Retinal bioavailability and functional effects of a synthetic very-long-chain polyunsaturated fatty acid in mice.

Rare, nondietary very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are uniquely found in the retina and a few other vertebrate tissues. These special fatty acids play a clinically significant role in retinal degeneration and development, but their physiological and interventional research has been hampered because pure VLC-PUFAs are scarce. We hypothesize that if Stargardt-3 or age-related macular degeneration patients were to consume an adequate amount of VLC-PUFAs that could be directly used in the retina, it may be possible to bypass the steps of lipid elongation mediated by the retina's ELOVL4 enzyme and to delay or prevent degeneration. We report the synthesis of a VLC-PUFA (32:6 n-3) in sufficient quantity to study its bioavailability and functional benefits in the mouse retina. We acutely and chronically gavage fed wild-type mice and Elovl4 rod-cone conditional knockout mice this synthetic VLC-PUFA to understand its bioavailability and its role in visual function. VLC-PUFA-fed wild-type and Elovl4 conditional knockout mice show a significant increase in retinal VLC-PUFA levels in comparison to controls. The VLC-PUFA-fed mice also had improvement in the animals' visual acuity and electroretinography measurements. Further studies with synthetic VLC-PUFAs will continue to expand our understanding of the physiological roles of these unique retinal lipids, particularly with respect to their potential utility for the treatment and prevention of retinal degenerative diseases.

Systemic Effects of Prenatal Carotenoid Supplementation in the Mother and her Child: The Lutein and Zeaxanthin in Pregnancy (L-ZIP) Randomized Trial -Report Number 1.

BACKGROUND: Adding carotenoids, particularly lutein (L) and zeaxanthin (Z), to prenatal micronutrient formulations has been promoted to enhance infant visual and neural development and to maintain maternal health. Although these claims are biologically plausible, they are not yet supported by a compelling prospective trial. OBJECTIVE: We investigated the effect of prenatal carotenoid supplementation on biomarkers of maternal and infant systemic carotenoid status. METHODS: We randomly assigned 47 first trimester pregnant subjects by 1:1 allocation to receive standard-of-care prenatal vitamins plus a 10 mg L and 2 mg Z softgel (the Carotenoid group) or standard-of-care prenatal vitamins with a placebo softgel (the Control group) for 6-8 mo. Maternal carotenoid concentrations in the serum and skin at the end of each trimester and postpartum were measured with HPLC and resonance Raman spectroscopy, respectively. Infants' systemic carotenoid status was assessed using similar techniques but optimized for infants. Repeated measures and paired t-tests were determined, and a P value < 0.05 was considered statistically significant. RESULTS: After supplementation, there was a statistically significant increase in maternal serum L + Z concentrations, serum total carotenoid concentrations, and skin carotenoid status (P < 0.001 for all) in the Carotenoid group relative to the Control group at all study time points. Similarly, infants whose mothers were in the Carotenoid group had a significant 5-fold increase in cord blood L + Z concentrations, over a 3-fold increase in cord blood total carotenoids, and a 38% increase in skin carotenoids compared with the Control group (P < 0.0001 for all). In addition, there was a strong positive, statistically significant correlation between postpartum maternal and infant systemic carotenoid status (P < 0.0001). CONCLUSION: Prenatal carotenoid supplementation significantly increased maternal and infant systemic (skin and serum) carotenoid status, which may benefit pregnant women and their infants' health. This trial was registered at clinicaltrials.gov as NCT03750968.

Mechanism for the selective uptake of macular carotenoids mediated by the HDL cholesterol receptor SR-BI.

The macular carotenoids lutein and zeaxanthin are taken up from the bloodstream into the human retina through a selective process, for which the HDL cholesterol receptor scavenger receptor BI (SR-BI) in the cells of retinal pigment epithelium (RPE) is thought to be a key mediator. However, the mechanism of SR-BI-mediated selective uptake of macular carotenoids is still not fully understood. Here, we investigate possible mechanisms using biological assays and cultured HEK293 cells, a cell line without endogenous SR-BI expression. Binding affinities between SR-BI and various carotenoids were measured by surface plasmon resonance (SPR) spectroscopy, which shows that SR-BI cannot bind lutein or zeaxanthin specifically. Overexpression of SR-BI in HEK293 cells results in more lutein and zeaxanthin taken up than ß-carotene, and this effect can be eliminated by an SR-BI mutant (C384Y) whose cholesterol uptake tunnel is blocked. Next, we determined the effects of HDL and hepatic lipase (LIPC), SR-BI's partners in HDL cholesterol transport, on SR-BI-mediated carotenoid uptake. HDL addition dramatically reduced lutein, zeaxanthin, and ß-carotene in HEK293 cells expressing SR-BI, but the cellular lutein and zeaxanthin are higher than ß-carotene. LIPC addition increases the uptake of all three carotenoids in HDL-treated cells, and promotes the transport of lutein and zeaxanthin better than ß-carotene. Our results suggest that SR-BI and its HDL cholesterol partner HDL and LIPC may be involved in the selective uptake of macular carotenoids.

