Folic acid (FA) is the synthetic form of folate, commonly used in vitamin supplements, food fortification and therapy. Currently, there is concern about the possibility of potential adverse effects of high doses of FA. In Brazil, daily use of FA 5 mg is recommended for several population groups, including pregnant women and patients with hemolytic anemia. This dose is five times greater than the tolerable upper intake level for healthy people. Both FA and heme iron are absorbed into duodenal enterocytes by the proton coupled folate transporter/heme carrier protein 1 (PCFT/HCP1). This transporter has a higher affinity for FA than heme iron, and we therefore surmised that high doses of FA might interfere with heme iron absorption.


The aim of this study was to test the effects of high levels of FA on the absorption of hemin (source of heme iron) using Caco-2 cell monolayers as an in vitro model of intestinal absorption.

Material and methods

Cells grown in 6-well plates were incubated with 25 µM hemin and varying FA concentrations (0, 5, 10, 31.25, 62.50 and 125 µM) for 12 and 24 h. Intracellular iron was measured by atomic absorption spectrometry and total protein was measured by the Bradford method.


At both time points, cells incubated with 125 μM of FA showed lower concentrations of intracellular iron compared with cells without FA in the medium (0 µM). Cells incubated for 12 h with 62.5 μM and 125 μM of FA had 1.50 and 1.56-fold lower intracellular iron concentrations, respectively, than cells incubated without FA (125 μM: p= 0.002; 62.5 μM: p=0.007). Decrease of intracellular iron in Caco-2 cells was greater at 24 h than at 12 h of incubation. After 24 h, cells incubated with 125 μM of FA showed an intracellular iron concentration 3.08-fold lower than cells with 0 µM FA (p = 0.047).


High levels of FA simulating intraluminal intestinal concentrations of FA following 5 mg FA ingestion were associated with lower intracellular iron content in Caco-2 cells due to decreased hemin absorption. These changes indicate that high doses of FA may impair heme iron absorption through their shared uptake via the PCFT/HCP1 transporter.

Financing: CNPq 4826412012-6, CNPq 401586/2014-6, CNPq 208204/2015-6 and FAPESP 2012/12912-1


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.