Xanthophylls are preferentially taken up compared with β-carotene by retinal cells via a SRBI-dependent mechanism*
2008; Elsevier BV; Volume: 49; Issue: 8 Linguagem: Inglês
10.1194/jlr.m700580-jlr200
ISSN1539-7262
AutoresAlexandrine During, Sundari Doraiswamy, Earl H. Harrison,
Tópico(s)Retinal Diseases and Treatments
ResumoThe purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. β-carotene(β-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7–9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than β-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for β-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable β-C 15,15′-oxygenase activity or convert exogenous β-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of β-C and ZEA were significantly decreased (40–60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (∼90%), which resulted in decreased β-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for β-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina. The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. β-carotene(β-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7–9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than β-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for β-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable β-C 15,15′-oxygenase activity or convert exogenous β-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of β-C and ZEA were significantly decreased (40–60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (∼90%), which resulted in decreased β-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for β-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina. Several lines of evidence suggest a protective role of the xanthophylls lutein (LUT) and zeaxanthin (ZEA) against age-related macular degeneration (AMD), the leading cause of blindness among elderly people (1Moeller S.M. Parekh N. Tinker L. Ritenbaugh C. Blodi B. Wallace R.B. Mares J.A. Associations between intermediate age-related macular degeneration and lutein and zeaxanthin in the Carotenoids in Age-Related Eye Disease Study (CAREDS): ancillary study of the Women's Health Initiative.Arch. Ophthalmol. 2006; 124: 1151-1162Crossref PubMed Scopus (213) Google Scholar, 2Snodderly D.M. Evidence for protection against age-related macular degeneration by carotenoids and antioxidant vitamins.Am. J. Clin. Nutr. 1995; 62 (6Suppl.): 1448S-1461SCrossref Scopus (690) Google Scholar, 3Mozaffarieh M. Sacu S. Wedrich A. The role of the carotenoids, lutein and zeaxanthin, in protecting against age-related macular degeneration: a review based on controversial evidence.Nutr. J. 2003; 11: 20-28Crossref Scopus (144) Google Scholar). Carotenoids are not synthesized in the human body and therefore must be obtained from the diet. Of the 50 carotenoids typically consumed in the human diet, only 20 of them are found in human blood and tissues, and β-carotene (β-C), α-carotene, lycopene, β-cryptoxanthin, LUT, and ZEA are the six most predominant (4Parker R.S. Carotenoids in human blood and tissues.J. Nutr. 1989; 119: 101-104Crossref PubMed Scopus (217) Google Scholar). The carotene β-C is the most common carotenoid found in the human diet and body and the most studied carotenoid for its provitamin A activity. LUT and ZEA are widely distributed in plants, mainly in dark green leafy vegetables (e.g., kale, spinach, broccoli, zucchini, green pea) and in yellow to orange fruits and vegetables (e.g., carrot, papaya, squash, peach) (5Goodwin T.W. Britton G. Distribution and analysis of carotenoids.In Plant Pigments, T.W. Goodwin, editor. Academic Press, New York. 1988; : 61-127Google Scholar, 6Holden J.M. Eldridge A.L. Beecher G.R. Buzzard M.I. Bhagwat S. Davis C.S. Douglass L.W. Gebhardt S. Haytowitz D. Schakel S. Carotenoid content of US foods: an update of the database.J. Food Compos. Anal. 1999; 12: 169-196Crossref Scopus (424) Google Scholar). In foods, LUT and ZEA