Kelly Nelson

Surface Plasmon Resonance (SPR) Studies on the Interactions of Carotenoids and Their Binding Proteins

The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carotenoid preparations that are nano-dispersed in aqueous buffers, and then we used a new-generation surface plasmon resonance (SPR) protocol called FastStep™, which is significantly faster than conventional SPR assays. We have explored carotenoid-binding interactions of five proteins: human serum albumin (HSA), β-lactoglobulin (LG), steroidogenic acute regulatory domain proteins (StARD1, StARD3) and glutathione S- transferase Pi isoform (GSTP1). HSA and LG showed relatively weak interaction with carotenoids (K(D)>1 μM). GSTP1 evidenced high affinity and specificity towards zeaxanthin and meso-zeaxanthin with K(D) values 0.14±0.02 μM and 0.17±0.02 μM, respectively. StARD3 expressed a relative high specificity towards lutein with a K(D) value of 0.59±0.03 μM, whereas StARD1 exhibited a relatively low selectivity and affinity (K(D)>1 μM) towards the various carotenoids tested.

Blue-light reflectance imaging of macular pigment in infants and children.

PURPOSE While the role of the macular pigment carotenoids in the prevention of age-related macular degeneration has been extensively studied in adults, comparatively little is known about the physiology and function of lutein and zeaxanthin in the developing eye. We therefore developed a protocol using a digital video fundus camera (RetCam) to measure macular pigment optical density (MPOD) and distributions in premature infants and in children. METHODS We used blue light reflectance to image the macular pigment in premature babies at the time of retinopathy of prematurity (ROP) screening and in children aged under 7 years who were undergoing examinations under anesthesia for other reasons. We correlated the MPOD with skin carotenoid levels measured by resonance Raman spectroscopy, serum carotenoids measured by HPLC, and dietary carotenoid intake. RESULTS We enrolled 51 infants and children ranging from preterm to age 7 years. MPOD correlated significantly with age (r = 0.36; P = 0.0142), with serum lutein + zeaxanthin (r = 0.44; P = 0.0049) and with skin carotenoid levels (r = 0.42; P = 0.0106), but not with dietary lutein + zeaxanthin intake (r = 0.13; P = 0.50). All premature infants had undetectable macular pigment, and most had unusually low serum and skin carotenoid concentrations. CONCLUSIONS Our most remarkable finding is the undetectable MPOD in premature infants. This may be due in part to foveal immaturity, but the very low levels of serum and skin carotenoids suggest that these infants are carotenoid insufficient as a consequence of low dietary intake and/or severe oxidative stress. The potential value of carotenoid supplementation in the prevention of ROP and other disorders of prematurity should be a fruitful direction for further investigation.

Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina

Fatty acids (FAs), including long-chain and very long-chain polyunsaturated fatty acids (LC-PUFAs, C12-22; VLC-PUFAs, C24-38), play an important role in retinal function and health. Deficiencies in LC-PUFAs and VLC-PUFAs, as well as mutations in the enzyme responsible for elongation of very long-chain fatty acids (ELOVL4), have been associated with macular dystrophies and degenerations. Published analytical methods, including high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and gas chromatography-MS (GC-MS), can quantify VLC-PUFAs but require at least an entire human retina which limits the ability to understand physiologically relevant variations in lipids that can occur at a regional level within the retina. Until now, quantification of VLC-PUFAs in just the human macula, the cone-rich region of the central retina responsible for high acuity vision, has not been feasible due to its small size (4-5mm in diameter). In this study, we have developed a sensitive GC-MS method using newer generation enhanced GC-MS detector sensitivity which for the first time quantifies not only 14 VLC-PUFAs and 26 LC-FAs but also n-3/n-6 ratios of PUFAs in 4mm punches of human retina or a single pair of mouse retinas. Our results showed that saturated LC-FAs are higher in the human peripheral retina than in the macula, while unsaturated LC-FAs are higher in the macula than in the peripheral retina. On the other hand, the VLC-PUFAs are higher in the peripheral retina compared to macula. There is no difference in n-3/n-6 ratios of PUFAs observed between human macula and peripheral retina, while mouse retina has almost ten times more VLC-PUFAs than human macula and peripheral retina (2.27% versus 0.25% and 0.32%, respectively) and much higher n-3/n-6 ratios compared to human retina (9:1 versus ∼0.9:1). This high sensitivity analytical technique provides a valuable new tool for studies on the role of FAs in the pathological processes of macular degenerations and dystrophies.

