Brunescent Cataract Research Articles

Cataract surgery in eyes with iridoschisis using the Perfect Pupil iris extension system

To evaluate the use of the Perfect Pupil system (Milvella Pty. Ltd.) in 3 eyes in cataract surgery with iridoschisis. Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany. Patient 1 was an 83-year-old man with a nuclear cataract, pseudoexfoliation syndrome, and iridoschisis. He was also having systemic anticoagulation therapy. Preoperative best corrected visual acuity (BCVA) was 0.05 in the right eye and 0.08 in the left eye. Patient 2 was a 73-year-old mentally retarded man with a brunescent cataract in the right eye. Preoperative BCVA was 0.1. In the first patient, cataract surgery was performed under topical anesthesia in both eyes through a clear corneal incision. Intraoperative pupil size was 3.0 mm. The Perfect Pupil device was inserted manually and fixated at the iris rim, leading to pupil dilation of 6.0 mm. Uneventful cataract surgery was performed without damage to the iris. Hydrophobic acrylic foldable intraocular lenses (IOLs) were implanted with forceps. Postoperative BCVA was 0.5 in the right eye and 0.4 in the left eye. The second patient was operated on under general anesthesia. Pupil dilation was successfully achieved. The brunescent cataract was removed and a hydrophobic acrylic IOL was implanted with an injector, resulting in postoperative uncorrected visual acuity of 0.3. In iridoschisis patients, there is risk of aspiration of iris fibers during cataract surgery. In these 2 patients, this was prevented using the Perfect Pupil system.

K2P—A Novel Cross‐Link from Human Lens Protein

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC-MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure as being 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues and a five-carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of water-soluble [WS] protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross-link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

The aqueous humour antioxidative capacity in different types and color of the age-related cataract

Oxidative stress results from increased oxidative processes, decreased antioxidative protection, or both processes simultaneously. Photooxidative stress, as a form of oxidative stress, induced by the energy of solar radiation, today is considered as crucial in the age-related cataractogenesis. Other known and unknown, endogenous and egsogenous factors that contribute to the oxidative stress intensity, can influnce the cataract type and brunescence. Thus the oxidative stress intensity and its form might determine the cataract type and brunescence, and also make the efforts in cataract prevention more complex. Hence, the objective of the present paper was to investigate the current amount of antioxidative capacity in aqueous humour during the cataract genesis of different types and pigmentation of cataract. Transversal review of 80 samples of humour aqueous obtained during extracapsular cataract extraction. Aqueouses were analyzed by tiobarbituric acid (TBA) method for the total antioxidant activity estimation, expressed as %iMDA, and by using 0.1 ml of aqueous. The mixed type of cataract showed the statistically significantly lower values of the intensities of antioxidative protection in aqueous humour compared to cortical and nuclear cataracts (p < 0.001, respectively). Between pure nuclear and cortical cataracts we found the small differences of the investigated parameter, but they pointed to the decreased level of antioxidative protection, i.e. the increased intensity of the aqueous humour oxidative stress in the cortical cataract type. A significant correlation betweer the cortical cataract maturation and the %iMDA (p < 0.05) was found. The role of the oxidative stress, here expressed as the antioxidative capacity of aqueous humour, could not be the same for all the cataract types. The lower level of antioxidative protection of aqueous in brunescent and mixed cataracts may point to the higher intensity of oxidative stress in those cataract types. The correlation betweer the cortical cataract maturity and %iMDA points to the significant decrease of the aqueous antioxidative protection in the cataract progression.

Late postoperative opacification of MemoryLens hydrophilic acrylic intraocular lenses Case series and review

Purpose: To report clinical and spectrographic analyses of 2 explanted hydrophilic acrylic intraocular lenses (IOLs). Setting: John A. Moran Eye Center, Salt Lake City, Utah, USA, and Johannes Gutenberg-University, Department of Ophthalmology, Mainz, Germany. Methods: We report 6 cases of opacification of MemoryLens ® IOLs (Ciba Vision) that occurred approximately 2 years after implantation. The anterior and posterior surfaces of the IOLs had a white, frosted appearance, and the IOLs' interior looked brown, similar to the appearance of a brunescent cataract. Two of the IOLs were explanted because of a significant decrease in visual acuity. The IOLs were sent for further clinicopathologic analysis including scanning electron microscopy and energy dispersive x-ray spectroscopy (EDX). Results: Microscopic analysis revealed multiple, fine, granular deposits of various sizes on the surface of the lens optics. The EDX analysis showed the presence of calcium within the deposits. Conclusions: Our cases show that hydrophilic acrylic IOLs can opacify even years after implantation. Analysis of the explanted IOLs revealed calcification.

