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Assessing iron bioavailability from commercial supplements containing iron.

Abstract.--Iron deficiency affects up to 70% of the world's population. Iron supplementation is one approach for controlling this global problem. The objective of this study was to compare iron bioavailability among commercial micronutrient and iron supplements. An in vitro cell model (Caco-2) was used in this study. Micronutrient or iron supplements were subjected to an in vitro digestion and presented to the Caco-2 cells, a mammalian intestinal cell line. The formation of cell ferritin, which is an index of iron bioavailability, was quantified; values were normalized using cell protein. Among the four supplements tested with ferrous fumarate as the iron source, iron supplement F and the micronutrient supplement C demonstrated higher iron uptake in Caco-2 cells, relative to micronutrient supplements A or B. Among five iron supplements, a commercial ferrous gluconate source demonstrated higher Caco-2 cell iron uptake than brands D (heme and non-heme iron), E (carbonyl iron), F (ferrous fumarate) or G (ferrous sulfate). Ferrous gluconate was the most effective supplement for providing iron, as determined by the in vitro Caco-2 cell culture assay.


Iron deficiency (ID) is the most prevalent micronutrient deficiency, affecting 30-70% of the world's population (Stoltzfus 2001; 2003). Symptoms of ID include lethargy, decreased cognitive function and growth, anemia, poor reproductive outcomes, and if left untreated, death (CDC 1998). While ID is a severe problem in developing nations, it is also prevalent among certain population groups in industrialized countries (Etcheverry et al. 2005). In the U.S., 14% of toddlers, 3.7% of children and 9.2% of women of childbearing age have ID (Cogswell et al. 2009).

One approach for controlling this global problem is through iron supplementation (Allen 2002). Iron supplements exist in several forms: as ferrous or ferric salts, tablets or liquid and as single nutrients or as part of multiple micronutrient supplements (Allen 2002). While the most common iron forms are ferrous sulfate, ferrous fumarate, and ferrous gluconate (Allen 2002; Brittenham 2009), there have been no studies comparing the bioavailability (i.e. absorbability) of the different iron salts. Furthermore, little is known about the relative bioavailability of iron from multivitamin/multiminerals compared to that of iron supplements. The presence of iron absorption inhibitors (such as calcium and zinc), and iron absorption enhancers (such as ascorbic acid) might affect iron bioavailability in these micronutrient supplements.

In this study, the iron bioavailability of three commercial multivitamin/multiminerals (containing ferrous fumarate as its iron source) was compared to that of an iron supplement also in the form of ferrous fumarate. Iron bioavailability among different commercial forms of iron supplements was also compared. The goal of this study was to determine which of the supplementation forms provided the most bioavailable iron. To carry out these objectives, an in vitro digestion/Caco-2 cell model was used. Caco-2 cells, which belong to a human colonic adenocarcinoma cell line, differentiate into enterocytes when grown in culture (Zweibaum 1993; Meunier et al. 1995). Ferritin, a cytosolic iron storage protein and an index of iron bioavailability (Glahn et al. 1998), was quantified.


Cell Culture.--The Caco-2 cells were donated by the USDA/ARS Plant, Soil and Nutrition Laboratory (Ithaca, NY). The cell cultures were maintained in Dulbecco's Modified Eagle's Medium at pH 7.4 (GIBCO, Grand Island, NY) supplemented with 10% FBS, 25 mM HEPES and 1% antibiotic/antimycotic solution (GIBCO). Cells were kept in a 37[degrees]C incubator supplied with 5% C[O.sup.2]. The medium was replaced every 2 d. For uptake experiments (passage 30-35), cells were grown in collagen-treated 6-well plates at an initial seeding of 50,000 cells/[cm.sup.2]. Uptake experiments were conducted 13 d post seeding.

Description of Samples.--The multivitamin/multiminerals consisted of brands A, B and C. These micronutrient supplements were compared to a commercial iron supplement (brand F). The multivitamin/multiminerals and the supplement F all contained iron in the ferrous fumarate form. Additional iron supplements consisted of brands D, E, G and H. The iron form and amount in each of these supplements as well as the calcium, zinc, and ascorbic acid contents are shown in Table 1.

