1,25 dihydroxycholecalciferol-mediated calcium absorption and gene expression are higher in female than in male mice

Recent advances in bone and calcium (Ca) metabolism have relied upon genetically modified mice. However, although human studies have identified gender as an important modulator of Ca metabolism, its effect on Ca metabolism has not been examined in mice. Here we examined basal and vitamin D-regulated Ca absorption (in situ ligated loops) and mRNA levels for the apical membrane calcium channel, TRPV6, and the calcium binding protein, calbindin D(9k) (CaBP) mRNA levels (real-time PCR) in duodenum of female and male mice. At 2 mo of age, females fed a 5 g Ca/kg diet had higher Ca absorption (62.3 +/- 4.8 vs. 47 +/- 3.6%) and TRPV6 mRNA levels than males even though plasma 1,25 dihydroxyvitamin D [1,25(OH)(2) D] was not different. In mice fed high (20 g/kg), normal (5 g/kg), or low (0.2 g/kg) Ca diets for 7 d to alter plasma 1,25(OH)(2) D (91 +/- 12, 322 +/- 25, and 587 +/- 43 pmol/L, respectively), the relation between Ca absorption (slope = 0.116 vs. 0.084, P = 0.021) or duodenum TRPV6 mRNA (slope = 0.042 vs. 0.025, P = 0.034) and circulating 1,25(OH)(2) D was steeper in females. After a single 1,25(OH)(2) D injection (200 ng/100 g body weight), peak induction of TRPV6 mRNA was 2-fold greater (at 6 h) and CaBP mRNA was 20% higher in females (at 16 h). Duodenal vitamin D receptor mRNA levels did not differ between genders. Our data indicate that female mice are more sensitive to changes in serum 1,25(OH)(2) D levels than males and that this must be considered when using mice to study calcium and bone biology.     

Intestinal calcium and lead absorption: effects of dietary lead and calcium

The combined effects of dietary calcium (Ca) and lead (Pb) status on intestinal Ca and Pb absorption and related parameters were investigated in young growing chicks. Dietary Pb intake resulted in two remarkable, apparently independent and essentially opposite effects on intestinal Ca and Pb absorption, depending on dietary Ca and Pb levels and duration of treatment. The initial response (1 week) to Ca deficiency was stimulated Ca absorption and calbindin-D level, regardless of dietary Pb intake. The later response (2 weeks) was a reversal, by Pb, of the early phase stimulation. Intestinal Pb absorption was similarly enhanced by Ca deficiency initially, and this response was also inhibited by prolonged dietary Pb intake. Ingestion of Pb by chicks fed adequate Ca resulted in generally elevated intestinal Ca absorption and calbindin-D levels after both 1 and 2 weeks. Intestinal Pb absorption was also increased in the adequate Ca situation, but only after 2 weeks at the lower levels of dietary Pb. The results underscore the complicated nature of Pb-Ca interactions and demonstrate the importance of thorough characterization of the animal model system.     

Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA

To study the role of calbindin D(9k) (CaBP) and epithelial calcium channel ECaC2 in intestinal calcium (Ca) absorption, vitamin D receptor knockout (KO) and wild-type (WT) mice were fed either 0.5% Ca or a 2.0% Ca rescue diet starting at 21 d of age. Ca absorption and parameters involved in this process were measured at 60 or 90 d of age. Compared with WT, KO mice fed the 0.5% Ca diet had higher plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], and lower plasma Ca and insulin-like growth factor-I (IGF-I). Duodenal Ca absorption (% Ca absorbed) in KO mice was reduced 71% relative to WT mice and was associated with 55% lower CaBP mRNA, 47% lower CaBP protein and 95% lower ECaC2 mRNA levels. Compared with WT mice, the percentage of Ca absorbed in KO mice fed the 0.5% Ca diet was inappropriately low for the level of duodenal CaBP. The 2% Ca rescue diet normalized plasma Ca, prevented osteomalacia, increased growth and plasma IGF-I levels, but did not normalize plasma PTH or 1,25(OH)(2)D(3) in KO mice. In addition, the relationship between CaBP protein and the percentage of Ca absorbed was normalized, whereas ECaC2 mRNA fell to near zero. Our data demonstrate that higher CaBP levels do not ensure high rates of duodenal Ca absorption and that transcellular Ca absorption can occur even when ECaC2 gene expression is very low. In addition, our data suggest that the 2% Ca diet promotes a vitamin D receptor-independent anabolic effect on bone formation and calcium absorption, leading to improved calcium balance even in the presence of high PTH levels.     

