Antioxidant activity of L-carnosine, a natural histidine-containing dipeptide in crystalline lens

Lipid peroxidation was shown to be an initiatory cause of cataract development in some cases. It has been established that injection into the vitreous body of the rabbit eye of a suspension of liposomes prepared from phospholipids containing lipid peroxidation products induces the development of posterior subcapsular cataract. Such modelling of cataract is based on a type of clouding of the crystalline lens similar to that observed in cataract resulting from diffusion of toxic lipid peroxidation products from the retina to the lens through the vitreous body on degeneration of the photoreceptors. Saturated liposomes (prepared from dipalmitoylphosphatidylcholine) did not cause clouding of the lens, which demonstrated the peroxide mechanism of the genesis of this form of cataract. Clouding of the lens was accompanied by accumulation of fluorescing lipid peroxidation products in the vitreous body, aqueous humor and the lens and also by a fall in the concentration of reduced glutathione in the lens. The ability of L-carnosine (beta-alanyl-L-histidine) to interact directly with lipid peroxidation products suggested its anticataract properties. The effect of L-carnosine on inhibiting or reversing the formation of cataract induced by the administration of lipid peroxidation products was discovered. This phenomenon appeared to be related with normalization of the peroxide metabolism parameters in the crystalline lens. In view of the data, an aqueous solution of L-carnosine is physiologically acceptable in effective nonsurgical treatment of cataracts.     

Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

Antioxidative enzyme activity and metabolism of peroxide compounds in the crystalline lens during cataractogenesis

Lens antioxidative enzyme activity (catalase, superoxide dismutase, glutathione peroxidase) in cataract as well as the possibility of cataract induction by the lipid peroxidation products and their influence on the content of reduced thiols (oxy-red balance) were studied. It was shown that the rate of the H2O2 decomposition by the human cataract lenses is lowered in comparison with the normal lenses. This is not due to the lowered catalase or glutathione-peroxidase 1 activity, but depends on the deficiency of reduced glutathione in the lens. Activity of superoxide dismutase and glutathione peroxidase metabolizing organic hydroperoxides is significantly lowered in the cataract lenses. Lipid peroxidation products injected into the rabbit vitreous induce posterior subcapsular cataract, which is accompanied by depletion of reduced glutathione level in the lens. The conclusion is made that two interrelated processes: accumulation of H2O2 and of lipid peroxides induce aggregation of the soluble proteins and the fragmentation of the membrane structures in cataract lenses.     

Vitreoretinal influences on lens function and cataract

The lens is composed of a thin metabolically active outer layer, consisting of epithelial and superficial fibre cells. Lying within this outer shell are terminally differentiated, metabolically inactive fibre cells, which are divided into an outer cortex and central nucleus. Mature fibre cells contain a very high protein concentration, which is important for the transparency and refractive power of the lens. These proteins are protected from oxidation by reducing substances, like glutathione, and by the low-oxygen environment around the lens. Glutathione reaches the mature fibre cells by diffusing from the metabolically active cells at the lens surface. With age, the cytoplasm of the nucleus becomes stiffer, reducing the rate of diffusion and making nuclear proteins more susceptible to oxidation. Low pO(2) is maintained at the posterior surface of the lens by the physical and physiological properties of the vitreous body, the gel filling the space between the lens and the retina. Destruction or degeneration of the vitreous body increases exposure of the lens to oxygen from the retina. Oxygen reaches the lens nucleus, increasing protein oxidation and aggregation and leading to nuclear cataract. We suggest that maintaining low pO(2) around the lens should prevent the formation of nuclear cataracts.     

