Impaired uptake of β-carotene by Caco-2 human intestinal cells in the presence of iron

Impaired uptake of b-carotene by Caco-2 humanintestinal cells in the presence of iron

ANTON BENGTSSON, NATHALIE SCHEERS, THOMAS ANDLID,

MARIE LARSSON ALMINGER, ANN-SOFIE SANDBERG &

ULF SVANBERG

Department of Chemical and Biological Engineering, Food Science, Chalmers University of

Technology, Goteborg, Sweden

AbstractAt present, there are conflicting data regarding whether or not b-carotene has a positive effect oniron absorption. This study was undertaken to evaluate possible interactions involved in theuptake of b-carotene and iron in a human intestinal cell model (Caco-2). The Caco-2 cells wereincubated with test solutions containing different amounts of ferrous chloride (10�50 mM) andb-carotene (0.3�2.5 mM) incorporated in synthetic micelles. In the absence of iron, cellularaccumulation of b-carotene from synthetic micelles was proportional (r2�0.97, PB0.001) tothe b-carotene concentration in the test solution. However, with addition of ferrous chloride(30 mM), the b-carotene uptake was significantly reduced (PB0.05), on average by 22%. Therewas also an inverse relationship between the b-carotene uptake and iron concentration inthe test solution (r2�0.93, PB0.05). Iron provided in physiological amounts inhibited theuptake of b-carotene in the in vitro Caco-2 cell system.

Keywords: Caco-2 cells, b-carotene, iron uptake, micelles, ferritin, high-performance liquid

chromatography

Introduction

Both vitamin A deficiency and anaemia are public health problems that affect millions

of people worldwide, particularly in developing countries (World Health Organization

2001; Sommer and Davidson 2002). While vitamin A deficiency essentially is caused

by an insufficient intake of vitamin A and provitamin A carotenoids from foods,

anaemia may be a result of several disorders such as iron deficiency, folate deficiency,

and infectious diseases including malaria and intestinal parasites (Lee 1983; Viteri

1997; van den Broek and Letsky 2000). Vitamin A deficiency is also recognized as a

cause of anaemia (Bloem 1995). The mechanisms by which vitamin A deficiency

induces anaemia are not clear, but impaired iron metabolism and erythropoiesis as

well as decreased resistance to infection have been suggested in previous reviews

(Fishman et al. 2000; Semba and Bloem 2002). Vitamin A and provitamin A

(b-carotene) have also been proposed to influence iron absorption. In two Venezuelan

Correspondence: Anton Bengtsson, Department of Chemical and Biological Engineering, Food Science,

Chalmers University of Technology, SE-412 96 Goteborg, Sweden. Fax: �46 31 772 38 30. E-mail:

[email protected]

ISSN 0963-7486 print/ISSN 1465-3478 online # 2009 Informa UK Ltd

DOI: 10.1080/09637480802641270

International Journal of Food Sciences and Nutrition,

September 2009; 60(S5): 125�135

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studies, supplementation of iron-fortified cereal meals with either vitamin A or

b-carotene enhanced the absorption of non-heme iron in humans (Layrisse et al.

1997; Garcia-Casal et al. 1998). On the contrary, in a Swiss/Swedish study no effect of

vitamin A was noticed on iron absorption in adults who consumed test meals of corn

bread (Walczyk et al. 2003). In addition, erythrocyte incorporation of iron decreased

significantly from a maize porridge supplemented with vitamin A given to iron-

deficient and vitamin A-deficient children in Cote d’Ivoire (Davidsson et al. 2003).

The contradictory findings in these studies might be explained by differences in

vitamin A, iron or disease status between the study groups. To what extent food iron

might influence the absorption of vitamin A or provitamin A carotenoids is not known.

Hence, there is an obvious lack of knowledge of the possible interactions between

vitamin A, b-carotene, iron, and their respective uptake systems during absorption

and the subsequent metabolism of these nutrients. The metabolic interactions of these

two micronutrients may have implications for the design of intervention programs

aimed at populations with both vitamin A deficiency and iron-deficiency anaemia.

The following study was designed to evaluate possible interactions involved in the

uptake of b-carotene and iron in a Caco-2 human intestinal cell model under

conditions of iron deficiency. The Caco-2 cell line is a human colon adenocarcinoma

cell line that differentiates into polarized duodenal-like cells in culture, thus displaying

many characteristics of normal enterocytes (Pinto et al. 1983; Hidalgo et al. 1989).

