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Journal of the American College of Nutrition, Vol. 18, No. 4, 309-315 (1999)
Published by the American College of Nutrition

Original Paper

ß-Carotene 15,15'-Dioxygenase Activity is Responsive to Copper and Iron Concentrations in Rat Small Intestine

Alexandrine During, PhD, Meira Fields, PhD, FACN, Charles G. Lewis, PhD and J. Cecil Smith, PhD

Phytonutrients Laboratory (A.D., J.C.S.), USDA-ARS, Beltsville Human Nutrition Research Center, Beltsville, Maryland
Nutrient Requirements and Functions Laboratory (M.F., C.G.L.), USDA-ARS, Beltsville Human Nutrition Research Center, Beltsville, Maryland

Address reprint requests to: A. During, Ph.D., USDA-ARS, Beltsville Human Nutrition Research Center, Phytonutrients Laboratory, Bldg. 307, Beltsville, MD 20705.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: Previous in vitro studies have suggested that ß-carotene 15,15'-dioxygenase is an iron-dependent enzyme. However, in vivo, it is difficult to alter iron tissue concentration by varying dietary iron because of homeostatic control. On the other hand, an interaction between iron and copper has been shown, i.e., copper-deficiency results in an increase of iron in rat liver. Therefore, we hypothesized that intestinal iron concentration could be increased by copper-deficiency. Our objective was to examine the effects of iron as affected by dietary copper on ß-carotene 15,15'-dioxygenase activity in the small intestine.

Methods: Weanling male Sprague-Dawley rats (40 to 45g) were divided into four dietary groups: two copper-adequate groups (6.0 µg Cu/g diet) and two copper-deficient groups (0.6 µg Cu/g) combined with either normal iron (44 µg Fe/g) or high iron (87 µg Fe/g). Iron and copper concentrations were determined by atomic absorption spectrophotometry and the dioxygenase activity by reverse phase HPLC.

Results: Intestinal copper concentration was significantly reduced (40%) by the consumption of the copper-deficient diets, but intestinal iron was not changed by doubling dietary iron in rats fed either copper-adequate or copper-deficient diets. However, as hypothesized, the two copper-deficient groups exhibited higher intestinal iron concentration (>=137%, p<0.001) than the copper-adequate controls. In addition, intestinal ß-carotene 15,15'-dioxygenase activity was increased by 27% and 106%, respectively, for copper-deficient rats fed either normal or high iron diets, compared to the respective copper-adequate controls (p<0.01). The dioxygenase activity was not significantly affected by dietary iron in either copper-adequate or copper-deficient groups. Finally, the enzyme activity was positively correlated (r=0.67, p<0.0001) with iron concentration and negatively correlated (r=-0.49, p<0.01) with copper concentration in small intestine.

Conclusions: Intestinal ß-carotene 15,15'-dioxygenase may be an iron-dependent enzyme sensitive to copper status in vivo.

Key words: carotenoid metabolism, vitamin A, copper, iron, rats


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Vitamin A is an essential micronutrient, which is not synthesized by mammals. Thus, it must be provided by dietary sources as either intact molecule, retinol, or by plant-derived precursors such as ß-carotene. This compound, C40H56, is the most common carotenoid found in fruits and vegetables. In mammals, the oxidative central cleavage of ß-carotene to retinal (direct precursor of vitamin A) is catalyzed by the cytosolic enzyme, ß-carotene 15,15'-dioxygenase, located primarily in small intestine. Although that reaction was first suggested over 65 years ago [1], in vivo investigations continue in an effort to characterize the enzyme and factors that influence its regulation.

In the mid 1960’s, reports from the laboratories of Olson and Goodman indicated that in vitro ß-carotene dioxygenase activities from rat intestine, rat liver and porcine intestine were inhibited by {alpha}'-dipyridyl and o-phenanthroline, two effective chelators of ferrous iron (Fe2+) [2–4]. Moreover, in 1974, Singh and Cama [5] found that the intestinal enzymes from guinea pig and rabbit were also inhibited by these chelating agents. Additionally, these authors showed that increasing Fe2+ concentrations in vitro stimulated the dioxygenase activities, whereas iron in the ferric state (Fe3+) had no effect [5]. These data were recently confirmed by Dmitrovskii et al. [6–7], who reported similar results: e.g., the enzyme activity from rabbit intestine was increased by the addition of Fe2+ in vitro but not affected by Fe3+. Furthermore, these authors were first to demonstrate an inhibition of the enzyme by copper ions [6].