Opposing effects of ethanol and nicotine on hippocampal calbindin-D28k expression.

Long-term ethanol exposure produces multiple neuroadaptations that likely contribute to dysregulation of Ca(2+) balance and neurotoxicity during ethanol withdrawal. Conversely, nicotine exposure may reduce the neurotoxic consequences of Ca(2+) dysregulation, putatively through up-regulation of the Ca(2+)-buffering protein calbindin-D(28k). The current studies were designed to examine the extent to which 10-day ethanol exposure and withdrawal altered calbindin-D(28k) expression in rat hippocampus. Further, in these studies, we examined the ability of nicotine, through action at alpha(7)(*)-bearing nicotinic acetylcholine receptors (nAChRs), to antagonize the effects of ethanol exposure on calbindin-D(28k) expression. Organotypic cultures of rat hippocampus were exposed to ethanol (50-100 mM) for 10 days. Additional cultures were exposed to 500 nM (-)-nicotine with or without the addition of 50 mM ethanol, 100 nM methyllycaconitine (an alpha(7)*-bearing nAChR antagonist), or both. Prolonged exposure to ethanol (>/=50 mM) produced significant reductions of calbindin-D(28k) immunolabeling in all regions of the hippocampal formation, even at nontoxic concentrations of ethanol. Calbindin-D(28k) expression levels returned to near-control levels after 72 h of withdrawal from 10-day ethanol exposure. Extended (-)-nicotine exposure produced significant elevations in calbindin-D(28k) expression levels that were prevented by methyllycaconitine co-exposure. Co-exposure of cultures to (-)-nicotine with ethanol resulted in an attenuation of ethanol-induced reductions in calbindin-D(28k) expression levels. These findings support the suggestion that long-term ethanol exposure reduces the neuronal capacity to buffer accumulated Ca(2+) in a reversible manner, an effect that likely contributes to withdrawal-induced neurotoxicity. Further, long-term exposure to (-)-nicotine enhances calbindin-D(28k) expression in an alpha(7)* nAChR-dependent manner and antagonizes the effects of ethanol on calbindin-D(28k) expression.     

The effect of marine oil-derived n-3 fatty acids on transepithelial calcium transport in Caco-2 cell models of healthy and inflamed intestines

Marine oil-derived n-3 fatty acids have been shown to stimulate intestinal Ca absorption in animal studies, but the effects of such fatty acids on Ca absorption in human subjects are relatively unknown. In particular, n-3 fatty acids may be of therapeutic value for some Crohn's disease patients who experience Ca malabsorption. Therefore, the aim of the present study was to investigate the effect of 20 : 5n-3 and 22 : 6n-3 on transepithelial Ca transport across monolayers of healthy Caco-2 cells as well as of TNF-alpha-treated Caco-2 cells (an in vitro model of Crohn's disease). Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers, which were treated with 80 microM-20 : 5n-3, 80 microM-22 : 6n-3, or 40 microM-20 : 5n-3 + 40 microM-22 : 6n-3 for 6 or 8 d, with or without co-treatment with TNF-alpha (10 ng/ml) (n 11-15 monolayers per treatment). On day 16, transepithelial and transcellular transport of 45Ca and fluorescein transport (a marker of paracellular diffusion) were measured. Treatment of healthy and inflamed Caco-2 cells with 20 : 5n-3, 22 : 6n-3 and both fatty acids combined for 8 d significantly (P < 0.005-0.01) increased total transepithelial Ca transport compared with that in control, effects which were mediated by an enhanced rate of transcellular Ca transport. The effects of n-3 fatty acids on Ca absorption after 6 d were less clear-cut. In conclusion, the present in vitro findings highlight the need to investigate the effect of marine oil-based n-3 fatty acids on Ca absorption in vivo in studies of healthy human subjects as well as of Crohn's disease patients.     

Plasma 1,25-(OH)2-vitamin D concentrations and net intestinal calcium, phosphate, and magnesium absorption in humans.

