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Original Research Communications |
1 From the Department of Food Science, Chalmers University of Technology, and the Department of Clinical Nutrition and Internal Medicine II, University of Göteborg, Sahlgrenska Hospital, Göteborg, Sweden
2 Supported by grants 50.0474/91 and 50.0023/94 from the Swedish Council for Forestry and Agricultural Research.
3 Address reprint requests to A-S Sandberg, Chalmers University of Technology, Department of Food Science, Box 5401, SE-402 29 Göteborg, Sweden. E-mail: Ann-Sofie.Sandberg{at}FSc.Chalmers.se.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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Objectives: The objective was to investigate the effects of inositol tri-, tetra-, and pentaphosphates (IP3, IP4, and IP5, respectively) on iron absorption in humans.
Design: Iron absorption was measured in 5 experiments from single meals by extrinsic labeling with 55Fe and 59Fe and determination of whole-body retention and the erythrocyte uptake of isotopes. In experiments 1–3 the meals contained white-wheat rolls to which 10 mg P as IP5, IP4, or IP3 , respectively, was added. Inositol 1,2,6-triphosphate [Ins(1,2,6)P3] and a mixture of isomers of IP4 and IP5 were studied. White-wheat rolls contained 10 mg P as IP3 + IP4 and 2 mg P as IP5 + IP6 in experiment 4 and 20 mg P as IP3 + IP4 and 3 mg P as IP5 + IP6 in experiment 5; inositol phosphates were obtained via fermentation of sodium phytate. Each experiment had 8–11 subjects.
Results: In experiment 1, iron absorption was reduced by 39%, whereas there was no significant effect on iron absorption in experiments 2 and 3. In experiments 4 and 5, iron absorption was reduced by 54% and 64%, respectively, suggesting that IP3 and IP4 contributed to the inhibitory effect.
Conclusions: IP5 has an inhibitory effect on iron absorption, whereas IP3 and IP4 in isolated form have no such effect. IP3 and IP4 in processed food contribute to the negative effect on iron absorption, presumably by binding iron between different inositol phosphates. To improve iron absorption from cereals and legumes, degradation of inositol phosphates needs to be to less-phosphorylated inositol phosphates than IP3.
Key Words: Phytate • inositol phosphates • iron absorption • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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No effect on zinc and calcium absorption of aqueous solutions of pure fractions of inositol phosphates with <5 phosphate groups, produced via acid-catalyzed hydrolysis, was observed in suckling rats (10). Added separately to white bread, IP4 produced via acid-catalyzed hydrolysis had no effect on zinc absorption in humans, whereas IP5 and IP6 depressed zinc absorption (11). In vitro addition of isolated IP5 and IP6 to a white-wheat roll under simulated physiologic conditions had a strong negative effect on iron solubility (12). The results suggested that the inhibitory effect of IP5 was five-sixths that of equimolar amounts of IP6, thus corresponding to the number of phosphate groups. The addition of IP3 and IP4 produced via acid-catalyzed hydrolysis slightly increased the iron solubility under simulated physiologic conditions, but it was found to be strongly dependent on pH, in contrast with the addition of IP5 and IP6. Iron solubility decreased markedly when the pH was increased from 6 to 7, making the effect of IP3 and IP4 on iron absorption difficult to predict. In a human study of iron absorption from different bread meals, a strong negative correlation was found between iron absorption and the sum of IP3, IP4, IP5, and IP6, suggesting that IP3 and IP4 also contributed to the negative effect on iron absorption (13). In the present study, the effect of inositol phosphates on iron absorption was investigated in humans.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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± SEM: 139 ± 4 g/L), a transferrin saturation of 12–54% ( ± SEM: 28.9 ± 2.4%), and a serum ferritin concentration of 10–127 µg/L ( ± SEM: 39 ± 9 µg/L). The volunteers were given oral and written information about the aims and procedures of the study. The project was approved by the Ethical Committee of the Medical Faculty of the University of Göteborg.