The Caco-2 cell line is recognized as an established model for studies of intestinal iron

uptake (Fairweather-Tait et al. 2005) and it has also been used to measure the

incorporation of b-carotene and other carotenoids (Garrett et al. 1999; During et al.

2002). The specific objective of the present study was to explore the effects of

interactions*direct or indirect*between b-carotene in synthetic micelles and iron on

the uptake of these nutrients by Caco-2 cells.

Materials and methods

Chemicals

Synthetic crystalline b-carotene (]95% all-trans), lysophosphatidylcholine, sodium

glycocholate, sodium glycodeoxycholate, sodium taurocholate, sodium taurodeox-

ycholate, cholesterol, butylated hydroxytoluene, minimal essential medium (MEM),

and radioimmunoprecipitation assay lysis buffer were purchased from Sigma-Aldrich

(Schnelldorf, Germany). High-purity (99%) monoolein, oleic acid, and phosphati-

dylcholine were obtained from Larodan Fine Chemicals (Malmo, Sweden). Media,

supplements, and other reagents for cell culture maintenance were purchased from

Fischer Scientific GTF (Vastra Frolunda, Sweden).

Cell culture

Caco-2 cells (HTB-37) were obtained from American Type Culture Collection

(Rockville, MD, USA) at passage 18. Stock cultures were maintained in Dulbecco’s

modified Eagle medium supplemented with 16% (v/v) foetal bovine serum, 1% (v/v)

non-essential amino acids, and antibiotic solution (100 units/ml penicillin and 100 mg/

ml streptomycin) at 378C in a humidified atmosphere of 95% air and 5% CO2. The

growth medium was changed every second to third day. Monolayers were subcultured

at �80% confluence using 0.5 g/l trypsin with 0.5 mM ethylenediamine tetraacetic

126 A. Bengtsson et al.

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acid (EDTA) in Dulbecco’s phosphate-buffered saline (PBS). Cells at passages 27�35

were seeded at a density of 50,000 cells/cm2 in collagen-treated 12-well plates

(Corning Incorporated Life Sciences, Lowell, MA, USA).

Preparation of synthetic micelles containing b-carotene

Synthetic micelles were prepared based on several studies (Canfield et al. 1990;

Chitchumroonchokchai et al. 2004; Reboul et al. 2005) with some minor modifica-

tions. Stock solutions of monoolein, oleic acid, phosphatidylcholine, and cholesterol in

chloroform, and lysophosphatidylcholine in chloroform:methanol (9:1, v/v) were

prepared, blanketed with nitrogen and stored at �208C. A stock solution of all-

trans-b-carotene was prepared in n-hexane. Aliquots of these compounds were

combined in a 20 ml glass vial to obtain the following final concentrations: 0.5 mM

monoolein, 1.5 mM oleic acid, 0.1 mM phosphatidylcholine, 0.3 mM lysophosphati-

dylcholine, and 0.15 mM cholesterol. b-Carotene was added to yield micellar

concentrations ranging from 0.3 to 2.5 mM in the final test solutions. The transfer

efficiency of crystalline b-carotene into micelles was lower with higher added

concentrations of b-carotene in the preparation of micelles (data not shown). Solvents

were evaporated by a stream of nitrogen at room temperature. MEM containing

25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 0.85 mM

glycocholate, 0.45 mM glycodeoxycholate, 0.45 mM taurocholate, and 0.25 mM

taurodeoxycholate was added to the vial and the mixture was sonicated in a water

bath (Branson Ultrasonics Corporation, 8510; Danbury, CT, USA) at room

temperature for 30 min. The mixture was filtered and sterilized by passage through a

0.22 mm filter to remove microcrystalline b-carotene particles, and the resulting

micellar solution was optically clear. All b-carotene preparations were protected from

light. High-performance liquid chromatography (HPLC) analysis was used to

determine the b-carotene content in the final micellar solutions. No significant

degradation of b-carotene was detected after sonication and filtration. In the uptake

experiments with added iron, no differences were observed in b-carotene content

between micellar test solutions containing iron added at different concentrations (10�50 mM) measured after 2 h incubation. Micellar solutions containing b-carotene were

prepared immediately prior to the uptake experiments.

