Copper deficiency increases iron absorption in the rat

Carla Thomas and Phillip S. Oates

Physiology School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, 6009, Australia

Submitted 2 December 2002 ; accepted in final form 17 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin (Hp) and ceruloplasmin (Cp), respectively. In swine, copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced, as evidenced by the loss of copper-dependent plasma ferroxidase I activity, and in enterocytes, CD reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced, and liver iron was doubled. CD reduced duodenal mucosal iron and ferritin, whereas CD increased iron absorption. Duodenal mucosal DMT1-IRE and ferroportin1 expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron-deficient anemic animals, strong correlations were found among mucosal iron, ferritin, and iron absorption, suggesting that the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp, as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased, it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also, the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages, because Kupffer cells of the liver and nonheme iron levels of the spleen were normal during copper deficiency, suggesting another role for Cp in these cells.

ceruloplasmin; ferrous; hephaestin; intestine; ferritin; ferroxidase; efflux


THE BODY'S USEABLE IRON pool is determined by the amount absorbed by the intestine plus that released from storage sites such as macrophages and hepatocytes (18). It has been shown that adequate copper stores are necessary for the efflux of iron from the liver. Thus, during copper deficiency in rats, iron accumulated within hepatocytes (7, 8, 13, 14, 24, 25, 30, 33, 45). The finding that liver iron was increased in proportion to dietary iron content suggests that iron absorption may be normal or even increased by copper deficiency. However, direct measurements of iron absorption in copper-deficient rats are inconclusive (5, 7, 9).

In contrast to rodents, copper deficiency in swine results in decreased levels of total liver iron, suggesting that iron absorption is impaired (17, 23). In fact, in these animals, the enterocytes accumulate large amounts of ferritin-bound iron (23), suggesting that iron import into enterocytes is normal but export is impaired. Supporting this, direct measurement showed that iron absorption was reduced in swine (23). Irrespective of these differences between the swine and rodent, as plasma iron levels fall, anemia follows (7, 8, 13, 14, 17, 21, 24, 25, 30, 34, 35, 45, 46).

The discovery of the copper-containing plasma protein ceruloplasmin (Cp) and its subsequent characterization as a ferroxidase led to the hypothesis that in hepatocytes, macrophages, and enterocytes, iron is oxidized during efflux to bind to plasma transferrin (19, 29). However, Williams and co-workers (46) showed that the ferroxidase activity of Cp varies widely between species, being lowest in the rat and 10-fold higher in swine, which led the authors to conclude that this action may not be the major function of Cp but that it only enhances the export of iron. Certainly, intact Cp is necessary for the export of iron, because iron accumulated in the liver of individuals with aceruloplasminemia and in Cp knockout mice (18, 47).

In Cp knockout mice, intestinal iron transport was not affected (18, 47) nor was Cp effective in augmenting iron efflux from enterocytes in whole tissue (9) or in cell lines (40, 49), suggesting the functioning of an alternative protein to Cp. Indeed, it was recently shown that for optimal efflux of iron, the enterocyte requires the Cp homolog hephaestin (Hp) (16, 42), and Attieh and co-workers (2) recently showed that it has ferroxidase activity comparable with that of ceruloplasmin. Therefore, it might be expected that if Hp operates by way of its ferroxidase activity in iron absorption, then this will be impaired by copper deficiency to a similar extent as Cp in hepatocytes. The function of Hp in iron absorption is, in part, based on studies in mice with sex-linked anemia (sla), in which three exons of the Hp gene are missing (1, 42). The intestinal phenotype of sla mice is similar in appearance to copper-deficient swine; that is, the export of iron from the intestine is impaired, and there is an accumulation of ferritin-bound iron within the enterocyte (3, 1012, 22, 31, 32). However, when these mice were fed an iron-deficient diet for several days during which time a new population of enterocytes with low cellular iron enters the absorptive zone of the villus, iron absorption increased, and in some studies, this was to levels seen in normal animals fed the same diet (11, 36, 39, 41). This finding suggests that the mutant Hp can function normally during iron deficiency of the enterocyte and raises the question as to whether Hp operates by way of its ferroxidase activity or by another mechanism that is compromised by the mutation.

