Human Cataractous Lenses Research Articles

A Rapid, Comprehensive Liquid Chromatography-Mass Spectrometry (LC-MS)-based Survey of the Asp Isomers in Crystallins from Human Cataract Lenses

Cataracts are caused by clouding of the eye lens and may lead to partial or total loss of vision. The mechanism of cataract development, however, is not well understood. It is thought that abnormal aggregates of lens proteins form with age, causing loss of lens clarity and development of the cataract. Lens proteins are composed of soluble α-, β-, and γ-crystallins, and as long lived proteins, they undergo post-translational modifications including isomerization, deamidation, and oxidation, which induce insolubilization, aggregation, and loss of function that may lead to cataracts. Therefore, analysis of post-translational modifications of individual amino acid residues in proteins is important. However, detection of the optical isomers of amino acids formed in these proteins is difficult because optical resolution is only achieved using complex methodology. In this study, we describe a new method for the analysis of isomerization of individual Asp residues in proteins using LC-MS and the corresponding synthetic peptides containing the Asp isomers. This makes it possible to analyze isomers of Asp residues in proteins precisely and quickly. We demonstrate that Asp-58, -76, -84, and -151 of αA-crystallin and Asp-62 and -96 of αB-crystallin are highly converted to lβ-, dβ-, and dα-isomers. The amount of isomerization of Asp is greater in the insoluble fraction at all Asp sites in lens proteins, therefore indicating that isomerization of these Asp residues affects the higher order structure of the proteins and contributes to the increase in aggregation, insolubilization, and disruption of function of proteins in the lens, leading to the cataract.

DNA damage in lens epithelium of cataract patientsin vivoandex vivo

DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.

Discrepant Expression of MicroRNAs in Transparent and Cataractous Human Lenses

Age-related cataracts are considered to be a pathological condition that arise as senescence progresses. However, little is known about the function of microRNAs (miRNAs) in the formation of age-related cataracts. The purpose of this study was to identify possible differences in miRNA expression in the central epithelium of transparent and age-related cataractous human lenses. Microarrays were used to determine the miRNA expression profiles of both transparent and cataractous lenses. The results were analyzed by significance analyses performed by the microarray software, and the results were confirmed by stem-loop RT-PCR. Algorithms were used to predict the target genes of the differentially expressed miRNAs. Two hundred and six miRNAs were identified in all human lenses. The top eight miRNAs according to expression levels were miR-184, let-7b, miR-923, miR-1826, miR-125b, miR-1308, miR-26a, and miR-638 in transparent lenses. In contrast, the top eight miRNAs in cataractous lenses were miR-184, miR-1826, let-7b/c, miR-24, miR-23b, miR-923, and miR-23a. The expression levels of 20 miRNAs were increased and the levels of 12 miRNAs were decreased by more than 2-fold in transparent lenses relative to the levels in cataractous lenses. These findings were confirmed by stem-loop RT-PCR. In addition, several genes that were predicted to be targets of the identified miRNAs have been reported to be involved in lens development or cataract formation. The authors report, for the first time, the distinct expression profiles of miRNAs in the central epithelium of transparent and age-related cataractous human lenses. Significant differences in miRNA expression were identified, and the genes targeted by the relevant miRNAs were predicted. The differential expression of miRNAs suggests that these miRNAs have potential roles in lens development and/or cataract formation.

Racemization of Two Proteins over Our Lifespan: Deamidation of Asparagine 76 in γS Crystallin Is Greater in Cataract than in Normal Lenses across the Age Range