Uptake experiments

Iron and b-carotene uptake experiments were conducted 14 days post seeding. Two

days before the uptake experiment, the growth medium was exchanged for MEM

supplemented with 3.5 g/l glucose and antibiotic solution (100 units/ml penicillin and

100 mg/ml streptomycin) in order to induce iron deficiency in the cells. This medium

did not contain detectable levels of iron (B0.5 mM) as determined by a high-

performance ion chromatography method developed by Fredriksson et al. (2002).

The mean background ferritin level in the cells after 2 days of iron-free incubation was

5.5 ng ferritin/mg protein. Prior to the experiments, the integrity of the monolayers

was examined by optical microscopy. To initiate the experiments, cells were washed

once in fresh MEM before adding 1 ml test solution containing 0.5 ml MEM and

0.5 ml micellar solution. The total b-carotene concentrations in the different test

solutions varied between 0.3 and 2.5 mM, and iron as ferrous chloride (FeCl2�4H2O)

was added at a concentration of 30 mM. Ferrous iron is commonly provided as

Impaired Caco-2 cell uptake of b-carotene 127

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a dietary fortification agent. It is also extensively used in human absorption studies as

well as in Caco-2 cell studies. Cells incubated with test solutions devoid of added iron

were used to obtain baseline ferritin levels. The pH of the test solutions was �7.2 and

remained stable during the incubation period. The incubation with test solutions was

carried out for 2 h at 378C in a humidified atmosphere of 95% air and 5% CO2. After

this incubation period, the test solutions were removed from the wells. The cells were

washed once with PBS containing 1% (w/v) bovine serum albumin to reduce non-

specific adsorption of micelles and twice with PBS only. Fresh MEM was added to the

cells before the plates were returned to the incubator for overnight incubation (22 h)

to allow for ferritin formation.

A separate experiment was performed to test the stability of incorporated b-carotene

up to 24 h by comparing cells harvested both directly after the 2 h incubation with the

test solutions and after the additional 22 h incubation with fresh medium (MEM).

Four different concentrations of iron were used in this experiment (0, 10, 30, and

50 mM ferrous chloride). The complete experimental outline for the uptake experi-

ments is presented in Table I.

Harvesting of Caco-2 cells

After 24 h incubation, the medium was removed by aspiration and cells were washed

once in ice-cold PBS. The cells were harvested in 300 ml ice-cold radioimmunopre-

cipitation assay lysis buffer mixed with EDTA-free protease inhibitor cocktail (40 ml/ml

lysis buffer; Roche Diagnostics, Rotkreutz, Switzerland) and divided into aliquots for

protein, ferritin and b-carotene analysis. Samples were stored at �808C until analysis.

The ferritin concentration was determined on an aliquot of the lysate with a solid-

phase direct sandwich enzyme-linked immunosorbent assay method (Ferritin ELISA;

Calbiotech, Spring Valley, CA, USA). Caco-2 cells have been shown to synthesize

ferritin in response to increases in the intracellular iron content (Glahn et al. 1998).

Hence, the ferritin/protein ratio of Caco-2 cells, in units of nanograms of ferritin/

milligrams of protein, was used as a measure of cellular iron uptake (Yeung et al.

2005). The total protein content of the cell samples was estimated by the

Table I. Summary of experimental outline.

Micellar b-carotene concentration

in test solutions (mM)a

Iron concentration in test

solutions (mM)b

Incubation time

(h)c

0 0/30 24

0.31 0/30 24

0.40 0/30 24

0.67 0/30 24

0.86 0/30 24

1.16 0/30 24

1.28 0/30 24

2.06 0/10/30/50 2/24

2.51 0/30 24

aSynthetic micelles were prepared containing the above-mentioned b-carotene concentrations as measured

with HPLC.bIron as ferrous chloride was added to the test solutions in the presented concentrations.cCells were incubated for 2 h with test solutions containing iron and b-carotene followed by a subsequent

incubation for 22 h with fresh MEM not containing any b-carotene or iron.


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bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL, USA) using

bovine serum albumin as a standard.

