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ARVO 2014 Annual Meeting Abstracts

347 Retinoids, Carotenoids and Macular PigmentTuesday, May 06, 2014 11:00 AM–12:45 PMExhibit/Poster Hall SA Poster SessionProgram #/Board # Range: 3466–3492/D0066–D0092Organizing Section: Biochemistry/Molecular BiologyContributing Section(s): Clinical/Epidemiologic Research, Retina

Program Number: 3466 Poster Board Number: D0066Presentation Time: 11:00 AM–12:45 PMHow to obtain a rhodopsin spectrum from a turbid suspension - beating the light scatter problemFederico Gonzalez-Fernandez1, Richard DeSa2. 1Med Res Svc/Veterans Affairs, SUNY at Buffalo, Buffalo, NY; 2Olis Inc., Bogart, GA.Purpose: Spectroscopy of cellular suspensions is severely limited by light scattering. Our long-term goal is to monitor retinoid delivery and removal from suspensions of physiologically active photoreceptors. Being able to follow these processes in living cell suspensions could complement studies using isolated photoreceptors.Methods: Outer segments were prepared by shaking dark-adapted bovine retina in PBS. This was followed by filtration through a wire mesh to enrich for the broken outer segments. Absorbance spectra were obtained using: 1) A rectangular quartz cuvette in a standard configuration (1 cm path-length) with a HP 8452 diode array spectrophotometer, and 2) An 8 ml spherical quartz cuvette surrounded by a tightly packed proprietary white powder serving to maximize diffuse reflectance of light on the exterior walls of the flask. Light was delivered using an Olis RSM 1000 UV/Vis[NIR] rapid-scanning spectrophotometer. The apertures to the reflecting sphere through which the measuring light entered and the transmitted/scattered light exited to the photomultiplier tube were at 90 degree angles. A stir bar within the chamber facilitated mixing and suspension of any particulate matter. The absorbance / cm was calculated using algorithms described by Fry et al. (2010, Applied Optics 49:575).Results: The outer segment suspension was turbid with visible particulate material. The absorbance spectrum generated using the HP 8452 diode array showed a high absorbance exponential curve providing no information content due to the extensive light scattering. In contrast, spectra generated using the spherical chamber showed low overall absorbance and definite peaks at 405 and 503 nm. The later peak disappeared upon bleaching in the presence of 100 mM hydroxylamine.Conclusions: To our knowledge this is the first time an absorption spectrum of rhodopsin has been obtained from an outer segment suspension. The spherical cuvette designed for total internal reflectance was nearly immune to light scattering. The higher effective path-length within such reflectance cells enhances sensitivity and can be accounted for mathematically allowing determination of the absorbance / cm. The overall approach described here circumvents the problem of light scattering, and may open the way for the use of photoreceptor suspensions such as metabolically active RIS / ROS suspensions in physiological assays of the visual cycle.Commercial Relationships: Federico Gonzalez-Fernandez, None; Richard DeSa, Olis (I), Olis (P)Support: Merit Review Award I01BX007080 from the Biomedical Laboratory Research & Development Service of the Veterans Affairs Office of Research and Development, NIH RO1 EY09412; an Unrestricted Research Grant from Research to Prevent Blindness to the Department of Ophthalmology at SUNY at Buffalo.

Program Number: 3467 Poster Board Number: D0067Presentation Time: 11:00 AM–12:45 PMRetinoid Uptake, Processing and Secretion in Human iPS-RPE Support the Visual CycleAlberto Muniz1, Whitney Greene1, Mark Plamper1, Jae Hyek Choi1, Anthony J. Johnson1, Andrew T. Tsin2, Heuy-Ching H. Wang1. 1Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX; 2Department of Biology, The University of Texas at San Antonio, San Antonio, TX.Purpose: Retinal pigmented epithelium (RPE) derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation, in-vitro disease models, and drug screening. For this reason it is essential to determine the functional competence of iPS-RPE. One essential role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.Methods: iPS-RPE was derived from human iPS cells. RT-PCR, immunocytochemistry and western blot analysis were used to detect expression of RPE- genes LRAT, RPE65, CRALBP and PEDF. All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. Retinoids were extracted and analyzed by HPLC.Results: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with NEM prevented retinyl ester synthesis. Additionally, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.Conclusions: iPS-RPE develops classical RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells’ ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in induced pluripotent stem cell-derived RPE.Commercial Relationships: Alberto Muniz, None; Whitney Greene, None; Mark Plamper, None; Jae Hyek Choi, None; Anthony J. Johnson, None; Andrew T. Tsin, None; Heuy-Ching H. Wang, NoneSupport: U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP)

Program Number: 3468 Poster Board Number: D0068Presentation Time: 11:00 AM–12:45 PMEarly Morphological Changes in IRBP Knockout MiceShannon Getz, Micah A. Chrenek, Natecia Williams, Jeffrey H. Boatright, J M. Nickerson. Ophthalmology, Emory University, Atlanta, GA.Purpose: Because IRBP knockout (KO) mice develop profound myopia and show a slow retinal degeneration after P23, we posit that IRBP plays an essential role in eye development and setting the number of retinal cells. Excessive ocular enlargement for IRBP KO begins some time between P7 and P10 (Wisard et al. IOVS. 2011; 52:5804-11). This coincides with “inner rod” development and death (Young, RW. J Comp Neurol. 1984; 229:362-73). To more accurately define when myopic elongation begins in IRBP KO mice, we measured globe dimensions, ophthalmic, and histological features

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in IRBP KO and C57BL/6J (WT) mice each day from postnatal (P) day 5 to 10.Methods: IRBP KO and WT mice were sacrificed from P5 to P10. Axial, nasal-temporal (N-T) and superior-inferior (S-I) measurements were conducted with a laser micrometer. The eyes were stained and the number of nuclei and retinal layer thickness were measured. For IRBP mRNA localization, fluorescence in situ hybridization was performed with IRBP KO and WT at P60. TUNEL staining was used to evaluate apoptosis. SD-OCT and fundus images were taken at P15 and P30.Results: IRBP mRNA was found exclusively in the inner segments and the outer nuclear layer in WT, but not in the IRBP KO. At each age, the mouse body weight of WT and KO strains was the same. From P5 to P7, the eye weight, axial, N-T, and S-I measurements were the same in both mice but increases (in IRBP KO over WT) were first detected at P8. Each retinal layer showed a slight decrease in thickness in the KO. The full thickness of the retinal layers showed slight thinning in the KO, beginning at P8. SD-OCT confirmed histological thickness measures at P15 & P30. A trend towards fewer “Inner rods” was found in the IRBP KO. No difference in fundus appearance was detected.Conclusions: Abnormalities in the IRBP KO began at P8 with a distinct increase of eye size in all three dimensions and eye weight. The WT and IRBP KO have the same body weight, demonstrating that the changes in eye size do not correspond to a change in body weight. IRBP KO nuclear counts and retinal layer thicknesses were less than WT, but not significantly. These changes suggest that IRBP plays an important developmental role in eye shape and size, affecting phenotype gradually beginning at a very discrete age, P8, with minimal impact early on retinal thickness, which may imply an excess total number of retinal cells, that are lost later (P23 and later).Commercial Relationships: Shannon Getz, None; Micah A. Chrenek, None; Natecia Williams, None; Jeffrey H. Boatright, None; J M. Nickerson, NoneSupport: NH Grant EY016470, NH Grant EY021592, NH Grant EY006360, Research to Prevent Blindness, Abraham & Phyllis Katz Foundation

Program Number: 3469 Poster Board Number: D0069Presentation Time: 11:00 AM–12:45 PMEvaluation of mutant retinol cycle enzymesMarkus N. Preising, Annabella Janise, Hoffmann Ina, Weber Julia, Birgit Lorenz. Department of Ophthalmology, Justis-Liebig University, Giessen, Germany.Purpose: We reported Fundus albipunctatus in patients caused by mutations in RDH5R238W and A294P and RPE65I115T (Retina 2010 and Ophthalmology 2011). We hypothesize disturbed interactions of the retinol cycle enzymes RDH5, RLBP1, and RPE65 as a possible reason for the reduced rate of conversion to 11-cis-retinal. Here we report on investigations of RDH5, RLBP1, and RPE65 under heterologous expression of wild type and mutant RDH5 and RPE65.Methods: RDH5, RLBP1, and RPE65 were cloned into pCDNA3.1 vectors and mutations were introduced into RDH5 and RPE65 by mutation specific primers and PCR. The constructs were expressed in HeLa cells, and probed with anti-human RDH5 (Santa Cruz Biotechnologie), anti-human RLBP1, and RPE65 (Novus Biologicals) and Alexa Fluor coupled secondary antibodies (Invitrogen).Protein was extracted from human RPE specimen using the Proteo Extract Native Membrane Protein Kit (Merck, Darmstadt) and after heterologous expression of constructs in HEK293 cells. The protein samples were resolved on Clear Native (CN)-PAGE gels (Serva).

