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National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, Japan and Department of Bioresources Chemistry, Graduate School of Fisheries Science, Hokkaido University, Hakodate, Japan *
2To whom correspondence should be addressed. E-mail: nagao{at}nfri.affrc.go.jp
ABSTRACT
Despite the interest in the beneficial roles of dietary carotenoids in human health, little is known about their solubilization from foods to mixed bile micelles during digestion and the intestinal uptake from the micelles. We investigated the absorption of carotenoids solubilized in mixed micelles by differentiated Caco-2 human intestinal cells, which is a useful model for studying the absorption of dietary compounds by intestinal cells. The micelles were composed of 1 µmol/L carotenoids, 2 mmol/L sodium taurocholate, 100 µmol/L monoacylglycerol, 33.3 µmol/L fatty acid and phospholipid (0–200 µmol/L). The phospholipid content of micelles had profound effects on the cellular uptake of carotenoids. Uptake of micellar ß-carotene and lutein was greatly suppressed by phosphatidylcholine (PC) in a dose-dependent manner, whereas lysophosphatidylcholine (lysoPC), the lipolysis product of PC by phospholipase A2 (PLA2), markedly enhanced both ß-carotene and lutein uptake. The addition of PLA2 from porcine pancreas to the medium also enhanced the uptake of carotenoids from micelles containing PC. Caco-2 cells could take up 15 dietary carotenoids, including epoxy carotenoids, such as violaxanthin, neoxanthin and fucoxanthin, from micellar carotenoids, and the uptakes showed a linear correlation with their lipophilicity, defined as the distribution coefficient in 1-octanol/water (log Pow). These results suggest that pancreatic PLA2 and lysoPC are important in regulating the absorption of carotenoids in the digestive tract and support a simple diffusion mechanism for carotenoid absorption by the intestinal epithelium.
KEY WORDS: • Caco-2 cells • carotenoids • lysophosphatidylcholine • phosphatidylcholine • phospholipase A2
Carotenoids are thought to contribute to the inverse relationship
between fruit and vegetable consumption and the risk of major clinical
diseases, such as cancer (1
–3
), cardiovascular disease
(4
–6
) and age-related macular degeneration
(6
, 7
). To increase our understanding of the potential
health benefits of carotenoids, we need greater insight into the
bioavailability of dietary carotenoids and their metabolic conversion.
The absorption of carotenoids requires several steps, including
disruption of the food matrix to release the carotenoids, dispersion in
lipid emulsion particles, solubilization into mixed bile salt micelles,
movement across the unstirred water layer adjacent to the microvilli,
uptake by the cells of intestinal mucosa and incorporation into
lymphatic lipoproteins (8
–10
). The steps up to
solubilization in mixed micelles are dependent mostly on the
physicochemical properties of foods and carotenoids and on the micelle
formation from bile and lipid hydrolysates (11
). Cellular
uptake of carotenoids is mediated by a simple diffusion mechanism, as
previously shown in perfused rat intestine and in hBRIE 380 rat
intestinal cells (12
, 13
). Several studies in vitro have
evaluated the cellular absorption of carotenoids solubilized in
micelles under conditions simulating those in the intestinal lumen
(11
–16
). Mixed micelles formed in intestinal lumen play
an essential role not only in the digestion and absorption of
triacylglycerols but also in the uptake of other lipophilic compounds.
Recent studies have indicated that phospholipids present in mixed
micelles and pancreatic phospholipase A2
(PLA2)3
(3
) modulate the cellular uptake of cholesterol and
-tocopherol (17
–20
), but the relationship between the
absorption of micellar carotenoids and mixed micellar components such
as bile salts, fatty acid, monoacylglycerol, cholesterol and
phospholipids has not been reported. Thus, the absorption of micellar
carotenoids and their subsequent secretion to lymphatic fluid in the
epithelial cells of the intestine is not well understood. An
understanding of these processes would be very useful in clarifying why
the accumulation of carotenoids in animals varies largely among
species.