Because of the impaired iron absorption reported in copper-deficient swine and sla mice, it is expected that copper-deficient rodents will respond similarly, but direct and indirect measurements do not support this (5, 7, 9) Furthermore, it was recently shown that in copper-deficient Caco-2 cells, iron absorption was increased (49), although others (38) have found that in these cells, exposure to copper increased the activity of the basolateral transporter ferroportin1 and cellular efflux of iron. In view of these discrepancies, we studied iron absorption in copper-deficient rats, and these data correlated with the expression of the apical and basolateral iron transporters DMT1 and ferroportin1, respectively, along with mucosal ferritin and nonheme iron, which are important factors known to contribute to the mechanism of iron absorption.


    MATERIAL AND METHODS
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals

The Animal Welfare Committee of the University of Western Australia has approved all procedures dealing with the handling of animals described in this study.

Normal, weanling outbred female Wistar rats were obtained from the Animal Resource Center (Murdoch, Western Australia). The rats were placed on one of three diets, each containing identical protein, fat, carbohydrate, complete vitamin, and fiber supplements. The diets were produced in accordance with the recommendations of the American Institute of Nutrition (AIN 93) (43). The diets also contained a balanced mineral mix differing only in the iron and copper contents. The copper-deficient diet contained no added copper but had a normal level of iron (70 mg/kg as finely ground ferric citrate). The control diet contained normal levels of copper [6 mg Cu(II)/kg] and iron (43), and the iron-deficient diet had normal levels of copper but no added iron.

Iron Absorption

Preparation of iron absorption test solution. The Fe(II) test dose consisted of a 200-µl solution containing 200 nmol of iron in the form of FeSO4 to ascorbate at a molar ratio of 1:100 in 50 mM HEPES and 80 mM NaCl. This solution was prepared immediately before use by mixing 59FeSO4 in 50 mM HCl with 100-fold molar excess of unlabeled FeSO4 in 50 mM HCl and then the required amounts of ascorbic acid. After it was mixed, NaCl, HEPES (pH 7.0), and NaOH were added in amounts required to give the correct iron, HEPES, and NaCl concentrations and to neutralize the acid in the iron solutions to pH 7.0. The Fe(II)-to-ascorbate molar ratio of 1:100 was chosen as suitable for maintaining the iron in a soluble, absorbable form in the ferrous state (26).

Animals were lightly anesthetized with Halothane before gavaging with Fe(II). Whole carcass radioactivity was then measured immediately and again 5 days later, at which time carcass radioactivity had stabilized. Absorption represented the percentage radioactivity remaining in the carcass divided by the total amount injected.

Isolation of Villus Enterocytes

The proximal small intestine was removed, and the enterocytes along the crypt-villus axis were separated into 10 fractions as previously described (28). Fractions 2–4, containing enterocytes derived from the midvillus region, were pooled and washed three times in PBS to remove extracellular mucous. Total cell copper concentration was measured by atomic absorption spectrometry and corrected for variations in cellular protein (40). Total copper-dependent oxidase activity was measured as described previously (40).

Recovery of Tissue for Analyses

Five days after gavage, the animals were anesthetized with pentobarbital sodium (Nembutal) at a dose of 12 mg/100 g body wt. After deep anesthesia, a midline abdominal incision was made into the abdomen and then into the thorax. A blood sample was taken from the heart for the measurement of hematocrit and plasma iron. The animal was then exsanguinated. The liver was removed, weighed, and a 200-mg portion of the right lobe was placed in 10% buffered formal saline (BFS). The duodenum was removed, and a ~5-mm segment from the proximal end was fixed in BFS. The remaining duodenum was opened longitudinally, and then a superficial scraping of the duodenal mucosa was homogenized in lysis buffer consisting of 25 mM Tris · HCl (pH 7.5), 150 mM NaCl plus 0.5% Triton X-100, and protein inhibitors PMSF, leupeptin, and Trasyol for Western blot analysis and the measurement of nonheme iron. A piece of liver was also prepared for Western blot analysis.

