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Instituto Venezolano de Investigaciones Científicas, Carretera Panamericana Km. 11 Altos de Pipe, Apartado Postal 21827, Caracas 1020-A. Venezuela.
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • iron uptake • vitamin A • ß-carotene • Caco-2 cells • phytates • polyphenols
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This line differentiates in culture
as polarized enterocytes exhibiting many properties of normal
absorptive epithelium. Caco-2 cell cultures show morphological and
functional features typical of normal absorptive cells. Some of the
reported characteristics are polarization, differentiation after
arresting growth, brush border membrane, tight junctions, basal
localization of nucleus and transferrin receptors, secretion of brush
border associated enzymes and dome formation (Hauri et al. 1985
, Hidalgo et al. 1989
, Hughson et al. 1990
, Pinto et al. 1983
, Ramond et al. 1985
, Rousset 1986
, Vanchon et al. 1992
).
These cells can be grown in culture flasks or microporous membrane
inserts and have been used to perform uptake and transport studies.
Caco-2 cells are capable of performing many of the metabolic functions
of normal enterocytes. They can actively transport bile salts,
vitamins, amino acids and drugs (Artursson 1990
,
Dix et al. 1990
, Hidalgo and Borchardt 1990
, Vincent et al. 1985
), synthesize
metallothionein (Rafaniello and Wapnir 1991
) and
transport and metabolize fatty acids (Levin et al. 1992
,
Trotter and Stroch 1993
).
Since 1980, several reports have validated the use of the Caco-2 cell
culture system as a suitable model to study iron metabolism. It has
been demonstrated that Caco-2 cells growing on microporous membranes in
bicameral chambers transport iron from the apical to the basal chamber
in a process dependent on iron valence, concentration and the iron
status of the cell (Alvarez-Hernandez et al. 1991
,
Halleux and Scheider 1994
, Han et al. 1995a
). This cell system also synthesizes transferrin,
ferritin, and transferrin receptor and secretes apo-transferrin
(Halleux and Scheider 1989
). Iron uptake by Caco-2 cells
is affected by substances that inhibit or enhance iron absorption in
humans such as phytates, ascorbic acid and meat proteins
(García et al. 1996
, Han et al. 1994
, and Han et al. 1995b
).
A severe economic crisis that started in Venezuela in 1983
progressively reduced the quantity and quality of food consumed by the
Venezuelan population. A survey carried out during 1990 in the low
socioeconomic stratum of the population showed that the prevalence of
iron deficiency and anemia were 14.1 and 3.6%, respectively. Two years
later (1992), iron deficiency and anemia more than doubled: the
prevalence of iron deficiency was 36.6% and anemia 19%
(Layrisse et al. 1996
).
A nation-wide fortification program started in 1993. Precooked corn
and white wheat flours were enriched with iron and vitamins. The iron
source added to the fortification mixture was ferrous fumarate. It also
contained riboflavin, niacin, thiamin and vitamin A for precooked corn
flour but not for wheat flour. After 1 y of fortification there
was an impressive reduction in the prevalence of iron deficiency
(15.8%) and anemia (9.3%), compared to the prevalence found in 1992
(Layrisse et al. 1996
). Later studies in humans
(García-Casal et al. 1998
, Layrisse et al. 1997
) demonstrated that vitamin A was responsible for the
rapid improvement of the iron status of Venezuelan population. It was
shown that ß-carotene also could improve iron absorption from rice,
wheat and corn. Vitamin A and ß-carotene could overcome the
inhibition by phytates present in cereals and polyphenols present in
coffee infusions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Caco-2 cells, a human colon adenocarcinoma cell line (HTB 37) were donated by Dr. F. Liprandi (Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela). Cells were grown in Falcon T-75 tissue culture flasks (Beckton-Dickinson, Lincoln Park, NJ) in Dulbecco’s modified Eagle’s medium with high glucose (DMEM)3 plus 1 mmol/L nonessential amino acids, antibiotics/antimycotics, 2 mmol/L glutamine, 5 mmol/L HEPES, 0.4 mmol/L pyruvate and 15% fetal bovine serum (Gibco BRL, Gaithersburg, MD). After reaching 80–90% confluence, cells were trypsinized and seeded into bicameral chambers (Falcon cell culture inserts and plates, 25-mm diameter, 0.4-µm pore size. Beckton-Dickinson, Lincoln Park, NJ) or T-25 flasks (Beckton-Dickinson, Lincoln Park, NJ) in supplemented DMEM at a density of 5 x 104 cells/cm2 and fed every 2 d with supplemented DMEM. Some experiments were performed at 8 d in culture, after the cells (growing in flasks or inserts) formed a confluent monolayer. Other experiments were performed at 16 d in culture, 1 wk after the cells reached confluence. All experiments were done on passages 19–45.
