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4 Laboratory of Human Nutrition and 5 Laboratory of Food Biotechnology, ETH Zurich, Zurich 8092, Switzerland and 6 Department of Chemistry and Department of Biochemistry, National University of Singapore, Singapore 117543
* To whom correspondence should be addressed. E-mail: mhoppler{at}ethz.ch.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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Increasing the ferritin content of seeds is currently considered the most promising approach to biofortifying staple foods, such as rice, wheat, cassava, or beans, with iron (9). Ferritin serves as an iron storage protein in virtually all living organisms, including bacteria, plants, and animals. It consists of 24 subunits that are assembled into a spherical shell that can store up to 4500 Fe(III) atoms in its inner cavity in the form of an iron oxyhydroxide-phosphate mineral. Lines of transgenic rice and maize that express soybean ferritin (Glycine max) or common bean ferritin (Phaseolus vulgaris) have already been developed (10–12). However, a basic requirement for the successful implementation of a biofortified crop is an adequate bioavailability of the respective nutrient. For ferritin-iron, contradictory results have been published from several human studies, either showing good (13–16) or poor (17–21) iron absorption from ferritin. These differences have been associated with isotopic labeling of ferritin-iron. It has been suggested that labeling of animal ferritin under inflammatory conditions, as employed in the studies showing poor iron bioavailability, may have led to a ferritin molecule that releases iron less readily (9). Recently, it was proposed that ferritin is resistant to digestion in the gastrointestinal tract and that it might even be absorbed intact by the mucosal cell (14). Such a mechanism would protect ferritin-iron from interactions with iron absorption inhibitors, like phytic acid or polyphenols, which are largely responsible for the low bioavailability of iron from plant sources (22,23). In this study, we evaluated the iron bioavailability of ferritin by monitoring the degradation of the ferritin molecule and the release of iron from its mineral core during cooking and simulated gastric conditions. If ferritin is degraded and ferritin-iron released during cooking and gastric digestion, ferritin-iron would be expected to enter the common nonheme iron pool and be absorbed to the same extent as other nonheme food iron.
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Materials and Methods |
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We have described the expression and isolation of recombinant bean (P. vulgaris) ferritin in detail elsewhere (24). Briefly, Escherichia coli cells were transformed with the expression plasmid pET 28a(+) (Novagene) containing ferritin cDNA from P. vulgaris (25). The bacterial culture was grown overnight at 37°C in M9 medium containing kanamycin (30 mg/L) and chloramphenicol (34 mg/L). Expression of ferritin was induced by adding isopropyl β-D-thiogalactopyranoside to the medium to a final concentration of 1 mmol/L together with iron at a concentration of 0.2 mmol/L for incorporation into ferritin. Recombinant bean ferritin was isolated by a 2-step procedure by ion exchange chromatography using a diethylaminoethyl (DEAE) Sepharose column (10 by 1.6 cm; GE Healthcare) followed by gel filtration using a Sephacryl S-300 column (2.6 by 60 cm). The purified ferritin was stored in 50% glycerol at –20°C. The protein concentration of the ferritin solution was determined by the Bradford method (26).
Quantification of total iron. The iron content of legumes and ferritin were analyzed by graphite furnace atomic absorption spectrophotometry (GF-AAS, AA240Z, Varian). Legumes were ground under liquid nitrogen with a rotor mill (ZM1, Retsch) using a titanium sieve (0.25 mm mesh) and successively mineralized by microwave digestion (MLS ETHOS plus, MLS GmbH) using an HNO3/H2O2 mixture. A commercial iron standard (Titrisol, Merck) was used for external calibration.
Iron speciation and quantification of ferritin-iron.
