CROHNS-01011; No of Pages 6 Journal of Crohn's and Colitis (2014) xx, xxx–xxx
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Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease☆ Ingo Mecklenburg a,1 , Diana Reznik a,1 , Elizaveta Fasler-Kan a,b , Jürgen Drewe a , Christoph Beglinger a , Petr Hruz a,⁎, On behalf of the Swiss IBD Cohort Study Group a
Department of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland University of Applied Sciences Northwestern Switzerland (FHNW), Institute of Chemistry and Bioanalytics, Muttenz, Switzerland b
Received 7 March 2014; received in revised form 7 April 2014; accepted 21 April 2014 KEYWORDS Hepcidin; Ulcerative colitis; Crohn's disease; Inflammatory bowel disease; Anemia
Abstract Background and aims: Anemia is a frequent complication of inflammatory bowel disease (IBD). Hepcidin, a key mediator in this anemia, is up-regulated by high iron levels and inflammation, and serum levels are elevated in IBD. However, the extent of inflammatory activity and iron deficiency for the regulation of hepcidin is not known. This study aimed to evaluate serum hepcidin levels in anemic and non-anemic IBD patients, with iron or non-iron deficiency, and active or inactive disease. Methods: This retrospective, observational study analyzed serum hepcidin levels from 247 patients with IBD (130 Crohn's patients and 117 with ulcerative colitis) recruited at Swiss Inflammatory Bowel Disease Cohort Study centers. Patients were divided into 5 different groups using criteria of active and inactive diseases (C-reactive protein, and CDAI/MTWAI = disease activity-index), anemia (hemoglobin) and iron deficiency (ferritin) and compared to healthy controls with no signs of anemia and normal ferritin levels. Hepcidin was measured using enzyme-linked immunosorbent assay. Results: Independent of inflammatory activity, all patients with decreased ferritin (b 30 μg/L) had significantly lower hepcidin levels when compared to patients and healthy controls having
Abbreviations: UC, Ulcerative colitis; CD, Crohn's disease; IBD, Inflammatory bowel disease; SIBDCS, Swiss Inflammatory Bowel Disease Cohort Study; ACD, Anemia of chronic disease; CRP, C-reactive protein; CDAI, Crohn's disease activity index; MTWAI, Modified Truelove and Witts activity Index; ELISA, Enzyme-linked immunosorbent assay. ☆ Financial disclosure: The authors have no financial relationships relevant to this article to disclose. ⁎ Corresponding author at: Department of Gastroenterology and Hepatology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland. Tel.: +41 61 328 66 09; fax: + 41 61 265 53 52. E-mail address: [email protected] (P. Hruz). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.crohns.2014.04.008 1873-9946/© 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved. Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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I. Mecklenburg et al. normal ferritin (N 30 μg/L). A significant correlation between serum ferritin levels and serum hepcidin was found (Spearman's Rho = 0.491; p b 0.001). A backward multi-linear stepwise regression analysis showed that only ferritin, and none of the inflammatory markers or age and sex correlated significantly (p = 0.005) with hepcidin. Conclusion: This retrospective analysis suggests that iron deficiency is the key trigger for hepcidin regulation in IBD patients with anemia. © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel disease (IBD) defined as chronic inflammatory and frequently relapsing disease of the gastrointestinal tract, implies two major disorders, Crohn's disease (CD) and ulcerative colitis (UC). CD is characterized by a discontinuous, transmural inflammation that can occur anywhere in the gastrointestinal tract, whereas UC shows a more superficial, continuous colonic inflammation that affects only the mucosal and submucosal layers.1 Both diseases are idiopathic, but the chronic inflammatory condition of the gut is related to a combination of genetic and environmental factors that impact on normal host–microbe interactions.2 The prevalence of CD in Europe varies from 1.52 to 213 cases per 100,000 persons, whereas the prevalence of UC is slightly higher and varies from 2.42 to 294 cases per 100,000 persons.3 Recent studies have demonstrated that anemia represents the most common systemic complication of IBD patients.4,5 In an analysis of the Swiss IBD Cohort Study, anemia was found in 21.2% of all IBD patients, with a higher rate in patients from tertiary referral centers (28.8%) when compared to general practice patients (12.9%).6 The cause of anemia in chronic inflammatory diseases is multifactorial. Most often anemia in IBD results in iron deficiency anemia (IDA), caused by reduced iron uptake from enterocytes and obvious chronic blood loss due to mucosal inflammation. Other causes that can aggravate anemia in IBD patients include vitamin B12 deficiency, folate deficiency and drug-induced anemia. On the other hand, in IBD patients, anemia of chronic diseases (ACD) can also occur, which is mediated via inflammatory mechanisms with the consequence of decreased iron levels in the circulation and limited iron availability for erythroid cells. In this latter cause of anemia, pro-inflammatory cytokines induce the formation of hepcidin, a key regulator of iron homeostasis. Hepcidin is a small antimicrobial peptide produced in the liver and transcriptionally up-regulated by high iron levels as well as inflammation.7 Hepcidin reduces the amount of circulating iron by binding to ferroportin, a divalent iron transporter, located on the cell surfaces of hepatocytes, macrophages and the basolateral membranes of enterocytes.8,9 Ferroportin blocks cellular iron (Fe2+) export from macrophages and inhibits the transfer of absorbed iron from the basolateral membranes of enterocytes into the circulation. The reduced circulating iron results in a limited availability for erythroid progenitors and impaired erythropoiesis. Various studies in patients having IBD and anemia exposed a relationship between hepcidin and Crohn's disease and ulcerative colitis.10–13 Hepcidin is upregulated by inflammation through IL6-mediated STAT3 signaling.14,15 It triggers an anti-inflammatory response in macrophages and, through its antimicrobial activity, hepcidin may be involved in the
control of microbial growth. These characteristics make hepcidin an interesting target of IBD research as these suggest a dual role in host defense and iron homeostasis.10 Hepcidin is significantly elevated in patients with CD and UC.13 However, the exact contribution of inflammatory activity (hepcidin's function as an acute phase protein) and iron deficiency (hepcidin's function as gatekeeper of iron homeostasis) in the regulation of hepcidin in patients with IBD is not known. With this observational study, we aimed to assess serum hepcidin levels in IBD patients with active and inactive IBDs and to correlate these levels with C-reactive protein (CRP), ferritin and hemoglobin.
