Received: 12 December 2018 | First decision: 8 January 2019 | Accepted: 16 January 2019

DOI: 10.1111/apt.15173

Review article: iron disturbances in chronic liver diseases

other than haemochromatosis – pathogenic, prognostic, and
therapeutic implications

Albert J. Czaja

Division of Gastroenterology and

Hepatology, Mayo Clinic College of Summary
Medicine and Science, Rochester, Background: Disturbances in iron regulation have been described in diverse chronic
Minnesota
liver diseases other than hereditary haemochromatosis, and iron toxicity may wor-
Correspondence sen liver injury and outcome.
Prof. Albert J. Czaja, Division of
Gastroenterology and Hepatology, Mayo Aims: To describe manifestations and consequences of iron dysregulation in chronic
Clinic College of Medicine and Science, liver diseases apart from hereditary haemochromatosis and to encourage investiga-
Rochester, MN.
Email: czaja.albert@mayo.edu tions that clarify pathogenic mechanisms, define risk thresholds for iron toxicity, and
direct management
Funding information
None. Methods: English abstracts were identified in PubMed by multiple search terms.
Full length articles were selected for review, and secondary and tertiary bibliogra-
phies were developed.
Results: Hyperferritinemia is present in 4%‐65% of patients with non‐alcoholic fatty
liver disease, autoimmune hepatitis, chronic viral hepatitis, or alcoholic liver disease,
and hepatic iron content is increased in 11%‐52%. Heterozygosity for the C282Y
mutation is present in 17%‐48%, but this has not uniformly distinguished patients
with adverse outcomes. An inappropriately low serum hepcidin level has charac-
terised most chronic liver diseases with the exception of non‐alcoholic fatty liver
disease, and the finding has been associated mainly with suppression of transcrip-
tional activity of the hepcidin gene. Iron overload has been associated with oxida-
tive stress, advanced fibrosis and decreased survival, and promising therapies
beyond phlebotomy and oral iron chelation have included hepcidin agonists.
Conclusions: Iron dysregulation is common in chronic liver diseases other than
hereditary haemochromatosis, and has been associated with liver toxicity and poor
prognosis. Further evaluation of iron overload as a co‐morbid factor should identify
the key pathogenic disturbances, establish the risk threshold for iron toxicity, and
promote molecular interventions.

The Handling Editor for this article was Dr Colin Howden, and this uncommissioned review
was accepted for publication after full peer‐review.

Aliment Pharmacol Ther. 2019;1–21. wileyonlinelibrary.com/journal/apt © 2019 John Wiley & Sons Ltd | 1
2 |
1 | INTRODUCTION
Disturbances in iron regulation have been described in diverse
chronic liver diseases other than hereditary haemochromatosis. 1–5
Serum ferritin levels are increased in cohorts with non‐alcoholic
fatty liver disease (NAFLD), 6–12
chronic alcoholic liver disease
(ALD),2,6 chronic hepatitis B,13 chronic hepatitis C,14–19 alpha‐1
anti‐trypsin deficiency,20–22 and autoimmune hepatitis.23,24 Stainable
iron has been present in 32% of cirrhotic livers of various
causes,25–27 and serum hepcidin levels that regulate the duodenal
absorption and tissue storage of iron have been low in autoimmune
hepatitis,24,28 primary biliary cholangitis (PBC),28 primary sclerosing
cholangitis (PSC),28 alcohol‐related cirrhosis,29 chronic hepatitis
B‐related cirrhosis, 30
and alpha‐1 anti‐trypsin deficiency. 22
In con-
trast, circulating hepcidin concentrations have been increased in
NAFLD.31,32
Hyerferritinemia has been associated with normal or near normal
serum transferrin saturations (≤45%) in most patients with chronic
liver disease and iron dysregulation, and further testing for the point
mutations (C282Y and H63D) of the haemochromatosis gene (HFE
[high Fe gene]) has not been warranted. 33,34
In those patients tested
for the HFE mutations, the frequency of genetic haemochromatosis
25
has been low; and the association of the heterozygous mutations
with the hyperferritinemia and liver disease has been inconsistent
and controversial. 4,11,18,19,35–42
The disturbances in iron homeostasis in chronic liver disease may
be a collateral manifestation of active inflammation and parenchymal
damage3,39,43,44 or a pathological mechanism by which iron toxicity
worsens the liver injury and outcome. 45,46
The goals of this review
are to present the findings in diverse chronic liver diseases that sug-
gest disturbed iron regulation, describe possible mechanisms that
could account for the manifestations, indicate how the dysregulation
of iron homeostasis could affect disease severity, and encourage
investigations that clarify pathogenic mechanisms, identify risk
thresholds for iron toxicity, and evaluate evolving molecular
interventions.
2 | MATERIALS AND METHODS
English abstracts were identified in PubMed using the primary search
words, “iron and chronic liver disease,” “iron and non‐alcoholic fatty
liver disease,” “iron and autoimmune hepatitis,” “iron and chronic
hepatitis C,” “iron and chronic hepatitis B,” and “iron and alcoholic
liver disease.” Abstracts judged pertinent to the review were identi-
fied; key aspects were recorded; and full‐length articles were
selected from relevant abstracts. A secondary bibliography was
developed from the references cited in the selected full‐length arti-
cles, and additional PubMed searches were performed to expand the
concepts developed in these articles. The discovery process was
repeated, and a tertiary bibliography was developed after reviewing
selected articles from the secondary bibliography. Over 1500
abstracts and 90 full length articles were reviewed.
CZAJA
3 | RESULTS
Hyperferritinemia has been the principal manifestation of a disturbed
iron homeostasis in chronic liver disease,1–4 but other findings have
included inappropriate serum hepcidin levels,22,24,28–32,47 increased
serum iron concentrations,11,24 stainable iron in hepatic
tissue,1,11,13,24–27,32,42,48,49 normal or moderately increased transfer-
rin saturations,11,24 and mutations of the HFE gene.14,18,36 The simi-
larity of the findings across a broad spectrum of different chronic
liver diseases and resolution of the abnormalities during treatment
suggest that the disturbances are consequences of liver injury.39,43,50
Alternatively, the association of the iron disturbances with disease
severity51–53 and the recognised hepatotoxicity of iron over-
load45,46,54,55 compel consideration of iron as a pathogenic factor.
3.1 | Normal iron metabolism
Iron is derived from dietary sources that contain heme (≤10%)
and nonheme (>90%) products.56,57 Intestinal absorption is the pri-
mary mechanism for acquiring iron, and 1‐2 mg are absorbed daily
by enterocytes located mainly in the duodenum.56,57 Nonheme
iron is ingested in the oxidised ferric (Fe3+) state which renders
the iron insoluble and biologically inert. Transport across the duo-
denal epithelium requires reduction of ferric (Fe3+) iron to ferrous
(Fe2+) iron by a ferric reductase in the brush border of the ente-
rocytes.58 Transportation of the ferrous iron into the enterocyte is
accomplished by the transporter protein, divalent metal transporter
1 (DMT1), and the bound ferrous iron is delivered to ferroportin
on the basolateral membrane of the enterocyte.59,60 Ferroportin is
a transmembrane protein located on enterocytes, macrophages,
hepatocytes and adipocytes, and it is the main transporter of
intracellular iron to the bloodstream.61–63 Hepcidin inhibits
ferroportin activity by promoting its lysosomal degradation (ubiqui-
tination), and it thereby modulates the intestinal absorption of
iron.64–68
The extracellular ferrous iron is oxidised to ferric iron by an oxi-
dising ferroxidase (hephaestin) that is homologous to ceruloplasmin,
and the inactive ferric iron is bound to transferrin.69 Transferrin
accepts iron from reticuloendothelial cells ingesting senescent ery-
throcytes, parenchymal tissue, and intestinal mucosal cells, and it
transports the ferric iron to the bone marrow for erythropoiesis and
to other sites for storage or synthesis of iron‐containing pro-
teins.45,70,71 Transferrin can also modulate hepcidin activity as part
of a complex system that senses plasma iron levels.71
Ferritin is the principal storage protein for iron, and the liver is
the principal storage site of ferritin.45 The 24 subunits that comprise
ferritin form a sphere containing 4500 atoms of iron that are depos-
ited as hydroxy‐phosphates.45,57,72 The iron is stored and detoxified
in the ferritin until it is exported back to the circulation by ferro-
portin in response to signals that reflect iron deficiency.73 Ferritin is
an acute phase protein that can be increased during inflammation,43
whereas transferrin is down‐regulated during inflammation74 and
reduced in advanced liver disease.75,76
CZAJA
Ferritin that has been iron‐overloaded, especially after haemoly-
sis or blood transfusion, can undergo incomplete lysosomal degrada-
tion, and the denatured ferritin can be deposited within cells as
57,77,78
haemosiderin. Haemosiderin provides storage for excess
iron,79 but its iron stores cannot be mobilised under physiological
conditions.80 Transferrin that has been iron‐overloaded can release
toxic nontransferrin‐bound iron (NTB1) into the plasma, and this
unbound ferrous iron is passively removed by the liver where it may
5,81
accumulate and induce toxicity. There are no regulated mecha-
nisms for iron excretion, and the desquamation of intestinal epithelia,
minor bleeding, and losses in sweat, urine, and faeces account for an
iron loss of 1 mg daily.57
The key regulatory molecules that maintain iron homeostasis are
transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2) that
mediate the uptake of transferrin‐bound iron by the liver,
haemochromatosis protein (HFE) that regulates TfR1 activity, hep-
cidin that modulates ferroportin activity, and ferroportin that moves
iron from storage sites within enterocytes, hepatocytes, and macro-
phages to the circulation.56,57,73 Genetic and epigenetic factors con-
trol the expression of these regulatory proteins, and deficiencies in
gene expression or the diverse molecular regulators that maintain
critical signalling pathways may disrupt iron homeostasis and account
for the iron disturbances found in some patients with chronic liver
disease (Figure 1).
3.2 | Iron disturbances in NAFLD
Serum ferritin levels have been increased in 58% of patients with
82 37,83
NAFLD and most patients with insulin resistance, and serum
transferrin saturations have been increased in 35%‐58% (Table 1).37,82
Serum ferritin levels greater than 1.5‐fold the upper limit of the nor-
mal range (ULN) have been associated with male gender, increased
serum aminotransferase concentrations, stainable iron in liver tissue,
increased NAFLD activity score, advanced hepatic fibrosis, and histo-
11
logical features of non‐alcoholic steatohepatitis (NASH). Obesity in
association with a serum ferritin level greater than 240 ng/mL has
been associated with NASH in 62% of individuals with normal serum
alanine aminotransferase (ALT) concentrations.9 Furthermore, a body
mass index (BMI) greater than 28.2 and serum ferritin level greater
than 240 ng/mL have identified patients with hepatic fibrosis (sensi-
tivity, 82%; specificity, 79%).9
Homozygosity for the C282Y mutation has been present in 3%
36
of patients with NASH, and heterozygosity for this mutation has
been associated with hyperferritinemia in 17% of white North Amer-
ican patients with NASH36 and 45% of Italian patients.84 Further-
more, patients with NASH and the C282Y mutation have had higher
serum ALT levels and more hepatic fibrosis than patients without
36
this mutation. Parenchymal iron deposition and moderate‐severe
hepatic fibrosis have also been associated with heterozygosity for
C282Y, homozygosity for H63D, and beta‐globin mutations charac-
teristic of the β‐thalassemia trait in 63% of Italian patients with
85
biopsy‐proven NAFLD. In the Italian population, the parenchymal
deposition of iron has been independent of age, gender, BMI, alcohol
| 3
intake, serum ferritin level, and transferrin saturation, and the stron-
gest association has been with the β‐thalassemia trait.85 The overall
findings suggest that hepatic iron accumulation in NAFLD is often
associated with genetic factors and that this association may
contribute to hepatic fibrosis.
Importantly, many patients with NAFLD and hyperferritinemia
lack an HFE mutation,39,86 and the severity of liver fibrosis42 and the
frequency of serum ferritin levels >1.5‐fold ULN11 have been similar
between patients with and without an HFE mutation. Furthermore,
the clinical and pathological effects of iron dysregulation in NAFLD
have been contested,87–89 especially when patients are matched for
age, obesity, diabetes, and aminotransferase levels.88,89 These obser-
vations have suggested that the HFE mutations are not the primary
basis for iron dysregulation in most patients with NAFLD and that
the disturbances in iron homeostasis may not be directly associated
with advanced fibrosis, progression to cirrhosis, or liver‐related mor-
tality.87
Studies in a genetically obese mouse model suggest that iron
overload associated with dietary supplementation has diverse patho-
genic effects and a pathological consequence.90 Iron overloaded
genetically obese mice develop manifestations of hepatic oxidative
stress, enhanced gene expression of inflammatory cytokines (inter-
leukin‐6 [IL‐6] and tumour necrosis factor‐alpha [TNF‐α]), and
increased production of markers associated with innate and adaptive
immune cell responses.90 Furthermore, the dietary iron overload
results in hepatocyte injury (ballooning) that has been associated
with human NASH.90–93 Similar findings have also been described in
a rat model of NASH supplemented with high dietary fat and iron.94
3.2.1 | Dysmetabolic iron overload syndrome
The dysmetabolic iron overload syndrome (DIOS) describes patients
with NAFLD who have high serum ferritin levels, normal transferrin
saturations, and hepatic iron deposition.37,48,84,95 The syndrome is
typically accompanied by one or more metabolic abnormalities,
including increased BMI, hypertension, dyslipidemia, insulin resis-
tance, and NASH, and it affects more than 30% of patients with
NAFLD.48,96,97 The amount and distribution of iron within the liver
(parenchymal vs nonparenchymal deposition) have been associated
with the frequency of hepatic fibrosis, but results have differed
between studies, possibly because of ethnic and environmental
differences.48,49,95
Semi‐quantitative assessments of hepatic iron deposits have
scored iron accumulations in hepatocytes and in sinusoidal and portal
locations in Italian patients with DIOS.48 Hepatic fibrosis has corre-
lated with the total amount of hepatic iron deposition (P = 0.01), espe-
cially in the portal hepatocytes (P < 0.0001).48 In contrast, stainable
iron has been present in 34% of North American patients with
NAFLD, and advanced hepatic fibrosis, portal inflammation, hepato-
cyte ballooning, and steatohepatitis have been associated mainly with
stainable iron in reticuloendothelial cells.49 Despite these differences,
each study has indicated that the presence and pattern of hepatic iron
deposition are possible factors in the progression of NAFLD.48,49
4 | CZAJA

