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Biochemical and Molecular Actions of Nutrients

Duodenal Ascorbate Levels Are Changed in Mice with Altered Iron Metabolism^1,2

Bisera Atanasova^*, Ian S. Mudway^,**, Abas H. Laftah^**, Gladys O. Latunde-Dada^**, Andrew T. Mckie^**, Timothy J. Peters, Kamen N. Tzatchev^* and Robert J. Simpson^**,3

^* Department of Clinical Laboratory and Clinical Immunology, Medical University, Sofia, Bulgaria; Lung Biology, Department of Health and ^** Department of Life Sciences, King’s College London, UK; and Department of Clinical Biochemistry, GKT School of Medicine, King’s College, Denmark Hill Campus, London, UK

³To whom correspondence should be addressed. E-mail: robert.simpson{at}kcl.ac.uk.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Ascorbate has long been thought to play an important role in intestinal iron absorption. The recent identification of a possible ascorbate-dependent duodenal ferric reductase suggests a role for intracellular ascorbate in the control of iron absorption. We set out to determine whether duodenal ascorbate concentrations are altered by treatments known to alter the rate of iron absorption and whether ascorbate levels affect duodenal reductase activity. Duodenal ascorbate was extracted and assayed by HPLC and/or a chemical assay. Ferric reductase was assayed in vitro with ferric nitrilotriacetate or nitroblue tetrazolium as substrates. Duodenal ascorbate concentrations were increased by iron deficiency, genetic hypotransferrinemia, and hypoxia. Parenteral iron overload increased iron stores but did not affect duodenal ascorbate concentrations. Hemolytic anemia induced in mice by phenylhydrazine injection also did not affect duodenal ascorbate concentrations. In vitro studies with incubated duodenum showed that decreased tissue ascorbate was associated with decreased mucosal ferric reductase activity, whereas incubation with dehydroascorbate prevented both the decrease in ascorbate concentration and reductase activity. Mouse duodenum ascorbate concentrations changed in response to treatments that altered iron absorption rates; in particular, ascorbate levels generally increased when iron absorption was increased by iron deficiency, hypoxia, or genetic hypotransferrinemia. We conclude that changes in ascorbate levels are associated with changes in ferric reductase activity. These findings are consistent with the proposal that duodenal ascorbate plays a role in intestinal iron absorption.

KEY WORDS: • iron absorption • ferric reductase • iron overload • iron deficiency • hypotransferrinemia

Iron is of vital importance for many biological processes, mainly because of its ability to undergo rapid, reversible valence changes. As the capacity of the body to excrete iron is limited, body iron levels are held within narrow limits primarily by controlling the proximal intestinal absorption of dietary iron. Iron balance is thus maintained by regulating iron absorption so that it approximates the uncontrolled losses of iron (1–2 mg/d) through exfoliation of epithelial mucosal cells and gastrointestinal and menstrual blood loss. Failure to maintain homeostasis leads to either iron deficiency or iron overload. The duodenum is the principal site of regulated iron absorption (1). Recent advances have identified several gene products as being involved in iron absorption and its regulation (1–5). These gene products collectively provide a framework for transcellular iron trafficking in the duodenum (1).

Ascorbic acid (AsA; Vitamin C)⁴ is a vitamin in humans and guinea pigs that has important functions in hydroxylation reactions, antioxidant defense, and metal metabolism (6). Ascorbic acid has long been known to enhance absorption of iron from test meals (7), and recent work showed that ascorbate levels are correlated with iron absorption for a range of typical American meals (8). Ascorbate seems to enhance iron absorption more strongly when meals do not include meat (9), but the effects of ascorbate appear to diminish for complex meals and over longer periods of study (10). Epidemiological studies have also demonstrated a correlation between dietary ascorbate and iron stores (11). A recent study of the effects of ascorbate on iron absorption in rats suggested that ascorbate has effects in addition to the accepted one of solubilizing iron in the intestinal lumen (12). Earlier work on iron absorption in scorbutic guinea pigs by Glover et al. (13) also led to the suggestion that intracellular ascorbate could regulate iron absorption. Recently a duodenal transplasma membrane reductase, duodenal cytochrome B (Dcytb), was determined to be associated with the mucosal surface reduction of ferric iron (3). Structural analysis and work with cultured cells that express Dcytb (3,14,15) suggest that the intracellular electron source for this reductase is cytosolic ascorbate. This mechanism might explain the interaction between intracellular ascorbate levels and the rate of iron absorption.

To further investigate the hypothesis that intracellular ascorbate concentrations affect the regulation of iron absorption, we assayed ascorbate levels in the duodenal mucosa of mice with altered iron metabolism.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Reagents.

