Title: Hepcidin in the management of patients with mild non-hemochromatotic iron overload: Fact or fiction?
Abstract: Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretionJournal of HepatologyVol. 49Issue 5PreviewThe aim of this study is to determine the clinical relevance of hepatic producing iron regulatory hormone-hepcidin, on iron overload in patients with chronic hepatitis C (CHC). Full-Text PDF Several non-hereditary liver diseases are associated with mild hepatic iron overload such as viral hepatitis, excessive alcohol consumption, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) [1Fernandez-Real J.M. Lopez-Bermejo A. Ricart W. Cross-talk between iron metabolism and diabetes.Diabetes. 2002; 51: 2348-2354Crossref PubMed Scopus (452) Google Scholar, 2Wrede C.E. Buettner R. Bollheimer L.C. Scholmerich J. Palitzsch K.D. Hellerbrand C. Association between serum ferritin and the insulin resistance syndrome in a representative population.Eur J Endocrinol. 2006; 154: 333-340Crossref PubMed Scopus (116) Google Scholar, 3Moirand R. Mortaji A.M. Loreal O. Paillard F. Brissot P. Deugnier Y. A new syndrome of liver iron overload with normal transferrin saturation.Lancet. 1997; 349: 95-97Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 4Bozzini C. Girelli D. Olivieri O. Martinelli N. Bassi A. De Matteis G. et al.Prevalence of body iron excess in the metabolic syndrome.Diabetes Care. 2005; 28: 2061-2063Crossref PubMed Scopus (143) Google Scholar, 5Turlin B. Mendler M.H. Moirand R. Guyader D. Guillygomarc'h A. Deugnier Y. Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients.Am J Clin Pathol. 2001; 116: 263-270Crossref PubMed Scopus (119) Google Scholar, 6Bacon B.R. Iron and hepatitis C.Gut. 2007; 41: 127-128Crossref Scopus (10) Google Scholar, 7Purohit V. Russo D. Salin M. Role of iron in alcoholic liver disease: introduction and summary of the symposium.Alcohol. 2003; 30: 93-97Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. In fact, the term insulin resistance or dysmetabolic iron overload syndrome was introduced to describe the association of body iron overload and NAFLD [[8]Mendler M.H. Turlin B. Moirand R. Jouanolle A.M. Sapey T. Guyader D. et al.Insulin resistance-associated hepatic iron overload.Gastroenterology. 1999; 117: 1155-1163Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar]. The contribution of HFE-mutations to the development of iron overload in these diseases remains debated [9George D.K. Powell L.W. Losowsky M.S. The haemochromatosis gene: a co-factor for chronic liver diseases?.J Gastroenterol Hepatol. 1999; 14: 745-749Crossref PubMed Scopus (30) Google Scholar, 10Bonkovsky H.L. Lambrecht R.W. Shan Y. Iron as a co-morbid factor in nonhemochromatotic liver disease.Alcohol. 2003; 30: 137-144Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar] (NAFLD/NASH [8Mendler M.H. Turlin B. Moirand R. Jouanolle A.M. Sapey T. Guyader D. et al.Insulin resistance-associated hepatic iron overload.Gastroenterology. 1999; 117: 1155-1163Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 11Bonkovsky H.L. Jawaid Q. Tortorelli K. LeClair P. Cobb J. Lambrecht R.W. et al.Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis.J Hepatol. 1999; 31: 421-429Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 12Chitturi S. Weltman M. Farrell G.C. McDonald D. Kench J. Liddle C. et al.HFE mutations, hepatic iron, and fibrosis: ethnic-specific association of NASH with C282Y but not with fibrotic severity.Hepatology. 2002; 36: 142-149Crossref PubMed Scopus (214) Google Scholar]) (HCV [13Snover D.C. Hepatitis C, iron, and hemochromatosis gene mutations A meaningful relationship or simple cohabitation?.Am J Clin Pathol. 2000; 113: 475-478Crossref PubMed Google Scholar, 14Pietrangelo A. Hemochromatosis gene modifies course of hepatitis C viral infection.Gastroenterology. 2003; 124: 1509-1523Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 15Piperno A. Vergani A. Malosio I. Parma L. Fossati L. Ricci A. et al.Hepatic iron overload in patients with chronic viral hepatitis: role of HFE gene mutations.Hepatology. 