Genetic mutations Concurrent variables Iron overload

C282Y HJV SLC40A1 ETOH
H63D HAMP HCV Fe3+ Fe3+
Hepatocyte
TfR2
Fe3+
Ferritin
+ HFE
Fe3+
BMP6 Fe3+
Ferroportin TfR2
Oxidative stress
Hypoxia
SMAD
mRNA mRNA
Hepcidin Hepcidin
HAMP
TMPRSS6
Nucleus sHemojuvelin
HGF
mRNA
STAT3 CREB3L3
Inflammation Metabolic stress
IL-1 Hepcidin Infection
IL-6 Inflammation
Erythropoiesis Nutrient loss
Anemia

F I G U R E 1 Factors contributing to iron dysregulation in chronic liver diseases outside the diagnosis of hereditary haemochromatosis.
Heterozygous genetic mutations of the HFE (High Fe) gene (C282Y and H63D) have been associated with iron overload in non‐alcoholic fatty
liver disease (NAFLD) and chronic hepatitis C, and they may impair transcriptional activity of the HAMP gene and reduce hepcidin production
(blunted arrow). Other genetic mutations, including (haemojuvelin [HJV], hepcidin [HAMP], transferrin receptor 2 [TfR2], and ferroportin
[SLC40A1]), are unstudied in these chronic liver diseases, but the genetic variants could increase iron overload by decreasing hepcidin
production or reducing ferroportin activity (blunted arrows). Concurrent host‐specific factors, especially alcohol consumption (ETOH) and
infection with the hepatitis C virus (HCV), can also suppress transcriptional activity of HAMP (blunted arrow) and increase iron overload. Iron
overload can activate bone morphogenetic proteins (BMPs), especially BMP6, which increase hepcidin production by stimulating HAMP activity
through the SMAD (sons of mothers against decapentaplegic) pathway (sharp arrow). The SMAD pathway is activated by the complex of HFE
protein, TfR2, and ferric iron (Fe3+) on the hepatocyte membrane. This stimulatory effect can be counterbalanced by disease‐associated
factors, including oxidative stress and tissue hypoxia, which can suppress hepcidin production by increasing molecular inhibitors of HAMP,
including transmembrane protease serine 6 (TMPRSS6), soluble haemojuvelin (sHaemojuvelin), and hepatocyte growth factor (HGF) (blunted
arrow). Metabolic stress on the endoplasmic reticulum can activate the transcription factor, CREB3L3 (cyclic adenosine monophosphate
response element binding protein 3‐like 3), which up‐regulates hepcidin production, and the pro‐inflammatory cytokines, interleukin (IL)‐1 and
IL‐6, can also contribute by activating the signal transducer and activator of transcription 3 (STAT3) pathway (sharp arrows). Disease‐related
up‐regulation of erythropoiesis because of an acquired anemia can contribute by dampening hepcidin production and favouring iron overload
(blunted arrow). The net effect on iron stores probably reflects the counterbalance between genetic, disease‐associated, and patient‐related
factors