Separation of the yellow chromophores in individual brunescent cataracts

Quantitative changes in the 330 nm absorbing chromophores and 350/450 nm fluorophores of water-soluble (WS) and water-insoluble (WI) proteins of individual human cataract lenses were characterized and compared with aged normal human lens. Twenty-five brunescent cataract lenses from India were selected from five different stages (types I–V) based upon the color of the lens. The WS and WI proteins from each lens were collected and subjected to an extensive enzymatic digestion procedure under argon. The lens protein digests were separated by Bio-Gel P-2 size-exclusion chromatography and individual peaks were analyzed further by reversed-phase HPLC. The total WI proteins increased and the total WS protein decreased with the development of cataract, especially in the late stages of cataract (III–V). The total 330 nm absorbance and 350/450 nm fluorescence of the WI fraction also increased, however, the A 330 and fluorescence per mg lens protein were constant except for type V (black) lenses. Bio-Gel P-2 chromatography separated the chromophores and fluorophores into four fractions. The main fraction (designated as peak 2+3) from the cataract WI proteins was several times higher than that present in aged normal human lens WI proteins. A significant increase of this fraction was observed in WI proteins, but not in WS proteins with cataract development. Similarly, fractions 1 and 4 in the WI proteins also increased gradually but fraction 5 did not. Reversed-phase HPLC resolved fraction (2+3) of the water-insoluble sonicate supernatant proteins into four 330 nm absorbing peaks and eight fluorescent peaks. Among these peaks, a late-eluting peak (peak 8) increased 10 to 15-fold with the progress of cataract, and accounted for 80% of the total chromophores in type V lenses. This peak may represent limit digests of advanced glycation end-products (AGEs) derived protein cross-links. HPLC profiles of fraction 5 from both WS and WI proteins showed numerous new peaks which were not observed in either WS protein from cataract or WI proteins from aged normal human. The severe coloration and the higher levels of numerous novel chromophores and fluorophores in brunescent cataractous lenses reveal the possibility that a different chemistry occurs during cataract development.

Studies on Singlet Oxygen Formation and UVA Light-mediated Photobleaching of the Yellow Chromophores in Human Lenses

The protein-bound chromophores, which increase with aging in the human lens, act as UVA sensitizers, producing almost exclusively singlet oxygen in vitro. Direct irradiation of whole, aged human lenses with high intensity UVA light (200mWcm−2 for 24hr), however, failed to produce singlet oxygen damage, as evidenced by the lack of either His or Trp photodestruction. Total homogenates of human lenses prepared in a cuvette under air did show destruction of His and Trp residues by UVA light, but no destruction was seen when equivalent homogenates were prepared under argon. These data are consistent with the idea that the low oxygen levels in the lens prevent singlet oxygen damage in vivo.UVA irradiation of aged human lenses in culture caused an extensive photobleaching of the yellow chromophores. A time course indicated that the photobleaching increased with time, with significant color loss apparent after 6hr. Homogenization of the irradiated and dark control lenses in 6M guanidine–HCl, followed by determination of the difference spectrum, showed approximately 50% bleaching of compounds with a λmax at 355nm. Similarly, fluorophores with a λmax for excitation of 355nm and for emission of 420nm were 50% destroyed by the UVA light. Similar results were obtained in vitro by the anaerobic irradiation of a sonication-solubilized WI fraction from type II brunescent cataracts and from aged human lenses. In this system, there was an initial bleaching of 15% after 30min of irradiation, followed by a slow increase over the next 6hr to a final bleaching of 30%. The addition of 1.0m M ascorbic acid, but not 1.0m M glutathione (GSH), increased the photobleaching to 60% under argon, and the loss of ascorbate could be detected under these anaerobic conditions. In the presence of air, UVA light produced no photobleaching, but rather caused a three-fold increase in absorbance at 345nm, which was prevented by the inclusion of 1.0m M ascorbic acid and almost 50% inhibited by 1.0m M GSH.The data are consistent with the conversion of the triplet state of the sensitizers to anion and cation radicals in the absence of oxygen. Photobleaching may occur either by dismutation of the anion radical or by reduction of the anion radical by ascorbate via type I chemistry. UVA irradiation of an enriched fraction of sensitizers from a proteolytic digest from type II cataract lenses produced a 63% bleaching at 330nm in the absence of oxygen, and the almost complete loss of the A330 absorbing and 350/450nm fluorescent peaks upon HPLC separation. This loss correlated with the loss of the ability of the irradiated fraction to produce singlet oxygen in vitro upon subsequent UVA irradiation.