However, a few in vivo studies using rats have failed to show an effect of trace mineral status on the ß-carotene conversion as reflected by liver vitamin A concentration. That is, the ß-carotene conversion to retinol was not affected by dietary copper- and/or selenium-supplementation [8], iron-deficiency [9] or copper-deficiency [10].

The in vitro data suggest that iron, probably in the ferrous state, and/or copper may be involved in the catalytic activity of the ß-carotene 15,15'-dioxygenase. In contrast, in vivo studies have failed to demonstrate these effects. However, it is difficult to modulate iron concentrations in tissues by varying dietary iron alone because iron absorption by the intestinal mucosal cells is well regulated and depends on the host’s requirements [11]. Several reports [12–14] have indicated that copper-deficiency in experimental animals results in hepatic iron accumulation due to an interaction between copper and iron. Therefore, we hypothesized that intestinal iron concentration could similarly be increased by feeding a copper-deficient diet. The localized increase of iron might affect intestinal ß-carotene dioxygenase activity.

Therefore, the objective of this in vivo investigation was to examine the effects of iron concentration as affected by copper on ß-carotene 15,15'-dioxygenase activity in small intestine using the rat as an experimental model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Materials
All-trans ß-carotene (type IV), all-trans retinal and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). d-a-Tocopherol was obtained from Dr. James Clark of Henkel (LaGrange, IL). HPLC-grade solvents were purchased from Fisher Scientific (Pittsburgh, PA). All-trans retinol was prepared by reducing all-trans retinal with NaBH4. All-trans ß-carotene, all-trans retinal and all-trans retinol were purified as described previously [15].

Animals and Diets
Weanling male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN, USA) weighing approximately 40 to 45 g were used. Rats were randomly divided into four dietary groups and fed two levels each of copper and iron: Group 1—copper-adequate, normal iron; Group 2—copper-adequate, high iron; Group 3—copper-deficient, normal iron; and Group 4—copper-deficient, high iron.

The basal diet used in this study met the nutrient requirements of laboratory rats, except for copper and iron, which were omitted from the mineral mix (Table 1). To the basal diet prepared in our laboratory, iron (ferric citrate) was added at two concentrations; 44 and 88 µg Fe/g, respectively, for normal and high iron diets. The copper-adequate diets were prepared by adding cupric carbonate to yield a final concentration of 6.0 µg Cu/g diet. The copper-deficient diets contained 0.6 µg Cu/g. Concentrations of copper and iron in the four diets were confirmed by flame atomic absorption spectrometry following a combination of dry heat and acid digestion procedures [18].


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  		TABLE 1. Composition of the Basal Diet

 
Rats were individually housed in stainless steel cages in a room with 12 hours light/dark cycle. The room was maintained at 22°C and 50 to 60% humidity. Rats were fed their respective diets for four weeks; the diets and deionized water were consumed ad libitum. At the end of the treatment periods, after overnight food deprivation, the rats were decapitated and liver and intestine collected. All procedures followed those outlined in the Guide for the Care and Use of Laboratory Animals [19].

Preparation of Organ Samples andTissue Homogenates
Liver was cut into small pieces (ca. 2 g) and stored at -20°C prior to analysis. The upper half of intestine (ca. 25 cm) was rinsed free of ingesta using an ice-cold 0.9 g/L NaCl solution. Intestinal mucosa was exposed and gently scraped off with a glass microscope slide and immediately frozen at -80°C until used. The tissues (liver and intestinal mucosa) were homogenized with a Potter-Elvehjem homogenizer in five volumes (wt/v) of ice-cold 50 mmol/L HEPES-KOH buffer (pH 7.4) containing 1.15 g/L KCl, 1 mmol/L EDTA and 0.1 mmol/L dithiothreitol (DTT). The homogenates were then divided into aliquots for analyses of vitamin A (retinol) ({approx}0.5 mL) and copper and iron concentrations ({approx}1 mL). An aliquot ({approx}5 mL) of intestinal mucosa homogenate was centrifuged at 10,000xg for 30 minutes (4°C). The resultant supernatant was applied to a Sephadex G-25M column equilibrated with ice-cold 10 mmol/L HEPES-KOH buffer (pH 7.4) containing 50 mmol/L KCl, 0.1 mmol/L EDTA and 0.1 mmol/L DTT. The eluted fraction was immediately assayed for ß-carotene dioxygenase activity. Protein concentrations of the tissues were determined by the method of Bradford [20] using bovine serum albumin as the standard.