Plasma ferroxidase I activity

Plasma ferroxidase I (ceruloplasmin) was measured by incubating plasma with 9.2 mM p-phenylenediamine in 0.2 M acetate buffer pH 5.2 in the presence or absence of 1.5 M sodium azide at 37°C. The reaction product was measured at 540 nm, and the plasma ferroxidase activity was converted to grams per liter expressed as units (29).

Nonheme Iron Levels in Duodenal Mucosa, Liver and Plasma

Nonheme iron was measured in plasma, liver and intestinal mucosa (20).

Expression of DMT1-IRE, ferritin, and ferroportin1 by Western Blot Analysis

The protein content of samples of the liver and intestinal mucosa was estimated by the biuret method. One hundred micrograms of total cellular protein were electrophoresed on an 8% sodium dodecyl sulfate-polyacrylamide gel as described previously (40). Staining of the gel with Ponceau S was performed to show equal loading of lanes before Western blotting. The blots were immunoreacted with polyclonal antibodies generated against human DMT1-IRE (NRAMP22-S, Alpha Diagnostics International), rat ferroportin1 (40), and rat L&H ferritins (40) and processed for Western blot analysis as described previously (40). The human DMT1-IRE recognizes rat DMT-1 antibody as evidenced by immunodetection of a protein migrating at the same rate from total protein derived from rat IEC-6 and rat enterocytes (unpublished observation). The density of the signal and background were determined by densitometry using the National Institutes of Health Image 1.62 to estimate the level of expression.

Detection of Iron and Ferritin/Hemosiderin in the Duodenum and Liver

Five-micrometer paraffin sections of duodenum and liver were stained for iron by the Perl's staining method. Staining was performed using 1% potassium ferrocyanide dissolved in either 2% HCl to identify hemosiderin deposits or in 5% acetic acid to detect soluble-labile iron (6).

Detection of Ferritin in the Duodenum

Sections were also prepared for immunohistochemistry to detect ferritins (40) as previously reported (27).

Statistics

Data were analyzed by either the Student's t-test for unpaired samples or when three conditions were analyzed collectively by analysis of variance with partitioning according to Tukey's test. Significance was considered at P < 0.05.


    RESULTS
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 RESULTS
 DISCUSSION
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Validation of the Model

There was no significant difference in plasma ferroxidase I activity between control and iron-deficient rats (Table 1). However, plasma ferroxidase I activity was significantly reduced from these levels in copper-deficient rats (Table 1). Copper deficiency of duodenal enterocytes was confirmed because copper concentration was reduced to 32% normal levels (Table 1) and total enterocyte copper-dependent oxidase activity was reduced to 46% normal levels (Table 1).


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Table 1. Plasma ferroxidase I activity, enterocyte copper levels, total enterocyte copper-dependent oxidase activity, body and liver weights, plasma iron, hematocrit, expression of DMT1-IRE, ferritin, and ferroportin1 in 12-wk-old rats fed a copper-deficient, normal, or iron-deficient diet to female rats for 8 wks from weaning

 

Body and Tissue Weights and Hematological Indexes

Total body and liver weights were not different between the three conditions (Table 1). Plasma iron and hematocrit were significantly reduced from controls in animals made either copper deficient or iron deficient (Table 1).

Liver and Spleen Nonheme Iron

Compared with controls, liver nonheme iron was increased with copper deficiency and significantly reduced with iron deficiency (Fig. 1A). Also, because there was no difference in the weight of the livers among the three groups, the same pattern was seen when the iron concentrations were corrected for slight variations in weight (Fig. 1A).



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Fig. 1. Liver (A) and spleen nonheme iron (B) expressed as either total or corrected for variations in weight (Fe/g) tissue. The results are the mean ± SE. Twenty-two copper-deficient and control rats and five iron-deficient rats were used to obtain these measurements. *Significant difference between control and iron-deficient or copper-deficient groups. 1Significant difference between control and iron-deficient group only.

