Determination of protein deposits on RGP lenses by X-ray photoelectron spectroscopy.

PURPOSE: To establish a novel and objective screening method for evaluating the cleaning efficacy of contact lens care solutions for removal of protein deposits on single rigid gas permeable (RGP) lenses. METHODS: New and unworn RGP lenses containing no nitrogen atoms were incubated in a standard tear protein test solution. Protein deposition on lenses was determined by X-ray photoelectron spectroscopy (XPS) before and after standardized cleaning with commercially available care solutions (daily cleaners A, B) and a wetting/soaking solution or with 0.1w/v% sodium dodecyl sulfate (SDS) as a control. Cleaning efficacy was calculated based on the percentage of nitrogen atoms present in the total elements of the measured XPS scan spectrum. RESULTS: Nitrogen atoms originating from residual protein deposits on RGP lenses after cleaning were determined by XPS. The cleaning efficacy of the wetting/soaking solution was lower (74%) than that of daily cleaners A and B (95%) or SDS (96%) controls. CONCLUSIONS: XPS determination of residual protein deposits is a novel and direct evaluation method for determining the cleaning efficacy of RGP lens care solutions for single lenses.

Effects of RGP lens extended wear on glucose-lactate metabolism and stromal swelling in the rabbit cornea.

PURPOSE: To assess the chronic effects of rigid gas-permeable (RGP) contact lenses on corneal swelling and glucose-lactate metabolism in the rabbit cornea during 1 month of continuous extended wear and to establish the relationship between these effects and the oxygen transmissibility (Dk/L) of the test lens polymer. METHODS: Four RGP lenses of varying Dk/L were tested in 8 rabbits per test group (left eyes served as controls). After 7 days and 1 month extended wear, the concentrations of lactate and glucose in the corneal epithelium, stroma and aqueous humor were determined by enzyme assay; and epithelial and stromal ATP concentrations were separately measured by bioluminescence techniques. Corneal thickness was measured at a standard morning time by ultrasonic pachymetry before and after 1, 7, 15 days and 1 month extended wear. RESULTS: After 7 days and 1 month extended wear, generalized decreases were found in aqueous humor lactate levels for all test lenses, while concomitant increased aqueous glucose concentrations were observed. Total epithelial lactate levels correlated inversely with decreasing Dk/L levels for lower oxygen transmissible lenses (R = 0.951, P = 0.0051); and remained unchanged after extended wear of the hyper-oxygen transmissible Dk/L 125 test lens. By contrast, stromal lactate levels consistently decreased at all time points measured forextended wear of all test lenses. As expected, both epithelial and stromal ATP concentrations simultaneously decreased in extended wear. Overnight corneal swelling values after 24 hours wear of Dk/L = 27, 43, 70 and 125 test lenses were increased by 9.8, 7.1, 5.5, and 5.2% while persistent (residual) stromal swelling after one month extended wear was 16.8, 10.1, 8.6, and 5.6% respectively, in excess of baseline values. CONCLUSIONS: Chronic RGP contact-lens induced hypoxia is associated with altered glucose-lactate metabolism in the cornea and aqueous humor with excess production of increased levels of lactate in the epithelium for lower Dk/L test lenses, but decreased lactate concentration in the stroma and aqueous humor. Extended wear of the hyper-oxygen transmissible test lens (Dk/L = 125) however, produced no increase in epithelial lactate levels. Expected lens-induced decreases in epithelial and stromal ATP were not dependent on lens-oxygen transmissibility. Despite the persistence of lower than normal stromal levels of lactate during 1 month of extended wear for all test lenses, residual corneal swelling values remained consistently elevated above baseline values. Taken together, these data establish that increased stromal lactate accumulation cannot account for persistent stromal edema in chronic extended wear of RGP lenses; and that this effect appears to be independent of lens-oxygen transmissibility and may thus represent the prolonged mechanical effect of lens wear itself.

Fluorophotometric assessment of rabbit corneal epithelial barrier function after rigid contact lens wear.

