Hepcidin levels in humans are correlated with hepatic iron stores, hemoglobin levels, and hepatic function
Le´naı¨ck De´tivaud, Elizabeta Nemeth, Karim Boudjema, Bruno Turlin, Marie-Be´renge`re Troadec, Patricia Leroyer, Martine Ropert,
Sylvie Jacquelinet, Brice Courselaud, Tomas Ganz, Pierre Brissot, and Olivier Lore´al
Hepcidin, a key regulator of iron metabolism, is synthesized by the liver. Hepcidin binds to the iron exporter ferroportin to regulate the release of iron into plasma from macrophages, hepatocytes, and enterocytes. We analyzed liver samples from patients undergoing hepatic surgery for cancer or receiving liver transplants and
analyzed correlations between clinical parameters and liver hepcidin mRNA and urinary hepcidin concentrations. Despite the many potential confounding inﬂuences, urinary hepcidin concentrations signiﬁcantly correlated with hepatic hepcidin mRNA concentrations, indicating that hepcidin quantiﬁcation in urine is a
Hepcidin is a peptide found in human plasma and urine1,2 and synthesized in the liver.1-3 Studies in mice, humans, and cell cultures demonstrated that hepcidin mRNA levels were regulated by iron stores,3 inﬂammation,3,4-6 anemia, and hypoxia,4 and inﬂuenced by the mouse strain and sex and transcription factors involved in hepatocyte differentiation.7,8 Hepcidin-deﬁcient mice accumulated iron in the liver and pancreas9 and mice engineered to overexpress hepcidin were born with severe iron deﬁciency that was not corrected by iron supplementation.10 Taken together these data suggested that hepcidin was a hormone involved in the regulation of iron metabolism.9 Studies of patients with iron disorders demonstrated the involvement of hepcidin in regulation of iron metabolism during juvenile hemochromatosis,11 classical HFE-related hemochromatosis,12,13 and chronic inﬂammatory diseases.5,14 Hepcidin appears to act by inhibiting cellular iron export through binding directly to the iron exporter ferroportin and inducing its internalization and degradation.15
Recent understanding of iron metabolism in mice is based on measurements of hepatic hepcidin mRNA, whereas the corresponding human studies examine urinary hepcidin concentrations. We examined the correlation between hepcidin mRNA and urinary hepcidin concentrations in the context of human liver disease.
Thirty-six patients, who were operated on for primary or secondary liver carcinoma or received transplants for cirrhosis, were included in this study.
valid approach to evaluate hepcidin expression. Moreover, we found in humans that hepcidin levels correlated with hepatic iron stores and hemoglobin levels and may also be affected by hepatic dysfunction. (Blood. 2005;106:746-748)
© 2005 by The American Society of Hematology
The patients with known HFE genetic hemochromatosis, expected to exhibit abnormal hepcidin regulation,12,16 were excluded. The study was approved by the local ethics committee (CCPPRB) and informed consent was obtained from the patients.
From each patient we collected clinical data and serum, urine, and nontumoral liver samples. Liver tissues were frozen in liquid nitrogen or ﬁxed in formalin for histologic studies. The hepatic ﬁbrosis status was evaluated according to the Metavir score17; 20 samples had no or slight ﬁbrosis, 7 samples were moderately ﬁbrotic, and 9 samples were strongly ﬁbrotic or cirrhotic.
Clinical laboratory studies. Clinical laboratory assays were performed at the Rennes University Hospital. The liver iron concentration (LIC) was evaluated as described previously.18
Quantitative RT-PCR. Total RNAs were extracted using the SV Total RNA Isolation System (Promega, Madison, WI). Quality-checked RNA (1 fg) was used for reverse transcription (RT) following the manufacturer’s protocol (Advantage RT-for-PCR Kit, Clontech, Palo Alto, CA). We performed polymerase chain reactions (PCRs) in triplicate to evaluate the hepcidin expression in each sample compared with the RNA 18S expression and with a standard set using the qPCR-Core-kit according to the manufacturer’s instructions (Eurogentec, Seraing, Belgium). Primer and probe sequences used for the hepcidin ampliﬁcation were: forward primer 5′-TCCCACAACAGACGGGACAA-3′, reverse 5′-AGCAGCCGCAGCAGAAAAT-3′ and FAM/TAMRA probe 5′-CCATGTTCCAGAGGCGAAGGAGGC-3′. The ampliﬁed 137-bp PCR fragment was checked by sequencing. For RNA 18S ampliﬁcation we used the 18S genomic control kit (Eurogentec). The PCR was run on ABI PRISM 7000 sequence detection system (Applied Bioscience, London, United Kingdom): 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.