1998; 28: 1105-1109Crossref PubMed Scopus (127) Google Scholar]) (alcohol [[16]Grove J. Daly A.K. Burt A.D. Guzail M. James O.F. Bassendine M.F. et al.Heterozygotes for HFE mutations have no increased risk of advanced alcoholic liver disease.Gut. 1998; 43: 262-266Crossref PubMed Scopus (88) Google Scholar]), but is probably slight. The underlying molecular mechanism centers on dysregulation of the iron regulatory hormone hepcidin. Hepcidin is primarily produced by hepatocytes, but there is also evidence for production by macrophages [[17]Peyssonnaux C. Zinkernagel A.S. Datta V. Lauth X. Johnson R.S. Nizet V. TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens.Blood. 2006; 107: 3727-3732Crossref PubMed Scopus (277) Google Scholar] and fat cells [[18]Bekri S. Gual P. Anty R. Luciani N. Dahman M. Ramesh B. et al.Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH.Gastroenterology. 2006; 131: 788-796Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar]. Hepcidin internalizes and subsequently degrades the cellular iron exporter ferroportin, present on the cell surface of macrophages and enterocytes, resulting in hypoferremia. Hepcidin is upregulated by inflammation, by the presence of increased body iron levels and inhibited by increased erythropoietic activity. These respective regulatory pathways are thought to protect the body from (i) extracellular proliferating pathogens, (ii) the harmful effects of iron overload and to (iii) ensure that body iron availability matches iron needs. Recent evidence has demonstrated that perturbation of hepcidin underlies non-hereditary (or non-hemochromatotic or acquired) mild iron overloading hepatic diseases (NHIOD). Hepcidin expression is affected by the upstream stimulatory factor 1 (USF1), a well established susceptibility gene for familial combined hyperlipidemia, a complex disorder of lipid metabolism [[19]Pajukanta P. Lilja H.E. Sinsheimer J.S. Cantor R.M. Lusis A.J. Gentile M. et al.Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1).Nat Genet. 2004; 36: 371-376Crossref PubMed Scopus (259) Google Scholar]. USF1 together with USF2 controls cis and trans regulation of hepcidin expression [[20]Bayele H.K. McArdle H. Srai S.K. Cis and trans regulation of hepcidin expression by upstream stimulatory factor.Blood. 2006; 108: 4237-4245Crossref PubMed Scopus (55) Google Scholar]. Other circumstantial evidence of a link between hepcidin, glucose and lipid metabolism comes from hepcidin producing hepatic adenomas in severe anemic type 1a glycogen storage disease (GSD1a) patients [[21]Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (541) Google Scholar], and hepcidin production by fat cells [[18]Bekri S. Gual P. Anty R. Luciani N. Dahman M. Ramesh B. et al.Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH.Gastroenterology. 2006; 131: 788-796Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar]. Interestingly, many of the diseases that are part of the metabolic syndrome have a disrupted iron homeostasis [3Moirand R. Mortaji A.M. Loreal O. Paillard F. Brissot P. Deugnier Y. A new syndrome of liver iron overload with normal transferrin saturation.Lancet. 1997; 349: 95-97Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 4Bozzini C. Girelli D. Olivieri O. Martinelli N. Bassi A. De Matteis G. et al.Prevalence of body iron excess in the metabolic syndrome.Diabetes Care. 2005; 28: 2061-2063Crossref PubMed Scopus (143) Google Scholar] (e.g. diabetes [1Fernandez-Real J.M. Lopez-Bermejo A. Ricart W. Cross-talk between iron metabolism and diabetes.Diabetes. 2002; 51: 2348-2354Crossref PubMed Scopus (452) Google Scholar, 2Wrede C.E. Buettner R. Bollheimer L.C. Scholmerich J. Palitzsch K.D. Hellerbrand C. Association between serum ferritin and the insulin resistance syndrome in a representative population.Eur J Endocrinol. 2006; 154: 333-340Crossref PubMed Scopus (116) Google Scholar, 8Mendler M.H. Turlin B. Moirand R. Jouanolle A.M. Sapey T. Guyader D. et al.Insulin resistance-associated hepatic iron overload.Gastroenterology. 