Long-lived proteins are widespread in man, yet little is known about the processes that affect their function over time, or their role in age-related diseases. Racemization of two proteins from normal and cataract human lenses were compared with age using tryptic digestion and LC/mass spectrometry. Asp 151 in αA crystallin and Asn 76 in γS crystallin were studied. Age-dependent profiles for the two proteins from normal lenses were different. In neither protein did the modifications increase linearly with age. For αA crystallin, racemization occurred most rapidly during the first 15 years of life, with approximately half of L-Asp 151 converted to D-isoAsp, L-isoAsp, and D-Asp in a ratio of 3:1:0.5. Values then changed little. By contrast, racemization of Asn 76 in γS crystallin was slow until age 15, with isoAsp accounting for only 5%. Values remained relatively constant until age 40 when a linear increase (1%/year) took place. When cataract lenses were compared with age-matched normal lenses, there were marked differences in the time courses of the two crystallins. For αA crystallin, there was no significant difference in Asp 151 racemization between cataract and normal lenses. By contrast, in γS crystallin the degree of conversion of Asn 76 to isoAsp in cataract lenses was approximately double that of normals at every age. Modification of Asn and Asp over time may contribute to denaturation of proteins in the human lens. An accelerated rate of deamidation/racemization at selected sites in proteins, such as γS crystallin, may contribute to cataract formation.

Promoter demethylation of Keap1 gene in human diabetic cataractous lenses

Age-related cataracts (ARCs) are the major cause of visual impairments worldwide, and diabetic adults tend to have an earlier onset of ARCs. Although age is the strongest risk factor for cataracts, little is known how age plays a role in the development of ARCs. It is known that oxidative stress in the lens increases with age and more so in the lenses of diabetics. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. We hypothesized that hyperglycemia will lead to a dysfunction of the Nrf2-dependent antioxidative protection in the lens of diabetics. We studied the methylation status of the CpG islands in 15 clear and 21 diabetic cataractous lenses. Our results showed significant levels of demethylated DNA in the Keap1 promoter in the cataractous lenses from diabetic patients. In contrast, highly methylated DNA was found in the clear lens and tumorized human lens epithelial cell (HLEC) lines (SRA01/04). HLECs treated with a demethylation agent, 5-aza-2′deoxycytidine (5-Aza), had a 10-fold higher levels of Keap1 mRNA, 3-fold increased levels of Keap1 protein, produced higher levels of ROS, and increased cell death. Our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. Decreased Nrf2 activity represses the transcription of many antioxidant enzyme genes and alters the redox-balance towards lens oxidation. Thus, the failure of antioxidant protection due to demethylation of the CpG islands in the Keap1 promoter is linked to the diabetic cataracts and possibly ARCs.

Osmotic stress induced oxidative damage: Possible mechanism of cataract formation in diabetes

Chronic hyperglycemia causes increased level of reactive oxygen species which is thought to be involved in the pathogenesis of diabetes associated complications including cataract. In diabetic cataractous lens, over production of free radicals and decreased capacity of antioxidant defense system are the major contributors to oxidative damage by polyol pathway and advanced glycation end products. The current study focused on analysis of factors associated with osmotic imbalance and oxidative stress in aging and diabetic human cataractous lenses. We examined activities of polyol pathway enzymes, G6PD and glutathione system in lenses from subjects suffering from cataract due to aging and diabetes. We observed elevated activities of aldose reductase and sorbitol dehydrogenase while G6PD and glutathione system enzyme activities were found to be lower in cataractous subjects suffering from diabetes. The findings from the current study support the premise that osmotic imbalance, AGEs formation and oxidative stress contribute synergistically to the development of lens opacity in hyperglycemia.

Photosensitized reactions mediated by the major chromophore arising from glucose decomposition, result in oxidation and cross-linking of lens proteins and activation of the proteasome

Glucose solutions incubated at low oxygen concentration gave rise to the appearance of an absorption band in the UVA-visible region after 10days. Further characterization evidenced that this band was composed by a single chomophore with maximum absorption bands at 335 and 365nm. HPLC/MS and UV spectroscopy assays indicated that this product is composed by five unities of furan. Importantly, the presence of a compound with identical spectral and chromatographic properties was observed in the water-soluble fraction of cataractous human eye lenses. The photo-biological effects of this glucose-derived chromophore (GDC) have been addressed using targets of biological relevance, such as water-soluble proteins from eye lens and the proteasome present in this protein mixture. Increased protein oxidation and protein crosslinking was observed when lens proteins were exposed to UVA-visible light in the presence of GDC under a 5% and 20% oxygen atmosphere. In addition, an increased proteasome peptidase activity was also observed. However, the use of D2O resulted in decreased proteasome activity, suggesting that singlet oxygen promotes the impairment of proteasome activity. Our results suggest that the species generated by Type I and Type II mechanisms have opposite effects on proteasome activity, being Type I a positive activator while Type II lead to impairment of proteasome function.

