Rubus suavissimus S. Lee Extract Alleviates Oxidative Stress and Inflammation in H2O2-Treated Retinal Pigment Epithelial Cells and in High-Fat Diet-Fed Mouse Retinas.

BACKGROUND: Age-related macular degeneration (AMD) is the most common cause of visual disorders in the aged population and is characterized by the formation of retinal pigment epithelium (RPE) deposits and dysfunction/death of the RPE and photoreceptors. It is supposed that both oxidative stress and inflammation play a critical role in the pathogenesis of AMD. The development of therapeutic strategies against oxidative stress and inflammation in AMD is urgently needed. Rubus suavissimus S. Lee (RS), a medicinal plant growing in the southwest region of China, has been used as an herbal tea and medicine for various diseases. METHODS: In this project, we evaluate the therapeutic potential of RS extract for AMD. We prepared RS extracts from dried leaves, which contained the main functional compounds. RESULTS: RS extract significantly increased cell viability, upregulated the expression of antioxidant genes, lowered the generation of malondialdehyde and reactive oxygen species, and suppressed inflammation in H2O2-treated human RPE cells. In the in vivo study, treatment with RS extract attenuated body weight gain, lowered cholesterol and triglyceride levels in the liver and serum, increased antioxidant capacity, and alleviated inflammation in the retina and RPE/choroid of mice fed a high-fat diet. CONCLUSIONS: Our findings suggest that RS extract offers therapeutic potential for treating AMD patients.

Anti-Apoptotic Effect of Chrysophanol Isolated from Cassia tora Seed Extract on Blue-Light-Induced A2E-Loaded Human Retinal Pigment Epithelial Cells.

The seeds of Cassia tora (C. tora) species mainly contain anthraquinone, anthraquinone glycoside, and naphthalene derivatives. We investigated the anti-apoptotic effects of C. tora seed extract and its isolated compounds on blue-light-induced lipofuscin (A2E)-loaded human retinal pigment epithelial (RPE) cells. For analysis of the C. tora extract, high-performance liquid chromatography method was used. A2E-loaded human retinal pigment epithelial cells and blue light were used to create excessive photo-oxidation to induce cell death. Lactate dehydrogenase (LDH) assay was used to measure cell cytotoxicity, and the mRNA expression of genes involved in apoptosis was examined to evaluate the mechanism of cell death. C. tora extract, n-hexane fraction, and chrysophanol were found to inhibit apoptotic cell death. Additionally, C. tora extract, n-hexane fraction, and chrysophanol reduced the mRNA expression of genes involved in the apoptosis pathway. C. tora and chrysophanol were considered to inhibit apoptosis and oxidative stress response. The major component of C. tora has a protective effect against apoptosis. The ingredients of C. tora can be used as therapeutic substances or to prevent diseases caused by the excessive oxidation of A2E substances in the retina, such as in age-related macular degeneration.

An Evaluation of the Novel Biological Properties of Diterpenes Isolated from Plectranthus ornatus Codd. In Vitro and In Silico.

Plectranthus ornatus Codd, the genus Plectranthus of the Lamiaceae family, has been used as traditional medicine in Africa, India and Australia. Pharmacological studies show the use of this plant to treat digestive problems. In turn, leaves were used for their antibiotic properties in some regions of Brazil to treat skin infections. The present study examines the anti-inflammatory, antioxidant and cytotoxic effects of the halimane and labdane diterpenes (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (HAL) and 1α,6ß-diacetoxy-8α,13R*-epoxy-14-labden-11-one (PLEC) and the forskolin-like 1:1 mixture of 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin (MRC) isolated from P. ornatus on lung (A549) and leukemia (CCRF-CEM) cancer cell lines, and on normal human retinal pigment epithelial (ARPE-19) cell line in vitro. Additionally, molecular docking and computational approaches were used. ADMET properties were analysed through SwissADME and proTox-II-Prediction. The results indicate that all tested compounds significantly reduced the viability of the cancer cells and demonstrated no cytotoxic effects against the non-neoplastic cell line. The apoptosis indicators showed increased ROS levels for both the tested A549 and CCRF-CEM cancer cell lines after treatment. Furthermore, computational studies found HAL to exhibit moderate antioxidant activity. In addition, selected compounds changed mitochondrial membrane potential (MMP), and increased DNA damage and mitochondrial copy number for the CCRF-CEM cancer cell line; they also demonstrated anti-inflammatory effects on the ARPE-19 normal cell line upon lipopolysaccharide (LPS) treatment, which was associated with the modulation of IL-6, IL-8, TNF-α and GM-CSF genes expression. Docking studies gave indication about the lowest binding energy for 1,6-di-O-acetylforskolin docked into IL-6, TNF-α and GM-CSF, and 1,6-di-O-acetyl-9-deoxyforskolin docked into IL-8. The ADMET studies showed drug-likeness properties for the studied compounds. Thus, halimane and labdane diterpenes isolated from P. ornatus appear to offer biological potential; however, further research is necessary to understand their interactions and beneficial properties.

