from [1-14C]erythrose by reduction and selective oxidation by Gluconobacter oxidans. 17 Similarly, [U-14C]d-glucose was reduced to glucitol and converted to [U-14C]l-sorbose by cultures of G. oxidans, and then converted into [U-14C]l-ascorbic acid by the method of Reichstein and Grüssner as described in Crawford and Crawford 17 and as we have described previously. 18 Two preparations of [U-14C]ascorbate were synthesized in this manner, with specific activities of 1.6 and 7.8 mCi/millimole. Aliquots of the [U-14C]ascorbate preparations were chromatographed over a Resex RNM-carbohydrate column in the sodium form (Phenomenex, Torrance, CA), to remove any remaining residual impurities and were used as such for the protein incorporation studies. When a sample of the [U-14C]ascorbate was purified in this manner and treated with ascorbate oxidase, no radioactivity eluted at the original position of ascorbate, indicating no impurities were present in the pooled peak. All phosphate buffers were pretreated with 10 g/L resin (200–400 mesh; Chelex; Bio-Rad Laboratories, Richmond, CA), to remove heavy metal ion contaminants, and were filtered through a 0.2-μm nitrocellulose filter before use. 
Ascorbate Oxidation
Incubation mixtures were prepared, containing 50 μM ascorbic acid, 1.0 mg/mL lens proteins, 4.0 M guanidine-HCl, 50 mM phosphate buffer (pH 7.0), 0.1 mM diethylenetriaminepentaacetic acid (DTPA), and 1.0 mL was placed in a 4.0-mL quartz cuvette with a screw cap containing a rubber septum seal. Each cuvette with a loosened cap was subjected to a stream of argon through a needle aimed at the surface of the liquid. This was continued with simultaneous stirring of the solution with a magnetic stir bar for a minimum of 10 minutes to remove oxygen. In most cases, the cuvette was further sealed by dipping the top into melted paraffin wax and then cooled before irradiation. UVA irradiation was performed on triplicate samples for each protein preparation with a 1000-W Hg/Xe lamp (Oriel Corp., Stratford, CT) after passing through a 10-cm water-jacketed filter containing a 5% copper sulfate solution and through a 338-nm cutoff filter. The filter and the cuvette holder were kept at 17°C by a circulating water bath. This system delivered 200 mW/cm2 of light at the cuvette’s face. Light measurements showed that 86 mW/cm2 of light was absorbed by the protein solutions. Ascorbate oxidation was determined by the loss of absorbance at 265 nm, which was measured at 15-minute intervals over 1 hour and compared with an equivalent cuvette kept at room temperature in the dark. Oxygen measurements, made with an oxygen electrode (Ocean Optics, Dunedin, FL) showed no detectable oxygen after the argon treatment, and the addition of sodium dithionite to chemically remove any remaining oxygen, caused no further decrease in the electrode response. 
Action Spectra
Samples containing 50 μM ascorbic acid, 1.0 mg/mL of a whole-lens homogenate from aged human lenses, 4.0 M guanidine-HCl, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA were prepared and irradiated with the incident light from a spectrofluorometer (model F2500; Hitachi, Ltd., Tokyo, Japan). Irradiations were performed at 10-nm intervals (20-nm bandwidth) from 320 to 400 nm, and spectra were collected after each 1-hour irradiation. The UVA-dependent loss of absorbance at 265 nm and at 350 nm was measured to determine the loss of ascorbate and the bleaching of lens chromophores, respectively. 8 The amount of UVA light absorbed was determined at each wavelength setting, and the final data reported as nanomoles of ascorbate oxidized per Joule of absorbed light. Action spectra were determined in duplicate with a WISS preparation from aged human lenses. 
Whole Lens Irradiation
One lens was removed from a donor human eye, placed into a cuvette containing 1.3 mL of artificial aqueous humor (AAH), 19 and irradiated for 2 hours with UVA light. The paired lens was treated similarly but kept in the dark. Each lens was homogenized in 0.8 mL of 0.1% m-phosphoric acid containing 0.1 mM DTPA, and 0.4 mL of this homogenate was mixed with 0.4 mL of 8% m-phosphoric acid containing 0.1 mM DTPA, vortexed, and placed in ice. After centrifugation at 27,000g for 10 minutes to remove the precipitated proteins, the supernatant was diluted twofold with 0.1 mM DTPA and passed through a 5-kDa cutoff centrifuge filter (M-0286; Amicon, Danvers, MA) at 5000g. An aliquot of the filtrate was injected onto a 7.8 × 300-mm Resex RNM-carbohydrate column and eluted isocratically with 1% m-phosphoric acid and 0.1 mM DTPA at a flow rate of 0.3 mL/min. The column eluate was monitored at 243 nm, which is the λmax of ascorbate at the acidic pH of the m-phosphoric acid solution. Confirmation of the ascorbate peak was made by pretreating aliquots of the homogenate with 1.0 unit/mL of ascorbate oxidase (catalog no. A 0157; Sigma-Aldrich) before acid precipitation. A standard curve was prepared by subjecting known amounts of ascorbate to HPLC chromatography on the Resex RNM carbohydrate column and quantifying the area under the ascorbate peak. 
Whole human eyes of aged donors were treated with several drops each of 1% tropicamide (Mydriacyl; Alcon, Fort Worth, TX) and 2.5% phenylephrine (AK-Dilate; Akorn, Inc., Buffalo Grove, IL) to dilate the pupils. After 10 to 20 minutes to allow for maximum dilation, the eyes were placed in AAH, and 4 to 5 drops of each compound were added. The eyes were then subjected to 2 hours of UVA irradiation in a plastic chamber with a circular quartz window, using the UVA apparatus described earlier. After irradiation at 20°C, the lenses were removed from each eye and homogenized, and the acid-soluble supernatants were assayed for ascorbate levels, using the Resex RNM-carbohydrate column. These values were compared with the paired eyes kept in AAH in the dark. 
UVA-Mediated Incorporation of [U-14C]Ascorbic Acid
Reaction mixtures of 1.0 mL contained 2.0 mg of dialyzed WISS proteins, 1.4 μCi (0.2 micromole) [U-14C]ascorbate in 50 mM phosphate buffer (pH 7.0) and 0.1 mM DTPA. Quartz cuvettes (4.0 mL) and septum screw caps were exposed to UV irradiation overnight in a hood (Steril-GARD; Baker, Sanford, ME), to kill bacteria. A large reaction mixture was sterile filtered and 1.0-mL aliquots placed into each of seven sterile cuvettes. The solution in each cuvette was exposed to a stream of argon through an oxygen trap filter for at least 10 minutes to remove oxygen, and the cuvettes were closed and dipped in paraffin to seal out air. One cuvette was frozen at −70°C and considered time zero. Three cuvettes were subjected to 2 hours of UVA irradiation. All the reaction mixtures were then incubated in the dark, and cuvettes were removed daily over 3 days and frozen. At the end of the experiment, all the aliquots were thawed and 50-μL aliquots from each were spotted in triplicate on filter paper discs. The discs were stirred for 30 minutes each with cold 10% trichloroacetic acid (TCA), cold 5% TCA, 70°C 5% TCA, a 2:1 ethanol-ether mixture, and finally anhydrous ether. 16 The ether was evaporated from the discs in a fume hood and the radioactivity incorporated into the protein determined in a scintillation spectrometer (Perkin-Elmer-Packard, Meriden, CT). 
Human lens pairs were also individually placed in AAH as described earlier, 1.4 μCi of [U-14C]ascorbate was added to the medium, and the lenses were allowed to take up the [14C]ascorbate for 24 hours. The lenses were then rinsed with AAH and placed in fresh AAH medium without ascorbate. One lens from each pair was irradiated for 2 hours, with the paired lens kept in the dark. The lenses were homogenized after 24 hours, and the incorporation of radioactivity into protein was determined by the filter paper disc method described earlier. 
Analysis of Enzymatic Digests of [U-14C]Ascorbate–Labeled Proteins
