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American Journal of Clinical Nutrition, Vol. 78, No. 6, 1168-1177, December 2003
© 2003 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

High-, but not low-bioavailability diets enable substantial control of women’s iron absorption in relation to body iron stores, with minimal adaptation within several weeks1,2,3,4

Janet R Hunt

1 From the US Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND.

2 Supported by the US Department of Agriculture, with additional support from the North Dakota Beef Commission. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

3 The US Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity and affirmative action employer, and all agency services are available without discrimination.

4 Address reprint requests to JR Hunt, USDA, ARS, GFHNRC, PO Box 9034, Grand Forks, ND 58202-9034. E-mail: jhunt{at}gfhnrc.ars.usda.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Adaptation of iron absorption in response to dietary iron bioavailability is less likely in premenopausal women, who generally have lower iron stores, than in men.

Objective: The objective of the study was to ascertain whether iron absorption in women adapts to dietary iron bioavailability and whether adaptation reflects altered absorptive efficiency or adjustment to specific inhibitors or enhancers of absorption.

Design: Heme- and nonheme-iron absorption from either high- or low-bioavailability diets was measured at 0 and 10 wk in premenopausal women as they consumed one of the diets for 12 wk (randomized 2 x 2 factorial design). The high- and low-bioavailability diets contained similar amounts of total iron, as 13.1 and 14.8 mg/d nonheme and 2.0 and 0.3 mg/d heme iron, respectively, and they differed in contents of meat, ascorbate, whole grains, legumes, and tea.

Results: In premenopausal women, the efficiency of nonheme-iron absorption (P = 0.06, two-tailed test), but not of heme-iron absorption, tended to adapt in response to a 12-wk difference in dietary iron bioavailability, whether absorption was tested with high- or low-bioavailability menus. Bioavailability, but not adaptation, substantially influenced total iron absorption ({approx}6-fold). In contrast with iron absorption from the low-bioavailability diet, that from the high-bioavailability diet consistently was inversely associated with serum ferritin.

Conclusion: Only the high-bioavailability diet enabled women to absorb more iron in relation to their low iron stores. Women consuming the high-bioavailability diet absorbed up to 4.5 mg (30–35%) dietary iron with minimal influence of the diet consumed during the previous 10 wk.

Key Words: Gastrointestinal adaptation • nonheme iron • heme iron • iron absorption • dietary bioavailability • iron requirements • serum ferritin • fecal ferritin • ascorbic acid • meat • phytic acid • tea


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dietary factors that influence iron bioavailability include the biochemical form (heme or nonheme) and concurrently consumed enhancers (eg, ascorbic acid and an unidentified "meat" factor) or inhibitors (eg, phytic acid, polyphenols, phosphates, calcium, and egg) (1, 2). Although dietary iron bioavailability influences iron absorption from single meals by as much as 10-fold (2), longitudinal studies lasting weeks or months indicate little or no responsiveness of body iron stores (estimated from serum ferritin) to changes in dietary iron bioavailability, including changes in intakes of ascorbic acid (3–6), calcium (7, 8), and meat (9). Despite documented 6- to 8-fold differences in the amount of iron initially absorbed from whole, controlled diets, consumption of such diets for 7–12 wk did not affect the serum ferritin concentrations of premenopausal women (10) or of men (11).

In the latter study, nonheme-iron absorption in the men partially adapted to differences in iron bioavailability; the difference in total iron absorption between high- and low-bioavailability diets was reduced from 8-fold to 4-fold when initial absorption was compared with the absorption tested after consumption of the diets for 10 wk (11). It was not clear whether the partial adaptation of iron absorption to dietary iron bioavailability was related to altered absorptive efficiency (responding to the amount of iron recently absorbed without altering serum ferritin) or to adjustments to specific inhibitors or enhancers of absorption. However, 2 studies suggested that extensive exposure does not modify the influence of dietary factors that enhance or inhibit nonheme-iron absorption. Dietary phytate inhibited nonheme-iron absorption from single meals to a similar degree in long-term vegetarians and control subjects (12), and ascorbic acid enhanced nonheme iron absorption to a similar degree before and after 16 wk of ascorbic acid supplementation (3).