2. Methods 2.1. Study patients In this observational study, we evaluated serum hepcidin levels from 247 patients with IBD (130 patients with CD and 117 with UC), recruited at the centers participating in the Swiss Inflammatory Bowel Disease Cohort Study (SIBDCS)—a national prospective clinical cohort that started in 2006. A full description of the cohort profile has been published previously.16 Patients were recruited through their gastroenterologists in private practices, regional hospitals and tertiary centers. Inclusion criteria were diagnoses of CD or UC established at least 4 months before inclusion and confirmed by radiological, endoscopic or surgical assessment, or after at least one recurrence of the disease. CD case ascertainment was made based on Lennard-Jones criteria.17 Patients were excluded if they suffered from another form of colitis, were not regularly followed up for CD or UC, had no permanent residency in Switzerland, or if they did not sign the informed consent form. Patients with UC and CD included in this analysis were divided into 5 different groups, based on criteria for active and inactive diseases, presence or absence of anemia, and iron deficiency. Allocation into groups with active or inactive disease was based on measurement of C-reactive protein (CRP N 5 mg/L) and the clinical disease activity indices, CDAI (Crohn's disease activity index) for CD and MTWAI (Modified Truelove and Witts activity index) for UC. In addition, anemia (serum hemoglobin level: men Hb b 140 g/L; for women Hb b 120 g/L) and iron deficiency (serum ferritin b 30 μg/L) were used for further group stratification. A group of 21 healthy subjects with no laboratory or clinical signs of acute or chronic inflammation was used as the control group. These subjects had no laboratory signs of anemia and had normal iron status parameters. The study was approved by the local ethical committee.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
Hepcidin in IBD
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2.2. Determination of serum hepcidin levels Hepcidin was measured with enzyme-linked immunosorbent assay (ELISA) using Cusabio Human Hepcidin ELISA kit (Cusabio Biotech, Wuhan, China, catalog number: CSB-E13062h) according to the manufacturer's instructions.
2.3. Statistical analysis Demographic data are presented as median and range. Hepcidin values were compared among the patient groups by general linear model analysis. Pair-wise comparison between the groups used Scheffe's multi-comparison test to account for the multiplicity of testing. Local linear regression analysis of ferritin values b 380 μg/L was performed by non-parametric robust regression analysis (Matlab software, version R2013, Mathworks Inc., Natick, MA, USA). The predictive value of different demographic and laboratory parameters (e.g., age, sex, albumin, CRP, ferritin, hemoglobin, leucocyte count, thrombocyte counts) for the serum concentration of hepcidin was analyzed by backward multi-linear stepwise regression. If predictors were not normally distributed as indicated by Kolmogorov-Smirnov test, regression analysis was performed on logarithmically transformed data. For statistical analysis, IBM SPSS statistics, version 21 was used. A p-value b 0.05 was considered statistically significant.
3. Results Analysis of serum hepcidin levels in patients with chronic inflammatory bowel disease (CD and UC) was performed and compared to healthy controls. The demographic data of patients with CD and UC are presented in two tables. In Table 1, data from CD are shown. As defined, patients with active disease (groups 1, 2 and 3) had signs of inflammation with elevated inflammatory parameters CRP, leucocytes and Table 1
CDAI when compared to patients in disease remission (groups 4 and 5). Lower albumin levels were observed in the active disease groups. In Table 2, data are presented for patients with UC. Similarly to CD patients, UC patients with active disease (groups 6, 7 and 8) had signs of inflammation as shown for the inflammatory parameters CRP, leucocytes and the disease activity score MTWAI when compared to UC patients in disease remission (groups 9 and 10). Furthermore, lower albumin levels were observed in the active disease groups. For both diseases (CD and UC) as defined, Hb levels were normal in patients in the non-anemic groups and were decreased in the groups with anemia, respectively. As a measure of the iron storage, ferritin levels were considered as normal when N 30 μg/L and decreased when b 30 μg/L. Because of small group sizes, hepcidin serum levels of CD and UC patients were combined for the statistical analysis (Fig. 1). This analysis demonstrated that, for non-anemic (groups 2 and 7) or anemic (groups 3 and 8) patients with active disease and a ferritin level N 30 μg/L, hepcidin levels comparable to non-anemic patients with inactive disease (groups 5 and 10) and healthy controls were seen. In contrast, anemic patients with active disease (groups 1 and 6) and patients in remission (groups 4 and 9) with a ferritin b 30 μg/L showed significantly decreased hepcidin levels when compared to patients and healthy controls with a normal ferritin value N 30 μg/L. A significant correlation between serum ferritin levels and serum hepcidin was found (Spearman's Rho = 0.491; p b 0.001, Fig. 2). The optimal fit calculated for ferritin values was ≤ 380 μg/L. These data suggest that, in the regulation of hepcidin, ferritin levels may be more important than the inflammatory parameters. Therefore, the predictive values of different demographic and laboratory parameters (e.g., age, albumin, CRP, ferritin, hemoglobin, leucocyte count) for the concentration of serum hepcidin were analyzed by backward multi-linear stepwise regression and showed that only ferritin—and none of the inflammatory markers—was significantly (p = 0.005)