PURPOSE: The effect of overnight wear of graded rigid gas-permeable (RGP) contact lenses on the rabbit corneal epithelial barrier function was assessed by fluorophotometry. METHODS: RGP lenses with optimal fitting were worn by rabbits for 24 h. The values of oxygen transmissibility (Dk/L) of lenses used were 0, 10, 27, 43, 84, and 109 x 10(-9) (cm/s)(ml O2/ml mm Hg); lens thickness (0.15 mm) and diameter (14.0 mm) were standardized. Corneal epithelial barrier function was measured by a slit-lamp fluorophotometer. Corneal surface lesions by fluorescein staining were observed by a slit-lamp biomicroscope with the aid of fluorescein filter paper and were scored. Corneal epithelial structure was observed by scanning electron microscopy (SEM). RESULTS: After 24-h wear of RGP lenses, fluorescein uptake by the cornea increased, and this increase depended on the Dk/L value of lenses worn; mean +/- standard error, 1,601+/-454 (Dk/L 0), 347+/-226 (Dk/L 10), 144+/-42 (Dk/L 27), 100+/-25 (Dk/L 43), 56+/-18 (Dk/L 84), 51+/-15 (Dk/L 109), and 49+/-4 ng/ml (control). There was a significant correlation between the fluorophotometric value and the biomicroscopic scores of corneal lesions. SEM showed progressive desquamation of epithelial superficial and wing cells for lower-value Dk/L lens groups. CONCLUSION: This study demonstrated for the first time that overnight wear of Dk/L 84 and 109 lenses does not alter the barrier function of the rabbit corneal epithelium. In contrast, wear of a PMMA lens greatly alters the corneal epithelial barrier function. It is suggested that RGP lenses with a Dk/L >80 can provide safe overnight wear, based on the epithelial barrier function.

Determination of oxygen tension on rabbit corneas under contact lenses.

PURPOSE: To determine oxygen tension (PO2) on rabbit corneas beneath rigid gas permeable (RGP), hydrogel, and silicone elastomer lenses under open- and closed-eye conditions and to demonstrate the relationship between PO2 and overnight corneal swelling response in the rabbit model. METHODS: An improved PO2 monitoring system (PO-2080) with a platinum-micro-wire-electrode was used to measure PO2. An ultrasonic pachymeter (DGH-2000) was used to measure corneal thickness after overnight wear. RESULTS: The relationship between PO2 and oxygen transmissibility (Dk/ L) of the contact lens was linear for Dk/L between 0 and 70 x 10(-9) (cm/ sec)(mLO2/mL x mmHg). For Dk/L greater than 70 x 10(-9), PO2 gradually reached a plateau at 120 mmHg for open-eye conditions and 20 mmHg for closed-eye conditions. PO2 was inversely related to the overnight corneal swelling, ranging from 5.1% swelling at PO2 113.5/17.5 mmHg (open/closed-eye) for a hyper Dk/L lens (125 x 10(-9)) to 15.1% swelling at PO2 10.4/5.1 mmHg for a low Dk/L lens (11.5 x 10(-9)). CONCLUSIONS: Polarographic determination of PO2 provides reliable information about the amount of oxygen available to the cornea under a lens for both open-eye and closed-eye conditions. The data demonstrate that it is not possible to achieve normal oxygen levels with contact lens wear, even when hyper Dk/L lenses are worn.

Effects of rigid lens extended wear on lactate dehydrogenase activity and isozymes in rabbit tears.