The main purpose of the present study was to determine whether a partial adaptation to modify the influence of dietary iron bioavailability occurs in premenopausal women, who generally have lower iron stores than do men and who may be less likely to have attained stable body iron equilibrium. An additional objective was to determine whether adaptation to dietary iron bioavailability reflects altered absorptive efficiency or adjustments to specific inhibitors or enhancers of absorption. Further objectives were to add to the limited number of measurements of iron absorption from whole human diets and to test various indexes of iron status after women consumed these typical Western diets for several weeks.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General protocol
Heme-iron absorption and nonheme-iron absorption were determined from radiolabeled diets consumed by healthy nonanemic premenopausal women (n = 36). Each volunteer consumed either a high- or low-bioavailability maintenance diet for 12 wk, and her iron absorption was measured from the entire 2-d cyclic menu of one of these 2 diets at 0 and 10 wk (Figure 1Go). The same high- or low-bioavailability menus were used for the radiolabeled test diets and the 12-wk maintenance diets, but the menus were randomly assigned to volunteers in a 2 x 2 factorial design. Blocking was used to obtain similar serum ferritin values for volunteers in 4 treatment groups. The 4 treatments tested iron absorption from a) the high-bioavailability diet before and after adaptation to the same diet, b) the high-bioavailability diet before and after adaptation to the low-bioavailability diet, c) the low-bioavailability diet before and after adaptation to the high-bioavailability diet, and d) the low-bioavailability diet before and after adaptation to the same diet. This allowed testing for adaptation with time as influenced by the maintenance diet, the radiolabeled test diet, and any interaction between the test and maintenance diet. Blood iron indexes were measured at 0, 2, 10, and 12 wk and tested for the effect of the maintenance diet. Fecal ferritin excretion was determined during a 3-d baseline period before the controlled diets began and for days 1–12 and 72–83.



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  		FIGURE 1. Experimental design, designating the general schedule for each subject and the subjects’ assignment to 1 of 4 dietary treatment groups.

 
Subjects
Healthy nonpregnant women were recruited through public advertising and selected after an interview and blood analysis helped determine that they were 20–44 y of age, had no apparent underlying disease, did not routinely use medications besides hormonal contraceptives, had not donated blood or used iron supplements exceeding 20 mg/d for >= 6 mo before the study, did not participate in rigorous physical training, and had blood hemoglobin concentrations of >= 120 g/L. Each applicant agreed to discontinue all nutrient supplements when her application was submitted, generally 6–12 wk before the beginning of the study. Because the study involved radioisotope administration, the women were requested to use some form of birth control or to abstain from sexual intercourse, and they were tested for pregnancy before each set of radiolabeled meals.

Study participants were 36 women who were (mean ± SD) 32 ± 7 y old (range: 20–44 y) and had a body weight of 65 ± 11 kg (52–89) and a body mass index (BMI; in kg/m2) of 24 ± 4. One additional woman completed the study, but her data were eliminated because an infection and the use of antibiotics toward the end of the study greatly increased her serum ferritin and C-reactive protein concentrations and lowered her second measurement of iron absorption. Because this study recruited volunteers concurrently with an unrelated study that eliminated women with low iron stores, the women in the present study tended to have low serum ferritin, which was considered advantageous to the study. Participants’ serum ferritin values at the time of recruitment were used as a blocking factor to modify the random assignment of experimental treatments; geometric mean initial serum ferritin values were 21 µg/L (range: 4–73 µg/L).

The participants gave written informed consent. The Radioactive Drug Research Committee and Institutional Review Board of the University of North Dakota and the Human Studies Review and Radiological Safety Committees of the US Department of Agriculture reviewed and approved this human study.

Diets
Two weighed, experimental diets in a 2-d menu cycle were planned by registered dietitians with the use of ordinary foods but with varying food selections and serving sizes to minimize or maximize iron bioavailability (Tables 1 and 2GoGo). The diets were similar but not identical to those used in a similar experiment with men (11), and proportionately less food was served in accordance with the women’s energy requirements. The high-iron-bioavailability diet contained generous quantities (294 g, or {approx}10.5 oz/d) of meat or poultry (2/3 as beef or pork and 1/3 as chicken), refined-cereal and -grain products, no coffee or tea, and foods with >= 75 mg ascorbic acid in each of the 3 main daily meals. The low-iron-bioavailability diet contained no meat, limited amounts (46 g, or {approx}1.7 oz/d) of poultry (chicken) and fish (shrimp), plenty of legumes and whole-grain cereal and bread products, tea (from 1 g dry, black instant) at each meal, and foods with ascorbic acid just sufficient to meet the Recommended Dietary Allowance (60 mg/d when the study was conducted; 16), distributed over several meals.