Confluence assay.
The integrity of the monolayer was tested by optic microscopy and by the addition of 2 mL phosphate buffered saline (PBS) containing phenol red to the apical chamber and 2 mL PBS without phenol red to the basal chamber. After 2 h incubation at 37°C, the optical density of the basal chamber contents was measured at 546 nm to detect any leakage of the phenol red through the intercellular spaces.
Uptake experiments.
Unless otherwise indicated, experiments were performed in T-25 flasks containing Caco-2 cells cultured for 8 or 16 d. Cells were rinsed with PBS and incubated at 37°C for 1 h with 6 mL of an incubation mixture containing PBS adjusted to pH 5.5, 37 kBq 59Fe (NEN Life Sciences Products, Boston MA) and 3 µmol/L of iron as ferrous fumarate (equivalent to approximately 25x the iron content of the isotope, with slight variations depending on the specific activity of 59Fe). After 60 min incubation, media was removed, cells rinsed once with PBS containing 1 mmol/L Na2-EDTA, detached from flasks with trypsin-EDTA (0.5% trypsin + 5.3 mmol/L EDTA; Gibco BRL, Gaithersburg, MD) and counted in a Compugamma LKB gamma counter to determine iron uptake. Results were calculated as pmol Fe/mg cell protein (3.55 mg protein/flask and 0.7 mg protein/insert) and also as percentage of uptake based on iron initially added to the incubation media (37 kBq for flasks and 9.25 kBq for inserts).
Effect of ascorbic acid and days in culture. Iron uptake was evaluated in Caco-2 cells immediately after reaching confluence (8 d in culture). Ascorbic acid (AA) was added to the incubation media in 0, 0.6, 15 and 30 µmol/L (1:0 to 1:10 Fe:AA molar ratios) (n = 5). For cells cultivated during 16 d, experiments (n = 6) were performed at pH 5.5 with 0 or 6 µmol/L ascorbic acid (1:0 and 1:2 Fe:AA molar ratios).
Iron uptake from ferrous fumarate as a function of cold iron in the incubation media. The effect of increasing concentrations of cold iron in the incubation media was evaluated in Caco-2 cells after 2 wk in culture. To 6 mL incubation media, iron was added as ferrous fumarate at 0.6, 1.2, 3, 6, 12, 30, 60 and 120 µmol/L, corresponding to 59Fe:cold Fe molar ratios from 1:5 to 1:1000 (n = 3).
Effect of vitamin A, ß-carotene, tannic acid and phytic acid on iron uptake. Concentrations of vitamin A (vit A), tannic acid (TA) and phytic acid (Phy) for experiments with Caco-2 cells were calculated to maintain the proportions used in Venezuela to fortify precooked corn flour and prepare coffee (4 g coffee powder/50 mL water). For human iron absorption studies the administration of a bread prepared with 100 g of this flour provided 5 mg Fe, 0.55 mg vit A, 161.6 mg Phy and 215.8 mg TA (equivalent to 1:0.01 Fe:vit A, 1:2 Fe:Phy and 1:1.4 Fe:TA molar ratios). ß-carotene (ßC) was calculated to provide retinol equivalents similar to vit A (1.8 mg ßC/100 g flour. Fe: ßC 1:0.02). For Caco-2 experiments, a range of concentrations were evaluated based on these variables.
Caco-2 cell monolayers (8 or 16 d in culture) were incubated with 3, 6, 15, 30 and 60 µmol/L vit A as water soluble retinol palmitate (Roche Laboratories. Caracas, Venezuela) equivalent to 1:1, 1:2, 1:5, 1:10 and 1:20 iron:vit A molar ratios (n = 3 for 8 d in culture; n = 4 for 16 d in culture).
