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Time-resolved spectroscopic techniques are used to determine the primary photoprocesses of A2E in solution. Comparison of the absorption and excitation spectrum of A2E in methanol solution indicates excitation at 400 nm populates the S2 excited state. Transient absorption signals decaying with a time constant of 0.9 ps were observed probing around 800 nm. These signals are attributed to the S2→Sn transition and reveal the S2→S1 relaxation occurs on the subpicosecond time scale. Transient absorption data probing at shorter wavelengths (480 and 550 nm) are attributed to the S1→Sn absorption. These signals exhibit an exponential decay with a time constant of 11 and 13 ps, respectively. Time-resolved emission measurements of the corresponding S0←S1 decay reveal a nonexponential decay; however, >95% of the signal amplitude is described by an exponential decay with a time constant of 12.4 ps. Both time-resolved emission and absorption experiments therefore indicate repopulation of the ground electronic state occurs with a time constant of ~12 ps. A weak transient absorption probing in the blue (400 nm) persists onto the nanosecond time scale and is attributed to the T1→Tn absorption of A2E. Photoacoustic spectroscopy establishes the quantum yield for intersystem crossing of A2E in methanol solution is at most 0.03. The emission quantum yield of A2E in ethanol is determined to be 0.01, and so, nonradiative relaxation is the dominant primary event. The quantum yield for the generation of singlet molecular oxygen following 355 nm excitation of A2E in acetonitrile was determined to be 0.02, consistent with a low production of the excited triplet state. These results establish A2E is not an efficient photogenerator of reaction oxygen species in solution.

Novel high-resolution microscopic techniques have been utilized to characterize the spatial distribution of orange emitting fluorophores, e.g., A2E, in lipofuscin granules isolated from human retinal pigment epithelium cells. Granules have been imaged using atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM). Near-field scanning optical microscopy images of lipofuscin show that the orange fluorophores, including A2E, are not major components of the granule. These results suggest that the orange fluorophores may not be the dominant photoactive species in lipofuscin.

The radical cations of five dietary carotenoids have been shown to react with dopamelanin (a model of the black eumelanin) and cysteinyldopamelanin (a model of the red/yellow pheomelanin) in both micellar and homogeneous environments. It is suggested that such repair reactions show previously unidentified protective roles for melanin/carotenoid combinations, especially where the maintenance of the (small) carotenoid concentration is vital to avoid deleterious effects, such as in the retina of the eye.

Nine areas of the brain were studied by electron paramagnetic resonance spectroscopy and total reflection X-ray fluorescence spectroscopy to measure paramagnetic metal ions, free radicals (neuromelanin), and total metal content. We also determined the extent of accumulation of metal ions by melanins incubated in homogenates of a region of the brain (putamen). The electron paramagnetic resonance spectra of metal ions varied considerably among areas of the brain. There was no correlation between total content of particular metal ions (iron was especially pertinent) and the observed electron paramagnetic resonance spectra, except that the substantia nigra appeared more consistently to have a prominent g = 4 electron paramagnetic resonance signal characteristic of ferric iron in a rhombic state. Only the substantia nigra, and to a lesser extent the locus coeruleus, had a free radical signal consistent with that of neuromelanin. This signal was much more prominent in the unprocessed substantia nigra but when metal ions were removed (reducing the amount of suppression of the electron paramagnetic resonance signal of neuromelanin due to dipole-dipole broadening from nearby metal ions), the electron paramagnetic resonance spectrum of neuromelanin of the locus coeruleus increased much more than that of the substantia nigra. This suggests that the structure of the pigment may differ in these two regions.

Iron and lipid peroxidation are believed to be involved in the degeneration of pigmented neurons in Parkinson′s disease. Melanin-iron interaction is thought to play a role in iron accumulation and reactivity. The purpose of this study was to examine antioxidant properties of isolated natural and synthetic neuromelanin. Effect of neuromelanin from substantia nigra and its synthetic model, dopamine melanin, on Lipid peroxidation, induced by ferrous ions and free-radical initiators, has been studied in methyl linoleate aqueous dispersions, 2,2′-azobis(amidinopropane)dihydrochloride and 2,2′-azobis(2,4-dimethyl-valeronitrile) were used as water-soluble and lipid-soluble radical initiator, respectively. Rate of oxidation was followed quantitatively by measuring oxygen uptake and accumulation of lipid hydroperoxides. Melanin had a distinct protective effect on lipid peroxidation induced by ferrous ions or water-soluble free-radical initiator but was relatively inefficient when peroxidation was initiated with lipid-soluble compound. It also inhibited iron-catalyzed decomposition of methyl linoleate hydroperoxides in the presence of ascorbate. Extent of the inhibition depended on the ratio of melanin to iron. Taken together, these results provide strong support for the idea that neuromelanin of pigmented neurons can act as a natural antioxidant by sequestering redox-active metal ions.

A characteristic feature of both Parkinson's disease (idiopathic paralysis agitans) and normal aging is loss of pigmented neurons in the substantia nigra. This has been found to correlate with the accumulation of neuromelanin and with oxidative stress in this brain region, but a clear association between these factors has not been established. Based on our recent demonstration that neuromelanin is a true melanin, containing bound metal ions in situ, we present a general model for its accumulation in vivo and the hypotheses (1) that it has a cytoprotective function in the sequestration of redox-active metal ions under normal conditions but (2) that it has a cytotoxic role in the pathogenesis of Parkinson's disease. Thus, neuromelanin accumulates normally through the autooxidation of catecholamines and serves tightly to bind redox-active metal ions, processes which would accelerate under conditions of intracellular or extracellular oxidative stress. Based on the known properties of melanin, however, neuromelanin also has the potential for exacerbating oxidative stress, eg by generating H2O2 when it is intact or by releasing redox-active metal ions if it loses its integrity; these reactions also would modulate the reactivity of the neuromelanin. By overwhelming intracellular antioxidative defense mechanisms, such a positive-feedback cycle could turn a condition of chronic or repeated oxidative stress in vulnerable neurons into an acute crisis, leading to cellular death. If the cumulative stress in duration and/or degree is severe enough, neuronal depletion could be sufficient to cause Parkinson's disease during life. One possible trigger for this cascade is suggested by the increased nigral iron contents in postmortem parkinsonian brains and the correlation of this disease with urban living where exposure to heavy metal ions is high: the saturation of neuromelanin with redox-active metal ions. Parkinson's disease therefore may be a form of accelerated aging in the substantia nigra associated with environmental toxins in which neuromelanin has a central, active role.

Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.

NADPH consumption and esr spectroscopy have been used to study the rate of formation and signal intensity of free radicals produced by various anthracycline anti-tumour drugs in rat liver microsomal extract. The drugs investigated were Adriamycin, 4'Deoxyadriamycin, Daunorubicin, 4 Demethoxydaun-orubicin and Carminomycin. Pulse radiolysis was also used to determine the ease of reduction of each of the analogues to its semiquinone radical and some kinetic properties of the radicals produced. It is believed that the Occurrence of reactions other than dismutation could be responsible for the shortened lifetimes of the semiquinone radicals observed in biological systems.