Hepcidin‐induced ubiquitination of ferroportin would be

3.2.2 | Hepcidin levels in NAFLD
The liver is the principal source of hepcidin,98,99 but adipose tissue
and macrophages also contribute.100,101 Serum hepcidin levels have
been increased in patients with NAFLD with and without DIOS, and
the amount of messenger ribonucleic acid (mRNA) for hepcidin in
liver tissue and the level of hepcidin in serum have correlated with
32
hepatic iron content but not the degree of steatohepatitis. Similar
findings have been reported in morbidly obese women with and
without histological features of NAFLD.31 In patients with severe
obesity, the level of mRNA for hepcidin in adipose tissue has corre-
lated with indices of inflammation, including IL‐6 and C‐reactive
protein (CRP), but it has not correlated with the presence of dia-
betes or NASH.100 These findings suggest that the excessive produc-
tion of hepcidin is associated with hepatic iron retention but not the
liver disease.
expected to protect against iron overload in NAFLD, but semi‐
quantitative histological studies suggest that parenchymal and
nonparenchymal iron stores can be overloaded in NAFLD.48,49
Hepcidin has been unable to limit iron absorption in patients
with DIOS following an oral challenge with iron, and this resis-
tance to hepcidin may contribute to the iron overload.47 A
similar study assessing the hepcidin response to oral iron in
patients with DIOS has suggested that the increased hepcidin
level is normal but delayed.102 In this instance, a decreased sen-
sitivity to mild changes in iron load may have resulted in a
delayed or deficient hepcidin response.103 Future investigations
must fully characterise the apparent disconnect between the
hepcidin level and iron overload in NAFLD and continue to clar-
ify the role of hepatic iron retention or excess in progressive
hepatic fibrosis.
CZAJA | 5

T A B L E 1 Iron dysregulation in chronic liver diseases other than haemochromatosis

Chronic liver disease Manifestations of iron dysregulation Clinical associations
NAFLD Hyperferritinemia in 58%82 Male11
Increased transferrin saturation in 35%‐58%37,82 Increased serum ALT level11,36
Stainable hepatic iron in 34%‐100%49,84 Increased NAFLD activity score11
Homozygosity for C282Y in 3%36 Advanced hepatic fibrosis9,11,36
Heterozygosity for C282Y in 17%‐45%36,84 Histological features of NASH11,84
Inappropriate high serum hepcidin levels31,32 Insulin resistance37,83
Increased HAMP mRNA in liver tissue31,32 Possible hepcidin resistance47
Autoimmune hepatitis Hyperferritinemia in 65%24 Serum ferritin >2.09 ULN and serum
Increased serum iron level in 58%24 IgG <1.89 ULN associated with complete
High serum transferrin saturation in 48%24 treatment response24
Stainable hepatic iron in 38%24 Hyperferritinemia and hepatic iron load
Inappropriate low serum hepcidin level24,28 resolve during treatment24
Excess HAMP mRNA in liver tissue28 Hepcidin translation defect28
Chronic hepatitis C Hyperferritinemia in 22%‐27%19,107 Increased serum AST, ALT, GGT109
High serum transferrin saturation in 20%19 HCC in transgenic mice115
Hepatic iron deposition in 17%‐42%19,107,108 Severe hepatic steatosis19
HFE mutation in 34%‐48%14,108 Hepcidin deficiency reversible50
Inappropriate low serum hepcidin level124–126 Hepcidin transcription defect50
Decreased hepcidin mRNA in liver50,124,128
Chronic hepatitis B Abnormal iron markers in 41%13 HDV‐induced liver iron overload13
Hepatic iron deposition in 35%13 HDV‐related greater liver fibrosis13
Inappropriately low serum hepcidin level30 Early mortality (3 mo)140
Chronic alcoholic liver disease Hyperferritinemia in 63%40 Advanced liver fibrosis144
High serum transferrin saturation in 29%40 Shortened survival145
Hepatic iron deposition in 22%‐52%40,143 HCC associated with C282Y147
C282Y mutations in 8%‐16%40,142,147 Increased risk of HCC and death168
Inappropriately low serum hepcidin level154 Hepcidin transcription defect154
Decreased hepcidin mRNA in liver154

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma‐glutamyl transferase; HAMP, hepcidin antimicrobial peptide; HCC, hepato-
cellular carcinoma; HDV, hepatitis delta virus; HFE, high Fe gene; IgG, immunoglobulin G; mRNA, messenger ribonucleic acid; NAFLD, non‐alcoholic fatty
liver disease; NASH, non‐alcoholic steatohepatitis; ULN, upper limit of the normal range.