Lycium barbarum L. and Salvia miltiorrhiza Bunge protect retinal pigment epithelial cells through endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum L. and Salvia miltiorrhiza Bunge (Gouqi and Danshen, LS) are traditional herbs for the treatment of retinal degeneration in China. LS have been integrated into pharmacopoeia and health care system of many countries around the world. However, the mechanisms by which LS protect retina are not fully clarified. AIM OF THE STUDY: We aimed at exploration of the effect of LS on retinal pigment epithelium (RPE) cells apoptosis as well as the endoplasmic reticulum (ER) stress mechanisms. MATERIAL AND METHODS: ARPE-19 cells were exposed to tunicamycin to induce ER stress, followed by LS treatment for 24 h. The cell morphology was photographed using the Incucyte S3 instrument, and the potential cytotoxic effect and viability were evaluated by CCK-8 assays. The Annexin V-FITC/PI staining and TUNEL assay were conducted to detect cells apoptotic. Western blot and digital PCR were used to detected related protein and gene expression. RESULTS: The ARPE-19 cells are increased in number and aligned after treating with LS. 1 mg/ml is the LS high dose group dose and treatment with LS increased cell vitality. LS significantly inhibit ARPE-19 cells apoptosis. Moreover, LS were markedly decreased the expression levels of ER stress-related factors in the ARPE-19 cells. CONCLUSIONS: This study reveals that LS relieve ARPE-19 cells apoptosis by inhibiting ER stress, and here we can speculate that LS have a certain protective effect on retina.

Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells.

BACKGROUND: Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics. METHODS: We used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins. RESULTS: We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells. CONCLUSIONS: Our studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.

Serum and Macular Carotenoids in Relation to Retinal Vessel Caliber Fifteen Years Later, in the Second Carotenoids in Age-Related Eye Disease Study.

Purpose: We investigated whether dietary carotenoids lutein and zeaxanthin (L/Z) in the serum and macula were associated with central retinal arteriole and venule calibers in a follow-up ancillary study among older women in the Women's Health Initiative. Methods: Among 390 women who participated in Carotenoids in Age-Related Eye Disease Study 2 (CAREDS2) (2016-2019), we investigated associations between serum L/Z at Women's Health Initiative baseline (1994-1998), and macular pigment optical density (MPOD) at CAREDS baseline (2001-2004), with central retinal vessel caliber in CAREDS2. MPOD was measured using heterochromatic flicker photometry (0.5° from the foveal center) in CAREDS baseline and CAREDS2. Vessel calibers were measured from fundus photographs (CAREDS2). We also explored associations in women with stable MPOD (±0.10 optical density units) over 15 years (n = 106), given the long-term increases in MPOD related to diet patterns and supplement use. Associations were investigated using linear modeling. Results: In the full sample (n = 390), higher serum L/Z (tertile 3 vs. 1) was positively associated with arteriole caliber (mean ± SE, 145.0 ± 1.4 µm vs. 140.8 ± 1.4 µm; P = 0.05) and venule caliber (214.6 ± 2.2 µm vs. 207.5 ± 2.2 µm; P = 0.03). MPOD was also associated with wider vessel calibers (tertile 3 vs. 1), but the trend was only statistically significant for venules (144.4 ± 1.4 µm vs. 141.1 ± 1.4 µm [P = 0.12] and 213.3 ± 2.1 µm vs. 206.0 ± 2.1 µm [P = 0.02], respectively.) Most associations were strengthened in women with stable MPOD over 15 years, including between MPOD and arteriole caliber (149.8 ± 2.6 µm vs.135.8 ± 3.0 µm; P = 0.001). Conclusions: Higher L/Z status in serum and retina was associated with larger central retinal vessel calibers. Prospective studies and clinical trials are needed to elucidate whether L/Z supplementation prevents vision loss through increasing blood flow.

