Protecting the Eye Lens from Oxidative Stress through Oxygen Regulation.

Molecular oxygen is a primary oxidant that is involved in the formation of active oxygen species and in the oxidation of lipids and proteins. Thus, controlling oxygen partial pressure (concentration) in the human organism, tissues, and organs can be the first step in protecting them against oxidative stress. However, it is not an easy task because oxygen is necessary for ATP synthesis by mitochondria and in many biochemical reactions taking place in all cells in the human body. Moreover, the blood circulatory system delivers oxygen to all parts of the body. The eye lens seems to be the only organ that is protected from the oxidative stress through the regulation of oxygen partial pressure. The basic mechanism that developed during evolution to protect the eye lens against oxidative damage is based on the maintenance of a very low concentration of oxygen within the lens. This antioxidant mechanism is supported by the resistance of both the lipid components of the lens membrane and cytosolic proteins to oxidation. Any disturbance, continuous or acute, in the working of this mechanism increases the oxygen concentration, in effect causing cataract development. Here, we describe the biophysical basis of the mechanism and its correlation with lens transparency.

An Overview of Lutein in the Lipid Membrane.

Lutein, zeaxanthin, and meso-zeaxanthin (a steroisomer of zeaxanthin) are macular pigments. They modify the physical properties of the lipid bilayers in a manner similar to cholesterol. It is not clear if these pigments are directly present in the lipid phase of the membranes, or if they form complexes with specific membrane proteins that retain them in high amounts in the correct place in the retina. The high content of macular pigments in the Henle fiber layer indicates that a portion of the lutein and zeaxanthin should not only be bound to the specific proteins but also directly dissolved in the lipid membranes. This high concentration in the prereceptoral region of the retina is effective for blue-light filtration. Understanding the basic mechanisms of these actions is necessary to better understand the carotenoid-membrane interaction and how carotenoids affect membrane physical properties-such as fluidity, polarity, and order-in relation to membrane structure and membrane dynamics. This review focuses on the properties of lutein.

How Do Xanthophylls Protect Lipid Membranes from Oxidative Damage?

Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes.

Spin-Lattice Relaxation Rates of Lipid Spin Labels as a Measure of Their Rotational Diffusion Rates in Lipid Bilayer Membranes.

The spin-lattice relaxation rate (T1-1) of lipid spin labels obtained from saturation recovery EPR measurements in deoxygenated membranes depends primarily on the rate of the rotational diffusion of the nitroxide moiety within the lipid bilayer. It has been shown that T1-1 also can be used as a qualitative convenient measure of membrane fluidity that reflects local membrane dynamics; however, the relation between T1-1 and rotational diffusion coefficients was not provided. In this study, using data previously presented for continuous wave and saturation recovery EPR measurements of phospholipid analog spin labels, one-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine in 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/cholesterol membranes, we show that measured T1-1 values are linear functions of rotational diffusion of spin labels. Thus, these linear relationships can be used to transfer T1-1 values into spin label rotational rates as a precise description of membrane fluidity. This linearity is independent through the wide range of conditions including lipid environment, depth in membrane, local hydrophobicity, and the anisotropy of rotational motion. Transferring the spin-lattice relaxation rates into the rotational diffusion coefficients makes the results obtained from saturation recovery EPR spin labeling easy to understand and readily comparable with other membrane fluidity data.

Multilamellar Liposomes as a Model for Biological Membranes: Saturation Recovery EPR Spin-Labeling Studies.

EPR spin labeling has been used extensively to study lipids in model membranes to understand their structures and dynamics in biological membranes. The lipid multilamellar liposomes, which are the most commonly used biological membrane model, were prepared using film deposition methods and investigated with the continuous wave EPR technique (T2-sensitive spin-labeling methods). These investigations provided knowledge about the orientation of lipids, their rotational and lateral diffusion, and their rate of flip-flop between bilayer leaflets, as well as profiles of membrane hydrophobicity, and are reviewed in many papers and book chapters. In the early 1980s, the saturation recovery EPR technique was introduced to membrane studies. Numerous T1-sensitive spin-label methods were developed to obtain detailed information about the three-dimensional dynamic membrane structure. T1-sensitive methods are advantageous over T2-sensitive methods because the T1 of spin labels (1-10 µs) is 10 to 1000 times longer than the T2, which allows for studies of membrane dynamics in a longer time-space scale. These investigations used multilamellar liposomes also prepared using the rapid solvent exchange method. Here, we review works in which saturation recovery EPR spin-labeling methods were applied to investigate the properties of multilamellar lipid liposomes, and we discuss their relationships to the properties of lipids in biological membranes.