No data on the structure-absorption relationship in the absorption
of carotenoids are available, although more than forty carotenoids are
typically ingested (21
). In particular, epoxy carotenoids,
such as violaxanthin, neoxanthin and fucoxanthin, are widely
distributed in nature and constitute the major dietary carotenoids in a
number of fruits, vegetables and edible algae. However, the digestion,
absorption and metabolism of the dietary epoxy carotenoids are not well
understood (22
), although recent reports have shown their
biological actions related to health benefits (23
–26
).
The object of the present study was to characterize the absorption of
micellar carotenoids through the use of differentiated cultures of
Caco-2 cells derived from human colonic carcinoma. The Caco-2 cells
have been a useful model for studying the metabolism and transport of
drugs and dietary compounds by intestinal absorptive cells
(27
, 28
). The present study focused on the effects of mixed
bile micelles containing phospholipids on carotenoid absorption into
intestinal epithelial cells and the structure-absorption
relationship among a wide variety of dietary carotenoids.
MATERIALS AND METHODS
Materials.
ß-Carotene, -carotene, phosphatidylcholine
(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine),
lysophosphatidylcholine
(1-palmitoyl-sn-glycero-3-phosphocholine), monoolein,
sodium taurocholate and porcine pancreas PLA2 were
purchased from Sigma Chemical (St. Louis, MO). ß-Cryptoxanthin and
zeaxanthin were purchased from Extrasynthese (Genay, France).
Canthaxanthin and astaxanthin were kindly donated by Nippon Roche
(Tokyo, Japan). Capsanthin was kindly provided by Kagome (Tokyo,
Japan). Lutein was kindly donated by Kyowa Hakko Kogyo (Tokyo, Japan).
Brown algae (Undaria pinnatifida) and spinach
(Spinacia oleracea L.) were purchased from a local
market in Tsukuba, Japan. Tomato oleoresin (LYC-O-Mato TM6%) was
kindly donated by Ajinomoto Takara (Tokyo, Japan). LDH-Cytotoxic
Test kit was purchased from Wako Pure Chemical Industries (Osaka,
Japan). Other chemicals and solvent were of reagent grade.
Preparation of carotenoids.
Fucoxanthin was extracted and refined from brown algae
(29
). The acetone extract from the brown algae was applied
to a silica gel (Keisel gel 60, Merck, Darmstadt, Germany) column and
was eluted by stepwise elution with a hexane:ethyl acetate mixture
(10:0–4:6, v/v). Fucoxanthin was recovered in the hexane:ethyl acetate
fraction (5:5–4:6, v/v). The fucoxanthin-rich fraction was further
subjected to flash column chromatography on a LiChroprep RP-18 (40–63
µm, 11 x 240 mm; Merck) with acetonitrile/methanol/water
(75:15:10) containing 0.1% ammonium acetate to isolate pure
fucoxanthin.
Neoxanthin and violaxanthin were isolated from spinach
(30
). The acetone extract from spinach leaves was
saponified with 5% potassium hydroxide in 95% ethanol at room
temperature overnight. The ether extract after saponification was
applied to a neutral alumina III column. After being washed with
increasing amounts of ethyl acetate in hexane (5:5–1:9, v/v),
violaxanthin- and neoxanthin-rich fractions were eluted with ethyl
acetate and ethyl acetate/ethanol (9:1, v/v), respectively. To isolate
pure violaxanthin and neoxanthin, each fraction was further subjected
to a flash column chromatography as described above. -Carotene and
ß-carotene were purified by passage through a neutral alumina III
column in hexane. Lutein, ß-cryptoxanthin and canthaxanthin were
applied to an alumina III column equilibrated with hexane and then were
fractionated using ether/ethanol (99:1, v/v), hexane/ether (4:1, v/v)
and hexane/ether (7:3, v/v), respectively. Astaxanthin and capsanthin
were purified by a silica gel column equilibrated with hexane and then
were fractionated using dichloromethane/hexane (7:3–9:1, v/v) and
ethyl acetate/hexane (2:8–3:7, v/v), respectively. Acyclic carotenoids
(lycopene, 
-carotene, phytofluene and phytoene) were prepared from
tomato oleoresin as described previously (31
).