Group stratification and laboratory parameters of patients with Crohn's disease. Active disease (CDAI N 150, CRP N 5)
Inactive disease (CDAI ≤ 150, CRP ≤ 5)
Hemoglobin (g/L)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Ferritin (μg/L) Group size (n =) Age Male:female Parameter median (range) CDAI Leucocytes (× 109/L) CRP (mg/L) Albumin (g/L) Hemoglobin (g/L) f Hemoglobin (g/L) m Ferritin (μg/L) Hepcidin (ng/mL) Group
Ferritin b 30 8 28 (20–46) 1:7
Ferritin ≥ 30 27 36 (21–62) 12:15
Ferritin ≥ 30 25 35 (20–73) 15:10
Ferritin b 30 15 35 (17–46) 12:3
Ferritin ≥ 30 55 36 (19–72) 30:25
180 (155–251) 7.9 (4.2–13.3) 18.0 (7–58) 29 (26–34) 100 (90–120) 136 (136–136) 16 (4–29) 97.1 (0–2125) 1
217 (152–378) 9.0 (4.8–15.4) 12.1 (5.6–193) 38 (24–45) 129 (123–139) 147 (140–158) 86 (31–259) 388.7 (9.8–1219) 2
210 (158–279) 8.3 (5.1–15.3) 30.1 (6–260) 31 (17–45) 108 (82–118) 128 (94–139) 130 (30–1111) 557.7 (2.7–1822) 3
64 (9–143) 5.9 (4.0–7.6) 3.0 (0.3–5.0) 40 (31–46) 117 (109–118) 133 (105–137) 18 (6–26) 93.1 (0–742) 4
57 (0–126) 7.1 (2.6–12.4) 2.3 (0.3–5.0) 40.5 (29–48) 139 (122–153) 151 (142–168) 103 (30–636) 457.6 (8.8–1822) 5
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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I. Mecklenburg et al. Table 2
Group stratification and laboratory parameters of patients with ulcerative colitis. Active disease (MTWAI ≥ 5, CRP N 5)
Inactive disease (MTWAI ≤ 5, CRP ≤ 5)
Hemoglobin (g/L)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Ferritin (μg/L) Group size (n =) Age Male:female Parameter median (range) MTWAI Leucocytes (× 109/L) CRP (mg/L) Albumin (g/L) Hemoglobin (g/L) f Hemoglobin (g/L) m Ferritin (μg/L) Hepcidin (ng/mL) Group
Ferritin b 30 15 42 (20–62) 9:6
Ferritin ≥ 30 18 40 (20–75) 10:8
Ferritin ≥ 30 27 35 (19–65) 19:8
Ferritin b 30 12 35 (18–47) 8:4
Ferritin ≥ 30 45 45 (20–82) 19:26
8 (6–15) 7.9 (4.1–19.0) 17.0 (7–100) 33 (21–42) 111 (103–119) 113 (83–139) 15 (4–29) 16.3 (0–1914) 6
9 (6–15) 9.1 (5.8–15.6) 9.2 (6–54) 39.5 (30–47) 129 (122–142) 147 (140–163) 98.5 (43–590) 577.2 (7.9–2157) 7
11 (6–16) 7.1 (2.1–16.0) 35 (6–222) 31 (17–44) 105 (85–115) 121 (70–140) 122 (36–1210) 698.2 (0–2251) 8
3 (0–5) 6.4 (5.0–11.1) 3.0 (0.5–5.0) 42 (32–48) 113 (111–118) 128 (105–137) 13 (4–18) 54.9 (0–1479) 9
1 (0–5) 6.4 (3.5–13.7) 3.0 (0.3–5.0) 44 (29–49) 133 (122–147) 152 (141–175) 97 (31–1400) 464.1 (0–1512) 10
correlated with hepcidin. The results of the regression analysis are given in Tables 3a and 3b.
4. Discussion Our results show that hepcidin serum levels are significantly decreased in anemic IBD patients with depleted iron storage, as evidenced by decreased levels of ferritin independent of their disease activity status. Anemic as well as non-anemic IBD patients with active disease and non-anemic patients in remission, but all with normal ferritin, presented with similar hepcidin levels and had levels comparable to healthy
Figure 1 Correlation of hepcidin and ferritin levels in patients with ulcerative colitis (n = 117) and Crohn's disease (n = 130). The analysis was performed for ferritin values ≤ 380 μg/L by non-parametric robust regression analysis. The non-parametric Spearman's Rho correlation coefficient (ρ) was calculated. The ellipse represents the 95% confidence interval.
subjects. A multi-linear regression analysis showed that only ferritin and none of the inflammatory markers was significantly (p = 0.005) correlated with hepcidin. These results suggest that primarily the iron storage level, and not the inflammatory activity, contributes to hepcidin regulation in our cohort of IBD patients. Several other studies have recently addressed levels of hepcidin and the precursor protein, prohepcidin, in patients with IBD. Oustamanolakis found significantly increased levels of hepcidin and significantly decreased levels of prohepcidin
Figure 2 Distribution of serum hepcidin levels in patients with IBD (ulcerative colitis, n = 117 and Crohn's disease, n = 130) and healthy controls (n = 21) is shown. The allocation of patients to the respective groups is described in the Methods section. Patient characteristics and laboratory parameters are shown in Tables 1 and 2. Boxes indicate the interquartile range with median value. Bars show the 5th and 95th percentiles and data points representing the 1st and 99th percentile are shown as a star. In addition, all individual values are shown.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
Hepcidin in IBD Table 3a
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Regression coefficients. Unstandardized coefficients
Standardized coefficients
Model
B
Standard error
Beta
(Constant) Log ferritin a
1.106 0.694
0.465 0.230
a
0.482
t
p-Value
95% Confidence intervals for B Lower bound
Upper Bound
2.378 3.016
0.024 0.005
0.156 0.224
2.056 1.165
Data were not normally distributed and logarithmically transformed.
