Phospholipids Affect the Intestinal Absorption of Carotenoids in Mice Vallikannan Baskaran, Tatsuya Sugawara, and Akihiko Nagao* National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
ABSTRACT: Previously, we have shown that uptake of carotenoids solubilized with mixed micelles by human intestinal Caco-2 cells is enhanced by lysophosphatidylcholine (lysoPC) and suppressed by PC. This study determined the effect of PC and lysoPC in mixed micelles on the accumulation of β-carotene and lutein in mice in order to elucidate the roles of micellar phospholipid in the intestinal uptake of carotenoids in vivo. Mixed micelles were composed of 2.5 mM monooleoylglycerol, 7.5 mM oleic acid, 12 mM sodium taurocholate, 200 µM carotenoid, and 3 mM phospholipid in PBS. The mice were fed single doses of β-carotene or lutein solubilized in PC (PC group), lysoPC (LPC group), and no phospholipid (NoPL group) micelles. The β-carotene responses in the plasma and liver of the PC group were markedly lower than those of the other two groups, whereas no differences were noticed between the LPC and NoPL groups. The average level of lutein in the plasma of the PC group after administration was significantly (P < 0.05) lower than those of the other groups. Moreover, the average level of lutein in the liver was significantly (P < 0.05) different among the groups in the order of LPC > NoPL >PC. Thus, the results clearly indicate that PC suppressed the accumulation of β-carotene and lutein in plasma and liver and that lysoPC enhanced the accumulation of lutein in liver. These results suggest that the hydrolysis of PC to lysoPC plays an important role in the intestinal uptake of carotenoids solubilized in mixed micelles. Paper no. L9219 in Lipids 38, 705–711 (July 2003).
Carotenoids are known to have beneficial functions in human health against major clinical diseases such as cancer, cardiovascular diseases, and age-related macular degeneration (1–4), although the mechanisms of their action at the molecular level still remain unknown (5,6). More information on the absorption and metabolic conversion of carotenoids is required to further understand their biological actions. The absorption of dietary carotenoids from foods involves several steps: the breakdown of the food matrix to release the carotenoids, dispersion in lipid emulsion particles, solubilization in mixed micelles, movement across the unstirred water layer adjacent to the microvilli, uptake by the cells of intestinal mucosa, and incorporation into chylomicron (7–9). Thus, the carotenoids Present address of the first author: Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore-570 013, India. *To whom correspondence should be addressed. E-mail:
[email protected] Abbreviations: AUC, area under the curve; LPC group, group fed mixed micelles containing lysoPC; lysoPC, lysophosphatidylcholine; NoPL group, group fed mixed micelles containing no phospholipid; PC group, group fed mixed micelles containing PC. Copyright © 2003 by AOCS Press
must be solubilized in mixed micelles before cellular uptake. The mixed micelles are composed of bile acids, cholesterol, and PC, which are secreted as bile fluid. Dietary fat induces the secretion of bile, and its hydrolysates, such as MAG and FA, are also included in the mixed micelles. The micelles have a disklike structure, where phospholipids and FA form a bilayer with bile acids located at the edges of the disk (10,11). Thus, the processes up to the solubilization in the mixed micelles are dependent mostly on the physicochemical properties of food and carotenoids, and on bile secretion and dietary components such as fats, sterols, and fiber (12–18). However, the detailed absorption processes of carotenoids after solubilization in mixed micelles have not been fully revealed. The early findings in perfused rat intestine and in hBRIE380 rat intestinal cells (19,20) support a simple diffusion mechanism for the cellular uptake of carotenoids. That mechanism was also supported by