RDH5 and RLBP1 were probed ON Western-blots with primary antibodies and HRP-labled secondary antibodies (Sigma-Aldrich).Results: Wild type RDH5 and RLBP1 were comparably distributed throughout the cytoplasma. RDH5R238W and RDH5A294P did not considerably change the intracellular localization but showed additional local accumulation of immunoreactivity close to the plasma membrane. RPE65 was distributed throughout the cytoplasma but focused close to the nucleus. RPE65I115T did not change this distribution.CN-PAGE revealed immunopositive bands comparable with dimeric RDH5 in protein extracts from human RPE specimen and additional bands in the higher molecular range. RLBP1 immunoreactivity was detected predominantly at sizes according to monomeric protein and at smaller amounts in the higher molecular range. Heterologous expression revealed immunoreactivity at sizes according to dimeric and higher molecular weight complexes with RDH5 from wild type and mutant constructs overlapping with RLBP1 immunopositive signals.Conclusions: We could show that RDH5 forms dimeric and higher molecular weight complexes which were localized throughout the cytoplasma in HeLa cells. The interactions and localizations were not completely abolished by the mutant proteins. Mutant RPE65 localized comparable to wild type RPE65 in HeLa cells. The higher molecular weight complexes detected in CN-PAGE require further work-up.Commercial Relationships: Markus N. Preising, None; Annabella Janise, None; Hoffmann Ina, None; Weber Julia, None; Birgit Lorenz, None

Program Number: 3470 Poster Board Number: D0070Presentation Time: 11:00 AM–12:45 PMThe Investigation of Human Retinal Lipofuscin Fluorophores in Association with Age Related Macular DegenerationJennifer Tournear1, James P. Dillon1, Elizabeth R. Gaillard1, 2. 1Chemistry and Biochemistry, Northern Illinois University, Dekalb, IL; 2Biology, Northern Illinois University, Dekalb, IL.Purpose: To investigate the chemical composition and fluorophores of human retinal lipofuscin for a better understanding of age related macular degeneration (AMD). Additional purpose is to examine the differences in composition of extracts of donor tissue diagnosed as wet and dry AMD.Methods: Human retinal lipofuscin is extracted from human donor eyes diagnosed with either wet or dry AMD as previously described by Feeney-Burns. The organic soluble lipofuscin is collected, dried, and reconstituted with methanol for use in high performance liquid chromatography tandem mass spectrometry (LC/MS) coupled with a fluorescent detector (Surveyor LC with PDA, Thermo Finnigan LCQ Advantage MS, Surveyor FL). Mass spectrometry data is analyzed in parallel with fluorescence data to determine novel fluorophores for use in diagnostic techniques. Tandem mass spectrometry data is analyzed for the investigation of chemical composition specific to wet and dry AMD.Results: Lipofuscin extracts from human donor tissue diagnosed as wet and dry AMD have been subjected to LC/MS. Total ion chromatograms observed from LC/MS analysis suggests unique compositions for wet and dry AMD. The extensively studied fluorophore, A2E, was not observed in lipofuscin extracts diagnosed as dry AMD. However, A2E and its derivatives were observed in extracts diagnosed as wet AMD and verified by analysis of fragmentation patterns.Conclusions: Some fluorophores of lipofuscin from AMD diagnosed tissue have been determined. The lipofuscin extracted from tissue diagnosed as wet and dry AMD suggest different chemical




composition. This proposes the possibility that wet and dry AMD are actually two different diseases. Analysis of an undefined AMD lipofuscin extract shows similarity to data obtained for dry AMD. Understanding the chemical composition and fluorophores found in these samples can aid in furthering the treatment, diagnosis and prevention of wet and dry AMD.Commercial Relationships: Jennifer Tournear, None; James P. Dillon, None; Elizabeth R. Gaillard, None

Program Number: 3471 Poster Board Number: D0071Presentation Time: 11:00 AM–12:45 PMP25L knock-in of mouse Rpe65 shows little change in visual function under normal low light conditions but confers protection against acute high intensity light damageT M. Redmond1, Todd Duncan1, Yichao Li2, Haohua Qian2, Lijin Dong3, Yan Li1. 1Lab. of Retinal Cell & Molecular Biology, National Eye Inst/NIH, Bethesda, MD; 2Visual Function Core, National Eye Institute, Bethesda, MD; 3Genetic Engineering Core, National Eye Institute, Bethesda, MD.Purpose: RPE65 is the RPE visual cycle retinol isomerase and various mutations of RPE65 in human lead to the retinal dystrophy termed Leber congenital amaurosis 2 (LCA2) that result in blindness. One such mutation is the homozygous P25L found in a LCA2 patient with mild phenotype including well-preserved cone function. We previously found that the hypomorphic RPE65 P25L missense mutant enzyme retains about 8% of wildtype (WT) activity in vitro. We generated the Rpe65/P25L knock-in mouse model to understand how this missense mutation affects retinal biochemistry and physiology.Methods: Retinal/RPE integrity of the homozygous Rpe65 /P25L knock-in mice (referred as KI/KI) and WT siblings (both Leu at aa450) were studied by optical coherence tomography (OCT) in vivo, and by histology. Visual function was examined by single flash ERG at different stimulus intensities. The ocular retinoid levels of KI/KI and WT mice were compared by reverse-phase HPLC. KI/KI mice and WT siblings were subjected to acute light damage treatment (10, 000 lux for 30 minutes) and consequent retinal degeneration was monitored.Results: Both histology and OCT showed no significant morphological differences between KI/KI and WT mice maintained under standard colony management protocols. HPLC retinoid analysis revealed comparable levels of 11-cis retinal and other retinoids in KI/KI and WT retinae and eyecups. Scotopic ERG responses of KI/KI mice also were similar to those observed in WT mice. The recovery of dark adaptation after light bleach, however, was slightly delayed in KI/KI mice, suggesting altered kinetics of visual cycle and/or rhodopsin regeneration. Significantly, P25L KI/KI mice showed increased resistance to light-induced retinal damage compared to WT animals.Conclusions: Taken together, our data suggest that under modest light intensities (<100 lux; such as experienced in standard mouse husbandry), mice with the P25L mutation have ample time to replenish chromophore, which thus manifests in a minimal phenotype in the KI/KI mice. Only when the retina is stressed with acute high light intensity is the biochemical effect of this mutation seen at the physiological level, when the reduced expression and catalytic activity of the hypomorphic P25L RPE65 protects the retina from the successive cycles of opsin regeneration that drive the light damage outcome.Commercial Relationships: T M. Redmond, None; Todd Duncan, None; Yichao Li, None; Haohua Qian, None; Lijin Dong, None; Yan Li, NoneSupport: Intramural Research Program of the NEI, NIH

Program Number: 3472 Poster Board Number: D0072Presentation Time: 11:00 AM–12:45 PMInterphotoreceptor retinoid-binding protein promotes delivery of all-trans retinol into rat Müller Cells in cultureBrandi S. Betts-Obregon1, Federico Gonzalez-Fernandez2, Andrew S. Mendiola1, Andrew T. Tsin1. 1Biology, University of Texas at San Antonio, San Antonio, TX; 2Med Res Svc/Veterans Affairs, SUNY at Buffalo, Buffalo, NY.Purpose: Interphotoreceptor Retinoid-Binding Protein (IRBP), a major component of the interphotoreceptor matrix, is thought to thought to function in the cone visual cycle. However, the mechanism that IRBP facilitates cone – Müller cell retinoid trafficking is unknown. IRBP binds to the pericellular matrix of the Müller cell villi. We hypothesize that IRBP facilitates the delivery of all-trans retinol (ATOL) into rMC-1 cells in culture.Methods: IRBP was purified from bovine retina by a combination of ion exchange, ConA affinity, and size exclusion chromatography. The concentration of the purified IRBP was confirmed by amino acid analysis. Rat Müller cells (rMC-1) were seeded into 6-well plates at 2.5 x 105 cells per mL per well. BSA or apo-bovine IRBP (bIRBP) was pre-incubated with ATOL before the ATOL/protein mixture was introduced into the culture medium at a final concentration of 10mM ATOL and 2mM BSA or bIRBP. rMC-1 homogenates and culture media were collected at 0, 12 and 24 hours, followed by retinol extraction and HPLC analysis.Results: After incubation with ATOL and BSA, ATOL in the cell media decreased from 2200 pmol/mL at 0 hour to 1700 pmol/mL at 12 hours, and then to 1132 pmol/mL at 24 hours (n=6). After incubation with ATOL and bIRBP, ATOL decreased from 2200 pmol/mL at 0 hours to 1200 pmol/mL at 12 hours of incubation, and then to 704 pmol/mL at 24 hours (n=9). Homogenates from Müller cells without the addition of ATOL to cell media or with the addition of ATOL without BSA or bIRBP (i.e. ATOL in ethanol dispersed directly into the cell media at a final concentration of 10 mM) did not yield ATOL. However, homogenates from Müller cells incubated with ATOL and BSA for 12 hours yielded 1788 pmol/mg protein, and this level increased to 3452 pmol/mg at 24 hours (n=6). After incubation of ATOL and bIRBP for 12 hours, cell homogenate yielded 2100 pmol/mg protein, and this level increased to 6900 pmol/mg protein at 24 hours (n=9).Conclusions: Incubation of ATOL and BSA or bIRBP resulted in a time-dependent increase of ATOL delivery into rat Müller cells in culture. In comparison to BSA, bIRBP facilitated a 2-fold higher accumulation of ATOL within a 24 hour incubation period. This may be attributable to the higher binding specificity and protective effect of retinol by bIRBP, and/or ability of IRBP to interact with the Müller cell.Commercial Relationships: Brandi S. Betts-Obregon, None; Federico Gonzalez-Fernandez, None; Andrew S. Mendiola, None; Andrew T. Tsin, NoneSupport: National Center for Research Resources 5G12RR013646-12, National Institutes of Minority Health and Health Disparities (G12MD007591), Merit Review Award I01BX007080 from the Biomedical Laboratory Research & Development Service of the Veterans Affairs Office of Research and Development, NIH RO1 EY09412 and an Unrestricted Research Grant from Research to Prevent Blindness to the Department of Ophthalmology at SUNY at Buffalo.