From the Institut National de la Sante et de la Recherche Medicale (INSERM), U522, Rennes, France; IFR 140, Universite´ de Rennes 1, Rennes, France; Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA; and Department of Visceral Surgery, Department of Pathology, Laboratory of General Biochemistry and Enzymology, and Liver Disease Department, University Hospital Pontchaillou, Rennes, France.
Submitted December 22, 2004; accepted March 18, 2005. Prepublished online as Blood First Edition Paper, March 29, 2005; DOI 10.1182/ blood-2004-12-4855.
Supported by a PHRC regional grant (R09-01) and by the QLRT-2001-00444 EC contract.
Reprints: Olivier Loreal, Unite´ INSERM U522, Hopital Pontchaillou, 35033 Rennes, France; e-mail: [email protected]
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
BLOOD, 15 JULY 2005 · VOLUME 106, NUMBER 2
BLOOD, 15 JULY 2005 · VOLUME 106, NUMBER 2 HEPCIDIN LEVELS IN HUMANS 747
Data analysis. For each sample, hepcidin mRNA threshold cycle (Ct) value was normalized with 18S RNA Ct value and compared to value of a standard sample (mix of 4 liver samples exhibiting readily detectable level of hepcidin mRNA as previously analyzed by Northern blot). Results were expressed in log 2 of ratio sample versus standard and noted for convenience in arbitrary units (AU).
Hepcidin assay. Urinary hepcidin assay was performed as previously described5 and hepcidin concentration in urine expressed as nanograms hepcidin per milligrams creatinine. Because the lower detection limit of the urinary hepcidin assay is 1 ng/mg creatinine, we attributed a value of 1 ng/mg creatinine to samples with lower value.
Statistical analysis. The statistical analysis was performed on Statview software (SAS institute, Cary, NC) using nonparametric tests. P less than .05 was considered signiﬁcant.
Results and discussion
Hepcidin mRNA expression is correlated with the urinary hepcidin level
Hepcidin mRNA and urinary hepcidin levels appeared to be positively correlated (Figure 1A), with levels of both parameters increasing in parallel. This result demonstrates that the urinary hepcidin assay can be considered a valid reﬂection of hepatic hepcidin production. The correlation, however, was not ideal, possibly related to the inclusion of patients with ﬁbrosis, which could have modiﬁed the relationship due to the heterogeneity of the liver in such cases. This interpretation is supported by improved correlation between hepcidin mRNA and urinary hepcidin (Figure 1B) when patients with more than slight ﬁbrosis were excluded. In the nonﬁbrotic group, the correlation could be partly obscured by (1) additional regulatory mechanisms during translation, secretion, and urinary excretion processes; (2) detection limits of the present urinary hepcidin assay; and (3) the acute effects of anesthesia and surgery that could selectively inﬂuence hepatic hepcidin mRNA concentrations but not urinary hepcidin concentrations.
Hepatic hepcidin mRNA level, urinary hepcidin level, and clinical parameters
As judged by laboratory studies, our patients displayed a broad range of inﬂammation status and iron loading (Table 1). The analysis of the relationship between hepcidin mRNA, urinary hepcidin, and other clinical data led us to identify signiﬁcant correlations, involving iron status, hematologic parameters, and hepatic functional status (Table 1).
Thus, LIC appeared to be signiﬁcantly correlated not only with hepcidin mRNA, as previously reported in untreated hemochromatotic patients,12 but also with urinary hepcidin levels (Figure 1D). In addition, urinary hepcidin was correlated with ferritinemia as previously documented.5 In a group of patients not selected for diseases of iron metabolism12 or inﬂammatory disorders,5 these observations document a relationship between hepatic iron and urinary hepcidin levels. However, in our population the 2 hepcidin parameters did not signiﬁcantly correlate with either serum iron concentration or with transferrin saturation.