WI proteins from aged human lenses (4.0 mg/2.0 mL reaction) were irradiated with 5.0 μCi (0.67 micromoles) [U-14C]ascorbate, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA for 2 hours and then incubated in the dark for 24 hours in a 37°C incubator. This reaction mixture was then subjected to the same irradiation and dark incubation procedure twice more, and the labeled proteins extensively dialyzed against 5 mM phosphate buffer. The proteins were digested with a series of proteolytic enzymes as described. 4 The digest was fractionated on a Bio-Gel P2 column (Bio-Rad Laboratories, Hercules, CA) in 25 mM formic acid, and the major peak of radioactivity was pooled and chromatographed on an analytical Prodigy HPLC column (Phenomenex) with a sample of yellow chromophores released from a digest of aged human lens WISS proteins (peaks 2 + 3 as described previously). 4 The column eluent was monitored for both radioactivity with a radiochromatography detector (Flo-One/Beta series A-500; Perkin-Elmer-Packard), and for the absorbance at 330 nm. Chromatographic conditions were identical with those described previously. 4  
Ascorbate Oxidation Products
[U-14C]ascorbate was irradiated with aged human lens WI proteins for 2 hours in the absence of oxygen. The acid-soluble supernatant after photolysis was subjected to Resex chromatography, as described earlier, except the column eluate was also monitored for radioactivity. To identify the peaks, samples of known ascorbate oxidation products were chromatographed in the same system, and aliquots from each fraction were spotted sequentially on a thin-layer chromatography (TLC) plate and visualized by spraying with 2,4-dinitrophenylhydrazine, as described previously. 16 The elution positions of oxalate and l-erythrulose were determined with carbon 14-labeled compounds. 
Ambient Light Experiments
Reaction mixtures (1.0 mL each) were prepared containing 2.0 mg/mL WISS proteins from aged human lenses, 1.4 μCi [U-14C]ascorbate, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA and sterile filtered into UV-sterilized septum cuvettes. Each cuvette was subjected to a vigorous stream of argon for at least 10 minutes, sealed in paraffin wax, placed in a water-jacketed sample holder, and exposed to ambient sunlight on the roof of the laboratory. The cuvette holder faced north and was shaded to avoid any direct sunlight. Therefore, each cuvette was exposed to only reflected sunlight after passing through a combination of two 338-nm cutoff filters and a pane of window glass to ensure removal of all UVB light. Measurements of UVA light (338–475 nm) at the cuvette’s face from 11 AM to 3 PM averaged 1.1 J/h per square centimeter. The temperature was kept at 17°C by a circulating water bath for approximately 8 to 9 hours of sunlight exposure each day, after which, the cuvettes were removed and placed in a 37°C incubator in the dark for an additional 15 to 16 hours. After 5 to 7 days of this exposure regimen, the incorporation of radioactivity into proteins was determined by the filter paper disc assay and compared with the incorporation in dark control preparations kept in the laboratory. 
Results
Anaerobic Ascorbate Oxidation by UVA Light and Lens Proteins
The WI proteins from aged human lenses were solubilized by sonication and 1.0-mg/mL solutions were irradiated with UVA light in the presence of 50 μM ascorbic acid. These experiments were performed in the absence of oxygen, but in the presence of 4.0 M guanidine-HCl to expose the sensitizers in the WISS protein aggregates. 20 Spectra were gathered at various times to determine the decrease in absorbance at 265 nm (A265) over 1 hour, and this value was converted to nanomoles of ascorbate lost using a molar extinction coefficient of 1.5 × 104. Figure 2 shows a continual increase in nanomoles of ascorbate oxidized in the presence of an aged WI fraction, reaching 45 nanomoles/mL of ascorbate lost after 1 hour, which was 90% of the ascorbate present. Little or no loss of ascorbate was present in the dark control. A second experiment with another aged WI preparation yielded 34 nanomoles/mL of ascorbate oxidized after 1 hour. When ascorbate levels were measured on duplicate samples by HPLC analysis of the acid-soluble supernatant, ascorbate loss was 33.0 nanomoles/mL after 1 hour of irradiation, compared with 31.4 nanomoles/mL based on the decrease in the A265 of the irradiated solution. These data confirmed that A265 values are an accurate measure of ascorbate loss. 
Dialyzed WS proteins from the same lenses had only half the apparent ascorbate oxidizing activity, and even lesser activity in the WS fraction from young human lens (Fig. 2) or with calf lens proteins (not shown). A difference spectrum after irradiation was identical with that of ascorbate, indicating that the spectral decreases were due specifically to the loss of ascorbate. Only one dark control curve is shown in Figure 2 , but all proteins tested showed little or no loss of ascorbate in the absence of UVA. A definite ascorbate loss (7 nanomoles/mL per hour) occurred during UVA irradiation in the absence of any added protein. A similar loss of ascorbate occurred in phosphate buffer alone and with several different commercial ascorbate preparations. Therefore, the actual protein-mediated ascorbate oxidation was 26, 9, 2, and 1 nanomoles/mL per hour for the aged WISS, aged WS, young WS, and calf lens proteins, respectively. 
Action Spectrum for Ascorbate Loss and Protein Photobleaching
The scheme shown in Figure 1 proposes that ascorbate induces photobleaching by donating an electron to the triplet state of lens sensitizers, thereby forming ascorbyl free radical. Figure 3 shows the action spectrum for both ascorbate loss (decrease in A265) and photobleaching (decrease in A350 absorbance) in the presence of 50 μM ascorbate and in the absence of oxygen. Irradiation was performed at various wavelengths, using the monochromator for incident light in the spectrofluorometer. Ascorbate loss and photobleaching were present throughout the UVA region. The spectra were very similar, showing maximum activity at 340 to 350 nm. Whole lens homogenates in 4.0 M guanidine-HCl were used, however, similar spectra were obtained with a washed WISS fraction. Wavelengths below 320 nm were not used to avoid any direct photolysis of ascorbate. Solutions containing 1.0 mM ascorbate exhibited an absorbance of 15 at 265 nm, but no absorbance above 325 nm. Therefore, all the ascorbate loss shown in Figure 3 was sensitizer mediated. 
Although the data in Figure 3 are consistent with an oxidation of ascorbate by a type I mechanism, we wanted to eliminate the possibility that traces of oxygen could be present in sufficient amounts to cause a singlet oxygen–mediated ascorbate oxidation. Therefore, ascorbate loss was measured in the presence and absence of 20 mM sodium azide, a singlet oxygen quencher. As can be seen in Figure 4 , the addition of azide had no inhibitory effect on ascorbate loss, giving values of approximately 40 nanomoles/h of ascorbate oxidized in both reactions with identical kinetics. 
UVA-Mediated Incorporation of [U-14C]Ascorbate into Lens Proteins
The aerobic oxidation of ascorbate forms products capable of glycating proteins. 16 To determine whether the UVA-mediated photoproducts also exhibit glycating activity, [U-14C]ascorbate was irradiated for 2 hours with 2.0 mg/mL WI proteins from aged human lenses and then incubated for 3 days in the dark at 37°C to allow glycation to proceed. Figure 5 shows a rapid incorporation of radioactivity into protein during the first day after irradiation, which reached a plateau at days 2 and 3. Only a slight incorporation occurred in the dark control, confirming a light-dependent incorporation. The total incorporation was equivalent to 4.7 nanomoles of ascorbate. Repeat experiments with different WI protein preparations produced 6.1 and 7.0 nanomoles of ascorbate incorporated, using the same protocol. Dark controls incorporated 0.9, 1.3, and 1.3 nanomoles, respectively, for these preparations. Similar experiments were performed with the various protein fractions shown in Figure 2 . After 3 days, the ascorbate incorporation was 4.8, 3.3, 1.3, and 1.8 nanomoles/2.0 mg protein for the aged WISS, aged WS, young WS, and calf lens proteins respectively, after correction for the dark control values. Time 0 shows the incorporation before UVA irradiation. 