Program Number: 3473 Poster Board Number: D0073Presentation Time: 11:00 AM–12:45 PMHyper Phototransduction of Cone and Rod Photoreceptors in Cntf-/- Mice is Underpinned by Upregulated Opsins and RPE65Minghao Jin1, Songhua Li1, Kota Sato1, Michael Sendtner2. 1Ophthalmology & Neuroscience, LSU Health Sciences Center, New Orleans, LA; 2Institute for Clinical Neurobiology, University of Würzburg, Würzburg, Germany.Purpose: Ciliary Neurotrophic Factor (CNTF) is a potent neurosurvival factor and has been applied to clinical trials for curing retinal degenerative diseases. CNTF exerts its function mainly through the gp130-STAT3 pathway that regulates gene expression. However, the role of CNTF in regulation of genes involved in the phototransduction and the visual cycle remains poorly understood. The purpose of this study is to define the role of CNTF in vision by identifying key phototransduction and visual cycle genes altered in Cntf-/- mice.Methods: We generated Cntf-/- mice with homologous Leu450 alleles of the Rpe65 gene. Visual function of cone and rod photoreceptors in 129S2/Sv and Cntf-/- mice was analyzed by photopic and scotopic electroretinography (ERG). The dark-adaptation kinetics in these mice was evaluated by recording scotopic ERG responses under different dark-adaptation conditions. We measured the flow of retinoids in the retinas and RPE to compare the regeneration rates of the 11-cis retinal chromophore in 129S2/Sv and Cntf-/-retinas. Expression levels of photoreceptor specific proteins and the visual cycle enzymes were determined by quantitative immunoblot analysis. Retinoid isomerase activity in the RPE was measured by monitoring synthesis of 11-cis retinol from all-trans retinol substrate. Retinoids were analyzed by high-performance liquid chromatography.Results: Cone and rod ERG responses in overnight dark-adapted Cntf-/- mice were significantly higher than those in 129S2/Sv mice under the same light condition. The recovery kinetics of rod photoreceptor light sensitivity measured by scotopic ERG was faster in the Cntf-/- mice compared to that in the wild-type (WT, 129S2/Sv) mice. Consistent with this result, the regeneration rate of the visual chromophore in the Cntf-/- retina was faster than that in WT mice. The outer nuclear layers stained with DAPI were at least 20% thicker in Cntf-/- mice than those in WT mice. Expression levels of cone opsins, rod opsin, and RPE65 in Cntf-/- mice at p10 and/or p21 were significantly higher than those in WT mice at the same ages. The retinoid isomerase activity in the Cntf-/- RPE was at least 15% higher than that in WT RPE.Conclusions: CNTF negatively regulates expression of cone opsin, rod opsin, and Rpe65, which play a pivotal role in photoreceptor light perception and degeneration of photoreceptors induced by light damage.Commercial Relationships: Minghao Jin, None; Songhua Li, None; Kota Sato, None; Michael Sendtner, NoneSupport: NIH Grant EY021208, RPB

Program Number: 3474 Poster Board Number: D0074Presentation Time: 11:00 AM–12:45 PMInhibition of RPE65 retinol isomerase activity by inhibitors of lipid metabolismAbdulkerim EROGLU, Susan Gentleman, Eugenia Poliakov, T M. Redmond. Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, MD.Purpose: RPE65 is the key isomerase in the RPE visual cycle that catalyzes the conversion of all-trans retinyl ester (ATRE) into 11-cis retinol. Recent crystal structures of RPE65 and site-directed mutagenesis studies have revealed aspects of its catalytic mechanism,

especially with respect to retinyl moiety isomerization, but other aspects remains to be determined. We are particularly interested in potential parallels between the RPE65 catalytic mechanism and that of lipid metabolism enzymes. We tested inhibitors of lipid metabolism to determine their effect on RPE65 isomerase activity.Methods: HEK 293-F cells were transiently transfected with expression vectors for visual cycle proteins (RPE65, LRAT, CRALBP and RDH5) and isomerase activity was measured in cellulo in the presence of added substrate and inhibitors. Membrane preparations of transfected cells were used to test the effect of additives on isomerase activity in vitro. Isomerase activity was determined by normal phase HPLC of isomeric retinols extracted from these cells or membranes.Results: In experiments to determine potential interactions between RPE65 and acyl-CoA synthases, we discovered that fatty acyl analog inhibitors of fatty acid CoA ligases (ACSLs), also potently inhibited RPE65 isomerase activity in HEK293-F cells transfected with RPE65, LRAT, CRALBP and RDH5. LRAT activity was not affected. RPE65 protein expression was not affected by any concentration of these inhibitors. Testing with a range of ATROL substrate concentrations revealed the mode of inhibition to be generally competitive in nature. To determine if these fatty acid analogs compete with the ATRE substrate specifically, we incubated membranes prepared from transfected cells with liposomes containing a range of ATRE concentrations. Our results also indicated a competitive nature of inhibition.Conclusions: We have identified inhibitors of ACSLs used in studies of lipid metabolism as potent inhibitors of RPE65 and that compete with the ATRE substrate of RPE65 for binding, thereby inhibiting its isomerase activity. The mode of inhibition of these agents likely depends directly on competition with the fatty acyl moiety of the retinyl ester in binding to the enzyme. The effects of this class of inhibitors provide further insight into the catalytic mechanism of RPE65 retinol isomerase.Commercial Relationships: Abdulkerim EROGLU, None; Susan Gentleman, None; Eugenia Poliakov, None; T M. Redmond, NoneSupport: Intramural Research Program of the National Eye Institute, NIH

Program Number: 3475 Poster Board Number: D0075Presentation Time: 11:00 AM–12:45 PMPSMD13 Promotes Degradation of Disease-Causing RPE65s via the Ubiquitin-Proteasome PathwaySonghua Li1, Tadahide Izumi2, Jane Hu3, Samuel G. Jacobson4, Dean Bok3, Minghao Jin1. 1Ophthalmology and Neuroscience, LSU Health Sciences Center, New Orleans, LA; 2Toxicology, University of Kentucky, Lexington, KY; 3Jules Stein Eye Institute, UCLA, Los Angeles, CA; 4Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA.Purpose: Mutations in the RPE65 gene have been associated with Leber’s congenital amaurosis (LCA). Many LCA-associated missense mutations result in rapid degradation of mutant RPE65 by an unknown mechanism. Pathogenicity of these mutations may depend on the degradation rate of mutant RPE65. The purpose of this study is to identify the molecular mechanism underlying rapid degradation of mutant RPE65s and to determine whether the 26S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, mediates degradation of mutant RPE65s.Methods: LCA-associated RPE65s generated by site directed mutagenesis were expressed in cultured human primary RPE and ARPE19 cells. Protein and mRNA contents of wild-type (WT) and mutant RPE65s in the cells were determined by quantitative immunoblotting and RT-PCR, respectively. To determine if mutant




RPE65s are degraded in the proteasome, cells were treated with proteasome inhibitors (MG115 & MG132) or pepstatin A, a lysosome inhibitor. Over-expression and siRNA-mediated knockdown were applied to test whether PSMD13 mediates degradation of WT and mutant RPE65s. RPE65s ubiquitinated with ubiquitin-His6 were purified with Ni-NTA beads and subjected to immunoblot analysis. Inhibition of protein ubiquitination was carried out using an inhibitor of ubiquitin-activating enzyme E1. Formation of disulfide bond-mediated protein complexes was determined using reducing agents. Aggresomes of mutant RPE65s in the cells were observed by confocal microscopy.Results: Expression levels of mutant RPE65s were 70-90% lower than that of WT RPE65 in the cells whereas the mRNA contents of mutant RPE65s were similar to that of WT RPE65. Proteasome inhibitors, but not pepstatin A, significantly increased expression levels of mutant RPE65s. Co-expression of PSMD13 resulted in a decrease in protein content of mutant RPE65s whereas knock down of PSMD13 rescued expression levels of the mutant RPE65s. WT and mutant RPE65s were ubiquitinated in ARPE19 cells. Inhibition of protein ubiquitination promoted formation of high molecular weight complexes (HMC) that contain mutant RPE65s. This HMC formation was significant in the absence of reducing reagents, but decreased in the presence of reducing reagents. Mutant RPE65s formed aggresomes in the RPE cells.Conclusions: PSMD13 regulates pathogenicity of RPE65 mutations by mediating degradation of mutant RPE65 in the proteasome.Commercial Relationships: Songhua Li, None; Tadahide Izumi, None; Jane Hu, None; Samuel G. Jacobson, None; Dean Bok, None; Minghao Jin, NoneSupport: NIH Grand EY021208, RPB

