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Nutrient Metabolism

Assessment of Lutein Bioavailability from Meals and a Supplement Using Simulated Digestion and Caco-2 Human Intestinal Cells^1,2

Chureeporn Chitchumroonchokchai^*, Steven J. Schwartz^*, and Mark L. Failla^*,**,3

^* Interdisciplinary Ph.D. Program in Nutrition Department of Food Science and Technology and ^** Department of Human Nutrition, The Ohio State University, Columbus, OH

³To whom correspondence should be addressed. E-mail: failla.3{at}osu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Lutein and zeaxanthin are selectively accumulated in the lens and macular region of the retina. It was suggested that these xanthophylls protect ocular tissues from free-radical damage that can cause cataracts and age-related macular degeneration. Insights regarding the absorption of dietary xanthophylls for delivery to ocular tissues are limited. Our primary objective was to examine factors affecting the transfer of lutein from foods to absorptive intestinal epithelial cells during digestion. Lutein and other carotenoids present in spinach purée and lutein from a commercial supplement were relatively stable during in vitro digestion. Micellarization of lutein and zeaxanthin during the small intestinal phase of digestion exceeded that of ß-carotene and was greater for xanthophylls in oil-based supplements than in spinach. Apical uptake of lutein from micelles by Caco-2 human intestinal cells was linear for at least 8 h, and accumulation from synthetic micelles exceeded that from micelles generated during simulated digestion. Stimulation of chylomicron synthesis resulted in the secretion of 7.6 ± 0.1% of cellular lutein into the triglyceride-rich fraction in the basolateral chamber. These data support the use of simulated digestion and the Caco-2 cell model as effective tools for identifying factors affecting absorption of dietary carotenoids.

KEY WORDS: • lutein • bioavailability • in vitro digestion • micelles • Caco-2 human intestinal cells

The dihydroxy-xanthophylls lutein and zeaxanthin are the predominant carotenoids that accumulate in the lens and the macular region of the retina (1,2). Because lutein and zeaxanthin efficiently absorb blue light and quench photochemically induced singlet oxygen, it was proposed that these pigments protect the lens and macula from insults that can induce development of cataracts and age-related macular degeneration (3–5). Accumulation of xanthophylls and other bioactive compounds from foods and supplements in peripheral tissues depends on intestinal absorption and first-pass metabolism by the intestinal epithelium and/or liver. Available data clearly demonstrate that carotenoid bioavailability is affected by numerous factors, including their physiochemical properties, the food matrix and processing, a variety of dietary components, nutritional status, gut health, and genotype (6,7). Thus, reliable prediction of their bioavailability from foods and meals remains problematic. Accurate assessment of carotenoid absorption from a single test food or meal requires isotopic tracer techniques (8,9). Carotenoid absorption also can be estimated by measuring the concentration of carotenoids and their metabolites in triglyceride-rich fractions of plasma at various times after ingestion of the test dose (10,11). However, these methods are labor-intensive and require sophisticated instrumentation.

We previously reported the development of an in vitro method for examining the digestive stability and micellarization of carotenoids and chlorophylls during simulated digestion of foods and meals (12–14). Caco-2 human intestinal cells were shown to accumulate carotenoids and chlorophyll derivatives from medium containing micelles generated during in vitro digestion. Recent studies (15–17) demonstrated that Caco-2 human intestinal cells are capable of converting ß-carotene to retinol and secrete ß-carotene, retinol, and retinyl esters. Moreover, secretion of all-trans-ß- and -carotene exceeded that of lutein, 9-cis-ß-carotene, and lycopene after their delivery to cells in Tween micelles (17). Other simple models have been used to define factors that affect the transfer of carotenoids from the food matrix to oil droplets and subsequent transfer into mixed micelles (18–21). Thus, in vitro methods are providing important insights about the effect of gastrointestinal processes on carotenoid bioavailability.

