European Journal of Clinical Investigation (2004) 34, 275–282 © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Rat duodenal IRP1 activity and iron absorption in iron deficiency and after H 2 O 2 perfusion K. Schümann * , K. Brennan † , M. Weiss § , K. Pantopoulos ‡ and M. W. Hentze † * Lehrstuhl für Ernährungsphysiologie der TUM, Weihenstephan, Germany, † European Molecular Biology Laboratory, Heidelberg, Germany, ‡ Lady Davis Institute for Medical Research and McGill University, Montreal, Canada, and § Pathologisches Institut, Ludwig-Maximilians-Universität, Munich, Germany Abstract Background Iron regulatory protein 1 (IRP1), a post-transcriptional regulator of iron metabolism, is activated in the duodenum of iron-deficient animals, which is associated with increased iron absorption. In cell cultures IRP1 was also activated by iron-independent signals, such as H 2 O 2 . Here we investigate whether luminal perfusion of rat duodenum with H 2 O 2 activates duodenal IRP1 and modulates duodenal iron absorption. Methods Duodena from iron-adequate Sprague-Dawley rats were luminally perfused with H 2 O 2 . Iron regulatory protein-1 activity was determined in duodenal mucosa or in villus and crypt preparations by an electrophoretic mobility shift assay. Duodenal 59 Fe absorption was measured in isolated, perfused duodenal segments ex vivo and in ligated loops in vivo. 59 Fe uptake from the blood side was assessed after i.v. injection of 59 Fe-nitrilotriacetic acid. Results Similar to iron deficiency, the perfusion with 0–50 mM of H 2 O 2 increases duodenal IRP1 activity along the entire crypt villus-axis in a dose-dependent manner. After H 2 O 2 treatment, IRP1 remains activated for 12–24 h in the tips and for 72 h in the crypts. In iron-deficiency, IRP activation correlates with increased 59 Fe absorption. However, the H 2 O 2 treatment fails to stimulate any increase in 59 Fe uptake, without promoting damage of mucosal architecture or impairing glucose and water transport. Conclusion Duodenal 59 Fe uptake is not affected by the H 2 O 2 -mediated activation of IRP1. Keywords Absorption, duodenum, iron, IRP1, oxidative stress. Eur J Clin Invest 2004; 34 (4): 275–282 Introduction Cellular iron uptake and storage are mediated by the trans- ferrin receptor (TfR) and by ferritin [1], respectively. The expression of both proteins is regulated post-transcriptionally by the IRE/IRP system. Ferritin and Tf R mRNAs contain ‘iron responsive elements’ (IREs) in their 5′ or 3′ untranslated regions (UTRs) which bind to two homologous cytoplas- mic ‘iron regulatory proteins’, IRP1 and IRP2. In iron starvation c-aconitase is converted to IRP1 by disassembly of a 4Fe-4S cluster while IRP2 is stabilized. These responses increase the IRE-binding activity of the IRPs, which inhibits ferritin translation and stabilizes TfR mRNA [2–4]. Iron regulatory protein-1 also responds to NO, hypoxia and oxidative stress [5,6] while IRP2 is not affected [9]. Exposure of intact cells in culture to hydrogen peroxide (H 2 O 2 ) activates IRP1 [7,8,10,11] and reduces the ferritin content while Tf R expression and 59 Fe-Tf uptake are increased [12]. Activation of IRP1 was also observed in rat liver after perfusion with a H 2 O 2 -generating system [13]. Not much is known about the physiological implications associated with IRP1 activa- tion by H 2 O 2 in the duodenum, which is the major site for intestinal iron absorption. Iron responsive element-containing mRNAs also encode the duodenal iron transporters DMT-1 Lehrstuhl für Ernährungsphysiologie der TUM, Weihenstephan, Germany (K. Schümann); European Molecular Biology Laboratory, Heidelberg, Germany (K. Brennan); Lady Davis Institute for Medical Research and McGill University, Montreal, Canada (K. Pantopoulos); Pathologisches Institut, Ludwig- Maximilians-Universität, München, Germany (M. Weiss). Correspondence to: Klaus Schümann, Lehrstuhl für Ernährungsphysiologie der TUM, Hochfeldweg 2, 85350 Freising-Weihenstephan, Germany. Tel.: +49–89–334115; fax: +49–89–33079576; e-mail: k.schuemann@lrz.uni-muenchen.de Received 3 September 2003; accepted 26 February 2004