the linear relationship found in our recent study between the cellular uptake of micellar carotenoid and its hydrophobicity (21). Thus, the size and lipid composition of the mixed micelles would affect the movement of carotenoids from micelles to the intestinal cells by diffusion. Recent studies have indicated that phospholipids in the mixed micelles and phospholipase A2 profoundly affect the cellular uptake of cholesterol and α-tocopherol (22–25). We also have found that PC suppresses the cellular uptake of carotenoids solubilized in mixed micelles by the human intestinal Caco-2 cells, whereas lysophosphatidylcholine (lysoPC) significantly enhances their uptake (21). Thus, phospholipids derived from bile and foods would affect the cellular uptake of carotenoids solubilized in mixed micelles formed in the intestinal tract. The present study was conducted to evaluate the accumulation of carotenoids in the plasma and liver of mice fed mixed micelles in order to clarify the in vivo effects of PC and lysoPC in mixed micelles upon intestinal uptake of carotenoids. MATERIALS AND METHODS Materials. All-trans-β-carotene (type IV), all-trans-retinol, retinyl palmitate, monooleoylglycerol, and sodium taurocholate were purchased from Sigma Chemical Co. (St. Louis, MO). Lutein was kindly donated by Kyowa Hakko Kogyo (Tokyo, Japan). d-α-Tocopherol was obtained from Eisai Co. (Tokyo, Japan). Egg-yolk PC and lysoPC were obtained from Q.P. Co. (Tokyo, Japan). HPLC grade acetonitrile and oleic acid (>99%) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Other chemicals and solvents were of reagent
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grade. All-trans-β-carotene (>98.8%) and lutein (>99%) were purified by passing them through a neutral alumina column (Brockman III; ICN Biomedicals, Eschwege, Germany) using hexane and methanol, respectively. Purified all-trans-βcarotene and lutein were dissolved in hexane and dichloromethane, respectively, and stored at −80°C until used. Retinyl palmitate was purified (>99.0%) by HPLC on a TSK gel ODS 80Ts column, 6.4 × 250 mm (Tosoh, Tokyo, Japan) with ethyl acetate/methanol (30:70, vol/vol) as the mobile phase. The purity of β-carotene, lutein, and retinyl palmitate was checked by HPLC, and the purity was estimated based on the peak area of components absorbing at specific wavelengths. Animals. Male ICR mice (7 wk old) obtained from Clea, Japan Inc. (Tokyo, Japan) were housed at 25°C with a 12-h light/dark cycle. The animals had free access to tap water and a commercial diet (MF, Oriental Yeast Co., Osaka, Japan). After 7 d of feeding, mice weighing 32 ± 2 g were deprived of food for 14–15 h before carotenoid administration. The mice were handled according to the guidelines for experimental animals of the National Food Research Institute, Ibaraki, Japan. Preparation of micelles and feeding. Mixed micelles in PBS containing 2.5 mM monooleoylglycerol, 7.5 mM oleic acid, 12 mM sodium taurocholate, and 200 µΜ β-carotene or lutein with either 3 mM PC, 3 mM lysoPC, or no phospholipid were prepared. Appropriate amounts of these chemicals dissolved in hexane or methanol were mixed to reach the final concentration. The solvent was evaporated using argon gas and the dried mixture was redissolved in PBS with vigorous mixing using a vortex mixer to obtain an optically clear solution. The micelle composition chosen was based on the composition of the clear layer obtained by ultracentrifugation of the duodenal content of healthy adult human subjects given a TG-rich meal (26). It would hypothetically produce a mixture of mixed micelles and small unilamellar vesicles, according to the phase diagram indicated by Staggers et al. (27). The vesicles can resolve spontaneously into the mixed micelles as the ratio of lipid to cholic acid decreases during the absorption process. Thus, the optically clear solutions obtained by the procedure described above were used as the mixed micelles in the present study. The concentrations of β-carotene