Program Number: 3476 Poster Board Number: D0076Presentation Time: 11:00 AM–12:45 PMIdentification of Key Residues Enhancing Isomerohydrolase Activity of Human RPE65 for More Efficient Gene TherapyYusuke Takahashi1, 3, Gennadiy P. Moiseyev2, 3, Jian-Xing Ma2,

3. 1Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 3Harold Hamm Diabetes Center, Oklahoma City, OK.Purpose: RPE65, retinoid isomerohydrolase, is a membrane-associated protein, which is predominantly expressed in the retinal pigment epithelium (RPE) and converts all-trans retinyl ester to 11-cis retinol, a key reaction in the retinoid visual cycle. We have previously reported that RPE65 in cone-dominant chicken (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits approximately 7 fold higher isomerohydrolase activity than that of bovine RPE65 and hRPE65. We expected that some key residues are responsible for the higher enzymatic activity of cRPE65. The purpose of this study was to identify the key residues responsible for the higher enzymatic activity of cRPE65.Methods: Based on the amino acid sequence comparison of mammalian and chicken RPE65, 8 residues of hRPE65 were replaced by their counterparts of cRPE65 using site-directed mutagenesis. The wt hRPE65, cRPE65 and the site-directed mutants were expressed in 293A-LRAT, a cell line stably expressing human lecithin retinol acyltransferase (LRAT). The expression levels of generated mutants were examined by Western blot analysis and semi-quantified by densitometry. The enzymatic activities of the mutants were measured by in vitro isomerohydrolase activity assay, and the generated retinoids were analyzed by HPLC.Results: Among the mutants analyzed, we identified that two single mutations of N170K and K297G in hRPE65 and the double

mutant, N170K/K297G, exhibited significantly higher catalytic activity than that of wt hRPE65. Further, when the amino terminal end (1Met-33Arg) fragment of the N170K/K297G double mutant of hRPE65 was replaced by the corresponding cRPE65 fragment, the isomerohydrolase activity and protein level were further increased to a level similar to that of cRPE65.Conclusions: We successfully engineered more efficient human isomerohydrolase. This finding contributes to the understanding of structural basis for isomerohydrolase activity and may improve the efficacy of RPE65 gene therapy for retina degeneration caused by RPE65 mutations.Commercial Relationships: Yusuke Takahashi, None; Gennadiy P. Moiseyev, None; Jian-Xing Ma, NoneSupport: NIH grants EY018659, EY012231, EY019309, P20GM104934, an IRRF grant and OCAST grants (HR12-103 and HR13-076)

Program Number: 3477 Poster Board Number: D0077Presentation Time: 11:00 AM–12:45 PMIdentification and in vitro Characterization of Novel Non-retinoid RPE65 InhibitorsGennadiy P. Moiseyev1, Jian-Xing Ma1, Konstantin Petrukhin2. 1Physiology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Ophthalmology, Columbia University, New York, NY.Purpose: Accumulation of A2E, a toxic non-degradable bisretinoid, a byproduct of the visual cycle, in the retinal pigment epithelium (RPE) plays a major role in pathogenesis of age-related macular degeneration (AMD) and Stargardt disease (STGD), causing dysfunction and death of RPE cells. Slowing the visual cycle by inhibition of the rate-limiting step catalyzed by RPE65 is a promising strategy to decrease the accumulation of A2E. The aim of this study was the identification of new non-retinoid compounds capable of inhibiting RPE65 and slowing the visual cycle.Methods: Using the available crystallographic model of bovine RPE65 we generated a docking model of its ligand-binding pocket and conducted the in silico screen of 350,000 compounds. Compounds with the highest QFIT score were characterized experimentally. All-trans-[3H]-retinol was used as a substrate to measure the activities of lecithin:retinol acyltransferase (LRAT) and isomerohydrolase in bovine RPE microsomes. The generated retinoids were analyzed by HPLC. To determine the inhibition mode of the compounds chicken RPE65 was used in a liposome isomerohydrolase assay.Results: A total of 65 compounds with the highest QFIT scores were synthesized for the experimental verification. Four compounds identified in the in silico docking completely inhibited bovine RPE65 when tested at 200 mM. IC50 for these compounds were determined from the concentration-dependent inhibition of RPE65 activity measured in bovine microsomes. IC50 for the most potent inhibitor CU239 was found to be 6 mM. LRAT activity was not affected by these inhibitors at a concentration as high as 200 mM. To analyze the inhibition type, all-trans retinyl palmitate was incorporated in liposomes and used as a substrate for chicken RPE65. The concentration dependence of RPE65 reaction was measured in the absence and presence of CU239. The Lineweaver-Burk graph demonstrated two lines intersected at Y-axis suggesting that CU239 inhibits the isomerohydrolase reaction in a competitive manner.Conclusions: We identified CU239, a small molecule compound, which potently and selectively inhibited conversion of all-trans-retinyl ester to 11-cis-retinol catalyzed by RPE65 isomerohydrolase. The competitive mode of the inhibition suggests that this inhibitor is likely bound to the RPE65 active site. This inhibitor may be used to




slow down the visual cycle and to prevent accumulation of A2E in STGD and AMD.Commercial Relationships: Gennadiy P. Moiseyev, None; Jian-Xing Ma, None; Konstantin Petrukhin, NoneSupport: NIH grants EY018659, EY012231, EY019309, P20RR024215, U01 NS074476 and R24 EY019861

Program Number: 3478 Poster Board Number: D0078Presentation Time: 11:00 AM–12:45 PMOver-expression of RBP4 Causes Progressive Retinal Degeneration in MiceKrysten M. Farjo1, Laura Otalora1, TJ Hollingsworth1, Rafal Farjo2, Alexander Quiambao2. 1Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2EyeCRO LLC, Oklahoma City, OK.Purpose: Elevated levels of serum retinol-binding protein (RBP4) contribute to the development of insulin resistance and type 2 diabetes. Moreover, patients with proliferative diabetic retinopathy have a significant increase in serum RBP4 levels compared to diabetic patients with mild or no retinopathy. We have previously shown that elevation of RBP4 induces inflammation in human retinal capillary endothelial cells by a retinol-independent mechanism, and thus may contribute to the formation of vascular lesions in diabetic retinopathy. The current study evaluates the physiologic effects of serum RBP4 elevation on the retina.Methods: We are using transgenic mice that constitutively over-express RBP4 (RBP4-Tg), resulting in a ~10-fold increase in serum RBP4 levels compared to wild-type mice, which is similar to the level of RBP4 elevation in patients with long-duration type 2 diabetes. RBP4-Tg mice and wild-type controls were evaluated from 1 to 9 months of age to assess retinal function and structure by electroretinography (ERG) and histological analyses, respectively. To determine the effect of RBP4 elevation on retinal retinoid uptake and visual cycle activity, retinoid levels in mouse eyecups were quantified by high performance liquid chromatography.Results: The ERG scotopic a-wave amplitude is consistently diminished by ~33% in RBP4-Tg mice between ages 3-9 months, whereas the scotopic and photopic b-wave amplitudes are initially reduced by 20-30% at 3 months and progressively decline with age to a 50-70% reduction by 9 months of age. Quantitative “spidergram” histological analyses showed significant thinning of the outer and inner nuclear layers (ONL and INL) of the retina, with the INL thinning being more pronounced. This coincides with the dominant ERG b-wave reduction, as well as a significant increase in TUNEL-positive nuclei within the INL and increased expression of glial fibrillary acidic protein (GFAP), a marker of retinal gliosis. There was no difference in cumulative steady-state dark-adapted retinal retinoid profiles between RBP4-Tg and wild-type controls, indicating that the visual cycle is intact in RBP4-Tg mice. Further analyses of visual cycle kinetics and retinal inflammation are ongoing.Conclusions: These results, in combination with our studies in human endothelium, suggest that elevation of serum RBP4 could contribute to both vascular lesions and neurodegeneration in diabetic retinopathy.