in unselected CD and UC patients.13 Some 42% of this group of IBD patients had anemia. In a multivariate analysis in patients with UC, hepcidin significantly correlated only with ferritin and disease activity, but not with anemia. Prohepcidin did not correlate with any of the examined parameters. In contrast, when compared to healthy controls, decreased serum levels of hepcidin were observed in IBD patients, independent of whether or not these patients were iron-deficient.10 Furthermore, prohepcidin levels did not differ significantly when compared to control subjects, and suggested that serum prohepcidin level may not be a satisfactory diagnostic or prognostic measure in anemia of chronic inflammatory disease.12 Current data from the literature point out the considerable difficulty in interpreting serum hepcidin and prohepcidin levels in IBD patients. Furthermore, in our analysis, we observed a high variability of hepcidin levels within the individual groups that may indicate complex pathophysiological mechanisms involved in hepcidin regulation. Indeed, hepcidin is associated with a variety of chronic inflammatory conditions. Inflammation due to infection, autoimmune disease or cancer stimulates the synthesis of pro-inflammatory cytokines and especially interleukin-6 (IL-6) which has been shown to induce hepcidin production.18–20 This IL-6-induced hepcidin up-regulation is mediated via STAT3.15 Patients with inflammation characterized by high C-reactive protein levels (N 10 mg/L) and patients with multiple myeloma (known to secrete excess IL-6) all have inappropriately elevated hepcidin levels.8 After injection of bacterial lipopolysaccharide into healthy humans, one observes an acute inflammation with marked IL-6 production that is correlated with increased hepcidin production.18 For IBD patients, only studies with few patients have been performed. In 17 CD patients with ACD, a strong positive correlation between serum IL-6 and hepcidin levels was demonstrated.11 In the same study, similar to our findings, a strong correlation Table 3b
Excluded variables.
Model predictors Log thrombocyte count Leucocyte count Logs hemoglobin a Gender Log age a Albumin Log CRP a
a
Beta
t
p-Value
− 0.209 − 0.117 0.070 − 0.156 0.097 0.198 0.018
− 1.248 − 0.722 0.405 − 0.969 0.592 1.249 0.109
0.222 0.476 0.688 0.341 0.558 0.222 0.914
a Data were not normally distributed and logarithmically transformed.
between serum ferritin and hepcidin levels was observed. However, in our analysis, we could not demonstrate any correlation with inflammatory disease markers. This may be related to the fact that IBD patients with an established diagnosis of CD or UC for at least 4 months are included in our Swiss IBD cohort, and patients with a severe flare-up may be underrepresented in our evaluation. An interesting study was performed in 19 pediatric patients with Crohn's disease. After an overnight fast, serum iron, hemoglobin levels, serum markers of inflammation and urine hepcidin were analyzed before and after oral administration of ferrous sulfate (1 mg/kg). Patients with active disease had significantly elevated CRP, IL-6 and urine hepcidin when compared to patients with inactive disease. In addition, impaired intestinal iron absorption correlated with disease activity and markers of inflammation, and indicated that the inflammatory effects have functional consequences.21 Blocking endogenous hepcidin production could therefore be an effective treatment for anemia of chronic disease.22 In conclusion, our analysis confirmed a modest correlation between serum hepcidin and ferritin levels as a measure of iron storage in patients with IBD. For our IBD patients, we did not find any correlation between serum hepcidin levels and inflammatory parameters.
Conflict of interest The authors have no conflicts of interest to disclose.
