Fluorescence emission and excitation spectra of fluorophores of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes

Visible light-induced changes in fluorescence characteristics of lipofuscin granules (LG) isolated from retinal pigment epithelium of human cadaver eyes are compared with the analogous age-related changes and correlated with the content and photooxidation of LG main fluorophore, A2E. We used HPLC to examine changes of LG fluorophore composition with donor age, as well as before and after visible-light irradiation (the latter HPLC tests were also done with synthetic A2E). Visible light induces oxidation of LG fluorophores. As a result, their fluorescence characteristics change: the emission spectrum is blue-shifted by 25–40 nm. The observed age-dependent changes in the relative content of LG fluorophores and their oxidized derivatives were qualitatively similar with those caused by irradiation. To improve the accuracy of a new noninvasive diagnostic method, fundus autofluorescence imaging, it is important to know the ratio of nonoxidized and oxidized fluorophore derivatives depending on age and eye pathology.     

Age-dependent photoionization thresholds of melanosomes and lipofuscin isolated from human retinal pigment epithelium cells

Melanosomes and lipofuscin were isolated from 14-, 59-, and 76-year-old, human retinal pigment epithelium specimens and examined. The morphological features of these samples were studied by scanning electron microscopy and atomic force microscopy, and the photoionization properties were examined by photoelectron emission microscopy. Ovoid- and rod-shaped melanosomes were observed. The size of the granules and the distribution between the two shapes show no significant age-dependent change. However, there is a higher occurrence of irregularly shaped aggregates of small round granules in older samples which suggests degradation or damage to melanosomes occurs with age. The melanosomes from the 14-year-old donor eye are well characterized by a single photoionization threshold, 4.1 eV, while the two older melanosomes exhibit two thresholds around 4.4 and 3.6 eV. Lipofuscin from both young and old cells show two thresholds, 4.4 and 3.4 eV. The similarity of the potentials observed for aged melanosomes and lipofuscin suggest that the lower threshold in the melanosome sample reflects lipofuscin deposited the surface of the melanosome. The amount, however, is not sufficient to alter the density of the melanosome, and therefore these granules do not separate in a sucrose gradient at densities characteristic of the typical melanolipofuscin granule. These data suggest that thin deposits of lipofuscin on the surface of retinal pigment epithelium melanosomes are common in the aged eye and that this renders the melanosomes more pro-oxidant.     

Enzymatic degradation of A2E, a retinal pigment epithelial lipofuscin bisretinoid

Some forms of blinding macular disease are associated with excessive accumulation of bisretinoid lipofuscin in retinal pigment epithelial (RPE) cells of the eye. This material is refractory to lysosomal enzyme degradation. In addition to gene and drug-based therapies, treatments that reverse the accumulation of bisretinoid would be beneficial. Thus, we have examined the feasibility of degrading the bisretinoids by delivery of exogenous enzyme. As proof of principle we report that horseradish peroxidase (HRP) can cleave the RPE bisretinoid A2E. In both cell-free and cell-based assays, A2E levels were decreased in the presence of HRP. HRP-associated cleavage products were detected by ultraperformance liquid chromatography (UPLC) coupled to electrospray ionization mass spectrometry, and the structures of the aldehyde-bearing cleavage products were elucidated by 18O-labeling and 1H NMR spectroscopy and by recording UV?vis absorbance spectra. These findings indicate that RPE bisretinoids such as A2E can be degraded by appropriate enzyme activities.     

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors

Electron spin resonance (ESR) examinations of human retinal pigment epithelium melanosomes isolated from eyes of young and old donors were carried out. The examined ESR signal was a single line, which is characteristic for free radicals of eumelanin o-semiquinones. The content of free radicals related to melanosomes dry weight for samples from older donors (ages over 45 years) were higher than for sample from younger donors (between 14 and 22 years). Simultaneously, the content of free radicals calculated for one melanosome is constant and does not depend on age. The homogeneous broadening of the recorded ESR lines shows that there are no isolated spin packets in all investigated melanin samples. Slow spin-lattice (T1 approximately 10(-5) s) and fast spin-spin (T2 approximately 10(-8) s) relaxation processes occur in these samples. Saturation of the ESR lines at low microwave power was measured. High concentration of free radicals in melanosome samples was responsible for the fast spin-spin relaxation process.     