 

In the spleen, copper deficiency did not alter nonheme iron stores compared with control, but iron deficiency reduced nonheme levels compared with copper deficiency and control animals (Fig. 1B). This was the case when the data were corrected for variations in weight (Fig. 1B).

Perl's Staining within the Liver

Little iron staining was seen in the livers of control animals (Fig. 2A) and none was detected in the livers of iron-deficient animals (data not shown). Liver iron staining was evident in copper-deficient animals, where it was highest within periportal hepatocytes and least in those surrounding the central vein region (Fig. 2B). Kupffer cells appeared negative for iron staining (Fig. 2).



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Fig. 2. Perl's staining for iron in the liver of normal (A) and copper-deficient (B) rats. Perl's positive granules stain weakly in hepatocytes and strongly in macrophages (arrows) of control animals (A), but with copper deficiency, hepatocyte iron loading is markedly increased, particularly in the periportal region of hepatocytes. Absent is macrophage iron loading (B; arrows).

 

Iron Absorption

Total iron absorption was significantly greater in animals made copper deficient compared with control animals. However, in iron deficiency, total iron absorption was greater than in copper-deficient and control animals (Table 1).

Duodenal Mucosal Nonheme Iron Concentration

Copper deficiency and iron deficiency significantly reduced mucosal nonheme iron concentration compared with control animals. Mucosal iron levels were significantly lowered in iron deficiency compared with copper deficiency (Table 1).

Comparisons Among Duodenal Mucosal Nonheme Iron Concentration, Ferritin Expression, and Iron Absorption

When these data from control, copper-deficient, and iron-deficient rats were pooled and correlations were performed between mucosal nonheme iron and ferritin expression (Fig. 3A), mucosal nonheme iron and total iron absorption (Fig. 3B), and ferritin expression and total iron absorption (Fig. 3C), strong correlations were found. The strongest correlation existed between mucosal nonheme iron and ferritin expression, but the other relationships were also significant.



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Fig. 3. Correlations between mucosal nonheme iron vs. mucosal ferritin (A) and total iron absorption (B). C: correlation between mucosal ferritin and total iron absorption. Data from normal ({diamond}), iron-deficient ({circ}), and copper-deficient ({square}) animals were pooled to perform these analyses (n = 13). Correlation coefficients to these regressions are 0.93, -0.87, and -0.84, respectively. Mucosal nonheme iron concentration is expressed as ng/µg protein, total iron absorption is expressed as %dose in body 5 days after gavage, and ferritin is expressed as arbitrary units/100 µg protein.

 

DMT1, Ferroportin1, and Ferritin Expression of Villus mucosa

The levels of DMT1-IRE and ferroportin1 expression were similar in copper-deficient and control conditions. DMT1-IRE and ferroportin1 were detected with molecular weights of ~66 and ~60 kDa, respectively (Fig. 4, A and B). The ferritins were detected as a single band migrating at ~20 kDa (Fig. 4C). Compared with control animals, mucosal ferritin was reduced in animals deficient in copper and, to a greater degree, by iron deficiency (Table 1, Fig. 4).



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Fig. 4. Western blot analysis of protein from duodenal mucosa of copper-deficient animals (-Cu) and control (Con) animals. One hundred micrograms of protein were electrophoresed on an 8% polyacrylamide gel. After transfer, the gel was immunoreacted with polyclonal antibodies against human DMT1-IRE (A), rat ferroportin1 (B), and rat ferritin (C).

 

Staining of Duodenum for Iron and Cellular Distribution of Ferritin

There was no detectable staining by the Perl's method in the duodenum of any condition studied (data not shown). This was the case when the reaction was carried out to reveal soluble iron using 5% acetic acid or stored iron in the form of hemosiderin using 2% hydrochloric acid (data not shown).

In control animals, ferritin expression commenced at the crypt-villus junction within enterocytes and reached maximal intensity at the villus tip (Data not shown). In copper-deficient animals, ferritin expression was less than in control animals. It was largely confined to the upper one-third of villus enterocytes (data not shown).