Role of xanthurenic acid 8-O-beta-D-glucoside, a novel fluorophore that accumulates in the brunescent human eye lens.

We have been able to identify a blue fluorophore from the low-molecular weight soluble fraction of human adult nondiabetic brunescent cataract lenses as xanthurenic acid 8-O-beta-D-glucoside (XA8OG) (excitation = 338 nm and emission = 440 nm). To determine the role of this fluorophore in the lens, we have examined its photophysical and photodynamic properties. We found XA8OG to have a fluorescence quantum yield (phi) of 0.22 and a major emission lifetime of 12 ns. We found it to be a UVA-region sensitizer, capable of efficiently generating singlet oxygen species but little of superoxide. We also demonstrated that XA8OG oxidizes proteins when irradiated with UVA light, causing photodynamic covalent chemical damage to proteins. Its accumulation in the aging human lens (and the attendant decrease of its precursor O-beta-D-glucoside of 3-hydroxykynurenine) can, thus, add to the oxidative burden on the system. XA8OG, thus, appears to be an endogenous chromophore in the lens, which can act as a cataractogenic agent.

Phacoemulsification of brunescent and black cataracts

Purpose: To evaluate the efficacy and safety of a step-by-step, chop in situ, lateral separation technique to remove brunescent and black cataracts. Setting: Iladevi Cataract and IOL Research Center, Ahmedabad, India. Methods: In this prospective study conducted between May 1997 and June 1998, 167 consecutive eyes were divided into 2 groups: Group 1, brunescent cataract (n = 123), and Group 2, black cataract (n = 44). Preoperative assessment included axial length (AL), slitlamp examination, corneal pachymetry, tonometry, and specular microscopy. During phacoemulsification performed by a single surgeon, a step-by-step, chop in situ, lateral separation technique was used to divide the nucleus. Intraoperatively, hydroxypropyl methylcellulose 2% was used and irrigation was by balanced salt solution (BSS®). Postoperatively, all eyes were assessed at 1, 7, 30, 90, 180, and 360 days. The results were evaluated using regression analysis, the chi-square test, and the Student t test. Results: The mean follow-up was 14.4 months (range 6 to 35 months) in Group 1 and 13.0 months (range 6 to 32 months) in Group 2. The AL was significantly greater in Group 2 ( P = .02). Corticapsular adhesions were present in 17.82% in Group 1 and 31.82% in Group 2. The mean cumulative dissipated energy was 2.03 and 3.12, respectively ( P = .0005). Wound site thermal injury occurred in 16 eyes (13.01%) in Group 1 and 4 eyes (9.09%) in Group 2. No serious intraoperative or postoperative complications were noted. One day postoperatively, the mean rise in intraocular pressure was 1.76 mm Hg in Group 1 and 4.15 mm Hg in Group 2 ( P = .012), and transient corneal edema was present in 24.40% and 34.10%, respectively. At 1 month, the endothelial cell loss was 10.06% in Group 1 and 9.22% in Group 2. Conclusion: The step-by-step, chop in situ, lateral separation technique was effective and did not produce serious complications such as zonulysis or posterior capsule rupture. However, the incidence of wound site thermal injury and endothelial cell loss was greater than after emulsification of standard cataracts.

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

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A 330 units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R f values for the long wavelength-absorbing fluorophores. Glycation with [U- 14C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A 330-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A 330-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A 330-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.

