Novel Insights to Iron Deficiency Anemia Chaim Hershko M.D.

1. Novel Insights to Iron Deficiency Anemia Chaim Hershko M.D. Department of Medicine, Shaare Zedek Medical Center and Hebrew University Hadassah Medical School Jerusalem, Israel

2. Metabolic Functions of Iron – a Transition Metal Heme Iron Compounds • Hemoglobin, Myoglobin • Cytochrome a,b,c (oxidative energy) • Cytochrome P450 (drug metabolism) • Catalase, Peroxidase Nonheme Iron Compounds • NAD dehydrogenase • Succinate dehydrogenase • Xanthine oxidase • Ribonucleotide reductase

3. Regulation of mRNA by IRE-IRP Interaction Low iron High iron Ferritin IRP IRP 40s coding coding 5’ (A)n (A)n 5’ 43s 60s Transferrin receptor IRP IRP IRP IRP (A)n coding 5’ 5’ coding RNAse Harford et al 1994

4. Proteins Involved in Iron Regulation Protein Role IRE DMT1; Nramp2 (Gunshin 1997, Fleming 1997) Duodenal brush border metal transporter Yes Ireg1; Ferroportin Basolateral transmembrane iron transporter Yes (McKie 1998) Cytochrome b558 (Pountney 1999) Duodenal brush border ferric reductase _ Hephaestin(Vulpe 1999) Transmembrane-bound ferroxidase _ HCP1(Shayeghi 2005) Duodenal brush border heme carrying protein Yes _

5. Cellular Control of Iron Transport in Duodenal Enterocyte Basolateral Brush border Fe(II) Fe(II) Fe(II) Ferroportin DMT1 Fe(II) Fe(II) Fe reductase Hephaestin Fe(II) Fe(II) Fe(II) Labile Iron Fe(II) Fe(II) Pool Fe(III) Fe(III) Fe(II) Fe(II) Fe(II) Tf Fe(II) Fe(III) HCP1 Fe(III) heme Fe(III) endocytic Fe(III) Tf Fe(III) vesicle Fe(III) Fe(III) TfR ferritin Fe(III) Tf : transferrin TfR: transferrin receptor

6. Cellular Control of Iron Transport in Duodenal Enterocyte Basolateral Brush border Hepcidin Fe(II) Fe(II) Fe(II) Ferroportin DMT1 Fe(II) Fe(II) Fe reductase Hephaestin Fe(II) Fe(II) Fe(II) Labile Iron Fe(II) Fe(II) Pool Fe(III) Fe(III) Fe(II) Fe(II) Fe(II) Tf Fe(II) Fe(III) HCP1 Fe(III) heme Fe(III) endocytic Fe(III) Tf Fe(III) vesicle Fe(III) Fe(III) TfR ferritin Fe(III) Tf : transferrin TfR: transferrin receptor

7. Milestones in the hepcidin saga

8. Inherited Hemochromatosis Syndromes All mutations are assocated with failure of Hepcidin expression

9. Hepcidin Feedback Mechanism Erythron Parenchyma HFE TfR2 R.E. Transferrin Hemojuvelin Ferroportin Hepcidin Ferroportin Iron depletion Hemolytic anemia Genetic hemochromatosis Iron overload Inflammation Gut

10. Basal regulation of hepcidin transcription BMP: bone morphogenetic protein Anderson & Frazer: Editorial for Babitt & Huang, Nat Genet. 2006 May HJV and BMP: family of repulsive guidance molecules

11. Basal expression Induction in inflammation Figure 1 Transcriptional regulation of hepcidin expression. Two pathways regulating hepcidin transcription are well characterized. Basal expression depends upon signaling through bone morphogenetic protein receptors (BMPR) and downstream SMAD factors. Hemojuvelin (HJV) acts as a BMP coreceptor. Hepcidin induction in inflammation results, at least in part, from the signaling of interleukin-6 (IL-6) through its receptor and STAT3 (signal transducer and activator of transcription 3). HJV: hemojuvelin Hemochromatosis: genetic iron overload resulting from mutations in any of five genes involved in iron homeostasis HFE: the gene mutated in classical hemochromatosis Transferrin receptor 2 (TFR2): a homolog of TFR1 of hepcidin expression through the interaction Signal transduction of hepcidin expression in iron excess Goswami T, Andrews NC. J Biol Chem. 2006;281: 28494-8.