Experimental design
Experiments 1–5
Each experiment included 8–11 subjects and iron absorption was measured in each subject by using 2 different radioiron tracers, 1 in the control meal and 1 in the test meal. Thus, each subject served as his or her own control. The control meal served in each experiment consisted of 2 white-wheat rolls, butter, and water. The test meals in the 5 experiments consisted of 2 white-wheat rolls, butter, and water, with added inositol phosphates as described below. IP5 (experiment 1), IP4 (experiment 2), and IP3 (experiment 3) were dissolved in a small amount of water and injected into the rolls after they were baked. The amount of inositol phosphates added to these 3 test meals was 10 mg P as inositol phosphates (Table 1
). In experiments 4 and 5, 1.00 and 1.71 mg P as phytate/g flour, respectively, were added as sodium phytate (Sigma Chemical Co, St Louis) to the dough before fermentation for 1.3 and 3 h, respectively. As a result, 
10 and 20 mg P as IP3 and IP4, and 2 and 3 mg P as IP5 and IP6 were obtained in the respective rolls.
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Experiment 6
IP1, IP2, IP3, IP4, IP5, and IP6 were analyzed and the isomeric forms of IP3 and IP4 were quantified in rye rolls containing native inositol phosphates in amounts comparable with those in experiment 5. These rye rolls were used in a previous iron-absorption study in which the test meal consisted of sourdough-fermented rye rolls and the control meal consisted of white-wheat bread (13).
Meals, roll making, and meal labeling
Each control or test meal consisted of 2 white-wheat rolls. In all experiments, the rolls were served with 20 g butter and 150 mL demineralized water. The calculated energy content of the meals was 1.8 MJ. Iron (3.7 mg) as ferrous sulfate was added to the dough of all rolls (Table 1
). The white-wheat rolls were made from 40 g unfortified white-wheat flour (55% extraction), yeast, sugar, table salt, and demineralized water. The amounts of yeast, sugar, salt, and water were 65, 35, 10, and 750 g/kg flour, respectively. The control and test doughs in experiments 1–3 were fermented for 3 h at 23°C to reduce the natural inositol phosphate content to undetectable concentrations. The test doughs in experiments 4 and 5 were fermented for 1.3 and 3 h, respectively, at 23°C after addition of 1.00 and 1.71 g P as phytate/g flour, respectively, to give 10 and 20 mg P as IP3 + IP4. The control and test doughs in experiments 1–5 were then kneaded again and weighed amounts were transferred to small aluminum forms, which were left standing for 20 min for further fermentation. The rolls were then baked at 250°C for 15 min.
In all studies, each meal was labeled with 46.2 kBq 59Fe or 55Fe by adding the liquid portion of the dough mixture. The isotope solutions of 55Fe (specific activity: 626 MBq/mg) and 59Fe (specific activity: 1127 MBq/mg) (Amersham International, Amersham, Buckinghamshire, United Kingdom), as ferric chloride in 0.1 mol/L, were prepared at the beginning of the study and checked for activity at the Radiation Physics Department, Sahlgren's Hospital, Göteborg, Sweden, before being used. In experiments 1–5, the test rolls were labeled with 55Fe and the control rolls with 59Fe.
Oral reference doses
A solution of 10 mL of 0.01 mol HCl/L containing 3 mg Fe as ferrous sulfate and 30 mg ascorbic acid labeled with 59Fe was used as a reference in all studies. The 10-mL vials containing the iron solution were rinsed twice with water and this rinse water was also consumed. Each subject received 2 reference doses on 2 consecutive mornings after an overnight fast. No food or drink was allowed for 3 h after the reference dose. Each subject received a total of 55.5 kBq 59Fe.