The estimation of purity was based on the peak area of all components
absorbed at each specific wavelength in HPLC separation. The purities
of all carotenoids prepared were >99%. The extinction coefficients of
respective carotenoids were used for quantification (32
).
Cell culture.
Caco-2 cells (American Type Culture Collection, Rockville, MD) were maintained in 10-cm dishes (Corning Glassworks, Corning, NY) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 4 mmol/L L-glutamine, 40,000 U/L penicillin, 40 mg/L streptomycin and 1% nonessestial amino acids. Cells were kept at 37°C in a humidified atmosphere of 95% air and 5% CO2. The growth medium was replenished every 2 or 3 d. Cells were reseeded when the cell monolayers became semiconfluent. For experiments, cells at passages 25–50 were seeded in 12-well plates at 1.2 x 105 cells/well and grown under the same conditions as those described above. The experiments were performed at 20–22 d post seeding.
Preparations of various carotenoids solubilized in micelles.
Carotenoids were delivered to cells as mixed micelles that were
prepared by modifying procedures previously described
(15
, 33
–35
). Briefly, appropriate volumes of stock
solutions of the compounds tested were transferred to glass tubes, and
the organic solvent was removed under a stream of argon. The residue
was dissolved in serum-free DMEM with a Vortex mixer. The final
concentration of each component in the medium was as follows: 2 mmol/L
sodium taurocholate, 100 µmol/L monoolein, 33.3
µmol/L oleic acid, 0–200 µmol/L
phospholipid and 1.0 µmol/L carotenoid. The resultant
solutions were optically clear. The medium was sterilized by passage
through a presterilized 0.22-µm filter. The
concentration of micellar carotenoids after filtration was confirmed to
be 1.0 ± 0.05 µmol/L by HPLC before the
carotenoids were used in the following experiments.
Cellular uptake of micellar carotenoids.
The differentiated monolayers of Caco-2 cells in a 12-well plate were
washed twice with 0.5 mL serum-free medium and then supplemented
with 1 mL of the medium containing micellar carotenoids. After
incubation in the cell culture section for the indicated time, the cell
culture plates were placed on ice, the media were removed and
monolayers were washed twice with 0.5 mL phosphate-buffered saline
(PBS) containing 10 mmol/L sodium taurocholate to remove
surface-bonded carotenoids followed by two additional washings with
0.5 mL PBS. The washed cells were harvested in 1 mL PBS and pelleted by
centrifugation at 1,000 x g for 5 min at 4°C. The
supernatants were discarded, and the cell pellets were homogenized with
a microtube homogenizer in 0.5 mL ice-cold PBS. An aliquot of each
cell homogenate was taken to determine the protein content, according
to the method of Lowry et al. (36
). To extract the
carotenoids, we added 1.5 mL dichloromethane/methanol (1:2, v/v)
containing 70 µmol/L -tocopherol to 0.4 mL of the
cell homogenate and mixed the solution with a Vortex mixer. Hexane
(0.75 mL) was mixed with the solution, and the resultant upper layer of
hexane-dichloromethane was withdrawn. The lower layer was similarly
extracted with 0.5 mL dichloromethane followed by 0.75 mL hexane. The
hexane-dichloromethane layer was combined with the initial extract.
The combined extract was dried under a stream of argon gas, dissolved
in 400 µL dichloromethane/methanol (1:4, v/v) and subjected to HPLC
analysis as described below. We also analyzed the concentration of
carotenoids in medium before and after incubation. An aliquot of medium
(100 µL) was mixed with 400 µL dichloromethane/methanol (1:4, v/v)
and subjected to HPLC analysis.
We evaluated the potential cytotoxicity of the micellar preparation on cultures in pilot studies. The morphological appearance of the monolayer and release of cytoplasmic lactate dehydrogenase (LDH) into the medium were similar in cultures incubated in serum-free DMEM with and without micelles for at least 6 h.
All procedures were carried out under dim yellow light to minimize degradation of the carotenoids by light irradiation.
HPLC analyses.