Molecular oxygen as a probe molecule in EPR spin-labeling studies of membrane structure and dynamics.

Molecular oxygen (O2) is the perfect probe molecule for membrane studies carried out using the saturation recovery EPR technique. O2 is a small, paramagnetic, hydrophobic enough molecule that easily partitions into a membrane's different phases and domains. In membrane studies, the saturation recovery EPR method requires two paramagnetic probes: a lipid-analog nitroxide spin label and an oxygen molecule. The experimentally derived parameters of this method are the spin-lattice relaxation times (T 1s) of spin labels and rates of bimolecular collisions between O2 and the nitroxide fragment. Thanks to the long T 1 of lipid spin labels (from 1 to 10 µs), the approach is very sensitive to changes of the local (around the nitroxide fragment) O2 diffusion-concentration product. Small variations in the lipid packing affect O2 solubility and O2 diffusion, which can be detected by the shortening of T 1 of spin labels. Using O2 as a probe molecule and a different lipid spin label inserted into specific phases of the membrane and membrane domains allows data about the lateral arrangement of lipid membranes to be obtained. Moreover, using a lipid spin label with the nitroxide fragment attached to its head group or a hydrocarbon chain at different positions also enables data about molecular dynamics and structure at different membrane depths to be obtained. Thus, the method can be used to investigate not only the lateral organization of the membrane (i.e., the presence of membrane domains and phases), but also the depth-dependent membrane structure and dynamics, and, hence, the membrane properties in three dimensions.

Factors Differentiating the Antioxidant Activity of Macular Xanthophylls in the Human Eye Retina.

Macular xanthophylls, which are absorbed from the human diet, accumulate in high concentrations in the human retina, where they efficiently protect against oxidative stress that may lead to retinal damage. In addition, macular xanthophylls are uniquely spatially distributed in the retina. The zeaxanthin concentration (including the lutein metabolite meso-zeaxanthin) is ~9-fold greater than lutein concentration in the central fovea. These numbers do not correlate at all with the dietary intake of xanthophylls, for which there is a dietary zeaxanthin-to-lutein molar ratio of 1:12 to 1:5. The unique spatial distributions of macular xanthophylls-lutein, zeaxanthin, and meso-zeaxanthin-in the retina, which developed during evolution, maximize the protection of the retina provided by these xanthophylls. We will correlate the differences in the spatial distributions of macular xanthophylls with their different antioxidant activities in the retina. Can the major protective function of macular xanthophylls in the retina, namely antioxidant actions, explain their evolutionarily determined, unique spatial distributions? In this review, we will address this question.

Factors determining barrier properties to oxygen transport across model and cell plasma membranes based on EPR spin-label oximetry.

This review is motivated by the exciting new area of radiation therapy using a phenomenon termed FLASH in which oxygen is thought to have a central role. Well-established principles of radiation biology and physics suggest that if oxygen has a strong role, it should be the level at the DNA. The key aspect discussed is the rate of oxygen diffusion. If oxygen freely diffuses into cells and rapidly equilibrates, then measurements in the extracellular compartment would enable FLASH to be investigated using existing methodologies that can readily measure oxygen in the extracellular compartment. EPR spin-label oximetry allows evaluation of the oxygen permeability coefficient across lipid bilayer membranes. It is established that simple fluid phase lipid bilayers are not barriers to oxygen transport. However, further investigations indicate that many physical and chemical (compositional) factor can significantly decrease this permeation. In biological cell plasma membranes, the lipid bilayer forms the matrix in which integral membrane proteins are immersed, changing organization and properties of the lipid matrix. To evaluate oxygen permeability coefficients across these complex membranes, oxygen permeation across all membrane domains and components must be considered. In this review, we consider many of the factors that affect (decrease) oxygen permeation across cell plasma membranes. Finally, we address the question, can the plasma membrane of the cell form a barrier to the free diffusion of oxygen into the cell interior? If there is a barrier then this must be considered in the investigations of the role of oxygen in FLASH.