    DISCUSSION
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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In this study, copper deficiency is evidenced by depleted duodenal enterocyte copper concentration and consequently enterocyte copper-dependent oxidase activity. It also eliminated plasma ferroxidase I activity, produced anemia, and doubled hepatic iron stores. These findings support previous studies on the effect and extent of copper deficiency on iron metabolism in the rat and mouse (7, 8, 13, 14, 24, 25, 30, 33, 45). Because liver iron levels are raised in copper-deficient rats, it suggests that total iron absorption is increased; however, previous direct measurements have produced conflicting results (5, 7, 9). In this investigation, we demonstrate that copper deficiency increased iron absorption. It should be pointed out that in these studies, absorption of Fe(II) was measured; however, it cannot be excluded that absorption of Fe(III) is reduced by copper deficiency. This is based on the dependence of a surface-localized reductase such as Dcytb to reduce Fe(III) to Fe(II) before uptake. Yu and Wessling-Resnick (48) have shown that copper deficiency reduced uptake of Fe(III), possibly suggesting impairment of reductase activity.

These results therefore differ from those reported in copper-deficient swine, in which iron absorption is impaired and, supporting this, liver iron levels are lower than normal (17, 23). It is unlikely that in the swine, the reduced liver iron is due to increased release from hepatocytes, because parenteral iron leads to hepatocyte iron overload despite severe anemia (23). The most likely reason for this difference is the greater dependence on copper-dependent ferroxidase activity in facilitating the release of iron from cells of swine compared with rats. In fact, rat and human Cp have significantly less biological activity than porcine Cp in increasing plasma iron levels in copper-deficient swine (35, 46) and in ferroxidase I activity (46). On the basis of the low ferroxidase activity of rat Cp, it was suggested that this function of Cp may be secondary to another more important function within Cp that exports iron from the liver (46).

It is also possible that Hp functions by means other than its ferroxidase activity. This is based on two observations. First, compared with other forms of stimulation of iron absorption, such as iron deficiency anemia and what occurs normally, with copper deficiency the mucosal iron levels and mucosal ferritin are low and appropriate for the increased level of iron absorption (see below). This suggests that there is no impairment to the release of iron from the enterocyte. Second, with copper deficiency, despite impaired Cp activity as evidenced by reduced plasma ferroxidase I activity and hepatocyte iron loading, mucosal iron levels are reduced, suggesting normal Hp activity. Thus, if the ferroxidase I activity of Hp is the main function of this protein, then enterocyte iron accumulation and impaired iron absorption would be seen. Because this was not observed, we suggest that in addition to copper-dependent ferroxidase activity, Hp may have another function that is lost by the mutation in sla mice. Recently, Syed and co-workers (37) modeled the NH2-terminus ectodomain of human Hp and suggested that the mutation to Hp found in sla mice would result in substantial nonfolding of the secondary structure of the protein that in turn would lead to impaired ferroxidase activity. However, it is also possible that another function residing within this region would also be compromised because of the mutation.

In copper-deficient rats, in contrast to increased iron levels within hepatocytes, macrophages have normal levels of iron as evidenced by normal Perl's staining of Kuppfer cells and normal spleen nonheme iron levels, a finding supported by others (7, 8, 24, 30). However, efflux of iron from the spleen clearly requires Cp activity, because in Cp knockout mice, spleen levels increased 250% compared with wild-type animals (18, 47). Thus it is possible that in the rodent, the efflux of iron from the macrophage also does not rely on ferroxidase I activity of Cp but on some other function performed by this protein.

