Lipids DOI 10.1007/s11745-015-4033-9
ORIGINAL ARTICLE
Glyceroglycolipids Affect Uptake of Carotenoids Solubilized in Mixed Micelles by Human Intestinal Caco‑2 Cells Eiichi Kotake‑Nara1 · Lina Yonekura1 · Akihiko Nagao1
Received: 22 October 2014 / Accepted: 1 May 2015 © AOCS 2015
Abstract We previously reported that phospholipids markedly affected the uptake of carotenoids solubilized in mixed micelles by human intestinal Caco-2 cells. In the present study, we found that two classes of dietary glyceroglycolipids and the corresponding lysoglyceroglycolipids affected uptake of β-carotene and lutein by differentiated Caco-2 cells. The levels of carotenoid uptake from micelles containing digalactosyldiacylglycerol or sulfoquinovosyldiacylglycerol were significantly lower than that from control micelles. On the other hand, the uptakes from micelles containing digalactosylmonoacylglycerol or sulfoquinovosylmonoacylglycerol were significantly higher than that from control micelles. In dispersed cells and Caco-2 cells with poor cell-to-cell adhesion, however, the levels of uptake from micelles containing these lyso-lipids were much lower than that from control micelles. The uptake levels from control micelles were markedly decreased depending on the development of cell-to-cell/cell–matrix adhesion in Caco-2 cells, but the uptake levels from the micelles containing these lyso-lipids were not substantially changed, suggesting that the intercellular barrier formed by cell-to-cell/cell–matrix adhesion inhibited the uptake from control micelles, but not from the lyso-lipid-containing micelles. The lyso-lipids appeared to enhance carotenoid uptake by decreasing the intercellular barrier integrity. The results showed that some types of glyceroglycolipids have the potential to modify the intestinal uptake of carotenoids.
* Eiichi Kotake‑Nara [email protected] 1
National Food Research Institute, National Agriculture and Food Research Organization, 2‑1‑12 Kannondai, Tsukuba, Ibaraki 305‑8642, Japan
Keywords Glyceroglycolipid · Lysoglyceroglycolipid · Carotenoid · β-Carotene · Lutein · Mixed micelles · Caco-2 · Lyso-lipid · Intercellular barrier integrity · Intestinal uptake Abbreviations CF 5(6)-carboxyfluorescein DGDG Digalactosyldiacylglycerol DGMG Digalactosylmonoacylglycerol DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide FBS Fetal bovine serum FWHM Full width at half-maximum GL Diacyl glyceroglycolipid(s) lysoGL Lysoglyceroglycolipid(s) lysoPtdCho Lysophosphatidylcholine MGDG Monogalactosyldiacylglycerol MGMG Monogalactosylmonoacylglycerol PBS Phosphate-buffered saline PtdCho Diacyl phosphatidylcholine SQDG Sulfoquinovosyldiacylglycerol SQMG Sulfoquinovosylmonoacylglycerol
Introduction Fruits and vegetables are major dietary sources of carotenoids, which have been reported to show anti-oxidant activities [1, 2] and other characteristic biological activities including anti-allergic [3, 4], anti-cancer [5], anti-inflammatory [6, 7], and anti-obesity [8, 9] effects. Thus, the activities of dietary carotenoids have attracted a great deal of attention in terms of human health. However, the bioavailability of carotenoids is known to be lower than that of the other lipophilic nutrients, such
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as triacylglycerol and α-tocopherol [10–13]. Carotenoids are absorbed after solubilization in the intestinal tract via several steps. First, carotenoids are released from the food matrix of, for example, fruits and vegetables, and dissolved into dietary lipids. The carotenoids are then dispersed in the digestive tract as the lipids are emulsified with bile [14]. As the digestion of the emulsified lipids progresses with pancreatic lipase, carotenoids are solubilized in mixed micelles consisting of bile salts, cholesterol, fatty acids, monoacylglycerols, and lysophospholipids. Although the mixed micellar carotenoids are accessible to uptake by epithelial cells in the intestine, it is thought that only a portion of them are actually taken up by the cells. Their intestinal uptake, as well as their solubilization into the micelles, is thus a critical factor in carotenoid bioavailability. We found previously that mixed micelle components such as phospholipids, fatty acids, and monoacylglycerol Fig. 1 Typical chemical structures of glyceroglycolipids, phosphatidylcholine, and their lyso-lipids. a PtdCho, b lysoPtdCho, c MGDG, d MGMG, e DGDG, f DGMG, g SQDG, h SQMG. R1OC and R2OC in these structures are fatty acid moieties
a
markedly affected the uptake of carotenoids by human intestinal Caco-2 cells in different ways [15–17]. In particular, phosphatidylcholine (PtdCho) suppressed the uptake, while lysophosphatidylcholine (lysoPtdCho) significantly enhanced it. Differences in the polar head group in phospholipids also affected the uptake [18]. These observations suggest that amphipathic lipids greatly influence the uptake of carotenoids solubilized in mixed micelles by intestinal cells. Diacyl glyceroglycolipids (GL), which are ingested together with carotenoids from green leafy vegetables, are also amphipathic lipids with a sugar moiety as a polar head group. We have hypothesized that GL can affect the cellular uptake of carotenoids solubilized in mixed micelles. Hence, in the present study, we determined the effects of three major dietary GL and the corresponding lysoglyceroglycolipids (lysoGL) (Fig. 1) on the uptake of β-carotene and lutein by Caco-2 cells.