3.3 | Iron disturbances in autoimmune hepatitis
Serum ferritin levels have been increased in 65% of untreated
24
patients with autoimmune hepatitis, and serum iron concentrations
and transferrin saturations have been abnormally increased in 58%
24
and 48%, respectively (Table 1). Furthermore, the serum ferritin
and immunoglobulin G (IgG) levels at presentation have predicted
the completeness of biochemical response to standard immunosup-
pressive therapy.24 Semi‐quantitative iron scores104 have demon-
strated mild hepatic iron deposition (range, 1‐30 on scale of 0‐60) in
approximately 38% of patients, but hepatic iron content has not cor-
related with the subsequent treatment response.24
A serum ferritin level >2.09‐fold ULN and serum IgG level
<1.89‐fold ULN at baseline have characterised those patients who
achieved persistent normalisation of serum aminotransferase concen-
trations and IgG level after conventional glucocorticoid treatment
24
(complete biochemical response). Serum ferritin levels have paral-
leled serum aminotransferase levels in those treated patients achiev-
ing laboratory resolution, and the hyperferritinemia and hepatic iron
deposition have resolved during treatment.24
The iron disturbances in untreated autoimmune hepatitis and
their subsequent resolution with treatment‐induced normalisation of
the liver enzyme and IgG abnormalities could reflect increased iron
release from damaged hepatocytes and their remediation after suc-
cessful treatment.43 Ferritin is an acute phase reactant, and its serum
level does correlate with serum concentrations of CPR and the pro‐
inflammatory cytokine, IL‐6.24,100 Alternatively, the hyperferritinemia
and increased transferrin saturation in some patients with autoim-
mune hepatitis could reflect inappropriate suppression of hepcidin
and increased intestinal absorption and tissue mobilisation of iron by
unfettered ferroportin activity.24
3.3.1 | Hepcidin levels in autoimmune hepatitis
Serum hepcidin levels are greatly reduced in untreated patients with
autoimmune hepatitis (median levels, <5% of normal),24 and unlike
healthy individuals in whom the serum hepcidin levels parallel the
serum ferritin levels,105 the patients with autoimmune hepatitis and
hyperferritinemia have inappropriately low serum hepcidin concen-
trations (Table 1).24 The laboratory indices of liver inflammation
(ALT, aspartate aminotransferase [AST], alkaline phosphatase, and
CRP) do not correlate with the circulating hepcidin level, and the
homeostatic relationship between the serum hepcidin concentration
and the standard iron parameters is not restored until induction of
6 |
biochemical remission.24 Furthermore, the hepcidin mRNA level in
liver tissue has correlated positively with the serum ferritin level but
not with the serum hepcidin level, and the serum hepcidin concen-
tration has remained low in some patients during a 2‐year period of
28
treatment.
The unexplained inappropriately low serum hepcidin level in
autoimmune hepatitis could reflect the severity of liver injury and
106
impairment of the hepatic production of hepcidin. Persistence of
a low hepcidin concentration despite protracted therapy in some
patients with autoimmune hepatitis suggests that the loss of hep-
cidin production by the liver is irreversible or that other factors sup-
28,106
pressing hepcidin production are persistent. Furthermore, the
observation that the hepatic content of mRNA for hepcidin does not
correlate with the serum hepcidin level suggests that translation of
the mRNA for hepcidin is impaired or that the delivery of hepcidin
to the circulation is compromised by post‐transcriptional processes.28
Future investigations of the iron disturbances in autoimmune hepati-
tis should explore genetic mutations, epigenetic factors, and other
mechanisms that could affect transcriptional activity of the hepcidin
gene (HAMP, hepcidin antimicrobial peptide), the translation of mRNA
into hepcidin, and the delivery of hepcidin to the circulation.
3.4 | Iron disturbances in chronic hepatitis C
Hyperferritinemia has been described in 4%‐27% of patients with
chronic hepatitis C (Table 1).16,19,107 Transferrin saturation has been
abnormally increased in 20%,19 and hepatic iron deposition has been
present in 11%‐42%.16,19,107,108 Of 14 462 participants in a national
health survey in the United States, individuals with chronic hepatitis
C have had higher mean serum levels of ferritin and iron than partic-
ipants without liver disease, and the serum levels of ferritin have
correlated with serum AST, ALT and γ‐glutamyl transpeptidase (GGT)
109
levels (P < 0.0001). Furthermore, 52%‐69% of patients with
chronic hepatitis C have had macrovesicular steatosis (mainly grade
1 steatosis) (Table 1).19,110 The steatosis may have constituted
a cytopathic effect associated with the hepatitis C virus (HCV),
especially genotype 3,111–113 or it may have been part of an HCV‐
independent, dysmetabolic syndrome associated with obesity.114
Hepatic iron deposition in patients with chronic hepatitis C has
been mainly in hepatocytes, and hepatic iron accumulation in the
sinusoidal and portal compartments has been variously associated
with increased serum ferritin levels, histological severity, hepatic
14–
fibrosis, cirrhosis, HFE mutations, and alcohol consumption.
17,19,107,108
The distribution of hepatic iron, especially within the
sinusoids, has also been associated with metabolic parameters,
including BMI, insulin resistance, serum low density lipoprotein (LDL)
19
cholesterol levels, and hepatic steatosis.
The association of hepatocellular carcinoma (HCC) with iron
overload in chronic hepatitis C remains uncertain. In male transgenic
mice expressing the HCV polyprotein, hepatic tumours, including
HCC, developed in 45% of animals fed a diet enriched in iron,
whereas none of the transgenic mice fed a normal diet manifested
these changes.115 In contrast, the occurrence of HCC in 45% of 139
CZAJA
patients with HCV and cirrhosis was not associated with iron
content or the C282Y mutation.116
At least one HFE mutation has been present in 34%‐48% of
patients with chronic hepatitis C,14,108 and individuals with the
C282Y mutation have had a higher mean hepatic iron concentration
and more hepatic fibrosis than patients without a HFE mutation
(Table 1).14 Heterozygosity for the C282Y or H63D mutation has
been an independent risk factor for liver fibrosis and cirrhosis,117–119
and the frequency of HFE mutations has been higher in patients with
hepatic steatosis than in patients without this finding (P = 0.03).19
Furthermore, serum ferritin levels and the frequency of HFE muta-
tions have increased with the severity of hepatic steatosis.19 In Italy,
heterozygosity for the beta‐globin mutations associated with β‐tha-
lassemia trait have been associated with hepatic iron accumulation
and fibrosis.120
Not all studies have linked HFE mutations with increased hepatic
iron accumulation in chronic hepatitis C,108 and the bases for the
associations between iron disturbances, steatosis, HFE mutations,
and advanced hepatic fibrosis remain speculative.41,121 Iron absorp-
tion may be favoured by heterozygous HFE mutations;63 iron‐in-
duced oxidative stress may promote hepatic steatosis and
fibrosis;115,122 and HCV may contribute by suppressing hepcidin pro-
duction and inducing metabolic abnormalities that promote hepatic
steatosis.123
3.4.1 | Hepcidin levels in chronic hepatitis C
Most studies have described low serum hepcidin levels in patients
with chronic hepatitis C, and the hepcidin response has been inap-
propriate for the associated findings of elevated serum iron concen-
tration, hyperferritinemia, and increased transferrin saturation.124–127
The mRNA for hepcidin has been decreased in the liver tissue of
these patients,124–126,128 and suppression of the transcriptional activ-
ity of the HAMP gene has been proposed as a basis for the iron
overload.125,126,129 The defect in hepcidin production has been
reversible with antiviral therapy and eradication of HCV.50
Reactive oxygen species (ROS) induced by HCV proteins have
suppressed the expression of hepcidin in transgenic mice by inhibit-
ing the binding of a transcription factor to the hepcidin promoter.130
In hepatoma cells, inhibition of gene transcription has been attribu-
ted to HCV‐induced ROS and to epigenetic changes associated with
increased histone deacetylase activity.131 The hypo‐acetylation of
histones within the nuclear chromatin has inhibited the binding of
transcription regulators to the hepcidin promoter, and this inhibition
has been reversed by anti‐oxidants and by inhibitors of histone
deacetylase activity.131 Oxidative damage of hepatic deoxyribonu-
cleic acid (DNA) has correlated strongly with serum ferritin levels
and total hepatic iron content,129 and the oxidative stress induced
directly by HCV could result in an iron overload that in turn sustains
or enhances the liver damage.
Hepcidin can also influence HCV replication. Hepcidin has had a
direct suppressive effect on HCV replication in cell culture in associ-
ation with activation of the STAT3 (signal transducer and activator
CZAJA
of transcription protein 3) signalling pathway.132 It has also had an
indirect effect on HCV replication by modulating the intracellular
iron content.133–135 Iron can inactivate HCV RNA polymerase
134
(NS5B) and inhibit HCV replication, and hepatic iron accumula-
tion may prevent progression of chronic hepatitis C.135 These direct
and indirect antiviral actions of hepcidin suggest a negative feedback
mechanism by which HCV replication can be modulated.
Hepcidin production and its antiviral action may be reduced by
HCV. Low serum hepcidin levels may in turn increase duodenal
absorption of iron by up‐regulating duodenal ferroportin,136,137 and
the increased intracellular iron content may suppress HCV replica-
134,135,138
tion in a negative feedback loop. The net effect of these
counterbalancing actions may depend on the relative strength of the
inherent response to raise hepcidin levels in response to iron over-
load and the prowess of HCV to circumvent the antiviral action of
hepcidin by suppressing transcriptional activity of the HAMP
gene.126
Importantly, the effects of hepcidin on HCV replication and hep-
atic iron accumulation may vary at different stages of chronic hepati-
tis C. Early in the disease, the antiviral action of hepcidin may
125
directly suppress HCV replication. Later in the disease, the persis-
tence of HCV infection may suppress hepcidin production and
increase hepatic iron deposition. The iron overload may then gener-
ate an increase in hepcidin expression and counterbalance the viral
125
effect. The hepcidin response to the hepatic iron concentration
has been shown to be appropriate in chronic hepatitis C,139 and
patients with chronic hepatitis C who achieve a sustained virological
response (SVR) to anti‐viral therapy correct their hepcidin defi-
ciency.50
Future investigations should evaluate serum ferritin and hepcidin
concentrations as prognostic biomarkers in different stages of
chronic hepatitis C and determine the value of these biomarkers in
guiding direct‐acting antiviral interventions to protect against pro-
gressive hepatic fibrosis and risk of HCC.
3.5 | Iron disturbances in chronic hepatitis B
Serological evidence of iron dysregulation has been demonstrated in
41% of patients with chronic hepatitis B, and hepatic iron deposits
have been recognised in 35% (Table 1).13 Hepatic iron accumulation
has been mild (grade 1 deposits in 60%), and they have been associ-
ated with male gender and increased serum levels of ferritin, GGT,
and alkaline phosphatase.13 A normal serum ferritin level has had a
negative predictive value of 90% for excluding mild‐moderate hep-
atic iron accumulation, and the major factor associated with higher
grades of hepatic iron deposition and more advanced stages of hep-
13
atic fibrosis has been co‐infection with hepatitis D virus (HDV).
These findings have supported speculation that the iron dysregula-
tion and hepatic iron deposition in chronic hepatitis B are manifesta-
tions of active inflammation or advanced stage disease.
Comparisons between chronic hepatitis C and chronic hepatitis B
have indicated more hepatic iron deposition and lower hepatic hep-
128
cidin mRNA levels in patients with chronic hepatitis C, and the
| 7
hepcidin‐to‐ferritin ratio has been significantly lower in patients with
chronic hepatitis C than in chronic hepatitis B.124 Iron overload by
brain imaging has been demonstrated in patients with chronic hep-
atitis B and cirrhosis, and the hepcidin levels have been inappropri-
ately low.30 Furthermore, hyperferritinemia has predicted 3‐month
mortality in patients with acute‐on‐chronic liver failure associated
with hepatitis B virus (HBV) infection.140 The findings suggest that
iron disturbances in patients with chronic hepatitis B have the same
pattern but less severity than in chronic hepatitis C. Future studies
are needed to determine if these similarities and differences are
attributable to a common pathogenic pathway that is modulated by
virus‐specific properties and if the iron disturbances reflect or
contribute to the disease severity.
3.6 | Iron disturbances in alcoholic liver disease
Serum ferritin levels and transferrin saturations are increased in 63%
and 29% of patients with alcoholic liver disease (ALD),40,141 and the
results are significantly higher than in healthy individuals (P < 0.05)
(Table 1).142 Stainable hepatic iron in parenchymal and reticuloen-
dothelial cells has been demonstrated in 52% of patients with
ALD,40 and severe (grade 3‐4) iron deposition has been present in
22% of liver explants.143 Stainable hepatic iron has been one of sev-
eral factors associated with liver fibrosis in ALD,144 and the hepatic
iron concentration has been predictive of shortened survival in alco-
holic cirrhosis.145
A HFE mutation has been detected more frequently in patients
with ALD and hepatic iron accumulation than in patients with ALD
and no hepatic iron accumulation (odds ratio, 17.23; 95% CI: 2.09‐
142.34; P = 0.008), but the HFE mutation has not been associated
with increased hepatic fibrosis, disease severity, or inflammatory
activity.142 Other studies have demonstrated that the occurrence of
a single HFE mutation in ALD has not increased the hepatic iron
content, frequency of fibrosis, or predisposition for severe
ALD.35,40,146 Heterozygosity for the C282Y mutation has been more
common in patients with alcoholic cirrhosis and HCC than in
patients without HCC (21% vs 4%, P = 0.002),147 but the basis for
this association remains speculative as other contributing factors,
including active alcohol consumption, serum and liver hepcidin levels,
and hepatic iron concentration, have not been fully analysed.
Alcohol down‐regulates the transcriptional activity of the hep-
cidin gene by decreasing the DNA binding of CCAAT/enhancer‐
binding protein α (C/EBPα).148,149 C/EBPα is a transcription factor
that interacts with regulatory sequences in the promoter and
enhancer regions of diverse genes to promote transcriptional activ-
ity,150 and its effect can be diminished by pro‐inflammatory cytoki-
nes (interferon‐γ, IL‐1, IL‐6, TNF‐α),150–152 bacterial
152,153
lipopolysaccharides, and certain toxins, including alco-
hol.148,150 The hepatic content of mRNA for hepcidin is diminished
in ALD,154 and serum hepcidin levels are inappropriately low in
patients with manifestations of iron overload.154,155 The duodenal
iron transporters are up‐regulated,148,156 and the duodenal absorp-
tion of iron is increased.149,155
8 |
The deposition of iron within the liver has been proposed as an
145
independent risk factor for survival in ALD (P = 0.007), and the
hepatic accumulation of ferrous iron may generate reactive oxygen
species (ROS) that promote progressive tissue injury, liver fibrosis,
and malignant transformation.157–159 In a murine model of combined
liver injury by iron overload, alcohol ingestion, and high fat diet, the
iron overload induced an unfolded protein response within the liver
and protein changes indicative of stress within the endoplasmic
reticulum that were accompanied by impaired degradation and recy-
cling of cellular components (autophagy).160 The findings suggested
that the iron toxicity increased hepatic stress by impairing adaptive
compensatory mechanisms.
Alcohol is also a strong promoter of tissue hypoxia which in turn
can increase oxidative stress within the liver.159,161,162 Alcohol can
reduce oxygen tensions in the liver possibly by increasing hepatic
159,161,162
metabolic activity and altering hepatic blood flow. Hypoxia
markedly reduces the liver expression of HAMP and inhibits the tran-
163–165
scription of hepcidin. Alcohol can also enhance the production
of ROS by inducing the activity of cytochrome P450 2E1
(CYP2E1)159,166 and by impairing the anti‐oxidant response of
167
mitochondria.
The risks of HCC (hazard ratio, 1.76; 95% CI: 1.01‐3.06;
P = 0.03) and death (hazard ratio, 1.63; 95% CI: 1.07‐2.44; P = 0.02)
have been greater in patients with alcoholic cirrhosis and low circu-
lating levels of hepcidin.168 The lack of a strong correlation between
serum hepcidin levels and hepatic iron accumulation suggests that
alcohol consumption (resulting in low serum hepcidin levels) rather