The KH₂PO₄ and K₂HPO₄ were purchased from BDH Chemicals; all other reagents were purchased from Sigma. The ascorbate oxidase (from Cucurbita species) had an activity of 205 U/mg. The AsA and FeCl₃ were ACS reagents.

Ascorbate and dehydroascorbate assay.

A spectrophotometric assay was used to determine the concentrations of AsA and dehydroascorbic acid (DAsA) in various biological samples, with the exception of some experiments with hypoxic mice, in which both spectrophotometric and HPLC assays were employed. The spectrophotometric assay was modified from one described by Kampfenkel (16) that employs the reduction of Fe³⁺ to Fe²⁺ by AsA and spectrophotometric detection of Fe²⁺ complexed with ferrozine. Dehydroascorbic acid was assayed by reducing it to ascorbic acid by preincubation of the samples with dithiothreitol (DTT). Excess DTT was removed with N-ethylmaleimide, then total ascorbic acid (i.e., DAsA + AsA) was assayed by the same procedure used for AsA. The concentration of DAsA was then calculated by difference (16). Ascorbic acid oxidase was used to correct for background non-AsA reducing activity. The calibration curve for the AsA assay showed that the color development was linear from 50 to 500 µmol/L. The detection limit for the assay was 50 µmol/L. All tissues had measured absorbance above the variation of the blank sample.

Tissue ascorbate concentrations were assayed by reversed-phase HPLC with electrochemical detection at 400-mV, 0.2-µA sensitivity, using established methodologies (17). Peak identities were confirmed by incubating unacidified samples with ascorbate oxidase (2 U per sample, 15 min at room temperature). Total ascorbate concentration (ascorbate + dehydroascorbate) was assayed by pretreating samples with reductant DTT according to the method of Esteve et al. (18). There was good correlation between the spectrophotometric assay and the HPLC method (R = 0.77, P < 0.001; n = 34).

Homogenization of the tissues.

A modified extraction method was used for the mouse intestinal samples, because it improved AsA recovery compared to published methods (16). Hepatic or duodenal tissue (100 mg) was put in a plastic homogenization tube and 1 mL of ice-cold 50 g/L metaphosphoric acid (MPA) was added. The samples were homogenized with a single 25-s burst, using an Ultra Turrax homogenizer (Janke & Kunkel Ika Werke). Homogenates were kept on ice and protected from direct light at all times. The homogenates were centrifuged for 5 min (17,900 xg) at 4°C in an Eppendorf 5417 R centrifuge. The supernatants were stored at -70°C for up to 2 wk. The reliability of the extraction procedure was checked by a recovery experiment; AsA recovery totaled 102 ± 1.0% of added AsA (SD, n = 4) when added to tissue extracts and 90 ± 9.0% (SD, n = 3) when carried through the whole extraction with tissue.

Animals.

Male mice, CD1 strain, 3 or 6 wk old, were obtained from Charles Rivers. The mice were housed in plastic and stainless steel cages in an animal facility with a 12 h light, 12 h dark cycle. To induce iron deficiency, 4-wk-old mice were fed a purified low-iron diet [<1 mg Fe/kg (19)] containing no AsA for 3 wk. Mice in the control group were fed the same diet supplemented with 62 mg/kg of iron as FeCl₃ · 6H₂O (19). The mice were kept in grid-bottom cages for the first 2 d after initiation of the purified diets.

Iron overload, phenylhydrazine, or hypoxic exposure treatment was performed on groups of mice that were 6 to 7 wk old and that had unrestricted access to a standard commercial mouse diet (Diet RM1; Scientific Diet Services) (19). This diet contained 160 mg Fe/kg and 2.7 mg ascorbate/kg (manufacturer’s data). Chronic hypoxia was induced in mice by placing them in a hypobaric chamber maintained at 53.3 kPa for 1 or 3 d (19). Iron overload was induced in 6-wk-old mice by i.m. and/or subcutaneous injection of 5 mg iron dextran (100 µL of 50 mg/mL, Vifor) on each of 2 consecutive days (mice in the control group received the same volume of 0.15 mol/L NaCl). These mice were then left for 2 wk to allow for iron redistribution. Phenylhydrazine treatment was performed as described previously (20). Mice were injected with 60 mg/kg body weight of neutralized phenylhydrazine by the intraperitoneal route on each of 2 consecutive days, then killed 4 d later.