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Materials and Methods Materials β-Carotene (type II), 1-oleoyl-rac-glycerol (monoolein), 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocholine (lysoPtdCho), lipase (from Rhizopus arrhizus), and 5(6)-carboxyfluorescein (CF) were purchased from SigmaAldrich (St. Louis, MO). Lutein was purchased from Extrasynthese (Genay Cedex, France). β-Carotene and lutein were purified by alumina column chromatography (neutral, grade III; MP Biomedicals, Solon, OH). The purified carotenoids were stored at −80 °C until use. 1-Palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (PtdCho) was purchased from Avanti Polar Lipids (Alabaster, AL). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo). Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS). GL, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), were purchased from Larodan Fine Chemicals (Malmö, Sweden). Ethyl β-apo-8′-carotenoate was purchased from Fluka Biochemica (Buchs, Switzerland). The other chemicals and solvents were of reagent grade. Preparation of LysoGL LysoGL, monogalactosylmonoacylglycerol (MGMG), digalac tosylmonoacylglycerol (DGMG), and sulfoquinovosylmonoacylglycerol (SQMG) were prepared from the corresponding GL using a previously described procedure with modifications [19]. In brief, 25 mg of GL was hydrolyzed by Rhizopus arrhizus lipase (105 units) in Tris–HCl buffer (25 mM, pH 7.7, 5 mL) containing 2.4 % Triton X-100 at 38 °C for 30 min. The enzymatic reaction was quenched with 6 N HCl (0.15 mL). After 5 mL of methanol was added, lysoGL were extracted twice with 10 mL of chloroform. The extracts were combined and then dried in a centrifugal evaporator. The residue was dissolved in 250 μL of dichloromethane/methanol (2:1, v/v) and subjected to purification by thin-layer chromatography on silica gel 60 with chloroform/methanol (3:1, v/v) as the developing solvent. The rate of flow (Rf) values were 0.53 for MGMG, 0.17 for DGMG, and 0.10 for SQMG. Fatty Acid Analysis of GL and LysoGL The acyl groups of GL and lysoGL (100–200 μg) were converted to methyl ester together with 100 nmol heneicosanoic acid (21:0) as an internal standard by an HCl/methanol method, and then the methyl ester was subjected to gas chromatographic analysis as previously described [20]. Based on the fatty acid compositions, we calculated the
average molecular weight of the GL and lysoGL (761 for MGDG, 931 for DGDG, 885 for SQDG, 487 for MGMG, 672 for DGMG, and 630 for SQMG), and determined the molar amounts. Cell Culture and Differentiation Caco-2 cells derived from human colon carcinoma were purchased from the American Type Culture Collection (Rockville, MD). The cells were routinely cultured in a medium consisting of DMEM, 10 % heat-inactivated FBS, 4 mM l-glutamine, 40 units/mL of penicillin, 40 μg/mL of streptomycin, and 0.1 mM nonessential amino acids at 37 °C in a humidified atmosphere of 5 % CO2 in air. The cells were grown by passaging 2 ×/week. To obtain fully differentiated Caco-2 cell monolayers, the cells at passages of 33–60 were seeded into 24-well plates at a density of 5 × 104 cells per well containing 0.5 mL of the culture medium, and the medium was replaced with fresh medium 2 ×/week for 20–22 days. The differentiated cells were used in carotenoid uptake experiments, unless otherwise stated. Preparation of Mixed Micelles Containing Carotenoids Mixed micelles were prepared as described previously [18]. All of the mixed micelle components and carotenoids (β-carotene or lutein) were dissolved in an appropriate solvent and transferred to glass test tubes. The solvent was then evaporated under a stream of argon and the solution was further dried in a centrifugal evaporator. The dry matter was dispersed into FBS-free DMEM, and the resulting clear solution was passed through a 0.2-μm filter to remove any carotenoids that were not solubilized into the micelles. The mixed micelles consisted of taurocholate (2 mM), oleic acid (33 μM), monoolein (100 μM), α-tocopherol (0.1 μM), GL/lysoGL/PtdCho/lysoPtdCho (0 or 50 μM), and carotenoid (1 μM), unless otherwise stated. The major fatty acids in GL and lysoGL were 16:3 and 18:3 (the total was 42.6–96.6 mol%), suggesting that the oxidative stability of these lipids would be very low. In a preliminary experiment, the amount of carotenoids in mixed micelles containing GL was significantly decreased upon the incubation without cells at 37 °C for 2 h (data not shown). α-Tocopherol was therefore added to all the samples in order to inhibit the oxidative degradation of GL/ lysoGL and carotenoids during the incubation. To confirm the initial concentration of micellar carotenoids in the medium before the addition to the Caco-2 cells, we diluted one part of the micellar carotenoids in the medium with nine parts of dichloromethane/methanol (1:4, v/v) for β-carotene [17] or dichloromethane/methanol (1:9, v/v) for lutein, and then subjected the solution to a
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high-performance liquid chromatographic (HPLC) analysis as described below. UV–VIS Absorbance Spectral Analysis of Carotenoids Solubilized in Mixed Micelles We measured the ultraviolet–visible (UV–VIS) spectra of carotenoids solubilized in the mixed micelles at 300– 600 nm with a spectrophotometer (Model U-3310; Hitachi, Tokyo) at room temperature. Phosphate-buffered saline (PBS) was used instead of FBS-free DMEM for dispersion of the mixed micelle components, because the phenol red in the DMEM would interfere with the measurement of the carotenoid spectra. The strength of the interaction of the carotenoids with GL/lysoGL/PtdCho/lysoPtdCho is expressed as the full width at half-maximum (FWHM), which was calculated from the height equivalent to one-half of the absorbance at the absorption maximum (λmax) of each spectrum. Evaluation of Carotenoid Uptake by Differentiated Caco‑2 Cells from Mixed Micelles Differentiated Caco-2 cells in a 24-well plate were washed twice with 0.5 mL of FBS-free DMEM and then treated with 0.5 mL of a medium containing micellar carotenoids in a CO2 incubator at 37 °C for 2 h. Immediately after the treatment, the medium was collected to evaluate the residual micellar carotenoids as described below. The cells were washed with 0.5 mL of 10 mM taurocholate in PBS and then washed twice with PBS. They were homogenized in 1 mL of PBS containing 0.01 mL of 0.2 mM α-tocopherol/ methanol as an antioxidant, with a probe-type sonicator (Ultra S, VP-5S; Taitec, Saitama, Japan). To evaluate the amount of carotenoid uptake by Caco-2 cells, we extracted the carotenoids from the cell homogenate as described previously [17]. The extract was then dissolved in 0.2 mL of dichloromethane/methanol (1:4, v/v) for β-carotene or dichloromethane/methanol (1:9, v/v) for lutein, and analyzed by HPLC as described below. Another aliquot of the cell homogenate was used to determine the protein content by the Lowry method [21]. To measure the residual micellar carotenoids in the medium at the end of the incubation, we analyzed aliquots of the medium after passing them through a filter as described above. The total combined amount of carotenoids in the micelles and cells reflects the amount of carotenoids accessible to the cells during the 2-h incubation [16]. We defined the ratio of the combined amounts of carotenoids in the cells and in the micelles to the initial amount of carotenoids incubated with the cells as the accessibility of carotenoids [17]. We used the ratio of cellular carotenoids to the accessible carotenoids as an index of the efficiency