In Vitro Digestion/Caco-2 Cell Iron Uptake-The in vitro digestion/Caco-2 cell procedure for iron uptake was used as described previously (Etcheverry et al. 2004) with some modifications. Briefly, supplements were ground and an amount containing 900 pg of iron was weighed and mixed with 15 mL of saline (140 mM NaCl, 5 mM KCl); 2 mL samples of this solution were then aliquotted into six 15 mL test tubes. Samples were adjusted to pH 2 using 1 N HCl; 0.25 mL of pepsin solution (0.8 g pepsin dissolved in 40 mL of 0.1 M HCl) was added to each 2 mL sample. Tubes were then agitated on a rocking platform at 37[degrees]C for 1 h. Following the peptic digestion, the pH of the samples was adjusted to 7 with 1 M NaHC[O.sub.3] and the volume was brought up to 6 mL with saline. Tubes were incubated at 37[degrees]C for 30 min to allow for pepsin inactivation and then centrifuged at 3600 RPM for 15 min. To each well of Caco-2 cells, 1.5 mL of the corresponding supernatant was added. Twenty four h later the cells were harvested in 2 mL of 18M[OMEGA] water, and stored at -20[degrees]C for future ferritin and protein analysis.

Controls in this experiment included an iron positive control (consisting of a 1:5 molar ratio of iron to ascorbic acid, with iron provided as ferric chloride and prepared using 3600 pg per 15 mL of saline), used as a quality control measure to demonstrate the proper functioning of the cells, and a blank (consisting of the saline solution), used to measure background levels of ferritin. Controls were digested as described above.

Ferritin and Protein Assays.--Ferritin concentration was quantified as the index of iron availability using a FER-IRON II Ferritin Assay (RAMCO Laboratories, Houston, TX), and normalized to cell protein concentration. A Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA) was used to determine protein concentration.

Statistical Analysis.--Data were analyzed by one-way ANOVA after testing for normality and equal variance using the Prism software (GraphPad Software, Inc., San Diego, CA). A Bonferroni post test was used to compare sample means, with means based on six replicates per treatment. Significance was at the level ofP < 0.05.


The iron availability study comparing multiple micronutrient supplements with an iron supplement, using equivalent amounts of iron and the Caco-2 cell model, demonstrated the highest cell ferritin concentrations in the Caco-2 cells when fed iron supplement F (82.7 ng/mg cell protein) or multivitamin/multimineral C (64 ng/mg cell protein), with no statistical difference between these two supplements (Fig. 1). Caco-2 cells exposed to the multivitamin/multimineral B produced an average ferritin per protein value of 35 ng/mg (42% of the iron supplement F value), while the multivitamin/multimineral A resulted in 3.6 ng ferritin/mg protein (4% of the iron supplement F value). The blank in this study had a ferritin concentration of 14 ng/mg cell protein.

Results of the study comparing iron bioavailability from different iron supplements are shown in Figure 2. Iron availability was highest from the commercial ferrous gluconate (brand H), as demonstrated by ferritin results of 228.3 ng/mg cell protein. It was followed by brands G (136.2 ng/mg), F (91 ng/mg), D (74.2 and E (70.8 ng/mg). The blank resulted in a ferritin level of 6 ng/mg protein.

The protein levels were not significantly different among the samples in each study. In the first study the average protein concentration was 1.85 [+ or -] 0.07 mg/mL (Fig. 3) and in the second study it was 1.66 [+ or -] 0.04 mg/mL (Fig. 4).


The fact that the iron supplement F led to the highest cell ferritin formation compared to most of the micronutrient supplements suggests that iron alone is more bioavailable than when in the presence of other micronutrients. Ferritin formation in cells exposed to the multivitamin/multimineral C was not statistically different from that of iron supplement F; however, brand C ferritin values were significantly higher than those obtained from the micronutrient supplements A or B. This could be due to 70% more ascorbic acid in brand C than in brands A or B (Table 1). On the other hand, the micronutrient supplement C had more than twice the amounts of calcium than that present in the two micronutrient competitors, which suggests that ascorbic acid is a more potent iron enhancer than calcium is an inhibitor. There were no significant differences in zinc content among the three commercial micronutrient supplements, but the presence of this iron inhibitor could explain the lower cell ferritin formation in the micronutrient supplements A, B and C relative to the iron supplement F, which contained no zinc.