For experiments using ßC (10% cold water soluble powder. Roche Laboratories. Caracas, Venezuela), cells (16 d in culture) were incubated with 0.01, 0.02, 0.3, 0.9, 1.8, 3.0 and 6.0 µmol/L ßC freshly prepared and completely dissolved in the incubation media described before. These concentrations included Fe:ßC molar ratios from 1:0 to 1:2 (n = 6). From these experiments, 0.9 µmol/L (Fe:ßC molar ratio 1:0.3) and 3 µmol/L (Fe:ßC molar ratio 1:1) were used to perform experiments with TA and Phy (n = 6).
To study the inhibitory effect of coffee on iron uptake and to evaluate the role of ßC in overcoming this inhibition, TA (Sigma Chemicals, St. Louis, MO) was added to the incubation mixture mentioned above, containing 0.9 µmol/L ßC and 2.1, 4.2, 7.5 and 15 µmol/L TA, equivalent to 1:0.7, 1:1.4, 1:2.5, 1:5 and 1:10 Fe:TA molar ratios (n = 3–10). Cells for these experiments were used at 16 d in culture.
Phytic acid (Inositol hexaphosphoric acid, sodium salt. Sigma Chemicals, St. Louis, MO) was also evaluated incubating cells (16 d in culture) with a mixture containing iron prepared as mentioned before, 3 µmol/L ßC and 6, 12, 24 and 48 µmol/L Phy (1:2 to 1:16 Fe:Phy molar ratios) (n = 3–6).
Uptake in flasks and inserts.
All experiments described were performed in T-25 flasks to evaluate uptake. Some experiments were performed using flasks and inserts to compare uptake from both systems after 16 d in culture. Cells were incubated as described above with 6 (flasks) or 1.5 mL (inserts) of the same freshly prepared incubation mixture containing PBS pH 5.5, 6.2 MBq/L 59Fe and 3 µmol/L iron (25x the iron content of the isotope) as ferrous fumarate. vit A, ßC and AA were added to some flasks and inserts in 1.5, 3.0 and 6 µmol/L (1:0.5, 1:1 and 1:2 Fe:compound molar ratios), respectively (n = 5). In this way, flasks and inserts received proportional amounts of the compounds to be tested, based on differences in surface area (approximately 5 times higher for flasks).
Statistical analysis.
All measurements were expressed as means ± SEM. ANOVA with Bonferroni as post-test was used to compare uptake values for each group. Values were considered significantly different if P < 0.05. In experiments with increasing concentrations of AA, all uptakes were compared to one another. For ferrous fumarate, comparisons were made between different iron concentrations versus the iron concentration that achieved maximal uptake. For ßC, comparisons were made between different concentrations added and no ßC addition and also between each concentration tested. In experiments with TA and Phy, comparisons were made to evaluate the effect of ßC for the same concentration of inhibitor and also the effect of different concentrations of inhibitors with or without ßC. Paired t-test was used for comparisons between cells growing for 8 or 16 d and also for comparisons between experiments performed in flasks or inserts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AA significantly (P < 0.05) improved iron uptake by Caco-2 cells. After 8 d in culture uptake increased from 28.8 ± 1.7 pmol/mg cell protein without AA to 78.8 ± 1.4, 81.6 ± 0.6 and 84.8 ± 1.2 pmol/mg cell protein, when 6, 15 and 30 µmol/L of AA were added, respectively, to the incubation mixture. There was no difference due to the concentrations of ascorbic acid tested.
When experiments were performed in cells after 16 d in culture, the same significant effect was achieved in presence of AA. Iron uptake increased from 58.3 ± 1.8 to 103 ± 5.1 pmol/mg cell protein. Although the enhancing effect of AA on iron uptake was achieved for both 8 and 16 d in culture, uptake was significantly higher for 16 d in culture. This effect of days in culture on iron uptake was evident not only in presence of AA (78.8 ± 1.4 pmol/mg cell protein for 8 d in culture and 103.5 ± 5.1 pmol/mg cell protein for 16 d in culture), but also when iron uptake was evaluated alone. For 16 d in culture, iron uptake was significantly higher (58.3 ± 1.8-pmol/mg cell protein) than uptake at 8 d in culture (28.8 ± 3.4 pmol/mg cell protein).
Preliminary experiments conducted to establish optimal uptake conditions, for both 8 and 16 d in culture, showed that there was a significantly higher iron uptake when incubation media was adjusted to pH 5.5 compared to pH 6.5 or 7.5 (data not shown).
Iron uptake from ferrous fumarate as a function of cold iron in the incubation media.