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of carotenoid transfer into the cells, as reported previously [17]. The effects of GL and lysoGL on the transfer of carotenoids from the micelles into the cells were evaluated based on both uptake and transfer efficiency. Influence of Cell‑to‑Cell/Cell–Matrix Adhesion in Caco‑2 Cells on Carotenoid Uptake To evaluate the influence of the degree of cell-to-cell/ cell–matrix adhesion on the carotenoid uptake by cells, we prepared two types of cells. The first type was prepared without cell-to-cell/cell–matrix adhesion by dispersing the cells with trypsin. The dispersed cells were washed twice with FBS-free DMEM and then seeded in 24-well plates at a density of 20 × 104 cells per well containing 0.5 mL of the FBS-free DMEM, for suspension cultures (SC plate; Greiner Bio-One, Frickenhausen, Germany). The other type of cells was prepared by shortening the culture period from 20–22 days to less than 7 days after the seeding described above. The cells of this type were thought to have poorer cell-to-cell adhesion than the differentiated Caco-2 cells. An aliquot (0.5 mL) of micellar carotenoids was added to adherent cells or dispersed cells in individual wells containing 0.5 mL of FBS-free DMEM, resulting in a total medium volume of 1.0 mL/well, and a micellar carotenoid concentration that was one-half of the initial concentration. After incubation in a CO2 incubator at 37 °C for 2 h, both types of cells were washed once with 2 mM taurocholate/ PBS and then twice with PBS, and then the cellular carotenoids and residual micellar carotenoids were evaluated by HPLC as described above. We also evaluated the β-carotene uptake by HP-100-1, a variant of HL-60 human promyelocytic leukemia cells. The suspension culture of these cells exhibited essentially no cell-to-cell/cell–matrix adhesion. Cells were cultured as described earlier [22], except that DMEM was used instead of RPMI1640. Here, cells were seeded in 24-well SC plates at a density of 50 × 104 cells per well containing 0.5 mL of the FBS-free DMEM. The mixed micelles were added to the cells at a concentration that was one-fifth of that used in the experimental conditions for the differentiated Caco-2 cells. The final medium volume was 1.0 mL per well. Incubation, washing, and carotenoid analyses were carried out as in the experiment using the dispersed Caco-2 cells. HPLC Analysis Carotenoids were analyzed by HPLC (using an LC20AT pump, an SPD-M10A photodiode array detector, and a CTO-10AS column oven at a constant temperature of 25 °C; Shimadzu, Kyoto, Japan) on an ODS-80Ts column (2.0 × 150 mm) with an ODS-S1 precolumn (2.0 × 10 mm; Tosoh, Tokyo). Solvent A was acetonitrile/
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methanol/water (75:15:10, v/v/v) containing 0.1 % ammonium acetate, and solvent B was methanol/ethyl acetate (70:30, v/v) containing 0.1 % ammonium acetate. An isocratic analysis was performed at a flow rate of 0.2 mL/min with solvent B for β-carotene and solvent A/B (50:50, v/v) for lutein. These carotenoids were quantified from the peak area at 450 nm using a calibration curve of the authentic standard. All experiments using carotenoids were conducted under dim yellow light to minimize isomerization and the degradation of carotenoids by light irradiation. Leakage of CF from PtdCho Liposomes We evaluated the influence of lysoPtdCho and three lysoGL on cell membrane permeability by measuring the leakage of CF from liposomes. The measurement of CF leakage using PtdCho liposomes is the standard method for determining cell membrane permeability [23]. CF-encapsulated PtdCho liposomes were prepared in the following manner. A chloroform solution of PtdCho was transferred to glass test tubes. The solvent was dried in a centrifugal evaporator. The dried film of PtdCho was dispersed with Tris–HCl buffer (20 mM, pH 7.4) containing 100 mM CF. PtdCho (2 mM) liposomes were further sonicated with a probe-type homogenizer and then passed through the gel-filtration column of a Sephadex G-50 to remove the un-encapsulated CF. Lyso-lipids/methanol were added at a final concentration of 0.1 mM to the liposomes, and the final concentration of methanol was 2 % (v/v). In this method, the fluorescence of CF is self-quenched at a high concentration in the liposome interior and increases when the CF is leaked from the liposome to the extracellular buffer. The leakage level of CF was monitored up to 6 h at 37 °C at Ex. 485 nm and Em. 535 nm with a GENios microplate reader (Tecan Group, Männedorf, Switzerland) and was expressed as the percentage of the total amount of the encapsulated CF, which was calculated by lysis of the liposome membrane with Triton X-100 at a final concentration of 0.2 % (w/v). Concomitant with CF leakage, the turbidity of the liposomal suspension was spectrophotometrically monitored at 595 nm with the GENios microplate reader. During the monitoring period, the lyso-lipids did not affect the turbidity of the liposomal suspension (data not shown), indicating that there was no lysis of the liposome membrane by the lyso-lipids. Statistical Analysis The data were tested for homogeneity of variances by the Bartlett test. When homogenous variances were confirmed,
we analyzed the data by a one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test. The values underwent log transformation before the test, when necessary. When heterogeneous variances were detected, we analyzed the data nonparametrically by using the Kruskal– Wallis test, and significant differences of means were evaluated by the Mann–Whitney U-test. p-values 70 %, Fig. 2b). MGDG realized only a slight enhancement of β-carotene uptake, compared with NoPL (Fig. 2a), but the transfer efficiency was slightly reduced (Fig. 2c), indicating that MGDG had no actual effect. In contrast, DGDG and SQDG significantly reduced both the uptake and transfer efficiency (Fig. 2a, c), like PtdCho. As shown in Fig. 2d–f, the effect of GL on lutein uptake followed a trend similar to that of GL on β-carotene uptake. Effect of LysoGL on Carotenoid Uptake LysoPtdCho markedly enhanced both the β-carotene uptake (by approximately fourfold) and the transfer efficiency (by approximately twofold) compared to NoPL (Fig. 3a, c). DGMG and SQMG also significantly enhanced both
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c b a
Effect of GL and LysoGL on the UV–VIS Absorption Spectrum of Carotenoids in the Mixed Micelles As shown in Fig. 4a, the absorption spectrum of β-carotene in hexane had the sharp peak with a λmax at 450 nm. The λmax of the β-carotene spectrum in the NoPL micelles showed a bathochromic shift to about 460 nm while maintaining the same spectral structure. However, the absorption peaks of the β-carotene spectra in the PtdCho, DGDG, and SQDG micelles were very broad compared to that in the NoPL micelles (Fig. 4a, b). In addition, the FWHM values
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the uptake and transfer efficiency (Fig. 3a, c), but their enhancement effects were lower than those of lysoPtdCho. No significant differences in enhancement effects were found between DGMG and SQMG on the uptake, but the effect of SQMG on transfer efficiency was higher than that of DGMG. MGMG slightly enhanced the uptake but did not affect the transfer efficiency, indicating that it had no actual effect. The effect of lysoGL on lutein uptake showed a trend similar to that of lysoGL on β-carotene uptake (Fig. 3d–f). However, the enhancement effect of DGMG and SQMG on the lutein uptake was almost the same as that of lysoPtdCho.