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography.

Application of femtotechnologies and terahertz spectroscopy methods in cataract diagnostics

We study the destructive action of femtosecond pulses (200 fs) on the human cataractous crystalline lens and the transmission of the cataractous lens in the terahertz spectral range of electromagnetic oscillations (0.2–1 THz) in relation to the density of the nucleus of the lens.

Advanced Drug Delivery of N-Acetylcarnosine (N-Acetyl-beta-alanyl-Lhistidine),Carcinine (Beta-alanylhistamine) and L-carnosine (Beta-alanyl-L-histidine) in Targeting Peptide Compounds as Pharmacological Chaperones for Use in Tissue Engineering, Human Disease Management and Therapy: From in vitro to the Clinic

A pharmacological chaperone is a relatively new concept in the treatment of certain chronic disabling diseases. Cells maintain a complete set of functionally competent proteins normally and in the face of injury or environmental stress with the use of various mechanisms, including systems of proteins called molecular chaperones. Proteins that are denatured by any form of proteotoxic stress are cooperatively recognized by heat shock proteins (HSP) and directed for refolding or degradation. Under non-denaturing conditions HSP have important functions in cell physiology such as in transmembrane protein transport and in enabling assembly and folding of newly synthesized polypeptides. Besides cellular molecular chaperones, which are stress-induced proteins, there have been recently reported chemical, or so-called pharmacological chaperones with demonstrated ability to be effective in preventing misfolding of different disease causing proteins, specifically in the therapeutic management of sight-threatening eye diseases, essentially reducing the severity of several neurodegenerative disorders (such as age-related macular degeneration), cataract and many other protein-misfolding diseases. This work reviews the biological and therapeutic activities protected with the patents of the family of imidazole-containing peptidomimetics Carcinine (β-alanylhistamine), N-acetylcarnosine (N-acetyl-β-alanylhistidine) and Carnosine (β-alanyl-L-histidine) which are essential constituents possessing diverse biological and pharmacological chaperone properties in human tissues.

Advanced glycation end products in diabetic and non-diabetic human subjects suffering from cataract

Advanced glycation end products (AGEs) play a pivotal role in loss of lens transparency, i.e., cataract. AGEs formation occurs as a result of sequential glycation and oxidation reaction between reducing sugars and protein. AGEs production takes place throughout the normal aging process but its accumulation is found to be more rapid in diabetic patients. In this study, we quantified AGEs and N-(carboxyethyl) lysine (CEL) in human cataractous lenses from non-diabetic (n=50) and diabetic patients (n=50) using ELISA. We observed significantly higher (p<0.001) levels of lens AGEs and CEL in diabetic patients with cataract as compared with their respective controls. The presence of AGEs and CEL was also determined by western blotting and immuno-histochemical analysis. Furthermore, isolated β-crystallin from cataractous lenses of non-diabetic and diabetic patients was incubated with different sugars to evaluate the extent of glycation in a time dependent manner. Our data indicated more pronounced glycation in patients suffering from diabetes as compared to non-diabetics subjects demonstrating the need to focus on developing normoglycemic approaches. Such studies may provide an insight in developing therapeutic strategies and may have clinical implications.

Mass Spectrometric Identification of Oxidative Modifications of Tryptophan Residues in Proteins: Chemical Artifact or Post-Translational Modification?

Oxidative modification of tryptophan to kynurenine (KYN) and N-formyl kynurenine (NFK) has been described in mitochondrial proteins associated with redox metabolism, and in human cataract lenses. To a large extent, however, previously reported identifications of these modifications were performed using peptide mass fingerprinting and/or tandem-MS data of proteins separated by gel electrophoresis. To date, it is uncertain whether NFK and KYN may represent sample handling artifacts or exclusively post-translational events. To address the problem of the origin of tryptophan oxidation, we characterized several antibodies by liquid chromatography-tandem mass spectrometry, with and without the use of electrophoretic separation of heavy and light chains. Antibodies are not normally expected to undergo oxidative modifications, however, several tryptophan (Trp) residues on both heavy and light chains were found extensively modified to both doubly oxidized Trp and KYN following SDS-PAGE separation and in-gel digestion. In contrast, those residues were observed as non-modified upon in-solution digestion. These results indicate that Trp oxidation may occur as an artifact in proteins separated by SDS-PAGE, and their presence should be carefully interpreted, especially when gel electrophoretic separation methods are employed.

