FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2508864126> ?p ?o ?g. }

• W2508864126 endingPage "1878" @default.
• W2508864126 startingPage "1865" @default.
• W2508864126 abstract "The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin. The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin. Age-related macular degeneration (AMD) is an incurable disease in adults 55 years of age and older and is the leading cause of vision loss in this population (1Zarbin M.A. Rosenfeld P. Pathway-based therapies for age-related macular degeneration: an integrated survey of emerging treatment alternatives.Retina. 2010; 30: 1350-1367Crossref PubMed Scopus (126) Google Scholar). A 2014 meta-analysis predicts that 196 million people will have AMD by 2020 increasing to 288 million by 2040 (2Wong W.L. Su X. Li X. Cheung C.M. Klein R. Cheng C.Y. Wong T.Y. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis.Lancet. 2014; 2: e106-e116PubMed Scopus (2414) Google Scholar). AMD occurs due to deterioration of the macula located in the retina of the eye impacting central vision and the ability to see fine detail. The xanthophylls, lutein and zeaxanthin (Fig. 1A, B), are dietary carotenoids of interest because they accumulate in the retina of the eye and may provide protection from AMD. Common xanthophylls in the human diet include lutein, zeaxanthin, and β-cryptoxanthin (3Kotake-Nara E. Nagao A. Absorption and metabolism of xanthophylls.Mar. Drugs. 2011; 9: 1024-1037Crossref PubMed Scopus (104) Google Scholar) and sources include corn, kale, spinach, eggs, and broccoli (4Reboul E. Borel P. Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes.Prog. Lipid Res. 2011; 50: 388-402Crossref PubMed Scopus (164) Google Scholar). Meso-zeaxanthin (Fig. 1C), a stereoisomer of zeaxanthin, is present in the macula of the eye but is not a common dietary component. Of the 700 carotenoids found in nature, only about 25 are found in the diet and human serum (5Namitha K.K. Negi P. Chemistry and biotechnology of carotenoids.Crit. Rev. Food Sci. Nutr. 2010; 50: 728-760Crossref PubMed Scopus (170) Google Scholar). The top six carotenoids in the human plasma include α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene (6Brevik A. Andersen L.F. Karlsen A. Trygg K.U. Blomhoff R. Drevon C.A. Six carotenoids in plasma used to assess recommended intake of fruits and vegetables in a controlled feeding study.Eur. J. Clin. Nutr. 2004; 58: 1166-1173Crossref PubMed Scopus (66) Google Scholar). Although the concentration of carotenoids in the serum varies widely among individuals, the typical order from highest to lowest is β-carotene > lutein > zeaxanthin (7Chung H.Y. Ferreira A.L. Epstein S. Paiva S.A. Castaneda-Sceppa C. Johnson E.J. Site-specific concentrations of carotenoids in adipose tissue: relations with dietary and serum carotenoid concentrations in healthy adults.Am. J. Clin. Nutr. 2009; 90: 533-539Crossref PubMed Scopus (92) Google Scholar, 8Peng Y.M. Peng Y.S. Lin Y. Moon T. Roe D.J. Ritenbaugh C. Concentrations and plasma-tissue-diet relationships of carotenoids, retinoids, and tocopherols in humans.Nutr. Cancer. 1995; 23: 233-246Crossref PubMed Scopus (131) Google Scholar). Xanthophylls comprise about 20% of the carotenoids in the human plasma with a lutein:zeaxanthin ratio between 2:1 and 4:1 (6Brevik A. Andersen L.F. Karlsen A. Trygg K.U. Blomhoff R. Drevon C.A. Six carotenoids in plasma used to assess recommended intake of fruits and vegetables in a controlled feeding study.Eur. J. Clin. Nutr. 2004; 58: 1166-1173Crossref PubMed Scopus (66) Google Scholar, 9Bone R.A. Landrum J.T. Friedes L.M. Gomez C.M. Kilburn M.D. Menendez E. Vidal I. Wang W. Distribution of lutein and zeaxanthin stereoisomers in the human retina.Exp. Eye Res. 1997; 64: 211-218Crossref PubMed Scopus (342) Google Scholar, 10Thomas J.B. Duewer D.L. Mugenya I.O. Phinney K.W. Sander L.C. Sharpless K.E. Sniegoski L.T. Tai S.S. Welch M.J. Yen J.H. Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum.Anal. Bioanal. Chem. 2012; 402: 749-762Crossref PubMed Scopus (22) Google Scholar). Xanthophylls accumulate in the macula of the retina, imparting the yellow macular pigment color named macula lutea. The functions of xanthophylls in the macula are not fully understood, but they are thought to filter light preventing damage to the macula and might provide protection as antioxidants. Unlike carotenoid concentrations in the blood, lutein and zeaxanthin represent 80% of carotenoids in the retina, while β-carotene is present in only trace amounts (11Schmitz H.H. Poor C.L. Gugger E.T. Erdman J.W. Analysis of carotenoids in human and animal tissues.Methods Enzymol. 1993; 214: 102-116Crossref PubMed Scopus (44) Google Scholar). In the peripheral macula, lutein dominates over zeaxanthin with a ratio of between 2:1 and 3:1 (9Bone R.A. Landrum J.T. Friedes L.M. Gomez C.M. Kilburn M.D. Menendez E. Vidal I. Wang W. Distribution of lutein and zeaxanthin stereoisomers in the human retina.Exp. Eye Res. 1997; 64: 211-218Crossref PubMed Scopus (342) Google Scholar, 12Bone R.A. Landrum J.T. Hime G.W. Cains A. Stereochemistry of the human macular carotenoids.Invest. Ophthalmol. Vis. Sci. 1993; 34: 2033-2040PubMed Google Scholar). Moving closer to the central macula from the peripheral macula, the ratio changes to predominantly zeaxanthin with a 1:2 ratio of lutein to zeaxanthin. About 50% of the zeaxanthin within the central macula is present as a stereo-isomer of zeaxanthin called meso-zeaxanthin. It is unclear what mechanisms are responsible for the selective accumulation of xanthophylls and, particularly, zeaxanthin in the retina. Meso-zeaxanthin is not found in detectable amounts in the blood, liver, or in the typical human diet to account for the concentrations found in the macula (13Khachik F. de Moura F.F. Zhao D.Y. Aebischer C.P. Bernstein P.S. Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models.Invest. Ophthalmol. Vis. Sci. 2002; 43: 3383-3392PubMed Google Scholar). It is hypothesized that lutein is converted to meso-zeaxanthin either enzymatically or induced by light (14Bernstein P.S. Li B. Vachali P.P. Gorusupudi A. Shyam R. Henriksen B.S. Nolan J.M. Lutein, zeaxanthin, and meso-zeaxanthin: the basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease.Prog. Retin. Eye Res. 2016; 50: 34-66Crossref PubMed Scopus (318) Google Scholar, 15Gorusupudi A. Shyam R. Li B. Vachali P. Subhani Y.K. Nelson K. Bernstein P.S. Developmentally regulated production of meso-zeaxanthin in chicken retinal pigment epithelium/choroid and retina.Invest. Ophthalmol. Vis. Sci. 2016; 57: 1853-1861Crossref PubMed Scopus (20) Google Scholar). Thus, primates given a xanthophyll-free diet from birth followed by a lutein supplement showed the presence of meso-zeaxanthin in the retina, while those provided no xanthophylls or with just zeaxanthin alone did not have meso-zeaxanthin in the retina (16Johnson E.J. Neuringer M. Russell R.M. Schalch W. Snodderly D.M. Nutritional manipulation of primate retinas, III: Effects of lutein or zeaxanthin supplementation on adipose tissue and retina of xanthophyll-free monkeys.Invest. Ophthalmol. Vis. Sci. 2005; 46: 692-702Crossref PubMed Scopus (170) Google Scholar). However, no studies have been able to show how this conversion occurs in the macula. Even so, the discovery of meso-zeaxanthin as an additional macular pigment xanthophyll has prompted supplement companies to promote products containing lutein, zeaxanthin, and meso-zeaxanthin for the prevention of AMD. When supplemented, meso-zeaxanthin is found in the human serum (17Thurnham D.I. Trémel A. Howard A.N. A supplementation study in human subjects with a