Our study also demonstrates a relationship between hemoglobin level and hepcidin mRNA expression (Figure 1E), supporting the hypothesis of an impact of anemia or hypoxia (or both) on hepcidin mRNA expression, as reported in mice.4 However, we did not ﬁnd a signiﬁcant correlation between erythroid parameters and urinary
Figure 1. Correlations between hepcidin mRNA levels, urinary hepcidin concentrations, and clinical parameters. Urinary hepcidin levels expressed in nanogram per milligram urinary creatinine were correlated with hepcidin mRNA levels expressed in arbitrary units (log 2 scale) in the whole population of the 36 patients (.; A) and within the subgroup of 20 patients with no or slight ﬁbrosis (E; B). In our total population (n � 36) LIC was correlated with hepcidin mRNA (C) and urinary hepcidin
(D) levels. Hepcidin mRNA level was correlated with blood hemoglobin value (E) as well as serum albumin level (F). Inverse correlations were found for hepatic ﬁbrosis status and hepcidin mRNA (G) and urinary hepcidin levels (H). Horizontal bars correspond to the mean values.
hepcidin concentration, suggesting again additional regulatory mechanisms.
Surprisingly, we did not ﬁnd a relationship between hepcidin expression and C-reactive protein (CRP) levels, an indicator of inﬂammatory state. The heterogeneity of our population, the small number of patients exhibiting high CRP levels, the possible lack of CRP sensitivity for expressing hepatic inﬂammatory status, and the presence of confounding regulatory factors could partly explain this result.
Finally, we found that parameters reﬂecting hepatic function were correlated with hepcidin levels (Figure 1F-H). Thus, serum albumin was positively correlated with hepcidin mRNA levels, whereas ﬁbrosis status was negatively correlated with hepcidin mRNA and urinary hepcidin levels. Taken together, these results suggest that hepatic function and the effects of disease processes on hepatocytes could modulate iron metabolism. This hypothesis is reinforced within the subgroup of 20 patients with no or slight ﬁbrosis, which exhibits an improvement of the correlations,
748 DE´ TIVAUD et al BLOOD, 15 JULY 2005 · VOLUME 106, NUMBER 2
Table 1. Correlations between clinical ﬁndings and hepcidin mRNA and urinary hepcidin levels
|Bioclinical parameters||Average||Range||Hepcidin mRNA||Hepcidinuria|
|Sex, no. male/no. female||28/8||—||NS||NS|
|LIC, fmol/g dry weight||34.6||1.9-194.6||0.438*||0.404†|
|Serum iron concentration, fM||15.8||3.2-32||NS||NS|
|Transferrin level, g/L||2.5||0.9-4.2||NS||NS|
|Transferrin saturation, %||28.9||5-94.7||NS||NS|
|Ferritin concentration, fg/L||319||9-1203||NS||0.337‡|
|Hemoglobin level, g/L||13.3||8.6-16.9||0.350‡||NS|
|Red blood cell count, x 1012/L||4.3||2.3-5.4||NS||NS|
|CRP level, mg/L||8.3||2.1-47.7||NS||NS|
|Serum albumin level, g/dL||39.4||21-54.4||0.450*||NS|
The clinical parameters are presented as average, maximum, and minimum values. The correlation (rho and P values) found between these parameters and hepcidin
mRNA and urinary hepcidin levels are shown.
NS indicates not signiﬁcant; —, not applicable.
*P < .01.
†P < .02.
‡P < .05. §P < .04.
especially those involving urinary hepcidin with LIC (rho 0.632; P < .01) and ferritin (rho 0.755; P < .001). In addition, a relationship between hepcidin mRNA and ferritinemia appears in this group (rho 0.515; P < .03), as previously described.13 Conversely, all these correlations disappeared in the patient subgroup exhibiting high levels of ﬁbrosis or cirrhosis.
In conclusion, our results demonstrate that urinary hepcidin levels reﬂect hepatic hepcidin mRNA expression. Moreover, despite the heterogeneity of our patients, they show in humans a relationship between hepcidin levels, hepatic iron stores, hemoglo-
bin level, and hepatic function. With further reﬁnement, hepcidin assays could contribute to the diagnosis and improved understanding of the pathophysiology of iron disorders.
We thank the Biological Resource Centre (BRC) of Rennes for the supplying of human biologic samples and Medhi Alizadeh for helpful discussion.
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