As increasing days in culture improved iron uptake from ferrous
fumarate at pH 5.5, we decided to evaluate iron uptake under these
conditions for other experiments. Iron uptake was greater when added
iron increased from 0.6 to 12 µmol/L in the incubation mixture
(Fig. 1
).Uptakes from media containing 6 and 12 µmol Fe/L did not differ. The
following experiments were performed using 3 µmol/L iron as ferrous
fumarate (1:25 59Fe:cold iron molar ratio), at pH 5.5 and
with cells one week after reaching confluence (16 d in culture).
|
Iron uptake from ferrous fumarate in presence of vit A was evaluated using Caco-2 cells immediately after reaching confluence (8 d). There was no significant improvement in iron uptake when vit A was added to the incubation media. With no vit A addition uptake was 18.2 ± 3.0 pmol/mg cell protein. When vit A was added at 3, 6, 15, 30 and 60 µmol/L (1:1 to 1:20 iron: vit A molar ratios), uptake was 18.2 ± 2.1, 16.8 ± 1.5, 17.4 ± 1.4, 19.2 ± 2.0 and 26.1 ± 3.3 pmol Fe/mg cell protein, respectively.
When experiments were performed on Caco-2 cells 2 wk after reaching confluence (16 d in culture), uptake increased significantly to 61.1 ± 12.2 pmol/mg cell protein without vit A addition. However, as at 8 d in culture, vit A (3 µmol/L) did not affect iron uptake (62.8 ± 9.4 pmol Fe/mg cell protein).
The addition of 0.01 to 0.3 µmol/L ßC did not affect iron uptake
(Fig. 2
).However, addition of 0.9 µmol/L ßC (1:0.3 Fe:ßC molar ratio),
significantly (P < 0.05) increased uptake compared to
no ßC addition. Further increases in ßC resulted in significant
(P < 0.05) increases in iron uptake compared to ßC
addition. Differences were also significant for 3 and 6 µmol/L ßC
compared to the other concentrations tested.
|
).ßC (0.9 µmol/L) overcame the inhibition of iron uptake produced by
2.1 and 4.2 µmol/L TA.
|
).When (3 µmol/L) was added to incubation mixtures without Phy, iron
uptake was significantly greater. Increasing Phy concentrations in
presence of ßC did not affect this enhancement.
|
Iron uptake from ferrous fumarate alone or in the presence of vit A, ßC or vitamin C was evaluated in Caco-2 cells growing for 16 d in T-25 flasks or inserts, and there was no difference in iron uptake when flasks or inserts were used. When experiments were performed in T-25 flasks, uptake was 61.4 ± 3.4 pmol/mg cell protein for ferrous fumarate, 61.6 ± 8.8 pmol/mg cell protein for ferrous fumarate + vit A, 101.8 pmol/mg cell protein for ferrous fumarate + ßC and 87.9 ± 4.2 for ferrous fumarate + vitamin C. Uptake values for experiments performed in inserts were 51.1 ± 7.5, 56.3 ± 4.8, 97.2 ± 3.3 and 85.2 ± 7.3 pmol/mg cell protein, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Lissoos et al. 1995
, Quick and Ong 1990
) have reported that Caco-2 cells posses the machinery
required to internalize and esterify retinol including
retinol-binding protein II, lecithin-retinol acyltransferase
and Acyl-CoA retinol acyltransferase. Metabolism of ßC is still
controversial. As in normal enterocytes it can be taken up intact by
Caco-2 cells. The presence of ßC 15, 15' dioxygenase, the enzyme that
catalyzes the conversion of ß-carotene to retinal has been reported
(Quick and Ong 1990
). However, cells seem to lose this
capability with cell passages and culture conditions. Recently
During et al. (1998)
did not detect ßC conversion in
parental Caco-2 cells, but the activity was present in 2 subclones
(PF11 and TC7) at different levels.
This work characterizes iron uptake by Caco-2 cells from ferrous
fumarate, the iron compound used in Venezuela in a successful
fortification program that reduced iron deficiency and anemia 50% in
only one year (Layrisse 1996
). Although this improvement
was not due only to iron, it is important to evaluate the compound
because it has low cost and high bioavailability. In preliminary
experiments, iron uptake from ferrous fumarate and ferrous sulfate were
similar, in spite of differences in solubility (data not shown).
Availability of iron from fumarate is comparable to ferrous sulfate and
cost is about the same. Still, ferrous fumarate is moderately soluble
in aqueous solutions, which could be a problem for some purposes.