Acknowledgments We would like to thank Ulrich Baumgartner and Viktoria Gillich for technical assistance. The Swiss National Science Foundation (33CSC0_134274) provided a research grant to support this study, but did not have an active role in the design, collection, analysis and interpretation of data, nor in the writing of the manuscript or in the decision to submit the manuscript for publication. PH and IM conceptualized and designed the clinical question, drafted the initial manuscript; DR evaluated the patient data, and drafted the initial manuscript; CB conceptualized, and critically reviewed the manuscript; EF performed laboratory analysis, and critically reviewed the manuscript; JD conceptualized, critically reviewed the manuscript and performed the statistical calculation. All authors read and approved the final manuscript as submitted.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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12. Nagy J, Lakner L, Poor VS, Pandur E, Mozsik G, Miseta A, et al. Serum prohepcidin levels in chronic inflammatory bowel diseases. J Crohns Colitis 2010;4:649–53. 13. Oustamanolakis P, Koutroubakis IE, Messaritakis I, Malliaraki N, Sfiridaki A, Kouroumalis EA. Serum hepcidin and prohepcidin concentrations in inflammatory bowel disease. Eur J Gastroenterol Hepatol 2011;23:262–8. 14. Wang L, Trebicka E, Fu Y, Ellenbogen S, Hong CC, Babitt JL, et al. The bone morphogenetic protein-hepcidin axis as a therapeutic target in inflammatory bowel disease. Inflamm Bowel Dis 2011;18: 112–9. 15. Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin expression through STAT3. Blood 2006;108:3204–9. 16. Pittet V, Juillerat P, Mottet C, Felley C, Ballabeni P, Burnand B, et al. Cohort profile: the Swiss Inflammatory Bowel Disease Cohort Study (SIBDCS). Int J Epidemiol 2009;38:922–31. 17. Lennard-Jones JE. Classification of inflammatory bowel disease. Scand J Gastroenterol Suppl 1989;170:2–6 [discussion 16–9]. 18. Kemna 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;106:1864–6. 19. Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, et al. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 2004;113:1271–6. 20. Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002;110:1037–44. 21. Semrin G, Fishman DS, Bousvaros A, Zholudev A, Saunders AC, Correia CE, et al. Impaired intestinal iron absorption in Crohn's disease correlates with disease activity and markers of inflammation. Inflamm Bowel Dis 2006;12:1101–6. 22. Theurl I, Schroll A, Sonnweber T, Nairz M, Theurl M, Willenbacher W, et al. Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats. Blood 2011;118:4977–84.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease☆ Ingo Mecklenburg a,1 , Diana Reznik a,1 , Elizaveta Fasler-Kan a,b , Jürgen Drewe a , Christoph Beglinger a , Petr Hruz a,⁎, On behalf of the Swiss IBD Cohort Study Group a
Department of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland University of Applied Sciences Northwestern Switzerland (FHNW), Institute of Chemistry and Bioanalytics, Muttenz, Switzerland b
Received 7 March 2014; received in revised form 7 April 2014; accepted 21 April 2014 KEYWORDS Hepcidin; Ulcerative colitis; Crohn's disease; Inflammatory bowel disease; Anemia
Abstract Background and aims: Anemia is a frequent complication of inflammatory bowel disease (IBD). Hepcidin, a key mediator in this anemia, is up-regulated by high iron levels and inflammation, and serum levels are elevated in IBD. However, the extent of inflammatory activity and iron deficiency for the regulation of hepcidin is not known. This study aimed to evaluate serum hepcidin levels in anemic and non-anemic IBD patients, with iron or non-iron deficiency, and active or inactive disease. Methods: This retrospective, observational study analyzed serum hepcidin levels from 247 patients with IBD (130 Crohn's patients and 117 with ulcerative colitis) recruited at Swiss Inflammatory Bowel Disease Cohort Study centers. Patients were divided into 5 different groups using criteria of active and inactive diseases (C-reactive protein, and CDAI/MTWAI = disease activity-index), anemia (hemoglobin) and iron deficiency (ferritin) and compared to healthy controls with no signs of anemia and normal ferritin levels. Hepcidin was measured using enzyme-linked immunosorbent assay. Results: Independent of inflammatory activity, all patients with decreased ferritin (b 30 μg/L) had significantly lower hepcidin levels when compared to patients and healthy controls having
Abbreviations: UC, Ulcerative colitis; CD, Crohn's disease; IBD, Inflammatory bowel disease; SIBDCS, Swiss Inflammatory Bowel Disease Cohort Study; ACD, Anemia of chronic disease; CRP, C-reactive protein; CDAI, Crohn's disease activity index; MTWAI, Modified Truelove and Witts activity Index; ELISA, Enzyme-linked immunosorbent assay. ☆ Financial disclosure: The authors have no financial relationships relevant to this article to disclose. ⁎ Corresponding author at: Department of Gastroenterology and Hepatology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland. Tel.: +41 61 328 66 09; fax: + 41 61 265 53 52. E-mail address: [email protected] (P. Hruz). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.crohns.2014.04.008 1873-9946/© 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved. Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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I. Mecklenburg et al. normal ferritin (N 30 μg/L). A significant correlation between serum ferritin levels and serum hepcidin was found (Spearman's Rho = 0.491; p b 0.001). A backward multi-linear stepwise regression analysis showed that only ferritin, and none of the inflammatory markers or age and sex correlated significantly (p = 0.005) with hepcidin. Conclusion: This retrospective analysis suggests that iron deficiency is the key trigger for hepcidin regulation in IBD patients with anemia. © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel disease (IBD) defined as chronic inflammatory and frequently relapsing disease of the gastrointestinal tract, implies two major disorders, Crohn's disease (CD) and ulcerative colitis (UC). CD is characterized by a discontinuous, transmural inflammation that can occur anywhere in the gastrointestinal tract, whereas UC shows a more superficial, continuous colonic inflammation that affects only the mucosal and submucosal layers.1 Both diseases are idiopathic, but the chronic inflammatory condition of the gut is related to a combination of genetic and environmental factors that impact on normal host–microbe interactions.2 The prevalence of CD in Europe varies from 1.52 to 213 cases per 100,000 persons, whereas the prevalence of UC is slightly higher and varies from 2.42 to 294 cases per 100,000 persons.3 Recent studies have demonstrated that anemia represents the most common systemic complication of IBD patients.4,5 In an analysis of the Swiss IBD Cohort Study, anemia was found in 21.2% of all IBD patients, with a higher rate in patients from tertiary referral centers (28.8%) when compared to general practice patients (12.9%).6 The cause of anemia in chronic inflammatory diseases is multifactorial. Most often anemia in IBD results in iron deficiency anemia (IDA), caused by reduced iron uptake from enterocytes and obvious chronic blood loss due to mucosal inflammation. Other causes that can aggravate anemia in IBD patients include vitamin B12 deficiency, folate deficiency and drug-induced anemia. On the other hand, in IBD patients, anemia of chronic diseases (ACD) can also occur, which is mediated via inflammatory mechanisms with the consequence of decreased iron levels in the circulation and limited iron availability for erythroid cells. In this latter cause of anemia, pro-inflammatory cytokines induce the formation of hepcidin, a key regulator of iron homeostasis. Hepcidin is a small antimicrobial peptide produced in the liver and transcriptionally up-regulated by high iron levels as well as inflammation.7 Hepcidin reduces the amount of circulating iron by binding to ferroportin, a divalent iron transporter, located on the cell surfaces of hepatocytes, macrophages and the basolateral membranes of enterocytes.8,9 Ferroportin blocks cellular iron (Fe2+) export from macrophages and inhibits the transfer of absorbed iron from the basolateral membranes of enterocytes into the circulation. The reduced circulating iron results in a limited availability for erythroid progenitors and impaired erythropoiesis. Various studies in patients having IBD and anemia exposed a relationship between hepcidin and Crohn's disease and ulcerative colitis.10–13 Hepcidin is upregulated by inflammation through IL6-mediated STAT3 signaling.14,15 It triggers an anti-inflammatory response in macrophages and, through its antimicrobial activity, hepcidin may be involved in the
control of microbial growth. These characteristics make hepcidin an interesting target of IBD research as these suggest a dual role in host defense and iron homeostasis.10 Hepcidin is significantly elevated in patients with CD and UC.13 However, the exact contribution of inflammatory activity (hepcidin's function as an acute phase protein) and iron deficiency (hepcidin's function as gatekeeper of iron homeostasis) in the regulation of hepcidin in patients with IBD is not known. With this observational study, we aimed to assess serum hepcidin levels in IBD patients with active and inactive IBDs and to correlate these levels with C-reactive protein (CRP), ferritin and hemoglobin.