Dry peas, white beans, red kidney beans, and soybeans were ground under liquid nitrogen and then suspended in a 10-fold volume of ice-cold phosphate buffer (50 mmol/L sodium phosphate, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulphonyl fluoride, 1% polyvinylpolypyrrolidone, pH 7). Proteins were extracted after homogenization with a Polytron (PT1200 E, Kinematica). After centrifugation at 20,000 x g for 20 min, the supernatant was separated from the sediment. For cooking and digestion experiments, the recombinant bean ferritin solution and legume extracts were analyzed for ferritin-iron content by size exclusion chromatography using a Superdex 200 column with a fractionation range of 10–600 kDa (1 x 30 cm, GE Healthcare) and phosphate buffer for elution (0.15 mmol/L sodium phosphate, 0.15 mmol/L NaCl, pH 7). Protein elution was monitored spectrophotometrically at 280 nm. Fractions of 4 mL were collected and iron content was measured in each fraction by GF-AAS. Ferritin-iron eluted from the column 
20 min after sample injection. For pea extract, it was verified whether all the iron collected in this fraction was ferritin-bound. The collected fraction was desalted and further purified by ion exchange chromatography using a DEAE column (GE Healthcare) with a NaCl gradient of 0–0.5 mol/L. Iron containing fractions after ion exchange chromatography were pooled and concentrated by ultrafiltration. The concentrated sample was applied to native PAGE. Iron in gels was stained as Prussian blue [2% K4Fe(CN)6 solution in 2% HCl]. For phosphorus quantification in recombinant bean ferritin, the phosphate buffer was removed and exchanged with 18 M
water (NANOpure system, Barnstead/Thermolyne) by using ultrafiltration spin columns (Vivaspin, Sartorius AG) and passing 18 M water 5 times through the columns. Phosphorus content was determined by multi-collector inductively coupled plasma MS (Neptune, Thermo Scientific) by external calibration using a gravimetrically prepared standard.
Ferritin stability during cooking.
To investigate the stability of ferritin during cooking, recombinant bean ferritin and different legume seeds were boiled in a 5-fold volume of 18 M water for 50 min (recombinant bean ferritin, dry peas, red kidney beans, and soybeans) or 100 min (white beans) until tender. Cooking experiments were repeated with 1 mmol/L EDTA (pH 7) in the cooking water. EDTA was added to allow the detection of ferrous or ferric iron in the soluble fraction. In the absence of a chelator, free iron was poorly soluble at neutral pH and would precipitate. The legumes were cooled down after cooking and a phosphate buffer was added to 10-fold the sample volume. Cooked legumes were blended using a hand blender and homogenized with a Polytron. The samples were centrifuged at 20,000 x g for 20 min at 4°C and the supernatant was loaded onto the gel filtration column for ferritin separation.
Ferritin stability at different pH and in the presence of EDTA. To assess ferritin stability during digestion, recombinant bean ferritin was exposed to a pH range that could be expected in the human stomach. Ferritin was warmed up to 37°C and pH adjusted to 7, 5, 3.5, and 2, respectively, by the addition of 0.1 mol/L HCl. Samples were flushed with argon to prevent oxidation and were incubated in a shaking water bath at 37°C in the dark. Aliquots were taken after 15, 30, 60, and 120 min, respectively, and pH adjusted to 7 using 0.1 mol/L NaOH. After centrifugation at 20,000 x g for 20 min at 4°C, the supernatant was applied to the gel filtration column for ferritin separation. For experiments with EDTA, ferritin stability in the presence of EDTA was verified by incubating recombinant bean ferritin at neutral pH in a shaking water bath at 37°C in a 1 mmol/L EDTA solution. Aliquots were taken after 15, 30, 60, and 120 min and ferritin was separated as described earlier.
Pepsin/HCl digestion. Gastric digestion of recombinant bean ferritin and red kidney bean extract was simulated in vitro using a modification of the procedure of Miller et al. (27). For digestion, 40 mg pepsin/g protein (pepsin from porcine stomach mucosa, Sigma-Aldrich) were added and pH was adjusted to 2 by adding 0.1 mol/L HCl. The mixture was incubated in a shaking water bath at 37°C for 2 h. After incubation, pH was adjusted to 7 using an aqueous NaOH solution. EDTA was then added to a final concentration of 1 mmol/L in the digest to prevent precipitation of solubilized iron at neutral pH. After centrifugation at 20,000 g for 20 min at 4°C, the supernatant was applied to the gel filtration column for ferritin separation.
Kinetics of ferritin degradation during simulated pepsin/HCl digestion. Recombinant bean ferritin and raw and cooked peas were subjected to an in vitro procedure that simulated gastric conditions. Pepsin was added in the same concentration as described above. The acidity of the solution was lowered stepwise to mimic a transient change, as reported for the human stomach during gastric digestion (28–30). Digestion was initiated by lowering the pH to 5 (t = 0) to imitate the higher pH due to the postprandial buffering effect of a meal in the stomach (29). The pH was then lowered in 3 steps by adding 0.1 mol/L HCl to pH 3.5 after 15 min, to pH 2.5 after 30 min, and to pH 2 after 60 min, respectively. Experiments were terminated at 120 min. Samples were incubated at 37°C in a shaking water bath in the dark and were kept under argon to protect samples from oxidation. Aliquots for ferritin-iron determination were taken after 15, 30, 60, and 120 min. After digestion, pH was adjusted to 7 using 0.1 mol/L NaOH to mimic pH conditions in the duodenum as the predominant stage of intestinal iron absorption. Samples were centrifuged at 20,000 x g for 20 min at 4°C and applied to the gel filtration column for ferritin separation.