The HPLC system consisted of an LC-10AD pump (Shimadzu, Kyoto, Japan),
an SPD-10A UV-VIS absorbance detector (Shimadzu), an AS-8020
autosampler (Tosoh, Tokyo, Japan) and a personal computer with EZChrome
Chromatography Data System software (Scientific Software, Pleasanton
CA). Carotenoids were separated on a TSK gel ODS-80Ts (Tosoh), 4.6
x 150 mm, attached to a precolumn (2 x 20 mm) of Pelliguard
LC-18 (Supelco, Bellefonte, PA). Solvent A was
acetonitrile/methanol/water (75:15:10, v/v/v) containing 0.1% ammonium
acetate, and solvent B was ethyl acetate/methanol (30:70, v/v)
containing 0.1% ammonium acetate. Isocratic analyses were performed at
1.0 mL/min with solvent B for -carotene, ß-carotene,
ß-cryptoxanthin and acyclic carotenes; with solvent A/B (5:5, v/v)
for canthaxanthin; with solvent A/B (7:3, v/v) for lutein, zeaxanthin,
astaxanthin, capsanthin and violaxanthin; and with solvent A for
fucoxanthin and neoxanthin. -Carotene, phytofluene and phytoene were
monitored at 400, 348 and 286 nm, respectively. Other carotenoids were
detected at 450 nm. The carotenoids were quantified from their peak
area by use of standard curves.
Thin-layer chromatography analyses.
To confirm that hydrolysis of phosphatidylcholine (PC) in medium after
treatment with PLA2 had occurred, the total lipids of the
medium were extracted by the method of Folch et al. (37
).
The total lipids were applied to a silica thin-layer chromatography
(TLC) plate (silica gel 60; Merck) and developed in
chloroform/methanol/water (65:25:4, by vol). Lipids were visualized by
spraying with 50% sulfuric acid followed by charring at 200°C.
Statistical analysis.
The data represent the mean ± SD. Statistical analyses were made by one-way ANOVA and Dunnett’s or Scheffé’s F test to identify significant differences between the groups.
RESULTS
ß-Carotene and lutein uptake by Caco-2 cells versus micellar phospholipid content.
The effects of micellar phospholipid content on carotenoid uptake were
examined by incubating differentiated Caco-2 cells in serum-free
DMEM with micelles containing 0–200 µmol/L PC or
lysophosphatidylcholine (lysoPC) for 2 h. ß-Carotene and lutein,
which are major carotenoids found in human plasma, were used as
representative carotenoids of hydrocarbon carotenoids and xanthophylls.
As shown in Figure 1
A and B, the phospholipid content of micelles had
profound effects on the cellular uptake of ß-carotene and lutein. PC
in the micelles greatly suppressed ß-carotene and lutein uptake from
micelles by Caco-2 cells in a dose-dependent manner. The cellular
content of ß-carotene and lutein after incubation with micelles
containing 200 µmol/L PC was 31 and 12%, respectively, of
the content with phospholipid-free micelles. On the other hand,
lysoPC, the lipolysis product of PC by PLA2,
markedly enhanced both ß-carotene and lutein uptake by the cells. The
amounts of both cellular ß-carotene and lutein reached maximum levels
when the cells were incubated with micelles containing 50
µmol/L lysoPC. In these cases, the maximal amounts of
cellular ß-carotene and lutein were 220 and 130%, respectively,
higher than those attained with phospholipid-free micelles. As
shown Fig. 1
C, the relative uptake of lutein to that of
ß-carotene was reduced by the presence of the phospholipids in the
mixed micelles. This reduction was larger in PC micelles than in lysoPC
micelles.
|
Effect of PLA2 on carotenoid uptake.