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  		TABLE 1 Menus for experimental diets

 

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  		TABLE 2 Diet composition, calculated1

 
The 2 diets were similar in calcium and total iron content, but the high-iron-bioavailability diet contained considerably more heme iron and ascorbic acid, slightly more vitamin A (calculated as retinol equivalents from retinol and ß-carotene combined) (13), and considerably less phytic acid (Table 2Go; 14) than did the low-iron-bioavailability diet. The refined bread and cereal products in the menus were commercially enriched with iron to the extent common in the United States [20 mg iron/lb (or 460 g) flour]; iron-fortified breakfast cereals were not used. The iron contributed from this fortification provided nearly one-third of the total iron in each diet. Most of this fortification iron was ferrous sulfate, which is known to be highly bioavailable, but {approx}1.5–2.0 mg/d was from elemental reduced iron (with a chemical valance of 0; reduced refers to the method of production), or the form was unspecified. Coffee was excluded from the diets. City water, a low-energy carbonated water, and chewing gum were consumed as desired, after analyses indicated minimal trace element content. Limited amounts of salt, pepper, and selected low-energy carbonated beverages were individualized to each volunteer’s preferences and then served consistently throughout the study.

All diet ingredients except water were weighed, prepared, and provided to the volunteers by the research center. Volunteers ate one meal every weekday at the research center and consumed the remaining foods, after some minimal re-heating, away from the research center. Foods were weighed to 1% accuracy and consumed completely with the use of spatulas and rinse bottles. To maintain individual body weights, energy intakes were adjusted in increments of 0.94 MJ (225 kcal) by proportionately changing the amounts of all foods.

Measurements of iron absorption
Heme-iron absorption and nonheme-iron absorption were measured by isotopically labeling the entire 2-d menu at the beginning (days 1 and 2) and after 10 wk (days 70 and 71) of the 12-wk controlled diet period, according to the diet assignment for the test diet. At each of these 2 times, the menu (3 meals/d for 2 d; evening snack foods were served with the third meal) was labeled with 18.5 kBq 55Fe-labeled hemoglobin and 37 kBq 59FeCl3. Radiolabeled hemoglobin was obtained by intravenously injecting 74 MBq 55Fe into an iron-deficient, pathogen-free rabbit, exsanguinating the animal 2 wk later, and removing the stroma by lysing and centrifugation (17). The isotopes were added to the diet in proportion to the heme- and nonheme-iron contents of the meals to yield constant specific activities (ratios of 55Fe to dietary heme iron and of 59Fe to nonheme iron) for all 6 meals. Accordingly, for the low-iron-bioavailability diet, 55Fe-labeled hemoglobin was added only to the one daily meal that included heme iron (Table 1Go); this added < 0.015 mg iron per each heme-labeled meal. Tracers were added to the foods that were the best sources of that form of iron in each meal. Meat, poultry, and fish dishes were cooked, cooled, radiolabeled, and then minimally reheated in the microwave just before consumption.

Although dietary energy was occasionally adjusted over time to maintain body weights, for each participant, the amount of energy in the radiolabeled meals was consistent for the 2 sets of labeled meals, separated by 10 wk. All labeled meals were consumed at the research center.

Absorption of nonheme iron was determined by whole-body scintillation counting, which detected only the gamma-emitting 59Fe radioisotope. The custom-made whole-body counter (18) used 32 crystal NaI(Tl) detectors, each 10 x 10 x 41 cm, arranged in 2 planes above and below the participant, who lay supine. The initial total body activity was calculated from the whole-body activity after 2 meals (determined >= 1 h after the second meal but before any unabsorbed isotope was excreted), divided by the fraction of the total activity contained in those 2 meals. The percentage of nonheme iron absorbed was determined as the portion of initial whole-body activity that remained after 2 wk (day 15), with correction for physical decay and for background activity measured 1–2 d before the meals. In a previous study (10), the slopes of semilogarithmic whole-body retention plots for 4 wk after isotope administration did not consistently differ significantly from zero, which indicates that iron excretion was minimal and that it was unnecessary to correct for endogenous excretion of iron during the 2 wk after isotope administration.

Radioisotope concentrations in blood (19) were also measured after 2 wk (day 15) and expressed as fractions of the administered radioisotopes, which were measured from aliquots prepared when the foods were labeled. All isotope determinations included corrections for physical decay and background activity. The blood retention of 55Fe and 59Fe, expressed as a percentage of the administered doses, was calculated from the blood radioisotope concentration and an estimate of total blood volume based on body height and weight (20). The blood incorporation of nonheme iron, expressed as a percentage of the absorbed nonheme iron, was calculated by dividing the fractional blood retention of 59Fe by the fractional absorption of 59Fe as measured by whole-body counting.