The ascorbate oxidation products produced by UVA light were analyzed by Resex chromatography immediately after the 2-hour irradiation (Fig. 6A) and again after 1 day of incubation in the dark (Fig. 6B) . The UVA irradiation produced several new peaks of radioactivity, whereas only ascorbate was detected in the dark control (not shown). By comparison with authentic standards, the major peaks were identified as DHA, DKG, and an early-eluting peak, which included in part, oxalic acid. The ascorbate peak after UVA accounted for only 62% of the total radioactivity, compared with 97% of the total in the dark control profile. This represented the oxidation of approximately 80 nanomoles of ascorbate during the 2-hour irradiation. After 24 hours in the dark, however, almost all the oxidation products disappeared, which correlated with the incorporation of radioactivity into protein, as shown in Figure 5
Demonstration of Formation of AGEs by UVA Light
To support the idea that the incorporation resulted from a glycation reaction between ascorbate oxidation products and lens proteins, the labeled proteins were dialyzed and digested with a series of proteolytic enzymes to release the modified amino acids. 4 The digests were separated by Bio-Gel P-2 chromatography, and these profiles are shown in Figure 7A . Definite peaks of radioactivity appeared in the UVA-irradiated protein digest, and the major peak of radioactivity eluted coincident with the major A330 peak present in the aged WI protein digest. This peak (fractions 28–33) was pooled and subjected to reversed-phase HPLC on an analytical Prodigy column. Several radioactive peaks corresponded to A330 peaks present in the human lens digests. The blackened peaks in Figure 7B represent those A330 peaks in the aged human lens digest, which corresponded to peaks of radioactivity in the ascorbate-labeled sample. Double peaks at 5, 14, and 20 minutes were represented by a peak and a shoulder in the radioactivity profile. Several of the A330 peaks, normally present in aged human WISS proteins, 21 eluted later than most of the labeled peaks. The labeled peaks may represent only early AGEs, because of the short labeling time used. 
Whole-Lens Irradiation and Ascorbate Loss
Whole lenses in AAH were irradiated for 2 hours with UVA light. The acid-soluble fractions of irradiated and paired dark control lenses were analyzed for ascorbate levels by HPLC on a Resex column. Figure 8A shows the A265 elution profile from a carbohydrate column of the acid-soluble fraction from a dark control lens before and after treatment with ascorbate oxidase. An ascorbate standard eluted at 31 minutes, and a major peak from the lens supernatant eluted at 30.7 minutes. This peak was almost completely destroyed by the addition of ascorbic acid oxidase, indicating no other WS compounds coeluted with the ascorbate peak. Figure 8B shows the same profiles from the 2-hour irradiated lens, which had a markedly reduced ascorbate peak. Four lens pairs were analyzed in this manner (data not shown). Ascorbate levels decreased 35% ± 9% due to the UVA irradiation of whole aged human lenses. In addition, two pairs of whole eyes were irradiated through the cornea and the lenses analyzed in the manner just described. Quantification of the ascorbate peaks from the HPLC column showed losses of 10% and 17% after 2.0 hours of UVA compared with paired eyes kept in the dark (data not shown). In control experiments, four pairs of donor lenses were directly compared for ascorbate content. Although the absolute values varied from 144 to 267 nanomoles/lens, the ascorbate content of lens pairs differed by only 3.8% (data not shown). 
Incorporation of [U-14C]Ascorbic Acid into Whole-Lens Proteins by UVA Light
Whole aged lenses were incubated with [U-14C]ascorbate in AAH for 24 hours, rinsed several times, and placed in fresh AAH without [U-14C]ascorbate. These lenses were irradiated for 2 hours with UVA light and incubated in the dark for 24 hours. Table 1 shows an almost threefold increase in [U-14C]ascorbate incorporation in three different lenses, due to UVA irradiation, when compared with paired lenses kept as dark controls (989 ± 64 disintegrations per minute [DPM] vs. 373 ± 117 DPM in the dark control). The limited amount of incorporation reflected the limited amounts of radioactivity taken up by the lens, the marked dilution of the specific radioactivity of the [U-14C]ascorbate within the lens and by the leakage of labeled compounds into the medium during the dark incubation in ascorbate-free medium. 
Glycation Due to Ambient Sunlight
The sequence of events after UVA irradiation argue that ascorbate glycation can represent a significant protein modification in vitro, even in the absence of oxygen. Whether ambient sunlight is sufficient to cause ascorbate glycation of the human lens proteins, however, cannot be assumed. Therefore, 2.0 mg/mL human lens WI proteins were incubated with [U-14C]ascorbate as described in Figure 2 , but in cuvettes mounted in a cell holder on the roof of the laboratory with a circulating water bath. Light at the cuvette face was determined to be 1.1 J/h per square centimeter on both sunny and slightly overcast days. This light was filtered to remove UVB light, and the apparatus was shaded. After exposure to reflected sunlight for 5 to 7 days, the incorporation into proteins was measured. Table 2 shows the labeling per milligram protein obtained compared with the dark control. In every case, ambient sunlight was capable of causing an increased incorporation of [U-14C]ascorbate into proteins. This increase was 2.4-, 1.4-, and 2.4-fold over the dark controls, which was equivalent to 0.52 ± 0.02 nanomoles of ascorbate incorporated. 
Discussion
Comparison of the spectral properties, 22 sensitizer activity, 22 and chromatographic fractionation 4 of the yellow chromophores in aged human lens WI proteins with samples from bovine lens proteins glycated by ascorbic acid in vitro for 4 weeks, showed them to be almost identical. The glycation reaction, however, requires the presence of oxygen to produce the glycation-active species, because ascorbate, per se, has not been reported to have glycating ability. 16 23 The extent of ascorbate glycation, therefore, may be greatly limited in vivo by the low levels of oxygen reported to be present in lens tissue. 9 10 The absorption of UVA light by the human lens yellow proteins has been shown to lead almost exclusively to the production of singlet oxygen, 6 and this reaction would also be severely limited by low oxygen levels in vivo. 
High doses of UVA irradiation caused a bleaching of aged human lens yellow chromophores in the absence of oxygen in vitro. This bleaching was markedly accelerated by the addition of ascorbic acid, suggesting a competition between ascorbic acid and oxygen for the triplet state of the activated human lens sensitizers. This mechanism is supported by results presented herein, showing that ascorbate is readily oxidized by UVA light in the absence of oxygen, but in the presence of aged human lens proteins. The extent of ascorbate loss mirrored the amount of yellow modifications present in various lens protein fractions and was strictly UVA and age dependent. We observed a surprising loss of 7 nanomoles/h ascorbate in the absence of protein. Recent experiments have shown that freshly prepared phosphate buffer causes the oxidation of only 0.8 to 0.9 nanomoles of ascorbate/h in the absence of protein and oxygen. However, this was increased to 6.2 and 7.0 nanomoles/h of ascorbate oxidized after filtration through a serum filter (GF/0.2 μm Supor; Acrodisc, Bridgeport, NJ). Filtration apparently dissolved unknown sensitizer(s) from the nitrocellulose. When serum filters (Acrodisc 25 with HT Tuffryn membranes; Acrodisc) were used to filter sterilize our reaction mixture minus protein, an oxidation of only 0.8 nanomoles of ascorbate was detected on UVA irradiation. Also, the use of the 338-nm filter on our light source prevented any direct UV photolysis of ascorbate (λmax = 265 nm at pH 7.0). 