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Fig. 2 Effects of GL on the uptake of carotenoids solubilized in mixed micelles by Caco-2 cells. Caco-2 cells were treated for 2 h with carotenoids solubilized in mixed micelles prepared as described in the “Materials and Methods”. a, d Carotenoid uptake by Caco-2 cells. b, e Accessibility of carotenoids: the ratio of the combined amounts of carotenoids in the micelles (open columns) and in the cells (solid columns) after 2-h incubation to the initial amount of carotenoids incubated with the cells, expressed as a percentage. Statistical analysis was carried out for the accessibility values. c, f Efficiency of carotenoid transfer. Data are the means ± SD of four wells in a single experiment. Replicate experiments showed a similar trend. Values not sharing a common letter were significantly different by the Tukey–Kramer test or Mann–Whitney U-test (p DGMG > SQMG. Influence of Cell‑to‑Cell/Cell–Matrix Adhesion in Caco‑2 Cells on Carotenoid Uptake We next investigated the participation of cell-to-cell/cell– matrix adhesion as another possible mechanism for the enhancement effects of lysoPtdCho, DGMG, and SQMG
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Fig. 3 Effects of lysoGL on the uptake of carotenoids solubilized in mixed micelles by Caco-2 cells. Caco-2 cells were treated for 2 h with carotenoids solubilized in mixed micelles prepared as described in the “Materials and Methods”. a, d Carotenoid uptake by Caco-2 cells. b, e Accessibility of carotenoids: See the Fig. 2 legend. c, f Efficiency of carotenoid transfer. Data are the means ± SD of four wells in a single experiment. Replicate experiments showed a similar trend. Values not sharing a common letter were significantly different by the Tukey–Kramer test (p SQMG > lysoPtdCho > DGMG, though the accessibility of β-carotene in NoPL micelles was low (Fig. 7b). The transfer efficiency of the NoPL micelles was thus the highest among the four micelles (Fig. 7c). Carotenoid uptake might be affected by cell-surface damage caused by the trypsin treatment for preparing dispersed Caco-2 cells. In order to rule out this possibility, we also evaluated the effect of PtdCho and lysoPtdCho on the β-carotene uptake by HP-100-1 cells, which are a variant of HL-60 human promyelocytic leukemia cells, as typical floating cells. The amount of β-carotene taken up from lysoPtdCho micelles was much lower than that taken up from NoPL micelles (Fig. 8a–c), which agreed with the effect of lysoPtdCho on carotenoid uptake by dispersed Caco-2 cells.
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Wavelength (nm) Fig. 4 Effects of GL/lysoGL on the UV–VIS absorption spectra of β-carotene in mixed micelles. β-Carotene was dissolved at a concentration of 1 μM in hexane, or solubilized in NoPL, PtdCho, lysoPtdCho (a), GL (b), or lysoGL micelles (c) at room temperature. The mixed micelles were prepared as described in the “Materials and Methods”
In addition to evaluating the β-carotene uptake in dispersed Caco-2 cells, we evaluated the uptake of β-carotene solubilized in NoPL, lysoPtdCho, DGMG, and SQMG micelles by Caco-2 cells with poor cell-to-cell adhesion (Fig. 9a–c). These cells were cultured for 1, 3, and 5 days and then incubated with β-carotene solubilized in the mixed micelles (marked by arrows at the top of Fig. 9). The adherent cells seemed to have already reached confluence after culturing for 3 days. Thus, cells cultured for 1 days were thought to have poor cell-to-cell adhesion.