Comparison of Non-Tryptophan Fluorophores in Protein-Free Extract of Brunescent and Non-Brunescent Human Cataract

Purpose: To investigate whether any of the fluorophores in the human lens nuclei might be responsible for human brunescent cataract formation. Methods: Human lens nuclei (non-brunescent, from 13; brunescent, from 8) were obtained after extracapsular cataract extraction in nondiabetic patients. Protein-free extract, prepared by filtrating the water-soluble fraction of each nucleus through a centrifugal ultrafilter (molecular weight < 5,000), was analytically separated by high-performance liquid chromatography. Results: No significant differences between non-brunescent and brunescent nuclei were observed in the concentrations (mean ± SD) of 3-hydroxykynurenine O-β-glucoside (0.67 ± 0.38 vs. 0.85 ± 0.62 μmol/g wet weight), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside (4.1 × 10 5 ± 2.9 × 10 5 vs. 6.3 × 10 5 ± 5.0 × 10 5 area unit/g wet weight), and kynurenine (0.016 ± 0.011 vs. 0.029 ± 0.021 μmol/g wet weight). A novel fluorophore that has not been identified so far was significantly present more in brunescent than in non-brunescent nuclei (brunescent: 1.5 × 10 5 ± 1.0 × 10 5 vs. non-brunescent: 2.6 × 10 3 ± 6.3× 10 3 area unit/g wet weight, P < .01). Digestion of the protein-free extract with β-glucosidase eliminated the peak corresponding to the novel unidentified fluorophore. Conclusion: The present results imply a novel protein-unbound fluorophore, presumably a β-glucoside, might possibly be involved in brunescent cataract formation.

Chromatographic Comparison of the UVA Sensitizers Present in Brunescent Cataracts and in Calf Lens Proteins Ascorbylated in vitro

The water-insoluble (WI) fraction from aged human lenses contains yellow chromophoric sensitizers, which generate reactive oxygen species (ROS) when irradiated with UVA light. The WI proteins from type I to V brunescent cataract lenses were assayed for UVA-dependent superoxide anion synthesis. Rates varied from 8.4–15 nMol h−1mg protein−1, but there was no significant difference in specific activity between cataract types. When calf lens soluble proteins were incubated with ascorbic acid for 4 weeks and dialyzed, they were capable of generating 30–40 nMol h−1mg protein−1superoxide anion when irradiated with UVA light. Two preparations each of brunescent cataract WI proteins and bovine lens proteins ascorbylated in vitro were extensively digested with proteolytic enzymes and the released amino acids separated by normal phase HPLC. The elution profiles of the digests were very similar based upon the absorbance at 330 nm and fluorescence at 350 nm excitation/450 nm emission. Each peak was pooled and analyzed for the UVA-dependent generation of both superoxide anion and singlet oxygen. Every peak exhibited sensitizer activity, and the UVA-dependent ROS generation was roughly proportional to the absorbance at 330 nm. In addition, the ratio of superoxide anion to singlet oxygen generated was similar with both preparations. These data argue that it is the brown, fluorescent compounds which accumulate during aging and cataract formation that are responsible for the UVA-dependent ROS formation, and that these browning products may be similar to the advanced glycation endproducts produced by ascorbylation of lens proteins under oxidative conditions. This work also presents an initial report of a chromatographic method to separate the UVA-sensitizers present in each of these protein preparations without the use of acid or base hydrolysis.

Spontaneous Generation of Superoxide Anion by Human Lens Proteins and by Calf Lens Proteins Ascorbylated In Vitro