1999; 117: 1155-1163Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar], hypertension [[22]Piperno A. Trombini P. Gelosa M. Mauri V. Pecci V. Vergani A. et al.Increased serum ferritin is common in men with essential hypertension.J Hypertens. 2002; 20: 1513-1518Crossref PubMed Scopus (138) Google Scholar] and elevated body mass index [[23]Rossi E. Bulsara M.K. Olynyk J.K. Cullen D.J. Summerville L. Powell L.W. Effect of hemochromatosis genotype and lifestyle factors on iron and red cell indices in a community population.Clin Chem. 2001; 47: 202-208PubMed Google Scholar]). Thus, we are only starting to discover the pleiotropic role of hepcidin. Alcoholic and hepatic viral infections are the most typical examples of acquired mild forms of iron overload. Liver biopsies in these disorders consistently show iron deposits in hepatocytes and Kupffer cells [5Turlin B. Mendler M.H. Moirand R. Guyader D. Guillygomarc'h A. Deugnier Y. Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients.Am J Clin Pathol. 2001; 116: 263-270Crossref PubMed Scopus (119) Google Scholar, 24Brunt E.M. Pathology of hepatic iron overload.Semin Liver Dis. 2005; 25: 392-401Crossref PubMed Scopus (51) Google Scholar]. Elevated liver iron deposits are also commonly observed in NAFLD [5Turlin B. Mendler M.H. Moirand R. Guyader D. Guillygomarc'h A. Deugnier Y. Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients.Am J Clin Pathol. 2001; 116: 263-270Crossref PubMed Scopus (119) Google Scholar, 24Brunt E.M. Pathology of hepatic iron overload.Semin Liver Dis. 2005; 25: 392-401Crossref PubMed Scopus (51) Google Scholar]. Iron can induce oxidative stress, which can cause tissue injury. Indeed, hepatic iron deposits have been associated with more advanced forms of NHIOD, including liver fibrosis, cirrhosis and hepatocellular cancer in some reports (NAFLD/NASH [9George D.K. Powell L.W. Losowsky M.S. The haemochromatosis gene: a co-factor for chronic liver diseases?.J Gastroenterol Hepatol. 1999; 14: 745-749Crossref PubMed Scopus (30) Google Scholar, 11Bonkovsky H.L. Jawaid Q. Tortorelli K. LeClair P. Cobb J. Lambrecht R.W. et al.Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis.J Hepatol. 1999; 31: 421-429Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 25George D.K. Goldwurm S. MacDonald G.A. Cowley L.L. Walker N.I. Ward P.J. et al.Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis.Gastroenterology. 1998; 114: 311-318Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar, 26Fargion S. Mattioli M. Fracanzani A.L. Sampietro M. Tavazzi D. Fociani P. et al.Hyperferritinemia, iron overload, and multiple metabolic alterations identify patients at risk for nonalcoholic steatohepatitis.Am J Gastroenterol. 2001; 96: 2448-2455Crossref PubMed Google Scholar] , HCV [27Fujita N. Horiike S. Sugimoto R. Tanaka H. Iwasa M. Kobayashi Y. et al.Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients.Free Radic Biol Med. 2007; 42: 353-362Crossref PubMed Scopus (77) Google Scholar, 28Furutani T. Hino K. Okuda M. Gondo T. Nishina S. Kitase A. et al.Hepatic iron overload induces hepatocellular carcinoma in transgenic mice expressing the hepatitis C virus polyprotein.Gastroenterology. 2006; 130: 2087-2098Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Diwakaran H.H. Befeler A.S. Britton R.S. Brunt E.M. Bacon B.R. Accelerated hepatic fibrosis in patients with combined hereditary hemochromatosis and chronic hepatitis C infection.J Hepatol. 2002; 36: 687-691Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar] , alcohol [30Nahon P. Sutton A. Rufat P. Ziol M. Thabut G. Schischmanoff P.O. et al.Liver iron, HFE gene mutations, and hepatocellular carcinoma occurrence in patients with cirrhosis.Gastroenterology. 2008; 134: 102-110Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 31Raynard B. Balian A. Fallik D. Capron F. Bedossa P. Chaput J.C. et al.Risk factors of fibrosis in alcohol-induced liver disease.Hepatology. 2002; 35: 635-638Crossref PubMed Scopus (317) Google Scholar]), but this was not confirmed in others (NAFLD/NASH [12Chitturi S. Weltman M. Farrell G.C. McDonald D. Kench J. Liddle C. et al.HFE mutations, hepatic iron, and fibrosis: ethnic-specific association of NASH with C282Y but not with fibrotic severity.Hepatology. 