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Wavelength (nm) Fig. 5 Effects of GL/lysoGL on the UV–VIS absorption spectra of lutein in mixed micelles. Lutein was dissolved at a concentration of 1 μM in ethanol, or solubilized in NoPL, PtdCho, lysoPtdCho (a), GL (b), or lysoGL micelles (c) at room temperature. The mixed micelles were prepared as described in the “Materials and Methods”
The results obtained by using cells cultured for 1 day were also very different from those obtained by using the differentiated cells that were cultured for 20–22 days (Fig. 3). The amount of β-carotene uptake from NoPL micelles by Caco-2 cells cultured for 1 day (the interstices between cell colonies were broad) was the highest among the four types of micelles tested (Fig. 9a), though the accessibility of β-carotene in the NoPL micelles was very low (Fig. 9b), indicating a very high transfer efficiency
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Fig. 6 Effect of lysoGL on CF leakage from PtdCho liposomes. Liposomes were prepared as described in the “Materials and Methods”. LysoPtdCho (open squares), MGMG (filled circles), DGMG (filled triangles), and SQMG (filled squares) dissolved in methanol were added to CF-encapsulated PtdCho liposomes. Methanol alone at a final concentration of 2 % (v/v) (open circles) was used as the vehicle control. The lyso-lipids dissolved in methanol were added at a final concentration of 0.1 mM to the liposomes. The CF leakage is expressed as the percentage of the total amount of encapsulated CF. Data are the means ± SD values of eight wells in a single experiment. Replicate experiments showed a similar trend. Values not sharing a common letter were significantly different by the Tukey– Kramer test or Mann–Whitney U-test (p
ORIGINAL ARTICLE
Glyceroglycolipids Affect Uptake of Carotenoids Solubilized in Mixed Micelles by Human Intestinal Caco‑2 Cells Eiichi Kotake‑Nara1 · Lina Yonekura1 · Akihiko Nagao1
Received: 22 October 2014 / Accepted: 1 May 2015 © AOCS 2015
Abstract We previously reported that phospholipids markedly affected the uptake of carotenoids solubilized in mixed micelles by human intestinal Caco-2 cells. In the present study, we found that two classes of dietary glyceroglycolipids and the corresponding lysoglyceroglycolipids affected uptake of β-carotene and lutein by differentiated Caco-2 cells. The levels of carotenoid uptake from micelles containing digalactosyldiacylglycerol or sulfoquinovosyldiacylglycerol were significantly lower than that from control micelles. On the other hand, the uptakes from micelles containing digalactosylmonoacylglycerol or sulfoquinovosylmonoacylglycerol were significantly higher than that from control micelles. In dispersed cells and Caco-2 cells with poor cell-to-cell adhesion, however, the levels of uptake from micelles containing these lyso-lipids were much lower than that from control micelles. The uptake levels from control micelles were markedly decreased depending on the development of cell-to-cell/cell–matrix adhesion in Caco-2 cells, but the uptake levels from the micelles containing these lyso-lipids were not substantially changed, suggesting that the intercellular barrier formed by cell-to-cell/cell–matrix adhesion inhibited the uptake from control micelles, but not from the lyso-lipid-containing micelles. The lyso-lipids appeared to enhance carotenoid uptake by decreasing the intercellular barrier integrity. The results showed that some types of glyceroglycolipids have the potential to modify the intestinal uptake of carotenoids.
* Eiichi Kotake‑Nara [email protected] 1
National Food Research Institute, National Agriculture and Food Research Organization, 2‑1‑12 Kannondai, Tsukuba, Ibaraki 305‑8642, Japan
Keywords Glyceroglycolipid · Lysoglyceroglycolipid · Carotenoid · β-Carotene · Lutein · Mixed micelles · Caco-2 · Lyso-lipid · Intercellular barrier integrity · Intestinal uptake Abbreviations CF 5(6)-carboxyfluorescein DGDG Digalactosyldiacylglycerol DGMG Digalactosylmonoacylglycerol DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide FBS Fetal bovine serum FWHM Full width at half-maximum GL Diacyl glyceroglycolipid(s) lysoGL Lysoglyceroglycolipid(s) lysoPtdCho Lysophosphatidylcholine MGDG Monogalactosyldiacylglycerol MGMG Monogalactosylmonoacylglycerol PBS Phosphate-buffered saline PtdCho Diacyl phosphatidylcholine SQDG Sulfoquinovosyldiacylglycerol SQMG Sulfoquinovosylmonoacylglycerol
Introduction Fruits and vegetables are major dietary sources of carotenoids, which have been reported to show anti-oxidant activities [1, 2] and other characteristic biological activities including anti-allergic [3, 4], anti-cancer [5], anti-inflammatory [6, 7], and anti-obesity [8, 9] effects. Thus, the activities of dietary carotenoids have attracted a great deal of attention in terms of human health. However, the bioavailability of carotenoids is known to be lower than that of the other lipophilic nutrients, such
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as triacylglycerol and α-tocopherol [10–13]. Carotenoids are absorbed after solubilization in the intestinal tract via several steps. First, carotenoids are released from the food matrix of, for example, fruits and vegetables, and dissolved into dietary lipids. The carotenoids are then dispersed in the digestive tract as the lipids are emulsified with bile [14]. As the digestion of the emulsified lipids progresses with pancreatic lipase, carotenoids are solubilized in mixed micelles consisting of bile salts, cholesterol, fatty acids, monoacylglycerols, and lysophospholipids. Although the mixed micellar carotenoids are accessible to uptake by epithelial cells in the intestine, it is thought that only a portion of them are actually taken up by the cells. Their intestinal uptake, as well as their solubilization into the micelles, is thus a critical factor in carotenoid bioavailability. We found previously that mixed micelle components such as phospholipids, fatty acids, and monoacylglycerol Fig. 1 Typical chemical structures of glyceroglycolipids, phosphatidylcholine, and their lyso-lipids. a PtdCho, b lysoPtdCho, c MGDG, d MGMG, e DGDG, f DGMG, g SQDG, h SQMG. R1OC and R2OC in these structures are fatty acid moieties
a
markedly affected the uptake of carotenoids by human intestinal Caco-2 cells in different ways [15–17]. In particular, phosphatidylcholine (PtdCho) suppressed the uptake, while lysophosphatidylcholine (lysoPtdCho) significantly enhanced it. Differences in the polar head group in phospholipids also affected the uptake [18]. These observations suggest that amphipathic lipids greatly influence the uptake of carotenoids solubilized in mixed micelles by intestinal cells. Diacyl glyceroglycolipids (GL), which are ingested together with carotenoids from green leafy vegetables, are also amphipathic lipids with a sugar moiety as a polar head group. We have hypothesized that GL can affect the cellular uptake of carotenoids solubilized in mixed micelles. Hence, in the present study, we determined the effects of three major dietary GL and the corresponding lysoglyceroglycolipids (lysoGL) (Fig. 1) on the uptake of β-carotene and lutein by Caco-2 cells.