2. Methods 2.1. Study patients In this observational study, we evaluated serum hepcidin levels from 247 patients with IBD (130 patients with CD and 117 with UC), recruited at the centers participating in the Swiss Inflammatory Bowel Disease Cohort Study (SIBDCS)—a national prospective clinical cohort that started in 2006. A full description of the cohort profile has been published previously.16 Patients were recruited through their gastroenterologists in private practices, regional hospitals and tertiary centers. Inclusion criteria were diagnoses of CD or UC established at least 4 months before inclusion and confirmed by radiological, endoscopic or surgical assessment, or after at least one recurrence of the disease. CD case ascertainment was made based on Lennard-Jones criteria.17 Patients were excluded if they suffered from another form of colitis, were not regularly followed up for CD or UC, had no permanent residency in Switzerland, or if they did not sign the informed consent form. Patients with UC and CD included in this analysis were divided into 5 different groups, based on criteria for active and inactive diseases, presence or absence of anemia, and iron deficiency. Allocation into groups with active or inactive disease was based on measurement of C-reactive protein (CRP N 5 mg/L) and the clinical disease activity indices, CDAI (Crohn's disease activity index) for CD and MTWAI (Modified Truelove and Witts activity index) for UC. In addition, anemia (serum hemoglobin level: men Hb b 140 g/L; for women Hb b 120 g/L) and iron deficiency (serum ferritin b 30 μg/L) were used for further group stratification. A group of 21 healthy subjects with no laboratory or clinical signs of acute or chronic inflammation was used as the control group. These subjects had no laboratory signs of anemia and had normal iron status parameters. The study was approved by the local ethical committee.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
Hepcidin in IBD
3
2.2. Determination of serum hepcidin levels Hepcidin was measured with enzyme-linked immunosorbent assay (ELISA) using Cusabio Human Hepcidin ELISA kit (Cusabio Biotech, Wuhan, China, catalog number: CSB-E13062h) according to the manufacturer's instructions.
2.3. Statistical analysis Demographic data are presented as median and range. Hepcidin values were compared among the patient groups by general linear model analysis. Pair-wise comparison between the groups used Scheffe's multi-comparison test to account for the multiplicity of testing. Local linear regression analysis of ferritin values b 380 μg/L was performed by non-parametric robust regression analysis (Matlab software, version R2013, Mathworks Inc., Natick, MA, USA). The predictive value of different demographic and laboratory parameters (e.g., age, sex, albumin, CRP, ferritin, hemoglobin, leucocyte count, thrombocyte counts) for the serum concentration of hepcidin was analyzed by backward multi-linear stepwise regression. If predictors were not normally distributed as indicated by Kolmogorov-Smirnov test, regression analysis was performed on logarithmically transformed data. For statistical analysis, IBM SPSS statistics, version 21 was used. A p-value b 0.05 was considered statistically significant.