can be recovered in different forms that partly determine their bioavailability (free molecule, bound to proteins, or esterified at one or both hydroxyl groups of the ionone rings) (5Goodwin T.W. Britton G. Distribution and analysis of carotenoids.In Plant Pigments, T.W. Goodwin, editor. Academic Press, New York. 1988; : 61-127Google Scholar). The daily xanthophyll intake varies between 0.5 and 2 mg/day in Western countries (7Curran-Celentano J. Hammond Jr., B.R. Ciulla T.A. Cooper D.A. Pratt L.M. Danis R.B. Relation between dietary intake, serum concentrations, and retinal concentrations of lutein and zeaxanthin in adults in a Midwest population.Am. J. Clin. Nutr. 2001; 74: 796-802Crossref PubMed Scopus (157) Google Scholar, 8Broekmans W.M. Klopping-Ketelaars I.A. Schuurman C.R. Verhagen H. van den Berg, F. J. Kok, and G. van Poppel H. Fruits and vegetables increase plasma carotenoids and vitamins and decrease homocysteine in humans.J. Nutr. 2002; 130: 1578-1583Crossref Scopus (166) Google Scholar). The xanthophylls account for 20–30% of total carotenoids in human plasma, and the ratio of LUT to ZEA is consistently between 4:1 and 5:1 (7Curran-Celentano J. Hammond Jr., B.R. Ciulla T.A. Cooper D.A. Pratt L.M. Danis R.B. Relation between dietary intake, serum concentrations, and retinal concentrations of lutein and zeaxanthin in adults in a Midwest population.Am. J. Clin. Nutr. 2001; 74: 796-802Crossref PubMed Scopus (157) Google Scholar, 9Stacewicz-Sapuntzakis M. Bowen P.E. Mares-Perlman J.A. Serum reference values for lutein and zeaxanthin using a rapid separation technique.Ann. N. Y. Acad. Sci. 1993; 691: 207-209Crossref PubMed Scopus (10) Google Scholar). Interestingly, in the human retina, LUT and ZEA represent ∼80% of the total carotenoid content of the retina, while β-C is found in trace amounts (10Handelman G.J. Snodderly D.M. Adler A.J. Russett M.D. Dratz E.A. Measurement of carotenoids in human and monkey retinas.Methods Enzymol. 1992; 213: 220-230Crossref PubMed Scopus (76) Google Scholar, 11Schmitz H.H. Poor C.L. Gugger E.T. Erdman Jr., J.W. Analysis of carotenoids in human and animal tissues.Methods Enzymol. 1993; 214: 102-116Crossref PubMed Scopus (44) Google Scholar). These xanthophylls are preferentially accumulated in the macula region of the retina to form a yellow spot (also named macula lutea) (12Bone R.A. Landrum J.T. Tarsis S.L. Preliminary identification of the human macular pigment.Vision Res. 1985; 25: 1531-1535Crossref PubMed Scopus (389) Google Scholar, 13Handelman G.J. Dratz E.A. Reay C.C. G. van Kuijk J. Carotenoids in the human macula and whole retina.Invest. Ophthalmol. Vis. Sci. 1988; 29: 850-855PubMed Google Scholar), and they are thus referred to as macular pigment. The highest xanthophyll concentration of the body was reported in the most central part (fovea) of the macula at 1 mM (14Landrum J.T. Bone R.A. Moore L.L. Gomez C.M. Analysis of zeaxanthin distribution within individual human retinas.Methods Enzymol. 1999; 299: 457-467Crossref PubMed Scopus (59) Google Scholar). This foveal xanthophyll concentration is >1,000-fold higher than the typical plasma xanthophyll concentration reported in the literature (between 0.1 and 0.6 μM) (15The Eye Disease Case-Control Study Group. 1992. Risk factors for neovascular age-related macular degeneration. Arch. Ophthalmol. 110: 1701–1708Google Scholar, 16Bone R.A. Landrum J.T. Dixon Z. Chen Y. Llerena C.M. Lutein and zeaxanthin in the eyes, serum and diet of human subjects.Exp. Eye Res. 2000; 71: 239-245Crossref PubMed Scopus (156) Google Scholar, 17Schalch W. Cohn W. Barker F.M. Kopcke W. Mellerio J. Bird A.C. Robson A.G. Fitzke F.F. J. van Kuijk F. Xanthophyll accumulation in the human retina during supplementation with lutein or zeaxanthin—the LUXEA (LUtein Xanthophyll Eye Accumulation) study.Arch. Biochem. Biophys. 