The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 646-650

Vitamin A and -Carotene Can Improve Nonheme Iron Absorption from Rice, Wheat and Corn by Humans^1,2

María Nieves García-Casal^*, Miguel Layrisse^{*, , 3}, Liseti Solano, María Adela Barón, Franklin Arguello, Daisy Llovera, José Ramírez^*, Irene Leets^*, and Eleonora Tropper^*

^* Centro de Medicina Experimental, Laboratorio de Fisiopatología, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas 1020A, Venezuela;  Unidad de Investigaciones en Nutrición, Universidad de Carabobo, Valencia, Venezuela; and  Universidad Central de Venezuela, Caracas, Venezuela

	ABSTRACT

Abstract Introduction Methods Results Discussion References

After the rapid decrease in the prevalence of iron deficiency and iron-deficiency anemia in the Venezuelan population when a national program for fortification of flours with iron and vitamins was instituted, we studied micronutrient interactions in Venezuelan diets. One hundred human adults were fed three cereal-based diets, labelled with either ⁵⁹Fe or ⁵⁵Fe in six studies. Each diet contained different concentrations of vitamin A (from 0.37 to 2.78 µmol/100 g cereal) or -carotene (from 0.58 to 2.06 µmol/100 g cereal). The presence of vitamin A increased iron absorption up to twofold for rice, 0.8-fold for wheat and 1.4-fold for corn. -carotene increased absorption more than threefold for rice and 1.8-fold for wheat and corn, suggesting that both compounds prevented the inhibitory effect of phytates on iron absorption. Increasing the doses of vitamin A or -carotene did not further significantly increase iron absorption. We measured the iron remaining in solution performing in vitro studies in which the pH of solutions was adjusted from 2 to 6 in the presence of vitamin A or -carotene. All of the iron from ferrous fumarate was soluble after changing the pH of the solution containing 3.4 µmol of -carotene to 6.0. Vitamin A was less effective. However, 78 ± 18% of iron was soluble in the presence of 3.3 µmol of vitamin A, whereas with no vitamin addition, only 26 ± 13% of iron was soluble (<0.05). Vitamin A and -carotene may form a complex with iron, keeping it soluble in the intestinal lumen and preventing the inhibitory effect of phytates and polyphenols on iron absorption.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Vitamin A is a vital nutrient for cellular differentiation, vision, bone growth, reproduction and integrity of the immune system (Olson 1984). It is also essential for erythropoiesis. Deficiency of this vitamin results in anemia in humans and animals that is reversed only by vitamin A supplementation (Bloem et al. 1989, Hodges et al. 1978, Mejía 1986, Mejía and Arroyave 1982, Mejía and Chew 1988, Mejía et al. 1979).

-carotene is the most abundant provitamin A in foods. Approximately 10-50% of the total -carotene consumed is absorbed in the gastrointestinal tract and within the intestinal wall is partially converted into vitamin A. The efficiency of -carotene absorption decreases as intake increases and conversion to vitamin A is regulated by the vitamin A status of the individual. -carotene accumulation is not toxic, so it is considered a safe source of vitamin A (Roche Laboratories 1994, Wang 1994).

After reporting the results on iron availability from diets consumed by different socioeconomic strata of the Venezuelan population (Taylor et al. 1995), we continued studies of micronutrient interactions on iron ab sorption, especially since 1993, when the fortification of precooked corn and white wheat flours with iron and vitamins was started (Layrisse et al. 1996).

The first experiments already were presented in two workshops (García-Casal and Layrisse 1996, Layrisse and García-Casal 1997); and published (Layrisse et al 1997, Layrisse and García 1997). The studies showed that addition of vitamin A (as retinol palmitate) and iron to corn or wheat flours used to prepare a typical Venezuelan breakfast (basal breakfast) containing corn or wheat bread, cheese, margarine and coffee or tea, diminished the inhibitory effect of phytates and polyphenols on iron absorption.