and lutein in the micelles were checked by HPLC before the mice were fed. The mice were randomly divided into seven groups. Six groups were fed with either β-carotene or lutein solubilized in the mixed micelles containing no phospholipid (NoPL group), PC (PC group), or lysoPC (LPC group). The other group was not fed mixed micelles (zero-time control). The mixed micelles (0.2 mL/mouse) were administered to the mice by direct intubation to the stomach. The volume size of intubation had no adverse effects on mice. Mice in the zero-time control (n = 5) and in each treatment group (n = 5/time point) at 1, 2, 3, 6, and 9 h after administration were anesthetized with diethyl ether and killed by exsanguination. Blood was collected from the caudal vena cava with a heparinized syringe. The livers were removed and washed with ice-cold isotonic saline. Blood was immediately separated into plasma by centrifugation at 1000 × g for 15 min at 4°C. The plasma and liver were Lipids, Vol. 38, no. 7 (2003)
immediately stored at −80°C until analyzed. The amount of carotenoid fed was calculated to be 0.671 mg/kg body weight and was comparable to the amounts reported in studies in which β-carotene was supplemented to human subjects. In a preliminary experiment in mice, one-tenth of the carotenoid level used in the present study was administered, but the accumulation of carotenoids in plasma was undetectable. Extraction from plasma and liver. β-Carotene, retinol, retinyl palmitate, and lutein were extracted from the plasma according to the procedures described previously by Sugawara et al. (21) with slight modifications. Briefly, the plasma (0.4 mL) was diluted to 0.8 mL with ice-cold deionized water, and 3 mL of dichloromethane/methanol (1:2, vol/vol) containing 0.2 µmol α-tocopherol was vigorously mixed with the plasma for 1 min using a vortex mixer. Then 1.5 mL of hexane was mixed with the solution. The resulting upper layer of hexane/dichloromethane was withdrawn. The extraction procedure was repeated for the lower layer two more times using 1 mL of dichloromethane and 1.5 mL of hexane. The combined extract was evaporated to dryness under a stream of argon gas. The plasma extract used for the analyses of βcarotene, retinol, and retinyl palmitate was dissolved in dichloromethane/methanol (2:1, vol/vol), and that used for the analysis of lutein was dissolved in methanol. Liver samples were homogenized with 9 parts ice-cold isotonic saline with a Potter–Elvehjem homogenizer, and 0.8 mL of the homogenate was used for extraction by the same procedure as described for the plasma. In the case of the groups fed lutein, the extract was further saponified by incubating in 2 mL of 10 M KOH at 60°C for 45 min. Subsequently, 2 mL of ice-cold deionized water was added, and lutein was extracted as described above. Samples were handled and homogenization and extraction were carried out on ice under dim yellow light to minimize isomerization and oxidation by light irradiation. HPLC analyses. β-Carotene, retinol, retinyl palmitate, and lutein in the extracts of plasma and liver were quantified with an HPLC system consisting of an LC-10AD pump, an SPD10A UV-vis absorbance detector (Shimadzu, Kyoto, Japan), an AS-8020 autosampler (Tosoh), and a personal computer equipped with EZChrome Chromatography Data System software (Scientific Software Inc., Pleasanton, CA). All the components were separated on a TSK gel ODS-80Ts column (Tosoh), 4.6 × 150 mm, attached to a precolumn (2 × 20 mm) of Pelliguard LC-18 (Supelco Inc., Bellefonte, PA). The column was kept in an oven at 20°C. Ethyl acetate/methanol (30:70, vol/vol) containing 0.1% ammonium acetate was used as a mobile phase for the analyses of β-carotene, retinol, and retinyl ester, and ethyl acetate/acetonitrile/methanol/water (21:23:53:3, by vol) containing 0.1% ammonium acetate was used for the analysis of lutein. An isocratic analysis was performed at a flow rate of 1 mL/min. β-Carotene and lutein were monitored at 450 nm with a UV-vis absorbance detector, and retinol and retinyl palmitate were monitored at 325 nm. They were quantified from their peak areas by use of the standard curves of reference compounds. The peak identity
