J. Bours

Lipid Peroxidation and Cataracts

AbstractCataract formation represents a serious problem in the elderly, with approximately 25% of the population aged >65 years and about 50% aged >80 years experiencing a serious loss of vision as a result of this condition. Not only do cataracts diminish quality of life, they also impose a severe strain on global healthcare budgets. In the US, 43% of all visits to ophthalmologists by Medicare patients are associated with cataract. Surgery represents the standard treatment of this condition, and 1.35 million cataract operations are performed annually in the US, costing

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C

US3.5 billion (year of costing, 1998).Unfortunately, the costs of surgical treatment and the fact that the number of patients exceeds surgical capacities result in many patients being blinded by cataracts worldwide. This situation is particularly serious in developing countries; worldwide 17 million people are blind because of cataract formation, and the problem will grow in parallel with aging of the population. In any event, surgical removal of cataracts may not represent the optimal solution. Although generally recognised as being one of the safest operations, there is a significant complication rate associated with this surgical procedure. Opacification of the posterior lens capsule occurs in 30–50% of patients within 2 years of cataract removal and requires laser treatment, a further 0.8% experience retinal detachments, approximately 1% are rehospitalised for corneal problems, and about 0.1% develop endophthalmitis. Although the risks are small, the large number of procedures performed means that 26 000 individuals develop serious complications as a result of cataract surgery annually in the US alone. Thus, risk and cost factors drive the investigation of pharmaceutical approaches to the maintenance of lens transparency.The role of free radical-induced lipid oxidation in the development of cataracts has been identified. Initial stages of cataract are characterised by the accumulation of primary (diene conjugates, cetodienes) lipid peroxidation (LPO) products, while in later stages there is a prevalence of LPO fluorescent end-products. A reliable increase in oxiproducts of fatty acyl content of lenticular lipids was shown by a direct gas chromatography technique producing fatty acid fluorine-substituted derivatives. The lens opacity degree correlates with the level of the LPO fluorescent end-product accumulation in its tissue, accompanied by sulfhydryl group oxidation of lens proteins due to a decrease of reduced glutathione concentration in the lens. The injection of LPO products into the vitreous has been shown to induce cataract. It is concluded that peroxide damage of the lens fibre membranes may be the initial cause of cataract development. N-acetylcarnosine (as the ophthalmic drug Can-C™), has been found to be suitable for the nonsurgical prevention and treatment of age-related cataracts. This molecule protects the crystalline lens from oxidative stress-induced damage, and in a recent clinical trial it was shown to produce an effective, safe and long-term improvement in sight. When administered topically to the eye in the form of Can-C™, N-acetylcarnosine functions as a time-release prodrug form of L-carnosine resistant to hydrolysis with carnosinase. N-acetylcarnosine has potential as an in vivo universal antioxidant because of its ability to protect against oxidative stress in the lipid phase of biological cellular membranes and in the aqueous environment by a gradual intraocular turnover into L-carnosine.In our study the clinical effects of a topical solution of N-acetylcarnosine (Can-C™) on lens opacities were examined in patients with cataracts and in canines with age-related cataracts. These data showed that N-acetylcarnosine is effective in the management of age-related cataract reversal and prevention both in human and in canine eyes.

Age-Related Changes in Water and Crystallin Content of the Fetal and Adult Human Lens, Demonstrated by a Microsectioning Technique

Abstract · Background: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. · Methods: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. · Results: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pIs) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was β-GlcNAc. Lactoferrin showed glycation with NANA-α-2–6-Gal or NANA-α-2–6-GalNAc, whereas the sugar specificity of sIgA was NANA-α-2–3-Gal. · Conclusions: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-α-2–3-Gal.

Age-dependent variations in the distribution of rat lens water-soluble crystallins, size fractionation and molecular weight determination

By means of a microsectioning technique, human fetal and adult lenses were divided into 8-16 fractions. The dry weight (DW), the water content, and the amounts of water-soluble (WS) crystallins and water-insoluble (WI) lens fraction were determined in each layer or fraction. Within the lens, the changes of these parameters are gradual and continuous. The lens nucleus contains the highest amounts of DW and WI fraction, and the lowest amounts of water and WS crystallins. There is a relative dehydration of the lens during development and ageing, most pronounced in the lens nucleus.

Protein profiles of microsections of the fetal and adult human lens during development and ageing.

In order to obtain deeper insights into the mechanisms that are responsible for the age-related changes in the eye lens, the water-soluble proteins of 6-day-old to over 3-year-old rat lenses were analyzed by high-performance gel-permeation chromatography. Using this technique eleven crystallin fractions could be discerned: HM-, alpha-, three beta H-, two beta L-, beta S- and three gamma-crystallins. The concentrations of the higher molecular weight crystallins (HM-, alpha- and beta H-crystallin) seem to increase with age while those of the lower molecular weight (beta L- and gamma-crystallin) decrease. Taking into account the gradual increase of water-insoluble protein with aging, the relative amount of alpha-crystallin decreases from an age of 0.5 year after an initial increase. Additionally, an age-dependent increase in its molecular weight was found: from 7 . 10(5) to over one million. It appears that the gamma-crystallins are directly involved in the insolubilization process, while alpha- and beta L-crystallin first take part in aggregation processes leading to HM- and beta H-crystallin aggregates. These aggregation and insolubilization processes proceed gradually with increasing age. A steep decrease in gamma-crystallin concentration in the early phase of life, which also causes the relative increase in alpha-crystallin content in this period, may originate from a decrease in biosynthesis of certain gamma-crystallins.

Age-related variations in the distribution of crystallins within the bovine lens.