Macular Pigment Carotenoids and Bisretinoid A2E.

Lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) are the three macular pigments (MP) carotenoids that uniquely accumulate in the macula lutea region of the human retina. L and Z are obtained by humans through dietary intake. The third MP, MZ, is rarely present in diet, and its abundance in the human fovea is due to the metabolic conversion of dietary L by the retinal pigment epithelium's RPE65 enzyme. The major functions of MP in ocular health are to filter high-intensity, phototoxic blue light and to act as effective antioxidants for scavenging free radicals. The pyridinium bisretinoid, N-retinylidene-N-retinylethanolamine (A2E), contributes to drusen formation in dry age-related macular degeneration (AMD) and to the autofluorescent flecks in autosomal recessive Stargardt disease (STGD1). Retinal carotenoids attenuate A2E formation and can directly and indirectly alleviate A2E-mediated oxidative damage. In this chapter, we review these more recently recognized interconnections between MP carotenoids and A2E bisretinoids.

HDL is the primary transporter for carotenoids from liver to retinal pigment epithelium in transgenic ApoA-I-/-/Bco2-/- mice.

Supplementation with antioxidant carotenoids is a therapeutic strategy to protect against age-related macular degeneration (AMD); however, the transport mechanism of carotenoids from the liver to the retina is still not fully understood. Here, we investigate if HDL serves as the primary transporter for the macular carotenoids. ApoA-I, the key apolipoprotein of HDL, was genetically deleted from BCO2 knockout (Bco2-/-) mice, a macular pigment mouse model capable of accumulating carotenoids in the retina. We then conducted a feeding experiment with a mixed carotenoid chow (lutein:zeaxanthin:ß-carotene = 1:1:1) for one month. HPLC data demonstrated that the total carotenoids were increased in the livers but decreased in the serum, retinal pigment epithelium (RPE)/choroids, and retinas of ApoA-I-/-/Bco2-/- mice compared to Bco2-/- mice. In detail, ApoA-I deficiency caused a significant increase of ß-carotene but not lutein and zeaxanthin in the liver, decreased all three carotenoids in the serum, blocked the majority of zeaxanthin and ß-carotene transport to the RPE/choroid, and dramatically reduced ß-carotene and zeaxanthin but not lutein in the retina. Furthermore, surface plasmon resonance spectroscopy (SPR) data showed that the binding affinity between ApoA-I and ß-carotene â‰« zeaxanthin > lutein. Our results show that carotenoids are transported from the liver to the eye mainly by HDL, and ApoA-I may be involved in the selective delivery of macular carotenoids to the RPE.

The emerging roles of the macular pigment carotenoids throughout the lifespan and in prenatal supplementation.

Since the publication of the Age-Related Eye Disease Study 2 (AREDS2) in 2013, the macular pigment carotenoids lutein (L) and zeaxanthin (Z) have become well known to both the eye care community and the public. It is a fascinating aspect of evolution that primates have repurposed photoprotective pigments and binding proteins from plants and insects to protect and enhance visual acuity. Moreover, utilization of these plant-derived nutrients has been widely embraced for preventing vision loss from age-related macular degeneration. More recently, there has been growing awareness that these nutrients can also play a role in improving visual performance in adults. On the other hand, the potential benefits of L and Z supplementation at very young ages have been underappreciated. In this review, we examine the biochemical mechanisms and supportive data for L and Z supplementation throughout the lifespan, with particular emphasis on prenatal supplementation. We propose that prenatal nutritional recommendations may aim at improving maternal and infant carotenoid status. Prenatal supplementation with L and Z might enhance infant visual development and performance and may even prevent retinopathy of prematurity, possibilities that should be examined in future clinical studies.