We evaluated the relationship between plasma concentrations of the renal hormone 1,25-(OH)2-vitamin D and net intestinal absorption of Ca, PO4, and Mg in vitamin D-replete patients eating similar diets, who had undetectable, normal or elevated plasma 1,25-(OH)2-D levels, Net intestinal Ca absorption was positively correlated to plasma 1,25-(OH)2-D concentrations: percentage dietary Ca absorbed = 10 + 0.17 x plasma total 1,25-(OH)2-3, pmole/liter, r = + 0.58; P less than 0.001. By contrast, there was no significant correlation between PO4 or Mg absorption and plasma 1,25-(OH)2-D concentrations. Moreover, significant quantities of PO4 and Mg were absorbed in the absence of detectable plasma 1,25-(OH)2-D. We conclude that net intestinal Ca absorption is critically dependent upon the availability of the renal hormone 1,25-(OH)2-D in vitamin D-replete humans when dietary Ca intake is normal. By contrast, other factors must play a dominant role in regulating net intestinal PO4 and Mg absorption.     

Net calcium absorption in premature infants: results of 103 metabolic balance studies.

Net calcium absorption was evaluated in 103 low-birth-weight preterm infants by a 72-h balance technique. At birth the infants had a mean (+/- SE) gestational age of 30.9 +/- 0.2 wk and weighed 1.43 +/- 0.03 kg. When tested 3 wk later, their net calcium absorption averaged 58 +/- 1% with an intake of 80 +/- 2 mg Ca.kg body wt-1.d-1. Of the 103 infants, 58 had been fed low-birth-weight formulas supplemented with vitamin D. The remainder received banked human milk, of whom 34 were supplemented with vitamin D and calcium; 11 infants received no supplementation. Calcium absorption in the four subgroups did not differ significantly, with neither vitamin D supplementation nor supplementation with vitamin D and calcium affecting percent absorption significantly. Net calcium absorption was a linear function of intake (40-130 mg Ca.kg body wt-1.d-1) with a zero intercept. Because vitamin D supplementation did not increase net calcium absorption, it is concluded that in preterm low-birth-weight infants calcium absorption proceeds by a nonsaturable route, with the transcellular, vitamin D-regulated mechanism not yet expressed.     

Enhancement of sulfhydryl group availability in the intestinal brush border membrane by deficiencies of dietary calcium and phosphorus in chicks

The reactivity and availability of sulfhydryl (-SH) groups in brush border membranes (BBM) from chicks adapted to a calcium-deficient (low Ca) or a phosphorus-deficient (low P) diet were determined. The calbindin-D28K concentrations of the intestinal mucosa of the low Ca and low P groups were both increased approximately 2.5-fold, demonstrating that adaptation to the mineral deficiencies had occurred. By the Ellman reaction, a threefold increment in -SH groups in BBM from both mineral-deficient groups was noted. By using DACM (N-7-dimethylamino-4-methylcoumarin-3-yl maleimide), a fluorescent probe for -SH groups, it was observed that fluorescence development was considerably greater with BBM from the low Ca and low P groups than with BBM from the controls, whether measured in the absence or presence of sodium dodecyl sulfate (SDS). In the absence of SDS, the pseudo-first-order reaction rate constants, k', calculated from the fluorescence data, were greater than the control group values, but in the presence of SDS, the k' values for all groups were about the same. Similar changes in BBM-SH groups were previously observed when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient chicks. The redox state of the sulfhydryl groups in enzymes and transport proteins is known to affect the level of their activity. The functional significance of the present observations concerning the -SH groups of chick intestinal BBM, particularly in relation to vitamin D-dependent calcium and phosphorus absorption, is not known but is under investigation.     

Effects of vitamin D on calcium regulation in vitamin-D-deficient pigs given a phytate-phosphorus diet

1. Vitamin-D-deficient pigs were fed on a phytate-phosphorus diet and treated with vitamin D3 (+D) to examine the time-course of adaptative changes in plasma minerals, vitamin D metabolites, parathyroid hormone (PTH) and calcium balance and intestinal Ca-binding protein (CaBP). 2. The 5-week vitamin D repletion (25 micrograms cholecalciferol/kg diet) regimen restored plasma Ca, P and alkaline phosphatase (EC 3.1.3.1) to normal, decreased PTH and markedly and rapidly increased plasma 25-hydroxycholecalciferol (25-OHD, sevenfold after 4 d) and 1,25-dihydroxycholecalciferol (1, 25(OH)2D3, 1.8-fold after 4 d). 3. CaBP concentrations were markedly elevated all along the digestive tract, especially in the distal regions. 4. Ca absorption and retention were enhanced (fourfold and sixfold respectively) by the +D diet. 5. The improved Ca absorption, coupled with increased CaBP and 1,25(OH)2D3 levels, suggest that vitamin D metabolism in phytate-P-fed pigs is sensitive to the depressed Ca availability due to phytate feeding. It also indicates that CaBP may play an important role in the adaptation of Ca absorption. 6. Persistent hypercalciuria indicates that mineral metabolism was still affected by the phytate nature of the dietary P in spite of the vitamin D treatment.     