Why is Zeaxanthin the Most Concentrated Xanthophyll in the Central Fovea?

Diet-based xanthophylls (zeaxanthin and lutein) are conditionally essential polar carotenoids preferentially accreted in high concentrations (1 mM) to the central retina, where they have the capacity to impart unique physiologically significant biophysical biochemical properties implicated in cell function, rescue, and survival. Macular xanthophylls interact with membrane-bound proteins and lipids to absorb/attenuate light energy, modulate oxidative stress and redox balance, and influence signal transduction cascades implicated in the pathophysiology of age-related macular degeneration. There is exclusive transport, sequestration, and appreciable bioamplification of macular xanthophylls from the circulating carotenoid pool to the retina and within the retina to regions required for high-resolution sensory processing. The distribution of diet-based macular xanthophylls and the lutein metabolite meso-zeaxanthin varies considerably by retinal eccentricity. Zeaxanthin concentrations are 2.5-fold higher than lutein in the cone-dense central fovea. This is an ~20-fold increase in the molar ratio relative to eccentric retinal regions with biochemically detectable macular xanthophylls. In this review, we discuss how the differences in the specific properties of lutein and zeaxanthin could help explain the preferential accumulation of zeaxanthin in the most vulnerable region of the macula.

Why Is Very High Cholesterol Content Beneficial for the Eye Lens but Negative for Other Organs?

The plasma membranes of the human lens fiber cell are overloaded with cholesterol that not only saturates the phospholipid bilayer of these membranes but also leads to the formation of pure cholesterol bilayer domains. Cholesterol level increases with age, and for older persons, it exceeds the cholesterol solubility threshold, leading to the formation of cholesterol crystals. All these changes occur in the normal lens without too much compromise to lens transparency. If the cholesterol content in the cell membranes of other organs increases to extent where cholesterol crystals forma, a pathological condition begins. In arterial cells, minute cholesterol crystals activate inflammasomes, induce inflammation, and cause atherosclerosis development. In this review, we will indicate possible factors that distinguish between beneficial and negative cholesterol action, limiting cholesterol actions to those performed through cholesterol in cell membranes and by cholesterol crystals.

The effect of carotenoids on the concentration of singlet oxygen in lipid membranes.

An effect of ß-carotene and its polar derivative, zeaxanthin, on a concentration of singlet oxygen in lipid membranes was studied in a model system. The carotenoids were incorporated into the membranes of small unilamellar liposomes at a concentration of 0.15 mol% with respect to lipid. Singlet oxygen was generated in a liposome suspension via photosensitization of toluidine blue, and its concentration in a membrane was detected with application of a specific fluorescence probe (singlet oxygen sensor green reagent) located in the lipid bilayer. The results show the carotenoid-dependent decrease in the concentration of singlet oxygen in the membranes formed with unsaturated lipids (egg yolk phosphatidylcholine and digalactosyldiacylglycerol) but not in the case of the membranes formed with a saturated lipid (dimyristoylphosphatidylcholine). The effect of carotenoids was about twice as high as in the case of cholesterol present in liposomes at the same concentration. The results suggest that carotenoids protect membranes formed with unsaturated lipids against singlet oxygen through combined activity of different mechanisms: modification of structural properties of the lipid bilayers, physical quenching of singlet oxygen and chemical reactions leading to the pigment oxidation. The latter conclusion is based on the analysis of the absorption spectra of liposomes before and after light exposure. An importance of the different modes of protection by carotenoids against single oxygen toxicity towards biomembranes is discussed.

Mechanisms enhancing the protective functions of macular xanthophylls in the retina during oxidative stress.