The question as to whether there is impaired iron absorption during copper deficiency also needs to be considered from another perspective, namely the nature of the systemic regulators. The significantly increased iron absorption produced by copper deficiency is the sum of two major but opposing stimuli, namely increased erythropoiesis and increased liver iron stores. The anemia of copper deficiency will stimulate the erythroid regulator and, in turn, this stimulates iron absorption. However, opposing this, increased iron stores, acting through the "stores" regulator possibly involving hepcidin, will inhibit iron absorption (15). Because the erythroid regulator is recognized to be the more potent of the two, a net but modest stimulation of iron absorption with copper deficiency was expected and found in this present study (15). To determine the effect of both of these stimulators operating cooperatively, we studied iron absorption in iron-deficient animals made anemic to a similar level as copper-deficient rats. These animals have increased erythropoiesis and markedly reduced hepatocyte iron stores. In addition, these animals also have markedly reduced enterocyte iron stores, which is another stimulator of iron absorption (4, 44). Under these circumstances, iron absorption was significantly raised above that of copper-deficient rats. In view of this comparison, it is likely that iron absorption in copper-deficient animals is operating appropriately for the level of stimulation from systemic regulators.

In summary, this study suggests in copper-deficient rodents that despite reduced copper-dependent ferroxidase activity of duodenal enterocytes, iron absorption is unimpaired, whereas plasma ferroxidase I activity is impaired, and there is hepatocyte iron accumulation. Macrophages also appear to rely on Cp for efflux but not on its ferroxidase I activity. In view of this, it will be of interest to determine whether the mutation to Hp in sla mice leads to the loss of its proposed copper-dependent ferroxidase activity or of a yet-to-be-determined function that resides within the region deleted by the mutation.


    DISCLOSURES
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 ABSTRACT
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This work was supported by a grant from the National Health and Medical Research Council of Australia.