The action spectra for ascorbate oxidation and chromophore bleaching were almost identical (Fig. 3) . Activity was noted throughout the UVA region, with a λmax of 340 to 350 nm, which was consistent with the difference spectrum for bleaching reported previously. 8 The data argue that bleaching and ascorbate oxidation are both linked to light absorption by a common set of sensitizers. A secondary peak of bleaching at 380 nm did not correspond to a peak of ascorbate loss. This peak may represent sensitizers that have an extremely short half-life for the triplet state, precluding type I chemistry, or it could be due to sensitizers that remain buried, even in 4.0 M guanidine, preventing the access of ascorbate. An example of the first type of sensitizer may be bound filter compounds, which are known to be photoinactive. 24  
In all the in vitro experiments presented herein, we measured the oxidation of ascorbic acid in the absence of oxygen. This was confirmed by oxygen measurements and by the fact that singlet oxygen, the preferred reactant with the triplet state of human lens sensitizers, made no contribution to the oxidation of ascorbate in vitro. Anaerobic conditions may represent an accurate model for the lens interior, because of the very low oxygen levels reported in vivo and because UVA light would very rapidly convert any residual oxygen to singlet oxygen in a matter of minutes. Because singlet oxygen forms adducts to Trp and His residues, it cannot be recycled. 25  
Ascorbate oxidation was rapid in the presence of high UVA fluence. Solutions containing 1.0 mg of WISS proteins oxidized 20 nanomoles of ascorbate in 15 minutes, which was approximately 1 nanomole/J of absorbed light. Ascorbate oxidation also proceeded readily when whole lenses were irradiated with UVA light. The absolute amount of ascorbate oxidized after 2 hours of UVA irradiation (37.2 nanomoles/lens), was not consistent with the increased light absorbed by the whole lens (800 J/2 h). The decreased efficiency (∼20-fold) could have been due to the aggregate nature of the proteins in the aged lenses, compared with the in vitro experiments, which were performed in 4.0 M guanidine-HCl to expose the sensitizers and to prevent light scattering. A smaller, but significant, amount of ascorbate was lost (12%, or 27 nanomoles/lens) when whole eyes were irradiated, probably limited by the small area of the lens irradiated through the pupil. The level of glutathione in the whole-lens experiments did not prevent (or completely reverse) the oxidation of ascorbate. This could have been because (1) reduced glutathione (GSH) levels are decreased in aged human lenses (0.4 mM in the aged human lens nucleus by our measurements), (2) GSH levels are further depleted by UVA light, and/or (3) because GSH cannot compete with ascorbate for the triplet state of the lens sensitizers. 8 Although GSH can rapidly reduce DHA back to ascorbate, GSH is a highly charged molecule and cannot penetrate into the high molecular weight aggregates of an aged WISS fraction, whereas ascorbate can. 20  
The UVA-mediated oxidation of ascorbate in the absence of oxygen produced the same products as seen without UVA but in the presence of oxygen. Therefore, whether lens nuclei contain low levels of oxygen or are completely anaerobic, they have no effect on the type of ascorbate oxidation products formed. The oxidation products disappeared during the ensuing 24 hours in the dark, which corresponded to the time when the incorporation of radioactivity into protein occurred. When the oxidation products were depleted, the incorporation ceased, consistent with the observation that ascorbate itself cannot glycate proteins. 16 23 The increased incorporation at days 2 and 3 after irradiation was quantitatively similar to the incorporation in the dark control preparations. The slow oxidation of ascorbate in the dark control could have been caused by leakage of oxygen over several days or by a chemical reaction of ascorbate with free radicals in the yellow proteins. 26 When larger amounts of labeled proteins were prepared, and the cycle of 2 hours’ UVA/24 hours’ incubation in the dark was repeated three times with a larger WISS reaction, an appropriate increase in incorporation was observed after each UVA irradiation. Clearly, unbleached, active sensitizers still remained after each UVA treatment. 
Proteolytic digests of aged WISS proteins and [U-14C]ascorbylated proteins were subjected to enzymatic digestion and cochromatographed. HPLC separation of the amino acid peak from a Bio-Gel P-2 column showed many labeled peaks that coeluted with “yellow” compounds present in the aged human lens WISS fraction. The late-eluting peak on the gel-permeation column represent aromatic amino acids and Trp oxidation products, which do not arise by ascorbate glycation. 21 The WISS fraction was more complex, which may reflect the difference between early AGE compounds formed over several days and AGE compounds formed over decades in the lens, which includes many protein cross-links. 
Calculations, based on our estimated data, showed that the irradiation of whole aged human lenses for 2 hours produced the oxidation of 37 nanomoles of ascorbate by 800 J of absorbed UVA light/cm2, or almost 0.05 nanomoles/J. Because reflected sunlight exposes lenses to approximately 10 J of UVA light/d, 27 28 ambient light levels could account for the oxidation of 3.5 nanomoles of ascorbate per week. Given the high rate of incorporation of ascorbate oxidation products into protein 18 and that there is no protein turnover in the lens, these data are consistent with the possibility that UVA-mediated ascorbylation is a viable mechanism for the accumulation of the AGEs present in the WI fraction. Experiments with aged lens proteins allowed us to show an incorporation of 0.52 nanomoles of [U-14C]ascorbate after 5 to 7 days of exposure to ambient sunlight (1.1 J/h per square centimeter). These data also suggest that ascorbate glycation could proceed in situ due to ambient sunlight. When WISS proteins were irradiated in vitro with of [U-14C]ascorbate, approximately 60% of the ascorbate (80 nanomoles) was oxidized over 2 hours. This resulted in the incorporation of 4.8 nanomoles of ascorbate into proteins over the next 24 hours, or 6% of the total ascorbate oxidized. Experiments with the aged WISS proteins after 5 to 7 days of exposure to ambient sunlight allowed us to show an incorporation of 0.52 nanomoles of [U-14C]ascorbate. If our estimate of potential ascorbate oxidation (3.5 nanomoles/wk) approaches the true level, the incorporation of ascorbate would be 15%. Given the varied methodologies used, this incorporation is not significantly different from the 6% in whole lenses. These data, therefore, are also consistent with the possibility that ascorbate glycation could be a viable mechanism for lens protein modification in situ. 
Although the yellow sensitizers in aged human lens proteins appear to be similar to ascorbate-generated AGEs, ascorbate oxidation by UVA light has not been confirmed when pure isolated compounds from both sources are used. Protein-bound filter compounds may also play a role in the UVA photochemistry described in this report. 29 30  
 