than iron overload is the major pathogenic factor.
Hepatic iron accumulation and chronic alcohol consumption are
each potential risk factors for progressive liver damage, HCC, and
diminished survival in ALD mainly through pathways of oxidative
stress. Future studies must continue to evaluate distinctions and
synergisms between these factors.
3.7 | Mechanisms of disrupted iron homeostasis in
chronic liver disease
Genetic and molecular factors are involved in maintaining total body
iron content, and one or more of these factors has been implicated
in the iron disturbances found in some patients with NAFLD,
autoimmune hepatitis, chronic hepatitis B, chronic hepatitis C, and
ALD.
3.7.1 | Genetic mutations
C282Y is the principal point mutation of the HFE gene associated
with hereditary haemochromatosis, and 90% of patients with heredi-
tary haemochromatosis are homozygous for C282Y (Figure 1).169
H63D and S65C are also polymorphisms of HFE, but they have not
been associated with iron overload unless heterozygous with C282Y
(compound heterozygosity).63,170–172 Other point mutations that can
cause iron overload are outside the HFE gene, and they are muta-
tions of the genes for haemojuvelin (HJV), hepcidin (HAMP),
CZAJA
transferrin receptor 2 (TfR2), and ferroportin (SLC40A1).34,63,173 Most
individuals homozygous for C282Y (as many as 99%) do not develop
haemochromatosis,169,174 and the occurrence of haemochromatosis
in individuals homozygous for C282Y probably relates to genetic
modifiers or environmental factors.63,175
Hereditary haemochromatosis has been excluded in most
patients with chronic liver disease and iron overload, but heterozy-
gosity for the HFE mutations has been present in some experiences.
The impact of simple heterozygosity on iron accumulation in patients
with chronic liver disease is unclear. Simple heterozygosity for the
HFE mutations in healthy individuals has not been associated with
iron excess,176 but undefined factors associated with the liver dis-
ease (aetiology, severity, duration) or individual (genetic modifiers,
alcohol abuse, nutritional status) may be critical for the clinical
expression of a heterozygous predisposition. Studies that suggest an
association between simple heterozygosity and hyperferritinemia in
chronic liver disease36,38,42,117,118 are counterbalanced by studies
that have found no association.11,42 Disease‐related or patient‐speci-
fic factors may account for these discrepant findings, and future
investigations must continue to define these risk factors in well‐
characterised patient populations.
3.7.2 | Disruptions in hepcidin transcription
Hepcidin production by the liver can be affected by factors that
affect the transcription of the hepcidin gene (HAMP) and the transla-
tion of the hepcidin mRNA. Transcription of the hepcidin gene is
regulated by the serum iron concentration which is sensed by two
transferrin receptors (TfR1 and TfR2) and the haemochromatosis
protein (HFE) (Figure 1).177 Holotransferrin (diferric transferrin) dis-
places HFE from TfR1 as its concentration increases, and the dis-
placed HFE can interact with TfR2 creating a complex that activates
pathways involving bone morphogenetic protein (BMP) and mitogen
activated protein kinase (MAPK).178–180
The binding of BMPs to BMP receptors activates the SMAD
(Sons of Mothers Against Decapentaplegic) pathway within the cyto-
sol of hepatocytes (Figure 1).177,181–183 The hepcidin gene (HAMP) is
up‐regulated, and hepcidin production is increased.184,185 BMP6 is
an iron‐specific ligand whose expression reflects the iron content of
hepatocytes, and it is the principal BMP that induces hepcidin pro-
duction.186,187 Haemojuvelin is a co‐receptor for BMP, and it can
enhance the hepcidin response by binding to BMP2, BMP4, and
BMP9.184,188–190
Hepcidin is an acute‐phase reactant with antimicrobial properties,
and pre‐treatment with hepcidin has protected mice from lethal
doses of lipopolysaccharides.191 The regulation of hepcidin produc-
tion by multiple intra‐ and extra‐cellular stress signals (iron stores,
inflammation, hypoxia, and erythropoiesis) may help modulate acute
inflammatory responses (Figure 1).192
Inflammation and infection promote hepcidin production by
increasing the secretion of IL‐1 and IL‐6193 and activating the pro-
moter region within the signal transducer and activator of transcrip-
tion 3 (STAT3) gene.194–197 CREB3L3 (cyclic adenosine
CZAJA
monophosphate response element binding protein 3‐like 3) is a
stress‐associated transcription factor in the endoplasmic reticulum of
cells that can also up‐regulate hepcidin production in response to
198,199 200
inflammation or metabolic stress. The CREB3L3 transcrip-
tion factor has been associated with hepcidin production and iron
retention in chronic inflammation and nutrient deprivation (diabetes,
insulin resistance, starvation) (Figure 1).37,201
Key factors that can inhibit hepcidin production are matriptase‐2,
soluble haemojuvelin, missense mutations of various genes involved
in hepcidin transcription and activity, and hepatocyte growth factor
(HGF) (Figure 1). Matriptase‐2, also named transmembrane protease
serine 6 (TMPRSS6), is a membrane protease whose overexpression
can cleave haemojuvelin, impair BMP binding, and suppress activa-
tion of the HAMP promoter.189,202 Soluble haemojuvelin is the extra-
cellular component of membrane haemojuvelin, and it can bind BMP,
competitively antagonise membrane haemojuvelin, and suppress
expression of hepcidin mRNA.185,203 Mutations in the HJV gene can
contribute by reducing the transcription of haemojuvelin, and muta-
tions in the transferrin receptor‐2 gene, ferroportin gene, and cerulo-
plasmin gene could alter the sensing of iron levels, efflux of iron from
4,173,204,205
cells, and iron absorption.
Hepatocyte growth factor (HGF) can inhibit the transcription of
206
hepcidin by countering the stimulatory effects of BMP6 and IL‐6,
and HGF may be a factor in reducing serum hepcidin concentrations
24
in autoimmune hepatitis (Figure 1). HGF is a plasminogen that is
secreted mainly by mesenchymal cells,207–210 and it may protect
against inflammatory and immune‐mediated responses by inhibiting
activation of the nuclear factor kappa‐light‐chain enhancer of acti-
vated B cells (NF‐κB), reducing the production of pro‐inflammatory
cytokines, increasing the secretion of IL‐10, and inducing the prolif-
eration of regulatory T cells (Tregs).210,211 Increased expression of
HGF could be a mechanism by which hepcidin production is
inhibited, iron absorption is increased, and hepatic iron toxicity
occurs.206 HGF may be a key modulatory factor altered by disease‐
related elements (cytokines, oxidative stress, hypoxia), and its role in
the suppression of hepcidin production in chronic liver diseases
24
must be clarified.
Disruption in the transcription of hepcidin has been suggested
as a mechanism for low serum hepcidin levels and iron overload
in chronic hepatitis C. Low levels of mRNA for hepcidin in liver
tissue have justified this consideration,128 and ROS and epige-
netic factors (increased histone deacetylase activity) in association
with the HCV infection may suppress the transcriptional activity
of the HAMP gene.124–126,131 The hepatic content of mRNA for
hepcidin is also reduced in patients with ALD, and suppression
of the transcriptional activity of the hepcidin gene by alcohol,
the reactive products of oxidative stress, and hypoxia within the
148,149,159
hepatic microenvironment have been implicated. Genetic
predispositions outside the HFE mutations may also influence the
mechanisms by which the serum iron level is sensed and moni-
tored in certain liver diseases, and they may contribute to fail-
ures in the proper physiological response of the HAMP gene to
iron excess.
| 9
Maintenance of the homeostatic axis regulating hepcidin produc-
tion is a pivotal management objective, and the salutary effects may
include prevention of hepatic fibrosis and protection from hepatic
steatosis. Hepcidin has protected against hepatic fibrosis in murine
models of toxic liver disease and bile duct ligation by suppressing
phosphorylation of the SMAD3 pathway,212 and it has protected
mice from diet‐induced obesity and hepatic steatosis by promoting
glucose tolerance, insulin sensitivity, lipolysis, and weight reduc-
tion.213 Future investigations in chronic liver disease must continue
to clarify the nature of the interactions between genetic predisposi-
tions, the diverse factors for iron overload (viruses, oxidative stress,
alcohol consumption, and toxins), and the deleterious effects of
hepcidin deficiency.
3.7.3 | Disruption in the translation and post‐
transcriptional processing of hepcidin mRNA
Disruption in the translation of hepcidin mRNA into hepcidin or
delivery of hepcidin to the circulation is suggested by hepatic con-
centrations of mRNA for hepcidin that exceed the serum level of
functional hepcidin (Figure 1).28 This disassociation has been
described in autoimmune hepatitis, PBC, and PSC,28 and the finding
may be a feature of iron overload in immune‐mediated liver disease.
The pathogenic mechanisms that disrupt the translation, trafficking,
binding, and delivery of hepcidin in certain chronic liver diseases
with iron overload are unknown, and the clarification of these mech-
anisms and determination of their disease specificity might improve
diagnostic and therapeutic algorithms.
3.7.4 | Liver inflammation and damage
The laboratory and histological manifestations of iron overload in
chronic liver disease could be consequences of the liver injury. The
intensity of inflammatory activity (resulting in hepatocyte and macro-
phage release of stored iron), the severity of liver injury (resulting in
impaired hepatic production of hepcidin), and the presence of ane-
mia and tissue hypoxia (resulting in ineffective erythropoiesis, sup-
pression of hepcidin production, and enhanced duodenal absorption
of iron) could erroneously suggest iron overload as a pathogenic fac-
tor.3,39,43,159,163
Multiple clinical observations support the possibility that the
iron disturbances in chronic liver disease are collateral manifesta-
tions of liver injury. The pattern and nature of the iron abnormali-
ties lack disease‐specificity,1,4,204,214 and serum ferritin levels have
correlated with markers of liver inflammation (serum AST, ALT
and GGT levels) in autoimmune hepatitis and chronic hepatitis
C.24,109 The serum ferritin level can parallel the circulating level of
CRP,24 daily alcohol consumption,142,215 and severity of liver injury
in the absence of iron overload.44 The iron abnormalities can
rapidly resolve with improvement of the liver disease24,50 or with-
drawal from alcohol,6 and the manifestations of iron overload
(HFE mutations, serum ferritin level, hepatic iron deposition, and
serum hepcidin concentrations) and features of liver injury
10 | CZAJA
(steatosis, fibrosis, HCC, and survival) have lacked a consistent
relationship.
Future investigations must validate that iron overload in chronic
liver diseases outside of hereditary haemochromatosis has a patho-
genic effect that warrants clinical assessment and management. Cur-
rent surrogate markers of iron overload can overestimate the hepatic
iron content and misrepresent its pathogenic significance.44 Hepatic
iron deposition must supersede the surrogate markers as the pivotal
finding in future studies, and it should be determined uniformly in all
study participants by tissue staining,216,217 direct measure-
ment,218,219 magnetic resonance imaging (MRI),220–222 or dual‐energy
computerised tomography (CT).223 The hepatic iron content can then
be correlated with clinical features and outcome irrespective of the
surrogate markers. Thresholds of clinical relevance can be deter-
mined, diagnostic algorithms refined, and management options con-
sidered.
Magnetic susceptometry is an emerging non‐invasive mechanism
by which to assess hepatic iron concentration, and preliminary stud-
ies with a room temperature susceptometer have demonstrated a
highly linear correlation (r2 = 0.998) between its signal and hepatic
224
iron concentration. The performance parameters of the suscep-
tometer for detection of hepatic iron overload (grade ≥2) have been
equal to those of atomic absorption spectroscopy and superior to all
serum iron markers. Furthermore, iron removal by phlebotomy could
224
be accurately monitored.
Magnetic susceptibility measures the attraction or repulsion of a
given material by a magnetic field, and it originally required a highly
complex and expensive device based on superconductor technology
and liquid helium for cooling. Magnetic susceptibility can now be
determined at the bedside by a helium‐free, room temperature sus-
224
ceptometer that could be cost‐effective compared to MRI. Room
temperature susceptometry at the bedside has the potential to facili-
tate future investigations of the pathogenic role of iron overload and
its removal in the chronic liver diseases that are not hereditary
haemochromatosis.
3.8 | Pathogenic consequences of iron overload
Most iron is tightly complexed with proteins that prevent its toxicity
45
by rendering it inactive and insoluble (Table 2). Iron resides mainly
in haemoglobin (67%), liver, spleen, and bone marrow (15%), iron‐
containing cytochromes and enzymes (10%), and muscle myoglobin
(8%).45,225 The conversion of stored iron from its bound, inactive, fer-
ric state to its unbound, reactive, ferrous state can trigger oxidative
stress,45,54 and it is this concern that has generated interest in evaluat-
ing and treating iron dysregulation in the chronic liver diseases that
are outside the diagnosis of hereditary haemochromatosis.
3.8.1 | Iron overload and oxidative stress
The release of iron from its binding complex and its conversion from
inactive ferric iron to reactive ferrous iron is triggered by iron over-
226,227 54
load. The unbound ferric iron is reduced to ferrous iron,
it is the ferrous iron that reacts with hydrogen peroxide and induces
the formation of reactive hydroxyl radicals (Table 2).54,55,226,227 The
generation of ROS (superoxide, hydrogen peroxide, hydroxyl radicals,
and singlet oxygen) can damage cell membranes, affect gene expres-
sion, impair mitochondrial function, induce hepatocyte injury, and
promote hepatic fibrosis.158 Oxidative stress has been implicated in
the pathogenesis of NAFLD,228–230 ALD,231,232 autoimmune hepati-
tis,233 and chronic hepatitis C,234,235 and the iron overload that can
occur in each of these diseases could be a deleterious factor that
warrants assessment and management.
3.8.2 | Ferritin as a cytoprotective agent and
pathogenic factor
Ferritin is an acute phase reactant whose production is stimulated
by the pro‐inflammatory cytokines, TNF‐α236–238 and IL‐1β.238
Oxidative stress increases the synthesis of ferritin,239–244 and the
NFκB pathway can increase cytokine production and up‐regulate the
expression of ferritin (Table 2).240,244 Under these circumstances,
hyperferritinemia could be a consequence of liver damage and oxida-
tive stress in the absence of hepatic iron overload.39 Ferritin also
detoxifies iron,45 and it may modulate inflammatory and immune
responses by suppressing the proliferation and differentiation of pro-
genitor cells within the bone marrow.245–247 Under these circum-
stances, hyperferritinemia could have a protective role in mitigating
iron‐induced tissue injury.
The ferritin molecule consists of heavy or heart (H) and light or
liver (L) subunits,57 and the H subunit has ferroxidase activity that can
oxidise ferrous iron to ferric iron and protect mitochondria from oxida-
tive damage.72,73,248–251 The hyperferritinemia in some patients with
chronic liver disease may constitute an anti‐oxidant response that
attenuates oxidative stress (Table 2).45,72,252–255 The H subunit of fer-
ritin can also modulate the response to apoptotic stimuli,255 and the
over‐expression of the H subunit in rat livers protects against ische-
mia‐reperfusion injury by inhibiting endothelial cell and hepatocyte
apoptosis.256 Furthermore, ferritin activates the promoters of genes
that transcribe proteins regulating haematopoiesis in cell lines, and this
direct up‐regulation of haemoglobin production could reduce the gen-
eration of ferrous iron.257 Acidic isoferritins also suppress the prolifer-
ation of granulocytes and macrophages in normal individuals,245 and
this myelosuppressive action in a murine model246,247 suggests that
hyperferritinemia might reduce the inflammatory and immune
responses that contribute to certain forms of tissue injury.
Hyperferritinemia is also an important prognostic marker that has
been associated with dire outcomes in advanced chronic liver dis-
ease,51–53,258,259 and it may have a nefarious role in some forms of
liver disease that counters its anti‐oxidant and cytoprotective
actions. Ferritin can promote Fas‐mediated apoptosis,260 activate
hepatic stellate cells (HSCs),261 increase hepatic fibrosis,11,261 and
behave as a pro‐inflammatory cytokine (Table 2).261 The association
of hyperferritinemia with low serum hepcidin levels and poor treat-
ment response in autoimmune hepatitis,24 increased early mortality
and in decompensated cirrhosis,51–53 shortened survival in acute‐on‐
CZAJA | 11