Relationship between Pentosidine and Pyridinoline Levels in Human Diabetic Cataract Lenses

The relationship between the levels of two different crosslink compounds, pentosidine and pyridinoline, in human diabetic cataract lenses was investigated to elucidate the pathogenic mechanism of diabetic cataract. Subjects were classified into diabetes mellitus (DM) group and non-DM group according to the presence or absence of DM. The levels of the crosslink compounds were determined using high-performance liquid chromatography and spectrofluorometry after acid hydrolysis. In the non-DM group the pentosidine level was significantly and positively correlated with the pyridinoline level and age. In the DM group the pentosidine level was not significantly correlated with either pyridinoline level or age. Pyridinoline levels and age were not significantly correlated in either group. The increase in crosslink compounds due to glycation and the relationship between the compounds are changed in DM lenses.

Therapeutic Uses of Drug-Carrier Systems for Imidazole-Containing Dipeptide Compounds That Act as Pharmacological Chaperones and Have Significant Impact on the Treatment of Chronic Diseases Associated With Increased Oxidative Stress and the Formation of Advanced Glycation End Products

The purpose of this study was to determine how the naturally occurring molecules N-acetylcarnosine, L-carnosine, and carcinine, which are chemical or pharmacological chaperones, affect the cells and biomolecules of patients with skin diseases, cosmetic skin lesions, or underlying clinically significant visual impairment such as age-related cataracts, age-related retinal degeneration, and ocular complications of diabetes. We evaluated and characterized the effects of cited pharmacological chaperones on enzyme activity, protein structure in tissues, and other biomarkers of diseases in skin cells and tissues or in ocular tissues (human cataractous and normal lenses) derived from ophthalmic patients or age-matched donors. The samples were used to test imidazole-containing peptidomimetic chemical/pharmacological chaperones in relation to oxidative stress induced by reaction with lipid peroxides or advanced non-enzymatic glycation processes. Chaperone function is characterized by interaction with other proteins, mediating their folding, transport, and interaction with other molecules, lipid peroxidation products, and membranes. Although these therapies remain on hold pending further investigation, we present growing evidence demonstrating the ability of N-acetylcarnosine (lubricant eye drops) or carcinine pharmacological chaperone therapy to act as novel treatments for age-related cataracts, age-related macular degeneration, and ocular complications of diabetes. Finally, we examine strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone and transglycating (de-glycation) types of activity in in vitro and in vivo models of human age-related eye diseases, such as cataracts, and advanced glycation tissue protein-engineered systems.

Potentiation of Intraocular Absorption and Drug Metabolism of N-Acetylcarnosine Lubricant Eye Drops: Drug Interaction with Sight Threatening Lipid Peroxides in the Treatment for Age-Related Eye Diseases