Figure 1.
 
Proposed scheme for the oxidation of ascorbate by the triplet state of the UVA sensitizers (*SIII) present in aged human lens.
Figure 1.
 
Proposed scheme for the oxidation of ascorbate by the triplet state of the UVA sensitizers (*SIII) present in aged human lens.
Figure 2.
 
The anaerobic oxidation of ascorbate by UVA light. Solutions containing 0.05 mM ascorbate, 1.0 mg/mL lens proteins, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA were irradiated with UVA light in the absence of air for 1 hour. Spectra were gathered at 15-minute intervals and compared with the spectra gathered from an identical cuvette kept in the dark. The loss of ascorbate was calculated from the decrease in A265 nm. WS, water-soluble proteins; WISS, water-insoluble sonicate supernatant proteins.
Figure 2.
 
The anaerobic oxidation of ascorbate by UVA light. Solutions containing 0.05 mM ascorbate, 1.0 mg/mL lens proteins, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA were irradiated with UVA light in the absence of air for 1 hour. Spectra were gathered at 15-minute intervals and compared with the spectra gathered from an identical cuvette kept in the dark. The loss of ascorbate was calculated from the decrease in A265 nm. WS, water-soluble proteins; WISS, water-insoluble sonicate supernatant proteins.
Figure 3.
 