The present study extends previous work by comparing micellarization of lutein and other carotenoids in spinach with a commercially available xanthophyll supplement during simulated digestion and their subsequent uptake and transport by Caco-2 human intestinal cells. In addition, we examined the micellar and intracellular stability and the secretion of lutein by Caco-2 cells to gain a better understanding of preabsorptive events as they relate to the bioavailability of this common dietary carotenoid.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Chemicals and standards. Unless stated otherwise, all reagents and materials were purchased from Sigma Chemical and Fisher Scientific. ³H-retinol (specific activity 2072 GBq/mmol) was purchased from Perkin-Elmer. Purified all-trans-lutein for use as a standard was a generous gift from Dr. Zoraida DeFreitas, Kemin Foods.

    Preparation of test foods for in vitro digestion. Fresh spinach (Spinacia oleracea) was purchased at a local supermarket. After the stems and ribs were removed, the leaves were washed with tap water, rinsed with deionized water, and drained. Spinach leaves were washed, weighed (125 g) and microwaved (750 W) for 3 min in the presence of deionized water (50 mL). Microwaving at moderate intensity was shown to destroy plant cell integrity without altering the carotenoid content and profile of spinach (22). The sample was cooled and homogenized with a kitchen blender (Osterizer Galaxie) for 4 min to produce a purée. Homogenized spinach was stored at –80°C under a blanket of nitrogen for use within 4 wk. The profile of the carotenoids was quantitatively and qualitatively stable during this period.

Soft-gel capsules containing 20 mg free lutein in corn oil were purchased from Vitamin World. The contents of the capsule were diluted 30-fold with corn oil. Lutein-rich corn oil was then diluted 50-fold in 25 g/L skim milk and homogenized (Ultra Turrax) on ice at 13,500 rpm for 7 min to prepare a stable emulsion (23).

    In vitro digestion. Details of the procedure for simulating the gastric and small intestinal phases of digestion were described previously (12). Digestion reactions (50 mL total volume) contained either 3.57 g pureed spinach with 75 mg virgin olive oil or 100 mg lutein-rich corn oil emulsified in 5 mL 25 g/L skim milk. The starting quantities of lutein in the spinach and supplement samples were 210 ± 4 and 80.6 ± 1 µg all-trans-lutein, respectively. Use of different quantities of lutein for digestion of the 2 test foods was based in part on the expectation that the extent of micellarization of lutein from the emulsified oil would exceed that from the more complex matrix of spinach. In addition, we were interested in comparing the digestive stability and micellarization of lutein with several of the less abundant carotenoids in spinach.

Previously, porcine bile extract at a final concentration of 2.4 g/L was used during the small intestinal phase of digestion (12–14). Bile salts represent 50% of this crude material (24), resulting in an estimated concentration of 2.5 mmol/L per reaction tube. Pilot studies were performed to evaluate replacement of the bile extract with one or more pure bile salts on micellarization of carotenoids during simulated digestion of spinach. Results showed that the efficiency of micellarization increased from 27 to 49% (P < 0.001) when a mixture providing final concentrations of 0.80 mmol/L glycodeoxycholate (GDC),⁴ 0.45 mmol/L taurodeoxycholate (TDC), and 0.75 mmol/L taurocholate (TC) was substituted for the bile extract. These 3 bile acids are among the most abundant in human bile (25) and were used in all studies described below.

After completion of the simulated digestion, the product is referred to as the "digesta." The digesta was centrifuged (Ti 50 rotor, Beckman Model L7–65) at 167,000 x g at 4°C for 35 min to separate the aqueous fraction containing mixed micelles from residual solids and oil droplets. The aqueous fraction was filter sterilized (cellulose acetate, 0.22-µm pore size; Gelman Science) to remove microcrystalline carotenoid aggregates and microbial contamination. Homogenized food, the digesta and the filtered aqueous fraction were stored at –80°C under nitrogen and analyzed within 1 wk. The aqueous fraction used as source of micellarized carotenoids was added directly to DMEM medium (see below).