Heme-iron absorption was calculated from the measurement of blood retention of 55Fe, by using the assumption that 80% of the absorbed heme iron was incorporated into blood (21). An alternative approach, multiplying the nonheme-iron absorption (determined by whole-body counting) by the ratio of 55Fe to 59Fe in the blood, mostly provided similar results but also resulted in a few more outlying values for heme iron absorption because it tended to exaggerate small errors between blood and whole-body 59Fe measurements.

Absolute absorption of heme and nonheme iron (mg/d) was calculated by multiplying percentage absorption values by the analyzed dietary content of heme and nonheme iron, respectively. Total iron absorption (mg/d) was calculated as the sum of heme- and nonheme-iron absorption, and the percentage of total dietary iron absorbed was calculated by dividing the total amount of iron absorbed (in mg) by the total iron content of the diet, multiplied by 100.

Chemical analyses
Fasting blood samples of 30 mL each were obtained at 0, 2, 10, and 12 wk. Duplicate diets were prepared for iron analyses. Feces were collected in 1-d composites for 3 d before the controlled diets were begun and for 12 d during the first and last 2 wk (days 1–12 and 71–82). Sample collection included precautions to avoid trace mineral contamination.

Aliquots of the diet composites were digested with concentrated nitric and 70% perchloric acids by method (II)A of the Analytic Methods Committee (22). The iron content of the digestates was determined by inductively coupled argon plasma emission spectrophotometry (ICAP; Perkin-Elmer, Norwalk, CT). Analytic accuracy was monitored by assaying the NIST Typical Diet (Standard Reference Material 1548a; National Institute of Standards and Technology, Gaithersburg, MD). Mean (±SD) measurements were 98 ± 7% of certified values for iron.

Heme iron in the meat-containing foods was measured previously (11), by difference, following the same digestion and ICAP methods to measure extracted nonheme iron (23). Our analyses indicated that cooking by our research procedures (generally, baking of individual dishes in closed containers) had negligible effects on the heme-iron content of beef or chicken dishes.

Hemoglobin, hematocrit, mean corpuscular volume (MCV), and erythrocyte distribution width were measured by using a Celldyne 3500 System (Abbott Laboratories, Abbott Park, IL). Serum iron was determined colorimetrically by using a Cobas Fara Chemistry Analyzer (Hoffmann-LaRoche Inc, Nutley, NJ) with a commercial chromogen (Ferene, Raichem Division of Hemagen Diagnostics, San Diego). Iron-binding capacity was similarly determined after adding a known amount of ferrous iron to the serum sample under alkaline conditions. Percentage transferrin saturation was calculated from serum iron and total-iron-binding capacity. To reduce analytic variation, each volunteer’s samples for either serum ferritin or fecal ferritin were stored frozen until they could be measured in a single analytic batch. Fecal ferritin was extracted from each lyophilized 1-d fecal composite as described by Skikne et al (24) and filtered with 5-µm membrane filters. Serum and fecal ferritin concentrations were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies (Abbott Laboratories) against human spleen ferritin, which mainly measure L-rich ferritin, the isoferritin primarily found in spleen and liver (25). The ferritin assay was calibrated against World Health Organization ferritin 80/602 First International Standard. Protein in fecal extracts was determined colorimetrically (26). C-reactive protein was measured by nephelometry (Behring Diagnostics Inc, Westwood, MA) to detect inflammation, which may be associated with increased serum ferritin. Serum C-reactive protein was substantially elevated in the volunteer who was eliminated because of infection and antibiotic treatment, but it did not exceed 7.5 mg/L in any other volunteers during the study.

Statistical analysis
Iron absorption, serum ferritin, transferrin saturation, transferrin receptor, and fecal ferritin data were logarithmically transformed, and geometric means are reported. All fecal ferritin data were increased by a negligible 0.1 µg/d to forgo transformation of some zero values when analyzing statistical relations. Dietary treatment effects were determined by using repeated-measures analysis of variance (ANOVA) and SAS software (27), with Tukey-Kramer contrasts. Simple linear regression analysis (27) was used to assess additional relations between variables. Probability values < 0.05 were accepted as significant. Values between 0.05 and 0.10 are also listed as tending to show differences; in this regard, it is noted that two-tailed tests were used by convention, but several P values that were between 0.05 and 0.10 in the hypothesized direction would be considered significant if one-tailed tests were applied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Iron absorption from the 2-d test diets
The women absorbed substantially more heme, nonheme, and total iron from the high-bioavailability test diet than from the low-bioavailability test diet (Table 3Go). Nonheme iron was nearly 5 times as efficiently absorbed (expressed as percentage absorption) from the high-bioavailability diet than from the low-bioavailability diet (11.1% and 2.3%, respectively; P < 0.0001), and because the nonheme-iron contents of the diets were nearly similar, this difference was reflected in the absolute amount of nonheme iron absorbed (1.3 and 0.3 mg, respectively; P < 0.0001). The efficiency of heme-iron absorption (expressed as percentage absorption) was not significantly affected by the test diet, but because the high-bioavailability diet contained much more heme iron from meat, poultry, and fish (Table 2Go), the absolute amount of heme iron absorbed from the high-bioavailability diet was substantially and significantly greater than that absorbed from the low-bioavailability test diet (0.43 and 0.016 mg, respectively; P < 0.0001).


