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,4
* Interdisciplinary Ph.D. Program in Nutrition and Department of Human Nutrition, The Ohio State University, Columbus, OH
4 To whom correspondence should be addressed. E-mail: failla.3{at}osu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bioavailability • carboxyl ester lipase • Caco-2 cells • wolfberry • zeaxanthin
Zeaxanthin (Zea)5 and lutein are the only dietary carotenoids present in the macular region of the retina and the lens (1–3). Some epidemiologic studies showed that the risks of age-related macular degeneration and cataracts are inversely correlated with dietary intake and the concentrations of these xanthophylls in the serum and macula (4,5). It was hypothesized that Zea and lutein contribute to the protection of ocular tissues by absorbing potentially harmful blue light and scavenging free radicals (6,7). This hypothesis is supported by the recent demonstration that supplementation of quail diet with Zea decreased the number of apoptotic photoreceptor cells after intermittent exposure to white light (8). Zea and lutein also dose dependently decreased lipid peroxidation and activation of the stress signaling mitogen-activated protein kinase (MAPK) pathway in cultures of human lens epithelial cells exposed to UVB (9).
The majority of commonly consumed fruits and vegetables contain much more lutein than Zea (10). However, elevated concentrations of Zea and particularly Zea esters were reported in several fruits and vegetables (11,12). For example, wolfberry (WB; Lycium chinense), a small fruit that is used to improve vision in traditional Chinese medicine, contains concentrations of Zea dipalmitate that can approach 1 g/kg wet weight (13). Recently, Breithaupt and associates (14) reported that the postprandial increase in plasma Zea was greater when subjects were fed a meal with Zea dipalmitate compared with the meal with an equivalent amount of free Zea. Similarly, increased and equivalent bioavailability of lutein and cryptoxanthin, respectively, were reported when subjects were fed meals supplemented with xanthophyll esters instead of free xanthophylls (15,16). Free, but not esterified, Zea and cryptoxanthin were present in the plasma of subjects fed meals enriched in either the free or esterified forms of the xanthophylls (14,16). Similarly, Wingerath et al. (17) also reported increases in the concentrations of free cryptoxanthin, Zea, and lutein in chylomicrons and sera from human subjects after they ingested tangerine juice concentrate that was rich in the esterified forms of these xanthophylls. These observations suggest that carotenoid esters are hydrolyzed before incorporation into nascent chylomicrons synthesized in absorptive intestinal epithelial cells. It is unclear whether the dietary carotenoid esters are hydrolyzed in the gastrointestinal lumen during digestion, after their uptake into enterocytes, or perhaps in both sites. We examined these possibilities by subjecting several foods rich in Zea to simulated gastric and small intestinal digestion and incubating Caco-2 human intestinal cells with micelles containing free and esterified Zea.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Preparation of test foods. One intensely colored orange pepper (Capsicum annum), one red pepper (Capsicum annum), and one butternut squash (Cucurbita moschata) with minimum surface damage were purchased from a local market. These vegetables were refrigerated overnight at 4°C before washing with tap water. The edible portions of each pepper were placed in a blender and processed for 1–3 min to prepare a purée. The squash (5 cm3 sections) was boiled in tap water for 15 min. The tender yellow pulp was transferred to the blender and puréed for 1 min. Vacuum-packed and frozen sun-dried wolfberry imported from China was purchased from a local store specializing in Asian products. Dried WB was hydrated in an equal volume of deionized water for at least 1 h before processing for 3 min to prepare a purée. All puréed foods were stored under nitrogen at –80°C.
Although Zea supplements are commercially available, lutein rather than Zea is the most abundant carotenoid in these preparations. Therefore, we prepared an oil enriched in Zea esters for an investigation of the effect of the WB matrix on micellarization. Olive oil (1 mL) was added to a glass vial containing 
2 mg Zea esters purified from WB (see below). The vial was sonicated in a bath for 30 min at 25°C. The mixture was centrifuged (16,000 x g, 4°C, 10 min) to remove Zea esters that were not solubilized. The concentration of Zea compounds in the oil was determined, and samples were stored under nitrogen in the dark at room temperature until used for in vitro digestion.