Effects of and recovery from continuous wear of four rigid gas permeable (RGP) contact lenses was assessed by noninvasive measurement of lactate dehydrogenase (LDH) activity and isozyme pattern in rabbit tears. Oxygen transmissibility (Dk/L) of lenses used was 27, 44, 84, and 97 x 10(-9) (cm/s)(ml O2/ml mm Hg); lens thickness (0.15 mm) and diameter (14.0 mm) were standardized. Lenses were worn continuously for 90 days; recovery was assessed 30 days after cessation of lens wear. LDH activity was measured by UV rate assay; isozyme subtypes were determined by agarose gel electrophoresis. Light and scanning electron microscopy (LM, SEM) were used with the determination of total protein as additional measures of lens effects. LDH levels were inversely correlated with lens Dk/L values; low Dk/L values increased the anaerobic (LDH4,5)/aerobic (LDH1,2,3) subtypen ratio indicating in vivo metabolic shift. SEM observations were consistent with these results. There was no significant difference in the total cell content of tears or total tear protein levels between control and RGP test-wear groups. Measurement of tear LDH activity and isozyme ratios appears to provide a sensitive, noninvasive assessment of the effects of RGP lens-induced hypoxia over time on the corneal surface. A level of Dk/L of > or = 84 appears best for maintaining corneal physiology during extended wear. Recovery from chronic lens-induced hypoxia is characterized by a return to normal tear LDH levels and isozyme subtypes.

Laser and tandem scanning confocal microscopic studies of rabbit corneal wound healing.

The process of corneal endothelial wound healing was studied using laser and tandem scanning confocal microscopy (LSCM and TSCM). Following transcorneal freeze (TCF) injury, rabbit corneas were observed using ex vivo LSCM and in vivo TSCM. LSCM revealed the intracellular actin filament organization which, stained with phalloidin-FITC, in migrating endothelial cells, transformed fibroblast-like cells, stroma keratocytes, and epithelial cells during wound healing in corneal tissue. The TSCM provided sequential spatial observation of morphologic changes from endothelium to epithelium of the cornea during in vivo cellular repair of wound healing noninvasively on the same cornea without animal sacrifice. Ex vivo LSCM supported the morphologic analysis of the in vivo TSCM observations.

The effects of daily wear of rigid gas permeable contact lenses treated with contact lens care solutions containing preservatives on the rabbit cornea.

We evaluated the effects on the rabbit cornea of daily wear of rigid gas permeable (RGP) contact lenses treated with preserved care solutions by measuring concomitant tear lactate dehydrogenase (LDH) activity followed by in vivo tandem scanning confocal microscopy (TSCM). In vivo morphologic changes were confirmed by in vitro scanning electron microscopy (SEM). Two standard commercial RGP lens wetting and soaking solutions from the same manufacturer were tested: solution A with 0.004% benzalkonium chloride (BAK) and solution B with 0.003% chlorhexidine digluconate (CHX) and 0.002% thimerosal. Two experimental PBS-based wetting and soaking solutions were also tested: solution C with 0.005% BAK and 2% hydroxypropylmethylcellulose (HPMC) and solution D with 0.005% BAK without HPMC. Instillation of solution A without contact lens wear caused significant (P < 0.01) increases in desquamation of the superficial corneal epithelium and tear LDH activity compared with control eyes. After 3 weeks of RGP contact lens daily wear (8 hours/day), modified Draize scores of ocular surface lesions on the eyes wearing RGP lenses treated with solution A increased according to the duration of lens wear. Solution B did not produce significant change. With daily wear for 4 days (8 hours/day), RGP lenses treated with solution C and solution D produced increased corneal epithelium desquamation and an increase of LDH activity in tears. These effects were greater with HPMC (solution C) than without HPMC (solution D).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo osmotic pertubation of intercellular fluid channels in the rabbit corneal endothelium.

An in vivo rabbit corneal model was used to evaluate morphological changes in the corneal endothelium associated with osmotically increasing fluid movement from the anterior chamber into the stroma. When the corneal stroma is rendered more hypertonic than normal by immersing the scraped epithelial side of the cornea in a hypertonic sucrose solution, intercellular channels and apical pores at the Y-junctions between endothelial cells become greatly enlarged. The foregoing changes are reversible and do not appear to result in damage to the corneal endothelium. These observations suggest that specific intercellular channels in the corneal endothelium may provide pathways for the movement of fluid from the aqueous humor into the stroma.

In vivo confocal microscopic studies of endothelial wound healing in rabbit cornea.