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Materials and Methods Materials β-Carotene (type II), 1-oleoyl-rac-glycerol (monoolein), 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocholine (lysoPtdCho), lipase (from Rhizopus arrhizus), and 5(6)-carboxyfluorescein (CF) were purchased from SigmaAldrich (St. Louis, MO). Lutein was purchased from Extrasynthese (Genay Cedex, France). β-Carotene and lutein were purified by alumina column chromatography (neutral, grade III; MP Biomedicals, Solon, OH). The purified carotenoids were stored at −80 °C until use. 1-Palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (PtdCho) was purchased from Avanti Polar Lipids (Alabaster, AL). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo). Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS). GL, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), were purchased from Larodan Fine Chemicals (Malmö, Sweden). Ethyl β-apo-8′-carotenoate was purchased from Fluka Biochemica (Buchs, Switzerland). The other chemicals and solvents were of reagent grade. Preparation of LysoGL LysoGL, monogalactosylmonoacylglycerol (MGMG), digalac tosylmonoacylglycerol (DGMG), and sulfoquinovosylmonoacylglycerol (SQMG) were prepared from the corresponding GL using a previously described procedure with modifications [19]. In brief, 25 mg of GL was hydrolyzed by Rhizopus arrhizus lipase (105 units) in Tris–HCl buffer (25 mM, pH 7.7, 5 mL) containing 2.4 % Triton X-100 at 38 °C for 30 min. The enzymatic reaction was quenched with 6 N HCl (0.15 mL). After 5 mL of methanol was added, lysoGL were extracted twice with 10 mL of chloroform. The extracts were combined and then dried in a centrifugal evaporator. The residue was dissolved in 250 μL of dichloromethane/methanol (2:1, v/v) and subjected to purification by thin-layer chromatography on silica gel 60 with chloroform/methanol (3:1, v/v) as the developing solvent. The rate of flow (Rf) values were 0.53 for MGMG, 0.17 for DGMG, and 0.10 for SQMG. Fatty Acid Analysis of GL and LysoGL The acyl groups of GL and lysoGL (100–200 μg) were converted to methyl ester together with 100 nmol heneicosanoic acid (21:0) as an internal standard by an HCl/methanol method, and then the methyl ester was subjected to gas chromatographic analysis as previously described [20]. Based on the fatty acid compositions, we calculated the
average molecular weight of the GL and lysoGL (761 for MGDG, 931 for DGDG, 885 for SQDG, 487 for MGMG, 672 for DGMG, and 630 for SQMG), and determined the molar amounts. Cell Culture and Differentiation Caco-2 cells derived from human colon carcinoma were purchased from the American Type Culture Collection (Rockville, MD). The cells were routinely cultured in a medium consisting of DMEM, 10 % heat-inactivated FBS, 4 mM l-glutamine, 40 units/mL of penicillin, 40 μg/mL of streptomycin, and 0.1 mM nonessential amino acids at 37 °C in a humidified atmosphere of 5 % CO2 in air. The cells were grown by passaging 2 ×/week. To obtain fully differentiated Caco-2 cell monolayers, the cells at passages of 33–60 were seeded into 24-well plates at a density of 5 × 104 cells per well containing 0.5 mL of the culture medium, and the medium was replaced with fresh medium 2 ×/week for 20–22 days. The differentiated cells were used in carotenoid uptake experiments, unless otherwise stated. Preparation of Mixed Micelles Containing Carotenoids Mixed micelles were prepared as described previously [18]. All of the mixed micelle components and carotenoids (β-carotene or lutein) were dissolved in an appropriate solvent and transferred to glass test tubes. The solvent was then evaporated under a stream of argon and the solution was further dried in a centrifugal evaporator. The dry matter was dispersed into FBS-free DMEM, and the resulting clear solution was passed through a 0.2-μm filter to remove any carotenoids that were not solubilized into the micelles. The mixed micelles consisted of taurocholate (2 mM), oleic acid (33 μM), monoolein (100 μM), α-tocopherol (0.1 μM), GL/lysoGL/PtdCho/lysoPtdCho (0 or 50 μM), and carotenoid (1 μM), unless otherwise stated. The major fatty acids in GL and lysoGL were 16:3 and 18:3 (the total was 42.6–96.6 mol%), suggesting that the oxidative stability of these lipids would be very low. In a preliminary experiment, the amount of carotenoids in mixed micelles containing GL was significantly decreased upon the incubation without cells at 37 °C for 2 h (data not shown). α-Tocopherol was therefore added to all the samples in order to inhibit the oxidative degradation of GL/ lysoGL and carotenoids during the incubation. To confirm the initial concentration of micellar carotenoids in the medium before the addition to the Caco-2 cells, we diluted one part of the micellar carotenoids in the medium with nine parts of dichloromethane/methanol (1:4, v/v) for β-carotene [17] or dichloromethane/methanol (1:9, v/v) for lutein, and then subjected the solution to a
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high-performance liquid chromatographic (HPLC) analysis as described below. UV–VIS Absorbance Spectral Analysis of Carotenoids Solubilized in Mixed Micelles We measured the ultraviolet–visible (UV–VIS) spectra of carotenoids solubilized in the mixed micelles at 300– 600 nm with a spectrophotometer (Model U-3310; Hitachi, Tokyo) at room temperature. Phosphate-buffered saline (PBS) was used instead of FBS-free DMEM for dispersion of the mixed micelle components, because the phenol red in the DMEM would interfere with the measurement of the carotenoid spectra. The strength of the interaction of the carotenoids with GL/lysoGL/PtdCho/lysoPtdCho is expressed as the full width at half-maximum (FWHM), which was calculated from the height equivalent to one-half of the absorbance at the absorption maximum (λmax) of each spectrum. Evaluation of Carotenoid Uptake by Differentiated Caco‑2 Cells from Mixed Micelles Differentiated Caco-2 cells in a 24-well plate were washed twice with 0.5 mL of FBS-free DMEM and then treated with 0.5 mL of a medium containing micellar carotenoids in a CO2 incubator at 37 °C for 2 h. Immediately after the treatment, the medium was collected to evaluate the residual micellar carotenoids as described below. The cells were washed with 0.5 mL of 10 mM taurocholate in PBS and then washed twice with PBS. They were homogenized in 1 mL of PBS containing 0.01 mL of 0.2 mM α-tocopherol/ methanol as an antioxidant, with a probe-type sonicator (Ultra S, VP-5S; Taitec, Saitama, Japan). To evaluate the amount of carotenoid uptake by Caco-2 cells, we extracted the carotenoids from the cell homogenate as described previously [17]. The extract was then dissolved in 0.2 mL of dichloromethane/methanol (1:4, v/v) for β-carotene or dichloromethane/methanol (1:9, v/v) for lutein, and analyzed by HPLC as described below. Another aliquot of the cell homogenate was used to determine the protein content by the Lowry method [21]. To measure the residual micellar carotenoids in the medium at the end of the incubation, we analyzed aliquots of the medium after passing them through a filter as described above. The total combined amount of carotenoids in the micelles and cells reflects the amount of carotenoids accessible to the cells during the 2-h incubation [16]. We defined the ratio of the combined amounts of carotenoids in the cells and in the micelles to the initial amount of carotenoids incubated with the cells as the accessibility of carotenoids [17]. We used the ratio of cellular carotenoids to the accessible carotenoids as an index of the efficiency
