Take home messages
- Hepcidin is the central regulator of systemic iron homeostasis, controlling dietary iron uptake and availability of iron for tissues and organs. Hepcidin expression levels are determined by a balance of signals arising from diverse physiological states, ranging from iron and inflammatory status to erythropoietic activity and hypoxia.
- Hepcidin measurement, either as a single index or in concert with other iron-related indices, may aid in diagnosis of iron-related disorders such as iron deficiency (ID), inflammatory anemia, hereditary hemochromatosis and iron-refractory iron deficiency anemia (IRIDA).
- Diagnostic test studies for hepcidin as a single index generally indicate moderate to strong performance in identifying ID, distinguishing iron deficiency anemia from inflammatory anemia, and predicting responsiveness to oral iron interventions.
- Routine implementation of hepcidin assessment in clinical practice and epidemiological surveys will be facilitated by assay standardization, reduced assay cost, application to high-throughput clinical analyzers, and development of robust normal ranges combined with evidence-based guidance in interpretation across clinical/public health settings.
The hormone hepcidin controls dietary uptake and availability of iron. It is regulated by a balance of signals related to iron status, inflammation, erythropoietic activity, and hypoxia, determining iron provision for erythropoiesis and other iron-demanding processes. Progress in understanding hepcidin's involvement in normal physiology and disease conditions,1 coupled with advances in quantification, make it an increasingly attractive candidate biomarker for assessing iron status and guiding iron intervention strategies.2
Current state of the art
Hepcidin regulates circulating iron concentrations by triggering ubiquitin-mediated degradation of the iron exporter ferroportin, but also by directly occluding ferroportin-mediated iron transport.3▪ Ferroportin is highly expressed by iron-recycling erythrophagocytic macrophages and duodenal enterocytes, so hepcidin activity restricts macrophage iron release and dietary iron uptake, causing hypoferraemia. In contrast, hepcidin suppression increases iron availability for tissues. Recent work also indicates a significant contribution of erythroblast-expressed ferroportin to systemic iron homeostasis.4▪
To maintain homeostasis, increases in iron stores and circulating iron are sensed by the liver leading to hepcidin upregulation via the BMP/SMAD signaling pathway. Hepcidin transcription is also directly upregulated during inflammation by JAK/STAT3 signaling. However, it is downregulated during ID, hypoxia and erythropoietic expansion. Expanded erythropoiesis increases expression of the erythropoietin-responsive hormone erythroferrone, which suppresses hepcidin through direct inhibition of BMP6 signaling5▪; in addition, erythropoietic activity also consumes iron, further reducing hepcidin stimulatory signals.6,7▪
Failures in hepcidin regulation cause iron-related pathology. Mutations in hepcidin itself, or more frequently in genes encoding components of the upstream BMP-SMAD signaling pathways (eg, HFE, BMP6, Hemojuvelin (HJV), Transferrin receptor 2 (TfR2)) that link iron sensing to hepcidin, result in inappropriately low hepcidin concentrations relative to iron status. These conditions, broadly termed hereditary hemochromatosis (HH), are characterized by toxic iron loading, predominantly affecting liver, pancreas, and heart. Chronically suppressed hepcidin due to ineffective erythropoiesis also explains secondary iron loading in conditions such as beta-thalassemia. In contrast, mutations in TMPRSS6 (encoding matriptase-2, a negative regulator of hepcidin) cause inappropriately elevated hepcidin, impaired iron absorption and consequent iron-refractory iron deficiency anemia (IRIDA).1,8
Hepcidin: a potential iron status biomarker?
ID refers to depletion of total body iron, primarily from reticuloendothelial and hepatic stores. Iron stores depletion leads to iron-restricted erythropoiesis and ultimately to iron deficiency anemia (IDA), its most prominent manifestation.9 Importantly, other functional consequences may precede anemia, including impaired brain development in young children.9
Absence of stainable reticuloendothelial iron in bone marrow is considered a gold standard means of diagnosing ID; however, being an invasive test, its applicability is limited. Several biochemical markers, together with hematological parameters (eg, MCV, reticulocyte-hemoglobin, hemoglobin), can aid identification of the different stages of iron limitation described above.9 Low ferritin can identify depleted iron stores effectively; however, ferritin is also an acute-phase protein switched on during inflammation, meaning it lacks sensitivity when inflammation is present; inflammatory status (eg, concurrent C-reactive protein (CRP) or alpha-1-acid glycoprotein (AGP)) must, therefore, be considered when interpreting ferritin concentrations.9 Serum iron or transferrin saturation (Tsat) indicate iron availability for tissues, but demonstrate notable diurnal variation and decrease during inflammation. Iron demand is frequently assessed by quantifying soluble transferrin receptor (sTfR) concentration: during iron-restricted erythropoiesis, iron-deficient erythroblasts increase TfR1 expression, correlating with increased serum concentrations of soluble transferrin receptor (sTfR).