Lutein and zeaxanthin reduce A2E and iso-A2E levels and improve visual performance in Abca4-/-/Bco2-/- double knockout mice.

Accumulation of bisretinoids such as A2E and its isomer iso-A2E is thought to mediate blue light-induced oxidative damage associated with age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1). We hypothesize that increasing dietary intake of the macular carotenoids lutein and zeaxanthin in individuals at risk of AMD and STGD1 can inhibit the formation of bisretinoids A2E and iso-A2E, which can potentially ameliorate macular degenerative diseases. To study the beneficial effect of macular carotenoids in a retinal degenerative diseases model, we used ATP-binding cassette, sub-family A member 4 (Abca4-/-)/ß,ß-carotene-9',10'-oxygenase 2 (Bco2-/-) double knockout (KO) mice that accumulate elevated levels of A2E and iso-A2E in the retinal pigment epithelium (RPE) and macular carotenoids in the retina. Abca4-/-/Bco2-/- and Abca4-/- mice were fed a lutein-supplemented chow, zeaxanthin-supplemented chow or placebo chow (~2.6 mg of carotenoid/mouse/day) for three months. Visual function and electroretinography (ERG) were measured after one month and three months of carotenoid supplementation. The lutein and zeaxanthin supplemented Abca4-/-/Bco2-/- mice had significantly lower levels of RPE/choroid A2E and iso-A2E compared to control mice fed with placebo chow and improved visual performance. Carotenoid supplementation in Abca4-/- mice minimally raised retinal carotenoid levels and did not show much difference in bisretinoid levels or visual function compared to the control diet group. There was a statistically significant inverse correlation between carotenoid levels in the retina and A2E and iso-A2E levels in the RPE/choroid. Supplementation with retinal carotenoids, especially zeaxanthin, effectively inhibits bisretinoid formation in a mouse model of STGD1 genetically enhanced to accumulate carotenoids in the retina. These results provide further impetus to pursue oral carotenoids as therapeutic interventions for STGD1 and AMD.

Lutein Encapsulated in PLGA-Phospholipid Nano-Carrier Effectively Mitigates Cytokines by Inhibiting Tumor Necrosis Factor TNF-α and Nuclear Factor NF-κB in Mice Retina.

Lutein, a photo- and thermo-labile macular pigment, prevents the retina from suffering ocular inflammation with its antioxidant and anti-inflammatory activity. However, its biological activity is poor due to poor solubility and bioavailability. Therefore, we developed a PLGA NCs (+PL), (poly (lactic-co-glycolic acid) nanocarrier with phospholipid) to improve the biological availability and bioefficacy of lutein in the retina of lipopolysaccharide (LPS)-induced lutein-devoid (LD) mice. The effect of lutein-loaded NCs with/without PL was studied in comparison with micellar lutein. The induction of inflammation by LPS significantly increased the production of nitrites in the LPS-induced group, revealing higher levels of nitric oxide (NO) in the serum (760%) and retina (891%) compared to the control group. Malondialdehyde (MDA) levels in the serum (93%) and retina (205%) of the LPS-induced group were higher compared to the control group. LPS induction resulted in increased protein carbonyls in the serum (481%) and retina (487%) of the LPS group compared to the control group. Further, to conclude, lutein-PLGA NCs (+PL) effectively down-regulated inflammatory complications in the retina.

Prenatal Carotenoid Supplementation With Lutein or Zeaxanthin Ameliorates Oxygen-Induced Retinopathy (OIR) in Bco2-/- Macular Pigment Mice.