Purification of Zea esters and free Zea. Puréed WB (10 g) was transferred to a 1 L separatory flask; 200 mL methanol:ethyl acetate:petroleum ether (1:1:1) was added and the mixture was shaken for 5 min. After standing for 2 min, the upper (organic) layer was collected and the lower layer was extracted 3 additional times. Pooled organic layers were passed through a Sep-pak C18 cartridge. The filtrate was transferred to 35-mL vials, dried under a stream of nitrogen, and stored at –80°C as WB extract. Zea mono- and diesters were purified from the WB extract using a preparative YMC C30 column (5-µm particles; 30 mm x 300 mm) as described below. Free Zea was purified similarly after treatment of the WB extract solubilized in ethanol with 30% (wt:v) KOH in methanol at room temperature for 1 h.
Hydrolysis of Zea esters by carboxyl ester lipase (CEL). CEL was shown to hydrolyze esterified astaxanthin, lutein, and ß-cryptoxanthin (18,19). The possibility that Zea esters from WB extract are a substrate for CEL was investigated using physiologically relevant emulsions as described by Tyssandier et al. (20). Zea esters from WB were incorporated into emulsified lipid droplets consisting of triolein and phosphatidylcholine (97.5:2.5). Samples with cholesteryl linoleate instead of Zea esters served as a positive control. Aliquots were added to 154 mmol/L NaCl containing 8 mmol/L bile salts and incubated in the absence or presence of porcine pancreatin, porcine pancreatin plus porcine colipase, bovine CEL (1000 units/L), or a combination of all of these enzymes. The activities of pancreatin and lipase plus colipase in reactions were similar to those used by others (19,20). Mixtures without enzymes served as a control. Mixtures were incubated with shaking at 37°C, and aliquots were removed periodically to quantify hydrolysis of the cholesterol ester by enzyme assay (Stanbio) and Zea ester by HPLC. As expected, there was a time-dependent increase in the concentration of free cholesterol in tubes containing CEL (1000 units/L) or the combination of CEL and the other enzymes. Pancreatin did not hydrolyze cholesteryl linoleate. Data for the cleavage of Zea esters are presented below.
In Vitro digestion. Reactions (50 mL final volume) contained puréed orange pepper (1 g wet weight with 440 nmol Zea equivalents), red pepper (1g with 230 nmol Zea equivalents), squash (1 g with 80 nmol Zea equivalents), WB (350 mg wet weight with 640 nmol Zea equivalents), or olive oil (30 mg) containing Zea esters (580 nmol Zea equivalents). Virgin olive oil (32 mg) was added to reaction tubes containing puréed foods as a source of exogenous fat. Digestion and isolation of the micellar fraction were simulated as described by Chitchumroonchokchai et al. (9), except that 50 units of bovine CEL also was present during the small intestinal phase of digestion once it was determined that the pancreatin lacked this activity (see below). The efficiency of micellarization represents the percentage of Zea in the starting material that partitions in the filtered aqueous fraction after simulated digestion.
Extraction and analysis of xanthophylls.
Xanthophylls were extracted from all samples as described previously (9), with the exception that 2 volumes of methanol:ethyl acetate: petroleum ether (1:1:1, by vol) containing 4.5 mmol/L BHT was used as the organic solvent. Free and esterified xanthophylls were quantified by HPLC as described by Weller and Breithaupt (12) using a Waters 2695 separation module with a 2996 photodiode array detector. Xanthophyll esters were identified by UV-visible absorption spectra and comparison of retention times to separations with a C30 column. The concentration of xanthophylls was calculated from a comparison of the area under the curve with known concentrations of the all-trans-Zea standard. The extinction coefficient, 
, at 450 nm in hexane was 2540 for all-trans and cis-zeaxanthin (21). Cis-isomers of Zea after digestion represented 10% of the total peak area that eluted with the appropriate spectral properties of Zea. The data presented represent the sum of all-trans and cis-Zea. Echinenone was used as an internal standard with recovery ranging from 95 to 103%.
Uptake and secretion of Zea by Caco-2 human intestinal cells. Caco-2 cells (HTB37, American Type Culture Collection; passages 26–28) were used for characterizing the uptake and transport of Zea from mixed micelles generated during simulated digestion and from mixed micelles prepared from chemical reagents. Details are described elsewhere (9). Cell morphology and metabolic integrity (22) were not compromised by exposing the monolayers of differentiated cells to medium containing 25% (v:v) filtered aqueous fraction with micelles generated during simulated digestion of test foods or synthetic micelles containing free and esterified Zea. The triglyceride-rich lipoprotein fraction of the medium in the basolateral compartment was isolated to determine the amount of Zea secreted from cells maintained on porous membrane inserts (9).