Solanum melongena L. Extract Protects Retinal Pigment Epithelial Cells from Blue Light-Induced Phototoxicity in In Vitro and In Vivo Models.

N-retinylidene-N-retinylethanolamine (A2E) accumulation in the retina is a prominent marker of retinal degenerative diseases. Blue light exposure is considered as an important factor contributing to dry age-related macular degeneration (AMD). Eggplant and its constituents have been shown to confer health benefits, but their therapeutic effects on dry AMD remain incompletely understood. In this study, we showed that an extract of Solanum melongena L. (EPX) protected A2E-laden ARPE-19 cells against blue light-induced cell death via attenuating reactive oxygen species. Transcriptomic analysis demonstrated that blue light modulated the expression of genes associated with stress response, inflammation, and cell death, and EPX suppressed the inflammatory pathway induced by blue light in A2E-laden ARPE-19 cells by inhibiting the nuclear translocation of nuclear factor kappa B and transcription of pro-inflammatory genes (CXCL8 and IL1B). The degradation of intracellular A2E was considered the major mechanism underlying the protective effect of EPX. Moreover, chlorogenic acid isolated from EPX exerted protective effects against blue light-induced cell damage in A2E-laden ARPE-19 cells. In vivo, EPX administration in BALB/c mice reduced the fundus damage and degeneration of the retinal layer in a blue light-induced retinal damage model. Collectively, our findings suggest the potential role of Solanum melongena L. extract for AMD treatment.

Astragalus polysaccharide attenuates metabolic memory-triggered ER stress and apoptosis via regulation of miR-204/SIRT1 axis in retinal pigment epithelial cells.

BACKGROUND: 'Metabolic memory' of early hyperglycaemic environment has been frequently suggested in the progression of diabetic retinopathy (DR). Retinal pigment epithelial (RPE) cells are crucial targets for DR initiation following hyperglycaemia. Astragalus polysaccharides (APS) has been long used as a traditional Chinese medicine in treating diabetes. In the present study, the preventive effects and mechanisms of APS on metabolic memory-induced RPE cell death were investigated. METHODS: The expressions of miR-204 and SIRT1 were determined by reverse transcription quantitative PCR (RT-qPCR). Dual luciferase assay was applied to detect the potential targeting effects of miR-204 on SIRT1. SIRT1, ER stress and apoptosis related proteins were monitored using Western blotting. Apoptosis was assessed by TUNEL assay and Annexin V/PI staining followed by flow cytometry analysis. MiR-204 mimics and shSIRT1 were applied for miR-204 overexpression and SIRT1 knockdown, respectively. RESULTS: High glucose exposure induced metabolic memory, which was accompanied with sustained dysregulation of miR-204/SIRT1 axis, high level of ER stress and activation of apoptotic pathway even after replacement with normal glucose. Pre-treatment with APS concentration-dependently reversed miR-204 expression, leading to disinhibition of SIRT1 and alleviation of ER stress-induced apoptosis indicated by decreased levels of p-PERK, p-IRE-1, cleaved-ATF6, Bax, cleaved caspase-12, -9, -3, and increased levels of Bcl-2 and unleaved PARP. The effects of APS on RPE cells were reversed by either miR-204 overexpression or SIRT1 knockdown. CONCLUSIONS: We concluded that APS inhibited ER stress and subsequent apoptosis via regulating miR-204/SIRT1 axis in metabolic memory model of RPE cells.