    Preparation of synthetic micelles containing lutein. Preparation of synthetic micelles was based on several in vitro studies (19,26,27). Aliquots of stock solutions of monoolein (MO), phosphatidylcholine (PC), and lyso-phosphatidylcholine (Lyso-PC) in chloroform, and -tocopherol and all-trans-lutein in ethanol were combined in a 35-mL glass vial. Solvents were removed under a stream of nitrogen at room temperature. A basal medium containing 0.8 mmol/L GDC, 0.45 mmol/L TDC, 0.75 mmol/L TC, and 1.5 mmol/L sodium oleate (OA) was added to the vial and the mixture was sonicated in a bath at room temperature for 30 min. The solution was filter sterilized (0.22-µm pores) to remove insoluble lutein and microbial contamination. Final concentrations of lipids were 1.5 mmol/L OA, 500 µmol/L MO, 200 µmol/L PC, 200 µmol/L lyso-PC, 10 µmol/L -tocopherol, and 1 µmol/L lutein.

    Uptake and secretion of lutein by Caco-2 human intestinal cells. Stock and test cultures of Caco-2 cells (HTB37, American Type Culture Collection; passages 26–34) were maintained as previously described (12–14). The uptake of lutein from natural and synthetic micelles was studied using cultures at 11–14 d postconfluency. The serum [fetal bovine serum (FBS)] content of the medium was decreased to 75 mL/L once cultures reached confluency.

To characterize uptake of micellarized lutein, spent medium was removed and monolayers were washed twice with basal medium at 37°C. DMEM containing 250 mL/L of either an aqueous fraction from simulated digestion (referred to as natural micelles) or synthetic micelles was added to wells containing the washed monolayers (2 mL/well). At indicated times, the medium was removed and monolayers were washed twice with ice-cold PBS containing albumin (2 g/L) to remove residual lutein adhering to the trans face of the cell surface (28) before washing twice with cold PBS. Cells were stored under nitrogen at –80°C for a maximum of 1 wk before analysis. Exposure of monolayers to micellar medium as described above does not adversely affect general cell morphology or metabolic integrity (12).

To examine the secretion of lutein accumulated by Caco-2 cells, inserts in 6-well dishes (24-mm diameter, 0.4-µm pore size) were seeded with 3.0 x 10⁵ cells. Cultures were used for experiments 21–25 d after reaching confluency because lipoprotein synthesis and secretion by Caco-2 cells are maximal at this time (29). DMEM (1.5 mL) containing 2.0 µmol/L lutein in synthetic micelles was added to the apical chamber, and DMEM (2.5 mL) containing 10 mL/L delipidated FBS was added to the basolateral chamber. The apical medium was removed after 6 h, and monolayers were washed twice with warm PBS containing albumin (2 g/L). Either DMEM (1.5 mL) containing 0.5 mmol/L phenol red or DMEM with 0.5 mmol/L phenol red plus 1.6 mmol/L OA, 0.5 mmol/L TC, and 0.05 mmol/L glycerol was added to the apical chamber. The addition of OA, TC, and glycerol to the apical chamber stimulates synthesis and secretion of chylomicrons (30). Fresh phenol red–free DMEM with 10 mL/L delipidated FBS was added to the basolateral chamber. After incubation for 20 h, the media in the apical and basolateral chambers and washed monolayers were collected and stored at –80°C for no longer than 1 wk.

Distribution of secreted lutein in the basolateral compartment was also determined using a modification of the procedure described by During et al. (17). Filtered DMEM (1.5 mL) with 0.5 mmol/L phenol red, synthetic micelles containing 2 µmol/L and 74 kBq ³H-retinol, 1.6 mmol/L OA, and 0.5 mmol/L TC was added to the apical compartment. The ³H-retinol tracer was included to confirm the synthesis of retinyl ester and its incorporation and secretion into a triglyceride-rich lipoprotein (TRL) fraction (see below). The basolateral compartment contained phenol red–free DMEM (2.5 mL) with 10 mL/L delipidated FBS. After continuous exposure of the monolayer to micellar lutein for 20 h, media from both compartments and washed cells were collected. The TRL fraction in the basolateral medium was isolated according to van Vliet et al. (31). ³H-retinol and ³H-retinyl esters in cells, basolateral medium, and the TRL fraction were separated using alumina column chromatography (16). To measure ³H, aliquots were solubilized in ScintiVerse (Fisher Scientific) and analyzed by liquid scintillation spectrometry (Beckman LS Model 3801).