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  		TABLE 3 Dietary heme- and nonheme-iron absorption, as well as incorporation of absorbed nonheme iron, measured from 2 d of high- and low-iron-bioavailability test diets, after 0 and 10 wk of consuming maintenance diets with high iron bioavailability (HBV) or low iron bioavailability (LBV)1

 
Short-term adaptation to dietary iron bioavailability
The women tended to absorb less iron over time with the high-bioavailability diet and more iron over time with the low-bioavailability maintenance diet. This tendency was reflected in the ANOVA for an interaction of maintenance diet x time (P < 0.06 to P < 0.08; Table 3Go). By convention, these probabilities are expressed as two-tailed tests; however, it is notable that the results were in the direction hypothesized and were consistent with the adaptation previously observed in men (11). Although this suggests that some adaptation occurred, the extent of the adaptation was low in magnitude; the adaptation tended to decrease the total iron absorbed by 0.15 and 0.09 mg (tested with the high- and low-bioavailability test diets, respectively) after 10-wk equilibration to the high-bioavailability maintenance diet and to increase it by 0.44 and 0.02 mg, respectively, after equilibration to the low-bioavailability maintenance diets (Table 3Go).

The tendency toward adaptation to slightly modify the influence of dietary iron bioavailability with time on the maintenance diet occurred whether absorption was tested with the high- or the low-bioavailability test diet (Table 3Go). This finding is consistent with a generalized adaptive response related to short-term differences in iron status, rather than with adaptation to specific dietary enhancers or inhibitors of iron bioavailability.

Incorporation of absorbed iron into erythrocytes
Although it was not strongly affected by dietary bioavailability per se, the erythrocyte incorporation of the absorbed nonheme 59Fe was significantly affected by the interaction of time with the maintenance diet. A tendency for greater incorporation with a longer time of consuming the low-bioavailability diet (Table 3Go; P < 0.01 for ANOVA interaction and P < 0.06 for Tukey-Kramer contrast of time consuming the low-bioavailability diet) suggests that body iron kinetics were sensitive to bioavailability-related differences in the amount of iron absorbed within 12 wk. Some measurement variation resulted in a few values > 100%; such values were not eliminated, as possible similar variation in the lowest values could not be identified. Despite this variation in a few measurements, mean erythrocyte incorporations of 75–87% (Table 3Go) were consistent with the common assumption of 80% incorporation that is used when whole-body counting is unavailable. An inverse correlation between ln (erythrocyte incorporation) and ln (serum ferritin) (R2 = 0.48; n = 19) was observed only for the initial administration of isotope to volunteers who were consuming the high-iron-bioavailability diet.

Blood indexes of iron status
Blood indexes of iron status were tested for treatment effects associated only with the 12-wk maintenance diets (not the 2-d test diets). Most blood indexes were unaffected by dietary treatment. The exception was MCV, which increased slightly with the high-bioavailability diet and decreased with the low-bioavailability diet during the study (Table 4Go). Although these changes were consistent with the microcytosis observed with iron deficiency anemia, the MCV values were well within the normal range (82–98 fL for this laboratory). The change in MCV was not accompanied by significant diet-associated differences in other erythrocyte variables, such as hemoglobin, hematocrit, mean corpuscular hemoglobin, or red blood cell distribution width (see Table 4Go for hemoglobin data; other data not shown).


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  		TABLE 4 Blood indexes of iron status during 12 wk of consuming maintenance diets with high (HBV) or low (LBV) iron bioavailability1

 
Serum ferritin and iron-binding capacity (IBC) changed significantly (P < 0.001) with time, independent of dietary treatment (Table 4Go). Most of these changes were between week 0 and week 2. The decrease in serum ferritin and the increase in IBC were consistent with a slight reduction in iron status associated with the procedural phlebotomy of 30 mL at each of 0, 2, 10, and 12 wk. Neither diet nor time affected transferrin saturation or transferrin receptor in these nonanemic women (Table 4Go).

