Photooxidation of A2-PE, a photoreceptor outer segment fluorophore, and protection by lutein and zeaxanthin

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Abstract

A2-PE is a pigment that forms as a byproduct of the visual cycle, its synthesis from all-trans-retinal and phosphatidylethanolamine occurring in photoreceptor outer segments. A2-PE is deposited in retinal pigment epithelial (RPE) cells secondary to phagocytosis of shed outer segment membrane and it undergoes hydrolysis to generate the RPE lipofuscin fluorophores, A2E, iso-A2E and other minor cis-isomers of A2E. We have demonstrated that A2-PE can initiate photochemical processes that involve the oxidation of A2-PE and that, by analogy with A2E are likely to include the formation of reactive moieties. We also show that potential sources of protection against the photooxidation of A2-PE are the lipid-soluble carotenoids zeaxanthin and lutein (xanthophylls), that constitute the yellow pigment of the macula. Irradiation of A2-PE in the presence of lutein or zeaxanthin suppressed A2-PE photooxidation and in experiments in which we compared the antioxidant capability of zeaxanthin and lutein to α-tocopherol, the carotenoids were more potent. Additionally, the effect with zeaxanthin was consistently more robust than with lutein and when α-tocopherol was combined with either carotenoid, the outcome was additive. Lutein, zeaxanthin and α-tocopherol were all efficient quenchers of singlet oxygen. We have also shown that lutein and zeaxanthin can protect against A2-PE/A2E photooxidation without appreciable consumption of the carotenoid by chemical reaction. This observation contrasts with the pronounced susceptibility of A2E and A2-PE to photooxidation and is of interest since lutein, zeaxanthin, A2E and A2-PE all have conjugated systems of carbon–carbon double bonds terminating in cyclohexenyl end-groups. The structural features responsible for the differences in quenching mechanisms are discussed. It has long been suspected that macular pigment protects the retina both by filtering high-energy blue light and by serving an antioxidant function. Evidence presented here suggests that the photochemical reactions against which lutein and zeaxanthin protect, may include those initiated by the A2-PE. Quantitative HPLC analysis revealed that in eyecups of C57BL/6J and BALB/cByJ mice, levels of A2-PE were several fold greater than the cleavage product, A2E. Taken together, these results may have implications with respect to the involvement of A2-PE formation in mechanisms underlying blue light-induced photoreceptor cell damage and may be significant to retinal degenerative disorders, such as those associated with ABCA4 mutations, wherein there is a propensity for increased A2-PE synthesis.

Introduction

Investigations on several fronts have shown that a substantial portion of the lipofuscin amassed in retinal pigment epithelial (RPE) cells originates in photoreceptor cells from fluorescent conjugates formed from visual cycle retinoids (Sparrow and Boulton, 2005). At least one of the retinoid adducts generated within photoreceptor outer segments is A2-PE (Fig. 1), a bis-retinoid compound that is the immediate precursor of the lipofuscin fluorophore A2E (Fig. 1) and its related photoisomers (Liu et al., 2000, Ben-Shabat et al., 2002). A2-PE is a product of the reaction of all-trans-retinal with phosphatidylethanolamine (2:1 ratio) and it has been isolated from bovine outer segments that are bleached to release endogenous all-trans-retinal (Liu et al., 2000, Ben-Shabat et al., 2002) and identified as the orange-colored fluorophore that accumulates in the photoreceptor outer segment debris of Royal College of Surgeon rats (Liu et al., 2000). A2-PE has also been detected in eyes of mice that are homozygous or heterozygous for null mutations in Abca4/Abcr (Mata et al., 2000, Mata et al., 2001), the gene that is causative for Stargardt macular degeneration (Allikmets et al., 1997). Additionally, an autofluorescent lipofuscin-like material, presumably A2-PE, has been described within the photoreceptor cell membrane in patients with Stargardt disease and retinitis pigmentosa (Szamier and Berson, 1977, Bunt-Milam et al., 1983, Birnbach et al., 1994). The structure of A2-PE, that of a phosphatidyl-pyridinium bisretinoid, was confirmed by collision-induced dissociation mass spectrometric analysis (FAB collision-induced dissociation mass spectrometry/MS) (Liu et al., 2000). Moreover, HPLC detection of A2E after enzyme-mediated hydrolysis of A2-PE established A2-PE as an intermediate in the A2E biosynthetic pathway (Liu et al., 2000, Ben-Shabat et al., 2002). Spectroscopic studies of A2-PE generated from dipalmitoy-l-α-phosphatidylethanolamine and all-trans-retinal (1:2 ratio), revealed two absorbance peaks with λmax 456 and 337 nm (Liu et al., 2000).