We examined the effects of increasing doses of vitamin A and -carotene on nonheme iron absorption. In addition, solubility changes of various iron compounds at pH 2 and 6 with and without vitamin A and -carotene are presented.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

One hundred and four adult subjects (27 men and 77 women from Valencia City, Venezuela) voluntarily participated in this study. The Committee for the Protection of Human Subjects of the Venezuelan Institute for Scientific Research approved the studies. The subjects were selected from a low socioeconomic stratum in a section of a Venezuelan city. They were in apparent good health, but some of the women had moderate iron-deficiency anemia (hemoglobin <120 g/L). Blood (30 ml) was taken from each subject in a tube with ethylenediamine tetraacetic acid (EDTA) and kept refrigerated at 4°C until used to measure hemoglobin concentration (Crosby et al. 1954), serum iron (International Committee for Standardization in Hematology 1978), unsaturated iron bin ding capacity (International Committee for Standardization in Hematology 1978), serum ferritin concentration (Flowers et al. 1986) and radioactivity measurements (see next section).

Three cereals were tested: corn, wheat and rice. The corn test contained bread from 100 g nonfortified precooked corn flour and 5 mg iron as ferrous fumarate; the flour was given with 10 g margarine and 50 g cheese. The wheat test contained a bread prepared from 100 g of commercial white wheat flour enriched with 0.15 mg thiamine, 0.2 mg riboflavin, 2 mg niacin and 2 mg iron as ferrous fumarate. It also contained 10 g margarine and 50 g cheese. The rice test contained 100 g polished rice and was administered with 10 g margarine.

Corn, wheat and rice tests were given alone or with different amounts of vitamin A or -carotene. The vitamin A used was water-soluble retinol palmitate, and -carotene was a 10% water-soluble powder. Both products kindly were supplied by Roche Laboratories of Venezuela (Caracas).

Absorption studies.  Six studies were performed. Each included ~20 randomly selected subjects. The studies were designed to determine iron absorption from each test given alone in meal 1, and with different amounts of vitamin A, -carotene or coffee in meals 2-4 (Table 1).

Each subject was included in only one study. Four meals were given to each individual. Radiolabeled iron was added to the water to prepare the cereal. Each meal contained either ⁵⁹Fe or ⁵⁵Fe (Table 1).

The first meal in each study was the corn, wheat or rice test given alone and administered after an overnight fast. The second meal was given in the afternoon of the same day. Blood was drawn 15 d later to determine the hematological profile of the subjects and measure the radioactivity of blood samples. The subjects were fed again in the morning and in the afternoon of d 15 with meals 3 and 4, and blood was drawn again on d 30 to measure the radioactivity in blood and serum ferritin.

Duplicate 10 ml blood samples and triplicate samples of the radioactive foods were prepared for scintillation counting using the technique of Dern and Hart (1961a and 1961b). Iron absorption from food was calculated from the radioactivity in the subject's blood using estimated blood volume based on sex, weight and height (Nadler et al. 1962).

The protocol for the administration of radioactive food in the morning after an overnight fast and the afternoon of the same day was based on experiments previously published (Taylor et al. 1995). Four-hour intervals between meals are sufficient for iron absorption studies.

View this table: [in this window] [in a new window]   Table 2. Changes in added Vitamin A and -carotene centration during preparation and cooking of meals1

Chemical analysis.  The total iron concentration of foods was determined by the digestion method (Bothwell et al. 1979), phytates by the method of Haug and Lantzsch (1983), tannate by the method of Price and Butler (1977), vitamin A by the method of Strohecker and Heming (1965) modified by Covenin (1989) and -carotene by the method of Strohecker and Heming (1967).

Phytate concentrations in precooked corn flour, wheat flour and rice were 257, 161 and 170 mg/100 g, respectively. The mean tannin concentration in coffee was 2500 mg/100 g. In studies in which coffee was included, each subject consumed coffee prepared by the infusion of 8 g of ground coffee beans.