The proteins isolated from aged human lenses and brunescent cataracts exhibit extensive disulfide bond formation. Diabetic rat lenses similarly contain disulfide-bonded protein aggregates. These observations are consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process. To assess whether the glycation-related modifications in human lens proteins spontaneously generate ROS, superoxide anion formation was measured using both cataractous lens proteins and calf lens proteins glycated in vitro with ascorbic acid (ascorbylated).The water-insoluble fraction from aged normal human lenses generated 0.3–0.6 nmol superoxide h−1mg protein−1, whereas the activity increased to 0.5–1.8 nmol h−1mg protein−1with the WI fraction from brunescent cataracts, and 2.3 nmol h−1mg protein−1with calf lens proteins ascorbylated for 4 weeks in vitro. The activity in the human lens proteins was observed in both the water-soluble and water-insoluble fractions, and was completely dependent upon the presence of oxygen. The pH optimum curve for superoxide formation increased from pH 6.5 to 10 with both the cataract and ascorbylated proteins. The superoxide-generating activity in human lens was completely bound to a boronate affinity column, but only partially bound with the ascorbylated proteins.The superoxide anion produced by a 5 m m solution of purifiedNϵ -fructosyl–lysine was barely detectable, and therefore, could not account for the superoxide formed by any of the lens protein preparations. Also, superoxide formation increased 10-fold at pH 8.8 with fructosyl–lysine, but only 1.3–1.8-fold with human lens proteins. The addition of copper-stimulated superoxide formation with glycated bovine serum albumin, but no stimulation was seen with cataractous proteins. Assays of specific compounds showed that catechol, hydroquinone, 3-OH kynurenine and 3-OH anthranylic acid exhibited the greatest activity for superoxide generation, but had a very short halflife. 2,3-Dihydroxypyridine and 4,5 dihydroxynaphthalene were one and two orders of magnitude less reactive.In long-term incubations at 37°, cataractous proteins retained the potential to produce superoxide anion, losing only half of the initial activity after 6–7 days. Therefore, the water-insoluble fraction from aged human lenses and dark brown cataracts are potentially capable of generating >100 nmol mg protein−1and >170 nmol mg protein−1of superoxide anion respectively, likely due to the presence of advanced glycation endproducts in human lens proteins. This spontaneous generation of superoxide anion in vivo could account for a major portion of the oxidation of sulfur amino acids seen during aging and cataract formation.

Singlet Oxygen Production Correlates with His and Trp Destruction in Brunescent Cataract Water-Insoluble Proteins

Singlet Oxygen Production Correlates with His and Trp Destruction in Brunescent Cataract Water-Insoluble Proteins

Phacoemulsification with a bevel-down phaco tip: Phaco-drill

We present a technique for in situ lens nucleus emulsification using low phaco power and high vacuum, a continuous curvilinear capsulorhexis, and hydrodelineation. Emulsification is done with the phaco tip slanted down 30 or 45 degrees. Cutting and aspiration do not cause an undesirable energy loss. This technique can be combined with the nuclear chopping or divide and conquer methods because of its ability to drill and hold the nucleus. Posterior capsular rupture is prevented because the separated epinucleus acts as a barrier between the nucleus and the cortex. The low power used minimizes the energy transfer to the corneal endothelium. This technique is particularly useful in eyes with brunescent cataract.

Calcium ATPase activity and membrane structure in clear and cataractous human lenses

PURPOSE. Maintenance of calcium homeostasis is imperative for the clarity of the lens. Ca 2+ -ATPase is essential for the removal of cytosolic calcium, either across the plasma membrane or through intracellular organelles such as the endoplasmic reticulum. In this study, membranes prepared from clear lens epithelium were compared to membranes prepared from cataractous lens epithelium. METHODS. Human lens membranes were prepared by a protocol utilizing homogenization and centrifugation. Ca 2+ -ATPase activity was measured biochemically using Gamma- 32 P labeled ATP. Lipid order was measured using infrared and Raman spectroscopy. RESULTS. Ca 2+ -ATPase activity was similar in membranes prepared from cataractous lenses that were classified as nuclear subcapsular, nuclear and brunescent cataracts. Ca 2+ -ATPase activity was approximately 50% less in membranes prepared from cataractous lenses in comparison to clear lenses. Because clear lenses from Indian donors was unavailable, clear human lenses were used as a qualitative control for the measurement for Ca 2+ -ATPase activity. Lipid order was measured in lens fibers from cataractous and clear lenses from the United States donors. Lipid order increased from 55% in the hydrocarbon chains from clear lens fibers to 84% in cataractous lens fibers. CONCLUSIONS. These findings support the hypothesis that membranes are deranged in cataractous tissue, which should lead to altered levels of calcium.

Racemization of tyrosine in the insoluble protein fraction of brunescent aging human lenses.

With increase in age, proteins of the eye lens undergo covalent modifications associated with interprotein cross-linking, loss of protein solubility, generation of protein-associated pigments, and racemization of some optically active residues. Precipitated protein aggregates localized in the nucleus of brunescent lenses are modified to such an extent that they become resistant to the commonly used methods of extraction by reducing agents or detergents. Thus, little information is available on the types of protein modifications that occur in the nuclear region of the lens. Here we report the simultaneous solubilization and digestion of the entire nuclear pellet of brunescent human cataract lenses into constituent amino acids and their derivatives through protease action at neutral pH. Fractionation of the resulting hydrolyzate by reverse phase chromatography, followed by spectroscopic and biochemical analysis of one of the purified fractions, showed the presence of racemic tyrosine in the amino acid mixture. Racemization of L-amino acid residues in protein chains is associated with the aging process. This is the first report of racemization of tyrosine in any system studied so far.