The hourly mean rate of phenol red flux from the apical to the basolateral compartment was 0.011 ± 0.003%/cm² and was independent of the composition of the medium in the apical chamber, demonstrating that monolayer integrity was not adversely affected by the various treatments.

    Extraction and analysis of carotenoids in test meals, media and cell pellet. Thawed samples (1–3 mL) of homogenized food, digesta, and the aqueous fraction were extracted by adding 3.0 mL petroleum ether:acetone (2:1) containing 4.5 mmol/L BHT, mixing on a vortex mixer for 1 min, and centrifuging (2000 x g for 5 min) to hasten phase separation. Thawed samples of apical and basolateral medium, and fractions collected after ultracentrifugation of the basolateral medium were processed similarly. The extraction procedure was repeated a total of 3 times and petroleum ether fractions were combined and dried at room temperature under a stream of nitrogen. The film was resolubilized in methyl-tert-butyl-ether (MTBE):methanol (MeOH) (1:1) and analyzed immediately.

Cell pellets were thawed before the addition of 10 g/L protease from Streptomyces griseus in PBS. After incubation at 37°C for 30 min, 0.5 mL of EtOH containing 34.6 mmol/L SDS and 4.5 mmol/L BHT was added before mixing on a vortex mixer for 1 min. After the addition of 1 mL petroleum ether:acetone (2:1), samples were mixed and centrifuged as above. The extraction was repeated a total of 3 times and pooled fractions of petroleum ether were dried under nitrogen at room temperature and resolubilized in MTBE:MeOH (1:1).

Carotenoids were quantified by HPLC according to Ferruzzi et al. (32). Confirmation of the identity of all-trans-carotenoids and their geometrical isomers was achieved by UV-visible absorbance spectral analysis using a photodiode array detector (Waters Model 996) and comparison of retention times with previous separations using a C₃₀ column (33,34)

    Miscellaneous assays. The protein content of cell samples was determined by the bicinchoninic acid assay (Pierce) using bovine serum albumin as a standard. The diffusion of phenol red across the monolayer from the apical to the basolateral compartment was determined by measuring absorbance at 546 nm 15 s after the addition of 20 µL of 1 mol/L NaOH to 150 µL basolateral medium at room temperature (35).

    Statistical analysis of data. All data were analyzed using Stata 8.0. Descriptive statistics including mean and SD were calculated for the efficiency of micellarization of carotenoids from digested foods, the stability of micellarized carotenoids in cell culture medium, and the uptake and secretion of carotenoids by Caco-2 cells. Means were compared using one-way ANOVA followed by Tukey’s test or the Bonferroni correction. Differences were considered significant at P < 0.05. All tests were conducted in triplicate and each experiment was repeated at least once to provide a minimum of 6 independent observations.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Composition of test materials. Spinach contained 75 mg carotenoids/kg fresh weight with all-trans-lutein and ß-carotene representing 94% of the total (Table 1). Small amounts of 13-cis-lutein, 9-cis-ß-carotene, and all-trans-zeaxanthin were detected. All-trans-lutein accounted for 95% of carotenoids in the supplement with the remainder detected as all-trans-zeaxanthin (Table 1).

View larger version (32K): [in this window] [in a new window]   FIGURE 1 Recovery (panel A) and efficiency of micellarization (panel B) of carotenoids in spinach and a lutein supplement after simulated digestion. Spinach purée and an aliquot of the contents of the soft-gel capsule were prepared and digested in vitro. Carotenoids were extracted from test food, digesta, and aqueous fraction and analyzed by HPLC. Digestive stability represents the percentage of carotenoid recovered after simulated gastric and small intestinal digestion. The efficiency of micellarization represents the percentage of the carotenoid in the test food that was transferred to the filtered aqueous fraction during simulated digestion. Data are means ± SD, n = 3 independent experiments each having 3 replicate digestion reactions per treatment. Within a panel, means that do not share a common letter above the error bar differ, P < 0.01.