Measurement of ß-Carotene 15,15'-Dioxygenase Activity in Intestinal Mucosa
ß-Carotene dioxygenase assay was carried out using the procedure described previously [21]. Briefly, the reaction medium (final volume of 0.2 mL) contained all-trans ß-carotene (3 nmol), N-tris-(hydroxy-methyl)-methylglycine (Tricine)-KOH buffer, pH 8.0 (40 µmol), DTT (0.2 µmol), Tween 40 (0.3 mg), sodium cholate (1.6 µmol), {alpha}-tocopherol (20 nmol) and the enzyme (less than 0.5 mg protein). The cleavage reaction was started by adding 40 µL of 75 µmol/L ß-carotene solubilized in 0.75 g/L Tween 40, incubated at 37°C for 60 minutes. The reaction was stopped by adding 50 µL of 37% (wt/wt) formaldehyde and the mixture again incubated at 37°C for 10 minutes. Then 500 µL of acetonitrile were added and the insoluble material was precipitated by centrifugation at 10,000xg for two minutes.

The supernatant (200 µL) was directly injected into the HPLC system consisting of a 114M pump (Beckman Instruments, Inc., California), a UV-970 uv-vis absorbance detector (Jasco, Tokyo, Japan), and the GOLD®software system for analyses (Beckman). Retinal, product of the reaction present in the incubation mixture after a complete reaction, was analyzed using a TSK gel ODS-80Ts C18 reverse phase column (5 µm particle size, 80-Å pore size, 4.6x150 mm) (TosoHaas, Tokyo, Japan), attached to a guard-column (2x20 mm) of Pelliguard LC-18 (Supelco Inc., Bellefonte, PA). Acetonitrile/water (90:10, v/v) containing 0.1% ammonium acetate was used as the mobile phase at a flow rate of 1.0 mL/minute. Retinal was monitored at 380 nm and eluted at approximately 7.5 minutes under these HPLC conditions. Retinal was quantified from its peak area using a standard curve of purified all-trans retinal (0.2 to 50 pmol/200 µL injected). Under these HPLC conditions, the detection limit of retinal was 0.2 pmol/0.2 mL of enzyme assay.

Incubation and extraction procedures were carried out under dim yellow light.

Determination of Total Retinol in Liver
Retinol was extracted from liver homogenates by the method of Ross [22]. Briefly, 100 µL of tissue homogenate were saponified at 60°C for 20 minutes in the presence of one mL of 95% ethanol/5% potassium hydroxide containing 1% pyrogallol. After saponification, 1 mL of water was added, samples were extracted three times with hexane (0.5 mL), the upper phases were combined and the hexane evaporated under nitrogen. Dried residues were then redissolved in 500 µL of acetonitrile/water (90:10, v/v) and 100 µL subjected to HPLC analysis. The analytical system was a Hewlett Packard, HP 1100 with a G1311A pump, a G1315A diode array detector and a G1329A thermostated autosampler (Hewlett-Packard GmbH, Waldbronn, Germany). Retinol was eluted on the TSK gel ODS-80Ts C18 reverse phase column, 4.6x250 mm, with acetonitrile/water (90:10, v/v) containing 0.1% ammonium acetate as the mobile phase (flow rate of 1.0 mL/min). Retinol was monitored at 325 nm, eluted at 10.5 minutes and quantified from its peak area by use of a standard curve of all-trans retinol (0.2 to 200 pmol/100 µL injected) in acetonitrile/water (90:10; v/v).