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of carotenoid transfer into the cells, as reported previously [17]. The effects of GL and lysoGL on the transfer of carotenoids from the micelles into the cells were evaluated based on both uptake and transfer efficiency. Influence of Cell‑to‑Cell/Cell–Matrix Adhesion in Caco‑2 Cells on Carotenoid Uptake To evaluate the influence of the degree of cell-to-cell/ cell–matrix adhesion on the carotenoid uptake by cells, we prepared two types of cells. The first type was prepared without cell-to-cell/cell–matrix adhesion by dispersing the cells with trypsin. The dispersed cells were washed twice with FBS-free DMEM and then seeded in 24-well plates at a density of 20 × 104 cells per well containing 0.5 mL of the FBS-free DMEM, for suspension cultures (SC plate; Greiner Bio-One, Frickenhausen, Germany). The other type of cells was prepared by shortening the culture period from 20–22 days to less than 7 days after the seeding described above. The cells of this type were thought to have poorer cell-to-cell adhesion than the differentiated Caco-2 cells. An aliquot (0.5 mL) of micellar carotenoids was added to adherent cells or dispersed cells in individual wells containing 0.5 mL of FBS-free DMEM, resulting in a total medium volume of 1.0 mL/well, and a micellar carotenoid concentration that was one-half of the initial concentration. After incubation in a CO2 incubator at 37 °C for 2 h, both types of cells were washed once with 2 mM taurocholate/ PBS and then twice with PBS, and then the cellular carotenoids and residual micellar carotenoids were evaluated by HPLC as described above. We also evaluated the β-carotene uptake by HP-100-1, a variant of HL-60 human promyelocytic leukemia cells. The suspension culture of these cells exhibited essentially no cell-to-cell/cell–matrix adhesion. Cells were cultured as described earlier [22], except that DMEM was used instead of RPMI1640. Here, cells were seeded in 24-well SC plates at a density of 50 × 104 cells per well containing 0.5 mL of the FBS-free DMEM. The mixed micelles were added to the cells at a concentration that was one-fifth of that used in the experimental conditions for the differentiated Caco-2 cells. The final medium volume was 1.0 mL per well. Incubation, washing, and carotenoid analyses were carried out as in the experiment using the dispersed Caco-2 cells. HPLC Analysis Carotenoids were analyzed by HPLC (using an LC20AT pump, an SPD-M10A photodiode array detector, and a CTO-10AS column oven at a constant temperature of 25 °C; Shimadzu, Kyoto, Japan) on an ODS-80Ts column (2.0 × 150 mm) with an ODS-S1 precolumn (2.0 × 10 mm; Tosoh, Tokyo). Solvent A was acetonitrile/
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methanol/water (75:15:10, v/v/v) containing 0.1 % ammonium acetate, and solvent B was methanol/ethyl acetate (70:30, v/v) containing 0.1 % ammonium acetate. An isocratic analysis was performed at a flow rate of 0.2 mL/min with solvent B for β-carotene and solvent A/B (50:50, v/v) for lutein. These carotenoids were quantified from the peak area at 450 nm using a calibration curve of the authentic standard. All experiments using carotenoids were conducted under dim yellow light to minimize isomerization and the degradation of carotenoids by light irradiation. Leakage of CF from PtdCho Liposomes We evaluated the influence of lysoPtdCho and three lysoGL on cell membrane permeability by measuring the leakage of CF from liposomes. The measurement of CF leakage using PtdCho liposomes is the standard method for determining cell membrane permeability [23]. CF-encapsulated PtdCho liposomes were prepared in the following manner. A chloroform solution of PtdCho was transferred to glass test tubes. The solvent was dried in a centrifugal evaporator. The dried film of PtdCho was dispersed with Tris–HCl buffer (20 mM, pH 7.4) containing 100 mM CF. PtdCho (2 mM) liposomes were further sonicated with a probe-type homogenizer and then passed through the gel-filtration column of a Sephadex G-50 to remove the un-encapsulated CF. Lyso-lipids/methanol were added at a final concentration of 0.1 mM to the liposomes, and the final concentration of methanol was 2 % (v/v). In this method, the fluorescence of CF is self-quenched at a high concentration in the liposome interior and increases when the CF is leaked from the liposome to the extracellular buffer. The leakage level of CF was monitored up to 6 h at 37 °C at Ex. 485 nm and Em. 535 nm with a GENios microplate reader (Tecan Group, Männedorf, Switzerland) and was expressed as the percentage of the total amount of the encapsulated CF, which was calculated by lysis of the liposome membrane with Triton X-100 at a final concentration of 0.2 % (w/v). Concomitant with CF leakage, the turbidity of the liposomal suspension was spectrophotometrically monitored at 595 nm with the GENios microplate reader. During the monitoring period, the lyso-lipids did not affect the turbidity of the liposomal suspension (data not shown), indicating that there was no lysis of the liposome membrane by the lyso-lipids. Statistical Analysis The data were tested for homogeneity of variances by the Bartlett test. When homogenous variances were confirmed,
we analyzed the data by a one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test. The values underwent log transformation before the test, when necessary. When heterogeneous variances were detected, we analyzed the data nonparametrically by using the Kruskal– Wallis test, and significant differences of means were evaluated by the Mann–Whitney U-test. p-values 70 %, Fig. 2b). MGDG realized only a slight enhancement of β-carotene uptake, compared with NoPL (Fig. 2a), but the transfer efficiency was slightly reduced (Fig. 2c), indicating that MGDG had no actual effect. In contrast, DGDG and SQDG significantly reduced both the uptake and transfer efficiency (Fig. 2a, c), like PtdCho. As shown in Fig. 2d–f, the effect of GL on lutein uptake followed a trend similar to that of GL on β-carotene uptake. Effect of LysoGL on Carotenoid Uptake LysoPtdCho markedly enhanced both the β-carotene uptake (by approximately fourfold) and the transfer efficiency (by approximately twofold) compared to NoPL (Fig. 3a, c). DGMG and SQMG also significantly enhanced both
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c b a
Effect of GL and LysoGL on the UV–VIS Absorption Spectrum of Carotenoids in the Mixed Micelles As shown in Fig. 4a, the absorption spectrum of β-carotene in hexane had the sharp peak with a λmax at 450 nm. The λmax of the β-carotene spectrum in the NoPL micelles showed a bathochromic shift to about 460 nm while maintaining the same spectral structure. However, the absorption peaks of the β-carotene spectra in the PtdCho, DGDG, and SQDG micelles were very broad compared to that in the NoPL micelles (Fig. 4a, b). In addition, the FWHM values
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the uptake and transfer efficiency (Fig. 3a, c), but their enhancement effects were lower than those of lysoPtdCho. No significant differences in enhancement effects were found between DGMG and SQMG on the uptake, but the effect of SQMG on transfer efficiency was higher than that of DGMG. MGMG slightly enhanced the uptake but did not affect the transfer efficiency, indicating that it had no actual effect. The effect of lysoGL on lutein uptake showed a trend similar to that of lysoGL on β-carotene uptake (Fig. 3d–f). However, the enhancement effect of DGMG and SQMG on the lutein uptake was almost the same as that of lysoPtdCho.