Homozygous hypotransferrinemic mice (trf^hpx/hpx, Balb/c strain) were bred and maintained at King’s College, London. The mice were fed a commercial diet (Diet CRM; Scientific Diet Services) that was similar to Diet RM1 [see Simpson (19)] and contained 184 g protein/kg, 34 g oil/kg, 151 g fiber/kg, 63 g ash/kg, 424 g starch/kg, 39 g sugar/kg, 131 mg Fe/kg, and 1.8 mg ascorbate/kg (manufacturer’s data), as described previously (21). Briefly, homozygotes were obtained from trf^hpx/+ x trf^hpx/+ matings and maintained by weekly injections of mouse serum (up to 0.3 mL, i.e., 1 mg of transferrin) until they were used at 3 wk of age. Normal (mixture of trf^+/+ and trf^hpx/+) same-sex littermate mice were used as controls.

The mice were killed by anesthetic overdose (halothane) followed by bleeding from the heart. Duodenum (and for some experiments also jejunum and ileum) samples were removed and rinsed with ice-cold 0.15 mol/L NaCl, then the tubes with the tissues were put into liquid N₂ immediately. The frozen samples were weighed and homogenized in 50 g/L MPA as described above. The blood was allowed to clot, then was centrifuged for 10 min at room temperature (1700 x g). The serum was mixed with an equal volume of 50 g/L MPA. The samples were processed as for tissues, and the supernatants were analyzed for AsA and DAsA. All mouse procedures were carried out under the authority of the appropriate UK Home Office licenses.

In vitro experiments.

To alter the duodenal ascorbate levels and evaluate the effect of this alteration on duodenal ferric reductase activity, duodenal fragments (20 mg) were obtained from freshly killed normal or hypoxic (1 or 3 d at 53.3 kPa) mice. Fragments were obtained immediately after killing mice by anesthetic overdose (halothane). The duodenum was opened longitudinally along the junction with the pancreas and blotted gently to remove mucous. Four sections (2 to 3 mm; 20 mg) were cut across the full width of the duodenum and incubated in oxygenated (95% O₂:5% CO₂) incubation buffer (pH = 7.4; 125 mmol/L NaCl, 3.5 mmol/L KCl, 1 mmol/L CaCl₂, 10 mmol/L MgSO₄, 16 mmol/L Na-HEPES, and 10 mmol/L D-glucose) at 37°C for 5 min. Three fragments were then placed in separate tubes containing incubation buffer and an addition of 5 mmol/L DAsA (to increase intracellular AsA concentration) or 2 mmol/L 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) (to decrease AsA concentration) or no addition (control group) as appropriate; the final volume of the incubation medium was 2.5 mL. The medium was oxygenated and the samples were incubated for 15 min at 37°C. Fragments were then rinsed and transferred to fresh incubation buffer containing either 1 mmol/L nitroblue tetrazolium (NBT) (to assay surface reductase activity) or, in a separate series of experiments, 250 µmol/L nitriloacetate (NTA) and 1 mmol/L ferrozine (to assay ferrireductase activity). The NBT reduction assays were performed for 5 min, then the tissue was rinsed and imaged using a dissecting microscope and a Polaroid Microcam. The FeNTA₂ reduction was performed for 5 min with the medium being sampled, and Fe²⁺ formation was assayed spectrophotometrically as described previously (22). The initial reduction rate for NBT or FeNTA₂ for each mouse was obtained by assaying the fourth fragment from each duodenum without the 15 min incubation step. The NBT reduction was measured by scanning densitometry as described previously (23). The FeNTA₂ reduction was calculated in units of pmol · mg wet wt^-1 · min^-1.

Hemoglobin and iron assays.

Liver nonheme iron and hemoglobin levels were measured as described previously (19).

Statistical analysis.

All results for the test groups are expressed as means ± SD. Data for the mice were compared by t test. The effects of in vitro incubation were analyzed by one-way ANOVA with post-hoc t tests. The effects of the intestinal location and hypotransferrinemia were assessed by two-way ANOVA. The spectrophotometric and HPLC assays were compared by regression analysis.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In vivo alterations in iron metabolism and tissue ascorbate levels.

There was some evidence for batch-to-batch variation in duodenal ascorbate levels in normal (control) mice, with the lowest levels in mice fed a purified diet containing no added ascorbate (see data for control groups in Tables 1, and 2). We assumed this was due to variations in the dietary ascorbate concentrations in different batches of diet and/or breeding strain, age, or genetic variations in the outbred CD1 mice. A similar variation was also apparent in the plasma ascorbate concentrations, which varied from 88 to 169 µmol/L. These concentrations were high, at plasma saturation levels probably reflecting the fact that the mice had not been deprived of food. All comparisons of alterations in iron metabolism were therefore carried out with age- and strain-matched control groups studied on the same day as the experimental groups.