View larger version (23K): [in this window] [in a new window]   FIGURE 2 Effect of the length of incubation and source of micelles on lutein accumulation by Caco-2 cells. Differentiated monolayers of Caco-2 cells were incubated with 2 mL of medium containing micelles with 1.0 ± 0.1 µmol/L lutein for the times indicated before determination of cellular lutein content. Micelles were either generated during simulated digestion of spinach or a supplement or prepared synthetically. Data (means ± SD) are representative of 3 independent experiments with 3–4 replicate samples per experiment, n = 9–12. Means that do not share a common letter above the error bar differ (P < 0.01) between incubation time and treatment groups.

Cellular accumulation of micellarized all-trans-ß-carotene from digested spinach was similar to that of all-trans-lutein with 16 and 31% of all-trans-ß-carotene initially in medium present in cells after 4 and 20 h of exposure, respectively. Cell all-trans-ß-carotene content was not altered by overnight incubation in medium without carotenoids.

    Stability of micellar carotenoids. The concentrations of all-trans-lutein, 13-cis-lutein, all-trans-zeaxanthin and all-trans-ß-carotene in medium containing micelles prepared by digestion of spinach were not affected by 4 h of incubation in cell-free dishes. However, all-trans-lutein decreased by 19 ± 2% (P < 0.05) after 20 h. This decline was associated with a slight (16%), but significant (P < 0.05), increase in the concentration of 13-cis-lutein, suggesting some conversion of all-trans to the cis isomer. Recovery of all-trans- and 9-cis-ß-carotene at 20 h was 99% (P > 0.05) and 91% (P > 0.05), respectively. Micellarized all-trans-lutein from digested lutein supplement was less stable than that from spinach meals. The concentration of all-trans-lutein declined linearly with 75% remaining after 16 h of incubation in the cell-free environment (P < 0.01). The losses were associated with the appearance of an equivalent quantity of 13-cis-lutein. In contrast to the partial isomerization of all-trans-lutein in natural micelles in the cell culture environment, recovery of all-trans-lutein from medium containing synthetic micelles exceeded 95% after 20 h.

The mean concentration of -tocopherol present in the medium containing synthetic micelles (10.0 ± 0.2 µmol/L) was higher (P < 0.001) than that in medium containing 250 mL/L aqueous fraction from either digested spinach (0.3 ± 0.1 µmol/L) or milk with emulsified lutein supplement (0.1 ± 0.04 µmol/L). This higher concentration of -tocopherol may have prevented degradation and isomerization of lutein in the synthetic micelles.

    Secretion of cellular lutein. Synthetic micelles served as vehicles for loading cells with lutein to investigate secretion into the basolateral compartment. Medium with micellar lutein (3 nmol) was added to the apical compartment for 6 h to facilitate cellular accumulation of the carotenoid. Cultures were then incubated overnight in medium without added carotenoid to determine retention and secretion of lutein. Caco cells retained 99 ± 2% of accumulated lutein when incubated in DMEM medium with 10 mL/L delipidated FBS for 20 h. The addition of 1.6 mmol/L OA, 0.5 mmol/L TC, and 0.05 mmol/L glycerol to the apical compartment to stimulate chylomicron synthesis and secretion resulted in the transfer of 6.3 ± 0.4% of the cellular lutein to the basolateral compartment after 20 h. Lutein was not detected in the apical compartment.