combination of meso-zeaxanthin, (3R,3′R)-zeaxanthin and (3R,3′R,6'R)-lutein.Br. J. Nutr. 2008; 100: 1307-1314Crossref PubMed Scopus (27) Google Scholar), leading researchers to investigate whether its supplementation could increase macular pigment and thus improve the outcome of retinal diseases like AMD. Human studies measuring serum concentration and macular pigment density have shown that taking a supplement predominantly composed of meso-zeaxanthin and small amounts of lutein and zeaxanthin results in the presence of all three xanthophylls in human serum and an increased macular pigment optical density compared with those unsupplemented (18Bone R.A. Landrum J.T. Cao Y. Howard A.N. Alvarez-Calderon F. Macular pigment response to a supplement containing meso-zeaxanthin, lutein and zeaxanthin.Nutr. Metab. (Lond.). 2007; 4: 12Crossref PubMed Scopus (75) Google Scholar). Because meso-zeaxanthin is found in the blood and may increase macular pigment, it is worth investigating the mechanisms of its retinal uptake. The retinal pigment epithelium (RPE) is similar to the blood-brain barrier for the retina in that it serves as a cellular and metabolic interface between the retina and the blood supply from the choroid (19Fliesler S.J. Bretillon L. The ins and outs of cholesterol in the vertebrate retina.J. Lipid Res. 2010; 51: 3399-3413Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). RPE cells display polarity with a basolateral side of tight junctions creating a barrier to the choriocapillaris and an apical side where villi extend and perform phagocytosis on photoreceptors of the retina. This unique position of the RPE allows it to provide nutrients to the photoreceptor cells, while also eliminating waste from the retina. Differentiated ARPE-19 cells are often used as a model for the study of retinal metabolism because they show structural and functional properties similar to the human RPE (20Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.Exp. Eye Res. 1996; 62: 155-169Crossref PubMed Scopus (1052) Google Scholar). ARPE-19 cells express lipoprotein receptors necessary for studying lipoprotein delivery of carotenoids, including scavenger receptor class B1 (SR-B1), SR-B2, LDL receptor (LDLR), lipid transporters cluster determinant 36 (CD-36), and ABCA1 (21Akanuma S. Yamamoto A. Okayasu S. Tachikawa M. Hosoya K. High-density lipoprotein-associated alpha-tocopherol uptake by human retinal pigment epithelial cells (ARPE-19 cells): the irrelevance of scavenger receptor class B, type I.Biol. Pharm. Bull. 2009; 32: 1131-1134Crossref PubMed Scopus (8) Google Scholar, 22Dunn K.C. Marmorstein A.D. Bonilha V.L. Rodriguez-Boulan E. Giordano F. Hjelmeland L.M. Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5.Invest. Ophthalmol. Vis. Sci. 1998; 39: 2744-2749PubMed Google Scholar, 23During A. Doraiswamy S. Harrison E.H. Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism.J. Lipid Res. 2008; 49: 1715-1724Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 24Gordiyenko N. Campos M. Lee J.W. Fariss R.N. Sztein J. Rodriguez I.R. RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.Invest. Ophthalmol. Vis. Sci. 2004; 45: 2822-2829Crossref PubMed Scopus (88) Google Scholar, 25Finnemann S.C. Silverstein R.L. Differential roles of CD36 and alphavbeta5 integrin in photoreceptor phagocytosis by the retinal pigment epithelium.J. Exp. Med. 2001; 194: 1289-1298Crossref PubMed Scopus (111) Google Scholar, 26Tserentsoodol N. Gordiyenko N.V. Pascual I. Lee J.W. Fliesler S.J. Rodriguez I.R. Intraretinal lipid transport is dependent on high density lipoprotein-like particles and class B scavenger receptors.Mol. Vis. 2006; 12: 1319-1333PubMed Google Scholar, 27Tserentsoodol N. Sztein J. Campos M. Gordiyenko N.V. Fariss R.N. Lee J.W. Fliesler S.J. Rodriguez I.R. Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.Mol. Vis. 2006; 12: 1306-1318PubMed Google Scholar). Based on analysis of the expression of lipoprotein transporters and receptors in the retina and two different RPE cell lines, including ARPE-19 cells, it was proposed that circulating LDL and HDL enter the basolateral side of the RPE via the LDLR and SR-B1 (27Tserentsoodol N. Sztein J. Campos M. Gordiyenko N.V. Fariss R.N. Lee J.W. Fliesler S.J. Rodriguez I.R. Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.Mol. Vis. 2006; 12: 1306-1318PubMed Google Scholar). Dietary carotenoids are incorporated into lipoproteins for distribution to various tissues in the human body. Previous work in our laboratory (28Romanchik J.E. Morel D.W. Harrison E.H. Distributions of carotenoids and alpha-tocopherol among lipoproteins do not change when human plasma is incubated in vitro.J. Nutr. 1995; 125: 2610-2617PubMed Google Scholar, 29Romanchik J.E. Harrison E.H. Morel D.W. Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation.J. Nutr. Biochem. 1997; 8: 681-688Crossref Scopus (38) Google Scholar) demonstrated that xanthophylls associated mostly with HDL, while β-cryptoxanthin, lycopene, and β-carotene predominantly associated with LDL in human serum. We also showed that zeaxanthin delivered by detergent micelles to ARPE-19 cells is preferentially taken up via an SR-B1-dependent mechanism compared with β-carotene (23During A. Doraiswamy S. Harrison E.H. Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism.J. Lipid Res. 2008; 49: 1715-1724Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The current work was designed to extend those studies and to investigate in detail the uptake and metabolism of lutein, zeaxanthin, meso-zeaxanthin, and β-carotene delivered in their physiologically relevant transport vehicles. Using differentiated ARPE-19 cells showing structural and functional properties similar to human RPE cells (20Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.Exp. Eye Res. 1996; 62: 155-169Crossref PubMed Scopus (1052) Google Scholar), we used human serum and the lipoproteins, LDL and HDL. We evaluated the kinetics of uptake, the possible interactions of the carotenoids, and the effects of the presence of other lipoproteins and specific inhibition of SR-B1. We demonstrate that xanthophylls, lutein, and zeaxanthin, transported in both HDL and LDL, show quite different uptake kinetics. Next, we show that meso-zeaxanthin is very similar to zeaxanthin in its uptake. Our experimental evidence provides strong support for HDL-dependent selective uptake of zeaxanthin and meso-zeaxanthin via SR-B1 in contrast to an LDL-mediated uptake of lutein and β-carotene. Finally, we found no evidence of xanthophyll conversion to meso-zeaxanthin or lutein conversion to apocarotenoids, providing evidence that RPE cells do not extensively metabolize xanthophylls. All-trans- lutein and all-trans-zeaxanthin [≥98% assay (UV) purity] were purchased from Indofine Chemical Co., Inc. (Hillsborough, NJ). All-trans-β-carotene and solvents used for HPLC were purchased from Sigma-Aldrich (St. Louis, MO). Meso-zeaxanthin was a gift from DSM Nutritional Products (Heerlen, The Netherlands). Recombinant human serum amyloid A (SAA) (human SAA1α except for the presence of an N-terminal methionine and substitution of asparagine for aspartic acid at position 60 and arginine for histidine at position 71) was purchased from PeproTech (Rocky Hill, NJ). Human and bovine serum albumins were purchased from Thermo Scientific. Blocking lipid transport 1 (BLT-1) (≥98% assay HPLC purity) and DMSO were purchased from Sigma-Aldrich. Human retinal pigment epithelial cells, ARPE-19 cells (ATCC®-CRL-2302™), were purchased from the American Type Culture Collection (Rockville, MD). ARPE-19 cells were maintained in Ham's F12 medium:DMEM (1:1) with 10% FBS (Gibco, Life