Commercial Relationships: Krysten M. Farjo, None; Laura Otalora, None; TJ Hollingsworth, None; Rafal Farjo, EyeCRO LLC (E); Alexander Quiambao, EyeCRO LLC (E)Support: NIH-NIGM Grant P20GM104934; BrightFocus Grant M2012057

Program Number: 3479 Poster Board Number: D0079Presentation Time: 11:00 AM–12:45 PMRole of plasma HDL remodeling and intestinal ABCA1 activity in the uptake of dietary lutein and zeaxanthinEric J. Niesor, Evelyne Chaput. F. Hoffmann-La Roche, Basel, Switzerland.Purpose: The mechanism of intestinal uptake and delivery to peripheral tissues of lutein (L) and zeaxanthin (Z), two antioxidants of dietary origin, is poorly understood. The intestinal absorption of these carotenoids is thought to involve high-density lipoproteins (HDL) and the ATP-binding cassette transporter A1 (ABCA1). We examined the effect of compounds known to modify HDL metabolism on intestinal uptake of L and Z.Methods: We first developed an animal model to study the intestinal uptake process by feeding hamsters increasing concentrations of a dietary supplement of L and Z given as 0.01 to 0.3% Floraglo® (n=10 per group). The selected 0.1% Floraglo®-supplemented diet was fed to animals treated for 14 days with/without the cholesteryl-ester transfer protein (CETP) modulator dalcetrapib, or inhibitor anacetrapib, as well as the ABCA1- inducer liver X receptor (LXR) agonist TO901317 as a 0.320%, 0.024% and 0.008% (w:w)




food admixture, respectively (n=10 per group). Plasma and liver levels of L and Z were determined by high-performance liquid chromatography mass spectrometry.Results: Supplementation of the diet with Floraglo® resulted in a dose-dependent elevation of plasma L and Z levels. L and Z concentrations in the liver were also dramatically raised, and tightly correlated with plasma levels. Treatment with dalcetrapib, which is known to increase HDL remodeling and pre-β-HDL formation, doubled plasma and liver L (+95% p<0.001 and +90%; p<0.001, respectively) and Z (+101%; p<0.001 and +109%; p< 0.001, respectively), without significantly increasing plasma HDL-cholesterol (HDL-C). Anacetrapib, which decreases CETP activity without allowing HDL remodelling, did not modify plasma and liver L and decreased plasma and liver Z (-32 and -31%; p<0.05, respectively) despite a 54% (p<0.001) increase in plasma HDL-C. Treatment with the LXR agonist TO901317 produced a marked increase in plasma L (+270%; p<0.001) and Z (+283%; p<0.001). However, liver concentrations of L and Z were marginally or not altered (+33%; p<0.0001, and +29%; ns, respectively) and occurred in the presence of expected hyperlipidemia.Conclusions: Results suggest that HDL remodelling, allowing the formation of pre-β-HDL, and activity of intestinal ABCA1, are functionally important in the uptake of dietary L and Z. Plasma HDL-C levels do not reflect this process, especially following complete CETP inhibition.Commercial Relationships: Eric J. Niesor, F. Hoffmann-La Roche (E); Evelyne Chaput, F. Hoffmann-La Roche (E)

Program Number: 3480 Poster Board Number: D0080Presentation Time: 11:00 AM–12:45 PMThree Characteristics of the Central Macula: Light, AMD, and Macular CarotenoidsRichard A. Bone1, Jorge C. Gibert2, Anirbaan Mukherjee1. 1Physics, Florida International University, Miami, FL; 2Natural Science, Health & Wellness, Miami-Dade College, Miami, FL.Purpose: To determine the cumulative distribution of light on the retina over extended periods under quasi-natural viewing conditions. Our hypothesis was that the light distribution would peak in the center of the macula, thereby offering a potential explanation for the role of light in age-related macular degeneration as well as the role of macular pigment in providing protection.Methods: An eye-tracker was used to obtain a time record of the spatial distribution of light in the subject’s field of view as well as the subject’s gaze position. A corresponding time record of the distribution of light on the retina was calculated. Five informed subjects were employed in feasibility tests and 58 naïve subjects participated in 5 experimental phases: 1) Subjects viewed a gray-scale image, 2) they observed a sequence of photographic images, 3) they viewed a video 4) they worked on a computer, 5) they walked around freely. The informed subjects were instructed to gaze at bright objects in the field of view and then at dark objects. Naïve subjects were allowed to gaze freely for all phases.Results: In Phase 4, light distributions peaked at the fovea for all subjects. Combining the results of the other phases, 74% of subjects showed an overall increase in retinal illuminance at or around the fovea (33% with a sharp increase at the fovea, 41% with a broader distribution) and 26% showed a peak illuminance elsewhere in the retina. Combining all phases, 77% of subjects spent more time viewing brighter objects in the field of view (39% showed a sharp increase in illuminance at the fovea, 38% with a broader distribution around the fovea). Under head-restraint conditions, subjects generally spent more time fixating bright features in the field of view. Under

unrestricted conditions (i.e. subjects walking freely), the distribution of light peaked in the inferior retina.Conclusions: Prior to this study, the distribution of light on the retina was unknown except under unnatural Ganzfeld illumination. Our results partially support our hypothesis that the cumulative light distribution is maximum in the central macula. In particular, using a computer consistently resulted in a peak at the center of the retina. The study has also shown that some individuals spend more time fixating on bright objects in the field of view. In these cases, there may be increased risk of AMD. By screening excessive light, macular pigment may lessen this risk.Commercial Relationships: Richard A. Bone, None; Jorge C. Gibert, None; Anirbaan Mukherjee, NoneSupport: NIH Grant SC3GM083671 and Four Leaf Japan Ltd

Program Number: 3481 Poster Board Number: D0081Presentation Time: 11:00 AM–12:45 PMA Virtual Model for Development of Macular Pigment in the Macaque RetinaJohn T. Landrum1, Vanesa Mendez1, Yisi Cao1, Martha Neuringer2. 1Chemistry and Biochemistry, Florida International University, Miami, FL; 2Division of Neuroscience, ONPRC, Oregon Health & Science University, Portland, OR.Purpose: Creation of a virtual model of macular pigment development from a data set of carotenoid levels in macaque retinas was undertaken to provide insight into the metabolic origin of R,S-zeaxanthin and the time-line of accumulation of the macular carotenoids.Methods: Macaque retinas were collected post-mortem from animals at the Oregon Primate Research Center. Sections were 2, 4, or 8 mm punches centered on the macula. Analysis of retinal sections for carotenoids was performed quantitatively by reversed-phase HPLC using UV-vis detection. Separation of zeaxanthin isomers was carried out using chiral column HPLC.Results: A linear regression fit for each of the macular carotenoids in retinal sections from macaques ranging in age from 133 days gestation to >10 years yields equations for the quantitative levels of carotenoids, lutein, R,R-zeaxanthin, R,S-zeaxanthin, S,S-zeaxanthin as a function of age. All carotenoid levels were shown to increase with increasing age but differential rates of increase were observed which lead to a variation in carotenoid ratios with age.Conclusions: Ratios of carotenoids vary with increasing age in the retina and are well modeled by a linear fit of individual carotenoid levels within retinal sections. Regression data for the levels of R,S-zeaxanthin are consistent with conclusion that this carotenoid is absent from the retina prior to birth and is formed in the retina from lutein. The rate of accumulation R,R-zeaxanthin in the macula is greater than that of the other carotenoids.Commercial Relationships: John T. Landrum, Guardion Health Sicences, LLC. (C); Vanesa Mendez, None; Yisi Cao, None; Martha Neuringer, NoneSupport: Financial support for this project was provided by Wyeth Nutrition and Four Leaf Japan.

Program Number: 3482 Poster Board Number: D0082Presentation Time: 11:00 AM–12:45 PMLong-Term Zeaxanthin Supplementation to the Mouse RetinaBinxing Li, Preejith P. Vachali, Aruna Gorusupudi, Zhengqing Shen, Kelly Nelson, Brian Besch, Paul S. Bernstein. Ophthalmology and Visual Sciences, Univ of UT Sch Med/Moran Eye Ctr, Salt Lake City, UT.Purpose: We have been able to reproducibly deliver the macular pigment (MP) carotenoids lutein and zeaxanthin to the retina of beta-




carotene oxygenase II knockout (BCO2-/-) mice by one month of oral carotenoid supplementation; however, the carotenoid amount in the supplemented mouse retina is still much lower than that in the human macula. In order to to try to deliver more macular pigment to the mouse retina, an 8-month zeaxanthin feeding study was carried out.Methods: 15 three-month-old BCO2 knockout (BCO2-/-) mice were fed with DSM zeaxanthin beadlets chow in this study (2.6 mg zeaxanthin / mouse / day). Retina, RPE, lens, liver and serum of 5 mice were collected at three different time points, 1 month, 4 months and 8 months, respectively. The zeaxanthin levels in the mouse tissues were analyzed by HPLC.Results: Zeaxanthin supplementation caused the retinas and many non-ocular organs of BCO2-/- mice to turn yellow in color, and the retinas of mice fed with zeaxanthin for 8 months showed the most intense yellow coloration. HPLC analysis of the retina showed a linear increase from 1 month to 8 month, reaching a maximum of 0.8 ng of zeaxanthin per pair of retinas, two times higher than the one- month level, and the RPE had an even higher level of 2.8 ng of zeaxanthin per eye. The carotenoid content in serum and liver reached the highest concentration of 768.3 ng/ml and 5496.6 ng/g at 4 month, respectively, and then dropped to 368.2 ng/ml and 4890.0 ng/g at 8 month.Conclusions: Long-term zeaxanthin supplementation for eight months can double the carotenoid content in the retina of the BCO2-/- mouse relative to one month of feeding. Further optimization is still needed to elevate the content of carotenoid in the mouse retina to a level comparable to that of the human macula.Commercial Relationships: Binxing Li, None; Preejith P. Vachali, None; Aruna Gorusupudi, None; Zhengqing Shen, None; Kelly Nelson, None; Brian Besch, None; Paul S. Bernstein, NoneSupport: NIH Grant EY11600; Research to Prevent Blindness