Corneal endothelial wound healing in living rabbit eyes after mechanical scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on the central corneal endothelium with an olive tip cannula; TCF injury was created using a 3-mm-diameter stainless steel probe cooled with liquid nitrogen. In vivo observation of wound healing using TSCM was correlated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed lamellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showing wide spaces between nuclei by TSCM. After 28 days, resurfaced endothelial cells showed normal hexagonal mosaic appearance with enlarged cells by TSCM and SEM. TCF injury produced fibroblastic changes in the endothelial cells with elongation and spreading by 24 h after injury. After 3 days, the wounded area was resurfaced with two cell types: (a) migrating endothelial cells at the peripheral area, which appeared polygonal in shape with wide intracellular spaces and (b) fibroblast-like cells at the center of the wound, which formed a retrocorneal fibrous membrane (RCFM). The RCFM was posteriorly covered with normal endothelium after 28-60 days. TSCM of the stroma demonstrated spindle-shaped, activated keratocytes migrating into the wounded stroma at 3-14 days. In conclusion, TSCM allows viewing of dynamic four-dimensional morphologic changes (x, y, z, and time) during in vivo cellular repair of corneal wound healing after either MS or TCF injury.

Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries.

PURPOSE: To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair. METHODS: Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM. RESULTS: At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous membrane was posteriorly covered with normal endothelium. CONCLUSIONS: These data support the hypothesis that endothelial wound healing involves two separate, injury-dependent, mechanisms--cell spreading and cell migration.

Morphological and biochemical evaluation for rigid gas permeable contact lens extended wear on rabbit corneal epithelium.

We studied the effects of 24-hour wear of rigid gas permeable (RGP) contact lenses of varying oxygen transmissibilities on the rabbit cornea by measuring concomitant lactate dehydrogenase (LDH) activity in tears and by in vivo tandem scanning confocal microscopy (TSCM). We used a PMMA lens and rigid gas permeable (RGP) lenses that had Dk/L values ranging from 7 to 64 x 10(-9) (cm/sec) (mL O2/mL mmHg) and a uniform 0.15 mm thickness. After 6- and 24-hour contact lens wear, rabbit tear LDH activity increased according to the decrease in the Dk of RGP lenses. Tear LDH activity after 24 hours of lens wear was higher than after 6 hours. The observed increase in tear LDH activity was correlated with in vivo corneal epithelial morphology by tandem scanning confocal microscopy. The observed severity of desquamation and swelling of corneal epithelial cells was dependent upon the Dk/Ltotal of contact lenses worn, which directly related to the contact lens induced corneal hypoxia. Based on the results of this study, we conclude that: 1) a nap or accidental overnight wear of contact lenses with less than 20 x 10(-9) Dk/Ltotal could cause severe corneal epithelial damage; 2) the ultra high Dk lens appeared to alter the ocular surface least; and 3) TSCM accompanied with tear LDH assay is an objective, non-invasive in vivo method to assess the effect of contact lens wear on the ocular surface over time at the cellular level.

A quantitative method for LDH, MDH and albumin levels in tears with ocular surface toxicity scored by Draize criteria in rabbit eyes.

We have established a quantitative method for evaluation of ocular surface lesions on the basis of lactate dehydrogenase (LDH) activity, malate dehydrogenase (MDH) activity, and albumin levels in rabbit tears. Lesions were produced with solutions of 0.005-0.02% benzalkonium chloride (BAK), 0.01-0.03% chlorhexidine digluconate (CHX), and 0.01-0.03% polyhexamethylene biguanide hydrochloride (PHMB), all of which are widely used in topical ophthalmic preparations. Two drops of test solution were instilled 15 times into rabbit eyes at 5 minute intervals. Sixty minutes after 0.02% BAK instillation, tear LDH activity increased from 1,840 U/L (without instillation) to 26,100 U/L, and concomitantly tear albumin levels rose from 0.11 mg/mL (without instillation) to 9.48 mg/mL. Instillation of 0.03% CHX and 0.03% PHMB caused smaller increases in LDH and MDH activity and albumin tear levels. LDH activity and albumin levels in tears were significantly correlated with the degree of total ocular surface lesions in both cornea and conjunctiva as observed by slit lamp biomicroscopy quantified using a modified Draize score. Based on the results of this study, we believe that LDH activity and albumin level in tears can be used as objective indicators for the quantitative evaluation of ocular surface lesions on both cornea and conjunctiva following application of topical ophthalmic preparations containing cytotoxic preservatives in animals and man.