To examine the distribution of lutein within the basolateral fraction, medium containing synthetic micelles with 4.3 nmol lutein plus OA, TC, glycerol, and ³H-retinol was added to the apical chamber. ³H-retinol tracer was included to confirm the presence of retinyl esters in the TRL fraction (16). After 20 h, 1.58 and 0.13 nmol lutein were present in the cells and basolateral chamber, respectively, i.e., 7.6 ± 0.1% of the lutein accumulated by cells was transferred to the basolateral compartment (Table 2). Similarly, 7.6 ± 0.5% of ³H accumulated by the cells was transferred to the basolateral compartment. Retinyl esters accounted for 60% of ³H in both the cells and the basolateral chamber. After centrifugation of the basolateral medium, all lutein (98% recovery) and 65% of ³H were present in the TRL fraction. Chromatographic analysis of ³H showed that retinyl ester and retinol accounted for >95% of ³H in TRL and other fractions, respectively (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The quantity of ingested carotenoid absorbed is dependent on its partitioning in micelles, delivery to enterocytes, and incorporation into chylomicrons (38). Results from human studies showed that carotenoids in oil-based supplements are absorbed more efficiently than from foods (39–41). Our demonstration that lutein in the soybean oil supplement was micellarized more efficiently than that in spinach offers a possible explanation for the increased absorption of lutein from supplements. Several human studies also suggested that apparent absorption of lutein is more efficient than that of ß-carotene. Van het Hof et al. (42) reported greater relative changes in plasma lutein than ß-carotene after feeding a high vegetable diet for several weeks compared with responses to pure carotenoid supplements. Similarly, serum lutein concentrations increased to a greater extent than ß-carotene after subjects consumed spinach products for 3 wk (40). Also, Gartner et al. (43) reported that the efficiency of absorption of lutein and zeaxanthin into the triglyceride-rich fraction exceeded that of ß-carotene after a single dose of Betatene, an algal extract in soybean oil enriched in ß-carotene. We speculate that the more efficient micellarization of lutein than ß-carotene from foods during digestion contributed to the above observations in human studies.

Many factors affect the micellarization of carotenoids during digestion. The higher efficiency of micellarization of lutein and zeaxanthin from the emulsified oil supplement compared with spinach is likely due to the absence of factors limiting transfer from chloroplasts to oil droplets (20). Observed differences between micellarization of the xanthophylls and ß-carotene from spinach are likely related to factors affecting transfer efficiency from the food matrix to the oil droplet and subsequent flux to the mixed micelle. Tyssandier et al. (19) observed that the transfer of carotenoids from oil droplets to mixed micelles is inversely proportional to the hydrophobicity of the pigments. Carotenes and lycopene are located deep within the lipid droplet, whereas xanthophylls reside at the surface and are kinetically active (18). Moreover, transfer of ß-carotene from the oil droplet to the mixed micelle was impaired when lutein was also present in the droplet (19). Lutein in disrupted spinach chloroplasts also has the potential to be transferred directly to micelles without the need for oil droplets to serve as an intermediate reservoir (21). Recently, Tyssandier et al. (44) examined carotenoid stability and micellarization after delivery of a liquefied meal containing vegetable purée by nasogastric tube. The relative quantity of lutein in micelles in the duodenum during the 3-h sampling period (5.6%) was higher than that of ß-carotene (4.7%). Although the percentages of micellarized lutein and ß-carotene in duodenum were well below those we observed in vitro, it is important to recognize that carotenoids are delivered to the brush border surface of enterocytes once micellarized within the small intestine. In contrast, carotenoids continue to accumulate in micelles in the in vitro digestion model because they are not delivered to target cells.

In the current study, spinach was processed and the bile composition modified (see Materials and Methods) to increase carotenoid content in micelles to facilitate examination of uptake by and stability within Caco-2 cells. Spinach was microwaved and puréed to destroy tissue structure and increase surface area (40,45). Micellarization of lutein and other carotenoids in spinach was almost doubled when several of the more abundant bile acids in humans (25) were substituted for crude bile extract. Previous investigators also reported effects of various bile salts on micellarization and intestinal uptake of ß-carotene (46,47). Cell accumulation of lutein and ß-carotene from micelles generated during digestion of spinach was similar to that previously reported (12,14). In contrast, Sugawara et al. (27) reported that apical uptake of carotenoids from synthetic micelles is proportional to the hydrophobicity of the carotenoid. Differences in the composition of the micelles may contribute to this discrepancy