The mean iron concentration was 6 mg/100 g in the fortified precooked corn flour, 3 mg/100 g in white wheat flour, and 4 mg/100 g in boiled rice. The molar ratio of iron to phytate was 0.29 in precooked corn flour, 0.23 in the wheat flour and 0.30 in boiled rice.

The mean tannin content in 8 g of coffee powder was 200 mg. The molar ratio of iron to tannin content in the corn test was 1.0, 0.50 in the wheat test, and 0.66 in the rice test.

View this table: [in this window] [in a new window]   Table 4. Iron absorption from meals containing rice, corn or wheat given alone or administered with Vitamin A or -carotene1

Because of the long distance between the laboratory and the place were the experiments were performed, the meals were administered to the subjects 1 d after the meals were prepared. Variations in added vitamin A and -carotene content due to cooking procedures are shown in Table 2. In the case of the administration of 1.1 µmol (1000 IU) of vitamin A, the wheat bread was prepared separately, and this amount of vitamin A was dissolved in 20 ml water and drunk while eating the baked bread. In a previous experiment, we demonstrated that >80% of the vitamin A is lost during preparation of the dough and baking procedures due to the effect of yeast and the prolonged heat (Layrisse et al. 1997).

Solubility of iron compounds at pH 2 and pH 6.  The effect of vitamin A and -carotene on iron solubility at pH 2 and 6 was measured. Four iron compounds were tested: ferrous sulfate, ferrous fumarate, an iron-amino acid chelate (Ferrochel) and Ferric-sodium-EDTA. Ferrous sulfate was studied because it is a reference compound for iron studies. Fumarate is the salt used in Venezuela for enrichment of corn and wheat flours. Ferric-sodium-EDTA and Ferrochel (Albion Laboratories, Clearfield, UT) were tested due to their stability at alkaline pH.

Iron solutions from each of the compounds mentioned, containing 5 mg of iron, were prepared in 0.1 mol/L HCl containing 0, 0.55, 1.1, 1.65, 2.2 and 3.3 µmol (0, 500, 1000, 1500, 2000 and 3000 IU) of vitamin A or 0, 0,85, 1.70, 2.55, 3,40 and 5.10 µmol (0, 500, 1000, 1500, 2000 and 3000 IU) of -carotene. A 2-ml aliquot was taken to measure soluble iron at pH 2, and to the remaining solution, the pH was adjusted to 6 with careful addition of NaOH.

After standing 10 min at room temperature, duplicate 1-ml aliquots from the top of the solution were taken, and iron was measured for all iron compounds with different concentrations of vitamin A and -carotene at pH 2 and 6 by the digestion method (Bothwell et al. 1979).

Statistical analysis.  Paired t test was performed for iron absorption studies comparing all meals within each study. It also was used to compare iron solubility at pH 2 and 6 in presence of vitamin A or -carotene.

	RESULTS

Abstract Introduction Methods Results Discussion References

Iron absorption studies.  The effect of increasing doses of vitamin A on nonheme iron absorption from meals containing rice, corn and wheat is shown in Table 3. Iron absorption from the meal given alone was significantly lower than when vitamin A was included. There were no significant differences in iron absorption from rice meals containing from 0.55 to 2.78 µmol (496-2526 IU) vitamin A. The same patterns of iron absorption were observed from the tests containing pre cooked corn flour and white wheat flour.

View this table: [in this window] [in a new window]   Table 6. Effects of -carotene content and pH on in vitro iron solubility

In study 4, iron absorption from the rice meal containing vitamin A (meal 2, Table 4), was more than double the absorption from the meal given alone. In the absorption test in which the meal contained 0.58 and 0.95 µmol (342 and 558 IU) -carotene, iron absorption was more than two and three times greater than the meal given alone. The same pattern of iron absorption was observed for corn and wheat tests. The addition of 8 g of coffee powder as a beverage to meal 3 for rice test (0.58 µmol -carotene) and meal 4 for corn and wheat tests (1.53 and 2.06 µmol -carotene, respectively) (Table 4), produced no difference in iron absorption compared with meals with lower or higher content of -carotene and without coffee, but the values were still significantly higher than those obtained when the meal alone was consumed.