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of these components was further confirmed by their characteristic UV-vis spectra, recorded with a model 1100 HPLC system equipped with a photodiode array detector (HewlettPackard, Palo Alto, CA). Statistical analysis. To quantify the postprandial βcarotene and lutein levels in the plasma and liver over 9 h, the area under the curve (AUC) was calculated by trapezoidal approximation. Data were tested for the homogeneity of variances by the Bartlett test. When homogenous variances were confirmed, the data were tested by ANOVA, and significant differences in means among groups and at different time intervals were evaluated by Tukey’s test. The values underwent log transformation before the tests if necessary. The differences between β-carotene and lutein levels were analyzed nonparametrically by the Kruskal–Wallis test, and significant differences in means were evaluated by the Mann–Whitney U test. Differences were considered significant at a level of P < 0.05. All analyses were performed using StatView software version 5.0J (SAS Institute Inc., Cary, NC). RESULTS Neither β-carotene nor lutein was detected in the plasma and liver of mice before the administration of micellar carotenoids. After the administration of β-carotene solubilized in the micelles, the β-carotene level in the plasma reached a maximum at 2 h in the PC and LPC groups and at 1 h in the NoPL group (Fig. 1A). The maximal levels in the PC, LPC, and NoPL groups were 2.6 ± 0.2, 36.4 ± 11.3, and 26.3 ± 10.4 nM, respectively. The β-carotene in the plasma then decreased to levels significantly lower than the maximal levels observed at 6 h after administration in all of the groups (P < 0.05). No significant differences were found between the NoPL and LPC groups by two-way ANOVA, whereas the βcarotene level of the PC group was markedly lower than those of the NoPL and lysoPC groups. The AUC of plasma βcarotene was calculated from the curves shown in Figure 1A. The NoPL and LPC groups had similar AUC values, whereas the PC group had an extremely low AUC value (Table 1). Lutein levels in the plasma after administration of micellar lutein are shown in Figure 1B. Levels in the NoPL and PC groups reached maxima of 4.75 and 4.30 nM, respectively, at 1 h after administration, whereas that in the LPC group reached TABLE 1 Area Under the Curve for β-Carotene and Lutein Levels in the Plasma and Liver of Mice over 9 h After Administration of Carotenoids Solubilized in Mixed Micellesa β-Carotene
Lutein
Group
Plasma (pmol/mL·h)
Liver (pmol/g.·h)
Plasma (pmol/mL·h)
Liver (pmol/g·h)
NoPL PC LPC
100.3 6.2 119.5
505.3 ND 467.7
26.8 15.6 26.6
109.5 78.4 148.4
a
NoPL, group fed mixed micelles containing no phospholipid; PC, group fed mixed micelles containing PC; LPC, group fed mixed micelles containing lysophosphatidylcholine; ND, not detected.
FIG. 1. β-Carotene and lutein levels in the plasma of mice after the administration of carotenoids solubilized in mixed micelles. Micelles were composed of 2.5 mM monooleoylglycerol, 7.5 mM oleic acid, 12 mM sodium taurocholate, and 200 µM β-carotene (A) or lutein (B) with 3 mM PC (▲ ▲ ), lysophosphatidylcholine (lysoPC) (● ● ), or no phospholipid (▲). Mice were fed a single dose of micelles (0.2 mL) and then sacrificed after various time intervals. β-Carotene and lutein in the plasma were analyzed by HPLC. Data represent the mean ± SD (n = 5). The values at each time point not sharing a common letter are significantly different (P < 0.05) between groups as determined by one-way ANOVA and Tukey’s test after log transformation. β-Carotene and lutein at zero hour, β-carotene at 6 and 9 h, and lutein at 9 h in the PC group were not detected (ND). Data on β-carotene in the PC group were not included in statistical analyses.