Crystalline lens induction of lipid peroxidation

The level of lipid peroxidation products (LPP) was determined in the aqueous humor from the anterior chamber of patients with cataract and donor eyes. The content of LPP in senile cataract aqueous humor was shown to be significantly increased. To determine the possible mechanism of LPP increase in aqueous humor, human lenses at different stages of cataract as well as transparent human and rabbit lenses were incubated for 3 hours in 3.0 ml medium containing liposomes (0.5 mg/ml) prepared from phospholipids from the egg yolk and 0.14 M NaCl + 0.01 M TRIS-HCl buffer, pH 7.4). Corrections were made for phospholipid autooxidation. The level of LPP accumulation in the medium was determined by MDA assay. The rate of LPP production increased significantly in transparent lenses and in early senile cataract, as compared to controls and advanced (mature) cataracts. EDTA (1 mM), superoxide dismutase (114 u/sample), catalase (900 u/sample), chelated iron (III): Fe3+-ADP addition to the incubation medium depressed the level of LPP accumulation. This suggests the participation of Fe2+, O2-., H2O2 in the mechanism of LPP production in the lens. The induction of lipid peroxidation in the lens can be significant for leukotriene and prostaglandin synthesis in the eye.     

Evidence of the oxidation of unsaturated fatty acids in cataracts

Gas chromatography analysis with the use of an electron captured detector including preparation of the halogen-substituted derivatives of fatty acids is a useful tool for the detection of lipid peroxidation products both in vitro and in vivo. This technique was applied to determine the content of fatty acid oxy-derivatives in lipid samples of transparent and completely opaque human lenses. At the stage of mature cataract a significantly increased level of oxyproducts was observed in the lens lipid fraction. It was concluded that accumulation of polar oxygroups in the lipid bilayer of plasma membranes of lens fibres is a plausible cause of their damage in cataracts.     

Lipid fluorophores of the crystalline lens in cataracts

Microcolumn liquid and column chromatography technique is conjunction with UV-spectrophotometry and spectrofluorescent analysis were used to study lipid peroxidation products accumulated in human lenses during cataract formation by means of chromatographic separation in regard to the molecular weight and polarity properties. Cataract is characterized by the appearance of certain substances changing UV-absorption lipid spectra in the region of 230 and 274 nm and having special fluorescence (excitation--320-370 nm), (emission--405-460 nm). The same changes were observed by ultrasoundinduced lipid peroxidation of model lipid samples. The accumulated lipid peroxidation products are concentrated in the same chromatographic fractions that are responsible for the change of UV-absorption and fluorescent spectra of lipids of cataractous lenses. It is the evidence of free radical lipid peroxidation products accumulation in human lenses at cataract formation. Along with the formation of diene and triene conjugates in the lens lipids, cataract is characterized by the formation of cetodienes and of low molecular weight lipid fluorescent products of fatty acids oxidation with low polarity due to the appearance of tetraene derivatives of polyunsaturated fatty acids. The particular features of mature cataract are an increased intensity of long-wave lipid fluorescence in the blue-green region (430-460 nm) of the spectrum, formation of high molecular weight fluorescent lipid peroxidation products with high polarity, and smooth decrease in absorbance in the region of 220-330 nm. During cataract formation products of deep lipid peroxidation resulting from radical phospholipids and fatty acids polymerisation are accumulated. It is supposed that lipid peroxidation is an initial phase of membrane desintegration and formation of HMW-proteins in cataract.     

The lens epithelium in ocular health and disease

The lens arises from invagination of head ectoderm during embryonic development and in the adult has a relatively simple structure, comprising just two cell types (epithelial and fibre cells). Its isolation from nerves and blood vessels in the adult make it a tractable model to investigate mechanisms that regulate epithelial cells. A major focus in lens research in the past 50 years has been on the differentiation of fibre cells from epithelial cells. Hence, there has been much interest in the role of signalling systems regulating fibre cell differentiation during development. In contrast, the signalling systems that control the formation and maintenance of the lens epithelium have, until recently, been largely ignored or incidental to studies on differentiation or cataract. One notable example has been the identification of signals that underlie epithelial-mesenchymal transition (EMT) that characterizes anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO). Recent data indicate that normal epithelial phenotype is regulated by several key signalling systems, including receptor tyrosine kinase receptors acting via the MAPK and Akt pathways, Wnt, Notch as well as extracellular matrix cues and possibly the Sal-Warts-Hippo pathway. Here we have shifted emphasis onto molecular mechanisms that regulate the establishment, maintenance and function of the lens epithelium.     