Preparation of inositol tri-, tetra-, and pentaphosphates
IP4 and IP5 were prepared according to the method of Sandberg et al (12). Sodium phytate (1.5 g; BDH Chemicals, Poole, United Kingdom) was hydrolyzed with 100 mL of 0.5 mol HCl/L for 7 h. The hydrolysate was evaporated to dryness at reduced pressure and a temperature of 40°C and then dissolved in 10 mL deionized water. The inositol phosphates formed were separated by anion-exchange chromatography. Glass columns (1 x 70 cm) containing 10 mL resin (AG 1-X8, 200–400 mesh; Bio-Rad Laboratories, Hercules, CA) were used. Fractions of 30 mL were eluted by a linear gradient from 0.05 to 0.5 mol HCl/L. The initial concentration of 0.05 mol HCl/L (2 x 30 mL) was used to separate IP1 and IP2 from the hydrolysate. IP3, but not IP4 and IP5, start to elute at a concentration >0.05 mol/L. The collected fractions of IP4 and IP5 were evaporated to dryness and dissolved in 10 mL mobile phase or water. The inositol phosphates were identified and quantified by high-performance ion-pair chromatography (12, 18). IP3 was prepared from sodium phytate and baker's yeast and the inositol 1,2,6-triphosphate [Ins(1,2,6)P3] isomer was separated by ion-exchange chromatography on an Aminex column (19). This fraction was prepared by Perstorp Pharma, Perstorp, Sweden.
Analysis of inositol phosphates and their isomers
The contents of IP3, IP4, IP5, and IP6 in white-wheat rolls; of isomers of IP4 and IP5 produced via acid-catalyzed hydrolysis; and of isomers of IP3, IP4, and IP5 in the white-wheat rolls used in experiments 4 and 5 and in the rye rolls used in experiment 6 were determined according to the method of Skoglund et al (20) after improvements to the anion exchanger were made (21). This method measures IP2, IP3, IP4, IP5, and IP6 in system 1 and IP1, IP2, and IP3 in system 2 with good separation of isomers. The method includes extraction of inositol phosphates from rolls with hydrochloric acid and separation of the inositol phosphates from the crude extract by anion-exchange chromatography. The inositol phosphate isomers were separated on high-performance ion-exchange columns with gradient elution (acidic gradient in system 1 and alkaline gradient in system 2) and were detected by using postcolumn reaction and ultraviolet detection (in system 1) or suppressed conductivity detection (in system 2). Furthermore, this improved method effectively separated the Ins(1,2,3)P3 and DL-Ins(1,2,6)P3 isomers in the white-wheat and rye rolls (21).
Analysis of iron
Rolls were freeze-dried and ground in a porcelain mortar. Duplicate samples (2.0 g) were analyzed for their iron content. All glassware was washed in 2.5 mol HCl/L and rinsed in deionized water before used. Iron was determined by atomic absorption spectrophotometry (model 360; Perkin-Elmer Co, Norwalk, CT) after dry-ashing at 450°C.
Statistical methods
All statistical analyses were made by using the STATVIEW II computer program (Abacus Concepts, Inc, Berkeley, CA). For statistical comparisons, means and SEs of the individual absorption ratios in each experiment were used. The possible statistical significance of the difference between the mean absorption and 1 was examined with an unpaired, two-sided t test. Differences were considered significant at P < 0.05. The recalculation of values to 40% of the absorption of the reference dose was used to compare the results of different iron-absorption experiments. The conclusion from each experiment was based on the results of pairwise comparisons in the same subjects without use of the reference dose results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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Experiments 1 and 2: pure fractions of IP4 and IP5
During acid-catalyzed hydrolysis of sodium phytate, a mixture of several isomers was formed as shown in Figure 1A. A similar sample was used to prepare the IP4 and IP5 fractions. The IP5 fraction was found to contain 24% Ins(1,3,4,5,6)P5, 41% DL-Ins(1,2,4,5,6)P5, 28% DL-Ins(1,2,3,4,5)P5, and 8% Ins(1,2,3,4,6)P5. The IP4 fraction was found to contain 7% DL-Ins(1,4,5,6)P4, 5% Ins(2,4,5,6)P4, 21% DL-Ins(1,2,5,6)P4, 14% DL-Ins(1,3,4,5)P4, 11% DL-Ins(1,2,4,5)P4, 6% Ins(1,3,4,6)P4, 23% DL-Ins(1,2,3,4)P4, 10% DL-Ins(1,2,4,6)P4, and 3% Ins(1,2,3,5)P4.