T A B L E 2 Mechanisms of iron toxicity in chronic liver diseases with iron overload

Mechanism Pathogenic actions Pathological consequences
Oxidative stress Generates unbound reactive ferrous iron54 Disrupts cell membranes158
Ferrous iron reacts with hydrogen peroxide54 Affects gene expression158
Superoxide, hydroxyl radicals develop158 Impairs mitochondrial function158
NFκB and cytokines stimulated240 Induces hepatocyte loss158
Ferritin synthesis increased239,242,243 Promotes hepatic fibrosis158
Hyperferritinemia Responds as acute phase reactant43 Promotes Fas‐mediated apoptosis260
(detrimental effects) Increased by inflammation, TNF‐α, IL‐1β238 Activates hepatic stellate cells261
Fails to retain inactive ferric iron226,227 Increases hepatic fibrosis11,261
Allows transformation to active ferrous iron54 Behaves as pro‐inflammatory cytokine261
Promotes generation of ROS54,227 Associated with poor outcome51–53,258,259
Hyperferritinemia Detoxifies and stores iron in cells45 Reduces oxidative stress242,243
(beneficial effects) Ferroxidase on heavy chain of ferritin248,249,251 Modulates inflammatory responses245–247
Transforms ferrous to ferric iron254 Mitigates iron‐induced tissue injury256
Anti‐oxidant effect242,243 Anti‐apoptotic in ischemia‐reperfusion256
Activates genes for erythropoiesis257
Suppresses granulocytes and macrophages246