2002; 36: 142-149Crossref PubMed Scopus (214) Google Scholar, 32Bugianesi E. Manzini P. D'Antico S. Vanni E. Longo F. Leone N. et al.Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver.Hepatology. 2004; 39: 179-187Crossref PubMed Scopus (377) Google Scholar, 33Younossi Z.M. Gramlich T. Bacon B.R. Matteoni C.A. Boparai N. O'Neill R. et al.Hepatic iron and nonalcoholic fatty liver disease.Hepatology. 1999; 30: 847-850Crossref PubMed Scopus (236) Google Scholar] , HCV [16Grove J. Daly A.K. Burt A.D. Guzail M. James O.F. Bassendine M.F. et al.Heterozygotes for HFE mutations have no increased risk of advanced alcoholic liver disease.Gut. 1998; 43: 262-266Crossref PubMed Scopus (88) Google Scholar, 30Nahon P. Sutton A. Rufat P. Ziol M. Thabut G. Schischmanoff P.O. et al.Liver iron, HFE gene mutations, and hepatocellular carcinoma occurrence in patients with cirrhosis.Gastroenterology. 2008; 134: 102-110Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar]). In NAFLD, there is increased urinary hepcidin excretion [[34]Barisani D. Pelucchi S. Mariani R. Galimberti S. Trombini P. Fumagalli D. et al.Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload.J Hepatol. 2008; 49: 123-133Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar] and elevated liver hepcidin expression [[35]Aigner E. Theurl I. Theurl M. Lederer D. Haufe H. Dietze O. et al.Pathways underlying iron accumulation in human nonalcoholic fatty liver disease.Am J Clin Nutr. 2008; 87: 1374-1383PubMed Google Scholar] but hepcidin levels are relatively low for the level of iron overload [[34]Barisani D. Pelucchi S. Mariani R. Galimberti S. Trombini P. Fumagalli D. et al.Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload.J Hepatol. 2008; 49: 123-133Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar], suggesting ineffective liver iron sensing. In this issue of the Journal of Hepatology, Fujita et al. [[36]Fujita N. Sugimoto R. Motonishi R. Tomosugi N. Tanaka H. Takeo M. et al.Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretion.J Hepatol. 2008; 49: 702-710Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] confirm that in chronic hepatitis C virus (HCV), hepatic hepcidin expression is inadequate for the body iron stores, but can be restored upon viral eradication. In addition, they found that hepatic hepcidin expression reflects circulating hepcidin levels. This finding suggests that serum hepcidin measurements might become a proxy parameter for hepatic hepcidin and aid in the management of patients with acquired forms of mild hepatic iron overload such as in HCV. Human hepcidin is mainly produced by hepatocytes as an 84 amino-acid (aa) pre-prohepcidin [[37]Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. et al.A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1398) Google Scholar]. Subsequent posttranslational processing results in the biologically active 25 aa form (hepcidin-25, molecular mass is 2789 Da) that is secreted in the plasma. Additional amino-acid degradation results in the production of two smaller isoforms (hepcidin-20 and -22) [38Kemna E.H. Tjalsma H. Podust V.N. Swinkels D.W. Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications.Clin Chem. 2007; 53: 620-628Crossref PubMed Scopus (190) Google Scholar, 39Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J Biol Chem. 2001; 276: 7806-7810Crossref PubMed Scopus (1717) Google Scholar], that have unknown biological significance. Progress in this field has been hampered by the difficulties many researchers face by measuring hepcidin. Until recently, only few tools were available to measure hepcidin. Largely because antibodies are hard to raise. Hepcidin has a small and compact structure and antigenic epitopes are scarce. In addition, hepcidin is conserved among a wide range of species which complicates the induction of antibodies in hosts such as rabbit. As a consequence, immunochemical methods based on specific anti-hepcidin antibodies are largely