Program Number: 3483 Poster Board Number: D0083Presentation Time: 11:00 AM–12:45 PMResponse of visual function to multiple xanthophyll supplementation in patients with retinal diseases.Roxanne R. Crosby-Nwaobi1, Kanom Bibi1, Tunde Peto1, 2, Philip G. Hykin1, Sobha Sivaprasad1. 1NIHR Clinical Research Facility, Moorfields Eye Hospital, London, United Kingdom; 2Reading Centre, Moorfields Eye Hospital, London, United Kingdom.Purpose: To assess the effect of multiple xanthophyll supplementation on visual function in patients with retinal diseases.Methods: 51 patients with retinal diseases attending Medical Retina clinics at Moorfields Eye Hospital were recruited to have xanthophyll supplementation (once daily for 6 months). Each participant underwent tests of refracted best corrected visual acuity (BCVA; ETDRS letters), Pelli-Robson contrast sensitivity (CS) and functional acuity contrast test (FACT; photopic and mesopic), glare disability (FACT), macular pigment densitometry (MPOD), ocular coherence tomography (OCT), dual wavelength autofluoresence (DWAF), 2 field fundus photographs, vision-related quality of life (NEI-VFQ25), health status (EQ-5D), drug compliance, ophthalmic and physical examination at baseline, month 3 and month 6. Univariate analysis to determine the mean change in BCVA, CS, FACT and MPOD; and correlation of drug compliance, visual function and OCT were conducted using SPSS v20. Significance set at p≤0.01 due to multiple testing.Results: 45 (88.2%) participants completed the study. Median drug compliance was 100% (IQR 6.7) at both 3 and 6 months. BCVA, CS and MPOD were not significantly different between baseline, month 3 and month 6. Mesopic FACT improved for 6.0cpd and 12.0cpd at month 3 (p≤0.01) and for all ranges of mesopic FACT at month 6 (p≤0.001). Photopic FACT demonstrated improvement at month 6

only for 12.0cpd and 18.0cpd (p≤0.006). Mesopic FACT under glare conditions improved for all the spatial frequencies with the exception of 18cpd at month 3 and month 6 (p≤0.009). Only spatial frequency 3.0cpd at month 3 improved in photopic FACT under glare conditions (p=0.007). Photopic FACT at 6.0cpd demonstrated a moderate correlation with total drug compliance (rs= 0.395, p=0.007). At 6 months, mesopic FACT at 1.5cpd and 3.0cpd was moderately correlated to OCT central macular thickness (rs=0.451 and rs=0.469, p=0.001 respectively).Conclusions: Multiple xanthophyll supplementation improved glare sensitivity in patients with retinal diseases. Improvement in mesopic contrast sensitivity was seen for all spatial frequencies at month 6. Supplementation may be recommended in persons suffering glare disability but larger studies are required.Commercial Relationships: Roxanne R. Crosby-Nwaobi, None; Kanom Bibi, None; Tunde Peto, None; Philip G. Hykin, Allergan (C), Allergan (F), Bayer (C), Bayer (F), Norvartis (C), Novartis (F); Sobha Sivaprasad, Allergan (C), Allergan (F), Bayer (C), Bayer (F), Novartis (C), Novartis (F)Support: The Howard FoundationClinical Trial: 2013-005286-39

Program Number: 3484 Poster Board Number: D0084Presentation Time: 11:00 AM–12:45 PMMajor American dietary patterns are related to risk of age-related macular degenerationChung-Jung Chiu1, 2, Min-Lee Chang1, Fang Fang Zhang3, Tricia Li4, Gary Gensler5, Molly Schleicher1, Allen Taylor1, 2. 1USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA; 2Department of Ophthalmology, School of Medicine, Tufts University, Boston, MA; 3Friedman School of Nutrition Science and Policy, Tufts University, Boston, MA; 4Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 5AREDS Coordinating Center, The EMMES Corporation, Rockville, MD.Purpose: We hypothesized that major American dietary patterns and differences in specific nutrient intakes, including vitamins C and E, beta-carotene, zinc, lutein/zeaxanthin, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), within the context of the patterns are associated with age-related macular degeneration (AMD) risk.Methods: 8,103 eyes from 4,088 eligible participants in the baseline Age-Related Eye Disease Study (AREDS) were classified into control (n=2,739), early AMD (n=4,599), and advanced AMD (n=765) according to the AREDS AMD Classification System. Using food consumption data collected by a 90-item food frequency questionnaire, two major dietary patterns, prudent and Western patterns, were identified by factor analysis based on 37 food groups. Applying the generalized estimating equation in a logistic regression, we tested our hypothesis by relating the two patterns and specific nutrients to AMD risk.Results: The “prudent pattern” was characterized by higher intake of vegetables, legumes, fruit, whole grains, and the “Western pattern” was characterized by higher intake of red meat, processed meat, high-fat dairy products, French fries, refined grains. After multivariate adjustment, higher quintiles of the prudent and Western pattern scores were strongly associated with reduced and increased risk of AMD, respectively. For early AMD, the odds ratio (OR) comparing the highest to lowest quintile of the prudent pattern score was ORE5P=0.74 (95% confidence interval (CI): 0.59–0.91; Ptrend=0.01), and the OR comparing the highest to lowest quintile of the Western pattern score was ORE5W=1.56 (95% CI: 1.18–2.06; Ptrend=0.01). For advanced AMD, the ORA5P was 0.38 (95% CI:




0.27–0.54; Ptrend<0.0001), and the ORA5W was 3.70 (95% CI: 2.31–5.92; Ptrend<0.0001). Our data also suggested benefit from higher dietary intakes of vitamin C, lutein/zeaxanthin, and beta-carotene, especially in the context of the Western pattern. In the context of the two major patterns, we did not observe a relationship between intake of vitamin E, zinc, DHA or EPA and AMD risk.Conclusions: This cross-sectional study indicates that diet plays an important role in the development of AMD and that the risk for AMD can be diminished by eating a prudent diet.Commercial Relationships: Chung-Jung Chiu, None; Min-Lee Chang, None; Fang Fang Zhang, None; Tricia Li, None; Gary Gensler, None; Molly Schleicher, None; Allen Taylor, NoneSupport: NIH Grants RO1EY021826, RO1EY013250, and RO1EY021212, and the USDA Grant under agreements 1950-5100-060-01A

Program Number: 3485 Poster Board Number: D0085Presentation Time: 11:00 AM–12:45 PMThe effect of short term nutritional supplementation on macular pigment optical densityAdam McGuinness, Frank Eperjesi, Hannah Bartlett. School of Optometry, Aston University, Birmingham, United Kingdom.Purpose: To investigate the effect of short term (22 days) ocular supplementation on macular pigment optical density (MPOD) of using a nutraceutical containing lutein (L), zeaxanthin (Z), vitamins and minerals (Nutrof® Total).Methods: Ten volunteers aged 50+ (range 51-70, average age 63.2), without diabetes or ocular pathologies that could interfere with measurement of MPOD were recruited from a community optometry practice in the UK. At baseline, the subject’s MPOD was measured, in the right eye where possible, by heterochromatic flicker photometry using the Macular Pigment Screener 9000. Subjects also completed a two day food recall diary. Participants were supplied with Nutrof® Total, a once daily supplement containing; 60mg vitamin C, 10mg vitamin E, 10mg zinc, 500mg copper, 25mg selenium, 330mg fish oil (132mg EPA, 66mg DHA, ≤16.5mg DPA), 10mg lutein, 2mg zeaxanthin and 1mg resveratrol. Participants returned on average 22 days later at which point their MPOD was measured again. This is part of a longer study in which patient numbers will increase to 40 and MPOD measurements and food diaries will be repeated at 3 and 6 months to ascertain longer term effects.Results: Results taken at baseline were compared to those taken on average 22 days later using a paired t test, and found to be non-significant (p=0.393).Conclusions: These preliminary results from an ongoing trial suggest that taking Nutrof® Total for 22 days has no detectable effect upon MPOD. This information is important for optometrists, indicating that short term supplementation is unlikely to be beneficial, those wishing to see a change in MPOD score may need to assess patients after a longer supplementation period.