12. Figure 5 Iron ingestion induces urinary hepcidin in humans. Five subjects collected their first morning urine for 9 days and took 65 mg of iron (as ferrous sulfate) in the morning of days 3, 4, and 5. Thick line represents the arithmetic mean. There is a significant increase in hepcidin excretion on day 4 (24 hours after the first dose of iron) compared with day 3 (P = 0.017 by paired Student's t test). Nemeth E et al J. Clin. Invest.113:1271-1276 (2004).

13. Causes of Iron Deficiency Increased physiologic requirements Limited supply Blood loss

14. Prevalence of Anemia in the World Region children children men women pregnant Age (y) 0-4 5-12 15-59 15-49 15-49 Africa 56 49 20 44 63 North America 8 13 4 8 - Latin America 26 26 13 17 30 East Asia 20 22 11 18 20 South Asia 56 50 32 58 68 Europe 14 5 2 12 14 Oceania 18 15 7 19 25 Developed regions 12 7 3 11 14 Developing regions 51 46 26 47 59 Cutoff values (g/dl): 11 12 13 12 11 DeMaeyer E & Adiels-Tegman M. World Health Statist. Quart. 38: 303, 1985

15. Diagnostic Workup of Iron Deficiency Anemia Hb, MCV, Tf saturation, ferritin, TfR, ZPP. %Hypo, CHr Initial workup: Males, Post-meno- pausal females Infants Pregnant women Young females Category: Detailed medical & gynecologic history Occult blood negative positive proceed to treatment complete GI workup Hershko et al Clinical Haematology 2005

16. Author (year) (n) Age mean (y) % females Blood loss Upper GI Blood loss Lower GI Menorrhagia No GI cause identified Coeliac Gastric surgery Mc Intyre & Long (1993) 111 63 68 40 18 2 3 7 37 Rockey & Cello (1993) 100 60 51 37 26 0 0 Excluded 38 Bini et al (1998) 186 44 100 Premeno-pausal 7 6 0 0 83 (probable) 83 Causes of iron deficiency among anemic subjects referred for GI evaluation (% of total)

17. Referral for hematologic evaluation is likely to take place when iron deficiency anemia persists despite a negative gastrointestinal workup or when anemia is unresponsive to standard oral iron treatment.

18. 14 y old male, referred for investigation of IDA

19. Suerbaum, Michetti Helicobacter Pylori Infection* N Engl J Med 347, 1175, 2002 * Australian physiologists Robin Warren and Barry Marshall were awarded the 2005 Nobel Prize in medicine

20. Prospective study 2002-2006: Causes of unexplained, or refractory IDA among subjects with no apparent GI disease Impact of screening for Celiac disease (endomysial, and gliadin antibodies) Autoimmune atrophic gastritis (gastrin, parietal cell antibodies, anti-IF) H pylori infection (antibodies and urease breath test)

21. Main diagnostic categories and coexistent findings in 300 IDA patients referred for hematologic evaluation Hershko et al Haematologica 2007 * 165 total H pylori

22. Significance of Autoimmune Atrophic Gastritis in Iron Deficiency Anemia

23. Autoimmune Atrophic Gastritis in IDA: Achylia Gastrica Revisited Achylia gastrica mit anamie: K Faber. Medizinishe Klinik 1909; 5: 1310 Idiopathic hypochromic anemia: Wintrobe MM, Beebe RT. Medicine 1933; 12: 187-243 Detection of latent pernicious anaemia in iron deficiency anaemia: Dagg JH et al. Brit J Med 1966; 2: 619-621

24. Autoimmune gastritis: mode of presentation, associated findings and results of endoscopy in 160 patients Hershko et al, Blood 2006

25. Effect of age on presenting features of autoimmune gastritis (mean± 1SD) Hershko et al, Blood 2006

26. Effect of age on hypergastrinemia, serum B12 and H pylori activity B12 pg/mL; Gastrin u/mL ; H pylori % positive Hershko et al, Blood 2006

27. Evidence supporting role of H pylori in pathogenesis of Autoimmune Atrophic Gastritis Antigenic mimicry between H pylori and gastric mucosa (Negrini et al, Gastroenterology 1996: 111. 655; Appelmelk et al, Infection and Immunity 1996; 64, 2031) Activated CD4+T cells from H. pylori -infected patients with autoimmune gastritis directed at H pylori epitopes recognize parietal cell H+K+ATPase as a major autoantigen (Amedei et al J Exper Med 2003; 198: 1147) In H pylori infectionthe emergence of parietal cell antibodies leads to progression of corpus atrophy and disappearance of H. pylori. (Valle et al, Scand J Gastroenterology 1996; 31, 546)