2007; 458: 128-135Crossref PubMed Scopus (98) Google Scholar), suggesting a selective uptake of xanthophylls by retinal tissue. The physiological significance of the presence of these xanthophyll pigments in the retina and particularly in the macula remains uncertain. LUT and ZEA are believed to help filter out damaging blue light, to improve visual acuity by attenuating light scattering and chromatic aberrations, or to quench harmful photochemically induced free radicals (18Haegerstrom-Portnoy G Short-wavelength-sensitive-cone sensitivity loss with aging: a protective role for macular pigment?.J. Opt. Soc. Am. A. 1988; 5: 2140-2144Crossref PubMed Scopus (83) Google Scholar, 19Schalch W Carotenoids in the retina—a review of their possible role in preventing or limiting damage caused by light and oxygen.EXS. 1992; 62: 280-298PubMed Google Scholar, 20Sperling H.G. Johnson C. Harwerth R.S. Differential spectral photic damage to primate cones.Vision Res. 1980; 20: 1117-1125Crossref PubMed Scopus (100) Google Scholar, 21Reading V.M. Weale R.A. Macular pigment and chromatic aberration.J. Opt. Soc. Am. 1974; 64: 231-234Crossref PubMed Scopus (128) Google Scholar). A direct neuroprotection of xanthophylls on photoreceptors was also reported recently (22Chucair A.J. Rotstein N.P. SanGiovanni J.P. During A. Chew E.Y. Politi L.E. Lutein and zeaxanthin protect photoreceptors from apoptosis induced by oxidative stress. Relation with docosahexaenoic acid.Invest. Ophthalmol. Vis. Sci. 2007; 48: 5168-5177Crossref PubMed Scopus (146) Google Scholar). These different activities of the xanthophylls may contribute to reducing the risk of AMD. How can LUT and ZEA accumulate preferentially to other carotenoids in the macula? Whenever a tissue exhibits a highly selective uptake for a compound, it is likely that one or more specific binding proteins are involved in the process. For instance, retinoids are highly concentrated in the retina as a result of the combined actions of retinoid binding proteins (23Saari J.C. Biochemistry of visual pigment regeneration.Invest. Ophthalmol. Vis. Sci. 2000; 41: 337-348PubMed Google Scholar) and of a membrane receptor recognizing plasma retinol binding protein (24Kawaguchi R. Yu J. Honda J. Hu J. Whitelegge J. Ping P. Wiita P. Bok D. Sun H. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.Science. 2007; 315: 820-825Crossref PubMed Scopus (591) Google Scholar). For carotenoids, much less is known about carotenoid binding proteins in the mammalian eye. Yemelyanov, Katz, and Bernstein (25Yemelyanov A.Y. Katz N.B. Bernstein P.S. Ligand-binding characterization of xanthophyll carotenoids to solubilized membrane proteins derived from human retina.Exp. Eye Res. 2001; 72: 381-392Crossref PubMed Scopus (92) Google Scholar) reported that LUT and ZEA bind saturably and specifically to solubilized membrane proteins from human retina, and the most highly purified preparations contained two major protein bands at 25 and 55 kDa that consistently coeluted with endogenous LUT and ZEA. Although these data provided the first evidence for the existence of specific xanthophyll binding protein(s) in the vertebrate retina, definitive identification and purification of the protein(s) clearly remain to be performed. Several recent reports have indicated that the intestinal transport of carotenoids is a facilitated process mediated by the scavenger receptor class B type I (SR-BI) in intestinal Caco-2 cells in culture (26Reboul E. Abou L. Mikail C. Ghiringhelli O. Andre 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 (213) Google Scholar, 27During A. Dawson H.D. Harrison E.H. Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with ezetimibe.J. Nutr. 2005; 135: 2305-2312Crossref PubMed Scopus (231) Google Scholar) and in mice (28Van Bennekum A. Werder M. Thuahnai S.T. Han C.H. Duong P. Williams D.L. Wettstein P. Schulthess G. Phillips M.C. Hauser H. Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol.Biochemistry. 