a maximum of 9.99 nM at 2 h. The average levels of lutein at 1 to 6 h after administration were significantly different (P < 0.05) among the three groups by two-way ANOVA. The average level of plasma lutein in the PC group (2.72 pmol/mL) during that time was significantly lower (P < 0.05) than those in the NoPL and LPC groups (3.64 and 4.80 pmol/mL, respectively). The AUC value of lutein in the PC group was lower than those in the NoPL and LPC groups, consistent with the average levels. The average level of lutein in the LPC group was not significantly different from that in the NoPL group, and the AUC value of the LPC group was the same as that of the NoPL group. However, the lutein in the LPC group reached the maximum, which was significantly higher than those of the NoPL Lipids, Vol. 38, no. 7 (2003)
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and PC groups at 2 h after administration (P < 0.05). The levels of β-carotene in the plasma at 1 and 2 h after administration were significantly higher than those of lutein in both the NoPL and LPC groups (P < 0.05). Consequently, the AUC value of β-carotene was approximately four times the value of lutein in the NoPL and LPC groups. The response of plasma retinyl palmitate to β-carotene administration is shown in Figure 2. No significant difference was found among the three groups by two-way ANOVA. In both the PC and NoPL groups, no significant difference was observed between time points. On the other hand, the plasma retinyl palmitate in the LPC group was significantly (P < 0.05) increased from the baseline level (24.7 ± 10.7 nM) to 65.8 ± 27.7 nM at 3 h after administration. There was no significant difference in the levels of free retinol in the plasma among the treatment groups (data not shown). β-Carotene levels in the liver after the administration of micellar β-carotene are shown in Figure 3A. No β-carotene was detected in the liver of the PC group at any time point (detection limit, 0.13 pmol/g liver). On the other hand, βcarotene levels in the liver of the LPC and NoPL groups reached maxima at 3 h after administration. The maximal levels in the NoPL and LPC groups were 79.4 ± 33.9 and 90.1 ± 44.6 pmol/g, respectively. The levels then decreased significantly at 9 h after administration (P < 0.05). No significant difference in the average levels of β-carotene was found between the LPC and NoPL groups by two-way ANOVA. The AUC value of liver β-carotene in the NoPL group was similar to that in the LPC group. Thus, the effects of micelles on the accumulation of β-carotene in the liver were similar to those observed in plasma. Lutein levels in the liver after the administration of micellar lutein are shown in Figure 3B. They reached maxima at
FIG. 2. Retinyl palmitate level in the plasma of mice after the administration of β-carotene solubilized in mixed micelles. Mice were fed micelles containing β-carotene and treated as described in Figure 1. Retinyl palmitate in the plasma was analyzed by HPLC. Data represent the mean ± SD (n = 5). Values not sharing a common letter in the lysoPC group are significantly different (P < 0.05) among time points as determined by one-way ANOVA and Tukey’s test. PC, ▲ ; lysoPC, ● ; no phospholipid, ▲. For abbreviation see Figure 1.
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FIG. 3. β-Carotene and lutein levels in the liver of mice after administration of carotenoids solubilized in mixed micelles. Mice were fed micelles containing β-carotene (A) or lutein (B) and treated as described in Figure 1. β-Carotene and lutein in the liver were analyzed by HPLC. Data represent the mean ± SD (n = 5). The values at each time point not sharing a common letter are significantly different (P < 0.05) among groups as determined by one-way ANOVA and Tukey’s test. β-Carotene and lutein at zero hour and β-carotene in the PC group at any of the time points were not detected (ND). PC, ▲ ; lysoPC, ● ; no phospholipid, ▲. For abbreviation see Figure 1.