Determination of Copper and Iron in Liver and Intestinal Mucosa
Copper and iron concentrations in tissue homogenates were determined according to the procedure of Hill et al. [18], using a flame atomic absorption spectrophotometer (Model 5000, Perkin-Elmer Corp., Norwalk, CT). Iron and copper concentrations in the sample were determined using standard curves of each mineral. Standard Reference Materials (bovine liver (SRM #1577b) and bovine serum (SRM #1598) of the National Institute of Standards and Technology, Gaithersburg, MD) were used to verify accuracy. These materials were analyzed concurrently with the tissue samples, beginning at the digestion phase.

Statistical Analysis
All data are expressed as mean±standard error of the mean (SEM). Data were tested for homogeneity of variances among groups using the Bartlett’s test (if necessary, data were log10-transformed to stabilize variances). Main effects and interactions of the two independent variables (dietary copper and iron) were tested by analysis of variances (two-way ANOVA), followed by a post hoc multiple comparisons of means using Fisher’s test. Relationships among variables were examined by simple-regression analysis. All statistical analyses were performed using StatView, Version 5.0 (SAS Institute Inc., Cary, NC). Differences were considered statistically significant at p<0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
The effects of doubling dietary iron concentrations (87 versus 44 µg Fe/g diet) were examined in rats in relation to their copper status (copper-deficient versus copper-adequate). The two groups of rats fed a normal iron level were considered as controls when iron effects were examined, and the two groups of rats fed an adequate copper level served as controls when copper effects were analyzed. Body and organ weights, hepatic retinol level and copper and iron concentrations of liver and intestine are presented in Table 2. The consumption of the copper-deficient diets resulted in a reduction of body weight (p<0.05 by ANOVA analysis). Liver weight was increased by high iron diets (p<0.01 by ANOVA analysis) (Table 2). The two copper-deficient diets resulted in a significant decrease of hepatic copper concentration (>=76%) compared to the copper-adequate controls (p<0.0001 by ANOVA analysis). In addition, copper-deficiency was associated with a significant elevation (>=80%) of iron concentration in liver (p<0.0001 by ANOVA analysis). The retinol concentration in liver was not changed by either dietary iron or dietary copper; however, this variable tended to be reduced with the consumption of high iron diets (but p>0.05 by ANOVA analysis). The statistical analysis by multiple comparisons revealed a significant difference in liver retinol concentrations between the copper-deficient rats fed high iron (87 µg Fe/g diet) and the copper-adequate rats fed normal iron (44 µg Fe/g) (p<0.05). Dietary iron and copper did not affect intestinal mucosa weight. As observed in liver, intestinal mucosa copper concentration was reduced by more than 40% (p<0.002 by ANOVA analysis) and intestinal mucosa iron concentration significantly increased by 137% to 227%, respectively, for the copper-deficient rats fed either normal or high iron diets (p<0.001 by ANOVA analysis), compared to the copper-adequate controls. In contrast, doubling dietary iron level (87 versus 44 µg Fe/g diet) did not affect intestinal mucosa iron concentration in either the copper-adequate or copper-deficient rats (Table 2).


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  		TABLE 2. Weights and Retinol, Copper and Iron Concentrations in Liver and Intestinal Mucosa of Rats Fed Norml Iron or High Iron Diets Combined with Either Copper-Adequate or Copper-Deficiency1

 
ß-Carotene 15,15'-dioxygenase activity was expressed as specific activity (pmol retinal/h.mg protein). The two copper-deficient groups exhibited higher specific activities by 27% and 106%, respectively, for rats fed normal or high iron diets, than those of the copper-adequate controls (p<0.01 by ANOVA analysis) (Fig. 1). However, the statistical analysis by multiple comparisons revealed a significant difference (p<0.02) only between the copper-deficient rats fed high iron (87 µg Fe/g diet) and the copper-adequate controls. Dietary iron did not have an effect on the ß-carotene conversion to retinal in intestinal mucosa (Fig. 1). By simple-regression analysis using individual values, ß-carotene dioxygenase activity [pmol/(h.mg protein)] was positively correlated (coefficient of correlation r=0.668, p<0.0001 for n=23 rats) with intestinal iron concentration (Fig. 2A) and negatively correlated (r=-0.493, p<0.01 for n=24 rats) with intestinal copper concentration (Fig. 2B).