unavailable. Lastly, quantitation of hepcidin is complicated by its tendency to aggregate [[40]Hunter H.N. Fulton D.B. Ganz T. Vogel H.J. The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis.J Biol Chem. 2002; 277: 37597-37603Crossref PubMed Scopus (332) Google Scholar] and stick to laboratory plastics, necessitating implementation of robust laboratory procedures. Currently, many studies use the serum-based commercial immunoassay that measures the hepcidin precursor prohepcidin rather than the bioactive peptide. The relevance of these data is controversial because prohepcidin levels fail to correlate with urinary and serum hepcidin [41Kemna E.H. Kartikasari A.E. van Tits L.J. Pickkers P. Tjalsma H. Swinkels D.W. Regulation of hepcidin: Insights from biochemical analyses on human serum samples.Blood Cells Mol Dis. 2008; 40: 339-346Crossref PubMed Scopus (96) Google Scholar, 42Kemna E. Pickkers P. Nemeth E. van der Hoeven H. Swinkels D. Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS.Blood. 2005; 106: 1864-1866Crossref PubMed Scopus (404) Google Scholar]. Furthermore, when measured in healthy controls, serum prohepcidin levels are around 40–50 times higher than those observed for bioactive hepcidin (≈180 ng/ml versus ≈4 ng/ml) (unpublished observations). This suggests that prohepcidin and hepcidin have different biological profiles [41Kemna E.H. Kartikasari A.E. van Tits L.J. Pickkers P. Tjalsma H. Swinkels D.W. Regulation of hepcidin: Insights from biochemical analyses on human serum samples.Blood Cells Mol Dis. 2008; 40: 339-346Crossref PubMed Scopus (96) Google Scholar, 42Kemna E. Pickkers P. Nemeth E. van der Hoeven H. Swinkels D. Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS.Blood. 2005; 106: 1864-1866Crossref PubMed Scopus (404) Google Scholar, 43Brookes M.J. Sharma N.K. Tselepis C. Iqbal T.H. Serum pro-hepcidin: measuring active hepcidin or a non-functional precursor?.Gut. 2005; 54: 169-170Crossref PubMed Scopus (28) Google Scholar, 44Roe M.A. Spinks C. Heath A.L. Harvey L.J. Foxall R. Wimperis J. et al.Serum prohepcidin concentration: no association with iron absorption in healthy men; and no relationship with iron status in men carrying HFE mutations, hereditary haemochromatosis patients undergoing phlebotomy treatment, or pregnant women.Br J Nutr. 2007; 97: 544-549Crossref PubMed Scopus (70) Google Scholar]. Ganz and Nemeth et al. have developed an immunodotblot assay using non-commercially available antibodies for the semi-quantitative measurement of the sum of all urinary hepcidin isoforms [[45]Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1162) Google Scholar]. The results of studies that employ this method have greatly improved our understanding of the regulation of hepcidin in patients with distorted iron homeostasis [[46]Ganz T. Hepcidin and its role in regulating systemic iron metabolism.Hematol Am Soc Hematol Edu Prog. 2006; 507: 29-35Crossref Scopus (179) Google Scholar]. More recently, new assays for the various hepcidin isoforms: hepcidin-25, -22 and -20 have been developed on a platform of novel technologies such as surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and liquid chromatography tandem MS (LC/MS–MS) [38Kemna E.H. Tjalsma H. Podust V.N. Swinkels D.W. Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications.Clin Chem. 2007; 53: 620-628Crossref PubMed Scopus (190) Google Scholar, 47Tomosugi N. Kawabata H. Wakatabe R. Higuchi M. Yamaya H. Umehara H. et al.Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System.Blood. 2006; 108: 1381-1387Crossref PubMed Scopus (236) Google Scholar, 48Murphy A.T. Witcher D.R. Luan P. Wroblewski V.J. Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry.Blood. 2007; 110: 1048-1054Crossref PubMed Scopus (135) Google Scholar, 49Murao N. Ishigai M. Yasuno H. Shimonaka Y. Aso Y. Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean-up.Rapid Commun Mass Spectrom. 