Reduced Disc Shedding and Phagocytosis of Photoreceptor Outer Segment Contributes to Kava Kava Extract-induced Retinal Degeneration in F344/N Rats.

There was a significant increase in the incidence of retinal degeneration in F344/N rats chronically exposed to Kava kava extract (KKE) in National Toxicology Program (NTP) bioassay. A retrospective evaluation of these rat retinas indicated a similar spatial and morphological alteration as seen in light-induced retinal degeneration in albino rats. Therefore, it was hypothesized that KKE has a potential to exacerbate the light-induced retinal degeneration. To investigate the early mechanism of retinal degeneration, we conducted a 90-day F344/N rat KKE gavage study at doses of 0 and 1.0 g/kg (dose which induced retinal degeneration in the 2-year NTP rat KKE bioassay). The morphological evaluation indicated reduced number of phagosomes in the retinal pigment epithelium (RPE) of the superior retina. Transcriptomic alterations related to retinal epithelial homeostasis and melatoninergic signaling were observed in microarray analysis. Phagocytosis of photoreceptor outer segment by the underlying RPE is essential to maintain the homeostasis of the photoreceptor layer and is regulated by melatonin signaling. Therefore, reduced photoreceptor outer segment disc shedding and subsequent lower number of phagosomes in the RPE and alterations in the melatonin pathway may have contributed to the increased incidences of retinal degeneration observed in F344/N rats in the 2-year KKE bioassay.

Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease.

The human macula uniquely concentrates three carotenoids: lutein, zeaxanthin, and meso-zeaxanthin. Lutein and zeaxanthin must be obtained from dietary sources such as green leafy vegetables and orange and yellow fruits and vegetables, while meso-zeaxanthin is rarely found in diet and is believed to be formed at the macula by metabolic transformations of ingested carotenoids. Epidemiological studies and large-scale clinical trials such as AREDS2 have brought attention to the potential ocular health and functional benefits of these three xanthophyll carotenoids consumed through the diet or supplements, but the basic science and clinical research underlying recommendations for nutritional interventions against age-related macular degeneration and other eye diseases are underappreciated by clinicians and vision researchers alike. In this review article, we first examine the chemistry, biochemistry, biophysics, and physiology of these yellow pigments that are specifically concentrated in the macula lutea through the means of high-affinity binding proteins and specialized transport and metabolic proteins where they play important roles as short-wavelength (blue) light-absorbers and localized, efficient antioxidants in a region at high risk for light-induced oxidative stress. Next, we turn to clinical evidence supporting functional benefits of these carotenoids in normal eyes and for their potential protective actions against ocular disease from infancy to old age.

Photooxidative damage in retinal pigment epithelial cells via GRP78 and the protective role of grape skin polyphenols.

Blue light induced oxidative damage and ER stress are related to the pathogenesis of age-related macular degeneration (AMD). However, the mechanism of blue light-induced damage remained obscure. The objective of this work is to assess the photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of ER stress associated apoptotic proteins, and investigate the mechanism underlying the protective effects of grape skin extracts. To mimic lipofuscin-mediated photooxidation in vivo, ARPE-19 cells that accumulated A2E, one of lipofuscin fluorophores, were used as a model system to investigate the mechanism of photooxidative damage and the protective effects of grape skin polyphenols. Exposure of A2E containing ARPE-19 cells to blue light resulted in significant apoptosis and increases in levels of GRP78, CHOP, p-JNK, Bax, cleaved caspase-9, and cleaved caspase-3, indicating that photooxidative damage to RPE cells is mediated by the ER-stress-induced intrinsic apoptotic pathway. Cells in which GRP78 had been knocked down with shRNA were more vulnerable to photooxidative damage. Pre-treatment of blue-light-exposed A2E containing ARPE-19 cells, with grape skin extracts, inhibited apoptosis, in a dose dependent manner. Knockdown GRP78 blocked the protective effect of grape skin extracts.