Purpose: Premature infants at risk of retinopathy of prematurity (ROP) miss placental transfer of the carotenoids lutein (L) and zeaxanthin (Z) during the third trimester. We previously demonstrated that prenatal L and Z supplementation raised carotenoid levels in infants at birth in the Lutein and Zeaxanthin in Pregnancy (L-ZIP) study (NCT03750968). Based on their antioxidant effects and bioavailability, we hypothesized that prenatal maternal supplementation with macular carotenoids would reduce the risk of ROP. To test this hypothesis, we utilized "macular pigment mice" genetically engineered to take up L and Z into the retina in a model of oxygen-induced retinopathy (OIR). Methods: Pregnant Bco2-/- mice were divided into nine experimental subgroups based on the type of supplementation (L, Z, or placebo) and on the maternal supplementation start date corresponding to the three trimesters of human fetal development (E0, E11, and P1). Pups and nursing mothers were exposed to 75% O2 for 5 days (P7-P12) and returned to room air for 5 days (P12-P17). Pups were killed at P12 and P17, and their retinas were analyzed for vaso-obliteration and intravitreal neovascularization. Results: Pups of pregnant mice supplemented with L or Z had significant reductions in areas of vaso-obliteration and intravitreal neovascularization compared to placebo. Prenatal carotenoid supplementation starting at E0 or E11 was significantly more protective against OIR than postnatal supplementation starting at P1. Conclusions: Prenatal supplementation with L and Z was beneficial in a mouse OIR model. We recommend testing prenatal L and Z supplementation in future human clinical trials to prevent ROP.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Extraction, detection, and imaging of the macular carotenoids.

The term "macular carotenoids" refers to the lutein, zeaxanthin, and meso-zeaxanthin that are highly concentrated at the center of the human retina. Intraretinal levels of these carotenoids are inversely associated with the risk of age-related macular degeneration (AMD), and oral supplementation with these carotenoids can significantly reduce AMD risk. To make macular carotenoid analysis more accessible, we systematically review the current methods for extraction, detection, and imaging of macular carotenoids in both basic and clinical research. We first introduce carotenoid extraction methods from the retina, retinal pigment epithelium (RPE)/choroid, serum, and liver of the human and animal models, such as mice and Japanese quails, as well as from algae, bacteria, and chicken egg yolks and cultured cells. We then review macular carotenoid detection by spectroscopy and HPLC, while particularly introducing carotenoid separation via cyano columns, chiral columns, and C30 columns. In the end, we summarize the common methods used to image carotenoids in living human eyes: resonance Raman spectroscopy, autofluorescence attenuation spectroscopy, and reflection spectroscopy, and we then review the utility of confocal resonance Raman microscopy to image the macular carotenoids in tissue sections of human and mouse retinas.

The macular carotenoids: A biochemical overview.

Among the more than 750 carotenoids identified in nature, only lutein, zeaxanthin, meso-zeaxanthin, and their oxidative metabolites are selectively accumulated in the macula lutea region of the human retina. These retinal carotenoids are collectively referred to as the macular pigment (MP) and are obtained only through dietary sources such as green leafy vegetables and yellow and orange fruits and vegetables. Lutein- and zeaxanthin-specific binding proteins (StARD3 and GSTP1, respectively) mediate the highly selective uptake of MP into the retina. Meso-zeaxanthin is rarely present in the diet, and its unique presence in the human eye results from metabolic conversion from dietary lutein by the RPE65 enzyme. The MP carotenoids filter high-intensity, short-wavelength visible light and are powerful antioxidants in a region vulnerable to light-induced oxidative stress. This review focuses on MP chemistry, absorption, metabolism, transport, and distribution with special emphasis on animal models used for MP study. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

What do we know about the macular pigment in AMD: the past, the present, and the future.

Carotenoids are lipophilic isoprenoid pigments with a common C40H56 core chemical structure that are naturally synthesized by many plants, algae, bacteria, and fungi. Humans and animals cannot synthesize carotenoids de novo and must obtain them solely through dietary sources. Among the more than 750 carotenoids in nature, only lutein, zeaxanthin, meso-zeaxanthin, and their oxidative metabolites selectively accumulate in the foveal region of the retina where they are collectively referred to as the macular pigment (MP) of the macula lutea. MP serves an ocular protective role through its ability to filter phototoxic blue light radiation and also via its antioxidant activity. These properties have led to the hypothesis that carotenoids may protect against the development of age-related macular degeneration (AMD), the most common cause of blindness in the aged population >60 years old. Epidemiological studies have supported this by showing that patients with lower concentrations of serum carotenoids and macular pigment optical density (MPOD) measurements are at a higher risk of developing AMD. Conversely, nutritional supplementation and diets rich in lutein and zeaxanthin readily impact MP concentrations and reduce the risk of progression to advanced AMD, and the AREDS2 supplement formulation containing 10 mg of lutein and 2 mg of zeaxanthin is the standard-of-care recommendation for individuals at risk for visual loss from advanced AMD. This article reviews the rich history of research on the MP dating back to the 1700s and outlines their potential for further therapeutic improvements for AMD in the future.