2007; 21: 4033-4038Crossref PubMed Scopus (100) Google Scholar]. With the use of internal standards these MS techniques enable reliable quantitative hepcidin analysis in urine and serum (Fig. 1) [50Swinkels D.W. Girelli D. Laarakkers C. Kroot J. Camprostrini N. Kemna E.H. et al.Advances in quantitative hepcidin measurements by time of flight mass spectrometry.PLoS ONE. 2008; 3: e2706Crossref PubMed Scopus (174) Google Scholar, 51van Dijk BA, Laarakkers CM, Klaver SM, Jacobs EM, van Tits LJ, Janssen MC, et al. Serum hepcidin levels are innately low in HFE-related haemochromatosis but differ between C282Y-homozygotes with elevated and normal ferritin levels. Br J Haematol, 2008 June 13. [Epub ahead of print] PMID: 18557745 [PubMed – as supplied by publisher].Google Scholar]. In fact, MS optimally uses an internal standard to correct for extraction, variable ionization and suppression of the analyte. Internal standards are spiked to the clinical samples and co-analysed. The ratio between the synthetic internal standard and endogenous peptide is determined by MS, allowing calculation of the absolute amount of hepcidin. In the current issue Fujita et al. exploit SELDI-TOF MS to (semi)quantify circulating hepcidin-25 levels [36Fujita N. Sugimoto R. Motonishi R. Tomosugi N. Tanaka H. Takeo M. et al.Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretion.J Hepatol. 2008; 49: 702-710Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 47Tomosugi N. Kawabata H. Wakatabe R. Higuchi M. Yamaya H. Umehara H. et al.Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System.Blood. 2006; 108: 1381-1387Crossref PubMed Scopus (236) Google Scholar]. This method shares the drawback of most currently available hepcidin analysis since it lacks the use of an internal standard. This precludes value assignment, necessitating expression in arbitrary units, and resulting in somewhat imprecise measurements. Nevertheless, their results provide important proof of principle insights in biologically relevant circulating hepcidin levels in HCV before and after treatment. Before measurement of hepcidin in urine and serum can be introduced into routine clinical practice, several issues need to be addressed. This starts with establishment of reference values among a wide variation of controls, and the assessment of a diurnal variation or fluctuation in relationship to meals. Proof of principle findings of a circadian (either intrinsic or diet related) variation with generally lower values in the fasted morning samples than in the afternoon [[38]Kemna E.H. Tjalsma H. Podust V.N. Swinkels D.W. Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications.Clin Chem. 2007; 53: 620-628Crossref PubMed Scopus (190) Google Scholar], may necessitate standardized sampling in the morning after an overnight fast (as was nicely done by Fujita et al.). Another relevant question is whether urine is a reliable alternative for serum to assess biologically relevant hepcidin levels. Urine hepcidin levels correlate with hepcidin expression in liver biopsies [[52]Detivaud L. Nemeth E. Boudjema K. Turlin B. Troadec M.B. Leroyer P. et al.Hepcidin levels in humans are correlated with hepatic iron stores, hemoglobin levels and hepatic function.Blood. 2005; 106: 746-748Crossref PubMed Scopus (178) Google Scholar], but discrepancies have been reported [[34]Barisani D. Pelucchi S. Mariani R. Galimberti S. Trombini P. Fumagalli D. et al.Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload.J Hepatol. 2008; 49: 123-133Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. Urine levels also correspond to circulating levels [[38]Kemna E.H. Tjalsma H. Podust V.N. Swinkels D.W. Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications.Clin Chem. 2007; 53: 620-628Crossref PubMed Scopus (190) Google Scholar] and nicely fit in the concept of hepcidin as the iron regulatory peptide [46Ganz T. Hepcidin and its role in regulating systemic iron metabolism.Hematol Am Soc Hematol Edu Prog. 2006; 507: 29-35Crossref Scopus (179) Google Scholar, 53Kemna E.H. Tjalsma H. Willems H.L. Swinkels D.W. Hepcidin: from discovery to differential diagnosis.Haematologica. 