Retinal accumulation of zeaxanthin, lutein, and β-carotene in mice deficient in carotenoid cleavage enzymes

Abstract Carotenoid supplementation can prevent and reduce the risk of age‐related macular degeneration (AMD) and other ocular disease, but until now, there has been no validated and well‐characterized mouse model which can be employed to investigate the protective mechanism and relevant metabolism of retinal carotenoids. &bgr;‐Carotene oxygenases 1 and 2 (BCO1 and BCO2) are the only two carotenoid cleavage enzymes found in animals. Mutations of the bco2 gene may cause accumulation of xanthophyll carotenoids in animal tissues, and BCO1 is involved in regulation of the intestinal absorption of carotenoids. To determine whether or not mice deficient in BCO1 and/or BCO2 can serve as a macular pigment mouse model, we investigated the retinal accumulation of carotenoids in these mice when fed with zeaxanthin, lutein, or &bgr;‐carotene using an optimized carotenoid feeding method. HPLC analysis revealed that all three carotenoids were detected in sera, livers, retinal pigment epithelium (RPE)/choroids, and retinas of all of the mice, except that no carotenoid was detectable in the retinas of wild type (WT) mice. Significantly higher amounts of zeaxanthin and lutein accumulated in the retinas of BCO2 knockout (bco2‐/‐) mice and BCO1/BCO2 double knockout (bco1‐/‐/bco2‐/‐) mice relative to BCO1 knockout (bco1‐/‐) mice, while bco1‐/‐ mice preferred to take up &bgr;‐carotene. The levels of zeaxanthin and lutein were higher than &bgr;‐carotene levels in the bco1‐/‐/bco2‐/‐ retina, consistent with preferential uptake of xanthophyll carotenoids by retina. Oxidative metabolites were detected in mice fed with lutein or zeaxanthin but not in mice fed with &bgr;‐carotene. These results indicate that bco2‐/‐ and bco1‐/‐/bco2‐/‐ mice could serve as reasonable non‐primate models for macular pigment function in the vertebrate eye, while bco1‐/‐ mice may be more useful for studies related to &bgr;‐carotene. HighlightsBCO1 and BCO2 regulate carotenoid delivery into the mouse retina.Zeaxanthin and lutein are preferentially accumulated in the mouse retina.Bco2‐/‐ mice can serve as “macular pigment mice” in study of eye disease prevention.

The Age-Related Eye Disease 2 Study: Micronutrients in the Treatment of Macular Degeneration

Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. With an increasingly aged population worldwide, the need for the prevention of AMD is rising. Multiple studies investigating AMD with the use of animal models and cell culture have identified oxidative stress-related retinal damage as an important contributing factor. In general, diet is an excellent source of the antioxidants, vitamins, and minerals necessary for healthy living; moreover, the general public is often receptive to recommendations made by physicians and health care workers regarding diet and supplements as a means of empowering themselves to avoid common and worrisome ailments such as AMD, which has made epidemiologists and clinicians enthusiastic about dietary intervention studies. A wide variety of nutrients, such as minerals, vitamins, ω-3 (n-3) fatty acids, and various carotenoids, have been associated with reducing the risk of AMD. Initial results from the Age-Related Eye Disease Study (AREDS) indicated that supplementation with antioxidants (β-carotene and vitamins C and E) and zinc was associated with a reduced risk of AMD progression. The AREDS2 follow-up study, designed to improve upon the earlier formulation, tested the addition of lutein, zeaxanthin, and ω-3 fatty acids. In this review, we examine the science behind the nutritional factors included in these interventional studies and the reasons for considering their inclusion to lower the rate of AMD progression.

RPE65 has an additional function as the lutein to meso-zeaxanthin isomerase in the vertebrate eye