2005; 44: 4517-4525Crossref PubMed Scopus (229) Google Scholar). Furthermore, the molecular basis for the blindness of a Drosophila mutant, ninaD, is a defect in the cellular uptake of carotenoids caused by a mutation in the ninaD gene that encodes for a scavenger receptor with a high homology to mammalian scavenger receptors [i.e., SR-BI and cluster determinant 36 (CD36)] (29Kiefer C. Sumser E. Wernet M.F. Von Lintig J. A class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila.Proc. Natl. Acad. Sci. USA. 2002; 99: 10581-10586Crossref PubMed Scopus (205) Google Scholar). This suggests that SR-BI could be involved in carotenoid uptake in vertebrate eyes as well. In the human retina, the retinal pigment epithelium (RPE) has been shown to play critical roles in the physiology of the underlying photoreceptors (i.e., functioning to transport nutrients from the vascular choroid). Therefore, RPE cells could be an important transfer point for LUT and ZEA uptake by the neural retina from the circulating blood. Dunn et al. (30Dunn 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 (1027) Google Scholar) have developed and characterized ARPE-19, a spontaneously arising human RPE cell line with differentiated structural and functional properties similar to those of RPE cells in vivo. In the current study, we demonstrate that xanthophylls are preferentially taken up by ARPE-19 cells and that SR-BI is involved in xanthophyll transport into RPE cells. All-trans β-C (type IV, >95% purity), Tween 40, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). β-C stock solution in hexane contained mainly all-trans β-C (cis isomers < 2%) as determined by HPLC. All-trans-LUT and all-trans-ZEA (>99.9% purity by TLC) were from Indofine Chemical Co., Inc. (Hillsborough, NJ). The rabbit polyclonal antibody against SR-BI (anti-SR-BI) and the mouse monoclonal antibody against human CD36 (anti-CD36) were purchased from Novus Biologicals (Littleton, CO). Nonimmunized rabbit anti-IgG was provided by DakoCytomation Corp. (Carpinteria, CA). ARPE-19 cells (human retinal pigment epithelial cells, passages 18–25) were obtained from the American Type Culture Collection (Rockville, MD). ARPE-19 cells at a seeding density of 1.7 × 106 cells/cm2 were grown in a 1:1 mixture of DMEM and Ham's F12 with 2.5 mM l-glutamine supplemented with 10% FBS and 1.5 g/l sodium bicarbonate (Gibco, Life Technologies, Inc.) and incubated at 37°C in a humidified atmosphere of 5% CO2. The medium was changed every 48 or 72 h. Caco-2 cells (passages 30–40) were obtained from the American Type Culture Collection and grown on Transwells (24 mm diameter, 3 μm pore size; Corning Costar Corp., Cambridge, MA) for 3 weeks in the presence of complete medium [DMEM plus 20% heat-inactivated FBS, 1% nonessential amino acids, and 1% antibiotics (Gibco) as described previously] (31During A. Hussain M.M. Morel D.W. Harrison E.H. Carotenoid uptake and secretion by CaCo-2 cells: beta-carotene isomer selectivity and carotenoid interactions.J. Lipid Res. 2002; 43: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). On the day of the experiment, the complete medium was replenished by the corresponding serum-free medium for either ARPE-19 or Caco-2 cells. β-C, LUT, and ZEA were delivered individually to cells using the "Tween" method (32During A. Albaugh G. Smith J.C. Characterization of β-carotene 15,15′-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2.Biochem. Biophys. Res. Commun. 1998; 249: 467-474Crossref PubMed Scopus (50) Google Scholar) unless specified otherwise. In a sterilized glass tube, required amounts of the carotenoid in hexane (for a final concentration of 1 μmol/l) and of Tween 40 in acetone (to obtain final concentrations of 0.1% and 0.05%, respectively, with Caco-2 and ARPE-19 cells) were introduced, solvents were evaporated, and the dried residue was solubilized in serum-free medium. β-C central cleavage enzyme activity was assayed according to the protocol of During et al. (32During A. Albaugh G. Smith J.C. Characterization of β-carotene 15,15′-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2.Biochem. Biophys. Res. Commun. 