Genetic hypotransferrinemia increased duodenal ascorbate levels (Table 2). Based on limited sampling, it also affected other regions of the intestine, although ileum ascorbate levels were lower than those of the duodenum (control duodenum, 1.58, 2.47; control ileum, 1.2, 1.5; hypotransferrinemic duodenum, 3.12, 2.9; hypotransferrinemic ileum, 2.2, 2.4 mmol/kg tissue; P < 0.05, n = 2; individual data values two-way ANOVA). The difference due to hypotransferrinemia was greater in the duodenum than in other tissues. Hypoxic exposure treatment had similar effects (ileum ascorbate levels of 1.4 ± 0.1 vs. 1.8 ± 0.2 mmol/kg in control vs. hypoxic mice; n = 6, P < 0.01). Iron deficiency also increased ileum ascorbate levels (1.8 ± 0.5 vs 2.7 ± 0.5 mmol/kg in control vs. iron-deficient mice; n = 5, P < 0.05; note that these values are for a different treatment group from those shown in Table 1).

In vitro alterations in duodenal ascorbate and duodenal reductase activity.

To experimentally alter duodenal ascorbate levels and evaluate the effects of this alteration on mucosal reductase activity, mouse duodenum was incubated in vitro and analyzed for ascorbate levels (Fig. 1). We previously reported that mouse tissue survived for 15 min in the incubation medium described with intact cellular ultrastructure and ATP levels (24). In the present study, the total incubation time was 25 min. Incubated tissue retained its morphology with no loss of epithelial cells in any case. Incubation of tissue caused the loss of ascorbate. The addition of dehydroascorbate prevented this loss of ascorbate, as in other cells (25). Incubation with Tempo further reduced ascorbate levels, as previously reported for red cells with similar reagents (26). The reduction of NBT, which we previously reported is catalyzed by the duodenal ferric reductase Dcytb (3), was inhibited by the same conditions (incubation of tissue and incubation with Tempo) that reduced ascorbate levels. The addition of DAsA to the incubation medium to prevent ascorbate depletion (Fig. 2) also prevented the decrease in reductase activity. Hypoxic mouse tissue was used for this experiment because NBT reduction by normal mouse duodenal tissue was minimal. Lowering tissue ascorbate levels by incubation also decreased ferric reductase activity in hypoxic mouse duodenal tissue (Fig. 3). The addition of DAsA to the medium prevented this effect. Tempo could not be used to lower ascorbate levels for ferric reductase assays because the reduction product of Tempo reacted with FeNTA₂, causing high apparent ferric reductase values (data not shown).

View larger version (10K): [in this window] [in a new window]   FIGURE 1 Effect of in vitro incubation on ascorbate levels in duodenal fragments from mice exposed to hypoxia for 3 d. Fragments were frozen immediately (No incub) or incubated for 15 min with buffer containing no additions (Buffer), 5 mmol/L DAsA, or 2 mmol/L Tempo, then frozen until ascorbate assay. Data are means ± SD, n= 6. Bars without a common letter differ, P < 0.05.

View larger version (8K): [in this window] [in a new window]   FIGURE 2 Effect of incubation with DAsA or Tempo on NBT reductase activity in duodenal fragments from a mouse exposed to hypoxia for 1 d. Fragments were incubated as described for Fig. 1, then assayed for NBT reductase. Data indicate peak villus optical density. Initial activity was measured in tissue that was not incubated prior to assay. Data are means ± SD, n = 7–15 villi. Bars without a common letter differ, P < 0.05. Data shown are for a representative experiment that was repeated twice.

View larger version (10K): [in this window] [in a new window]   FIGURE 3 Effect of incubation with DAsA on ferric reductase activity in duodenal fragments from mice exposed to hypoxia for 1 or 3 d. Fragments were incubated as described for Fig. 1, then rinsed in buffer (no additions), and assayed for ferric reductase by incubation for 5 min with 250 µmol/L FeNTA2 in the presence of 1 mmol/L ferrozine. Initial activity was measured in tissue that was not incubated prior to assay. Data are means ± SD, n = 4–6. Bars without a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Iron overload did not affect duodenal ascorbate concentrations in mice. Previous iron-overload studies using non-ascorbate synthesizing guinea pigs reported various tissue ascorbate responses; some studies reported increased ascorbate concentrations (30), whereas others reported no change (13). In contrast, studies in humans have suggested decreased ascorbate concentration under iron-overload conditions [for a review, see Roeser (26)].We found that the induction of iron deficiency in mice increased duodenal ascorbate concentrations; there were similar increases in mice exposed to hypoxia and in trf^hpx/hpx mice. Although tissue responses in these groups were consistent, serum ascorbate concentrations were unaffected in the iron-deficient and trf^hpx/hpx mice, but were markedly decreased in the hypoxic mice. The latter response may reflect the contradictory effects of hypoxia, which include both increased erythropoiesis and oxidative stress (31), whereas anatomical variations in the level and site of ROS production may explain why serum ascorbate concentrations are affected more than duodenal levels.