Action spectra for photobleaching and ascorbate oxidation. Cuvettes were prepared containing 1.0 mg/mL dialyzed lens homogenate from aged human lenses. Anaerobic irradiation was performed at each of the wavelengths, and the change in the absorbance at 350 nm (bleaching) and 265 nm (ascorbate loss) were determined.
Figure 3.
 
Action spectra for photobleaching and ascorbate oxidation. Cuvettes were prepared containing 1.0 mg/mL dialyzed lens homogenate from aged human lenses. Anaerobic irradiation was performed at each of the wavelengths, and the change in the absorbance at 350 nm (bleaching) and 265 nm (ascorbate loss) were determined.
Figure 4.
 
Effect of sodium azide on the UVA-dependent oxidation of ascorbate. Ascorbate loss was measured at 265 nm, as described in Figure 2 , with 1.0 mg/mL of a WISS fraction from aged human lenses, both in the presence and absence of 20 mM sodium azide.
Figure 4.
 
Effect of sodium azide on the UVA-dependent oxidation of ascorbate. Ascorbate loss was measured at 265 nm, as described in Figure 2 , with 1.0 mg/mL of a WISS fraction from aged human lenses, both in the presence and absence of 20 mM sodium azide.
Figure 5.
 
Incorporation of [U-14C]ascorbate into lens proteins after UVA irradiation. Solutions containing 2.0 mg/mL WISS proteins from aged human lenses were irradiated with UVA light in the presence of 1.4 μCi of [U-14C]ascorbate for 2 hours and incubated in the dark. Samples were removed daily to determine the incorporation of radioactivity into protein.
Figure 5.
 
Incorporation of [U-14C]ascorbate into lens proteins after UVA irradiation. Solutions containing 2.0 mg/mL WISS proteins from aged human lenses were irradiated with UVA light in the presence of 1.4 μCi of [U-14C]ascorbate for 2 hours and incubated in the dark. Samples were removed daily to determine the incorporation of radioactivity into protein.
Figure 6.
 
The oxidation products of [U-14C]ascorbate formed by UVA irradiation. After 2 hours of irradiation, as described in Figure 5 , the protein was precipitated with m-phosphoric acid and the supernatant fractionated on a Resex HPLC column (A). A similar irradiated reaction was incubated for 24 hours in the dark and the acid-soluble supernatant fractionated on a Resex HPLC column (B).
Figure 6.
 
The oxidation products of [U-14C]ascorbate formed by UVA irradiation. After 2 hours of irradiation, as described in Figure 5 , the protein was precipitated with m-phosphoric acid and the supernatant fractionated on a Resex HPLC column (A). A similar irradiated reaction was incubated for 24 hours in the dark and the acid-soluble supernatant fractionated on a Resex HPLC column (B).
Figure 7.
 
Chromatographic comparison of the enzymatic digest of [U-14C]ascorbate-labeled lens proteins with a digest of WISS proteins from aged human lenses. Lens proteins were labeled for 24 hours after a 2-hour UVA irradiation, as described in Figure 5 . This sequence was repeated twice more, and the proteins extensively dialyzed. The labeled proteins were digested with proteolytic enzymes and chromatographed on a gel-permeation column with a similar digest of WISS proteins from aged human lenses (A). The peak of radioactivity eluting in the amino acid region was pooled and chromatographed on an analytical HPLC column (B). The blackened A330 peaks correspond to peaks labeled in vitro by [U-14C]ascorbate.
Figure 7.
 
Chromatographic comparison of the enzymatic digest of [U-14C]ascorbate-labeled lens proteins with a digest of WISS proteins from aged human lenses. Lens proteins were labeled for 24 hours after a 2-hour UVA irradiation, as described in Figure 5 . This sequence was repeated twice more, and the proteins extensively dialyzed. The labeled proteins were digested with proteolytic enzymes and chromatographed on a gel-permeation column with a similar digest of WISS proteins from aged human lenses (A). The peak of radioactivity eluting in the amino acid region was pooled and chromatographed on an analytical HPLC column (B). The blackened A330 peaks correspond to peaks labeled in vitro by [U-14C]ascorbate.
Figure 8.
 
Ascorbic acid oxidation products after 2 hours of UVA irradiation of intact whole human lenses. A single lens was homogenized, and the acid-soluble fraction was fractionated on a carbohydrate HPLC column, before and after treatment with ascorbic acid oxidase (A). A paired lens was irradiated for 2 hours and subjected to the same analysis (B). The blackened peaks eluted at the position of authentic ascorbate.
Figure 8.
 
Ascorbic acid oxidation products after 2 hours of UVA irradiation of intact whole human lenses. A single lens was homogenized, and the acid-soluble fraction was fractionated on a carbohydrate HPLC column, before and after treatment with ascorbic acid oxidase (A). A paired lens was irradiated for 2 hours and subjected to the same analysis (B). The blackened peaks eluted at the position of authentic ascorbate.
Table 1.
 
Incorporation of [U-14C]Ascorbate into Whole Lens Proteins
Table 1.
 
Incorporation of [U-14C]Ascorbate into Whole Lens Proteins
Experiment # UVA Irradiated Dark Control
1 1060 370
2 1000 490
3 920 260
Average 990 ± 64 370 ± 117
Table 2.
 
[U-14C]Ascorbate Incorporation by Ambient Sunlight
Table 2.
 