Miscellaneous assays. Cell protein was determined by the bicinchoninic acid assay (Pierce) using bovine serum albumin as the standard. The barrier integrity of monolayers grown on inserts for examination of Zea secretion was determined by monitoring the rate of phenol red flux from the apical to the basolateral compartment (22). The flux of phenol red was 0.01 ± 0.003%/(cm2·h) and independent of medium composition in the apical compartment.
Statistical analysis of data.
A minimum of 3 independent observations was made for each group in an experiment and each experiment was repeated at least once to provide a minimum of n = 6 for determination of significant differences, unless stated otherwise. Statistical analysis was performed using SPSS/Win 13.0. Descriptive statistics including mean and SEM were calculated for the digestive stability and efficiency of micellarization of xanthophylls from digested foods. Significant differences were detected by 1-way ANOVA followed by Dunnett's post hoc test or paired t test, as appropriate. Differences were considered significant at P 
0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.1% of the wet weight of WB with lower concentrations of the xanthophyll present in orange pepper, red pepper, and squash (Table 1). The spectrum and retention time for the predominant (95%) carotenoid in WB extract were those of Zea dipalmitate (Fig. 1) as previously described (13). The elution profile of extracts from orange pepper showed the presence of similar quantities of free Zea (peaks 1–3), Zea monoesters (peaks 4–7) and Zea diesters (peaks 8–11) (Fig. 1, Table 1). Although capsanthin esters were the most abundant carotenoids in red pepper (not shown), free Zea and 5 distinct Zea esters were identified. Squash contained relatively low concentrations of both Zea monoesters and lutein monoesters, but no detectable amounts of either the free or diester forms of these xanthophylls.
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30% was hydrolyzed, with 27.1% and 2.7% of the products appearing as free Zea and Zea monoesters, respectively, after 3 h. In contrast, the concentration of free Zea did not increase (P > 0.05) in mixtures with pancreatin and lipase plus colipase. Thus, CEL (1000 units/L), as well as pancreatin and pancreatic lipase, was present during the small intestinal phase in all subsequent digestions.
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10%), but significant (P < 0.05) decrease in the concentration of Zea monoesters in cells exposed to medium with micelles generated during the digestion of WB and squash (Fig. 6). Zea esters from WB also were solubilized in Tween 20 (0.39 µmol Zea equivalents/L) and added to Caco-2 cell homogenates, and the total membrane and cytosol fractions (150,000 x g supernatant) to examine endogenous hydrolytic activity. Of the Zea esters, <3% were converted to free Zea in the cell sonicate. The limited hydrolytic activity of Zea diester was located primarily (> 90%) in the cytosol fraction.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our results suggest that ingested Zea esters, and other xanthophyll esters by inference, are processed in a manner similar to dietary cholesteryl esters in the lumen of the small intestine. Studies with differentiated cultures of Caco-2 intestinal cells (28) and CEL-null mice (29) showed that CEL enhances intestinal uptake and transport of cholesterol by cleaving cholesteryl esters both before and after their partitioning in micelles to increase the extracellular concentration of free cholesterol. In contrast, CEL does not affect apical uptake of either free cholesterol (28,29) or free Zea (Fig. 5). Once taken up by the enterocyte, the metabolism of cholesterol and the xanthophylls differs. Cholesterol is esterified by acyl-CoA:cholesterol acyltransferase and incorporated into nascent chylomicrons for secretion into lymph. In contrast, the concentration of free Zea remained relatively constant in Caco-2 cells, suggesting that enterocytes do not esterify xanthophylls before their transfer to nascent chylomicrons. It is unclear whether this lack of esterification reflects an inability to deliver xanthophylls to the acyltransferase, demonstrates that xanthophylls have low affinity for the substrate binding site of the enzyme, or suggests that the enzyme does not acylate the bound xanthophyll. The absence of reesterification by Caco-2 cells is in line with observations from several human studies showing that free, but not esterified, xanthophylls are present in the triglyceride-rich fraction of plasma after ingestion of a meal enriched with esterified zeaxanthin (14) and cryptoxanthin (16,17). Very low concentrations of xanthophyll esters were reported in the plasma of humans administered high doses (15 mg/d as mixed esters) of lutein for extended periods (30) and in human skin (31). Similarly, lutein esters were identified in plasma, skin, and liver of chickens chronically fed diets supplemented with free and esterified lutein (32,33). Together, these observations indicate the potential for postabsorptive acylation of xanthophylls.
