Dietary heme-iron is principally provided by meat, blood-derived products and other animal tissues. It is well known that heme-iron absorption is relatively unaffected by other dietary factors that are common inhibitors of mineral absorption, such as phytates and fiber (Torre et al., 1991
). The best known dietary factors that affect intestinal heme iron absorption are meat and calcium (Lopez and Martos, 2004
). Heme iron absorption is increased by the presence of meat (Carpenter and Mahoney, 1992
). In contrast, calcium is known to inhibit heme iron absorption in the same fashion that it inhibits non-heme iron at high concentrations (Hallberg et al., 1991
). Although most research on the relationship between dietary factors and iron absorption has focused on non-heme iron, few studies have been conducted on heme iron.
An early study indicated that heme iron absorption decreased due to the consumption of tea in humans (Disler et al., 1975
), and this finding was confirmed by our recent study (Ma et al., 2010
). We previously reported that dietary polyphenolic compounds (such as EGCG, a major polyphenolic compound in green tea, and GSE) markedly inhibited heme iron absorption when polyphenolic compounds were added at the concentration of 46 mg/L. Because many dietary factors modulate iron absorption in a dose-dependent manner (Hallberg et al., 1989
; Tuntawiroon et al., 1991
), we next investigated whether these selected bioactive polyphenolic compounds retained the ability to reduce heme iron absorption when added at lower concentrations. Our current data clearly indicated that EGCG and GSE inhibit heme iron absorption in a dose-dependent manner in human intestinal cells (). Similarly, GT, the natural source of EGCG, also significantly decreased heme iron absorption in a dose-dependent way. Even at very low concentrations (0.46 mg/L), EGCG, GSE and GT significantly reduced heme iron transport across the cell monolayer during a 7 h transport assay. According to the IC50
values, the transepithelial heme iron transport across the cell monolayer can be reduced 50% by 3.6, 3.0 and 5.1 mg/L of EGCG, GSE and GT, respectively.
Because the inhibitory effects of dietary factors on iron absorption can be offset or reversed by ascorbic acid, the most prominent dietary factor that enhances iron absorption, we next examined whether the inhibitory action of polyphenols on heme iron absorption can be counteracted by ascorbic acid. The addition of 100 μmol/L ascorbic acid completely reversed the inhibitory action of dietary polyphenols on heme iron absorption when the polyphenolic compounds were added at 0.46 mg/L. When the concentrations of polyphenols were increased to 4.6 mg/L, ascorbic acid was not able to counteract the inhibitory action of polyphenols on heme iron absorption, although the inhibition was reduced. However, ascorbic acid failed to have any positive effect on heme iron absorption when the polyphenols were added at a high (but still within physiological) level of 46 mg/L. These results imply that, while the inhibitory effect of low concentrations of bioactive polyphenols on heme iron absorption can be easily counteracted by ascorbic acid, the inhibitory action of high concentrations of polyphenolic compounds cannot be offset by regular consumption of dietary ascorbic acid.
Because the addition of ascorbic acid enhanced heme iron absorption above the control in the presence of the low level of polyphenolic compounds, we investigated the effect of ascorbic acid on heme iron absorption in the absence of dietary polyphenols. We found that ascorbic acid markedly enhanced heme iron transport across the cell monolayer without altering the apical uptake of heme. The applied 55
Fe- labeled heme remained intact because the free iron was not detected in solution containing heme-55
Fe. This finding is similar to a previous study on the effect of soy protein on heme iron absorption. Lynch et al. (1985)
showed a significant increase in heme iron absorption by soy protein in human subjects (Lynch et al., 1985
). However, the mechanism of the soy protein-mediated increase of heme iron absorption still remains to be explored.
To examine the possible mechanism by which ascorbic acid enhances heme iron absorption, we initially analyzed the expression of proteins involved in heme iron absorption and metabolism. Because these test solutions modulate heme transport across the cell monolayer without altering the apical heme uptake, we assessed the proteins involved in heme splitting, cellular iron metabolism and basolateral iron transport. Our western blot analysis data indicated that neither ascorbic acid nor selected polyphenolic compounds changed the expression of proteins involved in heme iron absorption and metabolism.
After heme enters the cell across the apical membrane of the enterocyte, it is then degraded by HOs to release ferrous iron and bilirubin, and the released iron enters into the soluble cytoplasmic pool of the enterocyte (Raffin et al., 1974
). It is likely that, after heme iron is disassembled by HOs, the liberated iron enters the same storage and export pathways as does non-heme iron. Duodenal HO activity was previously proposed as a limiting factor for heme iron absorption and shown to be linearly associated with heme iron absorption (Raffin et al., 1974
; Wheby and Spyker, 1981
). Therefore, to determine whether ascorbic acid and/or polyphenols modulate heme iron absorption by affecting the release of iron from heme, we assessed HO activity in cells treated with ascorbic acid and/or polyphenolic compounds. In the conventional HO assay, enzyme activity is measured by the rate of bilirubin formation, as iron release from heme is linearly associated with production of bilirubin (Raffin et al., 1974
). As indicated by , HO activity was not changed by ascorbic acid and polyphenolic compounds, suggesting that the addition of ascorbic acid and polyphenols modulated heme iron absorption without changing heme splitting in Caco-2 cells.
Our current data clearly indicate that ascorbic acid enhances heme iron absorption across the cell monolayer without modulating the apical heme uptake, indicating that ascorbic acid may affect cellular or basolateral events that increase heme-derived free iron export. The addition of ascorbic acid may enhance heme iron absorption by modulating several different steps in the cell, such as 1) by increasing the release of iron from heme, 2) by inducing basolateral iron export though an increase in FPN-1 expression, and 3) by facilitating the transfer of iron to the basolateral membrane. Because our western blot analysis data () and HO activity data () do not support the first two possible mechanisms, it is possible that ascorbic acid may increase the available iron pool for basolateral iron transporter or improve iron transport to the basolateral membrane.
It is believed that ascorbic acid mainly enhances non-heme iron absorption by reducing ferric to ferrous iron, a substrate of DMT-1 in the gastrointestinal lumen, and many studies have confirmed this conclusion (Raja et al., 1992
; Han et al., 1995
). A study (Han et al., 1995
) previously demonstrated that ascorbic acid enhanced non-heme iron absorption by reducing ferric to ferrous iron and then increasing the apical iron uptake. However, the iron transported across the basolateral membrane was also enhanced (Han et al., 1995
). Although the transepithelial iron transport across the Caco-2 cell monolayer was increased by 3.5-fold, the apical iron uptake was only elevated less than 2-fold compared with the control, indicating that ascorbic acid enhanced not only the apical uptake but also the basolateral transport. It is clear that ascorbic acid increases the apical uptake of non-heme iron, but it is unknown how ascorbic acid enhances the basolateral transport of iron. One proposed explanation is that ascorbic acid increases the basolateral iron transport by increasing the assimilation of iron into the cell (Han et al., 1995
). In the current study, ascorbic acid enhanced the transepithelial iron transport without changing the apical heme uptake, HO activity or the level of FPN-1, suggesting that ascorbic acid may facilitate iron transfer to the basolateral membrane, leaving more free 55
Fe available for the iron exporter FPN-1. Together, our results indicate that small amounts of polyphenolic compounds in foods are capable of reducing heme iron transport across the intestinal enterocyte. However, these inhibitory effects of dietary polyphenolic compounds on heme iron absorption can be offset by ascorbic acid, and they can possibly be avoided by decreasing consumption of polyphenols while simultaneously consuming ascorbic acid.