An important question that has not been addressed is whether the accumulation of the fluorescent pigment A2-PE impacts on the photoreceptor cell. For instance, being a bisretinoid molecule like A2E, A2-PE may also be susceptible to the same photooxidative change that we have demonstrated for A2E. In the latter case, we observed that upon irradiation (430 nm), A2E acts as a photosensitizer for generation of singlet oxygen and perhaps other reactive oxygen species that add to carbon–carbon double bonds of the retinoid-side arms of A2E (Ben-Shabat et al., 2002, Sparrow et al., 2002). The oxygen-containing moieties formed by the photoexcitation of A2E include epoxides that can rearrange to furanoid oxide structures and cyclic peroxides that are expected to be reactive (Ben-Shabat et al., 2002, Dillon et al., 2004, Jang et al., 2006). Experiments performed using an cell culture model, have shown that the photochemical events initiated by A2E can lead to modification of DNA and protein and can result in cell death (Sparrow and Cai, 2001, Sparrow et al., 2000, Sparrow et al., 2003, Sparrow et al., 2003; Zhou et al., 2005).

Potential sources of protection against the photooxidation of A2-PE are the dihydroxy carotenoids zeaxanthin and lutein (xanthophylls) (Fig. 1) which constitute the yellow macular pigment of the retina (Bone et al., 1985) and which absorb in the blue range of the visual spectrum (Junghans et al., 2001). These pigments are concentrated in the retina subsequent to dietary consumption of fruits and vegetables. They are also most abundant at the centre of the fovea and decline in amount toward the periphery, a pattern suggesting selective uptake by specific binding proteins (Bhosale et al., 2004). Lutein and zeaxanthin are incorporated into membranes (Landrum and Bone, 2001) and, in addition to the high levels that are present in photoreceptor cell axonal processes (Henle's fibres) (Snodderly et al., 1984), substantial quantities of lutein and zeaxanthin (10–25% of total retinal carotenoids) are present in photoreceptor outer segments (Sommerburg et al., 1999, Rapp et al., 2000). The macular pigment (λmax∼450) that is present in Henle's fibres is in a position to filter short-wavelength visible light, thereby decreasing chromatic aberration and scatter in the foveal image (Reading and Weale, 1974). In addition, the absorption of high energy blue light may prevent the latter from reaching the photosensitizers that are responsible for light damage to retina (Bone et al., 1997). These carotenoids are also suggested to serve as antioxidants (Khachik et al., 1997) and since they likely operate in a manner similar to other carotenoids (Landrum and Bone, 2001), it is possible that they quench singlet oxygen.

In the present work, we have observed that, for a pigment that has limited accretion because of conversion to A2E and related isomers, the levels of A2-PE in eyes of mice of different ages are appreciable relative to the amounts of A2E and iso-A2E. We have also sought evidence for the photooxidation of A2-PE and we have examined A2-PE as a photoreactive compound against which xanthophylls may act.

Section snippets

Reagents

Dulbecco's phosphate-buffered saline (DPBS) was purchased from Gibco (Grand Island, NY). HPLC grade solvents were purchased from Fisher Scientific (Fair Lawn, NJ). All-trans-retinal, ethanolamine, meta-chloroperoxybenzoic acid (mCPBA), phosphatidylethanolamine, α-tocopherol (vitamin E), trifluoroacetic acid, and all other chemicals were purchased from Sigma (St Louis, MO). Crystalline lutein and zeaxanthin (OPTISHARP™) were obtained as a gift from DSM Nutritional Products Ltd, Kaiseraugst,

A2-PE and A2E/iso-A2E in mice eyes

To demonstrate HPLC detection of A2-PE and A2E/iso-A2E and to compare A2-PE and A2E/iso-A2E in terms of age-dependent levels, we analysed these pigments in eyecups of C57BL/6J and BALB/cByJ mice at both 9 weeks and 8–9 months of age. Both strains were studied because we have previously shown that the levels of A2E are lower in C57BL/6J versus BALB/cByJ mice due to an amino acid variant in Rpe65 that slows the regeneration of 11-cis-retinal (Kim et al., 2004). The detection of A2E and A2-PE by

Discussion

We have previously shown that the excitation of A2E with blue light leads to the generation of singlet oxygen and perhaps other reactive oxygen species and that oxygen atoms are subsequently added to the A2E molecule (photooxidation) to create a reactive product with the potential for damaging cellular macromolecules such as protein and DNA (Sparrow and Cai, 2001, Sparrow et al., 2000, Sparrow et al., 2003, Zhou et al., 2005). Being a bisretinoid molecule like A2E it is not surprising that

Acknowledgements

This work was supported by NIH grant EY12951 (JRS) and by the American Health Assistance Foundation (JRS). JRS is a recipient of an Alcon Research Institute Award. Crystalline lutein and OPTISHARP™ zeaxanthin were gifts from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland.

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