[U-14C]Ascorbate Incorporation by Ambient Sunlight
Experiment # Sunlight Dark Control
DPM × 103/mg Nanomoles/mg DPM × 103/mg Nanomoles/mg
1 15.7 0.92 6.6 0.38
2 30.8 1.77 22.1 1.27
3 20.2 0.91 8.5 0.38
Ortwerth, BJ, Monnier, VM. (1997) Protein glycation by the oxidation products of ascorbic acid Packer, L Fuchs, J eds. Vitamin C in Health and Disease ,123-142 Marcel Decker, Inc. New York.
Nagaraj, RH, Sell, DR, Prabhakaram, M, Ortwerth, BJ, Monnier, VM. (1991) High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis Proc Natl Acad Sci USA 88,10257-10261 [CrossRef] [PubMed]
Nagaraj, RH, Monnier, VM. (1992) Isolation and characterization of a blue fluorophore from human eye lens crystallins: in vitro formation from Maillard reaction with ascorbate and ribose Biochim Biophys Acta 1116,34-42 [CrossRef] [PubMed]
Cheng, R, Lin, B, Lee, K-W, Ortwerth, BJ. (2001) Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation Biochim Biophys Acta 1537,14-26 [CrossRef] [PubMed]
Halliwell, B, Gutteridge, JMC, Cross, CE. (1992) Free radicals, antioxidants and human disease: where are we now? J Lab Clin Med 119,598-620 [PubMed]
Linetsky, M, Ortwerth, BJ. (1997) Quantitation of the singlet oxygen produced by UVA irradiation of human lens proteins Photochem Photobiol 65,522-529 [CrossRef] [PubMed]
Ortwerth, BJ, Casserly, TA, Olesen, PR. (1998) Singlet oxygen production correlates with His and Trp destruction in brunescent cataract water-insoluble proteins Exp Eye Res 67,377-380 [CrossRef] [PubMed]
Ortwerth, BJ, Chemoganskiy, V, Olesen, PR. (2002) Studies on singlet oxygen formation and UVA light-mediated photobleaching of the yellow chromophores in human lenses Exp Eye Res 74,217-229 [CrossRef] [PubMed]
Kwan, M, Niinikoski, J, Hunt, TK. (1972) In vivo measurements of oxygen Invest Ophthalmol 11,108-114 [PubMed]
Zander, R. (1975) Experimentelle Untersuchungen zur sauerstoffversorgung des auges Albrecht Graefes Arch Klin Exp Ophthalmol 195,215-230 [CrossRef]
Eaton, JW. (1991) Is the lens canned? Free Radic Biol Med 11,207-213 [CrossRef] [PubMed]
Benson, H, Bielski, J, Richter, HW. (1975) Some properties of the ascorbate free radical Ann NY Acad Sci 258,231-237 [CrossRef] [PubMed]
Gaillard, ER, Zheng, L, Merriam, JC, Dillon, J. (2000) Age-related changes in the absorption characteristics of the primate lens Invest Ophthalmol Vis Sci 41,1454-1459 [PubMed]
Ortwerth, BJ, Olesen, PR, Sharma, KK. (1986) Solubilization of the lens water-insoluble fraction by sonication Exp Eye Res 43,955-963 [CrossRef] [PubMed]
Ortwerth, BJ, Olesen, PR. (1992) Studies on the solubilization of the water-insoluble fraction from human lens and cataract Exp Eye Res 55,777-783 [CrossRef] [PubMed]
Slight, SH, Feather, MS, Ortwerth, BJ. (1990) Glycation of lens proteins by the oxidation products of ascorbic acid Biochim Biophys Acta 1038,367-374 [CrossRef] [PubMed]
Crawford, TC, Crawford, SA. (1980) Synthesis of L-ascorbic acid Adv Carbohydr Chem Biochem 37,79-155 [PubMed]
Lee, KW, Mossine, V, Ortwerth, BJ. (1998) The relative ability of glucose and ascorbate to glycate and crosslink lens proteins in vitro Exp Eye Res 67,95-104 [CrossRef] [PubMed]
Duncan, G, Jacob, TJC. (1984) Influence of external calcium and glucose on internal total and ionized calcium in the rat lens J Physiol 357,485-491 [CrossRef] [PubMed]
Linetsky, M, Ranson, N, Ortwerth, BJ. (1998) The aggregation in human lens proteins blocks the scavenging of UVA-generated singlet oxygen by ascorbic acid and glutathione Arch Biochem Biophys 351,180-188 [CrossRef] [PubMed]
Cheng, R, Lin, B, Ortwerth, BJ. (2002) Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue Biochim Biophys Acta 1587,65-74 [CrossRef] [PubMed]
Ortwerth, BJ, Linetsky, M, Olesen, PR. (1995) Ascorbic acid glycation of lens proteins produces UVA sensitizers similar to those in human lens Photochem Photobiol 62,454-462 [CrossRef] [PubMed]
Pischetsrieder, M, Larisch, B, Seidel, W. (1997) Immunochemical detection of oxalic acid monoamides that are formed during the oxidative reaction of L-ascorbic acid and proteins J Agric Food Chem 45,2070-2075 [CrossRef]
Krishna, CM, Uppuluri, S, Riesz, P, Zigler, JS, Jr, Balasubramanian, D. (1991) A study of the photodynamic efficiencies of some eye lens constituents Photochem Photobiol 54,51-58 [CrossRef] [PubMed]
Tomita, M, Irie, M, Ukita, T. (1969) Sensitized photooxidation of histidine and its derivatives: products and mechanism of the reaction Biochemistry 8,5149-5160 [CrossRef] [PubMed]
Linetsky, M, James, HL, Ortwerth, BJ. (1999) Spontaneous generation of superoxide anion by human lens proteins and by calf lens proteins ascorbylated in vitro Exp Eye Res 69,239-248 [CrossRef] [PubMed]
Sliney, DH. (1987) Estimating the solar ultraviolet radiation exposure to an intraocular lens implant J Cataract Refractive Surg 13,296-301 [CrossRef]
Sabburg, J, Parisi, AV, Wong, J. (2001) Effect of cloud on UVA and exposure to humans Photochem Photobiol 74,412-426 [CrossRef] [PubMed]
Berry, Y, Truscott, RJW. (2001) The presence of a human filter within the lens represents an oxidative stress Exp Eye Res 72,411-421 [CrossRef] [PubMed]
Vazguez, SJ, Aquilina, JA, Jamie, JF, Sheil, MM, Truscott, RJW. (2002) Novel protein modification by kynurenine in human lenses J Biol Chem 277,4867-4873 [CrossRef] [PubMed]
Figure 1.
 
Proposed scheme for the oxidation of ascorbate by the triplet state of the UVA sensitizers (*SIII) present in aged human lens.
Figure 1.
 
Proposed scheme for the oxidation of ascorbate by the triplet state of the UVA sensitizers (*SIII) present in aged human lens.
Figure 2.
 
The anaerobic oxidation of ascorbate by UVA light. Solutions containing 0.05 mM ascorbate, 1.0 mg/mL lens proteins, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA were irradiated with UVA light in the absence of air for 1 hour. Spectra were gathered at 15-minute intervals and compared with the spectra gathered from an identical cuvette kept in the dark. The loss of ascorbate was calculated from the decrease in A265 nm. WS, water-soluble proteins; WISS, water-insoluble sonicate supernatant proteins.
Figure 2.
 