The data indicate that micellar PC restricts carotenoid diffusion from
micelles to the cells (Fig. 1)
. To further confirm this observation, we
examined the effect of PC hydrolysis by pancreatic
PLA2 on carotenoid uptake by Caco-2 cells. The
time-courses of absorption of ß-carotene and lutein from micelles
containing 50 µmol/L PC by Caco-2 cells were determined in the
absence or presence of porcine pancreatic PLA2
(1,000 or 100 U/L). For comparison, incubation was also performed with
micelles containing 50 µmol/L lysoPC. Hydrolysis of
micellar PC in the medium by PLA2 was confirmed
by TLC. There was no PC spot on the TLC plate following incubation with
PLA2 (1,000 U/L) for 30 min or with
PLA2 (100 U/L) for 2 h. The lack of
morphological change and the low level of LDH released to the medium by
PLA2 treatment indicated that there was no
cytotoxic effect in the condition tested. As observed previously (Fig. 1)
, the uptake was greatly reduced in Caco-2 cells exposed to micelles
containing PC in comparison to the cells incubated with micelles
containing lysoPC (Fig. 2
). The restricted uptake of carotenoids was significantly reversed by
the addition of PLA2 (100 U/L) to the incubation
medium. The uptake of the carotenoids from micelles containing PC in
the presence of 1,000 U/L PLA2 was similar to
that from micelles containing lysoPC.
|
To evaluate the relationship between carotenoid structure and uptake by
Caco-2 cells, we compared the uptake of various micellar carotenoids
solubilized in the optimal conditions described above. Caco-2 cells
were incubated for 2 h with micelles containing 1.0
µmol/L of carotenoid in the presence of 50
µmol/L of lysoPC. All carotenoids including epoxy
carotenoids, such as violaxanthin, neoxanthin and fucoxanthin, were
detected in the cells incubated with micellar carotenoids (Fig. 3
). We observed a large variation of uptake among carotenoids. The amount
of ß-carotene uptake, which is the highest value in Fig. 3
, was
700% higher than that of neoxanthin, which suggests that the uptake
of various carotenoids may be dependent on their lipophilicity. Next,
we plotted amounts of carotenoid absorbed against octanol-water
partition coefficients (log Pow) calculated by
Cooper et al. (38
) as an index of lipophilicity
(Fig. 4
). Positive correlation (r2 = 0.8614) was
found between the amount of carotenoids absorbed by Caco-2 cells and
their log Pow. In this condition, the average
recovery of diverse carotenoids from the culture (cells and medium) was
82.2 ± 6.6%, close to the values of the experiments in Figure 1
.
The recoveries of diverse carotenoids were independent of their
lipophilicity (r2=0.1589) and cellular
uptake (r2 = 0.3207).
|
|
-carotene, phytofluene
and phytoene, were also examined under the same experimental condition
as described above, except for the micellar carotenoid concentration.
Because lycopene was easily crystallized and hardly solubilized in
micelles at 1.0 µmol/L, the uptake of acyclic carotenoids
by Caco-2 cells in the incubation with 0.4 µmol/L micellar
carotenoids was examined (Fig. 5
). The cellular lycopene level was approximately half that of other
acyclic carotenoids and ß-carotene.
|
DISCUSSION
Many factors are involved in the bioavailability of dietary carotenoids. Solubilization of carotenoids in mixed micelles is considered to be prerequisite for absorption by intestinal cells. However, the relationships of micellar composition and carotenoid structure to absorption have not been elucidated under well-defined conditions of intestinal models. In this study, we have evaluated the uptake of 15 carotenoids solubilized in mixed micelles by differentiated Caco-2 cells as a tissue culture model of the human intestinal epithelium. We demonstrated that phospholipids in the micelles greatly affected carotenoid uptake by Caco-2 cells, and that the cellular uptake was dependent on the lipophilicity of carotenoids.
In the present study, we measured the uptake of carotenoids into cells
by determining the amounts of carotenoids accumulated in the cells.
Insolubilization and oxidative degradation of carotenoids in medium
(39
) as well as metabolic conversion and oxidative
degradation in the cells might decrease the level of carotenoids
accumulated in cells. We have eliminated these effects of micelles by
reducing incubation time to 2 h in the standard condition. The
micellar carotenoids were quite stable in the medium alone, whereas a
small loss of carotenoid recovery from the culture (cells plus medium)
was observed in lysoPC micelles. However, the cellular levels of
diverse carotenoids accumulated in the cells were independent on the
recovery of carotenoids. The only well-known metabolic conversion
in mammals is the cleavage of provitamin A to vitamin A. However, the
conversion of provitamin A in Caco-2 cells during a short-time
incubation of 2 h would be too low to affect the cellular
carotenoid levels, because the ability of Caco-2 cells to produce
vitamin A is very low (40
–42
). Although we could not
eliminate the possibility of other unknown metabolic conversions or
oxidative degradation in the cells, a relatively short-time
incubation with carotenoids would decrease the effects of these
reactions on the cellular carotenoid level. Thus the apparent uptake of
diverse carotenoids observed in the present study indicates their
actual transport from medium to cells rather than unknown conversions
of carotenoids by the cells.