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. The main IP4 isomers in the white-wheat roll were DL-Ins(1,2,3,4)P4 (73%), DL-Ins(1,2,5,6)P4 (19%), and DL-Ins(1,3,4,5)P4 (4%); the IP3 isomers Ins(1,2,3)P3 and DL-Ins(1,2,6)P3 accounted for more than two-thirds of the total IP3 isomers. Furthermore, isomeric separation showed that there were almost equal amounts of these 2 IP3 isomers in the white-wheat rolls (21).(Figure 2C). Considerable amounts of less-phosphorylated inositol phosphates were found in the white-wheat rolls.
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). Ins(1,2,3)P3 and DL-Ins(1,2,6)P3 were present in equal amounts and accounted for 72% of total IP3 (Figure 2B). The latter isomer was used in experiment 3. The rye rolls were also found to contain considerable amounts of less-phosphorylated inositol phosphates. |
Iron absorption |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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. In experiment 1, iron absorption was significantly lower (by 39%) from the test roll, to which 10 mg P as IP5 was added, than from the control roll. Results from a previous study by Hallberg et al (22), who fed subjects a similar meal to which 10 mg P as IP6 had been added, showed 59% less iron absorption from the test meal than from the control meal. The inhibitory effect on iron absorption of IP5 was thus less marked than that of IP6. No significant effect on iron absorption was found when 10 mg P as IP4 (experiment 2) or Ins(1,2,6)P3 (experiment 3) was added to the test roll. In experiments 4 and 5, iron absorption was significantly lower (by 54% and 64%, respectively) from the test roll than from the control roll. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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10 mg) was added to the test rolls in experiments 1–3. A similar reduction in absorption was not found. The inhibiting effect on iron absorption was less for IP5 than that found previously by Hallberg et al (22) for IP6, and no significant inhibiting effect on absorption was seen with IP4 and IP3.
Thus, the results suggest that 
5 of the 6 possible sites on inositol needed to be phosphorylated for the inositol to exert an inhibitory effect. It is possible that this many phosphate groups are required to form the strong association between mineral ions and inositol phosphates that would be necessary to inhibit iron absorption. The inhibitory effect on absorption was due to the formation of an insoluble precipitate that cannot be absorbed. There is some evidence suggesting that mineral-binding capacity becomes progressively lower when phosphate groups are removed from IP6 (23, 24). In addition, the complexes formed by these derivatives become more soluble as the number of phosphate groups per molecule decreases (25). Thus, it is conceivable that no effects were observed when IP3 or IP4 was added to the test rolls because these inositol phosphates are more soluble and have a lower mineral-binding capacity for inositol phosphates than do IP5 and IP6.
In our previous study of iron absorption from many bread meals containing different amounts of various inositol phosphates, a strong negative correlation was found between the amounts of IP3, IP4, IP5, and IP6 and iron absorption (13), suggesting that these inositol phosphates contribute to the negative effect on iron absorption. In the present study, this effect was shown in experiment 6 from rolls baked from whole-rye flour and white-wheat flour containing 85% of the whole-wheat grain. Iron absorption from these rolls was 68% lower than that from the control roll despite the fact that there was only 4.9 mg P as IP5 + IP6 remaining after fermentation and baking. These rolls contained large amounts of IP3 and IP4 (25.8 mg P), with an approximate molar ratio of IP3 + IP4 to iron of 3.9:1. In experiment 5, iron absorption from the white-wheat test roll, which contained a mixture of inositol phosphates in amounts comparable with those in the rolls used in experiment 6 (21 mg P as IP3 + IP4 and 3 mg P as IP5 + IP6) and a molar ratio of IP3 + IP4 to iron of 3:1, was 64% lower than that from the control roll. This 64% lower absorption was likely not a result of the IP5 and IP6 content of the rolls alone.
The inhibition of nonheme-iron absorption by phytate was shown to be dose dependent (22). This relation between iron absorption and phytate content was composed of 2 regression lines with a point of intersection at 10 mg P as phytate. At this point of intersection, iron absorption was 60% less from the white-wheat test rolls than from the control rolls. On the basis of these regression lines, a test meal containing 3 mg P as IP6 would be expected to reduce iron absorption by 20–30%.