2 h after administration. The maximal levels in the NoPL, PC, and LPC groups were 16.9 ± 2.3, 18.0 ± 4.7, and 21.1 ± 2.3 pmol/g, respectively, without any significant difference among the groups. After reaching the maximal level, the lutein level of the LPC group remained elevated, whereas those of the other two groups decreased significantly by 3 h after administration (P < 0.05). The average values of lutein after administration in the NoPL, PC, and LPC groups were 13.05, 9.88, and 17.16 pmol/g, respectively, and were significantly different between the groups (P < 0.05, by two-way ANOVA). Consistent with the average values, the AUC values of liver lutein could be ordered by group as follows: LPC > NoPL > PC. The β-carotene levels in the liver were significantly higher than those of lutein at the time points from 1 to 6 h after administration in the NoPL and LPC groups (P <
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0.05), except for the 2-h point in the LPC group. As observed in plasma, the AUC value of β-carotene in the liver was about four times the value of lutein, except in the PC group. DISCUSSION The present study was conducted to elucidate the in vivo effects of phospholipids in mixed micelles on the intestinal uptake of carotenoids by monitoring the appearance of carotenoids in plasma and liver after feeding carotenoids and phospholipids to mice. To eliminate the uncertain solubilization state of carotenoids, the carotenoids were solubilized in the respective mixed micelles, and mice were then intubated with the micelles, instead of feeding diets mixed with carotenoids and phospholipids. β-Carotene levels in the plasma and liver after oral administration were not significantly different between the NoPL and LPC groups, but were markedly lower in the PC group. The AUC values and the average levels of lutein both in plasma and in liver were lower in the PC group than in the NoPL group. Thus, the results indicated clearly that PC in the mixed micelles suppressed the accumulation of lutein as well as β-carotene. The higher average level of lutein in the liver and its AUC value in the LPC group compared with those in the other two groups indicated that lysoPC enhanced accumulation of lutein in the liver. The lutein level in the plasma of the LPC group at 2 h after administration was higher than those of the other two groups, whereas the average level of plasma lutein was not significantly different between the NoPL and LPC groups. These results suggest that the lutein accumulated quickly in the liver, although its appearance in plasma was temporarily enhanced by lysoPC. The enhanced accumulation of β-carotene by lysoPC was not clearly observed from the data on β-carotene in the plasma and liver. However, plasma retinyl palmitate in the LPC group increased significantly to a level higher than the baseline level at 2 h after administration. The increase in retinyl palmitate might be due to the enhanced uptake of βcarotene in the intestinal cells, where β-carotene was converted to retinyl ester. The enhancement in the level of retinyl palmitate in the plasma was estimated to be 41.1 nM. This value corresponded to ca. 20 nM β-carotene, on the assumption that one molecule of β-carotene was converted to two molecules of retinal by the central cleavage enzyme in the intestinal cells (28). As the maximal β-carotene in the plasma of the LPC group was 36.4 nM, a considerable amount of βcarotene might have been converted to retinyl palmitate. However, it was not clear from the data on plasma retinyl palmitate whether lysoPC enhanced the uptake of β-carotene, because no significant differences in retinyl palmitate levels in the plasma were found between the groups. In particular, the levels of retinyl palmitate in the PC group were not significantly different from those of the other two groups, although the intestinal uptake of β-carotene was remarkably suppressed in the PC group. The large variance in the background level of retinyl palmitate made it difficult to evaluate the postprandial increase in retinyl palmitate. Taken together,
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these results suggest that PC suppresses the intestinal uptake of both β-carotene and lutein, whereas lysoPC enhances the uptake of lutein. However, the reason lysoPC had no effect on the level of β-carotene remains to be clarified. It is not certain whether the mixed micelles fed by direct intubation to the stomach in the present study reached the intestinal tract or were reconstituted as micelles in the intestinal tract after disintegrating in the stomach. Nonetheless, the results of the present study were basically consistent with those of our previous study, in which the carotenoids solubilized in micelles were directly incubated with cultured human intestinal Caco-2 cells. Moreover, in the present study, the accumulation of micellar lutein was lower than that of β-carotene in both the plasma and liver except in the PC group, even though a