Iron solubility with pH changes.  Changes in iron solubility when pH is raised from 2 to 6 in presence of vitamin A are shown in Table 5. For ferrous fumarate, pH increase produced a 75% decrease in iron solubility. However, when vitamin A was added and the pH raised to 6, iron solubility increased as a function of vitamin concentration. Iron solubility increased in ~20% with 1.1 µmol (100 IU) of vitamin A, and when 3.3 µmol (3000 IU) was added, solubility was ~80%.

Ferrous sulfate showed a similar pattern. Iron solubility decreased in 64% when pH was raised to 6. When vitamin A was added iron solubility increased, and at 3.3 µmol (3000 IU), iron solubility was near 90%.

When -carotene was added to ferrous fumarate solutions at pH 2 and then raised to pH 6, virtually all of iron remained in solution when 3.4 µmol (2000 IU) or 5.1 µmol (3000 IU) of -carotene was added (Table 6). For ferrous sulfate, 92% of iron remained in solution at pH 6 when 5.1 µmol (3000 IU) of -carotene was added, whereas only 36% was soluble at pH 6 without any provitamin addition.

Differences in percentage of soluble iron are not statistically significant between ferrous fumarate and ferrous sulfate for the same content of vitamin A or -carotene, except for 0.55 µmol vitamin A or 0.85 µmol -carotene (P < 0.05).

It was not possible to observe an effect of vitamin A or -carotene on iron solubility for EDTA and Ferrochel because iron is 100% soluble at pH 6 without any vitamin addition.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Vitamin A content is reduced to less than half during cooking procedures, but this amount of vitamin A significantly increased iron absorption from rice, corn and wheat meals compared with these meals given alone. It also shows that the increase in doses from 0.37 to 2.78 µmol (338 to 2526 IU) did not produce any further significant increase.

-carotene has the same effect on iron absorption than vitamin A. In the case of rice meal, it was shown that 0.95 µmol (558 IU) of -carotene increases iron absorption more than threefold compared with the meal given alone. Absorption from the other two cereals showed similar behavior. It seems that -carotene also prevents the inhibitory effect of polyphenols on iron absorption. When coffee was administered with the test meals containing -carotene, iron absorption did not show a significant decrease as expected. On the contrary, it increased from one- to twofold compared with the meal without -carotene or coffee.

Solubility tests demonstrated that vitamin A and -carotene are capable of solubilizing iron at pH 6. It seems that -carotene is more efficient that vitamin A at least for ferrous fumarate. This is may be due to a greater stability of -carotene to storage, handling and pH changes.

Results from solubility experiments as well as human absorption studies show an important role for vitamin A and -carotene in improving iron absorption especially from foods with a high content of inhibitors, which are the staple foods of many countries worldwide.

The unexpected behavior of vitamin A and -carotene of improving iron absorption requires further studies to discover the mechanism for this peculiar reaction. A previous publication (Layrisse et al. 1997) showed that spectrophotometric results, elution patterns from high-performance liquid chromatography (HPLC) and solubility of iron with vitamin A at pH 6 suggest that vitamin A binds iron liberated during the digestive process and forms a complex that acts as chelating agent preventing the inhibitory effect of phytates and polyphenols on nonheme iron absorption. This hypothesis agrees with the results of Mejia (1986), who demonstrated interactions between vitamin A and iron metabolisms. It also supported the dramatic reduction of the prevalence of iron deficiency after 1 y of an iron-fortification program in Venezuelan population, in which one food vehicle, precooked corn flour, was fortified with vitamin A (Layrisse et al. 1996).

Because iron and vitamin A deficiencies are the two main deficiencies in many developing populations (Bloem et al. 1989, Mejía and Arroyave 1982), it is recommended that both micronutrient should be used in fortification programs.

	FOOTNOTES

Manuscript received 4 June 1997. Initial reviews completed 3 July 1997. Revision accepted 12 November 1997.

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