Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis.

INTRODUCTION: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes. METHODS: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum. RESULTS: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1. CONCLUSION: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.

Changes in the expression of intestinal iron transport and hepatic regulatory molecules explain the enhanced iron absorption associated with pregnancy in the rat.

BACKGROUND: Iron absorption increases during pregnancy to cater for the increased iron requirements of the growing fetus. AIMS: To investigate the role of the duodenal iron transport molecules and hepatic regulatory molecules in coordinating the changes in iron absorption observed during pregnancy. METHODS: Rats at various days of gestation and 24-48 hours post-partum were examined for hepatic expression of hepcidin, transferrin receptors 1 and 2, and HFE (the gene mutated in the most prevalent form of hereditary haemochromatosis), and duodenal expression of divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), iron regulated mRNA (Ireg1), and hephaestin (Hp) by ribonuclease protection assay, western blotting, and immunohistochemistry. RESULTS: Decreased hepatic non-haem iron and transferrin saturation and increased expression of transferrin receptor 1 in the liver indicated a progressive reduction in maternal body iron stores during pregnancy. Duodenal expression of the iron transport molecules DMT1, Dcytb, and Ireg1 increased during pregnancy, and this corresponded with a reduction in hepcidin, HFE, and transferrin receptor 2 expression in the liver. Expression of all molecules returned towards control values by 24-48 hours post-partum. CONCLUSIONS: These data indicate that increased expression of key iron transport molecules is responsible for the elevated iron absorption associated with pregnancy, and implicate hepcidin, HFE, and transferrin receptor 2 in determining how the maternal iron homeostatic machinery responds to the increased iron demands accompanying gestation.

A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption.

BACKGROUND: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. AIMS: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. METHODS: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive (59)Fe. RESULTS: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. CONCLUSIONS: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.

Relationship between intestinal iron-transporter expression, hepatic hepcidin levels and the control of iron absorption.

Hepcidin is an anti-microbial peptide predicted to be involved in the regulation of intestinal iron absorption. We have examined the relationship between the expression of hepcidin in the liver and the expression of the iron-transport molecules divalent-metal transporter 1, duodenal cytochrome b, hephaestin and Ireg1 in the duodenum of rats switched from an iron-replete to an iron-deficient diet or treated to induce an acute phase response. In each case, elevated hepcidin expression correlated with reduced iron absorption and depressed levels of iron-transport molecules. These data are consistent with hepcidin playing a role as a negative regulator of intestinal iron absorption.

Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins.

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Electroanalytical exploitation of quinone-thiol interactions: application to the selective determination of cysteine.

Square wave voltammetry was applied to the detection of cysteine through the use of an indirect assay that exploits the reaction of the thiol with a quinone indicator. Voltammetric discrimination between unreacted quinone and the corresponding quinone-cysteine adduct is possible with clear resolution of the latter peak providing a linear response from 5 to 47 microM. The selectivity of the approach was assessed with no interference from cystine, lysine, paracetamol or 4-aminophenol. The response recorded in the presence of a massive excess of ascorbic acid was also investigated and the integrity of the approach confirmed. The effects of other sulfhydryl thiols, homocysteine and glutathione, were also assessed and found to present no appreciable change in the voltammetric profile. The practical utility of the approach was investigated through examining the response to cysteine in urine.

Laminated microelectrodes: a simple approach to the construction of inexpensive microelectrodes with a variety of geometries.

Designs for reusable microelectrodes, which are easy to assemble and so do not require special technical skills or services for their construction, are presented. Three types of microelectrodes are fabricated by lamination of metal foil, wire, and wire grids, producing microband, microdisk, and a linear array of microdisk geometries. The electrodes themselves do not need to be polished prior to their use but are observed to be slightly recessed from the surrounding insulating surface. Good agreement is observed between experimental steady-state voltammetric results in nonaqueous solutions and the relevant analytical theory for the voltammetric current.