1998; 249: 467-474Crossref PubMed Scopus (50) Google Scholar, 33During A. Nagao A. Hoshino C. Terao J. Assay of β-carotene 15,15′-dioxygenase activity by reverse-phase high-pressure liquid chromatography.Anal. Biochem. 1996; 241: 199-205Crossref PubMed Scopus (77) Google Scholar) using an enzyme preparation (supernatant S-9) from 7 week differentiated ARPE-19 cells obtained as previously described (32During A. Albaugh G. Smith J.C. Characterization of β-carotene 15,15′-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2.Biochem. Biophys. Res. Commun. 1998; 249: 467-474Crossref PubMed Scopus (50) Google Scholar). The product of the reaction (retinal) was analyzed by HPLC. The HPLC system was equipped with a 114M pump (Beckman Instruments, Inc.), a UV-970 UV-Vis absorbance variable-wavelength detector (Jasco, Tokyo, Japan), a 250 μl sample loop, and GOLD System for analyses (Beckman). Retinal was eluted on a TSK gel ODS-80Ts C18 reverse-phase column (5 μm particle size, 4.6 × 150 mm) (TosoHaas, Montgomeryville, PA) attached to a precolumn (2 × 20 mm) of Pelliguard LC-18 (Supelco, Inc., Bellefonte, PA) using acetonitrile-water (90:10, v/v) containing 0.1% ammonium acetate as mobile phase (flow rate of 1.0 ml/min). Retinal was monitored at 380 nm, and its retention time was ∼7.5 min. Retinal formed during the enzyme reaction was quantified from its peak area using a standard reference curve obtained using varying amounts of purified retinal. Gene expression of the lipid transporter SR-BI in ARPE-19 cells was blocked using the small interfering RNA (siRNA) or RNA interference (RNAi). Using the BLOCK-iT™ RNAi Designer, a double-stranded Stealth™ RNA (RNAi_1850; sense, 5′-UCA ACA AGC ACU GUU CUG GAA CCU U; antisense, 5′-AAG GUU CCA GAA CAG UGC UUG UUG A) was designed specifically for SR-BI (accession number NM_005505) (Invitrogen Corp., Life Technologies, Carlsbad, CA). A Stealth™ RNAi negative control (Medium GC Duplex; 48% GC; Invitrogen Corp.) was used as a control in RNAi transfection experiments. RNAi transfection into ARPE-19 cells was done using Lipofectamine 2000 (LP2000) (Invitrogen Corp.). Briefly, cells (passages 8–10) were platted on six-well plates (Corning Costar Corp.) at a high density (12.5 × 104 cells/cm2) to reach 40–60% confluence (24 h later) on the day of transfection. In a tube, RNAi-LP2000 complexes were formed by adding 5 μl of LP2000 and 250 pmol of RNAi in Opti-MEM® I Reduced Serum Medium (Gibco) and incubated for 20 min at room temperature. Then, RNAi-LP2000 complexes were added to cells and the cells were placed in a CO2 incubator at 37°C for 72 h. Cells were then incubated with carotenoids at 2 μM for 1 h using the mode of delivery described above. ARPE-19 cells were plated on 25 cm2 flasks at 12.5 × 104 cells/cm2 and then transfected with one of the following treatments: LP2000 alone, RNAi (or Stealth™ RNAi negative control)-LP2000 complexes, or RNAi_1850-LP2000 complexes. At 72 h after transfection, medium was removed, cells were washed three times with a saline solution, and proteins were extracted by using a total protein extraction kit (Chemicon International, Inc.). Protein concentration of samples was then determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories), and 25 μg of proteins was used for Western blot analysis. Western blot analyses were done according to the NuPAGE® technical guide (Invitrogen Corp.). Proteins were separated by SDS-PAGE, under reducing conditions, on a 4–12% NuPAGE® Novex Bis-Tris gel using the NuPAGE MOPS running buffer. After electrophoresis, proteins were transferred onto a 0.45 μm nitrocellulose membrane. Blotted membranes were then incubated with the anti-human IgG primary antibodies against SR-BI at 1:500, and immunodetection was performed using an anti-rabbit IgG secondary antibody according to the WesternBreeze Chromogenic kit (Invitrogen Corp.). Carotenoid extractions from cells and media were performed as described previously (34Barua 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, 35Barua A.B. Kostic D. Olson J.A. New simplified procedures for the extraction and simultaneous high-performance liquid chromatographic analysis of retinol, tocopherols and carotenoids in human serum.J. Chromatogr. 