Technologies, Inc.) as monolayers at 37°C with 5% CO2 in T-75 flasks. Cultures of ARPE-19 cells were seeded at 1.5 × 105 cells/cm2 with Ham's F12 medium:DMEM (1:1) with 10% FBS for experiments on 6-well flat bottom plates. Cells were plated and allowed to become confluent after 7 days. The cell medium was changed 2–3 times per week. Cells were used after differentiation for 6–8 weeks. Pilot experiments confirmed that maximal xanthophyll cell uptake occurs at this point of differentiation (results not shown). VLDL, LDL, and HDL were isolated from human serum (type AB, sterile; Valley Biomedical, Winchester, VA) using a method previously developed (30Ford T. Graham J. Rickwood D. Iodixanol: A nonionic iso-osmotic centrifugation medium for the formation of self-generated gradients.Anal. Biochem. 1994; 220: 360-366Crossref PubMed Scopus (124) Google Scholar, 31Graham J.M. Higgins J.A. Gillott T. Taylor T. Wilkinson J. Ford T. Billington D. A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol.Atherosclerosis. 1996; 124: 125-135Abstract Full Text PDF PubMed Scopus (77) Google Scholar). Briefly, chylomicrons were first removed by centrifugation at 100,000 g for 10 min in a Beckman Coulter Optima L-90K ultracentrifuge. Human serum was then mixed with OptiPrep™ (Sigma-Aldrich) (4:1 v/v, 12% iodixanol final concentration) and 3.5 ml was transferred to an OptiSeal™ tube (Beckman Coulter). The remaining tube was filled with PBS (Gibco, Life Technologies). The tube was capped and centrifuged in a Beckman Coulter Optima™ TLX ultracentrifuge at 350,000 g for 2.5 h at 16°C. Lipoprotein fractions were removed by tube puncture using a syringe. The syringe was inserted into the tube just below the lipoprotein band starting with VLDL at the top followed by LDL and then HDL at the bottom. The volumes were recorded and collected into separate vials. Lipoprotein fractions were confirmed using agarose gel electrophoresis and staining with Sudan black. Protein amounts in human serum and lipoproteins were measured using the Modified Lowry Method (Thermo Scientific Pierce Modified Lowry Method kit). Collected lipoproteins were used immediately following isolation. Whole human serum or lipoproteins isolated by centrifugation were enriched with carotenoids using a procedure previously reported (29Romanchik J.E. Harrison E.H. Morel D.W. Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation.J. Nutr. Biochem. 1997; 8: 681-688Crossref Scopus (38) Google Scholar). This method was previously shown to successfully enrich the lipoprotein with the intended carotenoid without influencing lipoprotein integrity or redistributing carotenoids among lipoproteins in whole serum when incubated in vitro (28Romanchik J.E. Morel D.W. Harrison E.H. Distributions of carotenoids and alpha-tocopherol among lipoproteins do not change when human plasma is incubated in vitro.J. Nutr. 1995; 125: 2610-2617PubMed Google Scholar). Carotenoids were added to human serum or lipoproteins dissolved in ethanol (zeaxanthin, meso-zeaxanthin, and lutein) or tetrahydrofuran (β-carotene) (<2% final volume) so that when diluted in serum-free medium they would meet the desired concentrations. The solution was mixed and incubated under nitrogen at 4°C in the dark on a mixer overnight for 24 h. Aliquots of the carotenoid-enriched human serum or lipoproteins were removed for analysis to confirm the initial concentration of carotenoids added to cells prior to experiments. After 6–8 weeks of cell differentiation, cell medium was removed and cells were rinsed three times with PBS. For cell uptake experiments, carotenoid-enriched human serum or isolated lipoproteins were added to serum-free cell medium (10% final volume unless otherwise described) to meet the desired carotenoid concentration and added to cells. For BLT-1 and SAA experiments, HDL