Effects of a lutein supplement on the plasma lutein concentration and macular pigment in patients with central serous chorioretinopathy.

PURPOSE: To investigate the effects of lutein supplementation on plasma lutein concentrations and the macular pigment optical density (MPOD) in central serous chorioretinopathy (CSC). METHODS: In this double-masked placebo-controlled study, 20 patients received lutein 20 mg/d and 19 received placebo. The plasma lutein concentration and MPOD using autofluorescence spectrometry (density unit, DU) were measured at baseline and 1 and 4 months. RESULTS: The mean plasma lutein concentrations and MPOD values in the lutein and control groups, respectively, were 91.5 and 78.2 ng/mL and 0.444 and 0.437 DU at baseline; 204.9 and 79.3 ng/mL and 0.460 and 0.442 DU at 1 month; and 228.0 and 78.4 ng/mL and 0.441 and 0.421 DU at 4 months. The plasma concentration in the lutein group was significantly higher than in controls at 1 and 4 months (P < 0.0001 for both comparisons); however, the MPOD values did not differ significantly between groups at 1 (P = 0.479) or 4 months (P = 0.883). In patients with a plasma lutein concentration below the mean level in 20 age-matched healthy subjects (mean 105.3 ng/mL; n = 13 in lutein group, n = 15 in control group), the control MPOD values significantly (P = 0.0430) decreased at 4 months (mean baseline, 0.437 DU; 4 months, 0.404 DU). The MPOD in the lutein group remained at the baseline level (mean baseline, 0.426 DU; 4 months, 0.438 DU) (P = 0.6542). CONCLUSIONS: The MPOD did not increase in patients with CSC with short-term lutein supplementation; however, among patients with low plasma lutein, supplemental lutein prevented a decline in MPOD that was observed in control subjects (www.umin.ac.jp/ctr number, UMIN000005849).

The effect of age and cataract surgery on macular pigment optic density: a cross-sectional, comparative study.

BACKGROUND: To analyze the effects of age, cataract surgery and postoperative period on macular pigment optical density (MPOD). METHODS: The study included cases referred to Ankara University Department of Ophthalmology, between April and June 2012, who had a transparent natural lens or had undergone cataract surgery at least a year ago with their best corrected visual acuity of ≥ 0.5 based on Snellen chart. Presence of an ocular disease that might affect lens, retina and optic nerve (cataract, macular degeneration, diabetic retinopathy, glaucoma etc.), cataract surgery within the previous year, light-colored iris, smoking and use of micronutrition supplementation were determined as exclusion criteria. After detailed opthalmologic examination of all patients, they were divided into three groups based on their age and their lens status as: group 1, patients < 50 years of age having a clear lens; group 2, patients > 50 years of age having a clear lens; and group 3, patients > 50 years of age who had cataract surgery. Age, gender, and postoperative period of the patients as well as the MPOD values of the eyes measured with heterochromatic flicker photometric (HFP) method (MacularMetricsTM) were included in the analysis. RESULTS: Sixty-eight eyes of 37 cases with a mean age of 53.4 ± 15.3 years were enrolled in the study. Group 1 included 20 eyes of 10 cases (mean age 29.4 ± 9.5); group 2 included 32 eyes of 16 cases (mean age 60.3 ± 6.8); and group 3 included 16 eyes of 11 cases (mean age 65.2 ± 9.7). The mean macular pigment optical density value of all cases was 0.511 ± 0.192 log unit, while the mean MPOD values of groups 1, 2 and 3 were 0.570 ± 170, 0.528 ± 203 and 0.400 ± 180 log units, respectively. The mean MPOD values of the patients with clear lens aged < 50 and aged > 50 years did not reveal a statisticially significant difference (p = 1). However, the mean MPOD value of the cataract surgery group (group 3) was found to be statistically significantly lower than the group 1 and group 2 (p = 0.022, p = 0.039, respectively). The correlations between MPOD values and postoperative periods of the patients in group 3 showed that a decrease in MPOD values in parallel with duration of the postoperative period and this negative correlation was found to be statistically significant (r: -0.66, p = 0.005). CONCLUSION: Our study has demonstrated that a significant correlation does not exist between age of the patients and MPOD values. MPOD values were lower than age-matched patients who had undergone cataract surgery and finally an inverse correlation existed between duration of the postoperative period after cataract surgery and MPOD values.