A study of the anharmonic effects on the vibrational spectra of a realistic retinal chromophore model

Ab initio and vibrational self-consistent field (VSCF) computations are used to investigate the vibrational normal coordinates of the protonated Schiff base (PSB) 4-cis-gamma,eta-dimethyl-C9H9 NH2+. The ground and the first excited states are investigated. Both harmonic and anharmonic frequencies for the first three overtones of the ground and first excited states are reported. Special attention is payed to the discussion of the normal coordinates modes that involve the central C=C bond which plays a significant role in the isomerization process.     

Environmental effects on the photochemistry of A2-E, a component of human retinal lipofuscin

Several retinal dystrophies are associated with the accumulation of lipofuscin, a pigment mixture, in the retinal pigment epithelium (RPE). One of the major fluorophores of this mixture has been identified as the bis-retinoid pyridinium compound, A2-E. Because this compound absorbs incident radiation that is transmitted by the anterior segment of the human eye, photophysical and photochemical studies were performed to determine if A2-E could photosensitize potentially damaging reactions. Steady-state fluorescence measurements indicate that the fluorescence emission maximum and quantum yield are very sensitive to the chemical environment and a correlation between these two parameters and the solvent dielectric constant is observed. Time-resolved absorption experiments of A2-E in pure organic solvents showed no formation of transient species on the timescale of our experiments. However, when these measurements were repeated for A2-E in Triton X-100 micelles, a short-lived (tau approximately 14 microseconds), weak absorption was observed. This species is quenched by oxygen (k = 2 x 10(9) M-1 s-1) and by the addition of the antioxidants, cysteine and N,N,N',N'-tetramethylphenylenediamine. Quenching of this species by 2,3,5-trimethylhydroquinone results in the formation of the 2,3,5-trimethylsemiquinone free radical and an increase in yield of the A2-E-derived species. Sensitization of the A2-E triplet excited state indicates that the species observed in micelles upon direct excitation is not consistent with the triplet excited state. Based on these data we tentatively assign this absorption to a free radical. In the RPE these initial processes can ultimately lead to damage to the tissue through the formation of peroxides and other oxidized species.     

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.     

The role of A2E in prevention or enhancement of light damage in human retinal pigment epithelial cells

The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.     

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.     

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

Comparisons of the structural and chemical properties of melanosomes isolated from retinal pigment epithelium, iris and choroid of newborn and mature bovine eyes

Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1-0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.     

A multitechnique study of liposome adsorption on Au and lipid bilayer formation on SiO2

We have used a new setup for parallel quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) measurements to measure the detailed kinetics of vesicle-to-bilayer transformation on SiO2 and vesicle adsorption on Au, respectively. The combination of SPR and QCM-D, complemented by atomic force microscopy measurements, has enabled a complete, time-resolved separation of vesicle and bilayer coverages, and thus, for the first time, allowed precise quantification of the critical surface coverage of vesicles needed for rupture. We furthermore demonstrate and quantify a previously undetected vesicle-size- and concentration-dependent loss of lipid material during the later stages of the process.     

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells

The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 microM) and exposed to UVA (5 J cm(-2)). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J cm(-2)) both berberine (10 microM) and palmatine (10 microM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (lambda > 400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal are estimated to contain approximately 1 mM berberine, while in tinctures the alkaloid concentration may be more than 10 times higher. Our findings show that eyewashes and lotions derived from Goldenseal or containing berberine must be used with caution when the eyes are exposed to bright sunlight but that oral preparations are not likely to cause ocular phototoxicity.     

A model study for the breaking of N2 from CNx within DFT

A hypothetical CN₂ structure was investigated as a model to study the release of N₂ from the octahedral hole of 3D carbon based ultra hard compounds, which is the most important drawback in the attempts to synthesize ultra hard compounds like C₃N₄ and C₁₁N₄. Full structure relaxations using DFT methods led to a structure at the energy minimum showing a significantly enlarged NN distance of 1.34 Å compared to the molecular N₂ (1.09 Å). While for small volume changes a high hardness for CN₂ of 405 GPa is calculated, we found that enlargements of the cell constant lead to the release of N₂ that could be followed calculating the ELF and the charge transfer within the AIM theory. The whole procedure simulates an inverted “harpoon mechanism”.     