Various non-digestible saccharides increase intracellular calcium ion concentration in rat small-intestinal enterocytes

We have previously shown that non-digestible saccharides (NDS) stimulate intestinal Ca absorption via tight junctions. However, the cellular mechanisms activated by the NDS are not yet known. We investigated the effects of four NDS, difructose anhydride (DFA) III, DFAIV, fructo-oligosaccharides, and maltitol, on intracellular Ca signalling in isolated rat small-intestinal enterocytes. The changes in intracellular Ca(2+) concentration were measured before and after the addition of capric acid (7.5 or 15 mmol/l, a positive control), glycerol, or each NDS (1 or 10 mmol/l) to fura-2-loaded enterocytes. Treatment with capric acid or each NDS caused an immediate and dose-dependent rise in intracellular Ca(2+) concentration. Mechanical and osmotic stimulation achieved by adding glycerol had no effect on intracellular Ca(2+) concentration. The intracellular Ca(2+) concentration in enterocytes treated with DFAIII and fructo-oligosaccharides reached a peak level at about 30 s after stimulation, but those treated with DFAIV and maltitol showed further increases after the initial rapid rise. The maximum change in intracellular Ca(2+) concentration obtained by the application of maltitol was higher than that of DFAIII at 10 mmol/l. These findings suggest that each of the four NDS directly stimulates rat enterocytes, and increases intracellular Ca(2+) concentration. Thus, molecular structure may be more important than the size of the NDS in the induction of Ca signalling in the cells.     

Gastrointestinal absorption of lead in chicks: involvement of the cholecalciferol endocrine system

The role of dietary calcium and phosphorus in modifying the intestinal absorption of lead and also the effect of lead ingestion on the metabolism of cholecalciferol were studied in chicks. The efficiency of absorption of 203Pb and 47Ca was increased when the animals were fed a low calcium diet and treated with cholecalciferol. The synthesis of the vitamin D-induced calcium-binding protein (CaBP) was correspondingly increased. When the chicks were depleted of vitamin D and repleted with 1,25-dihydroxycholecalciferol [1,25(OH)2D3] as their only source of the vitamin, the absorption of both 47Ca and 203Pb was unaffected by dietary calcium levels, and no change in CaBP levels occurred. Low dietary intake of phosphorus resulted in an increase in 47Ca and 203Pb absorption and in CaBP synthesis when the animals were treated with cholecalciferol. However, when the birds were repleted with 1,25(OH)2D3, the intestinal absorption of 47Ca and of 203Pb was increased, as well as the intestinal CaBP levels. Intracardial injection of increasing doses of 1,25(OH)2D3 to rachitic chicks resulted in a concomitant increase in 203Pb absorption in a manner that correlated with the degree of synthesis of CaBP. Ingestion of lead by the chicks was found to impair growth and renal production of 1,25(OH)2D3, resulting in lowered circulating and intestinal content of the hydroxylated metabolites of cholecalciferol.     

Effects of habitual chitosan intake on bone mass,bone-related metabolic markers and duodenum CaBP D9K mRNA in ovariectomized SHRSP rats