Candidate gene study of macular response to supplemental lutein and zeaxanthin.

Supplementation with carotenoids is proposed to protect against age-related macular degeneration. There is, however, considerable variability in retinal macular pigment response, which may be due to underlying genetic variation. The purpose of this study was to determine whether genetic factors, which have been previously associated with cross-sectional macular pigment levels in the retina or serum lutein, also influence response to supplementation. To this end we conducted an association study in 310 subjects from the TwinsUK cohort between variants in 8 candidate genes and serum lutein and retinal macular pigment optical density (MPOD) levels before and after supplementation. Four variants were associated with MPOD response to supplementation (p < 0.05): rs11057841 (SCARB1), rs4926339 (RPE65), rs1929841 (ABCA1) and rs174534 (FADS1). We also confirmed previous associations between rs6564851 near BMCO1 (p < 0.001) and rs11057841 within SCARB1 (p = 0.01) and baseline measures of serum lutein; while the latter was also associated with MPOD response, none of the BMCO1 variants were. Finally, there was evidence for association between variants near RPE65 and ELOVL2 and changes in lutein concentration after supplementation. This study is the first to show association between genetic variants and response to carotenoids supplementation. Our findings suggest an important link between MP response and the biological processes of carotenoids transport and fatty acid metabolism.

Long term effects of lutein, zeaxanthin and omega-3-LCPUFAs supplementation on optical density of macular pigment in AMD patients: the LUTEGA study.

BACKGROUND: The primary objective of LUTEGA is to determine the long-term effect of a supplementation with fixed combination of lutein, zeaxanthin, omega-3-longchain-polyunsaturated-fatty-acids (O-3-LCPUFAs) and antioxidants on macular pigment optical density (MPOD) in patients with non-exudative age-related macular degeneration (AMD). METHODS: The LUTEGA study is a double-blind, placebo-controlled clinical trial. 172 patients with non-exudative AMD were enrolled and randomized to three treatment arms. Supplementation included either once (dosage D1) or twice daily (dosage D2) of 10 mg L / 1 mg Z/ O-3-LCPUFAs (thereof 100 mg DHA, 30 mg EPA)/ antioxidants, or placebo (P). After best-corrected visual acuity (BCVA) test, blood sample was collected and MPOD was measured using the 1-wavelength-reflection method and recording reflection images at 480 nm (modified Visucam(NM/FA), Carl Zeiss Meditec, Germany). During 1 year of intervention, AMD patients were followed up after 1, 3, 6 and 12 months. 145 AMD patients (D1 = 50, D2 = 55, P = 40) completed the study. RESULTS: After 12 months of intervention, the MPOD parameters (volume, area, maxOD, meanOD) increased significantly in treatment arms D1 and D2 (p < 0.001). Volume of MPOD showed the highest within-group difference and increased significantly in D1 and D2, and decreased significantly in P (p = 0.041). Between-group comparison of absolute changes of all MPOD parameters were significantly different between D1 and P as well as D2 and P with p < 0.001 at end point (t = 12). BCVA, measured in log MAR, improved in D1 and in D2 (p < 0.001). After 12 months of intervention, the mean improvement in BCVA was significant in D2 (p = 0.006) and D1 (p = 0.038) compared to P. CONCLUSIONS: The supplementation of L, Z, O-3-LCPUFAs and antioxidants resulted in considerable increase in MPOD. There was no difference in accumulation of MPOD between both dosages. Thus, we believe that the used supplementation with L and Z seems to reach a saturation level in retinal cell structure. Additionally, the constant supplementation of L, Z, O-3-LCPUFAs and antioxidants in AMD patients seems to be useful, because MPOD reduces without supplementation. We conclude that the supplementation caused an increase of MPOD, which results in an improvement and stabilization in BCVA in AMD patients. Thus, a protective effect on the macula in AMD patients is assumed.