The effect of AA and days in culture was evaluated for ferrous fumarate
uptake. The enhancing effect of AA on iron uptake was reconfirmed
showing that uptake can be doubled when AA is added at optimal pH;
slight changes in pH widely modify uptake. In preliminary experiments
with ferrous fumarate, the highest uptake was achieved performing
experiments at pH 5.5. Uptake from ferrous sulfate is also optimal at
this pH (García et al. 1996
). Days in culture
are also an important issue to control in iron uptake experiments. As
shown with AA and vit A experiments, uptake was much higher when
performed at 16 d in culture, maybe due to a greater
differentiation, but even when there is not a complete differentiation
after 8 d in culture, iron uptake was greater in the presence of
AA. Moreover, although uptake values were higher at 16 d in
culture, results were comparable and consistent, i.e. if uptake was low
for a particular condition, it was low for both, 8 and 16 d in
culture. However, it is important to highlight that experiments should
be performed after 2 wk in culture when cells have achieved greater
differentiation.
For uptake experiments, culture flasks or inserts have been extensively used. In this work, experiments were planned to measure uptake the same day, under similar experimental conditions at the same cell passage using flasks and inserts. We found that there were no significant differences in uptake when either one was used. Maybe for longer incubation periods, when accumulation of metabolites could occur or because of by-product inhibition, the use of flasks is not advisable. However, for shorter incubations and to evaluate uptake, flasks can be the choice because they are less expensive and easier to handle.
In the experiments performed, concentrations of vit A, Phy, TA and ßC were calculated to maintain iron: compound molar ratios similar to those used in Venezuelan fortification program. ßC was added to provide a concentration similar (as retinol equivalents) to vit A.
ßC had a direct effect on iron uptake, probably due to a chelating effect that increased iron solubility and/or availability. There was a more than 100% increase in uptake when ßC (3 and 6 µmol/L) was included in the incubation mixture. Moreover, even in the presence of potent inhibitors of iron absorption, ßC was able to overcome this effect.
Our results did not show any significant improvement on iron uptake
when vit A was added to the incubation media, although vit A was shown
to have an enhancing effect in human studies (Layrisse et al. 1997
). Further experiments should be conducted under different
conditions and handling of vit A to definitely conclude that the
enhancing effect clearly showed in humans does not occur in Caco-2
cells. It is possible that some vit A degradation could occur during
experimental procedures or storage.
Vit A has been reported to have a direct effect on iron metabolism
(Koessler et al. 1925, Sure et al. 1929).
Deficiency of this vitamin produces anemia that can be reversed by the
administration of vit A without changes in iron intake
(Mejía and Arroyave 1982a
). This effect is due
to a reduction in iron incorporation into erythrocytes with elevation
of iron stores (Mejía and Arroyave 1982b
).
Previously we reported a new role of vit A and ßC in improving iron
absorption in humans (García-Casal et al. 1998
,
Layrisse et al. 1997
) and this study corroborates the
effect of ßC in Caco-2 cells.
The impressive decrease in iron deficiency and iron deficiency anemia
reported in Venezuela after only 1 y of a National fortification
program (Layrisse et al. 1996
) was difficult to explain
only because iron was administered in the fortification mixture. Later,
it was confirmed that vit A could keep iron in solution in the presence
of iron absorption inhibitors (phytates from flour and polyphenols from
coffee) (Layrisse et al. 1997
). ßC also has this
capability and is more effective that vit A in this regard
(García-Casal et al. 1998
).
The mechanisms responsible for this enhancing effect are still unknown. There is a clear effect of vit A and ßC in keeping iron soluble. This effect has been demonstrated in tests performed in our laboratory when soluble iron is measured after pH changes from 2 to 6 in the presence or absence of vit A and ßC. If iron associates with these compounds during the digestive process, iron would be less available to bind absorption inhibitors such as phytates and polyphenols. Also, it has to be demonstrated if these compounds have a role in the absorption process itself.
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FOOTNOTES |
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3 Abbreviations used: AA, ascorbic acid; ßC,
ß-carotene; DMEM, Dulbecco’s modified Eagle’s medium; Phy, phytic
acid; TA, tannic acid, Vit A, vitamin A.
Manuscript received February 17, 1999. Initial review completed April 22, 1999. Revision accepted September 3, 1999.
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