In vitro evaluation of biocompatibility of surface-modified poly(methyl methacrylate) plate with rabbit lens epithelial cells.

Collagen type I was immobilized onto a poly(methyl methacrylate) (PMMA) plate by covalent bonding following surface modification by two methods. One method introduced amino groups by aminolysis with N-lithioethylenediamine (PMMA-NH2) and the other introduced carboxyl groups by graft copolymerization of acrylic acid (AAc) and acrylamide (AAm) (PMMA-COOH). Lens epithelial rabbit cells were cultured on the PMMA plate which was immobilized with collagen. Polygonal cells with a mosaic appearance were observed on the PMMA-COOH plate immobilized with collagen type I, whereas pleomorphic cells were present on the virgin PMMA and on the PMMA-NH2 plate immobilized with collagen type I. We concluded the PMMA-COOH plate immobilized with collagen type I provided a more comfortable atmosphere for lens epithelial cells, causing no metaplasia, than the other plates used in this cell culture model experiment.

Effects of increasing Dk with rigid contact lens extended wear on rabbit corneal epithelium using confocal microscopy.

The effects of 24-h wear of various Dk-rigid gas-permeable (RGP) contact lenses on the rabbit corneal epithelium were studied by in vivo tandem scanning confocal microscopy (TSCM), and confirmed by scanning electron microscopy (SEM). Lenses used were polymethylmethacrylate (PMMA) (Dk/L = 0), RGP experimental A lens (siloxanylmethacrylate-fluoromethacrylate-methylmethacrylate , 33), experimental B (siloxanylmethacrylate-fluoromethacrylate, 56), and experimental C (siloxanylstyrene-fluoromethacrylate copolymer, 64 x 10(-9)) (cm/s) (ml O2/ml mm Hg) with 0.15-mm thickness (Dk/L measured by polarograph including boundary layer effect). After 24-h PMMA lens wear, TSCM showed no superficial epithelial cells but only exposed, underlying wing cells. The cornea with experimental A showed partial superficial epithelial desquamation. With experimental B wear, slight superficial epithelial cell swelling and desquamation were observed on the surface of the cornea. No changes were observed for the eye with experimental C and control. The observed severity of desquamation of superficial epithelial cells was dependent on the oxygen transmissibility (Dk/L) of RGP lenses worn. All in vivo findings were confirmed by SEM observations. Based on the results of this study, we conclude that (a) although Dk/L = 56 lens B shows no residual overnight corneal swelling, surface damage is still produced; (b) Dk/L = 64 lens C is best for epithelium showing the same corneal images as control; and (c) TSCM is a good way to evaluate the contact lens safety and efficacy in vivo at the cellular level noninvasively.

Confocal microscopic studies of living rabbit cornea treated with benzalkonium chloride.

The effects of benzalkonium chloride (BAK) on the living rabbit cornea were studied by in vivo Tandem scanning confocal microscopy (TSCM) and confirmed by conventional scanning electron microscopy (SEM). Two drops of saline or phosphate-buffered saline (PBS) containing BAK in concentrations of 0.02, 0.01, and 0.005% was applied to rabbit eyes 15 times at 5-min intervals. The solutions were pH 5.5-5.9 (saline) and pH 7.5 (PBS), and osmolarity was 275-280 (saline) and 300-307 mOsm (PBS). Immediately after application of 0.02 and 0.01% BAK, no normal corneal superficial epithelial cells could be imaged by in vivo TSCM. No swelling of the superficial epithelial cells was observed for the control solution without BAK; however, there was a small amount of desquamation. Application of as little as 0.005% BAK caused the superficial epithelial cells to swell and desquamate. The observed desquamation of corneal superficial epithelial cells increased with higher BAK concentrations applied to the eye. One hour after final drug application, inflammatory cells appeared on the surface of the cornea treated with 0.02% BAK. These findings were correlated with SEM observations. Based on the results of this study, we believe that BAK used frequently can produce clinical corneal toxicity and that the cytotoxicity of any topical ophthalmic solutions can be evaluated by in vivo TSCM.