IL‐1β, interleukin‐1 beta; NFκB, nuclear factor kappa‐light‐chain enhancer of activated B cells; ROS, reactive oxygen species; TNF‐α, transforming growth
factor‐alpha.

chronic liver failure,140 adverse pre‐ and post‐liver transplant out-
comes,259,262 and advanced fibrosis in NAFLD9,11 bespeak a patho-
genic role that requires clarification. Future investigations that clarify
the actions of ferritin outside its roles as an intracellular iron storage
protein and iron detoxifier may elucidate pathogenic mechanisms
that can predict outcome and be favourably manipulated.
iron chelation therapy has been a substitute for phlebotomy in indi-
viduals intolerant of the procedure.34,274,275 Iron removal by phle-
botomy has decreased the surrogate markers of oxidative stress
(modified serum proteins) and reduced the serum level of the pro‐
fibrotic cytokine, transforming growth factor‐beta 1 (TGF‐β1).276
Phlebotomy has also been performed in patients with iron overload
and NAFLD263–265 or chronic hepatitis C.266–268

3.9 | Management strategies

Management of chronic liver diseases with iron dysregulation out-
side of hereditary haemochromatosis is challenging. The pathogenic
bases for the iron excess are still being evaluated; the threshold cri-
teria for instituting treatment are incompletely defined; and the ideal
method for improving the iron disturbance is still under study. Inter-
ventions can be directed at reducing the iron excess by restricting
84
iron consumption (dietary measures), removing iron (phlebotomy
or oral iron chelation),263–270 correcting the underlying mechanisms
for the iron overload (low serum hepcidin levels),99,103 or treating
the underlying liver disease (NAFLD, ALD, autoimmune hepatitis,
chronic viral hepatitis) and associated risk factors (alcohol consump-
tion, obesity, metabolic syndrome, diabetes) (Table 3).44
The appropriate approach depends in part on the nature and
severity of the iron disturbance. A serum ferritin level >1000 ng/mL
has been proposed as a threshold indication for iron reduction ther-
apy as this level has been associated with reduced survival.258,271
Confirmation of hepatic iron overload by tissue sampling or imaging
is a requisite to support the treatment decision.44 Iron removal by
phlebotomy and stimulation of hepcidin production by hepcidin ago-
nists are being actively evaluated or considered.
3.10 | Phlebotomy
Phlebotomy has been the standard method of treating iron overload
in patients with hereditary haemochromatosis,34,63,216,272,273 and oral
3.10.1 | Phlebotomy in NAFLD
Phlebotomy has improved fasting and glucose‐stimulated plasma
insulin levels by 40%‐55% and decreased serum ALT levels in 17
patients with NAFLD (Table 3).263 Phlebotomy has reduced insulin
resistance in 64 patients with NAFLD and hyperferritinemia com-
pared to patients managed by lifestyle modifications,264 and it has
normalised serum ALT levels in patients maintained on lifestyle mod-
ifications.277 The NAFLD activity score and individual histological
features (lobular inflammation, steatosis, and hepatocyte ballooning)
have improved during a phase II clinical trial of phlebotomy in
NAFLD, but the size of the positive effect has been insufficient to
justify a phase III clinical trial.265 Iron depletion by deferoxamine in a
hepatoma cell line and murine model of fatty liver has increased the
activity of the insulin receptor and up‐regulated the glucose trans-
porter, but oral iron chelation therapy has not been evaluated in
patients with NAFLD.269
3.10.2 | Phlebotomy in chronic hepatitis C
Biweekly phlebotomies for 3 months have improved serum ALT
levels in 33 patients with chronic hepatitis C,266 and a phlebotomy
regimen for 44‐144 months combined with a low iron diet has been
associated with laboratory improvement and reduced risk of HCC in
35 patients with moderate‐severe hepatic fibrosis and failure or
intolerance of interferon therapy (Table 3).267 Serum ALT levels
12 | CZAJA

T A B L E 3 Evolving management strategies for iron overload in the absence of haemochromatosis

Strategy Rationale NonHaemochromatotic Experience
Phlebotomy Standard treatment for HH216,272,275 Experience in NAFLD patients:
Reduces hepatic iron content in HH34,63 ! Improves plasma insulin levels
263

Decreases oxidative stress in HH276 ! Reduces insulin resistance

264

Reduced profibrotic TGF‐β1 in HH276 ! Decreases serum ALT levels

263,277

! Improves histological features

265

! Small positive effect in phase II trial

265

Experience in chronic hepatitis C patients:

! Improves serum ALT level
266,267

! Reduces risk of HCC

267

! Improves frequency of SVR

268

! Antedates direct‐acting antiviral therapy

268

Oral iron chelation Substitute for phlebotomy in HH34,274,275 Deferoxamine experience in NAFLD model:
Deferasirox reduces iron burden in HH274 ! Up‐regulated insulin receptor
269

! Increased glucose transporter

269

Deferiprone in murine model of thalassemia:

! Removes iron with hepcidin agonists
286,287

279
Minihepcidins Truncated bioactive hepcidin derivative Murine models of β‐thalassemia and PV:
N‐terminus motif inhibits ferroportin279 ! Increased erythropoiesis
282

Reduced iron overload in HH model283 ! Improved anemia

282

Used in hepcidin deficiency models283 ! Decreased iron overload

282

TMRPSS6 inhibitors siRNA and ASOs inhibit TMPRSS6280,285
siRNA delivered in lipid nanoparticles280
Designed to up‐regulate hepcidin gene189
ASOs decreased iron in HH model285
Recombinant BMP6 Stimulates SMAD signalling182,184
Promotes hecidin gene expression184
Increases hepcidin in murine HH289
Small hepcidin agonists Identified by molecular screening278,290
Progesterone and genistein identified291,292
Increased hepcidin in thalassemia model280
Decreased iron load in thalassemia model280
Titratable, durable, reversible effect280
Unassessed outside haemochromatosis289
Causes injection site calcification289
Unassessed278
Lack specificity and proven efficacy278

ALT, alanine aminotransferase; ASOs, anti‐sense oligonucleotides; BMP6, bone morphogenetic protein 6; HH, hereditary haemochromatosis;
NAFLD, non‐alcoholic fatty liver disease; PV, polycythemia vera; siRNA, small interfering ribonucleic acids; SMAD, sons of mothers against
decapentaplegic pathway; SVR, sustained virological response; TGF‐β1, transforming growth factor‐beta 1; TMPRSS6, transmembrane protease
serine 6.