Interestingly, ferritin produced in response to the multivitamin/multimineral A was lower than that of the blank (Fig. 1). Ferritin measured in the blanks represents both baseline Caco-2 cell ferritin and any newly generated ferritin resulting from exogenous iron sources (such as the minimal amounts contributed as contaminants in the pepsin enzyme preparation). The fact that the ferritin measured in response to brand A feeding was lower than that of the blank suggests the presence of one or more components in the micronutrient supplement A that may have chelated both exogenous and endogenous iron, thus making it less available for the cells to absorb. Components in this supplement could also be affecting the expression level of brush border proteins involved in iron acquisition by the cells. Alternatively, these components could be increasing the overall cell protein expression, thus reducing the fractional ferritin to protein value. However, the fact that cell protein was not statistically different among the samples (Fig. 3) suggests that the lower ferritin values from brand A are not due to an increase in cell protein expression.

The comparative results among the different iron supplements revealed that the commercial ferrous gluconate source (brand H) provided the most bioavailable iron followed by brand G. Brand G (ferrous sulfate) contributed more bioavailable iron than brand E (carbonyl iron). A similar result between these two iron salts has also been observed in vivo (Swain et al. 2003). Brand D is a supplement that provides iron in a heme and non-heme form. While heme iron is more bioavailable when consumed in its unprocessed form, i.e. as part of hemoglobin, it becomes less bioavailable when it is separated from the protein. This is because pure heme iron can form large insoluble polymers even at a low pH (Conrad et al. 1966; Vaghefi et al. 2002). In general, the differences observed in cell ferritin formation among the samples could be attributed to pH stability, solubility, and chelating agents that might be present in the tablets.

This study compares iron bioavailability of commercial iron and micronutrient supplements. Based on the Caco-2 cell model, iron consumed alone appears to be more bioavailable than when consumed as part of several multivitamin/multimineral supplements. Among the iron supplements, ferrous gluconate provided the most bioavailable iron, followed by ferrous sulfate. Future studies could compare iron bioavailability between ferrous fumarate and ferrous gluconate in commercial micronutrient supplements.


This work was funded in part by funds from USDA-ARS under agreement No. 58-6250-0-008 to M.A.G. and from the Gates Grand Challenges in Global Health Program (sub-award from University of Freiburg, Germany) under agreement No. 37879 to M.A.G. This paper reports research conducted by D. A.-D. as part of his summer research experience in the NIH STEP-UP Program, administered by Charles R. Drew University of Medicine and Science, Los Angeles, CA. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does any mention of trade names, commercial products or organizations imply endorsement by the U.S. Government.

Literature Cited

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MAG at:

David Amaro-Driedger (1), Lori A. Center (2), Paz Etcheverry (2) and Michael A. Grusak (3)

(1) Bishop T. K. Gorman High School, Tyler, Texas 75701.

(2) Baylor College of Medicine, Houston, Texas 77030.

(3) USDA/ARS Children's Nutrition Research Center Baylor College of Medicine, Houston, Texas 77030.

Table 1. The iron, calcium, zinc and ascorbic acid amount in each
of the supplements, as provided by each manufacturer's fact
sheet, is presented, along with the weight of each tablet. The
daily dose of the micronutrient supplement C is two tablets (as
opposed to one for the other supplements), thus the values shown
here represent the weight and amount of the micronutrients in
that dose.

                                               Amount per
                                   Tablet      tablet (mg)
Supplement                         weight                     Ascorbic
Brand             Iron form         (g)     Fe    Ca    Zn      Acid

A             Ferrous fumarate     1.277    18    200   11       60
B             Ferrous fumarate     1.479    18    200   15       60
C             Ferrous fumarate     3.629    18    500   15      200
             21% heme iron, 79%
D            polysaccharide iron   0.504    28     0     0       0
E               Carbonyl iron      0.628    45     0     0       0
F             Ferrous fumarate     0.759    50     0     0       0
G              Ferrous sulfate     0.342    45     0     0     Trace
H             Ferrous gluconate    0.394    27     0     0       0
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Author:Amaro-Driedger, David; Center, Lori A.; Etcheverry, Paz; Grusak, Michael A.
Publication:The Texas Journal of Science
Date:Feb 1, 2012
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