A correlated ab initio study of the X2A1 and A2E states of MgCH3

The X2A1 and A2E states of the MgCH3 radical have been studied with correlation consistent basis sets and the coupled cluster method RCCSD(T) in order to compare with two recent experimental efforts [M. A. Anderson and L. M. Ziurys, Astrophys. J. 452, L157 (1995); R. Rubino, J. M. Williamson, and T. A. Miller, J. Chem. Phys. 103, 5964 (1995)]. The best computed values [RCCSD(T)/cc-pCVTZ] for the X2A1 state are (experimental results in parentheses): Ae = 160.433 GHz, Be = 10.948 GHz (B0 = 11.008 GHz), and Mue = 1.011 D. The Mg-CH3 bond is weak, 26.3 kcal/mol. Values for the A2E state are Ae = 154.648 GHz (A0 = 149.666 GHz), Be = 10.87 GHz (B0 = 10.932 GHz), and Mue = 1.022 D. The excitation energy (Te) for the A2E <-- X2A1 transition is 19 999 cm-1 (T00 = 20 030 cm-1). A brief discussion of bonding trends in Mg-containing radials is included.     

Effect of UV radiation and hydrogen peroxide on the antiradical and antioxidant activities of DOPA-melanin and melanosomes from retinal pigment epithelial cells

The effect of continuous UV radiation and hydrogen peroxide on destruction and antioxidant properties of synthetic DOPA-melanin (prepared by oxidation of 3,4-dihydroxyphenylalanine (DOPA)) and melanosomes isolated from cells of the retinal pigment epithelium (RPE) was investigated. The kinetics of melanin destruction was recorded based on the accumulation of fluorescent low-molecular-weight reaction products, the antiradical activity of melanin was determined by chemiluminescence method, the concentration of free radical products was measured by electron paramagnetic resonance, and the antioxidant activity of melanins was estimated by their inhibitory effect on lipid peroxidation. It was shown that UVC—UVA irradiation (up to 5 hours) of DOPA-melanin and melanosomes of retinal pigment epithelium decreased neither the latency period of luminol chemiluminescence nor the inhibitory action of pigments on Fe²⁺- and UV-induced peroxidation of cardiolipin liposomes. However, very long UV irradiation gave rise to fluorescent destruction products, decreased the concentration of paramagnetic centers in the pigment (especially light-dependent ones), and decreased the antiradical and antioxidant activities. For example, UV irradiation of DOPA-melanin during 52 h resulted in approximately a 2-fold decrease in the concentration of paramagnetic centers and decline of antiradical and antioxidant activities. However, even with such a hard irradiation the pigment retained significant inhibitory activity against lipid peroxidation. The oxidative destruction of DOPA-melanin in the presence of hydrogen peroxide in the dark resulted in complete destruction of the polymer and loss of its protective properties. It is assumed that destruction of RPE cell melanin is caused mainly by oxidative processes.     

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 μM of the fluorogenic substrate Mes-D-LGR-ANSN (C₂H₅)₂ (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0–1.6 U mL⁻¹. The statistically sensitive assay range was 0–0.4 U mL⁻¹ for enoxaparin and tinzaparin and 0–0.2 U mL⁻¹ for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-d-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.     

Comparative study of the cationic surfactants and their influence on the alkaline hydrolysis of acetylsalicylic acid

A kinetic study of the reaction of acetylsalicylic acid (aspirin) with sodium hydroxide has been studied in the presence of some conventional and novel cationic surfactants. The pseudo‐first‐order rate constant increases with the surfactant concentration initially and then decreases. In comparison to conventional cationic surfactants, i.e., cetyltrimethylammonium bromide and cetylpyridinium bromide, novel alkyldiethylethanolammonium bromide (R = C₁₆) surfactant accelerated the alkaline hydrolysis significantly. The pseudophase ion‐exchange model has been applied to fit the experimental results. Activation parameters have also been evaluated. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 43: 1–8, 2011