28. Significance of H pylori Infection

29. Proposed mechanism of refractory IDA in H pylori gastritis Occult GI bleeding (Yip et al, JAMA 1997: 277; 1135) Competition of H pylori for dietary iron and its storage in ferritin-like bacterial protein (Perez-Perez et al, Eur J Gastroenterology and Hepatology; 2000, 12, 1263) Interference with gastric acid and ascorbate secretion in H pylori gastritis and reversal by H pylori eradication. (Annibale et al, Gut 2003; 52, 496)

30. Long-term outcome of H pylori eradication in males with no bleeding source identified Hershko et al, Blood Cells Molec Dis 2006

31. Author (year) Site Group (n) Triple therapy Oral iron treatment Effect on anemia * H pylori eradication Konno et al (2000) Japan children 6 yes no 6/6 Cure at 27-50 months Choe et al (1999) Korea adolesc 22 yes yes no yes no yes n.a. n.a. n.a. Improved at 2 months Improved at 2 months no Ashorn et al (2001) Finland children 8 yes yes 7/8 Hb 7.4 to 12.0 at 1 month Annibale et al (1999) Italy women 30 yes no 89% Hb 10.2 to 13 at 6 months Choe et al (2000) Korea adolesc females 14 yes yes 85% Hb 8.6 to 11.3 at 6 weeks Choe et al (2001) Korea adolesc females 12 yes no no yes n.a. none Hb 10.3 to 12.5 at 2 months  Hb 10.0 to 10.2 at 2 months Response of refractory iron deficiency anemia to H pylori eradication* *Recommended by Maastricht-3-Consensus Report -2005for unexplained IDA www.helicobacter.org

32. Effect of H pylori eradication on serum gastrin Hershko et al, Blood 2006

33. Effect of H pylori eradication on parameters reflecting autoimmune gastritis A B Hershko et al, Blood Cells Molec Dis 2006

34. Causes of obscure iron deficiency revisited (% of total)

35. Modified Diagnostic Workup for Iron Deficiency Anemia Hb, MCV, Tf saturation, ferritin, TfR, ZPP,CHr Initial workup: Males, Post-meno- pausal females Infants Pregnant women Young females Category: Detailed medical & gynecologic history Occult blood negative positive proceed to treatment antiendomysial helicobacter parietal abs gastrin complete GI workup no finding no response

36. Conclusions • The favorable long-term clinical results of H pylori eradication offer strong evidence for a cause-and-effect relation between H pylori and IDA. A proportion of H pylori patients will develop autoimmune gastritis. • Our findings in autoimmune gastritis imply a disease presenting as IDA many years before the establishment of clinical cobalamin deficiency. • התוצאות החיוביות של טיפול בזיהום ההליקובקטר מהוות עדות תומכת חזקה בקשר סיבתי בין זיהום בחיידק זה לבין אנמיה של חסר ברזל. חלק מהחולים עם זיהום הליקובקטר עלולים לפתח בהמשך גסטריטיס אטרופית אוטואימונית. • הממצאים שלנו בחולי גסטריטיס אטרופית מעידים על מחלה המתגלה כאנמיה של חסר ברזל שנים רבות לפני ההתפתחות של חסר קובלאמין קליני

37. It is caused by an autoimmune process likely triggered by antigenic mimicry between H pylori epitopes and major autoantigens of the gastric mucosa. • Recognition of the respective roles of H pylori and autoimmune gastritis in the pathogenesis of iron deficiency should have a strong impact on the diagnostic workup and management of unexplained, or refractory iron deficiency anemia. • גטריטיס אטרופית אוטואימונית נגרמת על ידי תהליך המבוסס על חיקוי אנטיגני antigenic mimicry בין חיידק ההליקובקטר לבין האנטיגנים העיקריים של רירית הקיבה • ההכרה בתפקידם הבולט של זיהום ההליקובקטר והגסריטיס האוטואימונית בפתוגנזה של חסר ברזל, אמורה לשנות את כללי האבחון והטיפול באנמיה הבלתי מוסברת של חסר ברזל, או באנמיה העמידה לטיפול פומי.