Cataract is the dominant cause of blindness worldwide. Studies of the morphological structure and biophysical changes of the lens in human senile cataracts have demonstrated the disappearance of normal fiber structure in the opaque region of the lens and the disintegration of the lens fiber plasma membrane in the lens tissue. Morphological and biochemical techniques have revealed the regions in human cataractous lenses in which the plasma membrane derangement occurs as the primary light scattering centers which cause the observed lens opacity. Human cataract formation is mostly considered to be a multifactorial disease; however, oxidative stress might be one of the leading causes for both nuclear and cortical cataract. Phospholipid molecules modified with oxygen, accumulating in the lipid bilayer, change its geometry and impair lipid-lipid and protein-lipid interactions in lenticular fiber membranes. Electron microscopy data of human lenses at various stages of age-related cataract document that these disruptions were globules, vacuoles, multilamellar membranes, and clusters of highly undulating membranes. The opaque shades of cortical cataracts represent cohorts of locally affected fibres segregated from unaffected neighbouring fibres by plasma membranes. Other potential scattering centers found throughout the mature cataract nucleus included variations in staining density between adjacent cells, enlarged extracellular spaces between undulating membrane pairs, and protein-like deposits in the extracellular space. These affected parts had membranes with a fine globular aspect and in cross-section proved to be filled with medium to large globular elements. Lipid peroxidation (LPO) is a pathogenetic and causative factor of cataract. Increased concentrations of primary molecular LPO products (diene conjugates, lipid hydroperoxides, fatty acid oxy-derivatives) and end fluorescent LPO products were detected in the lipid moieties of the aqueous humor samples and human lenses obtained from patients with senile and complicated cataracts as compared to normal donors. Utilizing the pharmacokinetic studies and the specific purity N-acetylcarnosine (NAC) ingredient as a source of pharmacological principal L-carnosine, we have created an ophthalmic time-release prodrug form combined with a muco-adhesive lubricant compound carboxymethylcellulose and other essential corneal absorption promoter excipients tailoring the increased intraocular absorption of L-carnosine in the aqueous humor and optimizing its specific effect in producing the basic antioxidant activity in vivo and reducing toxic effects of lipid peroxides to the crystalline lens. L-Carnosine that finds its way into the aqueous humor can accumulate in the lens tissue for a reasonable period of time. However, administration of pure L-carnosine (1% solution) to the rabbit eye (instillation, subconjunctival injection) does not lead to accumulation of this natural compound in the aqueous humor over 30 min in concentration exceeding that in the placebo-treated matched eyes, and its effective concentration is exhausted more rapidly. The NAC prodrug eye drops optimize the clinical effects for the treatment of ophthalmic disorders (such as prevention and reversal of cataracts in human and animal [canine] eyes). The data provided predict a particular NAC ophthalmic prodrug's clinical effect; the suitable magnitude and duration of this effect suggest dose-related bioavailability of L-camosine released from NAC in the aqueous humor of the anterior eye segment. The ophthalmic NAC drug shows promise in the treatment of a range of ophthalmic disorders which have a component of oxidative stress in their genesis (including cataract and after-cataract, glaucoma, dry eye, vitreous floaters, inflammatory disorders, corneal, retinal and systemic diseases [such as diabetes mellitus and its ophthalmic complications]). The clinical efficacy of N-acetylcarnosine lubricant eye drops in ripe cataracts and retinal disorders can be enhanced in combined treatment with a patented oral formulation (Can-C Plus) of non-hydrolyzed carnosine including synergistic compounds (histidine, D-panthethine) with chaperone activity towards lens crystallins and oral supplementation with N-acetylcysteine providing an alternate means of boosting reduced glutathione (GSH) synthesis in the lens.

Effect of vitamin C and E activity on surgically removed cataractous human lens epithelium cells

Oxidative stress has been proposed as a common underlying mechanism of cataractogenesis. Experimental and observational data suggest that micronutrients like vitamin C and vitamin E with antioxidant capabilities may retard the development of age-related cataract. Effect of these factors on lens epithelium cells, center of lens metabolic activities, is not completely elucidated. The aim of present study was to examine the effect of vitamin C and E on surgically removed lens epithelium cells of patients with cataract. Capsulorhexis samples were collected from 170 patients, admitted for cataract surgery. Catalase specific activity was estimated in lens epithelium cells with and without vitamin (C or E) treatment at different concentration for different time duration. Student's t-test was employed for data analysis. We observed that in ex-vivo condition, a) both vitamin C and E bring about a decrease in catalase activity in lens epithelial cells. b) vitamin C showed toxic effect at high concentration. c) 100μM was the optimum concentration at which both vitamins showed maximum antioxidant activity. It was concluded that both vitamin C and E has direct effect on lens epithelium cells. At optimum concentration, they can reduce oxidative stress in these cells thus can support to prevent or delay cataract development.