We have demonstrated that the habitual intake of chitosan can decrease bone mass in ovariectomized (OVX) SHRSP rats fed a low-Ca diet (0.1%). In the present study, we examined both the etiology of bone loss induced by dietary chitosan and the preventive effect of vitamin C supplementation. Rats were OVX and maintained on one of the following diets for 6 wk: 10% cellulose (CE). 10% chitosan (CH) or 10% chitosan with sodium ascorbate (CHVC). CH caused a significant reduction in bone mineral density (BMD) and stiffness in femurs and the fourth lumbar vertebrae (L4). There was no significant difference in intestinal Ca absorption between CH and CE, whereas CH intake significantly reduced intestinal P absorption. The bone loss in CH rats was accompanied with an increase in urinary Ca excretion and a decrease in serum Ca as well as a significant increment In serum PTH and 1,25(OH)2D3. The vitamin D receptor and calcium binding protein D9K mRNAs were also significantly increased in the duodenum of CH rats. Vitamin C supplementation to CH caused an increase in the Ca and P contents of femurs as well as BMD of the L4, with a decrease in urinary Ca excretion. These results indicate that dietary chitosan with low Ca intake possibly induces the loss of bone mass by enhancing urinary Ca excretion rather than by inhibiting Ca absorption, and that vitamin C supplementation could prevent bone loss caused by chitosan through the increment of retained Ca followed by suppression of urinary Ca excretion.     

Bioavailability of vitamin B₁₂ in cows' milk

The natural source of vitamin B?? in human diets comes from animal products. For example, one glass (250 ml) of milk provides approximately 50 % of the RDA (2·4 μg/d). It was hypothesised that the provision of vitamin B?? from milk is more efficiently absorbed than the synthetic form used in vitamin supplements. Pigs (n 10) were used as a model for intestinal absorption of vitamin B?? in humans to compare the net fluxes of vitamin B?? across the portal-drained viscera (PDV; an indicator of intestinal absorption) after ingestion of meals complemented with conventional and vitamin B??-enriched (via injections to cows) milk (raw, pasteurised or microfiltrated) or with equivalent amounts of cyanocobalamin, the synthetic form used in supplements or unsupplemented. Net flux of vitamin B?? across PDV after the ingestion of milk was positive, though not influenced by milk enrichment (P>0·3) or technological processes (P = 0·8) and was greater than after ingestion of equivalent amounts of cyanocobalamin (cyanocobalamin v. all milk, P ≤ 0·003). In fact, net fluxes of this vitamin were not different from 0 after either cyanocobalamin or the meal devoid of vitamin B?? (unsupplemented v. cyanocobalamin, P = 0·7). The cumulative PDV fluxes during the 24 h following ingestion of meals complemented with milk varied from 5·5 to 6·8 μg. These values correspond to an efficiency of intestinal absorption of vitamin B?? from milk varying between 8 and 10 %. Therefore, vitamin B??, which is abundant in cows' milk, is also substantially more available than the most commonly used synthetic form of this vitamin.     

The effect of oestrogen and dietary phyto-oestrogens on transepithelial calcium transport in human intestinal-like Caco-2 cells

Recently, dietary phyto-oestrogens (PO) have been suggested as possible alternatives to oestrogen therapy (hormone replacement therapy) as a means of preventing bone loss associated with ovarian hormone deficiency. While PO, which exhibit oestrogen-like activity, act directly on bone cells, their protective effect on bone may be partly due to their ability to enhance Ca absorption. Therefore, the aim of the present study was to investigate the effect of 17beta-oestradiol and two commonly consumed soyabean PO (genistein and daidzein) on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 8-18 per treatment), grown in oestrogen-replete or -deplete media, were then exposed to 10 nm-17beta-oestradiol, 1 nm-1,25-dihydroxycholecalciferol, or 50 micro m-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of 45Ca and fluorescein transport (a marker of paracellular diffusion) were measured. As expected, 1,25-dihydroxycholecalciferol stimulated Ca absorption in Caco-2 cells, by up-regulating transcellular transport, whereas 17beta-oestradiol had no effect on Ca absorption. Unexpectedly, both PO decreased Ca absorption (by about 17-19 % compared with control, P<0.05), by reducing transcellular Ca transport in Caco-2 cells grown in oestrogen-replete media. This inhibitory effect disappeared when monolayers were grown in oestrogen-deplete media. In conclusion, PO at high luminal concentrations either had no effect or reduced Ca absorption in Caco-2 cells, dependent on oestrogen status. The effect of lower concentrations of these compounds needs to be investigated.     