Fecal ferritin excretion
In contrast with serum ferritin concentration, fecal ferritin excretion was significantly (P < 0.01) greater with the high-bioavailabity diet than with the low- bioavailability maintenance diet (Figure 2Go). This finding replicated that of our companion experiment in men (11), but it was extended in the present study to obtain baseline data. Accordingly, the women provided 2 fecal samples before the dietary treatments began. Fecal ferritin measurements at baseline were significantly greater than were subsequent measurements occurring as early as days 7–12 (P < 0.0001; Figure 2Go). Because fecal ferritin was similarly affected whether expressed per milligram of fecal protein (Figure 2Go) or per day (data not shown) for the controlled experimental diets, which differed considerably in dietary protein (Table 2Go), the greater baseline measurements standardized for protein are likely attributable to a greater ferritin excretion. This greater ferritin excretion could be related to greater iron fortification of the subjects’ self-selected diets; highly fortified foods, such as ready-to-eat cereals, were not included in the experimental diets. Although highly variable (range: 7–28 mg/d), the women’s mean (± SD) self-selected dietary iron intake estimated from 3-d food records was 16 ± 5. There was no correlation between ln serum ferritin and ln fecal ferritin at baseline.



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  		FIGURE 2. Least-squares mean (±SE) fecal ferritin excretion was significantly greater while subjects were consuming the high-bioavailability diet ({square}) than while they were consuming the low-bioavailability diet ({blacksquare}) (P < 0.01); with both diets, it was significantly reduced from baseline values as early as days 7–12 (P < 0.0001). n = 17–19 subjects for each mean.

 
Heme-, nonheme-, and total iron absorption in relation to body iron status
The high-bioavailability diet was needed for substantial adjustment of total iron absorption to the body iron stores in women. Initial (wk 0) total iron absorption from the high-bioavailability diet, but not from the low-bioavailability diet, was inversely related to serum ferritin (R2 = 0.81, P < 0.01, and 0.20, NS, respectively; Figure 3Go). At 10 wk, these correlations were significant if the subjects had consumed the high-bioavailability maintenance diet for 10 wk (R2 = 0.77 and 0.67 for absorption from the high- and low-bioavailability diets, respectively) or if they had been tested with the high-bioavailability diet (R2 = 0.48), but they remained insignificant if subjects were maintained on and tested with the low-bioavailability diet (R2 < 0.01).



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  		FIGURE 3. The relation of percentage total iron absorption from these whole diets (week 0 test diets) to body iron stores showed the effect of bioavailability. A high-bioavailability test diet was necessary to enable iron absorption in the women to respond to body iron stores.

 
The women with the lowest iron stores (but with normal hemoglobin concentrations >= 120 g/L) absorbed 30–35% of total dietary iron, whereas the women with the highest iron stores absorbed only 3% from the same high-bioavailability diet. In contrast, total iron absorption from the low-bioavailability diet ranged from 1% to 8%. In absolute amounts, the women absorbed 0.4–4.7 mg Fe/d, depending on body iron stores, from the high-bioavailability test diet and 0.1–1.0 mg Fe/d from the low-bioavailability test diet (Figure 4Go).



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  		FIGURE 4. The relation of iron absorption to serum ferritin at week 0 of the high-bioavailability test diet (A) and the low-bioavailability test diet (B). The contribution of heme iron to the total iron absorbed was relatively modest for most women with either diet, contributing substantially to the total iron absorbed only for those with the highest iron stores and considerable meat intake. The women with low iron stores (all nonanemic) absorbed <= 4.5 mg total iron/d from the high-bioavailability diet, mostly as nonheme iron.

 
Although the women absorbed heme iron more efficiently than nonheme iron, the greater total iron absorption by women with low iron stores was mainly attributable to nonheme iron. The initial absolute absorptions of heme, nonheme, and total iron from the high-bioavailability diet were all inversely related to serum ferritin (R2 = 0.57, 0.75, and 0.79, respectively; all P < 0.01), and heme iron accounted for {approx}33% of the iron absorbed from the high-bioavailability diet by the women with the highest iron stores but < 20% of that absorbed by the women with the lowest iron stores (Figure 4AGo). Of course, the contribution of heme iron to the total iron absorbed from the low-bioavailability diet was minimal (Figure 4BGo) because of the low heme-iron content of that diet (Table 2Go).