The absorption of carotenoids from a meal is affected by numerous factors including the manner in which the food is processed, the physical form, speciation, and molecular linkage of the carotenoid in the plant tissue, other components in the meal such as fat, the nutritional status and physiological state of the subject, and genetic background (34). During digestion, the carotenoid must be transferred from foods to mixed micelles to be delivered to the apical surface of absorptive epithelial cells. Differences in the efficiency of micellarization during simulated digestion were noted for the test foods. The efficiency of micellarization of Zea in orange pepper, red pepper, and squash ranged from 50–70%, whereas that from digested WB was only 24%. It is possible that differences in the processing of these foods before testing may have contributed to the discrepancy. The peppers were freshly prepared, the squash was cooked, and the wolfberry was dried and rehydrated before use. However, the factor that is striking is the Zea speciation in these foods. The efficiency of micellarization of Zea during digestion of WB, orange pepper, and red pepper was inversely associated with the relative amount of Zea diesters (P < 0.05, r = –0.92). The diesters accounted for 95, 64, and 36% of total Zea in WB, red pepper, and orange pepper, respectively. Solubilization of WB Zea esters in oil did not markedly enhance the efficiency of micellarization of Zea during simulated digestion, suggesting that the difference between WB and the peppers was not due simply to the matrix of the plant food. Rather, hydrolysis of Zea diesters during digestion may represent the limiting step for micellarization of this carotenoid from the tested sources in our in vitro model using 1 unit/mL CEL activity. Bowen et al. (15) proposed that the dissolution of lutein species in oil droplets generated during the digestion of foods or in the vehicle used for supplements, and not hydrolysis of the esters, is a key determinant affecting the bioaccessibility and degree of absorption of this xanthophyll. Roodenburg et al. (35) reported that efficient absorption of lutein from a lutein ester supplement required a higher amount of fat in the meal compared with efficient absorption of 
- and ß-carotene. Increased fat content in the meal increases the synthesis and secretion of pancreatic CEL and chylomicrons (23,24). It is noteworthy that Cheng et al. (36) recently reported that daily ingestion of 15 g wolfberry after the evening meal for 28 d increased plasma Zea concentrations 2.5-fold. This clearly demonstrates that Zea in wolfberry is bioavailable; together with other Zea rich foods such as those tested in the present study, it represents an appropriate sources for supplying this xanthophyll to ocular tissues.
Previous studies of the uptake of micellar carotenoids by differentiated Caco-2 cells and the TC7 clone showed that these cells accumulate similar amounts of lutein, -carotene, and ß-carotene from apical medium containing physiologically relevant levels of the pigments (9,22,37,38). However, transport across the basolateral membrane in chylomicrons is dependent on the carotenoid species when prandial conditions are mimicked (38). The present study provides the first assessment of Zea uptake and transport by Caco-2 cells. The results show that the cells preferentially took up free Zea from micelles and that the extent of transport (4.5%) in chylomicrons was similar to that reported for lutein in Caco-2 cells (9,38). This low amount is consistent with the estimated absorption of 3.3% of 5 mg Zea administered to human subjects (14). Several recent studies showed that the uptake of ß-carotene and lutein by Caco-2 cells is partially facilitated, with the scavenger receptor class B type 1 protein likely participating as the carrier (39,40). Studies are now required to determine whether, as expected, Zea uptake occurs in a manner similar to that of lutein and to examine possible interactions between Zea and the other dietary carotenoids that affect their bioaccessibility and absorption.
In summary, our results suggest that Zea esters are hydrolyzed to free Zea in the small intestine by CEL, that free Zea is preferentially taken up by intestinal epithelial cells, and that these cells appear to lack enzyme activity to hydrolyze acquired Zea monoesters. The results further support the usefulness of the coupled in vitro digestion/Caco-2 intestinal cell model for screening the bioaccessibility of carotenoids from foods and elucidating particular characteristics of carotenoid metabolism and transport in the gut.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the Ohio Agriculture Research and Development Center (OARDC) and the Virginia M. Vivian Endowment Fund (to C.C.).
3 Present address: Institute of Nutrition, Mahidol University, Sayala, Thailand.
5 Abbreviations used: CEL, carboxyl ester lipase; MAPK, mitogen-activated protein kinase; MTBE, methyl-tert-butyl-ether; PC, phosphatidylcholine; WB, wolfberry; Zea, zeaxanthin.
Manuscript received 12 August 2005. Initial review completed 6 September 2005. Revision accepted 22 December 2005.
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