protein was first measured using Lowry assay and carotenoids were added to 10 ug HDL protein per milliliter to reach the desired final concentration in serum-free medium. Extraction of cells and medium was performed as previously described with slight modifications (32Barua A.B. Olson J.A. Reversed-phase gradient high-performance liquid chromatographic procedure for simultaneous analysis of very polar to nonpolar retinoids, carotenoids and tocopherols in animal and plant samples.J. Chromatogr. B Biomed. Sci. Appl. 1998; 707: 69-79Crossref PubMed Scopus (84) Google Scholar, 33Barua A.B. Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues.J. Chromatogr. A. 2001; 936: 71-82Crossref PubMed Scopus (37) Google Scholar). For human serum or lipoproteins, 1 vol was treated with 3 vol 2-propanol-dichloromethane (2:1, v/v) and vortexed. All samples were kept on ice under yellow light to prevent oxidation during extraction. The mixture was centrifuged for 3 min at 2,000 rpm. The top layer was removed and dried under nitrogen. The resulting residue was resuspended in 200 ul of mobile phase, filtered through a 0.22 μm pore sized filter and injected on to the HPLC. For carotenoid extraction from cells, cell medium was removed and cells were rinsed with 2 ml of ice-cold PBS followed by the addition of 1 ml of 2-propanol-dichloromethane (2:1) for 30 min. This was performed three times at room temperature. Extracts were then collected, dried under nitrogen, resuspended in mobile phase, filtered through a 0.22 μM pore sized filter, and injected on to the HPLC. Lutein, zeaxanthin, and β-carotene were analyzed using an Agilent Technologies 1200 series diode array and multiple wavelength detector HPLC system (Santa Clara, CA) using a method previously described (30Ford T. Graham J. Rickwood D. Iodixanol: A nonionic iso-osmotic centrifugation medium for the formation of self-generated gradients.Anal. Biochem. 1994; 220: 360-366Crossref PubMed Scopus (124) Google Scholar, 34Aman R. Biehl J. Carle R. Conrad J. Beifuss U. Schieber A. Application of HPLC coupled with DAD, APcI-MS and NMR to the analysis of lutein and zeaxanthin stereoisomers in thermally processed vegetables.Food Chem. 2005; 92: 753-763Crossref Scopus (109) Google Scholar). A column C30 type carotenoid, 4.6 × 250 mm, 3 μm (YMC, Inc., Milford, MA) was used with methanol:methyl-tertiary-butyl-ether (MTBE) (90:10, v/v) at a flow rate of 0.9 ml/min as mobile phase. When only β-carotene was being measured, the same column was used, but with a gradient of 75:25, v/v methanol:MTBE and a flow rate of 1.4 ml/min. Carotenoids were monitored at 450 nm and quantified using external standard curves established for each carotenoid tested. Separation and identification of meso-zeaxanthin from zeaxanthin was analyzed using the same HPLC system described above except using a chiral column (ChiralPak AD, 25 cm length × 4.6 mm ID; Chiral Technologies, Exton, PA) and a method previously described, but with slight modifications (13Khachik F. de Moura F.F. Zhao D.Y. Aebischer C.P. Bernstein P.S. Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models.Invest. Ophthalmol. Vis. Sci. 2002; 43: 3383-3392PubMed Google Scholar). A three-step gradient was used starting with a mobile phase consisting of 94.5% hexanes and 5.5% 2-propanol for 40 min. From 40 to 50 min, 2-propanol was linearly increased to 15% while hexane was reduced to 85 percent. From 50 to 55 min the gradient of hexane and 2-propanol was changed to a 50:50 mixture and maintained for 15 min (70 min into the total run-time). At 70 min, the gradient was re-equilibrated to the initial gradient of 94.5% hexane and 5% 2-propanol from 70 to 80 min. The flow rate during the run was 0.7 ml/min and monitored at 453 nm. BLT-1 is a chemical inhibitor of lipid transport via the SR-B1 pathway (35Nieland T.J. Penman M. Dori L. Krieger M. Kirchhausen T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI.Proc. Natl. Acad. Sci. USA. 2002; 99: 15422-15427Crossref PubMed Scopus (180) Google Scholar). Differentiated ARPE-19 cells were pretreated with equal volumes (0.1% final volume) of either DMSO or BLT-1 dissolved in DMSO (10 μM) for 1 h in serum-free cell medium. This concentration of BLT-1 has been shown to be effective in inhibition of lipid transport from HDL to SR-B1 (36Moussa M. Landrier J.F. Reboul E. Ghiringhelli O. Comér C. Collet X. Fröhlich K. Böhm V. Borel P. Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not Niemann-Pick C1-like 1.J. Nutr. 2008; 138: 1432-1436Crossref PubMed Scopus (110) Google Scholar, 37Reboul E. Abou L. Mikail C. Ghiringhelli O. André M. Portugal H. Jourdheuil-Rahmani D. Amiot M.J. Lairon D. Borel P. Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI).Biochem. J. 2005; 387: 455-461Crossref PubMed Scopus (221) Google Scholar). After removal of cell medium, the cells received the previous treatment in addition to 0.1 μM of zeaxanthin- or lutein-enriched (final concentration when added to cell medium) HDL or LDL (∼10 ug protein per milliliter final volume), respectively, in serum-free cell medium for 3 h at 37°C. After the incubation, zeaxanthin or lutein was extracted from cells using the method previously described. Lipid-free SAA is an inhibitor of SR-B1-dependent binding and selective cholesterol uptake from HDL (38Cai L. de Beer M.C. de Beer F.C. van der Westhuyzen D.R. Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake.J. Biol. Chem. 2005; 280: 2954-2961Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). SAA or BSA (control) was added to differentiated cells at a concentration of 10 ug/ml along with 0.1 μM of zeaxanthin- or lutein-enriched HDL or LDL (∼10 ug/ml), respectively, in serum-free medium for 3 h at 37°C. The amounts of SAA and HDL added in this experiment have previously shown a significant decrease in SR-B1-specific HDL binding. After incubation, zeaxanthin or lutein was extracted from cells as described above. Statistical analyses were conducted using R data analysis software. Values are listed as mean ± SD. Results were analyzed using a mixed-effects ANOVA model. Where appropriate, this was followed by a multiple comparison of means using Tukey contrasts. P < 0.05 was considered significant. After centrifugation of human serum and separation and removal of lipoprotein fractions, the fractions were analyzed on agarose gel with Sudan black staining. Figure 2 shows the presence of only LDL and HDL staining in lanes 1 and 2, respectively, and the presence of all lipoproteins in whole serum in lane 3. After removal of lipoprotein fractions, carotenoids (β-carotene, lutein, and zeaxanthin) were extracted as described in the Materials and Methods and analyzed using HPLC. Each carotenoid was quanti" @default.
• W2508864126 created "2016-09-16" @default.
• W2508864126 creator A5033882770 @default.
• W2508864126 creator A5044010310 @default.
• W2508864126 date "2016-10-01" @default.
• W2508864126 modified "2023-10-18" @default.
• W2508864126 title "Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins" @default.
• W2508864126 cites W1130239587 @default.
• W2508864126 cites W1873700786 @default.
• W2508864126 cites W1907963035 @default.
• W2508864126 cites W1948539164 @default.
• W2508864126 cites W1970186060 @default.
• W2508864126 cites W1971090773 @default.
• W2508864126 cites W1972490310 @default.
• W2508864126 cites W1972969502 @default.
• W2508864126 cites W1974351089 @default.
• W2508864126 cites W1980064899 @default.
• W2508864126 cites W1980700229 @default.
• W2508864126 cites W1985413280 @default.
• W2508864126 cites W1986963574 @default.
• W2508864126 cites W1987154388 @default.
• W2508864126 cites W1989342601 @default.