Could hepcidin complement (or even replace) established biomarkers in indicating iron status? Hepcidin can predict response to oral iron,10 and so might provide a single-index guide for iron supplementation. Interestingly, the traditionally-used biomarkers could be regarded as proxies for key physiological inputs that control hepcidin expression: stored iron (proxy = ferritin) and circulating iron (proxy = Tsat) both induce hepcidin, involving BMP/SMAD signaling; inflammation (proxies = CRP/AGP) upregulates hepcidin via JAK/STAT3 signaling; iron demand (proxy = sTfR) associates with hepcidin suppression via erythroferrone-dependent/-independent BMP pathway restriction. Consistently, serum hepcidin typically correlates positively with ferritin/CRP/AGP, and negatively with sTfR. Moreover, evidence that opposing signals concurrently determine hepcidin expression is accumulating. For example, hepcidin upregulation by acute inflammation can be offset by increased erythroid drive or hypoxia11–14; enhanced erythropoiesis in beta-thalassemia overrides induction of hepcidin by iron loading; and in inflammatory anemias, chronic inflammation can increase hepcidin, despite the anemia.1,9 Overall, fine tuning of competing positive and negative signals controls hepcidin synthesis and consequent iron handling. However, for hepcidin to become widely adopted as an indicator of iron status/requirement, this biological appeal must be complemented by robust analytical performance and increased validation of its interpretation across clinical settings.
Hepcidin as a biomarker: analytical considerations, performance, interpretation
Hepcidin resists freeze-thaw, but adheres to laboratory plastics at room temperature potentially causing concentration underestimation; sample matrix (eg, plasma, serum) may also influence results. Hepcidin, like Tsat, exhibits diurnal variation, which should be accounted for. Several methods for hepcidin quantification are validated: mass spectrometry methods can distinguish hepcidin isoforms, while immunoassays typically offer greater flexibility. However, the various well-performing assays are not currently calibrated equivalently, so although data from different assays correlate well, absolute values returned differ.15 When comparing hepcidin data from different studies, knowing which assays were used is essential. Nevertheless, progress towards assay harmonization and ultimately standardization is advancing.15,16
Several studies have investigated hepcidin's performance as a single index for diagnosing ID, for distinguishing IDA from inflammatory anemia, and for predicting oral iron utilization/response (eg,17–22 studies reporting ROCAUC with N > 200; see also2). While the clinical/epidemiological settings and test definitions used vary considerably, these studies generally indicated moderate to strong test performance, in most cases at least comparable with established tests. Interestingly, independent reports from diverse settings evaluating hepcidin cutoffs for diagnosing ID found very similar optimal cutoffs19,22 that were also close to a cutoff identifying effective oral iron incorporation into erythrocytes.19
Hepcidin's inclusion in investigations related to iron is broadening, yet its establishment in routine clinical practice or epidemiological surveys requires several steps. Establishment of standardized reference ranges, implementation on high-throughput analyzers, and reducing assay cost will greatly facilitate its use. A greater breadth of prospective diagnostic test studies ideally with larger sample sizes, testing against gold standard definitions across diverse populations and disease conditions would be desirable. Determining whether a uniform cutoff for effective response to iron interventions exists, and whether hepcidin can indicate efficacy of oral versus intravenous iron interventions across different clinical settings will prove useful.
Despite diagnostic potential as a single index, clinical interpretation of hepcidin should consider the broader context, including its relationship with concurrent iron and hematological indices. This is especially so for investigations of iron-related disorders caused by inappropriate hepcidin regulation (eg, HH and IRIDA), as discussed above.1 Specifically, in the absence of inflammation, low hepcidin/ferritin ratios may contribute to diagnostic work-ups of genetically complex iron-loading disorders including HH,2,23 while low Tsat/hepcidin ratios may be indicative of IRIDA.8 Furthermore, hepcidin may bring diagnostic or disease management benefits in additional contexts besides these (see Fig. 1). In the longer term, hepcidin quantification may guide therapeutic hepcidin agonist and antagonist use in conditions ranging from hereditary hemochromatosis and beta-thalassemia, to inflammatory anemias. Prospective validation of the benefit of adding hepcidin analysis in each of these settings should be prioritized.
AEA and HD receive core funding through the MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford (MC_UU_12010/3). HD receives funding from the NIHR Biomedical Research Centre, Oxford. We also wish to acknowledge the authors of many other relevant and important studies, which we have not been able to cite here owing to space limitations.