Macular xanthophylls and ω-3 long-chain polyunsaturated fatty acids in age-related macular degeneration: a randomized trial.

IMPORTANCE: It has been shown that the functionality of the macula lutea depends on the nutritional uptake of lutein and zeaxanthin and that it is inversely associated with the risk of age-related macular degeneration (AMD). Additionally, ω-3 long-chain polyunsaturated fatty acids (LC-PUFAs) may also be protective. OBJECTIVE: To investigate the effect of a 12-month intervention with macular xanthophylls and ω-3 LC-PUFAs on xanthophylls and fatty acids in plasma, antioxidant capacity, and optical density of the macular pigment of patients with nonexudative AMD. DESIGN: The LUTEGA study was a randomized, double-blind, placebo-controlled, parallel clinical trial that was conducted for 12 months. SETTING: University Eye Hospital and Institute of Nutrition, Friedrich Schiller University Jena, Germany. PARTICIPANTS: A total of 172 individuals with nonexudative AMD. INTERVENTION: Individuals were enrolled and randomly divided as follows: placebo group, group 1 (a capsule containing 10 mg of lutein, 1 mg of zeaxanthin, 100 mg of docosahexaenoic acid, and 30 mg of eicosapentaenoic acid administered each day), and group 2 (same substances but twice the dose used in group 1). One hundred forty-five participants completed the study successfully. MAIN OUTCOME MEASURES: Plasma xanthophyll concentrations and fatty acid profiles, optical density of the macular pigment, and antioxidant capacity in plasma (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid [Trolox] equivalent antioxidant capacity and photochemiluminescence). RESULTS: The concentrations of the administered carotenoids in plasma as well as the optical density of the macular pigment increased significantly in the groups randomized to receive supplementary macular xanthophylls and ω-3 LC-PUFAs after 1 month of intervention and remained at this level through the end of the study. Use of the double dose resulted in a beneficial alteration of the fatty acid profile in the plasma of patients with AMD in comparison with the dose in group 1. The lipophilic antioxidant capacity in plasma was significantly elevated with the intervention. CONCLUSIONS AND RELEVANCE: A supplement containing a fixed combination of lutein, zeaxanthin, and ω-3 LC-PUFAs during 12 months significantly improved plasma antioxidant capacity, circulating macular xanthophyll levels, and the optical density of the macular pigment. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00763659.

Effects of formulation on the bioavailability of lutein and zeaxanthin: a randomized, double-blind, cross-over, comparative, single-dose study in healthy subjects.

PURPOSE: Lutein and zeaxanthin are macular pigments with a protective function in the retina. These xanthophylls must be obtained from the diet or added to foods or supplements via easy-to-use, stable formulations. The technique employed to produce these formulations may affect the bioavailability of the xanthophylls. METHODS: Forty-eight healthy volunteers were randomized into this double-blind, cross-over study investigating the plasma kinetics of lutein provided as two different beadlet formulations. Subjects (n = 48) received a single dose of 20 mg of lutein as either a starch-matrix ("SMB", FloraGLO® Lutein 5 %) or as a cross-linked alginate-matrix beadlet ("AMB", Lyc-O-Lutein 20 %) formulation. Plasma concentrations of lutein and zeaxanthin were measured at 0, 1, 3, 6, 9, 12, 14, 24, 26, 28, 32, 36, 48, 72, 168, and 672 h. RESULTS: The mean plasma AUC(0-72h), AUC(0-672h), and C(max) for total lutein and zeaxanthin and their all-E-isomers were significantly increased (p < 0.001) from pre-dose concentrations in response to SMB and AMB. There was no difference in lutein T max between the two test articles. However, by 14 h post-dose, total plasma lutein increased by 7 % with AMB and by 126 % with SMB. Total lutein AUC(0-72h) and AUC(0-672h) were 1.8-fold and 1.3-fold higher, respectively, for SMB compared to AMB. Both formulations were well tolerated by subjects in this study. CONCLUSION: These findings confirm that the bioavailability of lutein and zeaxanthin critically depends on the formulation used and document a superiority of the starch-based over the alginate-based product in this study.