Effect of contact-lens-induced hypoxia on lactate dehydrogenase activity and isozyme in rabbit cornea.

Lactate dehydrogenase (LDH) levels and subunit isozyme patterns in cornea were monitored in 36 albino rabbits wearing thick, rigid, gas-permeable contact lenses for periods of 24 h, 2 and 7 days, and 1 and 3 months. The oxygen transmissibility of the contact lens was 15.3 x 10(-9) (cm/s) (ml O2/ml mm Hg). The activity of LDH in corneal tissue decreased according to the duration of lens wear. The LDH isozyme patterns shifted with lens wear from LDH1,2,3 (heart type, aerobic) to LDH4,5 (skeletal muscle type, anaerobic). The cornea swelled 8.8% with overnight contact lens wear, with increased swelling (11-12%) after further continuous lens wear. After contact lens removal, however, the LDH activity and the isozyme pattern returned to normal, and the corneal thickness quickly returned to normal. Based on these observations, it is suggested that LDH in rabbit corneas was physiologically affected by lens-induced hypoxia, but these changes were reversible. These results might further suggest that tear LDH levels in the human contact lens wearer could provide an ongoing assessment of the tolerance of the lens by the ocular surface.

Tear lactate dehydrogenase levels. A new method to assess effects of contact lens wear in man.

Lactate dehydrogenase (LDH) activity in tears was measured in 202 myopic rigid gas permeable (RGP) and 79 hydrogel human contact lens wearers and 48 normal controls by noninvasive microcapillary sampling. The oxygen permeabilities (Dk) of five selected RGP contact lenses ranged between 0 and 230 x 10(-11) (cm2/s) ml O2/ml mm Hg), and the water content (WC) of the hydrogel lenses was 38, 72, and 80%. When normal diurnal variation of tear LDH activity and tear sample volume (0.3-0.6 microliters) were carefully controlled, the tear LDH activity of RGP lens wearers in daily and extended wear correlated as an inverse function of Dk/L, with the highest enzyme activity observed in wearers of polymethylmethacrylate daily wear lenses (347.4 U/L). The tear LDH activity in Menicon EX (Dk 108) lens wearers was higher in the extended wear (192.7 U/L) than in the daily wear group (161.9 U/L). There was no significant difference in tear LDH activity between the Menicon SF-P (Dk 230) extended wear group (132.0 U/L) and controls (133.5 U/L). In the hydrogel lens daily wear group, tear LDH activity of Experimental 72 (WC 72%) and Experimental 80 (WC 80%) was higher than that of hydroxyethylmethacrylate (HEMA) despite having high Dk value. Experimental 80 extended wearers showed lower LDH activity in tears sampled between 0.3 and 0.6 microliters than that of HEMA wearers. These results suggest that sequential measurement of LDH levels in tears may offer a new and unique method for the assessment of the physiologic effects of contact lens wear on the ocular surface, and provide a new clinical paradigm for the interaction of the contact lens with the cornea.

Changes of lens crystallins photosensitized with tryptophan metabolites.

The effect of UV irradiation on bovine lens soluble proteins (crystallins) in the presence of tryptophan metabolites was investigated in vitro. The cross-linking of crystallins by UV irradiation was accelerated by kynurenine, 3-hydroxykynurenine, anthranilic acid and 3-hydroxyanthranilic acid. On denaturation of crystallins by photooxidation, alpha-crystallin was characterized by the formation of water-soluble HMW (high molecular weight) protein, while water-insoluble HMW protein was produced from beta- and gamma-crystallin. These HMW aggregates showed cross-linking by non-disulfide covalent bonds. LMW (low molecular weight) peptides were formed by degradation of alpha- and beta-crystallin.