Other indexes of iron status also correlated (P < 0.05) with iron absorption, although generally not as strongly as they correlated with serum ferritin. [The following correlations were tested after grouping data according to the test diets (2-d) and time of the absorption measurement but without differentiating between the maintenance (12-wk) diet treatments.] Inverse correlations were observed between transferrin saturation and (logarithmically transformed) heme-, nonheme-, and total iron absorption only for the high-bioavailability test diet at wk 0 (data not shown). Transferrin receptor correlated directly with total iron absorption from the high-bioavailability test diet at both 0 and 10 wk and with that from the low-bioavailability diet at 10 wk; these correlations were not consistently attributable to heme or nonheme iron because both forms tended to correlate, but not always significantly (data not shown). Blood hemoglobin concentrations correlated significantly with nonheme and total iron absorption only when tested with the high-bioavailability diet at wk 10 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results emphasize the importance of diets with high iron bioavailability in meeting the iron requirements of premenopausal women with low iron stores. It is interesting to compare these data with the derivation of the newly established Dietary Reference Intakes (28). These iron recommendations for premenopausal women were strongly influenced by menstrual iron losses that are highly skewed (29); women at the 97.5th percentile were estimated to lose 2.32 mg Fe/d, averaged through the menstrual cycle (28). To replace menstrual and additional iron losses, menstruating women at the 97.5th percentile were estimated to require 3.15 mg absorbed iron/d, which together with an estimated 18% iron bioavailability from American and Canadian diets yielded a Recommended Dietary Allowance of 18 mg/d. The present results show that nonanemic women with low iron stores can absorb at least that much iron (Figure 4AGo) from a high-bioavailability diet containing {approx}13 mg iron (see Table 2Go).

The new Recommended Dietary Allowance is based on maintaining a serum ferritin concentration of 15 µg/L (28). The present data confirm that women with that serum ferritin concentration absorb {approx}18% of the iron from a high-bioavailability diet (Figure 3Go), but they also show that nonanemic women with lower serum ferritin can absorb considerably more iron—as much as 4.5 mg or 30–35% of the iron—from such a diet containing {approx}13 mg iron. The absolute absorption of nonheme iron may be slightly overestimated, because it is based on the generally accepted assumption that 59Fe labeled all of the nonheme iron in the diet (30), and this assumption probably does not hold true for elemental iron powders used in food fortification (31). The estimated 4.5 mg iron absorbed from the high-bioavailability diet by women with the lowest iron stores would be reduced to 4.0–4.25 mg/d if the fortification iron other than ferrous sulfate were only 0–50% as well absorbed as the other dietary nonheme iron. Even with this adjustment, these data indicate that women with the lowest iron stores absorbed considerable amounts of iron from the high-bioavailability diet—at least double the amount absorbed by women with a serum ferritin concentration of 15 µg/L (Figure 4AGo).

In this study, the high-bioavailability diet was probably more similar to typical US and Canadian diets than was the low-bioavailability diet, but the latter diet was not extremely different from many Western diets (Table 1Go) and was quite acceptable to the study volunteers. Societal trends and dietary recommendations to reduce the intake of red meat and to increase the consumption of whole grains, legumes, and tea are likely to reduce the iron bioavailability of typical Western diets. This study with whole diets confirms that the amount of dietary iron recommended is not nearly as critical as the dietary bioavailability.

The present results with whole diets generally support Cook’s observation (32), based mainly on experiments with single meals, that "humans can adapt successfully to a wide range of iron requirements and intakes." Although, in the present study, the tendency in menstruating women for an adaptation to dietary iron bioavailability to occur in 12 wk was of low magnitude (Table 3Go), the strong cross-sectional inverse relation between iron absorption from a high-bioavailability diet and body iron stores (Figures 3 and 4AGoGo) represents a much longer-term adaptation to control iron absorption in relation to body iron stores. The women in the present study and the men in a similar 12-wk study (11) showed the same tendency to adaptation of their nonheme-iron absorption, but the adaptation in the women was not as extensive as that in the men (Figure 5Go; 11). However, in another study, there was an adaptation in women to more substantial differences in iron intake. With iron supplementation, the reduction in the efficiency of nonheme-iron absorption by subjects with low iron stores (all women) was at least as substantial as that by those with higher iron stores (33).