decreased in all patients on the long‐term phlebotomy regimen (re-

turning to normal in 69%), and the frequency of HCC was 5.7% in
the phlebotomised patients at 5 years and 8.6% at 10 years com-
pared to 17.5% at 5 years and 39% at 10 years in the control
patients. Multivariate analysis demonstrated that the risk of HCC
was significantly reduced by the long‐term iron reduction therapy
267
(P = 0.03). Furthermore, a meta‐analysis of six prospective ran-
domised clinical trials has concluded that phlebotomy improves the
frequency of a SVR during interferon therapy (Odds ratio, 2.7; 95%
268
CI: 1.6‐4.5; P < 0.0001).
The findings of phlebotomy in NAFLD and chronic hepatitis C
have been encouraging, but they have been underwhelming in a
phase II clinical trial of NAFLD265 and obtained prior to the availabil-
ity of direct‐acting antiviral therapy for chronic hepatitis C. 268
Results may be improved in NAFLD by better selection criteria for
phlebotomy, and current direct‐acting antiviral regimens may render
phlebotomy unnecessary for chronic hepatitis C. Future investiga-
tions must establish the appropriate therapeutic target in chronic
liver diseases with iron overload outside of hereditary haemochro-
matosis and direct interventions to the iron overload, the primary
liver disease, or both.
3.11 | Hepcidin replacement or stimulation
Most chronic liver diseases with iron overload outside the diagnosis
of hereditary haemochromatosis have low serum hepcidin levels, and
correction of the hepcidin deficiency by replacement or stimulation
is an evolving management strategy.99,103,278 The objective is to
restore iron homeostasis by decreasing duodenal absorption of iron
and diminishing the release of ferric iron from intracellular storage
sites. The hepcidin molecule is difficult to synthesise because of its
complex secondary structure, and management by replacement of
the hepcidin molecule can be compromised by its rapid turn-
over.279,280 Small, biomimetic molecules that have hepcidin activity
and the development of hepcidin agonists promise to improve man-
agement opportunities.278
3.11.1 | Minihepcidins
The N‐terminus of the hepcidin molecule is critical for binding with
281
ferroportin, and stable, truncated, bioactive derivatives of hep-
cidin containing the critical 7‐9 amino acid motif at the N‐terminus
can inhibit ferroportin.279 These engineered peptides that have been
CZAJA
designed to limit iron efflux into the circulation have been named
minihepcidins. They have increased erythropoiesis, improved anemia,
and decreased iron overload in murine models of β‐thalassemia and
282
polycythemia vera (Table 3). In a hepcidin‐deficient murine model
of severe haemochromatosis, minihepcidin has prevented hepatic
iron deposition, reduced myocardial iron content, increased duodenal
iron retention, and redistributed iron from the liver to the spleen.283
Proprietary preparations of synthetic human hepcidin are also being
assessed in a similar fashion.278
3.11.2 | TMPRSS6 Inhibitors
Transmembrane protease serine 6 (TMPRSS6), also named matriptase‐
2, is a membrane‐bound protease in hepatocytes that cleaves
haemojuvelin from the hepatocyte surface, decreases activation of the
SMAD signalling pathway, blocks HAMP gene expression, and reduces
hepcidin production.189,284 The actions of TMPRSS6 can be inhibited
by small interfering RNA (siRNA)280 and anti‐sense oligonucleotides
(ASOs).285 The siRNAs are designed to have target‐specific
complementary nucleic acids that silence the intended gene by binding
to its mRNA and inducing cleavage and degradation of the
transcript.280 ASOs act similarly by targeting the mRNA of the
TMPRSS6 gene.285
TMPRSS6 siRNA that has been formulated in lipid nanoparti-
cles can be cleared by the liver, and the administration of this
preparation to a murine model of β‐thalassemia has increased hep-
280
cidin production and decreased iron loading (Table 3). Further-
more, the effect has been reversible, titratable, durable (80% gene
silencing after 14 days), relevant (hepatic hepcidin mRNA increased
by 2‐3 fold through 7 days), and appropriate (serum iron level and
transferrin saturation decreased by 50% through 14 days).280 Sec-
ond generation ASOs in murine haemochromatosis has also
decreased serum iron, transferrin saturation, and hepatic iron accu-
285
mulation through a similar mechanism of TPMRSS6 inhibition.
Both siRNA and ASO inhibitors of TMPRSS6 have been combined
with an iron chelator (deferiprone) to prevent secondary iron over-
load in a murine model of β‐thalassemia, and the combination has
the prospect of removing iron already deposited and preventing
further accumulation.286,287
3.11.3 | Recombinant BMP6
BMP6 stimulates the SMAD signalling pathway and increases hep-
cidin gene expression in hepatocytes.182,184 In a murine model of
haemochromatosis, the proteins participating in the SMAD pathway
for hepcidin signalling are inappropriately low for the iron burden
and the level of BMP6 mRNA, suggesting a block in BMP6 sig-
nalling.288 Supra‐physiological doses of recombinant BMP6 can over-
come impaired BMP6‐SMAD signalling in murine haemochromatosis
and increase hepcidin production (Table 3). 289
The effect can be
blocked by antibodies to BMP6. The major drawback has been the
propensity for bone production and calcification at injection sites
after long‐term use.289
| 13
3.11.4 | Small molecule hepcidin agonists
Multiple small molecules that activate hepcidin pathways and increase
hepcidin production have been identified by molecular screen-
ing.278,290 Progesterone and two other steroid molecules (epitiostanol
and mifepristone) have been identified in zebrafish that increase hep-
cidin production outside the BMP and STAT3 pathways
278,291
(Table 3). These steroid molecules require progesterone recep-
tor membrane component‐1 (PRMC‐1) to stimulate hepcidin produc-
tion. Genistein, the phytoestrogen in soybeans, is another small
molecule hepcidin agonist, and it can increase STAT3 phosphorylation
and up‐regulate hepcidin production in hepatoma cells.292 Small mole-
cule hepcidin agonists lack specificity for hepcidin expression, and
their ability to correct pathological deficiencies of hepcidin is unclear.
Nevertheless, they underscore the diversity of hepcidin agonists in the
environment, and the possibility that multiple factors outside the indi-
vidual can modulate the hepcidin response in certain disease states.
Hepcidin agonists, especially the minihepcidins and TMPRSS6
inhibitors, constitute an evolving armamentarium for the hepcidin‐
deficient clinical syndromes,278 and iron‐overloaded patients with
chronic liver diseases other than hereditary haemochromatosis may
become beneficiaries of the investigations now driven by the heredi-
tary anemias and genetic haemochromatosis. Investigations to
improve the manifestations of oxidative stress in the liver158 and
studies that directly target the synthesis or activation of ferro-
portin278 may also yield results that have applicability to highly
selected cases of iron‐overloaded chronic liver disease.
3.12 | Overview
Manifestations of iron dysregulation are common in NAFLD,11,82
autoimmune hepatitis,24 chronic viral hepatitis,13,19,30,107,113 and
ALD.40,141,142 Hyperferritinemia,1–4 increased serum iron concentra-
tions,11,24 elevated serum transferrin levels,11,24 stainable iron in liver
tissue,13,25,42 inappropriate serum hepcidin levels,24,28,30,31,124,154 and
mutations of the HFE gene14,18,36 are findings that justify concern that
iron overload is a pathogenic factor that could worsen outcome.
This concern has been supported by clinical experiences associat-
ing the manifestations of iron overload with histological severity and
hepatic fibrosis (NAFLD),9,11,49 poor response to glucocorticoid treat-
ment (autoimmune hepatitis),24 development of HCC (ALD),116,147
and reduced survival (ALD, chronic viral hepatitis).140,145,168 Studies
linking iron excess with oxidative stress and liver toxicity have incen-
tivised investigative efforts to better diagnose and manage the iron
disturbance.45,226,227
The pathogenic basis for the iron dysregulation in chronic liver
diseases other than hereditary haemochromatosis is unclear, but it
may relate to genetic (HFE and non‐HFE genes),63,173 epigenetic (tox-
ins, nutrition),293,294 and disease‐related (cytokine responses, tissue
hypoxia, hepatocyte damage)192,193,201 factors that disrupt iron
homeostasis. The serum hepcidin response to the liver injury may be
the pivotal factor in understanding the basis for the iron disturbance
and directing management.
14 | CZAJA
Inappropriately low serum hepcidin levels have been recognised
in most chronic liver diseases with iron overload,24,30,124–126,154 and
the hepcidin agonists alone (minihepcidins279,282,283 and TMPRSS6
inhibitors280,285) or in combination with an oral iron chelator286,287
are emerging as promising interventions in the hereditary anemias
and genetic haemochromatosis. Therapies that stimulate or replace
hepcidin production may have application in the hepcidin‐deficient
chronic liver diseases that are outside the diagnosis of hereditary
haemochromatosis. Importantly, the iron disturbances in these
chronic liver diseases are probably a consequence rather than a
cause of the liver disease, and successful management of the
primary liver disease remains a necessary, and possibly sufficient,
strategy for correcting the iron disturbance.44
Future investigations must continue to understand the
pathogenic association between the iron overload and liver
disease and develop confident, measurable thresholds of hepatic
iron toxicity that compel intervention. Studies based on hepatic
iron content must refine correlations with surrogate markers
of iron overload to eliminate overestimations of risk,44 and cost‐
effective non‐invasive evaluations of hepatic iron content, such as
the room temperature susceptometer,224 must continue to be
developed and applied.
ACKNOWLEDGEMENTS
Declaration of personal interests: None.
AUTHORSHIP
Guarantor of the article: Albert J. Czaja, MD.
Author contributions: Dr Albert J. Czaja conceived, researched,
designed, and wrote the manuscript without other writing assistance,
research support or financial aid from a funding agency or institu-
tion. The three fully referenced tables were developed by Albert J.
Czaja, MD exclusively for this review. The one colour figure is origi-
nal and previously unpublished. The figure was created by Dr Albert
Czaja exclusively for this review. Dr Albert J. Czaja has no conflicts
of interests to declare, and he has reviewed and approved the final
version of the manuscript prior to its submission, including the
authorship list.
ORCID
Albert J. Czaja https://orcid.org/0000-0002-5024-3065
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How to cite this article: Czaja AJ. Review article: iron
disturbances in chronic liver diseases other than
haemochromatosis – pathogenic, prognostic, and therapeutic
implications. Aliment Pharmacol Ther. 2019;00:
1–21. https://doi.org/10.1111/apt.15173