In addition, we found a 54% lower absorption of iron from the test roll with 10 mg P as IP3 + IP4 and a molar ratio of IP3 + IP4 to iron of 1:1 than from the control roll (experiment 4). The observed reduction in iron absorption was not a result of the content of 2 mg P as IP5 + IP6 in the roll only or of the content of the isolated form of 10 mg P as IP3 + IP4. In previous absorption studies with a similar design, 2 mg P as IP6 reduced iron absorption by 18% (22).
Hence, in the present study, IP3 and IP4 contributed to the negative effect on iron absorption from the rolls when IP5 and IP6 were also present, but not when isolated fractions of the inositol phosphates were added. One explanation for the results could be that certain isomers of IP3 and IP4 in processed foods other than those in the isolated fractions may have had an inhibitory effect, ie, the position of the phosphate groups on the inositol molecule influences its mineral-binding capacity and inhibitory effect. The isolated IP4 fraction added to the rolls in experiment 2 contained a mixture of isomeric forms, whereas one specific isomer of IP3, which predominates when sodium phytate is hydrolyzed by baker's yeast, was added to the rolls in experiment 3. Three of the 4 isomers in the isolated IP4 fraction were found to be formed in the rolls in experiments 4 and 5 and in the rye rolls used in the previous study (experiment 6; 13) as a result of phytate degradation by cereal phytase and yeast. The Ins(1,2,6)P3 isomer that was added to the rolls in the present study was 1 of the 2 predominant isomers formed in the white-wheat and rye rolls containing high concentrations of IP3 and IP4; the other predominant isomer was Ins(1,2,3)P3.
With use of potentiometric titration it was found that Ins(1,2,6)P3 forms stable complexes with Fe3+ at a pH of 7.0 (26). The 1,2,3-triphosphate grouping has, as well, been shown to be an orientation forming strong complexes with Fe3+. The iron uptake as a function of various isomeric forms of IP3 and IP4 in vitro was studied in Caco-2 cells, a highly differentiated enterocyte-like cell line (27). No effect on absorption due to different isomeric forms of inositol phosphates was found. Therefore, it does not seem likely that the isolated forms of the predominant isomers of IP3 and IP4 in the rolls had an inhibitory effect on iron absorption.
On the other hand, it is conceivable that a mixture of various inositol phosphates has effects different from those of the pure fractions alone. It is possible that IP3 and IP4, and the less-phosphorylated inositol phosphates (IP1 and IP2) that were found in considerable amounts (although not quantified in this study) in the rolls, contributed to the negative effect on iron absorption by interacting with the higher-phosphorylated inositol phosphates, resulting in an increase in mineral-binding capacity. One iron molecule presumably binds, with a high degree of covalency, 2 or 3 phosphate groups located on 1, 2, or 3 different inositol phosphate molecules. This presumption is strongly supported by the negative effect on iron absorption of the test rolls that containined a mixture of inositol phosphates with small amounts of IP5 and IP6 (2 or 3 mg P as inositol phosphates) and large amounts of IP3 and IP4 (10 or 21 mg P as inositol phosphates). The observed inhibition of iron absorption from these test rolls was related to the sum of the tri-, tetra-, penta-, and hexaphosphate groups.
In conclusion, addition of IP5 to white-wheat rolls inhibits iron absorption to a significantly lesser degree than does IP6, whereas IP4 produced via acid-catalyzed hydrolysis and Ins(1,2,6)P3 does not inhibit iron absorption significantly. Furthermore, the results suggest that IP3 and IP4 contribute to the negative effect on iron absorption of processed foods by interacting with the higher-phosphorylated inositol phosphates, resulting in an increase in mineral-binding capacity. These findings suggest that phytate degradation of not only IP5 and IP6, but also of IP3 and IP4 in cereals and soy products, for example, must occur in the home or in the manufacturing process to improve the iron absorption of such food products.
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSIron absorptionDISCUSSIONREFERENCES |
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