2008; 93: 90-97Crossref PubMed Scopus (242) Google Scholar]. Still, we caution against the uncritical use of urine concentrations as a universal surrogate for circulating levels since urinary secretion of hepcidin will depend on glomerular filtration and tubular re-absorption. In addition, hepcidin mRNA has been detected in the kidney, which suggests the potential for local production and release into the urine [[54]Kulaksiz H. Theilig F. Bachmann S. Gehrke S.G. Rost D. Janetzko A. et al.The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney.J Endocrinol. 2005; 184: 361-370Crossref PubMed Scopus (176) Google Scholar]. Increased hepatic, urinary and serum hepcidin levels are found in obesity [[18]Bekri S. Gual P. Anty R. Luciani N. Dahman M. Ramesh B. et al.Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH.Gastroenterology. 2006; 131: 788-796Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar], dysmetabolic iron overload syndrome [34Barisani D. Pelucchi S. Mariani R. Galimberti S. Trombini P. Fumagalli D. et al.Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload.J Hepatol. 2008; 49: 123-133Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 35Aigner E. Theurl I. Theurl M. Lederer D. Haufe H. Dietze O. et al.Pathways underlying iron accumulation in human nonalcoholic fatty liver disease.Am J Clin Nutr. 2008; 87: 1374-1383PubMed Google Scholar] and the metabolic syndrome (our unpublished observations). This (moderate) increase is likely due to a combination of (i) (low grade) inflammation and cytokine induced hepcidin production in hepatocytes [46Ganz T. Hepcidin and its role in regulating systemic iron metabolism.Hematol Am Soc Hematol Edu Prog. 2006; 507: 29-35Crossref Scopus (179) Google Scholar, 53Kemna E.H. Tjalsma H. Willems H.L. Swinkels D.W. Hepcidin: from discovery to differential diagnosis.Haematologica. 2008; 93: 90-97Crossref PubMed Scopus (242) Google Scholar, 55Chung B. Matak P. McKie A.T. Sharp P. Leptin increases the expression of the iron regulatory hormone hepcidin in HuH7 human hepatoma cells.J Nutr. 2007; 137: 2366-2370PubMed Google Scholar] and (ii) hepcidin production by fat cells [[18]Bekri S. Gual P. Anty R. Luciani N. Dahman M. Ramesh B. et al.Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH.Gastroenterology. 2006; 131: 788-796Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar] especially in severe obese. At first sight, these increased absolute levels contrast to the mild parenchymal overload as observed in NHIOD, because hepcidin inactivates ferroportin which, in theory, should decrease iron uptake by the intestine. However, the precise functional effect of circulating hepcidin levels on iron release of various cell-types, among them enterocytes, is unknown yet. There is some evidence of hepcidin-independent regulation of iron absorption (reviewed in [[56]Oates P.S. The relevance of the intestinal crypt and enterocyte in regulating iron absorption.Pflugers Arch. 2007; 455: 201-213Crossref PubMed Scopus (13) Google Scholar]). Other data suggest that hepcidin effects might be cell specific [[57]Chaston T. Chung B. Mascarenhas M. Marks J. Patel B. Srai S.K. et al.Evidence for differential effects of hepcidin in macrophages and intestinal epithelial cells.Gut. 2008; 57: 374-382Crossref PubMed Scopus (109) Google Scholar]. It is therefore plausible to postulate that in obesity related NHIOD (and possibly also in HCV and alcoholic liver disease) enterocytes are relatively resistant to circulating hepcidin, resulting in a relatively high intestinal iron absorption for the circulating hepcidin levels, while macrophages are more sensitive, leading to RES iron sequestration. Key in the report of Fujita et al. is their novel findings on reduced liver iron sensing in chronic HCV and especially its reversibility upon viral eradication. Similar to earlier studies in HH [51van Dijk BA, Laarakkers CM, Klaver SM, Jacobs EM, van Tits LJ, Janssen MC, et al. Serum hepcidin levels are innately low in HFE-related haemochromatosis but differ between C282Y-homozygotes with elevated and normal ferritin levels. Br J Haematol, 2008 June 13. 