The water-soluble proteins of the human fetal lens (175- and 285-day-old) contain HM-, pre-alpha-, alpha-, beta- and gamma-crystallins. Using the frozen-sectioning technique, it can be demonstrated that the fetal lens does not have an homogeneous distribution of crystallins, but there are gradual differences between the cortices and the nucleus. The frozen-sectioning technique shows for the adult lens significantly increasing amounts of beta-crystallins of pI 4.95-5.55, especially at the posterior supra-nuclear layer, increasing amounts of HM-crystallins and decreasing amounts of beta-crystallins of pI 5.80-7.05 in the nucleus. This microsectioning technique was correlated with Scheimpflug photographs of the fetal and adult lens. In the fetal lens, the anterior capsule and 2 peaks in the anterior and posterior supranuclear layers could be visualized after densitometry. In the adult lens 5 layers could be demonstrated, e.g. the anterior capsule, the anterior supranuclear layer, the nucleus, the posterior supranuclear layer and the anterior capsule.

Revival of the Lens Transparency with N-Acetylcarnosine

The native water-soluble proteins of equator, anterior cortex, posterior cortex and nucleus from bovine lenses in the age range 0.3-33.7 years were analyzed by high-pressure gel-permeation chromatography and high-pressure ion-exchange chromatography. Unlike the equator and cortices, the nucleus shows a gradual decrease in alpha-crystallin proportion with age which is not compensated for by an increase in HM-crystallin. The beta 6H-crystallin species, almost the only beta H-component in the youngest lens, is largely replaced by at least four fractions with higher and lower molecular weights in the older lenses. In the nucleus a beta L-component (39,000 MW) increasingly seems to replace the major beta L-crystallin (beta 2L, 50,000 MW). Moreover, a switch in the synthesis of monomeric crystallins is demonstrated. This study clearly reveals an age-related increase in the size heterogeneity of the native soluble crystallins with age.

Guidelines for the Classification of Lenses and the Characterization of Lens Proteins

The risk, cost and social requirement factors drive the investigation of pharmaceutical approaches to the management of cataracts. The role of free-radical-induced lipid oxidation (LPO) in the development of cataracts has been identified. Initial stages of cataract are characterized by the accumulation of primary (diene conjugates, cetodienes) LPO products,while in later stages there is a prevalence of LPO fluorescent end products. Reliable increase in oxiproducts of fatty acyl content of lenticular lipids was shown by a direct gas chromatography technique producing fatty acid fluorine- substituted derivatives. The lens opacity degree correlates with the level of the LPO fluorescent end product accumulation in its tissue, accompanied by SH group oxidation of lens proteins due to a decrease of reduced glutathione concentration in the lens. The injection of LPO products into the vitreous was shown to induce cataract. Peroxide damage of the lens fiber membranes may be the initial cause of cataract formation. The authors developed N-Acetylcarnosine ophthalmic drug with lubricant carboxymethylcellulose in eye drops (NAC, Can-C TM , Nu-Eyes TM ) suitable for the non-surgical prevention and treatment of age-related cataracts. The NAC ophthalmic drug protects the crystalline lens from oxidative stress-induced damages and in a recent clinical trial it was shown to produce an effective, safe and long-term improvement in sight. When administered topically to the eye, NAC drug functions as a time-release prodrug form of L- carnosine resistant to hydrolysis with carnosinase and significantly increases the intraocular uptake of L-carnosine in the aqueous humor. The mechanisms of prevention and reversal of cataracts with NAC ophthalmic drug are considered which include prevention by the intraocular released carnosine of free-radical-induced inactivation of proprietary lens antioxidant enzymes (superoxide dismutase), prevention of carbohydrate and metal-catalysed autooxidation of ascorbic acid -induced cross-linking glycation reactions to the lens proteins, universal antioxidant and scavenging activity towards lipid hydroperoxides, aldehydes and oxygen radicals, activation with L-carnosine ingredient of proteasome activity in the lens. In this study the clinical effects of a topical solution of NAC ophthalmic drug on lens opacities were examined in patients with cataracts and canines with age-related cataracts. The positive effect on lens clarity and clarifying modification of opacification zones is demonstrated. The data suggest a potential and show the efficacy of developed NAC ophthalmic drug for a positive effect of treatment (both reversal and prevention) of age-related cataracts. Innovative Vision Products, Inc. is a holder of the worldwide patent (including PCT International Publication Number WO 2004/028536 A1) for the application of N-acetylcarnosine for the treatment of ophthalmic disorders including cataracts.

The quantification of eight enzymes from the ageing rat lens, with respect to sex differences and special reference to aldolase

In November 1980 a workshop for the classification and characterization of lenses and lens proteins was held in Louvain-La-Neuve, Belgium. Some essential results are summarized in this paper.

Separation of lens proteins in rats with tryptophan deficiency cataracts

Eight enzymes, e.g. lactate dehydrogenase, malate dehydrogenase, fructose-diphosphate aldolase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were estimated quantitatively in the rat lens from 37 to 1,211 days of age, by spectrophotometric methods. The activity was expressed as mU/g LWW. All enzymes measured showed declining activities, but LDH, ALD, SDH, G-6-PDH, HK and PFK gave a significant decrease during ageing when plotted semi-logarithmically from 37 to 1,211 days. SDH and G-6-PDH showed a statistically significant difference between the enzymes from the male and the female lenses. The female lens always had a lower activity than the male lens. Of all enzymes the specific activity, expressed as mU/l mg protein, was calculated. This specific activity appeared to be rather constant during ageing, except for ALD. In the female lenses, the specific activity of 7 enzymes was lower than in the male lenses. For ALD the specific activity decreased significantly in the male lens from 5.32 at 37 days to 0.88 at 1,211 days. In the female lens this significant decrease was from 4.97 to 0.81.