Phosphorescent analysis of lipid peroxidation products in liposomes]

It was found that lipid peroxidation products incorporated into liposomes prepared from oxidized preparations of bovine heart phosphatidylcholine and the total lipid fraction of human erythrocyte membranes are able to phosphoresce at room temperature was studied. The temperature dependences of kinetic and spectral parameters of phosphorescence were measured. It is shown that mechanism of phosphorescence quenching of lipid chromophores has a dynamic nature. It is proposed to use endogenic molecules of the lipid peroxidation products capable of phosphorescence as intrinsic phosphorescence probes for studying the slow molecular dynamics of lipids in artificial and biological membranes in a millisecond range.     

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.     

Localization of lipid peroxidation products in liposomes after γ-irradiation

The distribution of lipid peroxidation products in liposomes after γ-irradiation at various doses was studied. Increases in thiobarbituric-acid-reactive substances, in the absorbance at 232 nm and in hydroperoxides were observed mainly in liposomal membranes after relatively low doses of irradiation, while carbonyl compounds were distributed both inside and outside the membranes. After higher doses of irradiation, however, the absorbance at 232 nm and the amount of hydroperoxides reached a maximal level in the membrane portion and then decreased when the decomposition products were released from the membranes. Under this condition, malondialdehyde and other carbonyl compounds were increased mainly in the medium of liposomal suspension. These results are discussed with reference to the lipid peroxidation process which is induced quantitatively by ionizing radiation.     

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.     

Lipid peroxidation contributes to age-related membrane rigidity

The aim of this work was to assess the relative contributions of lipid peroxidation and cholesterol content to the increase in membrane rigidity observed during senescence. Membrane fluidity was manipulated through exposure to peroxidized or cholesterol-loaded liposomes. Small unilamella liposomes were prepared and either peroxidized by Fe⁺⁺-ADP-ascorbic acid or loaded with cholesterol. After incorporation of the liposomes into rat liver microsomal membranes, membrane fluidity was quantitated by measuring changes in polarization. Membranes exhibited a greater sensitivity to peroxidation than cholesterol in that incorporation of peroxidized liposomes induced microsomal membrane rigidity substantially more than did cholesterol-loaded liposomes. Thus it is proposed, based on data from the present and earlier studies, that membrane fluidity can be modulated readily by lipid peroxidation of membrane phospholipids, irrespective of the influences of cholesterol. These results support the proposal that alterations of lipid structure are more potent and effective than compositional changes in cholesterol in inducing age-related increases in membrane rigidity.     

Extracellular matrix and integrin signaling in lens development and cataract

During development of the vertebrate lens there are dynamic interactions between the extracellular matrix (ECM) of the lens capsule and lens cells. Disruption of the ECM causes perturbation of lens development and cataract. Similarly, changes in cell signaling can result in abnormal ECM and cataract. Integrins are key mediators of ECM signals and recent studies have documented distinct repertoires of integrin expression during lens development, and in anterior subcapsular cataract (ASC) and posterior caspsule opacification (PCO). Increasingly, studies are being directed to investigating the signaling pathways that integrins modulate and have identified Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK) as downstream kinases that mediate proliferation, differentiation and morphological changes in the lens during development and cataract formation.     

Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.     

Intermembrane transfer and antioxidant action of alpha-tocopherol in liposomes

Intermembrane transfer and exchange of tocopherol are not well understood. To study this we tested the ability of alpha-tocopherol containing unilamellar donor liposomes to inhibit the accumulation of lipid peroxidation products in acceptor liposomes. With molar ratios of alpha-tocopherol:phospholipids from 1:100 to 1:1000 in donor liposomes prepared by sonication of lipid dispersions, alpha-tocopherol was incorporated into both monolayers and was homogenously distributed in monomeric form without forming clusters in the liposomes. Concentrations of alpha-tocopherol which completely prevented the peroxidation of lipids were chosen for donor liposomes. Hence inhibition of lipid peroxidation in mixtures of donor and acceptor liposomes was determined by the antioxidant effect of alpha-tocopherol in acceptor liposomes which resulted from intermembrane transfer and exchange of alpha-tocopherol. Evidence was obtained that this was not due to fusion of donor with acceptor liposomes. The efficiency of the "intermembrane" antioxidant action of tocopherol was more pronounced when donor liposomes contained unsaturated phospholipids, indicating that the presence of unsaturated fatty acids in the outer monolayer phospholipids facilitates intermembrane tocopherol exchange.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

Protection of liposomal lipids against radiation induced oxidative damage.