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These observations suggest that the hepcidin/ferritin ratio is only suitable as a measure to assess the adequacy of hepcidin regulation by the iron stores of parenchymal cells. This would implicate that a low ratio primarily inadequately predicts increased intestinal iron uptake when ferritin in the denominator reflects the iron levels in these parenchymal cells, such as occurs in various forms of HH. Hence, in NHIOD the decrease in hepcidin/ferritin ratio is likely to overestimate the reduction of iron sensing of parenchymal iron levels and the subsequent inadequate increase of intestinal iron absorption. It moreover fails to explain the observed Kupffer cell iron loading. It is therefore tempting to speculate that in HCV and other NHIOD local RES produced small amounts of inflammatory cytokines and hepcidin are involved in RES iron sequestration by decreasing ferroportin activity [62Theurl I. Theurl M. Seifert M. Mair S. Nairz M. Rumpold H. et al.Autocrine formation of hepcidin induces iron retention in human monocytes.Blood. 2008; 111: 2392-2399Crossref PubMed Scopus (221) Google Scholar, 63Ludwiczek S. Aigner E. Theurl I. Weiss G. Cytokine-mediated regulation of iron transport in human monocytic cells.Blood. 2003; 101: 4148-4154Crossref PubMed Scopus (340) Google Scholar]. Clearly, more work is warranted to unravel the biological mechanism explaining the distorted iron homeostasis in these patients to assess the suitability of the hepcidin/ferritin ratio or its alternative log transformations as a marker for inadequately elevated intestinal iron uptake. By assessing serum hepcidin levels in HCV Fujita et al. [[36]Fujita N. Sugimoto R. Motonishi R. Tomosugi N. Tanaka H. Takeo M. et al.Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretion.J Hepatol. 2008; 49: 702-710Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] corroborate previous findings on a decreased liver iron sensitivity as a uniform mechanism underlying mild hepatic iron overload. Down regulation of liver hepcidin expression by increased hepatic reactive oxygen species (ROS) by inhibition of the DNA-binding of C/EBPα might be an important causal factor in this apparent decrease in hepatocyte iron sensing (Fig. 1). Based on this role of hepcidin in NHIOD in general and chronic HCV in particular we propose that hepcidin may contribute to predicting and monitoring of iron overload in these patients. Relatively low hepcidin levels may indicate an increased risk of iron overload and normalization possibly points to the successful HCV eradication by antiviral regimes. Normalization of hepcidin levels by iron reduction (by e.g. dietary restriction, phlebotomy, iron chelation), antioxidants or hepcidin inducers or supplements, may perhaps be suitable as add-on treatment options for patients with NHIOD to improve the detrimental effects of iron on liver function. It is conceivable that in the case of HCV, hepcidin elevating therapies lead to an improved response to antiviral treatment. However, before we can translate these newly acquired insights into clinical practice, additional studies will be needed to further unravel critical cellular iron transporters involved in iron homeostasis in NHIOD. Finally, if hepcidin proves to be a useful marker in the management of non-hemochromatotic iron overload reliable, accessible, high throughput, affordable and reproducible methods will be needed to facilitate the measurements that hepatologists need. We thank the Nijmegen clinical chemistry hepcidin team for stimulating and fruitful discussions, which led to many of the thoughts mentioned above: Harold Tjalsma, April Kartikasari, Boukje van Dijk, Erwin Kemna, Joyce Kroot, Coby Laarakkers, Erwin Wiegerinck and Siem Klaver. D.W.S. and Harold Tjalsma steer the "www.hepcidinanalysis.com" initiative that serves the scientific community with quantitative hepcidin measurements by time-of-flight mass spectrometry.
Publication Year: 2008
Publication Date: 2008-11-01
Language: en
Type: review
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 27
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