• W2508864126 cites W1991380467 @default.
• W2508864126 cites W1994454511 @default.
• W2508864126 cites W1996186657 @default.
• W2508864126 cites W1996396197 @default.
• W2508864126 cites W2005044017 @default.
• W2508864126 cites W2007309421 @default.
• W2508864126 cites W2009948638 @default.
• W2508864126 cites W2013348589 @default.
• W2508864126 cites W2018167414 @default.
• W2508864126 cites W2022268067 @default.
• W2508864126 cites W2026636091 @default.
• W2508864126 cites W2028350502 @default.
• W2508864126 cites W2040218897 @default.
• W2508864126 cites W2041812991 @default.
• W2508864126 cites W2047393488 @default.
• W2508864126 cites W2053733211 @default.
• W2508864126 cites W2055541535 @default.
• W2508864126 cites W2058804420 @default.
• W2508864126 cites W2066483255 @default.
• W2508864126 cites W2072253833 @default.
• W2508864126 cites W2072613025 @default.
• W2508864126 cites W2073461406 @default.
• W2508864126 cites W2076490603 @default.
• W2508864126 cites W2081975579 @default.
• W2508864126 cites W2088976751 @default.
• W2508864126 cites W2095403143 @default.
• W2508864126 cites W2108732045 @default.
• W2508864126 cites W2114311313 @default.
• W2508864126 cites W2116977711 @default.
• W2508864126 cites W2123960592 @default.
• W2508864126 cites W2137793347 @default.
• W2508864126 cites W2139019608 @default.
• W2508864126 cites W2140211453 @default.
• W2508864126 cites W2144375280 @default.
• W2508864126 cites W2150473872 @default.
• W2508864126 cites W2151050370 @default.
• W2508864126 cites W2162462372 @default.
• W2508864126 cites W2165699456 @default.
• W2508864126 cites W2167405774 @default.
• W2508864126 cites W2169292626 @default.
• W2508864126 cites W2340339686 @default.
• W2508864126 cites W2345426343 @default.
• W2508864126 cites W2419255724 @default.
• W2508864126 doi "https://doi.org/10.1194/jlr.m070193" @default.
• W2508864126 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5036367" @default.
• W2508864126 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27538825" @default.
• W2508864126 hasPublicationYear "2016" @default.
• W2508864126 type Work @default.
• W2508864126 sameAs 2508864126 @default.
• W2508864126 citedByCount "57" @default.
• W2508864126 countsByYear W25088641262016 @default.
• W2508864126 countsByYear W25088641262017 @default.
• W2508864126 countsByYear W25088641262018 @default.
• W2508864126 countsByYear W25088641262019 @default.
• W2508864126 countsByYear W25088641262020 @default.
• W2508864126 countsByYear W25088641262021 @default.
• W2508864126 countsByYear W25088641262022 @default.
• W2508864126 countsByYear W25088641262023 @default.
• W2508864126 crossrefType "journal-article" @default.
• W2508864126 hasAuthorship W2508864126A5033882770 @default.
• W2508864126 hasAuthorship W2508864126A5044010310 @default.
• W2508864126 hasBestOaLocation W25088641261 @default.
• W2508864126 hasConcept C125575132 @default.
• W2508864126 hasConcept C178790620 @default.
• W2508864126 hasConcept C185592680 @default.
• W2508864126 hasConcept C2778024200 @default.
• W2508864126 hasConcept C2780827179 @default.
• W2508864126 hasConcept C55493867 @default.
• W2508864126 hasConcept C64584667 @default.
• W2508864126 hasConcept C86803240 @default.
• W2508864126 hasConcept C95444343 @default.
• W2508864126 hasConceptScore W2508864126C125575132 @default.
• W2508864126 hasConceptScore W2508864126C178790620 @default.
• W2508864126 hasConceptScore W2508864126C185592680 @default.
• W2508864126 hasConceptScore W2508864126C2778024200 @default.
• W2508864126 hasConceptScore W2508864126C2780827179 @default.

• next