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Fig. 2 Effects of GL on the uptake of carotenoids solubilized in mixed micelles by Caco-2 cells. Caco-2 cells were treated for 2 h with carotenoids solubilized in mixed micelles prepared as described in the “Materials and Methods”. a, d Carotenoid uptake by Caco-2 cells. b, e Accessibility of carotenoids: the ratio of the combined amounts of carotenoids in the micelles (open columns) and in the cells (solid columns) after 2-h incubation to the initial amount of carotenoids incubated with the cells, expressed as a percentage. Statistical analysis was carried out for the accessibility values. c, f Efficiency of carotenoid transfer. Data are the means ± SD of four wells in a single experiment. Replicate experiments showed a similar trend. Values not sharing a common letter were significantly different by the Tukey–Kramer test or Mann–Whitney U-test (p DGMG > SQMG. Influence of Cell‑to‑Cell/Cell–Matrix Adhesion in Caco‑2 Cells on Carotenoid Uptake We next investigated the participation of cell-to-cell/cell– matrix adhesion as another possible mechanism for the enhancement effects of lysoPtdCho, DGMG, and SQMG
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Fig. 3 Effects of lysoGL on the uptake of carotenoids solubilized in mixed micelles by Caco-2 cells. Caco-2 cells were treated for 2 h with carotenoids solubilized in mixed micelles prepared as described in the “Materials and Methods”. a, d Carotenoid uptake by Caco-2 cells. b, e Accessibility of carotenoids: See the Fig. 2 legend. c, f Efficiency of carotenoid transfer. Data are the means ± SD of four wells in a single experiment. Replicate experiments showed a similar trend. Values not sharing a common letter were significantly different by the Tukey–Kramer test (p SQMG > lysoPtdCho > DGMG, though the accessibility of β-carotene in NoPL micelles was low (Fig. 7b). The transfer efficiency of the NoPL micelles was thus the highest among the four micelles (Fig. 7c). Carotenoid uptake might be affected by cell-surface damage caused by the trypsin treatment for preparing dispersed Caco-2 cells. In order to rule out this possibility, we also evaluated the effect of PtdCho and lysoPtdCho on the β-carotene uptake by HP-100-1 cells, which are a variant of HL-60 human promyelocytic leukemia cells, as typical floating cells. The amount of β-carotene taken up from lysoPtdCho micelles was much lower than that taken up from NoPL micelles (Fig. 8a–c), which agreed with the effect of lysoPtdCho on carotenoid uptake by dispersed Caco-2 cells.
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Wavelength (nm) Fig. 4 Effects of GL/lysoGL on the UV–VIS absorption spectra of β-carotene in mixed micelles. β-Carotene was dissolved at a concentration of 1 μM in hexane, or solubilized in NoPL, PtdCho, lysoPtdCho (a), GL (b), or lysoGL micelles (c) at room temperature. The mixed micelles were prepared as described in the “Materials and Methods”
In addition to evaluating the β-carotene uptake in dispersed Caco-2 cells, we evaluated the uptake of β-carotene solubilized in NoPL, lysoPtdCho, DGMG, and SQMG micelles by Caco-2 cells with poor cell-to-cell adhesion (Fig. 9a–c). These cells were cultured for 1, 3, and 5 days and then incubated with β-carotene solubilized in the mixed micelles (marked by arrows at the top of Fig. 9). The adherent cells seemed to have already reached confluence after culturing for 3 days. Thus, cells cultured for 1 days were thought to have poor cell-to-cell adhesion.
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Wavelength (nm) Fig. 5 Effects of GL/lysoGL on the UV–VIS absorption spectra of lutein in mixed micelles. Lutein was dissolved at a concentration of 1 μM in ethanol, or solubilized in NoPL, PtdCho, lysoPtdCho (a), GL (b), or lysoGL micelles (c) at room temperature. The mixed micelles were prepared as described in the “Materials and Methods”
The results obtained by using cells cultured for 1 day were also very different from those obtained by using the differentiated cells that were cultured for 20–22 days (Fig. 3). The amount of β-carotene uptake from NoPL micelles by Caco-2 cells cultured for 1 day (the interstices between cell colonies were broad) was the highest among the four types of micelles tested (Fig. 9a), though the accessibility of β-carotene in the NoPL micelles was very low (Fig. 9b), indicating a very high transfer efficiency
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Fig. 6 Effect of lysoGL on CF leakage from PtdCho liposomes. Liposomes were prepared as described in the “Materials and Methods”. LysoPtdCho (open squares), MGMG (filled circles), DGMG (filled triangles), and SQMG (filled squares) dissolved in methanol were added to CF-encapsulated PtdCho liposomes. Methanol alone at a final concentration of 2 % (v/v) (open circles) was used as the vehicle control. The lyso-lipids dissolved in methanol were added at a final concentration of 0.1 mM to the liposomes. The CF leakage is expressed as the percentage of the total amount of encapsulated CF. Data are the means ± SD values of eight wells in a single experiment. Replicate experiments showed a similar trend. Values not sharing a common letter were significantly different by the Tukey– Kramer test or Mann–Whitney U-test (p
