James Dillon

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age‐related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time‐resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV‐visible absorption of longer‐lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady‐state and time‐resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time‐resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age‐related decline of RPE function and blue light damage.

The photophysics and photobiology of the eye

The eye consists of three major segments: the cornea, lens and retina. The main function of the anterior ocular tissue, the cornea and the lens is to transmit and focus light on the retina without distortion. They also filter out UV light (less than 400 nm) and prevent it from reaching the retina. Much of the light reaching the retina is used for sight. However, light can have numerous other effects on the constituents of the eye, both beneficial and deleterious. This article reviews the interaction of light with the eye, various protective mechanisms, the possible role of light in aging and disease states and the role of light in biological processes other than sight such as mood, hormonal secretions and the cyclic growth and phagocytosis of the rods and cones.

PHOTOCHEMICAL AND PHOTOPHYSICAL STUDIES ON HUMAN LENS CONSTITUENTS

Abstract— –The young human lens contains species (3‐hydroxy kynurenine; 3‐HK and its glucoside; 3‐HKG) which absorb most light between 300 and 400 nm. Photochemical studies have indicated that these compounds are relatively inefficient sensitizers of lens proteins. An investigation of the fluorescent properties of 3‐HKG indicate that it contains a fast deactivation pathway (ps) which would be expected to have minimal photochemical effect on the integrity of the lens. Further photophysical studies on 3‐HK indicates that it has an even faster fluorescent lifetime (< 10 ps) with a much lower quantum yield of fluorescence (0.001 vs 0.03 for 3‐HKG).

Non-tryptophan fluorescence associated with human lens protein; apparent complexity and isolation of bityrosine and anthranilic acid.

Abstract In order to characterize the age-related protein bound fluorescent compounds of human cataractous lenses, the insoluble yellow protein component was proteolytically digested and then fractionated using ion-exchange, gel filtration and high pressure liquid chromatography. A complex and heterogeneous mixture of fluorescent components was found to be associated with these lens proteins. In the course of this study 3,3′-bityrosine and anthranilic acid were structurally identified as constituents of the proteolytic hydrolysate. The latter compound appears to be a degradation product of an unstable precursor.

Antioxidant Properties of Melanin in Retinal Pigment Epithelial Cells

Abstract The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruchs membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light–induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.

Photolytic changes in lens proteins

Many of the age related and cataractous changes that occur in the human lens may be due to some photolytic process. These include either the direct photolysis of lens protein or photosensitized reactions involving 3-OH Kyn glucoside. The extent to which these photochemical reactions play a role is determined by the level of lens oxygen and the presence of quenchers endogenous to the lens.

THE PHOTOCHEMISTRY OF HUMAN RETINAL LIPOFUSCIN AS STUDIED BY EPR

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light‐induced contribution of these fluorophores to various diseases including age‐related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen‐free methanol led to the formation of a 5,5‐dimethylpyrroline‐N‐oxide (DMPO) spin‐trapped carbon‐centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform‐soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age‐related decline of RPE function and blue light damage in the retina.

The nitrite/collagen reaction: non-enzymatic nitration as a model system for age-related damage.

The effects of age seen in long-lived connective tissue proteins are thought to be the result of post-translational modifications by reactive molecules. One such molecule is the nitrite ion. Human nitrite exposure results predominately from endogenous production of nitric oxide as well as inhalation of cigarette smoke and ingestion of cured meats. Although nitrite reactions with various proteins have been studied previously with regard to carcinogenesis, the specific reaction with collagen and its role in age-related damage has never been examined. We describe the reaction of nitrite with type I collagen at neutral pH and body temperature. The incubation of collagen with nitrite results in an increase in cross-linking, the accumulation of a yellow chromophore, and a depletion of tyrosine residues. Similar changes also are found in aged human collagen. In addition, 3-nitro-tyrosine, which has recently been used as a marker for peroxynitrite mediated damage, is produced from this reaction. Thus, we propose non-enzymatic nitration as an in vitro model system for human collagen age-related damage.

LIGHT DAMAGE IN THE RAT RETINA: EFFECT OF A RADIOPROTECTIVE AGENT (WR‐77913) ON ACUTE ROD OUTER SEGMENT DISK DISRUPTIONS

Primary events in the course of light induced retinal lesions are still not fully elucidated. Under chronic conditions, lipid peroxidation in the retina and death of photoreceptor cells are observed. The radioprotective agent WR‐77913 scavenges singlet oxygen, hydrated electrons and free radicals. WR‐77913 was used to protect against acute light induced photoreceptor outer segment membrane disruptions in the rat retina. There was a partial but not complete protection at higher illuminance levels (800 lx for 30 min), whereas threshold lesions (400 lx for 30 min) were almost completely prevented. These observations indicate an involvement of photodynamic reactions in causing acute ohotoreceDtor lesions.

Oxidation of A2E Results in the Formation of Highly Reactive Aldehydes and Ketones

Abstract It has been reported that the photo-oxidation of A2E, a component of human retinal lipofuscin, leads to products that are toxic to cells via dark reactions. Because these compounds have been implicated in the development of various maculopathies such as age-related macular degeneration (AMD), it is important to determine the structures of those deleterious compounds. Both the photo-oxidation and auto-oxidation of A2E lead to the same complex mixture of products, some of which have lower molecular weights than the staring material. Because A2E is homologous to β-carotene, it was hypothesized that its oxidation would lead to products analogous to those found in oxidized β-carotene, namely, a series of cleavage products along the acyclic chain with the concomitant formation of aldehydes. This was found to be the case based upon 1) the formation of all of the aldehydes predicted from the oxidation of β-carotene, 2) the loss of 28 amu (carbonyl moiety) from the molecular ion, 3) the facile reaction of the aldehydes with nitrophenylhydrazines to form nitrophenylhydrazones and 4) the subsequent MS/MS cleavage of those derivatives at the N-N bond. If formed in vivo, these aldehydes would have toxic effects on any cell. Finally, the similarity in product mixtures from both the photo-oxidation and auto-oxidation strongly suggests that the intermolecular photo-oxidation of A2E results primarily from a radical process without the involvement of singlet oxygen. Any formation of singlet oxygen most likely arises from sensitization by the aldehyde oxidation products, as this process is well known for aldehydes, in general, and retinal, specifically.