    ACKNOWLEDGMENTS
 
We thank E. Morgan for helpful discussions during the writing of this manuscript and the technical assistance of A. Light.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Oates, Physiology School of Biomedical and Chemical Sciences, Univ. Of Western Australia, Crawley, 6009, Australia (E-mail: poates{at}cyllene.uwa.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Anderson GJ, Frazer DM, McKie AT, and Vulpe CD. The ceruloplasmin homologue hephaestin and the control of iron absorption. Blood Cells Mol Dis 29: 367-375, 2002.[ISI][Medline]
  2. Attieh ZK, Alaeddine RM, Su T, Anderson GJ, and Vulpe C. Identification of a ferroxidase activity of hephaestin. J Clin Gastroenterol 34: 370, 2002.
  3. Bedard YC, Pinkerton PH, and Simon GT. Ultrastructure of the duodenal mucosa of mice with a hereditary defect in iron absorption. J Pathol 104: 45-51, 1971.[ISI][Medline]
  4. Charlton RW, Jacobs P, Torrance JD, and Bothwell TH. The role of the intestinal mucosa in iron absorption. J Clin Invest 44: 543-554, 1965.[ISI][Medline]
  5. Chase MS, Gubler CJ, Cartwright GE, and Wintrobe MM. Studies on copper metabolism. IV. The influence of copper on the absorption of iron. J Biol Chem 757-763, 1952.
  6. Clark G. Staining Procedures. Baltimore: Williams and Wilkins, 1981, p. 202.
  7. Cohen NL, Keen CL, Hurley LS, and Lönnnerdal B. Determinants of copper-deficiency anemia in rats. J Nutr 115: 710-725, 1985.[ISI][Medline]
  8. Cohen NL, Keen CL, Lönnerdal B, and Hurley LS. Effects of varying dietary iron on the expression of copper deficiency in the growing rat: anemia, ferroxidase I and II, tissue trace elements, ascorbic acid, and xanthine dehydrogenase. J Nutr 115: 633-649, 1985.[ISI][Medline]
  9. Coppen DE and Davies NT. Studies on the roles of apotransferrin and caeruloplasmin (EC1.1631) on iron absorption in copper-deficient rats using an isolated vascularly- and luminally-perfused intestinal preparations. Br J Nutr 60: 361-373, 1988.[ISI][Medline]
  10. Edwards JA and Bannerman RM. Hereditary defect of intestinal iron transport in mice with sex-linked anemia. J Clin Invest 49: 1869-1871, 1970.[ISI][Medline]
  11. Edwards JA and Hoke JE. Effect of dietary iron manipulation and phenobarbitone treatment on in vivo intestinal absorption of iron in mice with sex-linked anemia. Am J Clin Nutr 28: 140-145, 1975.[ISI][Medline]
  12. Edwards JA, Hoke JE, Mattioli M, and Reichlin M. Ferritin distribution and synthesis in sex-linked anemia. J Lab Clin Med 90: 68-76, 1977.[ISI][Medline]
  13. Evans JL and Abraham PA. Anemia, iron storage and ceruloplasmin in copper nutrition in the growing rat. J Nutr 103, 196-201, 1973.[ISI][Medline]
  14. Fields M, Bureau I, and Lewis CG. Ferritin is not an indicator of available hepatic iron stores in anemia of copper deficiency in rats. Clin Chem 43: 1457-1459, 1997.[Free Full Text]
  15. Finch C. Regulators of iron balance in humans. Blood 84: 1697-1702, 1994.[Free Full Text]
  16. Frazer DM, Vulpe CD, McKie AT, Wilkins SJ, Trinder D, Cleghorn GJ, and Anderson GJ. Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins. Am J Physiol Gastrointest Liver Physiol 281: G931-G939, 2001.[Abstract/Free Full Text]
  17. Gubler CJ, Lahey ME, Chase MS, Cartwright GE, and Wintrobe MM. Studies on copper metabolism. III. The metabolism of iron in copper deficient swine. Blood 7: 1075-1092, 1952.[ISI]
  18. Harris ZL, Durley AP, Man TK, and Gitlin JD. Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl Acad Sci USA 96: 10812-10817, 1999.[Abstract/Free Full Text]
  19. Holmberg CG and Laurell CB. Investigations in serum copper. II. Isolation of the copper containing protein, and description of some of its properties. Acta Chem Scand 2: 550-556, 1948.[ISI]
  20. Kaldor I. Studies on intermediary iron metabolism. V. The measurement of non-haemoglobin tissue iron. Aust J Exp Biol Med Sci 32: 795-800, 1954.[ISI]
  21. Lahey ME, Gubler CJ, Chase MS, Cartwright GE, and Wintrobe MM. Studies on copper metabolism. II. Hematologic manifestations of copper deficiency in swine. Blood 7: 1053-1074, 1952.[ISI][Medline]
  22. Levy JE, Montross LK, and Andrews NC. Genes that modify the hemochromatosis phenotype in mice. J Clin Invest 105: 1209-1216, 2000.[Abstract/Free Full Text]
  23. Lee GR, Nacht S, Lukens JN, and Cartwright GE. Iron metabolism in copper-deficient swine. J Clin Invest 47: 2058-2069, 1968.[ISI][Medline]