3. Results Analysis of serum hepcidin levels in patients with chronic inflammatory bowel disease (CD and UC) was performed and compared to healthy controls. The demographic data of patients with CD and UC are presented in two tables. In Table 1, data from CD are shown. As defined, patients with active disease (groups 1, 2 and 3) had signs of inflammation with elevated inflammatory parameters CRP, leucocytes and Table 1
CDAI when compared to patients in disease remission (groups 4 and 5). Lower albumin levels were observed in the active disease groups. In Table 2, data are presented for patients with UC. Similarly to CD patients, UC patients with active disease (groups 6, 7 and 8) had signs of inflammation as shown for the inflammatory parameters CRP, leucocytes and the disease activity score MTWAI when compared to UC patients in disease remission (groups 9 and 10). Furthermore, lower albumin levels were observed in the active disease groups. For both diseases (CD and UC) as defined, Hb levels were normal in patients in the non-anemic groups and were decreased in the groups with anemia, respectively. As a measure of the iron storage, ferritin levels were considered as normal when N 30 μg/L and decreased when b 30 μg/L. Because of small group sizes, hepcidin serum levels of CD and UC patients were combined for the statistical analysis (Fig. 1). This analysis demonstrated that, for non-anemic (groups 2 and 7) or anemic (groups 3 and 8) patients with active disease and a ferritin level N 30 μg/L, hepcidin levels comparable to non-anemic patients with inactive disease (groups 5 and 10) and healthy controls were seen. In contrast, anemic patients with active disease (groups 1 and 6) and patients in remission (groups 4 and 9) with a ferritin b 30 μg/L showed significantly decreased hepcidin levels when compared to patients and healthy controls with a normal ferritin value N 30 μg/L. A significant correlation between serum ferritin levels and serum hepcidin was found (Spearman's Rho = 0.491; p b 0.001, Fig. 2). The optimal fit calculated for ferritin values was ≤ 380 μg/L. These data suggest that, in the regulation of hepcidin, ferritin levels may be more important than the inflammatory parameters. Therefore, the predictive values of different demographic and laboratory parameters (e.g., age, albumin, CRP, ferritin, hemoglobin, leucocyte count) for the concentration of serum hepcidin were analyzed by backward multi-linear stepwise regression and showed that only ferritin—and none of the inflammatory markers—was significantly (p = 0.005)
Group stratification and laboratory parameters of patients with Crohn's disease. Active disease (CDAI N 150, CRP N 5)
Inactive disease (CDAI ≤ 150, CRP ≤ 5)
Hemoglobin (g/L)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Ferritin (μg/L) Group size (n =) Age Male:female Parameter median (range) CDAI Leucocytes (× 109/L) CRP (mg/L) Albumin (g/L) Hemoglobin (g/L) f Hemoglobin (g/L) m Ferritin (μg/L) Hepcidin (ng/mL) Group
Ferritin b 30 8 28 (20–46) 1:7
Ferritin ≥ 30 27 36 (21–62) 12:15
Ferritin ≥ 30 25 35 (20–73) 15:10
Ferritin b 30 15 35 (17–46) 12:3
Ferritin ≥ 30 55 36 (19–72) 30:25
180 (155–251) 7.9 (4.2–13.3) 18.0 (7–58) 29 (26–34) 100 (90–120) 136 (136–136) 16 (4–29) 97.1 (0–2125) 1
217 (152–378) 9.0 (4.8–15.4) 12.1 (5.6–193) 38 (24–45) 129 (123–139) 147 (140–158) 86 (31–259) 388.7 (9.8–1219) 2
210 (158–279) 8.3 (5.1–15.3) 30.1 (6–260) 31 (17–45) 108 (82–118) 128 (94–139) 130 (30–1111) 557.7 (2.7–1822) 3
64 (9–143) 5.9 (4.0–7.6) 3.0 (0.3–5.0) 40 (31–46) 117 (109–118) 133 (105–137) 18 (6–26) 93.1 (0–742) 4
57 (0–126) 7.1 (2.6–12.4) 2.3 (0.3–5.0) 40.5 (29–48) 139 (122–153) 151 (142–168) 103 (30–636) 457.6 (8.8–1822) 5
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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I. Mecklenburg et al. Table 2
Group stratification and laboratory parameters of patients with ulcerative colitis. Active disease (MTWAI ≥ 5, CRP N 5)
Inactive disease (MTWAI ≤ 5, CRP ≤ 5)
Hemoglobin (g/L)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
Anemia (Hb b 120♀, 140♂)
No anemia (Hb ≥ 120♀, 140♂)
Ferritin (μg/L) Group size (n =) Age Male:female Parameter median (range) MTWAI Leucocytes (× 109/L) CRP (mg/L) Albumin (g/L) Hemoglobin (g/L) f Hemoglobin (g/L) m Ferritin (μg/L) Hepcidin (ng/mL) Group
Ferritin b 30 15 42 (20–62) 9:6
Ferritin ≥ 30 18 40 (20–75) 10:8
Ferritin ≥ 30 27 35 (19–65) 19:8
Ferritin b 30 12 35 (18–47) 8:4
Ferritin ≥ 30 45 45 (20–82) 19:26
8 (6–15) 7.9 (4.1–19.0) 17.0 (7–100) 33 (21–42) 111 (103–119) 113 (83–139) 15 (4–29) 16.3 (0–1914) 6
9 (6–15) 9.1 (5.8–15.6) 9.2 (6–54) 39.5 (30–47) 129 (122–142) 147 (140–163) 98.5 (43–590) 577.2 (7.9–2157) 7
11 (6–16) 7.1 (2.1–16.0) 35 (6–222) 31 (17–44) 105 (85–115) 121 (70–140) 122 (36–1210) 698.2 (0–2251) 8
3 (0–5) 6.4 (5.0–11.1) 3.0 (0.5–5.0) 42 (32–48) 113 (111–118) 128 (105–137) 13 (4–18) 54.9 (0–1479) 9
1 (0–5) 6.4 (3.5–13.7) 3.0 (0.3–5.0) 44 (29–49) 133 (122–147) 152 (141–175) 97 (31–1400) 464.1 (0–1512) 10
correlated with hepcidin. The results of the regression analysis are given in Tables 3a and 3b.