Chitosan-glycolipid nanogels loaded with anti-obese marine carotenoid fucoxanthin: Acute and sub-acute toxicity evaluation in rodent model.

Fucoxanthin (FUCO) is a light- and heat-sensitive marine xanthophyll carotenoid, present in brown algae that render physiological properties as anti-oxidants. In this study, nanoencapsulation is an approach adopted to improve bioavailability of FUCO by using ionic-gelation method with polymeric chitosan (CS) dispersed in glycolipid (GL), as a carrier. Further, the aim was to investigate adverse effect of acute and sub-acute toxicity of chitosan nanogels (CS-NGs) loaded with FUCO+GL in rats. In the acute toxicity study, FUCO was fed to rats at doses of 0.1, 1, 10 and 100 mg/kg body weight (BW). In the sub-acute toxicity study, FUCO was fed at doses of 1 and 10 mg/kg BW for 28 days. In both the studies, no mortality and abnormalities in gross morphology were observed. Acute toxicity study revealed that the LD50 of FUCO in CS-NGs is higher than 100 mg/kg BW. No postprandial plasma levels of FUCO were detected. However, fucoxanthinol (FUOH), a hydrolytic metabolite of FUCO was detected in a dose dependent manner (P < 0.01). Compared to the control group(s), no dose-related toxic effects of CS-NGs with FUCO + GL were found in haematological, histopathological, plasma biochemical indices, etc. The no-observed-adverse-effect level (NOAEL) for CS-NGs with FUCO + GL in rats was 10 mg/kg/day. To conclude, no apparent adverse effect of CS-NGs with FUCO + GL demonstrating CS could be a promising polymer matrix for safe delivery of FUCO. This is the first study to demonstrate the safety assessment of CS-NGs with FUCO + GL.

Biodegradable Poly (Lactic-co-Glycolic Acid)-Polyethylene Glycol Nanocapsules: An Efficient Carrier for Improved Solubility, Bioavailability, and Anticancer Property of Lutein.

Lutein bioavailability is limited because of its poor aqueous solubility. In this study, lutein-poly (lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) nanocapsules were prepared to improve the solubility, bioavailability, and anticancer property of lutein. The scanning electron microscopy and dynamic light scattering examination revealed that the nanocapsules are smooth and spherical with size ranging from 80 to 500 nm (mean = 200 nm). In vitro lutein release profile from nanocapsules showed controlled sustainable release (66%) up to 72 h. Aqueous solubility of lutein nanocapsules was much higher by 735-fold than the lutein. Fourier transform infrared spectroscopy analyses showed no chemical interaction among PLGA, PEG, and lutein, indicating possible weak intermolecular forces like hydrogen bonds. X-ray diffraction revealed lutein is distributed in a disordered amorphous state in nanocapsules. Postprandial plasma kinetics (area under the curve) of an oral dose of lutein from nanocapsules was higher by 5.4-fold compared with that of micellar lutein (control). The antiproliferative effect of lutein from nanocapsules (IC50 value, 10.9 µM) was higher (43.6%) than the lutein (IC50 value, 25 µM). Results suggest that PLGA-PEG nanocapsule is an efficient carrier for enhancing hydrophilicity, bioavailability, and anticancer property of lipophilic molecules such as lutein.

Promising interaction between nanoencapsulated lutein with low molecular weight chitosan: characterization and bioavailability of lutein in vitro and in vivo.

This study aims to develop water-soluble low molecular weight chitosan (LMWC) nanoencapsules with lutein to improve its bioavailability. Lutein-LMWC nanoencapsules were prepared, characterized and bioavailability was studied in vitro and in vivo with lutein in mixed micelles (control). The particle size ranged between 80-600 nm, which was confirmed by Atomic Force Microscope. The interaction between LMWC and lutein in nanocencapsules by (1)H and (13)C NMR showed the essentiality of water molecules to hold the lutein between LMWC chains of nanoparticle with a reversible weak bond. Bioavailability of lutein (200 µM) in vitro showed that lutein-LMWC nanoencapsules was significantly higher (27.7%) than control. Postprandial lutein level in the plasma (54.5%), liver (53.9%) and eyes (62.8%) of mice fed on nanoencapsulated lutein were higher than the control. LMWC may serve as novel carrier for enhancing the lutein bioavailability and can be suggested as the better dietary compound in food and pharmaceutical applications.