Protein-associated pigments that accumulate in the brunescent eye lens: Identification of a quinoline derivative

Brunescent (dark brown) cataract is particularly prevalent in the tropics. Enzymatic digestion of the insoluble protein fraction of brunescent cataractous eye lenses from India, followed by high performance liquid chromatographic separation of the pigments and spectroscopic investigations, have led to the identification of one of the pigments as 4-hydroxyquinoline-3-[α-aminoacetic acid] (compound A). The 4-hydroxyquinoline moiety is shown to be a photodynamic agent that generates O •− 2 and leads to protein crosslinking. This suggests that the compound A may play a long-term deleterious role in situ in the lens.

Regional distribution of oxidant scavengers in human brunescent cataracts from India

Regional distribution of oxidant scavengers in human brunescent cataracts from India

Pentosidine: A molecular marker for the cumulative damage to proteins in diabetes, aging, and uremia

Collagen undergoes progressive browning with age and diabetes characterized by yellowing, fluorescence, and cross-linking. The present research was undertaken in order to investigate the nature of the collagen-linked fluorescence. Human collagen was exhaustively cleaved into peptides by enzymatic digestion. Upon purification, a highly fluorescent chromophore was identified and purified from old human collagen. Structure elucidation revealed the presence of an imidazo [4,5-b] pyridinium-type structure acting as a cross-link between arginine, lysine, and a pentose. This advanced glycosylation end-product and protein cross-link results from the reaction of pentoses with proteins and was named pentosidine. Further work indicated that long-term glycosylation of proteins with hexoses also leads to pentosidine formation through sugar fragmentation. The proposed mechanism of pentosidine formation involves the dehydration of the pentose-derived Amadori compound to form an intermediate which is attacked under base catalysis by the guanido group of arginine. The strict requirement for the Amadori rearrangement is uncertain. However, oxidation is definitely involved since pentosidine is not formed in the absence of oxygen. Five-carbon sugars contributing to pentosidine formation could be formed from larger sugars by oxidative fragmentation or from trioses, tetroses, and ketoses by condensation and/or reverse aldol reactions. Pentosidine increases exponentially in human skin at autopsy. Mean age-adjusted skin levels were significantly increased in subjects with uremia and especially in type 1 diabetics with uremia vs. controls. In skin biopsy, levels were significantly elevated in all diabetic (type 1) vs. control subjects. The highest degree of association was with the cumulative grade of diabetic complication (retinopathy, nephropathy, arterial stiffness, and joint stiffness). Pentosidine also forms in various proteins other than collagen, although to a much lesser extent. In blood, pentosidine is mainly associated with plasma proteins and is highly elevated during uremia. In the lens, it is associated with both water-soluble and -insoluble protein fractions and is especially elevated during brunescent cataract formation. The origin of pentosidine in vivo is uncertain. Evidence suggests that the pentoses are the most reactive sugars in pentosidine formation in vitro; however, the origin and importance of free pentoses in vivo, especially during the diabetic state, are not certain. Possible origins include hemolysis and/or a defect in the primary pentose metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of pigmentation on glutathione redox cycle antioxidant defense in whole as well as different regions of human cataractous lens

‘Reactive oxygen species’, generated within the lens, are implicated in the deepening of nuclear pigmentation leading to browning of the human cataractous lens. The present study, which was carried out in senile brunescent cataracts, deals with changes in the antioxidant glutathione redox cycle defense system during the progression of browning (yellow to brown) in different regions of the human lens. A significant reduction in the levels of reduced glutathione was noted in the cortical and nuclear regions of the brown lens as compared to yellow lens, despite normal glutathione reductase activity. The rate limiting enzyme of the hexosemonophosphate shunt pathway, glucose 6-phosphate dehydrogenase, and the important oxidant scavenger enzyme, glutathione peroxidase, showed significant changes in opposite directions (glucose 6-phosphate dehydrogenase increased and glutathione peroxidase decreased), only in the cortical region of the brown lens as compared to yellow lens. The significance of the present findings in relation to the overall biochemical mechanisms underlying browning of the lens tissue in human cataracts is discussed.