Extraction and analysis of b-carotene

An aliquot of the cell suspension was extracted three times with 1.5 ml n-hexane and

0.5 ml methanol including 0.1% (w/v) butylated hydroxytoluene. Samples were

vortexed and centrifuged at 2,500 x g between each extraction to facilitate phase

separation. The combined hexanic phases were evaporated to dryness in a water bath

at 358C under a stream of nitrogen. The residues were dissolved in 100 ml mobile

phase (methanol:methyl tert-butyl ether, 1:1 (v/v)). b-Carotene was analysed by

reversed-phase HPLC using a Waters 600 system equipped with auto sampler

injector, degasser, pump and a Waters 996 UV�visible photodiode array detector

operating at 450 nm. The data were stored and processed by means of Millennium

4.00 Software (Waters, Stockholm, Sweden). Absorption spectra were recorded

between 250 and 500 nm. Separations were carried out on a C30 carotenoid column

(5 mm, 250 mm�4.6 mm i.d.: YMC Europe GMBH, Schermbeck, Germany). The

mobile phase used for isocratic elution consisted of methanol:methyl tert-butyl

ether:water (55:41:4, v/v/v). The flow rate was 1 ml/min and the injection volume

40 ml. Quantification was achieved using calibration curves with b-carotene at

450 nm. b-Carotene could not be detected in cells that had been exposed to micellar

solutions without any carotenoids.

Statistical analysis

The data for the b-carotene uptake as a function of different b-carotene concentra-

tions in the test solutions with and without iron are represented by the mean9

standard deviation (SD) of four incubations for each concentration. Incubations

were performed on separate plates and on two different days. The significance of

differences in b-carotene uptake was calculated by Student’s t-test. The data for the

b-carotene uptake as a function of different iron concentrations in the test solutions

are represented by the mean9SD of six incubations from two separate occasions.

These data were analysed by analysis of variance, and determination of significance of

differences was obtained by Tukey’s HSD test. The Pearson coefficients and their

significance levels were obtained from linear regression analysis. The statistical

evaluation was performed with SPSS (version 14.0; SPSS Inc., Chicago, IL, USA)

and PB0.05 was considered significant.

Results and discussion

b-Carotene uptake in the absence of iron

Synthetic micelles were used as the delivery vehicle for b-carotene to Caco-2 cells since

micelles facilitate the transfer of carotenoids in vivo across the unstirred water layer to

the intestinal brush border membrane, and thereby also the subsequent uptake

through the membrane (Erdman et al. 1993; Yonekura and Nagao 2007). In the

present study, cellular b-carotene content was directly proportional (r2�0.97, PB

0.001) to the b-carotene concentration in the test solutions without added ferrous

iron (Figure 1). The incorporated b-carotene content varied between 20.6 and


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268 pmol/mg protein for b-carotene concentrations of 0.31�2.51 mM in the test

solutions, respectively. Cellular uptake of b-carotene was examined by incubating

Caco-2 cells for 2 h in serum-free MEM containing synthetically prepared b-carotene

micelles. After an additional incubation with fresh medium (no b-carotene) for 22 h,

the b-carotene content in the Caco-2 cells was measured. The total experiment time

was therefore 24 h due to the time required for ferritin synthesis in response to iron

uptake. The b-carotene content was not significantly altered after the additional

incubation for 22 h with fresh MEM without b-carotene micelles either in the presence

or absence of iron. In fact, a corresponding overnight incubation has been shown to

have no significant effect on the cellular all-trans-b-carotene content (Ferruzzi et al.

2006). Representative HPLC chromatograms of b-carotene from the synthetically

prepared micelles and the Caco-2 cells after 24 h incubation are presented in Figure 2.

Our C30 column HPLC system did not reveal any formation of oxidation products or

any sign of further isomerization of the cell b-carotene content in relation to the small

amount of 13-cis-b-carotene already present in the freshly prepared micelles. Hence,

the b-carotene cell content after 24 h incubation was used as a measure of b-carotene

uptake in the present study.