Phenylhydrazine treatment did not increase either duodenum or serum ascorbate concentrations in mice. Glover et al. (13) also reported no increase in serum ascorbate in guinea pigs treated with phenylhydrazine, whereas Sengupta and Chatterjee (32) reported that phenylhydrazine decreased tissue ascorbate levels, although the latter authors used higher doses and allowed less time for tissue levels to recover after dosing than we did in the present study. Our data may therefore be consistent with both these previous studies, suggesting that although phenylhydrazine induces increased absorption of iron over time, it may also have a direct effect, as an oxidant, of initially decreasing tissue ascorbate levels.

Mice are not optimal models of ascorbate metabolism in humans, due to the presence of ascorbate-synthesizing mechanisms, especially in the liver (28). Mice have, however, been very useful in elucidating the fundamental mechanisms of iron absorption and its regulation in humans (1). As discussed above, there is evidence that the changes in ascorbate levels caused by altered iron metabolism in mice may also occur in humans and guinea pigs.

In all cases, duodenal ascorbate levels changed in the same direction as changes in iron absorption previously reported in these models (19–21,29), suggesting a positive association between duodenal ascorbate levels and iron absorption rates. Ileum ascorbate levels also increased when duodenum ascorbate levels increased, although the magnitude of the increase generally was greater in the duodenum. No changes were evident in the liver in any of the studied groups, presumably reflecting this organ’s capacity for regulated ascorbate synthesis in mice.

In vitro incubation generally caused a loss of ascorbate from the tissue, suggesting that the duodenum does not synthesize ascorbate rapidly from the glucose present in the medium. The addition of dehydroascorbate to the medium generally increased duodenal ascorbate levels. Presumably, dehydroascorbate is taken up by the duodenum via the glucose transporter and is reduced within the cells by glutathione, as previously suggested in studies of other cells (25).

In a previous study we reported that mucosal surface reductase activity is not mediated by the release of ascorbate into the medium, with reuptake and re-reduction of DAsA (33). The present data therefore further support the suggestion that intracellular ascorbate is the electron source for duodenal ferric reductase (14) and show that alterations in duodenal ascorbate levels can affect ferric reductase activity. The candidate gene for this activity, Dcytb, is a homologue of an ascorbate-dependent reductase, cytochrome b561, and the ascorbate binding sites on the protein are conserved in Dcytb (3,14). The present findings strongly support ascorbate as the intracellular electron donor for Dcytb activity and provide a molecular mechanism for an intracellular role for ascorbate in intestinal iron absorption. Previous work showed that genetic hypotransferrinemia, iron deficiency, and hypoxia increase duodenal Dcytb levels in mice (3); therefore, it seems that both the reductase and its substrate, ascorbate, are coordinately regulated. Factors that alter duodenal ascorbate levels (e.g., dietary intake of ascorbate, dehydroascorbate, or oxidants) may therefore alter the rate of iron absorption.

Duodenal ascorbate levels are increased by iron deficiency, hypoxia, and genetic hypotransferrinemia, and the changes suggest a positive association with iron absorption rates. In vitro alterations in ascorbate levels alter duodenal ferrireductase activity. These findings support an intracellular role for ascorbate in iron absorption.

	FOOTNOTES

 
¹ A preliminary report of some of this data was made in Atanasova, B., Simpson, R., Tzachev, K. & Peters, T. (2001) Assay of ascorbic and dehydroascorbic acid from mice tissues. Balk. J. Clin. Lab. 8: 72.

² This work was supported by an International Federation of Clinical Chemistry Professional Scientific Exchange Programme and the UK Medical Research Council.

⁴ Abbreviations used: AsA, ascorbic acid; DAsA, dehydroascorbic acid; Dcytb, duodenal cytochrome B; DTT, dithiothreitol; MPA, metaphosphoric acid; NBT, nitroblue tetrazolium; NTA, nitrilotriacetate; ROS, reactive oxygen species; Tempo, 2,2,6,6-tetramethylpiperidine-1-oxyl; trf^hpx/hpx, homozygous hypotransferrinemic mice.

Manuscript received 24 October 2003. Initial review completed 13 November 2003. Revision accepted 2 December 2003.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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