1. Muckenthaler MU, Rivella S, Hentze MW, et al. A red carpet for iron metabolism. Cell
2. Girelli D, Nemeth E, Swinkels DW. Hepcidin in the diagnosis of iron disorders. Blood
3.▪. Aschemeyer S, Qiao B, Stefanova D, et al. Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood
2018; 131:899.Functional analysis of disease-causing and structure-guided engineered mutations in ferroportin indicating that besides triggering ubiquitination, hepcidin also impairs ferroportin-mediated iron transport directly by occluding its central cavity.
4.▪. Zhang DL, Ghosh MC, Ollivierre H, et al. Ferroportin deficiency in erythroid cells causes serum iron deficiency and promotes hemolysis due to oxidative stress. Blood
2018; 132:2078.Experiments using erythroblast-specific ferroportin-knockout mice reveal that erythroblasts donate iron to serum via ferroportin, contributing to maintenance of systemic iron homeostasis while protecting erythrocytes from iron-induced oxidative stress.
5.▪. Arezes J, Foy N, McHugh K, et al. Erythroferrone inhibits the induction of hepcidin by BMP6. Blood
2018; 132:1473.Demonstration that hepcidin suppression by erythroferrone involves abrogation of BMP signalling through binding and inhibition of BMP6 and closely related BMP family members, BMP5 and BMP7.
6. Artuso I, Pettinato M, Nai A, et al. Transient decrease of serum iron after acute erythropoietin treatment contributes to hepcidin inhibition by ERFE in mice. Haematologica
7. Mirciov CSG, Wilkins SJ, Hung GCC, et al. Circulating iron levels influence the regulation of hepcidin following stimulated erythropoiesis. Haematologica
8. Donker AE, Schaap CC, Novotny VM, et al. Iron refractory iron deficiency anemia: a heterogeneous disease that is not always iron refractory. Am J Hematol
9.▪. Lynch S, Pfeiffer CM, Georgieff MK, et al. Biomarkers of nutrition for development (BOND)-iron review. J Nutr
2018; 148 (suppl_1):1001S.Highly comprehensive and up-to-date review of iron biology with emphases on clinical and public health implications of altered iron status, on the strengths and weaknesses of currently used iron biomarkers, and on the potential of newer biomarkers including hepcidin.
10. Prentice AM, Doherty CP, Abrams SA, et al. Hepcidin is the major predictor of erythrocyte iron incorporation in anemic African children. Blood
11. Kiers D, van Eijk LT, van der Hoeven JG, et al. Hypoxia attenuates inflammation-induced hepcidin production during experimental human endotoxaemia. Haematologica
12. Latour C, Wlodarczyk MF, Jung G, et al. Erythroferrone contributes to hepcidin repression in a mouse model of malarial anemia. Haematologica
13. Moretti D, Mettler S, Zeder C, et al. An intensified training schedule in recreational male runners is associated with increases in erythropoiesis and inflammation and a net reduction in plasma hepcidin. Am J Clin Nutr
14. Stoffel NU, Lazrak M, Bellitir S, et al. The opposing effects of acute inflammation and iron deficiency anemia on serum hepcidin and iron absorption in young women. Haematologica
15.▪. van der Vorm LN, Hendriks JC, Laarakkers CM, et al. Toward worldwide hepcidin assay harmonization: identification of a commutable secondary reference material. Clin Chem
2016; 62:993.Multicentre study evaluating and comparing the performance of widely-used hepcidin assays, combined with the development of reference calibration materials that should pave the way for improving equivalence of hepcidin assays and ultimately standardising hepcidin measurement.
16. Diepeveen LE, Laarakkers CMM, Martos G, et al. Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material. Clin Chem Lab Med
17. Bah A, Pasricha SR, Jallow MW, et al. Serum hepcidin concentrations decline during pregnancy and may identify iron deficiency: analysis of a longitudinal pregnancy cohort in the gambia. J Nutr
18. Kanuri G, Chichula D, Sawhney R, et al. Optimizing diagnostic biomarkers of iron deficiency anaemia in community dwelling indian women and preschool children. Haematologica
19. Pasricha SR, Atkinson SH, Armitage AE, et al. Expression of the iron hormone hepcidin distinguishes different types of anemia in African children. Sci Transl Med
20. Pasricha SR, McQuilten Z, Westerman M, et al. Serum hepcidin as a diagnostic test of iron deficiency in premenopausal female blood donors. Haematologica
21. Shin DH, Kim HS, Park MJ, et al. Utility of access soluble transferrin receptor (sTfR) and sTfR/log ferritin index in diagnosing iron deficiency anemia. Ann Clin Lab Sci
22. Wray K, Allen A, Evans E, et al. Hepcidin detects iron deficiency in Sri Lankan adolescents with a high burden of hemoglobinopathy: a diagnostic test accuracy study. Am J Hematol
23. Swinkels DW, Fleming RE. Novel observations in hereditary hemochromatosis: potential implications for clinical strategies. Haematologica