The anaerobic oxidation of ascorbate by UVA light. Solutions containing 0.05 mM ascorbate, 1.0 mg/mL lens proteins, 50 mM phosphate buffer (pH 7.0), and 0.1 mM DTPA were irradiated with UVA light in the absence of air for 1 hour. Spectra were gathered at 15-minute intervals and compared with the spectra gathered from an identical cuvette kept in the dark. The loss of ascorbate was calculated from the decrease in A265 nm. WS, water-soluble proteins; WISS, water-insoluble sonicate supernatant proteins.
Figure 3.
 
Action spectra for photobleaching and ascorbate oxidation. Cuvettes were prepared containing 1.0 mg/mL dialyzed lens homogenate from aged human lenses. Anaerobic irradiation was performed at each of the wavelengths, and the change in the absorbance at 350 nm (bleaching) and 265 nm (ascorbate loss) were determined.
Figure 3.
 
Action spectra for photobleaching and ascorbate oxidation. Cuvettes were prepared containing 1.0 mg/mL dialyzed lens homogenate from aged human lenses. Anaerobic irradiation was performed at each of the wavelengths, and the change in the absorbance at 350 nm (bleaching) and 265 nm (ascorbate loss) were determined.
Figure 4.
 
Effect of sodium azide on the UVA-dependent oxidation of ascorbate. Ascorbate loss was measured at 265 nm, as described in Figure 2 , with 1.0 mg/mL of a WISS fraction from aged human lenses, both in the presence and absence of 20 mM sodium azide.
Figure 4.
 
Effect of sodium azide on the UVA-dependent oxidation of ascorbate. Ascorbate loss was measured at 265 nm, as described in Figure 2 , with 1.0 mg/mL of a WISS fraction from aged human lenses, both in the presence and absence of 20 mM sodium azide.
Figure 5.
 
Incorporation of [U-14C]ascorbate into lens proteins after UVA irradiation. Solutions containing 2.0 mg/mL WISS proteins from aged human lenses were irradiated with UVA light in the presence of 1.4 μCi of [U-14C]ascorbate for 2 hours and incubated in the dark. Samples were removed daily to determine the incorporation of radioactivity into protein.
Figure 5.
 
Incorporation of [U-14C]ascorbate into lens proteins after UVA irradiation. Solutions containing 2.0 mg/mL WISS proteins from aged human lenses were irradiated with UVA light in the presence of 1.4 μCi of [U-14C]ascorbate for 2 hours and incubated in the dark. Samples were removed daily to determine the incorporation of radioactivity into protein.
Figure 6.
 
The oxidation products of [U-14C]ascorbate formed by UVA irradiation. After 2 hours of irradiation, as described in Figure 5 , the protein was precipitated with m-phosphoric acid and the supernatant fractionated on a Resex HPLC column (A). A similar irradiated reaction was incubated for 24 hours in the dark and the acid-soluble supernatant fractionated on a Resex HPLC column (B).
Figure 6.
 
The oxidation products of [U-14C]ascorbate formed by UVA irradiation. After 2 hours of irradiation, as described in Figure 5 , the protein was precipitated with m-phosphoric acid and the supernatant fractionated on a Resex HPLC column (A). A similar irradiated reaction was incubated for 24 hours in the dark and the acid-soluble supernatant fractionated on a Resex HPLC column (B).
Figure 7.
 
Chromatographic comparison of the enzymatic digest of [U-14C]ascorbate-labeled lens proteins with a digest of WISS proteins from aged human lenses. Lens proteins were labeled for 24 hours after a 2-hour UVA irradiation, as described in Figure 5 . This sequence was repeated twice more, and the proteins extensively dialyzed. The labeled proteins were digested with proteolytic enzymes and chromatographed on a gel-permeation column with a similar digest of WISS proteins from aged human lenses (A). The peak of radioactivity eluting in the amino acid region was pooled and chromatographed on an analytical HPLC column (B). The blackened A330 peaks correspond to peaks labeled in vitro by [U-14C]ascorbate.
Figure 7.
 
Chromatographic comparison of the enzymatic digest of [U-14C]ascorbate-labeled lens proteins with a digest of WISS proteins from aged human lenses. Lens proteins were labeled for 24 hours after a 2-hour UVA irradiation, as described in Figure 5 . This sequence was repeated twice more, and the proteins extensively dialyzed. The labeled proteins were digested with proteolytic enzymes and chromatographed on a gel-permeation column with a similar digest of WISS proteins from aged human lenses (A). The peak of radioactivity eluting in the amino acid region was pooled and chromatographed on an analytical HPLC column (B). The blackened A330 peaks correspond to peaks labeled in vitro by [U-14C]ascorbate.
Figure 8.
 
Ascorbic acid oxidation products after 2 hours of UVA irradiation of intact whole human lenses. A single lens was homogenized, and the acid-soluble fraction was fractionated on a carbohydrate HPLC column, before and after treatment with ascorbic acid oxidase (A). A paired lens was irradiated for 2 hours and subjected to the same analysis (B). The blackened peaks eluted at the position of authentic ascorbate.
Figure 8.
 
Ascorbic acid oxidation products after 2 hours of UVA irradiation of intact whole human lenses. A single lens was homogenized, and the acid-soluble fraction was fractionated on a carbohydrate HPLC column, before and after treatment with ascorbic acid oxidase (A). A paired lens was irradiated for 2 hours and subjected to the same analysis (B). The blackened peaks eluted at the position of authentic ascorbate.
Table 1.
 
Incorporation of [U-14C]Ascorbate into Whole Lens Proteins
Table 1.
 
Incorporation of [U-14C]Ascorbate into Whole Lens Proteins
Experiment # UVA Irradiated Dark Control
1 1060 370
2 1000 490
3 920 260
Average 990 ± 64 370 ± 117
Table 2.
 
[U-14C]Ascorbate Incorporation by Ambient Sunlight
Table 2.
 
[U-14C]Ascorbate Incorporation by Ambient Sunlight
Experiment # Sunlight Dark Control
DPM × 103/mg Nanomoles/mg DPM × 103/mg Nanomoles/mg
1 15.7 0.92 6.6 0.38
2 30.8 1.77 22.1 1.27
3 20.2 0.91 8.5 0.38
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×