significant amount of β-carotene might have been converted to vitamin A. This result was consistent with the previous finding in which a linear relationship between the uptake of carotenoids solubilized in lysoPC micelles and their hydrophobicity was observed in Caco-2 cells. The ratio between the AUC values of β-carotene and lutein was approximately 4 in both the plasma and liver except in the PC group. This finding indicates that there was no discrimination between β-carotene and lutein in the incorporation from the plasma to the liver once the carotenoids were absorbed. As the incorporation of carotenoids into the liver is mediated through a chylomicron remnant, whole carotenoids present in chylomicron would be incorporated into the liver (29). In contrast, micellar carotenoids would be discriminated in the uptake by intestinal cells, and some part of the βcarotene might be converted to retinyl ester in the cells. The decline in carotenoid levels in the plasma and liver after reaching maxima would reflect the distribution of carotenoids to other tissues. As the liver has the second-highest activity of β-carotene dioxygenase among the tissues (30), conversion of β-carotene to vitamin A may be involved in the decline of β-carotene levels in the liver. The results of the present study and the previous study with Caco-2 cells suggest that the hydrolysis of phospholipids in the intestinal tract by phospholipase A2 is required for the efficient uptake of carotenoids into intestinal cells, although PC plays an important role in the solubilization of carotenoids in lipid emulsions (31). The mechanism underlying these effects is not yet fully understood. PC, with two long-chain acyl moieties, is more hydrophobic than lysoPC, with one acyl moiety and a free hydroxyl group. Therefore, PC has a greater affinity for hydrophobic carotenoid molecules than does lysoPC (32). The uptake of PC itself by intestinal cells is known to be much lower than that of lysoPC (23,24). Thus, PC can strongly retain the carotenoid in mixed micelles so that the uptake of carotenoid to intestinal cells is suppressed. LysoPC can be taken up by the cells of the jejunum across the unstirred water layer, whereas bile acids are taken up later in the ileum. LysoPC might associate with the carotenoid and facilitate its diffusion across the water layer from micelles to the brush border membrane of intestinal cells. Moreover, the lysoPC taken up into the intestinal cells is quickly converted Lipids, Vol. 38, no. 7 (2003)
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to PC and TAG, which then stimulates the synthesis of TAG and the secretion of chylomicron (33–35). Therefore, the increased cellular level of lipids and their secretion into the lymph may shift the equilibrium of the carotenoid partition from the micelles toward the cells and lymph. The in vivo effects of phospholipids on carotenoid uptake observed in the present study were similar to those found on the uptake of cholesterol and α-tocopherol (23–25). The intestinal uptake of such highly hydrophobic substances may partly follow an identical mechanism. These properties of phospholipids would make it possible to modify the bioavailability of the hydrophobic substances with diets and supplements rich in phospholipids. Since carotenoids were fed to mice after direct solubilization in mixed micelles in the present study, it is uncertain whether phospholipids present in foods can influence the bioavailability of dietary carotenoids. However, the amount of phospholipids fed to mice in the present study was ca. 19 µmol/kg body weight, which was comparable to the daily ingestion of dietary PC (0.91–1.85 mmol) in the Western diet (36). Moreover, dietary supplementation with PC was reported to decrease cholesterol absorption in the human intestine (37). Therefore, dietary phospholipids have high potential to modify the bioavailability of carotenoids. The present study is the first report to indicate that PC in mixed micelles suppresses the accumulation of carotenoids in mouse plasma and liver while lysoPC enhances the accumulation of lutein in liver. Thus, our earlier in vitro results and the present in vivo results suggest that the hydrolysis of PC to lysoPC plays an important role in the intestinal uptake of carotenoids solubilized in the mixed micelles and that dietary phospholipids modify the bioavailability of carotenoids. The mechanism of these effects of phospholipids in mixed micelles on the intestinal uptake of carotenoids and, in particular, its relationship to intestinal lipid metabolism, deserve further study. ACKNOWLEDGMENTS This work was supported in part by the Kirin Co. Ltd. Fellowship Program of the United Nations University, by the Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the MAFF Nanotechnology Project.
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[Received December 16, 2002, and in revised form and accepted June 20, 2003]
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