4. Discussion Our results show that hepcidin serum levels are significantly decreased in anemic IBD patients with depleted iron storage, as evidenced by decreased levels of ferritin independent of their disease activity status. Anemic as well as non-anemic IBD patients with active disease and non-anemic patients in remission, but all with normal ferritin, presented with similar hepcidin levels and had levels comparable to healthy
Figure 1 Correlation of hepcidin and ferritin levels in patients with ulcerative colitis (n = 117) and Crohn's disease (n = 130). The analysis was performed for ferritin values ≤ 380 μg/L by non-parametric robust regression analysis. The non-parametric Spearman's Rho correlation coefficient (ρ) was calculated. The ellipse represents the 95% confidence interval.
subjects. A multi-linear regression analysis showed that only ferritin and none of the inflammatory markers was significantly (p = 0.005) correlated with hepcidin. These results suggest that primarily the iron storage level, and not the inflammatory activity, contributes to hepcidin regulation in our cohort of IBD patients. Several other studies have recently addressed levels of hepcidin and the precursor protein, prohepcidin, in patients with IBD. Oustamanolakis found significantly increased levels of hepcidin and significantly decreased levels of prohepcidin
Figure 2 Distribution of serum hepcidin levels in patients with IBD (ulcerative colitis, n = 117 and Crohn's disease, n = 130) and healthy controls (n = 21) is shown. The allocation of patients to the respective groups is described in the Methods section. Patient characteristics and laboratory parameters are shown in Tables 1 and 2. Boxes indicate the interquartile range with median value. Bars show the 5th and 95th percentiles and data points representing the 1st and 99th percentile are shown as a star. In addition, all individual values are shown.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
Hepcidin in IBD Table 3a
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Regression coefficients. Unstandardized coefficients
Standardized coefficients
Model
B
Standard error
Beta
(Constant) Log ferritin a
1.106 0.694
0.465 0.230
a
0.482
t
p-Value
95% Confidence intervals for B Lower bound
Upper Bound
2.378 3.016
0.024 0.005
0.156 0.224
2.056 1.165
Data were not normally distributed and logarithmically transformed.
in unselected CD and UC patients.13 Some 42% of this group of IBD patients had anemia. In a multivariate analysis in patients with UC, hepcidin significantly correlated only with ferritin and disease activity, but not with anemia. Prohepcidin did not correlate with any of the examined parameters. In contrast, when compared to healthy controls, decreased serum levels of hepcidin were observed in IBD patients, independent of whether or not these patients were iron-deficient.10 Furthermore, prohepcidin levels did not differ significantly when compared to control subjects, and suggested that serum prohepcidin level may not be a satisfactory diagnostic or prognostic measure in anemia of chronic inflammatory disease.12 Current data from the literature point out the considerable difficulty in interpreting serum hepcidin and prohepcidin levels in IBD patients. Furthermore, in our analysis, we observed a high variability of hepcidin levels within the individual groups that may indicate complex pathophysiological mechanisms involved in hepcidin regulation. Indeed, hepcidin is associated with a variety of chronic inflammatory conditions. Inflammation due to infection, autoimmune disease or cancer stimulates the synthesis of pro-inflammatory cytokines and especially interleukin-6 (IL-6) which has been shown to induce hepcidin production.18–20 This IL-6-induced hepcidin up-regulation is mediated via STAT3.15 Patients with inflammation characterized by high C-reactive protein levels (N 10 mg/L) and patients with multiple myeloma (known to secrete excess IL-6) all have inappropriately elevated hepcidin levels.8 After injection of bacterial lipopolysaccharide into healthy humans, one observes an acute inflammation with marked IL-6 production that is correlated with increased hepcidin production.18 For IBD patients, only studies with few patients have been performed. In 17 CD patients with ACD, a strong positive correlation between serum IL-6 and hepcidin levels was demonstrated.11 In the same study, similar to our findings, a strong correlation Table 3b
Excluded variables.
Model predictors Log thrombocyte count Leucocyte count Logs hemoglobin a Gender Log age a Albumin Log CRP a
a
Beta
t
p-Value
− 0.209 − 0.117 0.070 − 0.156 0.097 0.198 0.018
− 1.248 − 0.722 0.405 − 0.969 0.592 1.249 0.109
0.222 0.476 0.688 0.341 0.558 0.222 0.914
a Data were not normally distributed and logarithmically transformed.
between serum ferritin and hepcidin levels was observed. However, in our analysis, we could not demonstrate any correlation with inflammatory disease markers. This may be related to the fact that IBD patients with an established diagnosis of CD or UC for at least 4 months are included in our Swiss IBD cohort, and patients with a severe flare-up may be underrepresented in our evaluation. An interesting study was performed in 19 pediatric patients with Crohn's disease. After an overnight fast, serum iron, hemoglobin levels, serum markers of inflammation and urine hepcidin were analyzed before and after oral administration of ferrous sulfate (1 mg/kg). Patients with active disease had significantly elevated CRP, IL-6 and urine hepcidin when compared to patients with inactive disease. In addition, impaired intestinal iron absorption correlated with disease activity and markers of inflammation, and indicated that the inflammatory effects have functional consequences.21 Blocking endogenous hepcidin production could therefore be an effective treatment for anemia of chronic disease.22 In conclusion, our analysis confirmed a modest correlation between serum hepcidin and ferritin levels as a measure of iron storage in patients with IBD. For our IBD patients, we did not find any correlation between serum hepcidin levels and inflammatory parameters.
Conflict of interest The authors have no conflicts of interest to disclose.
Acknowledgments We would like to thank Ulrich Baumgartner and Viktoria Gillich for technical assistance. The Swiss National Science Foundation (33CSC0_134274) provided a research grant to support this study, but did not have an active role in the design, collection, analysis and interpretation of data, nor in the writing of the manuscript or in the decision to submit the manuscript for publication. PH and IM conceptualized and designed the clinical question, drafted the initial manuscript; DR evaluated the patient data, and drafted the initial manuscript; CB conceptualized, and critically reviewed the manuscript; EF performed laboratory analysis, and critically reviewed the manuscript; JD conceptualized, critically reviewed the manuscript and performed the statistical calculation. All authors read and approved the final manuscript as submitted.
Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
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Please cite this article as: Mecklenburg I, et al, Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease, J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.04.008