  24. Marston HR and Allen SH. Function of copper in the metabolism of iron. Nature 215: 645-646, 1967.[ISI][Medline]
  25. Marston HR, Allen SH, and Swaby SL. Iron metabolism in copper-deficient rats. Br J Nutr 25: 15-30, 1971.[ISI][Medline]
  26. Oates PS and Morgan EH. Defective iron uptake by the duodenum of Belgrade rats fed diets of different iron contents. Am J Physiol Gastrointest Liver Physiol 270: G826-G832, 1996.[Abstract/Free Full Text]
  27. Oates PS and Morgan EH. Ferritin gene expression and transferrin receptor activity in intestine of rats with varying iron stores. Am J Physiol Gastrointest Liver Physiol 273: G636-G646, 1997.[Abstract/Free Full Text]
  28. Oates PS, Thomas C, and Morgan EH. Characterization of isolated duodenal epithelial cells along a crypt-villus axis in rats fed diets with different iron content. J Gastroenterol Hepatol 12: 829-838, 1997.[ISI][Medline]
  29. Osaki S and Johnson DA. Mobilization of liver iron by ferroxidase (Ceruloplasmin). J Biol Chem 244: 5757-5765, 1969.[Abstract/Free Full Text]
  30. Owen CA. Effects of iron on copper metabolism and copper on iron metabolism in rats. Am J Physiol 224: 514-518, 1973.[Free Full Text]
  31. Pinkerton PH. Histological evidence of disordered iron transport in the X-linked hypochromic anaemia of mice. J Pathol Bacteriol 95: 155-165, 1968.[ISI][Medline]
  32. Pinkerton PH and Bannerman RM. Hereditary defect in iron absorption in mice. Nature 216: 482-483, 1967.[ISI][Medline]
  33. Prohaska JR. Repletion of copper-deficient mice and bridled mice with copper or iron. J Nutr 114: 422-430, 1984.[ISI][Medline]
  34. Ragan HA, Nacht S, Lee GR, Bishop CR, and Cartwright GE. Effect of ceruloplasmin on plasma iron in copper-deficient swine. Am J Physiol 217: 1320-1323, 1969.[Free Full Text]
  35. Roeser HP, Lee GR, Nacht S, and Cartwright GE. The role of ceruloplasmin in iron metabolism. J Clin Invest 49: 2408-2417, 1970.[ISI][Medline]
  36. Sorbie J, Hamilton DL, and Valberg LS. Effects of various factors on iron absorption in mice with X-linked anaemia. Br J Haematol 27: 559-569, 1974.[ISI][Medline]
  37. Syed BA, Beaumont NJ, Patel A, Naylor CE, Bayele HK, Joannou CL, Rowe PSN, Evans RW, and Srai SKS. Analysis of the human hephaestin gene and protein: comparative modeling of the N-terminus ectodomain based upon ceruloplasmin. Protein Eng 15: 205-214, 2002.[Abstract/Free Full Text]
  38. Tennant J, Stansfield M, Yamaji S, Srai SK, and Sharp P. Effects of copper on the expression of metal transporters in human intestinal Caco-2 cells. FEBS Lett 527: 239-244, 2002.[ISI][Medline]
  39. Thomson ABR and Valberg LS. Integrity of the iron transport process in mice with X-linked anaemia. Scand J Haematol 14: 347-354, 1975.[ISI][Medline]
  40. Thomas C and Oates PS. IEC-6 cells are an appropriate model of intestinal iron absorption in rats. J Nutr 132: 680-687, 2002.[Abstract/Free Full Text]
  41. Toskes PP, Smith GW, and Conrad ME. Cobalt absorption in sex-linked anemic mice. Am J Clin Nutr 26: 435-437, 1973.[ISI][Medline]
  42. Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N, Gitschier J, and Anderson GJ. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet 21: 195-199, 1999.[ISI][Medline]
  43. Werman MJ and Bhathena SJ. Copper-deficient and excess diets: theoretical considerations and preparations. In: Trace Elements in Laboratory Animals-Nutrition Trace Elements in Animal Nutrition, edited by Watson RR. Boca Raton, FL: CRC, 1996, p. 147-161.
  44. Wheby MS and Crosby WH. The gastrointestinal tract and iron absorption. Blood 22: 416-428, 1963.[ISI][Medline]
  45. Williams DM, Kennedy S, and Green BG. Hepatic iron accumulation in copper-deficient rats. Br J Nutr 50: 653-660, 1983.[ISI][Medline]
  46. Williams DM, Lee GR, and Cartwright GE. Ferroxidase activity of rat ceruloplasmin. Am J Physiol 227: 1094-1097, 1974.[Free Full Text]
  47. Yamamoto K, Yoshida K, Miyagoe Y, Ishikawa A, Hanaoka K, Nomoto S, Kaneko K, Ikeda S, and Takeda S. Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice. Biochim Biophys Acta 1588: 195-202, 2002.[ISI][Medline]
  48. Yu J and Wessling-Resnick M. Influence of copper depletion on iron uptake mediated by SFT, a stimulator of Fe transport. J Biol Chem 273: 6909-6915, 1998.49.[Abstract/Free Full Text]
  49. Zerounian NR and Linder MC. Effect of copper and ceruloplasmin on iron transport in the Caco-2 cell intestinal model. J Nutr Biochem 13: 138-148, 2002.[ISI][Medline]