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  		FIGURE 5. Women in the present study who were treated with the same maintenance (12 wk) and test (2 d) diets tended to have adaptations in a similar but much less pronounced pattern as was seen in the men in a companion study (11). The men, with their higher iron stores, absorbed considerably less nonheme iron ({square}) than did the women, and similar amounts of heme iron (({blacksquare}), even though the men’s greater energy requirements resulted in proportionately greater iron intake (13 mg total iron for women, 15–16 mg for men). The figures above each bar designate total iron absorbed (mg/d). For these data, 9 women and 14 men were tested with the high-bioavailability diet, and 7 women and 17 men were tested with the low-bioavailability diet. Diet x time interaction for total iron absorbed, P < 0.07 for women and P < 0.001 for men (separate ANOVAs for each sex).

 
In both the present study and the comparable study with men (11), 12-wk consumption of controlled diets with substantial differences in total absorbable iron did not affect serum ferritin or most other indexes of body iron status (with the exception of MCV in the present study), but it did influence fecal ferritin excretion. The unresponsiveness of serum ferritin in these and other studies (3–9) is in contrast to proposed algorithms (34)—based on cross-sectional, rather than kinetic, observations—that predict relatively rapid changes in serum ferritin with changes in dietary iron bioavailability.

Although serum ferritin was unresponsive to diet, it decreased significantly with time, which suggested sensitivity to phlebotomy in this study (Table 4Go) and in the study with men (11). IBC was affected by time (and phlebotomy) in the present study with women (Table 4Go) but not in the study with men (11), presumably because the women had lower iron stores and their iron status was low enough for IBC to be a sensitive indicator.

This study, together with the few other studies comparing iron absorption from controlled whole diets differing in bioavailability (10, 11, 35), confirms that differences in bioavailability can result in substantial differences in total iron absorption (6- to 7-fold in the present study; see Table 3Go). This is in contrast to the modest or negligible results observed when the diets were not controlled by the investigators but by subjects who self-selected their experimental diets according to the investigator’s instructions (36–38).

These data with whole diets also confirm observations (32) that most of the iron absorbed by people (usually women) with low iron stores is nonheme iron (Figure 4Go). This finding emphasizes the potential importance of dietary iron bioavailability, because concurrently consumed enhancing and inhibiting dietary constituents substantially influence the absorption of nonheme-iron (2) by increasing its solubility in the intestinal lumen (32). Iron deficiency up-regulates mucosal iron transporters, such as divalent metal ion transporter (DMT-1), which increases the mucosal uptake of nonheme iron (39, 40), but such regulation can be effective only if the iron is accessible at the brush border in a reduced, soluble form, and this is more likely with a high-bioavailability diet. Initial iron absorption from the low-bioavailability diet did not correlate significantly with serum ferritin concentrations in the narrow ranges of serum ferritin in the present study (Figures 3 and 4GoGo) or in the similar study with men (11). These variables did correlate significantly when the initial data were combined to include the broader range of serum ferritin concentrations associated with both sexes (data not shown). Such a correlation is consistent with other studies of diets with low iron bioavailability (10, 34, 41). However, women with the lowest iron stores absorbed up to 4.5 mg Fe/d from the high-bioavailability diet and <1 mg Fe/d from the low-bioavailability diet (Figure 4Go). The results suggest that diets with high iron bioavailability are needed for a substantial increase in iron absorption in women with low iron stores.

In conclusion, an adaptation occurred to reduce the influence of dietary iron bioavailability in women by altering their general absorptive efficiency, but the magnitude of the adaptation within several weeks was quite low. This tendency for short-term adaptation occurred whether iron absorption was tested with low- or high- bioavailability labeled diets, which suggests altered absorptive efficiency rather than adjustment to specific inhibitors or enhancers of absorption. These results, when added to the limited published measurements of iron absorption from whole diets, show that nonanemic women with low iron stores can absorb up to 4.5 mg or 30–35% of the iron from a high-bioavailability diet and that such a diet is necessary for substantially increased iron absorption by women with low body iron stores.


   ACKNOWLEDGMENTS
 
I gratefully acknowledge the contributions of members of the Grand Forks Human Nutrition Research Center human studies research team, particularly the work of Carol Ann Zito, who conducted blood radioiron analyses. In addition, Emily J Nielsen managed volunteer recruitment and scheduling; Bonita Hoverson supervised planning and service of the controlled diets; Sandy K Gallagher supervised clinical laboratory analyses; Glenn I Lykken designed and consulted in the use of the whole body counter; Fariba Roughead reviewed the manuscript and provided advice on fecal ferritin measurements; and LuAnn K Johnson performed the statistical analyses. The whole team is especially grateful for the women volunteers for their dedication and service.

The author has no financial or personal affiliation with the North Dakota Beef Commission.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication April 29, 2003. Accepted for publication June 17, 2003.




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