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Results |
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1,000 Fe atoms per ferritin molecule. This degree of iron saturation of ferritin is well within the range found in various plant ferritins (31–33). Phosphorus quantification showed a molar iron to phosphorus ratio of 4.2:1, which is in the typical range reported for plant ferritins (34,35). When purified recombinant bean ferritin was separated on a Superdex 200 column, it was recovered in fraction A3 20 min after injection (Fig. 1A). An amount of >95% of total iron of the purified recombinant bean ferritin solution was recovered in this fraction.
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Ferritin stability during cooking. Iron speciation was studied in recombinant bean ferritin and different legumes by gel filtration before and after cooking. Soybeans showed the lowest soluble ferritin-iron content of 18% of total seed iron, and peas the highest at 42% (Table 1). After cooking for 50–100 min in boiling water, the soluble ferritin-iron content of the 4 analyzed legumes was below the detection limit of the applied method of 5% relative to total iron content of the seeds. Similar observations were made for solutions of recombinant bean ferritin after cooking. Ferritin-iron that was recovered after cooking was found to be 8% for recombinant bean ferritin.
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40 min after sample injection in the presence of EDTA. This was observed for both the recombinant bean ferritin and the red kidney bean extract (Fig. 3B,D). In a separate experiment, we found that Fe-EDTA solution applied to the same gel filtration column was also quantitatively recovered 40 min after injection (data not shown).
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Our studies show that native legume ferritin is altered to a large extent during cooking. We measured ferritin-iron contents in different legumes before cooking (Table 1) and found ferritin-iron as a percentage of total iron to vary from 42% in dry peas to 18% in soybeans (Table 1). In a previous study where Mössbauer spectrometry was employed for iron speciation, 90% of soybean seed iron was found in a trivalent state and showed an isomer shift and quadrupole splitting resembling ferritin-iron (42). This led to the conclusion that over 90% of soybean iron occurs in the form of ferritin-iron (41). The value we measured for soybeans corresponds better with another study, which reported that soluble ferritin-iron is 15% of the total iron in soybeans, as specifically measured by immunoprecipitation (16). After cooking, however, no ferritin-iron could be detected in any of the legumes tested. There are several possible explanations for these findings: 1) the release of iron in an ionized form from ferritin during cooking; 2) ferritin becoming insoluble during cooking and thereby trapping ferritin-bound iron in the shell; and 3) the dissociation of the protein shell of the ferritin molecule and liberation of the mineralized core that would be insoluble at the pH of the food matrix. Reductive food components, such as ascorbic acid or polyphenols, which are both found in legumes, have been shown to liberate iron from ferritin in vitro (43,44), and we speculate that this may account for iron leakage during food preparation. Alternatively, denaturation of the ferritin protein hull is also likely. Temperatures during boiling exceeded denaturation temperatures of ferritin, which were reported to be in the range of 77–93°C (45).
Whereas cooking reduced the amount of total soluble iron, the soluble iron remaining was much higher when legumes and isolated ferritin were cooked in the presence of EDTA (Table 1). EDTA serves as a chelating agent and prevents precipitation of solubilized iron at neutral pH after its release from the food matrix and/or the ferritin molecule. At the same time, EDTA per se had no detectable effect on ferritin stability (Fig. 2E). Similar results were obtained when experiments were conducted with purified recombinant bean ferritin, although detectable amounts of intact ferritin were identified after cooking (Table 1). We cannot rule out that ferritin stability during cooking is higher in other plant foods due to different methods of food preparation or different composition. Should any ferritin survive cooking unaltered, it would still be unlikely that significant amounts of intact ferritin would arrive in the duodenum because our results indicate that ferritin-iron is released during gastric digestion (Figs. 2–4). We assume that the ferritin shell is dissociated by the acidic conditions in the stomach and that the mineral core is dissolved in the gastric contents.