Uptake of b-carotene from synthetic micelles by Caco-2 cells has been studied by

other groups (Garrett et al. 1999; Sugawara et al. 2001; Yonekura et al. 2006). A

linear relationship has previously been reported for b-carotene concentrations similar

to ours (Garrett et al. 1999; During et al. 2002). In one of these studies, the

b-carotene-containing micellar solution was diluted with micelles without b-carotene

and incubated for 20 h (Garrett et al. 1999). In the present study, the micellar

solutions with b-carotene were used as they were prepared. The exposure of such

micellar solutions to the cell monolayers for 2 h was intended to mimic the absorptive

conditions in the small intestine. The concentration range at the apical side of the cells

in the present study was within or close to the physiological level of b-carotene

y = 102xr 2 = 0.972

y = 79.5xr 2 = 0.966

0

50

100

150

200

250

300

350

0 0.5 1 1.5 2 2.5 3

β-carotene in test solution (µM)

β-ca

rote

ne u

ptak

e (p

mol

/mg

prot

ein)

30 µM ironNo added iron

Figure 1. b-Carotene uptake by Caco-2 cells from micelles with various concentrations of b-carotene in test

solutions incubated for 2 h with or without iron. Each point represents the mean9SD (indicated by error

bars) of four incubations for each concentration. Incubations were performed on separate plates and on two

different days.


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reported in the small intestine after a normal daily ingestion of 5 mg b-carotene

(During et al. 2002). These levels represent realistic amounts found in meals where b-

carotene is the most important vitamin A source (Mulokozi et al. 2004), which is the

case in many developing countries.

b-Carotene uptake in the presence of iron

b-Carotene uptake was significantly lower (PB0.05) in the presence of iron (30 mM)

in the test solutions, and these results are summarized in Figure 1. The mean

reduction in b-carotene uptake was 22%. There are possible explanations for this

negative effect of iron on the b-carotene uptake by Caco-2 cells. Iron may complex to

micellar components such as bile acids present on the micelle surface and thereby

interfere with the b-carotene release to the Caco-2 cells. In addition, the iron present

may act as a pro-oxidant, which may result in the degradation of b-carotene

by oxidation prior to the absorption (Solomons and Bulux 1993). However, the

b-carotene content in the micellar test solutions measured after 2 h incubation was not

affected by adding different iron concentrations (10�50 mM).

There was a significant decrease (r2�0.93, PB0.05) in the uptake of b-carotene

with increasing amounts of iron. The response in b-carotene uptake to different

concentrations of iron (10�50 mM) is shown in Figure 3. These iron concentrations

were within the physiological level as previously reported (Jacobs and Miles 1969).

The uptake of b-carotene in the absence of iron was significantly higher (PB0.001)

than the uptake of b-carotene at iron concentrations of 30 and 50 mM. Moreover,

there was a significant difference (PB0.05) in the b-carotene uptake from the test

solution containing 10 and 50 mM iron, respectively.

0 5 10 15 20 25Retention time (min)

Res

pons

e (A

U)

all-trans-β-carotene

13-cis-β-carotene

Figure 2. HPLC profile of representative chromatograms of b-carotene from the synthetically prepared

micelles (continuous line) and the Caco-2 cells (dashed line). b-carotene isomers were separated on a C30

carotenoid column and identified by absorption spectra. AU, absorbance units.


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Ferritin formation in the presence of b-carotene

The b-carotene concentration in the synthetically prepared micelles ranged from 0.3

to 2.5 mM. This concentration range was estimated to represent a realistic physiolo-

gical level of b-carotene in the small intestine (During et al. 2002). The formation of

ferritin was chosen as an estimate of iron uptake. Ferritin formation within the Caco-2

cells at different b-carotene concentrations is presented in Table II. Iron uptake from a

test solution containing 30 mM ferrous chloride was not significantly influenced by the

addition of micelles containing b-carotene at various concentrations. The mean

ferritin concentrations (after subtraction of the corresponding baseline ferritin levels,

on average 7.0 ng ferritin/mg protein, obtained after incubation with iron-free test

solutions) ranged from 7.2593.56 to 7.7396.51 ng ferritin/mg cell protein for test

solutions with or without b-carotene micelles. As can be noticed, the ferritin formation

was highly variable, which indicates that this cell system using ferritin formation may

r 2 = 0.934

0

50

100

150

200

250

300

0 10 20 30 40 50

Iron in test solution (µM)

β-ca

rote

ne u

ptak

e (p

mol

/mg

prot

ein)

aa, b

b, cc

Figure 3. Effect of different iron concentrations on the uptake of b-carotene by Caco-2 cells. The

concentration of b-carotene in the test solutions was 2.06 mM. Each point represents the mean9SD

(indicated by error bars) of six incubations from two separate occasions (measured at 2 h and 24 h,

respectively). Values not sharing the same letters (a�c) are significantly different (PB0.05) using analysis of

variance and Tukey’s HSD test.