Macular xanthophylls (MXs) are distinguished from other dietary carotenoids by their high membrane solubility and preferential transmembrane orientation. Additionally, these properties enhance the chemical and physical stability of MXs in the eye retina, and maximize their protective activities. The effectiveness of MXs' protection is also enhanced by their selective accumulation in the most vulnerable domains of retinal membranes. The retina is protected by MXs mainly through blue-light filtration, quenching of the excited triplet states of potent photosensitizers, and physical quenching of singlet oxygen. To perform these physical, photo-related actions, the structure of MXs should remain intact. However, the conjugated double-bond structure of MXs makes them highly chemically reactive and susceptible to oxidation. Chemical quenching of singlet oxygen and scavenging of free radicals destroy their intact structure and consume MXs. Consequently, their physical actions, which are critical to the protection of retina, are diminished. Thus, it is timely and important to identify mechanisms whereby the chemical destruction (bleaching) of MXs in retinal membranes can be reduced. It was shown that nitroxide free radicals (spin labels) located in membranes protect MXs against destruction, and their effect is especially pronounced during the light-induced formation of singlet oxygen. That should extend and enhance their positive action in the retina through physical processes. In this review, we will discuss possible applications of this new strategy during ophthalmological procedures, which can cause acute bleaching of MXs and damage the retina through oxidative processes.

Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Cholesterol Bilayer Domains in the Eye Lens Health: A Review.

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review.

Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

Can Xanthophyll-Membrane Interactions Explain Their Selective Presence in the Retina and Brain?

Epidemiological studies demonstrate that a high dietary intake of carotenoids may offer protection against age-related macular degeneration, cancer and cardiovascular and neurodegenerative diseases. Humans cannot synthesize carotenoids and depend on their dietary intake. Major carotenoids that have been found in human plasma can be divided into two groups, carotenes (nonpolar molecules, such as ß-carotene, α-carotene or lycopene) and xanthophylls (polar carotenoids that include an oxygen atom in their structure, such as lutein, zeaxanthin and ß-cryptoxanthin). Only two dietary carotenoids, namely lutein and zeaxanthin (macular xanthophylls), are selectively accumulated in the human retina. A third carotenoid, meso-zeaxanthin, is formed directly in the human retina from lutein. Additionally, xanthophylls account for about 70% of total carotenoids in all brain regions. Some specific properties of these polar carotenoids must explain why they, among other available carotenoids, were selected during evolution to protect the retina and brain. It is also likely that the selective uptake and deposition of macular xanthophylls in the retina and brain are enhanced by specific xanthophyll-binding proteins. We hypothesize that the high membrane solubility and preferential transmembrane orientation of macular xanthophylls distinguish them from other dietary carotenoids, enhance their chemical and physical stability in retina and brain membranes and maximize their protective action in these organs. Most importantly, xanthophylls are selectively concentrated in the most vulnerable regions of lipid bilayer membranes enriched in polyunsaturated lipids. This localization is ideal if macular xanthophylls are to act as lipid-soluble antioxidants, which is the most accepted mechanism through which lutein and zeaxanthin protect neural tissue against degenerative diseases.

Why has Nature Chosen Lutein and Zeaxanthin to Protect the Retina?

Age-related macular degeneration (AMD) is associated with a low level of macular carotenoids in the eye retina. Only two carotenoids, namely lutein and zeaxanthin are selectively accumulated in the human eye retina from blood plasma where more than twenty other carotenoids are available. The third carotenoid which is found in the human retina, meso-zeaxanthin is formed directly in the retina from lutein. All these carotenoids, named also macular xanthophylls, play key roles in eye health and retinal disease. Macular xanthophylls are thought to combat light-induced damage mediated by reactive oxygen species by absorbing the most damaging incoming wavelength of light prior to the formation of reactive oxygen species (a function expected of carotenoids in nerve fibers) and by chemically and physically quenching reactive oxygen species once they are formed (a function expected of carotenoids in photoreceptor outer segments). There are two major hypotheses about the precise location of macular xanthophylls in the nerve fiber layer of photoreceptor axons and in photoreceptor outer segments. According to the first, macular xanthophylls transversely incorporate in the lipid-bilayer portion of membranes of the human retina. According to the second, macular xanthophylls are protein-bound by membrane-associated, xanthophyll-binding proteins. In this review we indicate specific properties of macular xanthophylls that could help explain their selective accumulation in the primate retina with special attention paid to xanthophyll-membrane interactions.