PC is one of the emulsifiers essential for the solubilization of
lipophilic compounds, such as cholesterol and fat-soluble vitamins,
in the digestive tract. However, PC was suggested to suppress
cholesterol absorption despite the promotion of their solubilization
(17
–19
). The dominant hypothesis to emerge from previous
studies suggested that the limited cholesterol uptake associated with
micellar PC was due to a shift in the partitioning of cholesterol from
the aqueous phase to the micellar phase. The findings in the present
study were consistent with this hypothesis. Because of their high
hydrophobicity, carotenoids associate strongly with long-chain acyl
moieties of PC in mixed micelles, resulting in reduced uptake by Caco-2
cells. Absorption of polar lutein, with hydroxy groups, was reduced
more strongly than that of nonpolar ß-carotene. Gabrielska and
Gruszecki (43
) reported a strong rigidifying effect of
zeaxanthin (dihydroxy-ß-carotene) but not of ß-carotene, with
respect to both the hydrophobic core of egg yolk PC bilayer and the
polar head group of PC, and they indicated that the polar ends of
zeaxanthin were oriented to be in contact with the opposite polar zones
of the bilayer. Mixed bile micelles in the digestive tract have a
disk-like structure composed of phospholipids and fatty acids
forming a bilayer and of bile acids occupying the edge positions
(44
). Thus, xanthophylls with polar ends might have a
higher affinity against a bilayer of PC-mixed micelles than
hydrocarbon carotenoids.
There are many reports that the inhibitory effect of PC on the
absorption of cholesterol and -tocopherol is abolished when lysoPC
is substituted for PC (17
–20
). In the present study, we
found that lysoPC greatly enhanced carotenoid uptake by Caco-2 cells.
Micelle size is one of the factors that determines the absorption of
lipophilic substances solubilized in mixed micelles. LysoPC micelles
are smaller than PC micelles, but a comparison of cholesterol uptakes
from lysoPC and PC micelles similar in size showed that the uptake was
still lower in the latter (18
). Thus, some other factor is
more important than micelle size. It is well known that lysoPC uptake
by intestinal cells is much greater than PC absorption
(18
, 19
). LysoPC can be quickly converted to PC and
triacylglycerols in Caco-2 cells (45
). The increased
cellular level of lipids caused by the uptake of lysoPC may simply
shift the equilibrium of the carotenoid partition from the mixed
micelles to the cells. Furthermore, this effect of lysoPC may be caused
by its stimulation of intracellular processing of lipids within
intestinal cells, because it is well documented that PC restructured
from lysoPC facilitates the packaging of lipids as well as chylomicron
formation and its secretion (19
, 46
–48
). With regard to
pancreatic PLA2, several studies have shown that
addition of the enzyme in mixed micelles or a lipid emulsion enhances
cholesterol uptake by intestinal cells (19
, 49
). In this
study, PLA2 from porcine pancreas enhanced the
uptake of carotenoid from micelles containing PC by Caco-2 cells. Thus,
pancreatic PLA2 and lysoPC may be important in
regulating the absorption of carotenoids.
Many works have indicated the passive uptake of carotenoids dependent
on their concentration by rat everted gut sacs (14
),
perfused rat intestine (12
), intestinal cells in culture
(13
) and intestinal brush border membrane vesicles
(50
). The most common way to predict drug absorption by
passive diffusion is to determine the lipophilicity of the drug. The
prediction relies on the assumption that a more lipophilic drug will
partition faster into the lipid cell membrane (51
). In our
optimal condition using micelles containing 50 µmol/L
lysoPC, a linear relationship between the lipophilicity of carotenoids,
indicated as the distribution coefficient in 1-octanol/water (log
Pow), and the uptake of carotenoids by Caco-2 was
clearly demonstrated. These results strongly suggested a simple
diffusion mechanism for carotenoid uptake by intestinal epithelium.