Significance Carotenoids are plant-derived pigment molecules that cannot be synthesized de novo by higher organisms. These physiologically relevant compounds function as potent antioxidants and light screening compounds, and their supplementation has been shown to ameliorate the progression of such diseases as age-related macular degeneration. Hundreds of carotenoids are present in the plant world, but the primate macula contains only three: lutein, zeaxanthin, and meso-zeaxanthin. The presence of meso-zeaxanthin in the foveal region of primates is an unexplained phenomenon, given its lack of dietary sources. We show that RPE65 is responsible for the conversion of lutein to meso-zeaxanthin in vertebrates, a unique role for RPE65 in carotenoid metabolism beyond its well-known retinoid isomerohydrolase function in the vertebrate visual cycle. Carotenoids are plant-derived pigment molecules that vertebrates cannot synthesize de novo that protect the fovea of the primate retina from oxidative stress and light damage. meso-Zeaxanthin is an ocular-specific carotenoid for which there are no common dietary sources. It is one of the three major carotenoids present at the foveal center, but the mechanism by which it is produced in the eye is unknown. An isomerase enzyme is thought to be responsible for the transformation of lutein to meso-zeaxanthin by a double-bond shift mechanism, but its identity has been elusive. We previously found that meso-zeaxanthin is produced in a developmentally regulated manner in chicken embryonic retinal pigment epithelium (RPE)/choroid in the absence of light. In the present study, we show that RPE65, the isomerohydrolase enzyme of the vertebrate visual cycle that catalyzes the isomerization of all-trans-retinyl esters to 11-cis-retinol, is also the isomerase enzyme responsible for the production of meso-zeaxanthin in vertebrates. Its RNA is up-regulated 23-fold at the time of meso-zeaxanthin production during chicken eye development, and we present evidence that overexpression of either chicken or human RPE65 in cell culture leads to the production of meso-zeaxanthin from lutein. Pharmacologic inhibition of RPE65 function resulted in significant inhibition of meso-zeaxanthin biosynthesis during chicken eye development. Structural docking experiments revealed that the epsilon ring of lutein fits into the active site of RPE65 close to the nonheme iron center. This report describes a previously unrecognized additional activity of RPE65 in ocular carotenoid metabolism.

Developmentally regulated production of meso-zeaxanthin in chicken retinal pigment epithelium/choroid and retina

Purpose meso-Zeaxanthin is a carotenoid that is rarely encountered in nature outside of the vertebrate eye. It is not a constituent of a normal human diet, yet this carotenoid comprises one-third of the primate macular pigment. In the current study, we undertook a systematic approach to biochemically characterize the production of meso-zeaxanthin in the vertebrate eye. Methods Fertilized White Leghorn chicken eggs were analyzed for the presence of carotenoids during development. Yolk, liver, brain, serum, retina, and RPE/choroid were isolated, and carotenoids were extracted. The samples were analyzed on C-30 or chiral HPLC columns to determine the carotenoid composition. Results Lutein and zeaxanthin were found in all studied nonocular tissues, but no meso-zeaxanthin was ever detected. Among the ocular tissues, the presence of meso-zeaxanthin was consistently observed starting at embryonic day 17 (E17) in the RPE/choroid, several days before its consistent detection in the retina. If RPE/choroid of an embryo was devoid of meso-zeaxanthin, the corresponding retina was always negative as well. Conclusions This is the first report of developmentally regulated synthesis of meso-zeaxanthin in a vertebrate system. Our observations suggest that the RPE/choroid is the primary site of meso-zeaxanthin synthesis. Identification of meso-zeaxanthin isomerase enzyme in the developing chicken embryo will facilitate our ability to determine the biochemical mechanisms responsible for production of this unique carotenoid in other higher vertebrates, such as humans.

All three human scavenger receptor class B proteins can bind and transport all three macular xanthophyll carotenoids

Carotenoids are plant pigment molecules that are potent antioxidants. Carotenoids cannot be synthesized de novo; therefore, their dietary intake and transport to various tissues are essential to harness their health benefits. Two of the three scavenger receptor class B (SRB) proteins, SR-B1 and CD36, have been implicated as carotenoid transporters in lower species and in various tissues of higher animals. The function of the third SRB protein, SR-B2, in carotenoid transport is unknown. Using surface plasmon resonance (SPR) analyses, we have determined that all three human SRB proteins are capable of binding the macular xanthophyll carotenoids; lutein, zeaxanthin, and meso-zeaxanthin. By over-expressing human SRB proteins in cells that do not endogenously express SRBs, we have determined that lutein uptake is enhanced in the presence of LDL and is mediated by SR-B1 and CD36. SR-B1, SR-B2, and CD36 were able to take up significant amounts of zeaxanthin as well as meso-zeaxanthin, and uptake was increased in the presence of HDL. Our analyses revealed no apparent differences in protein expression profiles of SRBs in central and peripheral regions of human donor tissues, indicating that carotenoid-binding proteins rather than transporters are likely to mediate selective accumulation of carotenoids into the macula.