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  		Fig. 1. Effects of two levels of dietary iron (44 and 87 µg/g diet) in relation to the copper status on intestinal ß-carotene 15,15'-dioxygenase activity expressed as specific activity [pmol retinal/(h.mg protein)]. Weanling male rats fed one of the following diets for four weeks: diet 1—copper-adequate, normal iron (NFe); diet 2—copper-adequate, high iron (HFe); diet 3—copper-deficient, NFe; and diet 4—copper-deficient, HFe [with copper-adequate (6.0 µg Cu/g diet) and copper-deficient (0.6 µg Cu/g diet)]. Enzyme assays were conducted as described in MATERIALS AND METHODS. Values represent mean±SEM (n=4 rats each for diets 1 and 3, n=8 rats for diet 2, and n=9 rats for diet 4). Main effects and interactions of dietary copper and iron on the enzyme activity were examined by two-way ANOVA analysis; Cu effect, p<0.01; Fe effect, NS; CuxFe effect, NS with NS=non significant. Multiple comparisons of means among groups were assessed using Fisher’s test. Means with different letters were significantly different; (a,b) and (b,c): p<0.02 and (a,c): p<0.0001.

 


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  		Fig. 2. Linear relationships between ß-carotene 15,15'-dioxygenase activity [pmol retinal/(h.mg protein)] and either A) iron concentration or B) copper concentration in rat small intestine. Individual values are shown for the four dietary groups according to the following: ({circ}) copper-adequate, normal iron (NFe); ({triangleup}) copper-adequate, high iron (HFe); (bull;) copper-deficient, NFe; and ({blacktriangleup}) copper-deficient, HFe (see Fig. 1 for diet composition). The coefficients of correlation r1 (dioxygenase versus iron) and r2 (dioxygenase versus copper) were obtained by simple-regression analysis; test of significance of the linear relationships was determined by ANOVA.

 

   DISCUSSION
 
To examine potential effects of iron on ß-carotene 15,15'-dioxygenase activity, we hypothesized that iron concentration in the intestinal mucosa of small intestine could be modified by the copper status of rats due to the antagonistic relationship between these two trace minerals. Usually, dietary iron consists of both non-heme and heme iron. Here, inorganic iron (iron citrate) was added at two levels (44 and 87 µg Fe/g diet) in combination with either normal copper (6.0 µg Cu/g diet) or copper-deficient (0.6 µg Cu/g diet) levels. The data suggest that iron concentration of intestine could not be changed by simply altering dietary iron. Indeed, intestinal mucosa iron was not increased for either the copper deficient or adequate rats when the amount of dietary iron was doubled. These observations demonstrate that iron concentration in intestinal mucosa is tightly regulated, probably depending on the host’s current requirements for iron. As reported earlier [11], intestinal iron concentration is influenced by total body iron content, which may play a role in the transport of iron from the lumen of gut to blood, perhaps via the intervention of receptors that follow saturation kinetics.

Significant reductions of copper concentrations were observed in liver and intestinal mucosa of rats that were fed the copper-deficient diets, showing that the animals were copper-deficient. In addition, copper-deficiency resulted in an increase of iron concentration in both organs. Therefore, the iron-copper interaction previously described in liver [12,14] is also manifested in rat intestine. Indeed, it has been recognized since 1932 that copper affects the metabolism of iron; likewise, iron affects the metabolism of copper [13,23]. Thus, it is well established that these two minerals act in concert [23,13,14]. Copper-deficiency is accompanied by liver iron accumulation due to the immobilization of iron from storage sites to bone marrow for heme synthesis, leading to iron-deficient erythropoiesis. Different systems of transport, such as transferrin- and nontransferrin-bound iron, apparently are involved in iron transport as well as the reduction of Fe3+ to Fe2+, a requisite for transport of iron across membrane bilayers. Recently, it has been demonstrated that some of the transporters’ activity and function in mammalian cells are dependent on copper [24]; furthermore, in the absence of copper, iron transport is impaired [24,25]. Therefore, by its interaction on iron transport, which is related to the redox state of iron, copper-deficiency may result in the accumulation of iron in storage tissues, particularly as Fe2+.