Liposomes were prepared from phospholipids extracted from biological membranes. A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes. The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation. High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation. Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation. The naturally occurring compounds reduced glutathione (GSH) and vitamin E(alpha-T) and the powerful radiation protector cysteamine (MEA). All three molecules could protect the liposomes against peroxidation. The membrane-soluble compound vitamin E was by far the most powerful. About 50 per cent protection was achieved by using 5 X 10(-6) M alpha-T, 10(-4) M GSH and 5 X 10(-4) M MEA. The fatty acid composition of the lipids altered drastically as a result of the irradiation. Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids. Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes.     

Mitochondria induce oxidative stress, generation of reactive oxygen species and redox state unbalance of the eye lens leading to human cataract formation: disruption of redox lens organization by phospholipid hydroperoxides as a common basis for cataract disease

The aging eye appears to be at considerable risk from oxidative stress. Lipid peroxidation (LPO) is one of the mechanisms of cataractogenesis, initiated by enhanced promotion of oxygen free radicals in the eye fluids and tissues and impaired enzymatic and non-enzymatic antioxidant defenses of the crystalline lens. The present study proposes that mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian and human lens epithelial cells and that therapies that protect mitochondria in lens epithelial cells from damage and reduce damaging ROS generation may potentially ameliorate the effects of free radical-induced oxidation that occur in aging ocular tissues and in human cataract diseases. It has been found that rather than complete removal of oxidants by the high levels of protective enzyme activities such as superoxide dismutase (SOD), catalase, lipid peroxidases in transparent lenses, the lens conversely, possess a balance between peroxidants and antioxidants in a way that normal lens tends to generate oxidants diffusing from lenticular tissues, shifting the redox status of the lens to become more oxidizing during both morphogenesis and aging. Release of the oxidants (O(2)(-)·, H(2)O(2) , OH·, and lipid hydroperoxides) by the intact lenses in the absence of respiratory inhibitors indicates that these metabolites are normal physiological products inversely related to the lens life-span potential (maturity of cataract) generated through the metal-ion catalyzed redox-coupled pro-oxidant activation of the lens reductants (ascorbic acid, glutathione). The membrane-bound phospholipid (PL) hydroperoxides escape detoxification by the lens enzymatic reduction. The lens cells containing these species would be vulnerable to peroxidative attack which trigger the PL hydroperoxide-dependent chain propagation of LPO and other damages in membrane (lipid and protein alterations). The increased concentrations of primary LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moiety of the aqueous humor samples obtained from patients with cataract as compared to normal donors. Since LPO is clinically important in many of the pathological effects and aging, new therapeutic modalities, such as patented N-acetylcarnosine prodrug lubricant eye drops, should treat the incessant infliction of damage to the lens cells and biomolecules by reactive lipid peroxides and oxygen species and "refashion" the affected lens membranes in the lack of important metabolic detoxification of PL peroxides. Combined in ophthalmic formulations with N-acetylcarnosine, mitochondria-targeted antioxidants are promising to become investigated as a potential tool for treating a number of ROS-related ocular diseases, including human cataracts.     

Carotenoids are degraded by free radicals but do not affect lipid peroxidation in unilamellar liposomes under different oxygen tensions

It has been questioned whether carotenoids can act as antioxidants in biological membranes. Biological membranes can be modeled for studies of lipid peroxidation using unilamellar liposomes. Both carotenoid depletion and lipid peroxidation were increased with increasing oxygen tension in unilamellar liposomes. Carotenoids in such liposomes were found to be very sensitive to degradation by free radicals generated from iron and 2,2'-azobis(2-amidinopropane) dihydrochloride, but they were not protective against lipid peroxidation. Lycopene and beta-carotene were more sensitive to free radical attack than lutein, zeaxanthin, and beta-cryptoxanthin.