The effect of conjugated linoleic acid and medium-chain fatty acids on transepithelial calcium transport in human intestinal-like Caco-2 cells

Capric (10:0) and lauric (12:0) acid and conjugated linoleic acid (CLA) have been shown to increase paracellular permeability across human intestinal-like Caco-2 cell monolayers. While this has generated interest in terms of improved drug absorption and delivery, little has been done in terms of their potential effect on nutrient transport across the intestinal epithelium. Therefore, the objective of the present study was to investigate the effect of these fatty acids on transepithelial Ca transport in Caco-2 cells. Cells were seeded onto permeable transport membranes and allowed to differentiate, over 21 d, into intestinal-like cell monolayers. Monolayers (n 9 per treatment) were exposed to 0 (control) or 80 microm-10:0, 80 microm-12:0, 80 microm-18:2, 80 microm-CLA (mixed isomers), 80 microm-cis-9,trans-11 CLA or 80 microm-trans-10,cis-12 CLA for 22 d after seeding (chronic effect) or for 24 h before Ca transport studies (acute effect) on day 22. After exposure, transepithelial and transcellular transport of 45Ca, fluorescein transport (a marker of paracellular Ca transport) and transepithelial electrical resistance (TEER, an indicator of permeability) were measured. Overall Ca transport and TEER in Caco-2 cells were unaffected by exposure to any of the fatty acids for 24 h, or to 18:2, CLA blend or cis-9,trans-11 CLA for 22 d. Paracellular (but not total transepithelial and transcellular) Ca transport across Caco-2 cells was significantly increased (P<0.01, by about 1.5-fold relative to the control value) by exposure to 10:0, 12:0 and trans-10,cis-12 CLA for 22 d, suggesting that these non-esterified fatty acids could potentially enhance Ca absorption in vivo.     

Lack of dose-responsive effect of dietary phyto-oestrogens on transepithelial calcium transport in human intestinal-like Caco-2 cells

Ca absorption has been shown to be unaffected by high luminal concentrations of two commonly consumed soyabean phyto-oestrogens (PO) (genistein and daidzein) in Caco-2 cells grown under oestrogen-depleted conditions. However, these compounds exhibit dose-dependent biphasic effects in some tissues, such as reproductive tissue and bone. Thus, in light of this biphasic activity, the effect of lower concentrations of genistein and daidzein on Ca absorption requires further investigation. Therefore, the aim of the present study was to investigate the effect of a range of concentrations of genistein and daidzein on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 12 per treatment), grown in oestrogen-deplete media, were then exposed to 10 nm-1,25-dihydroxycholecalciferol (1,25 (OH)2D3), or 1, 10 and 50 microm-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of (45)Ca and fluorescein transport were measured. As expected, 1,25 (OH)2D3 stimulated Ca absorption in Caco-2 cells, by up regulating transcellular transport. Ca absorption was unaffected by either PO at luminal concentrations of 1, 10 or 50 microm, typical of intakes by Western and Asian populations as well as supplemental levels, respectively. The results of this model suggest that the proposed beneficial effects of supplemental levels of these PO compounds on bone mass in postmenopausal women more probably arise from direct effects on bone cells, and not by an indirect effect of these compounds on Ca absorption.     

Ascorbic acid offsets the inhibitory effect of bioactive dietary polyphenolic compounds on transepithelial iron transport in Caco-2 intestinal cells

We previously reported that (-)-epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) at high concentration nearly blocked intestinal iron transport across the enterocyte. In this study, we aimed to determine whether small amounts of EGCG, GSE, and green tea extract (GT) are capable of inhibiting iron absorption, to examine if ascorbic acid counteracts the inhibitory action of polyphenols on iron absorption, and to explore the mechanisms of polyphenol-mediated apical iron uptake and basolateral iron release. An(55)Fe absorption study was conducted by adding various concentrations of EGCG, GSE, and GT using Caco-2 intestinal cells. Polyphenols were found to inhibit the transepithelial (55)Fe transport in a dose-dependent manner. The addition of ascorbic acid offset the inhibitory effects of polyphenols on iron transport. Ascorbic acid modulated the transepithelial iron transport without changing the apical iron uptake and the expression of ferroportin-1 protein in the presence of EGCG. The polyphenol-mediated apical iron uptake was inhibited by membrane impermeable Fe(2+) chelators (P < 0.001), but at a low temperature (4°C), the apical iron uptake was still higher than the control values at 37°C (P < 0.001). These results suggest that polyphenols enhance the apical iron uptake partially by reducing the conversion of ferric to ferrous ions and possibly by increasing the uptake of polyphenol-iron complexes via the energy-independent pathway. The present results indicate that the inhibitory effects of dietary polyphenols on iron absorption can be offset by ascorbic acid. Further studies are needed to confirm the current findings in vivo.