LysoPC is formed from PC by pancreatic PLA2 in
the digestive tract at an amount equaling 80% of the total
phospholipids in the aqueous phase of duodenal content in humans
(52
). Thus, our present data obtained by using micelles
containing lysoPC simulating the mixed micelles formed in intestinal
lumen would be close to the in vivo absorption of carotenoid
solubilized in mixed micelles by intestinal epithelium. However, the
overall bioavailability of dietary carotenoids in vivo would be
determined not only by uptake of micellar carotenoids but also by other
factors, such as their release from food matrix and their dispersion to
lipid emulsions as well as the formation of mixed micelles
(8
–10
). In the present study, uptake of micellar lutein
was slightly smaller than that of ß-carotene, whereas von het Hof et
al. (53
) reported that the bioavailability of lutein from
vegetables is five times higher than that of ß-carotene in humans.
This discrepancy was probably caused by the difference in the
solubilization into mixed micelles from foods, because lutein is more
readily solubilized than ß-carotene in an in vitro digestion system
(11
, 16
). Oshima et al. (54
) reported that the
increase of capsanthin in human plasma tended to be lower than
increases of lutein and ß-cryptoxanthin following the ingestion of
paprika juice. This result would reflect our finding that polar
carotenoids in mixed micelles were poorly absorbed into cells. The
quite different effects of PC and LysoPC in mixed micelles on the
uptake of carotenoids suggest that intake of considerable amounts of
dietary PC might suppress the absorption of carotenoids, as reported in
a case of reduced cholesterol absorption by dietary PC in human
subjects (55
).
In the present study, the amount of lycopene absorption was half that
of other acyclic carotenes and ß-carotene. Previous studies have
shown that lycopene was less efficiently absorbed than canthaxanthin
and astaxanthin in rats (56
, 57
). Supplementation studies
with humans also suggested that lycopene is not efficiently absorbed
(58
, 59
). Poor absorption of lycopene in vivo would be due
to poor solubilization into mixed micelles (11
, 16
) and the
low uptake to cells observed in the present study. However, the reason
for the lower absorption of lycopene is unclear.
Recently, several reports have suggested that epoxy carotenoids have
beneficial effects on cancer chemoprevention (23
–26
).
However, there has been no report on the absorption and metabolism of
epoxy xanthophylls, such as fucoxanthin and neoxanthin. The present
study demonstrated for the first time the uptake of fucoxanthin,
neoxanthin and violaxanthin by differentiated Caco-2 cells, which
represent a good model of the human intestinal cell. Further
investigations are required to clarify the absorption and metabolic
conversion of epoxy xanthophylls in vivo.
In the present study, we focused on the absorption of micellar carotenoids by differentiated Caco-2 cells. We demonstrated that phospholipid in micelles greatly affected carotenoid uptake by Caco-2 cells, and that the uptake from lysoPC micelles was highly dependent on their lipophilicity. The mechanism of increased uptake of carotenoids in the presence of lysoPC and the subsequent secretion process of carotenoids to lymph in connection with lipid metabolism deserve future studies.
FOOTNOTES
1 Supported partly by the PROBRAIN project
"Regulation of oxidative stress with phytochemicals from foods" of
Bio-oriented Technology Research Advancement and by Special
Coordination Funds of the Ministry of Education, Culture, Sports,
Science and Technology, the Japanese Government. 
3 Abbreviations used: DMEM, Dulbecco’s modified
Eagle’s medium; LDH, lactate dehydrogenase; lysoPC,
lysophosphatidylcholine; PBS, phosphate-buffered saline; PC,
phosphatidylcholine; PLA2, phospholipase A2;
TLC, thin-layer chromatography.
Manuscript received 29 May 2001. Initial review completed 21 June 2001. Revision accepted 14 August 2001.
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