Sample curve displaying well defined minima and good consistency of results.Commercial Relationships: Adam McGuinness, None; Frank Eperjesi, None; Hannah Bartlett, NoneClinical Trial: ISRCTN79841039

Program Number: 3486 Poster Board Number: D0086Presentation Time: 11:00 AM–12:45 PMOptimization of Retinal Uptake of Lutein and Zeaxanthin in Transgenic MicePreejith P. Vachali, Binxing Li, Aruna Gorusupudi, Zhengqing Shen, Brian Besch, Kelly Nelson, Paul S. Bernstein. Moran Eye Center, University of Utah, Salt Lake City, UT.Purpose: The macular pigment carotenoids, lutein and zeaxanthin, exhibit low systemic and undetectable ocular bioavailability in wild-type (WT) mice. Recent studies from our laboratory suggest that this is due to the active carotenoid cleavage enzymes (BCO1 and BCO2) present in these WT mice. Here we report the reproducible delivery of both lutein and zeaxanthin in BCO double knockout mice.Methods: BCO1-/- / BCO2 -/- double KO mice (BCODKO) and WT mice (8-12 weeks) were used for this study. The mice were put on a vitamin-A deficient chow for 4 weeks, and after that the experimental groups (20-25 each) received zeaxanthin or lutein in DSM ActiLease beadlet chow (1g/kg) for another 4 weeks. The tissues were harvested after 4 weeks of feeding, and the samples were analyzed using HPLC.Results: The zeaxanthin levels in the liver samples of BCODKO and WT were 6584 ± 2179 and 5686 ± 1884 ng/g respectively. The serum zeaxanthin levels for BCODKO and WT were 1332 ± 435 and 925 ± 243 ng/ml respectively. The zeaxanthin levels in BCODKO RPE/choroid and retina were 4.73 ± 0.61 ng/pair and 1.16 ± 0.15 ng/pair respectively. The WT RPE/choroid had 0.94 ± 0.30 ng/pair zeaxanthin, and no zeaxanthin was detected in WT retina. The lutein levels in the liver samples of BCODKO and WT were 7242 ± 1571and 4792 ± 839 ng/g respectively. The serum lutein levels for BCODKO and WT were 1107 ± 295 and 394 ± 107 ng/ml respectively. Lutein level in BCODKO RPE/choroid and retina were 2.97 ± 0.45 ng/pair and 0.75 ± 0.19 ng/pair respectively. In the WT, RPE/choroid had 0.45 ± 0.10 ng/pair lutein and no lutein was detected in WT retina. There were no carotenoids present in the WT lens, whereas BCODKO lens had trace amounts (Lower than Quantitation).Conclusions: In BCODKO mice, we could detect both lutein and zeaxanthin in the retina. The results were statistically significant compared with the wild type mice. Studies in progress using BCO1-/- or BCO2 -/- single knockout mice suggest that the double knockout has a higher relative uptake of carotenoids into the retina. These results emphasize the importance of using transgenic mice lacking carotenoid cleavage enzymes for the successful retinal delivery of macular pigments when studying the ocular effects of lutein and zeaxanthin in mouse models of retinal disease.




Commercial Relationships: Preejith P. Vachali, None; Binxing Li, None; Aruna Gorusupudi, None; Zhengqing Shen, None; Brian Besch, None; Kelly Nelson, None; Paul S. Bernstein, NoneSupport: EY 11600 ; Research to Prevent Blindness

Program Number: 3487 Poster Board Number: D0087Presentation Time: 11:00 AM–12:45 PMZebrafish mutants as models for studying receptors involved in the intestinal uptake of dietary vitamin A for visionGlenn P. Lobo1, Brian D. Perkins1, 2, Joan Heath3, Johannes von Lintig4, Stephanie A. Hagstrom1, 2. 1Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH; 2Ophthalmology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH; 3Chemical Biology, Walter and Eliza Hall Institute, Melbourne, VIC, Australia; 4Pharmacology, Case Western Reserve University, Cleveland, OH.Purpose: To elucidate the biological function of the novel retinol binding receptor, RBPR2, in the intestinal uptake of endogenous preformed vitamin A and whether loss of this receptor impacts embryonic retinoid metabolism for vision and also retinoic acid-dependent developmental processes in the zebrafish.Methods: Whole-mount in-situ hybridization (WISH) staining was performed to evaluate RBPR2 mRNA expression patterns in staged zebrafish embryos. Biochemical and cell culture studies were performed to evaluate the retinol-binding capabilities of this receptor. Localization studies were performed by over-expressing recombinant zebrafish RBPR2 protein in NIH3T3 cultured cells.Results: WISH staining for RBPR2 mRNA showed expression patterns at the 8 somite embryo stage in the yolk syncytium and mesendodermal cells, becoming more restricted to the intestine, liver and pancreas between 4-6 days post fertilization. Biochemical studies using HPLC analysis for retinoids showed that NIH3T3 cells constitutively expressing zebrafish RBPR2 are capable of retinol uptake from its bound form. Finally, localization studies showed a predicted plasma membrane pattern for the overexpressed zebrafish RBPR2 protein in NIH3T3 cells.Conclusions: RBPR2 mRNA expression patterns and biochemical assessment in the zebrafish model suggest a functional role for this receptor in the intestinal uptake of retinol. By studying corresponding human homologous of proteins proposed in retinol uptake using animal models like the zebrafish we hope to obtain a more detailed molecular understanding of the regulatory principles of retinol uptake processes. This would aid in elucidating novel strategies for the prevention and therapy of blindness and retinopathies associated with vitamin A deficiency.Commercial Relationships: Glenn P. Lobo, None; Brian D. Perkins, None; Joan Heath, None; Johannes von Lintig, None; Stephanie A. Hagstrom, None

Program Number: 3488 Poster Board Number: D0088Presentation Time: 11:00 AM–12:45 PMBioavailability of α- and β-Cryptoxanthin in Serum, Liver, and Ocular Tissues of Japanese QuailPaul S. Bernstein1, Binxing Li1, Preejith P. Vachali1, Aruna Gorusupudi1, Fred Khachik2. 1Ophthal and Visual Sciences, University of Utah, Salt Lake City, UT; 2Chemistry and Biochemistry, University of Maryland, College Park, MD.Purpose: To determine the bioavailability of deuterium-labelled (3R)-β-cryptoxanthin-D2 and (3R,6’R)-α-cryptoxanthin-D2 in serum, liver, and retinas of Japanese quail and to investigate whether these carotenoids that are absorbed into ocular tissues of humans, can also be absorbed into eye tissues of a non-primate animal model for metabolic studies.

Methods: The serum, liver, retinas, RPE, and lens from 13 Japanese quail after 4 weeks of daily supplementation with 0.25 mg mixture of synthetic (3R)-β-cryptoxanthin-D2 (79%) and (3R,6’R)-α-cryptoxanthin-D2 (21%) were extracted and analyzed by normal phase and reversed phase HPLC.Results: (3R)-β-cryptoxanthin-D2 and (3R,6’R)-α-cryptoxanthin-D2 were detected in serum, liver, and retinas of the quail but not in RPE and lens. The serum carotenoids were identified as (3R)-β-cryptoxanthin-D2 (20%), (3R,6’R)-α-cryptoxanthin-D2 (14%), lutein (41%), and zeaxanthin (25%). The liver of quails contained nearly equal levels of (3R)-β-cryptoxanthin-D2 and (3R,6’R)-α-cryptoxanthin-D2 while lutein and zeaxanthin were not detected. The composition of carotenoids in the retina was: (3R)-β-cryptoxanthin-D2 (4%), (3R,6’R)-α-cryptoxanthin-D2 (8%), lutein (40%), and zeaxanthin (48%).Conclusions: The distribution of carotenoids in serum of quail suggest a significantly higher ratio of α-cryptoxanthin to β-cryptoxanthin in compare to the ratio of these carotenoids in the supplements provided to these birds. These carotenoids are almost equally distributed in the liver. However, the ratio of α-cryptoxanthin to β-cryptoxanthin in the retina is 2:1, suggesting a preferential absorption and transport of the former into the retina. Surprisingly, these carotenoids were not detected in the RPE. This could be due to the low levels of these carotenoids in this tissue. The levels of lutein and zeaxanthin in the retina of quail were consistent with our earlier analysis of these carotenoids in which we showed a nearly 1:1 ratio for these carotenoids in the retina. The serum of quail showed a higher concentration of lutein relative to zeaxanthin. This study clearly demonstrates that α-cryptoxanthin and β-cryptoxanthin are absorbed into serum, liver, and retina of quail. The fact that the level of β-cryptoxanthin in retina is half of that of α-cryptoxanthin also suggests that there is no in vivo conversion of the latter to the former carotenoid.Commercial Relationships: Paul S. Bernstein, None; Binxing Li, None; Preejith P. Vachali, None; Aruna Gorusupudi, None; Fred Khachik, NoneSupport: NIH Grant EY11600; Research to Prevent Blindness

Program Number: 3489 Poster Board Number: D0089Presentation Time: 11:00 AM–12:45 PMThe relationship between macular pigment and visual function among glaucoma subjects: A baseline evaluation of the Macular Pigment and Glaucoma TrialJames Loughman1, We Fong Siah2, Colm J. O’Brien2. 1Optometry, Dublin Institute of Technology, Dublin, Ireland; 2Ophthalmology Department, Institute of Ophthalmology, Dublin, Ireland.Purpose: Glaucoma patients commonly suffer from disability glare and the cause of this is poorly understood. There is emerging evidence that the macula is affected in early glaucoma. Macular pigment (MP) consisting of lutein (L), zeaxanthin (Z) and meso-zeaxanthin (meso-Z) is highly concentrated at the macula. MP possesses antioxidant properties and has a vital role in visual performance. It has been shown that dietary MP supplementation can improve glare symptoms in healthy individuals or those with age-related macular degeneration. Glaucoma subjects have recently been observed to exhibit significantly lower MPOD compared to age-matched healthy controls. This study comprises an analysis of the baseline visual function data collected as part of the Macular Pigment and Glaucoma Trial (ISRCTN56985060).Methods: All glaucoma participants underwent a detailed eye exam including MPOD (heterochromatic flicker photometry using the Macular Densitometer), contrast and glare sensitivity, photostress recovery time, Humphrey standard 10-2 visual fields and vision-




related quality of life questionnaires with particular emphasis on glaucoma and glare disability (TyPE SPEC and Glaucoma-Activity Limitation, GAL-9).Results: A total of 67 glaucoma subjects were recruited to the trial (38 male, 29 female). The mean age of participants was 64.8 years (range 36-84 years). There was a positive and statistically significant correlation between MPOD and mean deviation (MD) from the visual field analysis (r = 0.29, p = 0.04). Statistically significant relationships were observed between both the TyPE Spec self reported glare symptoms and mean deviation results with measures of glare disability. No statistically significant relationships were observed between MPOD and any other measure of visual function (p > 0.05 for all).Conclusions: Although there was no baseline association between MPOD and parameters of psychophysical function including acuity and contrast, it is interesting to note the significant relationship between MP and MD, a measure of severity of visual field loss. Furthermore, the inter-relationships observed between self reported glare symptoms, MD and glare disability suggest that lower MP levels in the presence of global central visual field loss as a potential factor in the development of glare symptoms among glaucoma subjects.Commercial Relationships: James Loughman, None; We Fong Siah, None; Colm J. O’Brien, NoneSupport: Howard FoundationClinical Trial: ISRCTN56985060