Comparison of macular pigment in patients with age-related macular degeneration and healthy control subjects - a study using spectral fundus reflectance.

PURPOSE: Previous studies have reported an age-dependent decline of macular pigment optical density (MPOD) as well as a relative lack of MPOD in age-related macular degeneration (AMD). Results are, however, strongly dependent on the technique used. In this study, we investigated the age dependence of MPOD using spectral fundus reflectance. In addition, we hypothesized that patients with AMD have a reduced MPOD as compared to healthy controls. METHODS: A total of 85 healthy subjects and 96 patients with AMD were included in this study. The healthy control subjects showed a wide range of ages (mean, 51.6 years; range, 21-79years). Patients with AMD were significantly older (mean, 71.2 years; range, 50-89 years). Spectral fundus reflectance of the fovea was measured in a 2.3° detection field with a custom built fundus reflectometer. Calculation of MPOD was based on a previously published fundus reflectance model. RESULTS: Patients with AMD showed a reduced MPOD (0.35 ± 0.12) as compared to the healthy control group (0.39 ± 0.12, p = 0.013 between groups). No age dependence of MPOD (r = -0.14, p = 0.19) was found in the healthy control group. In the AMD group, however, MPOD declined with age (r = -0.24, p = 0.019). CONCLUSIONS: This study indicates that MPOD is reduced in patients with AMD. In addition, the data of this study indicate that MPOD is age dependent in AMD patients, but not in healthy controls. Taken together with data indicating that lutein supplementation increases MPOD, this provides a rationale for supplementation of the macular pigments in patients with AMD, although long-term clinical outcome data are lacking.

Effect of lutein and zeaxanthin on macular pigment and visual function in patients with early age-related macular degeneration.

PURPOSE: To determine whether supplementation with lutein and zeaxanthin improves macular pigment and visual function in patients with early age-related macular degeneration (AMD). DESIGN: Randomized, double-masked, placebo-controlled trial. PARTICIPANTS: Participants with probable AMD who were 50 to 79 years of age were screened for study eligibility from the local communities. One hundred eight subjects with early AMD were recruited. INTERVENTION: Early AMD patients were assigned randomly to receive 10 mg/day lutein (n = 27), 20 mg/day lutein (n = 27), 10 mg/day lutein plus 10 mg/day zeaxanthin (n = 27); or placebo (n = 27) for 48 weeks. Macular pigment optical density (MPOD) and visual function variables were assessed at baseline, 24 weeks, and 48 weeks. MAIN OUTCOME MEASURES: The primary outcome was MPOD. Secondary outcomes were visual function variables including best-corrected visual acuity (BCVA), contrast sensitivity (CS), photorecovery time, and Amsler grid testing results. RESULTS: Macular pigment optical density increased significantly by a mean ± standard error of 0.076 ± 0.022 density unit in the 20-mg lutein group and 0.058 ± 0.027 density unit in the lutein and zeaxanthin group during 48 weeks. There was a significant dose-response effect for lutein supplementation, and the changes in MPOD from baseline to 48 weeks were correlated negatively with baseline MPOD in all active treatment groups (r = -0.56; P<0.001). At 48 weeks, a trend toward improvement was seen in BCVA, and there was a significant between-group difference in CS at 3 and 6 cycles/degree between the 20-mg lutein group and the placebo group. The increase in MPOD related positively to the reduction in the logarithm of the minimum angle of resolution BCVA (r = -0.31; P<0.01) and the increases in CS at 4 spatial frequencies (r ranging from 0.26 to 0.38; all P<0.05). CONCLUSIONS: Among patients with early AMD, supplementation with lutein and zeaxanthin improved macular pigment, which played a causative role in boosting visual function and might prevent the progression of AMD. Future studies are required to evaluate the effect of these carotenoids on the incidence of late AMD.