Two-photon excited fluorescence of the lens for the diagnosis of presbyopia

Presbyopia is a wide spread phenomenon in elder people and is caused by the hardening of the lens in human eyes. Research is performed to make such lenses again more flexible by application of geometrically optimised cuts through the lens with a femtosecond-laser. Different protein agglomerations are responsible for the flexibility reduction of the lens. Two-photon excited fluorescence of the lens can be used as a diagnostic tool to localise such protein accumulations. In in-vitro experiments with human cataract lenses and also lenses of the Philly-mouse it could be demonstrated that with age the fluorescence increases as presbyopia proceeds. The distribution of the fluorescing compounds are not homogeneous but rather cloudy. Discrimination of the compounds by fluorescence lifetime measurements in relation of the depth in the lens is possible.

Combined microwave energy and fixative agent for cataract induction in pig eyes

We describe a technique used in enucleated pig eyes to create a uniformly hardened lens nucleus and an anterior capsule similar to the cataractous human lens. After treatment with microwave heat and a fixative agent, the pig eyes have clear corneas, anterior capsules with less tension and elasticity, and harder lens nuclei, with a consistency similar to that of a nuclear sclerosis grade I to V in the Emery-Little classification. The eyes can be used by residents in training to practice various phacoemulsification techniques, including continuous curvilinear capsulorhexis, hydrodelineation, sculpting, divide and conquer, phaco chop, and nondividing phacoemulsification.

Quantification of Isotopically Overlapping Deamidated and 18O-Labeled Peptides Using Isotopic Envelope Mixture Modeling

A robust peptide quantification method was developed where overlapping peptide isotopic distributions were fit with predicted peptide isotopic envelope mixture models (IEMMs). Application to two difficult quantitative problems was demonstrated. The first was the quantification of deamidation, where masses of isotopic peaks differ by 1 Da, and the second was (18)O labeling, where the isotopic peaks are shifted 2 and 4 Da. In both cases, peptide quantification cannot be performed by simple integration of extracted ion chromatograms, because the isotopic envelopes of mass-shifted peptides are normally not resolved. To test the methodology for quantification of deamidation, several synthetic peptides and their corresponding deamidated forms were mixed at various ratios (1:0, 1:2, 2:1, 4:1, 10:1, and 20:1) and analyzed using the IEMM method, resulting in a high correlation (R(2) = 0.96) between measured and known percentages of deamidation. The IEMM method was then incorporated into a workflow for deamidation quantification in a large-scale proteomics experiment. A series of normal (3 day, 2 year, 35 year, and 70 year) and cataractous (93 year) human lenses were analyzed using two-dimensional liquid chromatography tandem mass spectrometry, and deamidation quantities of several gammaS-crystallin peptides ([N14-Q16], N53, [Q63-Q70], and N143) were determined. Two peptides (N53 and [Q63-Q70]) had more extensive deamidation in the water-insoluble portions of normal lens samples, and deamidation at N143 was more extensive in the 93 year water-insoluble cataractous sample. The utility of the technique for analysis of (18)O-labeled peptides was examined using mixtures of labeled BSA peptides in known (16)O/(18)O ratios (10:1, 4:1, 1:1, 1:4, and 1:10). The methodology allowed for accurate measurements of ratios of (16)O/(18)O peptides over the wide range of relative abundances.

Expression and activity of paraoxonase 1 in human cataractous lens tissue

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that is believed to be involved in the protection against oxidative stress. There is evidence that paraoxonase activity is reduced in patients with diabetes and cataract. In the current study, we analyzed mRNA expression of PON1 as well as other members of the paraoxonase family, PON2 and PON3, in human cataractous lens samples. Our results indicate that only PON1 is expressed at the gene and protein levels in human lens tissues. We quantified MDA levels and measured PON1 (paraoxonase/arylesterase) enzymatic activities in subjects suffering from cataract due to aging and diabetes. Decreased PON1 activity was more pronounced in diabetic patients (p< 0.001) compared to senile subjects, which may be due to glycation and increased oxidative insult. To examine the structural alterations that occur in response to glycation, we constructed a three-dimensional model of PON1 and its glycated variant. Glycation at Lys70 and Lys75 is predicted to cause hindrance in binding of substrate to the active site of the enzyme.