Program Number: 3490 Poster Board Number: D0090Presentation Time: 11:00 AM–12:45 PMThe relationship between macular pigment and glaucoma-related structural parameters: A baseline evaluation of the Macular Pigment and Glaucoma TrialWe Fong Siah1, James Loughman2, Colm J. O’Brien1. 1Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland; 2Optometry, Dublin Institute Technology, Dublin, Ireland.Purpose: To investigate whether there is any relationship between macular pigment optical density (MPOD) and glaucoma-related structural parameters including the ganglion cell complex (GCC), retinal nerve fibre layer (RNFL), foveal full and inner retinal thickness and standard automated Humphrey 10-2 for macular testing. This study comprises an analysis of the baseline structural data collected as part of the Macular Pigment and Glaucoma Trial (ISRCTN56985060).Methods: All glaucoma participants underwent a detailed slit-lamp exam, MPOD measurement (heterochromatic flicker photometry using the Macular Densitometer), and spectral domain optical coherence tomography (sdOCT) imaging of the optic nerve head and macula (SD-OCT, Optovue). Results were analyzed using the SPSS statistics software (version 21).Results: A total of 67 glaucoma subjects were recruited to the trial, of which 38 were male, and 29 female (35 primary open angle glaucoma, 23 low-tension glaucoma, 7 pseudoexfoliation glaucoma, 2 pigment dispersion glaucoma). The mean age of participants was 64.8 years (range 36-84 years). Participants with sub-normal GCC values exhibited statistically significantly lower MPOD (at 0.25 degrees and 1 degree of retinal eccentricity) values compared to those glaucoma subjects demonstrating normal GCC values [0.25 degrees - mean MPOD = 0.20±0.13 (sub-normal GCC group) versus 0.32±0.11 (normal GCC group); p=0.004); 1.0 degrees (0.11±0.06 vs 0.18±.09; p=0.014) respectively. There was no significant relationship observed between MPOD and RNFL. An inverse and statistically significant correlation between MPOD (at 0.25, 0.50 and 1.0 degrees of retinal

eccentricity) and glaucoma duration (r = -0.302 to -0.369; p = 0.032 to 0.039).Conclusions: There is emerging evidence that the macula is affected early in glaucoma. Our previous study has demonstrated that glaucoma patients have reduced MPOD. Here, our baseline results suggest a linear relationship between MPOD and GCC. Furthermore, the association between MPOD and duration of glaucoma is suggestive of a potential loss of MP over the time course of the condition, perhaps as a consequence of longitudinally increased oxidative stress or compromised ocular blood flow levels.Commercial Relationships: We Fong Siah, None; James Loughman, None; Colm J. O’Brien, NoneSupport: The Howard Foundation (UK)Clinical Trial: ISRCTN56985060

Program Number: 3491 Poster Board Number: D0091Presentation Time: 11:00 AM–12:45 PMAssociations between Dietary Intake of Lutein and Diabetic Retinopathy in the Atherosclerosis Risk in Communities (ARIC) StudyMichelle Sahli1, Ronald Klein3, Julie A. Mares3, Kristin J. Meyers3, Heather M. Ochs-Balcom1, William E. Brady2, 1, Barbara E. Klein3, Richard P. Donahue1, Amy E. Millen1. 1Social and Preventive Medicine, State University of New York at Buffalo, Buffalo, NY; 2Department of Biostatistics and Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY; 3Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI.Purpose: Lutein may protect against diabetic retinopathy (DR) due to its ability to absorb oxidizing blue light, its antioxidant properties and its location within the retina. Data from animal models and small studies conducted in humans (N<150) suggest that lutein may be inversely associated with DR. We hypothesized that dietary intake of lutein is inversely associated with DR and examined this association in the biracial ARIC cohort.Methods: We tested our hypothesis by analyzing data collected from 1,430 study participants with diabetes (n=957 White and n=473 Black). We used logistic regression to calculate crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for prevalent DR by quartile (Q) of energy-adjusted lutein intake. DR was assessed using a 45-degree nonmydriatic retinal photograph from one randomly chosen eye taken at visit 3 (1993-95). Dietary lutein intake was estimated using an interviewer administered 66-item food frequency questionnaire at visit 1(1987-89).Results: The median estimated daily lutein intake was 1370 (interquartile range 723 - 2627) mg/1000 kcals and the prevalence of DR was 21%. We found a crude association between lutein and prevalent DR [OR (95% CI) for Q4 (high intake) vs. Q1 (low intake) =2.12 (1.45 - 3.10); p for trend=0.0003]. This association was attenuated after adjustment for race, duration of diabetes, glycated hemoglobin levels, field center and energy intake [1.40 (0.86 - 2.27); p for trend=0.23]. Associations were similar when the analyses were stratified by race but the association was stronger in Blacks [2.29 (0.53 - 9.88); p for trend=0.53]. In analyses limited to people with a short duration of diabetes (<6 years), the association between lutein and DR no longer persisted [0.94 (0.33-2.65); p for trend=0.99] as compared to the association in those with a longer duration of diabetes (≥6 year) [1.54 (0.88-2.70); p for trend=0.24] We found no evidence of multiplicative interaction between lutein and duration of diabetes (p for interaction = 0.70).Conclusions: Contrary to our hypothesis, we found that the odds of higher lutein intake were greater among those with DR than those without DR. However, after adjusting for confounders, intake




of lutein was not associated with DR. This was a secondary data analysis and further investigation of this finding using data collected for this purpose is needed.Commercial Relationships: Michelle Sahli, None; Ronald Klein, None; Julie A. Mares, None; Kristin J. Meyers, None; Heather M. Ochs-Balcom, None; William E. Brady, None; Barbara E. Klein, None; Richard P. Donahue, None; Amy E. Millen, None

Program Number: 3492 Poster Board Number: D0092Presentation Time: 11:00 AM–12:45 PMAssociation of Macular Pigment Optical Density (MPOD) and age in ocular healthy adults of different ethnicities -A preliminary reportPinakin G. Davey1, 2, F. Carusone2, S. Alvarez1, P. Vyas1, Jack Greenan1, T. Thamsopit1, S. Zaczyk1, R. Shah1, C. Lievens2. 1College of Optometry, Western University of Health Sciences, Pomona, CA; 2Southern College of Optometry, Memphis, TN.Purpose: To evaluate the effect of age on the macular pigment as assessed by heterochromatic flicker photometry in a group of ocular healthy individuals of various ethnicities.Methods: The macular pigment study group is created to better understand the role of macular pigment in retinal diseases. This cross-sectional study was designed to understand the level of macular pigment optical density (MPOD) at different decades of life. To further asses if the different ethnic groups had different level of macular pigment when compared to each other. The study was conducted at two sites, Pomona California and Memphis, Tennessee. It is aims to recruit 600 qualified ocular healthy subjects; 150 in each ethnicity of Caucasian, African American, Asian Indian and Hispanic. The study began in March 2013 and anticipates completion around March 2015. This interim report summarizes the findings of MPOD measured on the dominant eye of the qualified 264 subjects of; 96 Caucasian, 78 African American, 22 Asian Indian and 68 Hispanic. The mean age of was 38, 43, 28, 39 years for Caucasian, African American, Asian Indian and Hispanic respectively. The mean age of each Asian Indians was significantly lower than the other groups.Results: The mean MPOD as measured by the heterochromatic flicker photometry was 0.41, 0.39, 0.56 and 0.50 for the groups I, II, III and IV respectively. The mean MPOD was significantly lower in the Caucasian and African American when compared to Asian Indian and Hispanic respectively (One way-ANOVA p<0.0001). There was a trend of lower MPOD with increase in age which did not reach statistical significance overall (r-square 0.005, linear regression p=0.2).Conclusions: The preliminary analysis indicates that the MPOD may be different in different ethnicities but overall MPOD may remain similar with increase in age. The complete report will shed better light on the effect of age on MPOD in various ethnicities.Commercial Relationships: Pinakin G. Davey, None; F. Carusone, None; S. Alvarez, None; P. Vyas, None; Jack Greenan, None; T. Thamsopit, None; S. Zaczyk, None; R. Shah, None; C. Lievens, NoneSupport: Unrestricted grant from Zeavision LLC to Western University of Health Sciences Principal Investigator Dr. Pinakin Davey


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