The lack of detection of ferritin-iron and concurrent detection of EDTA-bound iron after gastric digestion at pH 2 is an indication for the release and/or dissolution of ferritin-iron (Fig. 3B,D). EDTA itself did not have any influence on ferritin-iron solubilization in this experiment because it was only added after digestion to keep dissolved iron in solution at neutral pH. In experiments that were conducted without the subsequent addition of EDTA, any released or dissolved iron was probably rendered insoluble at pH 7 (Figs. 2, 4A). This was not the case in the digestion of dried peas, which resulted in >30% of nonferritin-bound iron remaining soluble at pH 7 (Fig. 4B) and could be due to soluble iron-binding pea components. The observed ferritin-oligomers (Fig. 2) seem to be minor intermediate products formed during digestion. It is unlikely that these play a special role for iron uptake because they are undetectable by the end of gastric digestion. Our results (Figs. 2–4) are in agreement with 2 recent studies, which showed that after in vitro digestion at pH 2, the ferritin protein hull was fully dissociated or degraded (46,47). In the study by Kalgaonkar and Lönnerdal (47), this effect was less pronounced when gastric digestion was simulated at pH 4 instead of pH 2. This result gives rise to the hypothesis that some ferritin may leak into the intestine in intact form at an early stage of gastric digestion. It can be argued, however, that these experiments were conducted with uncooked, intact ferritin. Ferritin-iron is mostly present in foods such as legumes that usually undergo long and harsh cooking procedures. As shown by our experiments, these conditions have a strong effect on ferritin integrity. Assuming that part of ferritin is altered to an insoluble form during cooking, there are indications that such ferritin-iron is also dissolved in the gastric juice: 20–30% of insoluble iron after cooking, possibly insoluble ferritin-iron, was solubilized by gastric digestion over 2 h (Table 2). Ferritin was also degraded during digestion when it was not previously extracted but embedded in the plant matrix, where it may be protected by plant cell components and plastids (Fig. 4B; Table 2). These findings imply that a possible ferritin-specific uptake mechanism, as has been proposed earlier (14,15,41), has a smaller impact on iron absorption than previously assumed.
The view that ferritin could protect its iron core from absorption inhibitors (41) was primarily based on the results of 2 iron absorption studies where intrinsically labeled soybeans were used (13,48). Those studies reported relatively high fractional absorption (20% and 27%, respectively) despite the presence of phytic acid in the test meals. When considering early and recent intrinsic/extrinsic tag studies (48–51), however, it seems unlikely that ferritin-iron is better absorbed than other nonheme iron. In these studies, iron in cereals and legumes was labeled intrinsically by adding a radioiron tag during growth, presumably also labeling ferritin-iron intrinsically. An extrinsic radioiron tag in the form of FeCl3 was added shortly before test meal administration. In all studies, the efficiency of intrinsic tag absorption from the labeled legume-based meals was similar to the extrinsic tag absorption. Even in the study of Sayers et al. (48), which showed high fractional iron absorption, the extrinsic tag of FeCl3 was as well absorbed as the intrinsically labeled soybean iron (including ferritin-iron). An evaluation of the data by Murray-Kolb et al. (13) in this context is difficult because no extrinsic tag was given for comparison. Therefore, these experiments do not provide any indication that ferritin-iron is absorbed to a different degree than other nonheme iron. This is also in line with 2 recent human studies that showed that iron from in vitro labeled plant and animal ferritin is as equally well absorbed as iron from ferrous sulfate (14,15). Furthermore, Caco-2 cell studies have demonstrated that after in vitro digestion, uptake of ferritin-iron was influenced by dietary factors such as phytic acid, tannic acid, calcium, and ascorbic acid (46,47). Another Caco-2 cell study has shown that iron absorption from common beans is influenced by the polyphenol content of the beans (52), which likewise does not indicate that the ferritin-iron in the beans is protected from absorption inhibitors.
In conclusion, the results from our study show that most plant ferritin is altered during cooking, either by release of iron or denaturation of the protein hull. Any remaining ferritin-iron, either in soluble form as intact ferritin, as insoluble ferritin after denaturation, or as the naked mineral core, is then released and dissolved in a pH-dependent manner during gastric digestion. Such released and dissolved iron would be taken up by the divalent metal transporter-1 pathway for nonheme iron and would display similar susceptibility to iron absorption enhancers and inhibitors. At the present stage, we cannot exclude the possibility that small amounts of ferritin may survive cooking and gastric passage intact. However, our experiments indicate that these amounts can be safely expected to be very small and are therefore unlikely to change overall absorption efficiency of plant iron.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: M. Hoppler, A. Schönbächler, L. Meile, R. F. Hurrell, and T. Walczyk, no conflicts of interest.
3 Supplemental Figure 1 is available with the online posting of this paper at jn.nutrition.org.
Manuscript received 26 October 2007. Initial review completed 27 November 2007. Revision accepted 24 February 2008.
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