Table II. Ferritin formation by Caco-2 cells exposed to ferrous chloride (30 mM) in response to various

concentrations of b-carotene.

Concentration of b-carotene

in test solution (mM)

Ferritin formation

(ng ferritin/mg protein)a

Significance

levelb

0 (n�8) 7.7396.51

0.31�0.86 (n�16) 7.2593.56 Not significant

1.16�2.51 (n�16) 7.6491.29 Not significant

aMean9SD after subtraction of the corresponding baseline ferritin levels. The mean baseline ferritin level

was 7.091.2 ng ferritin/mg protein.bThe significance level was calculated in comparison with the test solution containing no b-carotene.


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not be a suitable model for the investigation of possible effects of b-carotene on iron

absorption. However, with a modified Caco-2 cell system and with lower iron

concentrations, which may be the case in plant-based diets from developing countries,

any influence of b-carotene on iron uptake might be revealed.

One previous study addresses the question of whether b-carotene may influence

iron uptake by Caco-2 cells (Garcia-Casal et al. 2000). In contrast to the present

study, iron uptake was significantly increased in the presence of 0.9�6.0 mM

b-carotene. However, a number of differences in the experimental procedures make

a direct comparison difficult. In the study by Garcia-Casal et al. (2000), iron uptake

was measured as increased levels of radio iron in the incubated Caco-2 cells, while in

our study the formation of ferritin was used as a measure of cellular iron uptake.

Furthermore, cold water-soluble beadlets were used as the delivery vehicle for

b-carotene to the medium by Garcia-Casal et al. (2000). Micellar media prepared

with these water-soluble beadlets have been shown to be unstable, rendering orange

precipitations and hence a low micellar yield (Garrett et al. 1999). In addition, the

preparation of beadlets also contained additives such as sucrose and a-tocopherol,

which could possibly affect the uptake of nutrients in Caco-2 cells. In the present

study, b-carotene was presented to the Caco-2 cells in synthetically prepared micelles,

which more closely reflects the conditions in the duodenum.

To conclude, the presence of b-carotene, in concentrations ranging from 0.3 to

2.5 mM did not affect the uptake of iron in our cell assay. Our interpretation is that

there is no effect of b-carotene on the uptake of iron in cultured Caco-2 cells, and that

b-carotene converted to vitamin A is more likely to modulate iron utilization at

another level of iron metabolism. Animal studies have shown an impaired incorpora-

tion of iron into erythrocytes in vitamin A-deficient rats, suggesting that during this

condition iron is trapped in the liver and spleen and is not effectively released for

erythropoiesis by red bone marrow (Gardner et al. 1979; Mejıa et al. 1979).

At the chosen pH, suggested to be within the physiological pH range at the site for

absorption (Woodtli and Owyang 1995; Wright et al. 2008), there is a possibility of

some degree of iron precipitation, which may have compromised the effect of

b-carotene on iron uptake.

Conclusion

To our knowledge, the present study provides the first assessment of the impact of iron

on uptake of micellar b-carotene by Caco-2 cells. The uptake of b-carotene was

negatively affected by the presence of iron in the test solution provided to the cells,

and higher iron concentrations resulted in lower b-carotene uptake. However,

b-carotene in synthetic micelles did not increase iron uptake under the experimental

conditions employed in the present study. Our in vitro results that measures

accumulation of b-carotene in Caco-2 cells warrant further studies in humans using

stable isotope techniques that measures both uptake and consecutive metabolism of

b-carotene.

Acknowledgements

This work was supported by The Swedish Agency for Research Cooperation in

Developing Countries (Grant SWE-2004-005), The Swedish Council for Environment,


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Agricultural Sciences and Spatial Planning (Formas) (Grant No. 222-2004-1889),

and Wilhelm och Martina Lundgrens stiftelse.

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This paper was first published online on iFirst on 4 February 2009.


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Impaired uptake of β-carotene by Caco-2 human intestinal cells in the presence of iron

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