In the present study, ß-carotene 15,15'-dioxygenase activity was positively correlated with iron concentration in small intestine. These in vivo results are in agreement with previous in vitro studies [2–7], which suggested that ß-carotene dioxygenase could be iron-dependent, perhaps a metalloenzyme. This suggestion is supported by a general characteristic of dioxygenases; among the 35 dioxygenases previously studied, more than 80% have nonheme iron in their structure or require extrinsic iron for maximum activity [26]. However, ß-carotene 15,15'-dioxygenase has only been partially purified, and its structure, as well as its molecular weight, is unknown. Therefore, an obligate role of iron for the enzyme’s activity remains hypothetical.

Intestinal ß-carotene 15,15'-dioxygenase activity was higher in all copper-deficient groups compared to copper-adequate controls. Assuming that the enzyme is iron-dependent, copper-deficiency may affect the conversion of ß-carotene by its interaction with the metabolism of iron, in particular the accumulation of Fe2+ in tissues (as proposed above). Thus, if this hypothesis were true, ß-carotene dioxygenase would be correlated to tissue Fe2+ concentrations. The concept of Fe2+ as a cofactor necessary for the enzyme activity is supported by previous in vitro studies [5,7], demonstrating that ß-carotene conversion to retinal was stimulated by Fe2+, but not by Fe3+.

On the other hand, we found that ß-carotene dioxygenase activity was negatively correlated with copper concentration in the intestine, suggesting that copper ions may also play an indirect role in the reactional mechanism of the enzyme. Indeed, Dmitrovskii et al. [6] proposed that copper ions (Cu2+) may deactivate the enzyme-Fe2+ complex associated with ß-carotene peroxide radical. Referring to both the mechanism of linoleic acid oxidation by lipoxygenase [27] and the formation of free radicals in the nonenzymatic oxidation of ß-carotene [28], Dmitrovskii et al. [6] proposed a mechanism of the ß-carotene conversion by dioxygenase involving the participation of free radicals and a tripartite complex consisting of enzyme-Fe2+-substrate. According to this proposal, the enzyme-Fe2+ complex abstracts a hydrogen ion from ß-carotene to generate a ß-carotene free radical; this reacts with molecular oxygen to form ß-carotene peroxide radical, followed by the oxidation of Fe2+ to Fe3+ with an electron relocation on the substrate molecule, yielding two molecules of retinal.

In the present investigation, total retinol (free retinol+retinyl esters) was determined in rat liver (major organ of vitamin A storage) with the assumption that an increase of this parameter would be associated with increase of intestinal ß-carotene 15,15'-dioxygenase activity. However, we found an inverse relationship; the lowest hepatic retinol concentration was associated with the highest intestinal ß-carotene dioxygenase activity in copper-deficient rats fed high dietary iron (87 µg Fe/g diet). These findings are in agreement with those of Swanson and Parker [9], who reported that rats fed iron-deficient diet exhibited hepatic retinyl ester levels significantly higher than those of rats fed iron-adequate diet. Those authors [9] reported that changes in iron status in rats perturb many biological functions, in particular lipid and/or lipoprotein metabolism, which in turn may affect ß-carotene and vitamin A concentrations in the liver. In our study, rats were exposed to a combination of factors which could result in an oxidative stress. Indeed, copper-deficiency is associated with a reduction of superoxide dismutase activity, a copper-dependent enzyme, which normally counters oxygen radicals [29]. In addition, iron is a promotor of oxidative damage via the Fenton reaction. These profound oxidative conditions could lead to a hypermetabolic state causing a reduction of retinol in the liver. Given such conditions, we speculate that ß-carotene dioxygenase activity may increase to compensate for the increased turnover of retinol metabolism.

In conclusion, our results suggest that intestinal ß-carotene 15,15'-dioxygenase is iron-dependent and sensitive to the copper status of rats. Copper may influence the enzyme activity either by its action in the reactional mechanism such as by dissociation of the enzyme-Fe2+-substrate complex (hypothesis) and/or by its interaction with the metabolism of iron. In order to understand the mechanism involved, additional investigations are required to determine if similar data might be observed with a wider range of dietary iron.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
We are grateful to Dr. A.D. Hill of USDA-ARS, Beltsville Human Nutrition Research Center, for technical assistance of copper and iron analyses.

Received January 1, 1999. Accepted May 1, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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