The influence of lead on the biomechanical properties of bone tissue in rats.

INTRODUCTION AND OBJECTIVE: Environmental lead (Pb) is a serious public health problem. At high levels, Pb is devastating to almost all organs. On the other hand, it is difficult to determine a safe level of exposure to Pb. More than 90% of the Pb in the adult human body and 70% in a child's body is stored in the bones. In the presented study, the effects of lead exposure on bones were studied for rats treated orally with Pb acetate in drinking water for 14 days. The hypothesis was tested that lead exposure negatively affects bone structure. MATERIALS AND METHODS: Femur strength was measured in a three-point bending test, whereas infrared spectroscopy (FTIR) was used to measure molecular structural changes. RESULTS: Lead significantly decreased the ratio of area of two types of vibrational transitions, which are highly specific to mineral to matrix ratio. The results of the biomechanical study show that femurs of rats treated by Pb-acetate appeared to be weaker than bones of the control group, and may produce a condition for the development of higher risk of fractures. Additionally, a great difference in body mass was observed between control and the Pb acetate-treated groups. CONCLUSIONS: The lower bone mineral content and the weaker mechanical properties of bones from Pb-treated rats are associated with the pathologic state dependent of the exposure of lead.

Modeling gender effects on electrical activity of single ventricular myocytes.

In this study we investigate the mechanisms underlying gender differences in the generation of arrhythmias in the long QT and Brugada syndromes. Simulations were conducted at the single myocyte level using a detailed mathematical model of human ventricular myocytes. Given the scarce human data on the gender-related differences in single cardiac cells, we assumed gender-related differences in five ionic-current systems: fast sodium current (INa), slowly inactivating late sodium current (INal), transient outward potassium current (Ito), slow delayed rectifier potassium current (IKs), and calcium current through the L-type channel (ICa(L)), based on experimental results obtained in canine myocytes. Our modeling results suggest that in left ventricular myocytes, enhanced INal under conditions of reduced repolarization reserve results in sex-dependent development of early afterdepolarizations (EADs) in the post-pause action potentials (APs). Moreover, this modeling study demonstrates increased propensity for the development of the loss of the AP dome in male epicardial myocytes of the right ventricle compared with other types of myocytes from the left and right ventricles. Finally, we also found a slight effect of INal on gender-dependent loss of AP dome in epicardial right ventricular myocytes. In conclusion, at the cellular level, gender differences in the development of EADs and the propensity to develop the loss of the AP dome can be attributed to male/female related differences in INa, INal, Ito, IKs, and ICa(L).

Can macular xanthophylls replace cholesterol in formation of the liquid-ordered phase in lipid-bilayer membranes?

Lateral organization of membranes made from binary mixtures of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) and macular xanthophylls (lutein or zeaxanthin) was investigated using the saturation-recovery (SR) EPR spin-labeling discrimination by oxygen transport (DOT) method in which the bimolecular collision rate of molecular oxygen with the nitroxide spin label is measured. This work was undertaken to examine whether or not lutein and zeaxanthin, macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity, can parallel other structural functions of cholesterol, including formation of the liquid-ordered phase in membranes. The DOT method permits discrimination of different membrane phases when the collision rates (oxygen transport parameter) differ in these phases. Additionally, membrane phases can be characterized by the oxygen transport parameter in situ without the need for separation, which provides information about the dynamics of each phase. In gel-phase membranes, two coexisting phases were discriminated in the presence of macular xanthophylls - namely, the liquid-ordered-like and solid-ordered-like phases. However, in fluid-phase membranes, xanthophylls only induce the solitary liquid-ordered-like phase, while at similar concentrations, cholesterol induces coexisting liquid-ordered and liquid-disordered phases. No significant differences between the effects of lutein and zeaxanthin were found.