Lutein accumulation by Caco-2 cells was greater when the cells were exposed to synthetic micelles instead of micelles generated during digestion of spinach or a supplement. The different composition of natural vs. synthetic micelles may affect particle size and surface charge and hence the ability to interact with the brush border surface (38). Although it is possible that the presence of other carotenoids in the micelle affected apical uptake (48), this does not appear to contribute to the observed difference because micelles generated from digestion of the lutein supplement contained very low amounts of zeaxanthin. Phospholipid composition represents another possible factor. Lyso-PC was shown to stimulate lutein and ß-carotene uptake from micelles by Caco-2 cells and absorption by mice (27,49). We observed that lutein incorporation into synthetic micelles was markedly increased by the addition of equivalent concentrations of lyso-PC and PC compared with PC only. The effect of the lyso-PC:PC ratio on uptake was not tested because the number of micelles in the apical compartment would have to have been varied to introduce equimolar amounts of lutein. However, analysis of lyso-PC and PC in the micellar fraction after digestion of the spinach revealed that the ratio of lyso-PC and PC was almost identical to that in the synthetic micelles. Thus, increased cellular uptake of lutein from the synthetic micelles was due to factors other than phospholipid composition.

Micelles within the intestinal lumen and lipoproteins in circulation represent the physiologic carriers for carotenoids (38). Investigators reported that carotenoids are degraded when introduced into the cell culture medium with organic solvents, liposomes, and water-dispersible beadlets as the vehicle (26,50,51). We (this study) and others (26) found that carotenoids are relatively stable in medium containing synthetic micelles. However, there was some degradation of lutein in our study when micelles formed during the digestion of spinach were incubated in culture medium (12). Oxidizing compounds in spinach and the relatively low concentration of -tocopherol may have contributed to the loss of some of the micellarized lutein. Isomerization of all-trans-lutein was observed in micelles generated during digestion of the supplement. Durning et al. (17) also reported that all-trans-ß-carotene in Tween 40 micelles was partially isomerized to cis-ß-carotene in the apical compartment of Caco-2 cultures after 16 h. Light, acid, heat, photosensitizers, and oxidants can induce isomerization of carotenoids (52). Because all samples were handled in an identical manner, it is likely that unidentified oxidants in the digested spinach and oil supplements contributed to the partial degradation and isomerization of lutein.

Lutein secretion from Caco-2 cells required the presence of oleate and taurocholate in the apical compartment. The extent of secretion was similar when cells were exposed to micellar lutein either before or at the same time as oleate and taurocholate. Lutein in the basolateral chamber was localized in the TRL fraction, as were retinyl esters. These results are quite similar to those reported by Harrison and associates (16,17), who introduced carotenoids in Tween micelles and retinol in the presence of oleate and taurocholate.

The above in vitro observations provide additional support for the use of model systems of digestion and differentiated intestinal epithelial cells for investigating factors affecting the digestive stability, accessibility, and absorption of bioactive compounds from foods and supplements.

	ACKNOWLEDGMENTS

 
We thank Mario Ferruzzi for helpful conversations and his critical reading of the manuscript and Martha Belury for her suggestions for phospholipid analysis. The authors also thank Dr. Zoraida DeFreitas for the gift of all-trans-lutein.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 03, April 11–15, 2003, San Diego, CA [Chitchumroonchokchai, C., Schwartz, S. J. & Failla, M. L. (2003) Micellarization and uptake of xanthophylls by Caco-2 human intestinal cells. FASEB J. 17: A758 (abs.)].

² Supported by the U.S. Department of Agriculture, Initiative for Future Agriculture and Food Systems (USDA IFAES), The Ohio State Agriculture Research and Development Center (OARDC), and Virginia M. Vivian Endowment Fund (C.C.)

⁴ Abbreviations used: FBS, fetal bovine serum: GC, glycocholate; GDC, glycodeoxycholate; Lyso-PC, lyso-phosphatidylcholine; MO, monoolein; MTBE, methyl-tert-butyl-ether; OA, sodium oleate; PC, phosphatidylcholine; TC, taurocholate; TDC, taurodeoxycholate; TRL, triglyceride-rich lipoprotein.

Manuscript received 23 March 2004. Initial review completed 3 May 2004. Revision accepted 15 June 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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