1993; 617: 257-264Crossref PubMed Scopus (106) Google Scholar). Carotenoids were analyzed using a Waters HPLC system equipped with a model 717 Plus autosampler, a model 996 photo diode array detector, and a Millenium32 chromatography manager (Waters T system; Milford, MA). For β-C analyses, a TSK gel ODS 120-A C18 reverse-phase column, 4.6 × 250 mm, 5 μm (TosoHaas), was used with methanol-dichloromethane (84:16, v/v) at 1 ml/min as mobile phase. For xanthophyll analyses, column C30 Type Carotenoid, 4.6 × 250 mm, 3 μm (YMC, Inc., Milford, MA) was used with methanol-methyl-tert-butyl-ether (90:10, v/v) at 0.9 ml/min as mobile phase. Under these conditions, the two standards, LUT and ZEA, are well separated, with retention times of ∼12.5 min and ∼15.5 min, respectively (Fig. 1A). Carotenoids were monitored at 450 nm and quantified from their peak areas using external standard curves established for each carotenoid tested. Average recoveries of carotenoids in cells and media after incubation (16 or 20 h) were as follows: 98% for β-C, 94% for LUT, and 85% for ZEA. Values are means ± SD. Statistical analyses of the results were assessed using Statview, version 5.0 (SAS Institute, Cary, NC). Data were tested for homogeneity of variances by Bartlett's test and then analyzed by one-way ANOVA coupled with the posthoc Fisher's least significant difference test to identify means with significant differences. Relationships between two variables were examined by simple or logarithmic regression analyses. The choice of the regression (simple vs. logarithmic or exponential) was determined by the squared value of the regression coefficient (R2); the regression given the highest R2 value was chosen. P values of <0.05 were considered significant. After incubation with all-trans-LUT, differentiated ARPE-19 cells had a typical xanthophyll profile of 96 ± 3% all-trans-LUT, 4 ± 2% all-trans-ZEA, and 0–2% unidentified peaks, as shown on Fig. 1B, while the 20 h cell culture medium (data not shown) contained 86 ± 5% all-trans-LUT, 3 ± 1% all-trans-ZEA, and 11 ± 5% unidentified peaks (against 96 ± 1% LUT and 4 ± 1% ZEA only in the 0 h medium) (values are from four independent experiments, n = 4). The unidentified peaks present in the medium probably correspond to cis isomers of xanthophylls. Indeed, our HPLC conditions (C30 column) allow us to separate all-trans (all-E) from cis (Z) isomers of xanthophylls, as demonstrated in a previous study using the same HPLC conditions (36Aman 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 (106) Google Scholar). When cells were incubated with all-trans-ZEA, all-trans-ZEA was the main peak detected in cells with all-trans-LUT and another unidentified peak present in trace amounts as shown on Fig. 1C, while the 20 h cell culture medium (data not shown) contained 92 ± 3% all-trans-ZEA and 8 ± 3% all-trans-LUT (against 96 ± 2% ZEA and 3 ± 2% LUT at 0 h incubation) (n = 4). Thus, during incubation at 37°C, xanthophylls can be spontaneously isomerized and that isomerization was greater in cell culture media than in cells. All-trans-LUT is more subject to isomerization than all-trans-ZEA. Note that, under our analytical conditions, we were not able to distinguish between the configurational isomers (3R,3′R)-ZEA and (3R,3′S)-meso-ZEA, which presumably eluted together. To separate these two compounds, the use of a derivatizing method or a chiral HPLC column would be required (37Khachik F. Spangler C.J. Smith Jr., J.C. Canfield L.M. Steck A. Pfander H. Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum.Anal. Chem. 1997; 69: 1873-1881Crossref PubMed Scopus (460) Google Scholar). Previous data have shown that ARPE-19 cells, when cultured under the same conditions as used in the present study, started to develop RPE characteristics at around 5 weeks of a 12 week total culture period (38Janssen J.J. Kuhlmann E.D. H. van Vugt, H. J. Winkens, B. P. Janssen, A. F. Deutman, and C. A. Driessen A Retinoic acid delays transcription of human retinal pigment neuroepithelium marker genes in ARPE-19 cells.Neuroreport. 2000; 11: 1571-1579Crossref PubMed Scopus (20) Google Scholar). Therefore, the purpose of this study was to see whether the stage of differentiation of ARPE-19 cells (from 2 to 10 weeks after confluence) could affect the cellular uptake of carotenoids. When data were expressed per milligram of protein (Fig. 2A), the uptake of the three carotenoids all increased in a similar manner up to 4 weeks of differentiation. After that, we observed an ∼2-fold higher uptake for ZEA from 6 to 9 weeks (P < 0.02) and 1.6-fold higher uptake for LUT at 9 weeks (P < 0.05) compared with that of β-C at comparable stages of differentiation (Fig. 2A). A similar tendency was observed when data were expressed as percentage uptake of the initial dose of carotenoid added to the cell culture medium (Fig. 2B). For instance, at 9 weeks of differentiation, the percentage uptake was 1.6% for β-C, 2.5% for LUT, and 3.2% for ZEA (Fig. 2B). Note that RPE cell uptake of ZEA was higher than that of LUT, in agreement with a previous report (39Lornejad-Schäfer M.R. Lambert C. Breithaupt D.E. Biesalski H.K. Frank J. Solubility, uptake and biocompatibility of lutein and zeaxanthin delivered to cultured human retinal pigment epithelial cells in tween40 micelles.Eur. J. Nutr. 2007; 46: 79-86Crossref PubMed Scopus (19) Google Scholar). In sum, when ARPE-19 cells are well differentiated, the xanthophylls LUT and ZEA are better taken up by cells than the carotene β-C. β-C, LUT, and ZEA were delivered to ARPE-19 cells via either a carotenoid-detergent micellar suspension (Fig. 3A) or a more physiological mode of delivery, the carotenoid-enriched lipoproteins produced by Caco-2 cells (Fig. 3B). We reported previously that, under oleic acid-taurocholate supplementation in the presence of a carotenoid, highly differentiated Caco-2 cells were able to produce and secrete chylomicrons, but no HDL, and 90% of the total amount of the secreted carotenoid was associated with chylomicrons (31During A. Hussain M.M. Morel D.W. Harrison E.H. Carotenoid uptake and secretion by CaCo-2 cells: beta-carotene isomer selectivity and carotenoid interactions.J. Lipid Res. 2002; 43: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Therefore, in the present experiment (Fig. 3B), we conducted a coculture in which 3 week differentiated Caco-2 cells on membrane were placed on the top of 7 week differentiated ARPE-19 cells. At time 0 of the experiment, the carotenoid (10 μM) and oleic acid-taurocholate were added at the apical side of the Caco-2 cells. After 20 h of incubation, carotenoids were analyzed in media and both types of cells. As expected, ∼10% of the initial dose of the carotenoid passed through Caco-2 cells, making a basolateral concentration of ∼1 μM (Fig. 3B), comparable with that used with the Tween 40 experiment (Fig. 3A). Note that in the experiment of Fig. 3B, we assume that, in the basolateral medium, carotenoids were associated with large lipoproteins secreted by Caco-2. Identical uptakes for each of the three carotenoids by the ARPE-19 cells were observed when the carotenoid was delivered in either Tween 40 or lipoproteins (Fig. 3). Both LUT and ZEA exhibited higher uptake rates (∼1.5%) compared with those of β-C (0.3%), confirming that xanthophylls are preferen
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