Iron plays an essential role in many physiological functions, but whenever body iron exceeds its need and storage capabilities are saturated, toxicity due to iron overload may arise. Normal hepatic iron content (HIC) is usually below 35 umol/g of dry weight. HIC above 200-250 umol/g has been associated with liver fibrosis and cirrhosis, as seen in haemochromatosis and thalassemia (1). Even the presence of mild iron excess in the liver represents a risk for toxicity: iron can act as a comorbid factor (along with fat, hepatitis viruses and alcohol) and fuel oxidative stress-driven cell toxicity, or signaling pathways involved in fibrogenesis and carcinogenesis (2). In chronic liver diseases, iron deposits are found either in hepatocytes, Kupffer/sinusoidal cells or in both. Hepatocytic iron usually reflects increased iron influx as a consequence of circulatory iron excess. The cause of increased iron influx is hepcidin deficiency due to either genetic (e.g. C282Y HFE gene mutation) or acquired factors (e.g. alcohol, HCV etc.) (1). Excess iron deposits in Kupffer/sinusoidal cells may affect their immunomodulatory and antiinflammatory activity, cytokine biology, defense against infections, immunosurveillance of tumor growth or response to immunomodulatory drugs (3). Instestinal absorbtion of iron is regulated by hepcidin. Hepcidin is a 25-aminoacid peptide synthesized in the liver. Its binding to ferroportin which is an iron transporter in enterocytes and macrophages, results in internalization and degradation of ferroportin. In this way hepcidin leads to decreased systemic iron bioavailability and parenchymal iron stores but also to increased its macrophagic stores. Hepcidin production is up-regulated by body iron excess and inflammation and down-regulated by anemia and hypoxia. The others hepcidin modulators are: the bone morphogenic protein (BMP), the hemojuvelin ( HJV), HFE gene and transferrin receptor type 2. Mutations in genes encoding these proteins result in hemochromatosis. Many more genes and involved in hepcidin expression and still several questions remains to be answered (4).

The large spectrum of iron-related disease is now divided into 3 main groups (4):

1. Four types of hereditary haemochromatosis,

2. Conditions related to mutations in ferroportin, ceruloplasmin, transferrin or di-metal transporter 1 genes,

3. Acquired iron-overload syndroms.

Genetic iron-overload disorders are divided into haemochromatotic and non-haemochromatotic.

Hemochromatosis is considered to be the most common hereditary metabolic disorder in white adults. It is linked to HFE gene which was identified in 1996. Main mutations of HFE gene namely C282Y and H63D were described and their frequencies analyzed in various population of European descent (5). Most patients with clinical symptoms of haemochromatosis are homozygous for C282Y. This mutation has arised from a single Celt or Viking ancestor. Between 2000-2004 the other genes involved in iron homeostasis were intesively studied, leading to recognition of hepcidin ( HAMP) – the most important iron hormone, hemojuvelin ( HJV), transferin receptor 2 (TfR2) and ferroportin. Recent findings led to a novel hypothesis on potential digenic modes of inheritance or the involvement of modifier genes. Haemochromatotic phenotype is characterized by normal erythropoiesis, increased transferrin saturation (reflecting plasma pool of iron), increased ferritin (reflecting parenchymal pool of iron) with impaired production/regulation/activity of hepcidin (6). Based upon this definition four types of haemochromatosis can now be proposed:

Type 1 or classic, representing more than 90% of all iron-related syndromes, associated with mutations of HFE gene, transmitted as an autosomal recessive trait. Early symptoms are not specific (weakness, fatigue and arthralgia), but advanced disease is a result of iron-overload in several parenchymal organs leading to liver cirrhosis with high risk of HCC development, diabetes mellitus, dark discoloration of the skin, congestive heart failure with arrhythmias, osteoarthritis and hypogonadrotropic hypogonadism.

Type 2 – Juvenile haemochromatosis – is related to mutations on hemojuveline gene ( HJV, type 2A) i.e. G320V or hepcidin ( HAMP, type 2B). There are severe autosomal recessive disorders with early onset, affecting heart and endocrine organs. Patients usually die due to heart failure (7).

Type 3 is related to mutations in Transferrin receptor 2 ( TfR2) (i.e. Y250X, Q317X) and the symptoms of this autosomal recessive disease are similar to typical haemochromatosis (8).

Type 4 of haemochromatosis is associated with mutation of Ferroportin gene (SLC40A1). This condition – called Ferroportin disease type B – is transmitted as a dominant disease (9) and gives late articular and hepatic symptoms.

These conditions are summarized in table 1.

Nonhaemochromatotic iron-overload disorders include:

1. Ferroportin disease type A, an autosomal dominant disease, ill-named as type 4 haemochromatosis, caused by mutation in ferroportin gene (SLC40A1). Biochemical features of disease are different from classic one. Typically high levels of ferritin are observed with normal or slightly elevated transferrin saturation, which reflects mesenchymal iron-overload (9).

2. Hereditary a(hypo)ceruloplasminemia, related to mutations in the ceruloplasmin gene, an autosomal recessive disease with haematological (microcytic anaemia), neurological (retinal degeneration, extrapyramidal syndrome, cerebellar ataxia and dementia) and metabolic (diabetes) symptoms and both low-serum iron and transferrin saturation (10).

3. Hereditary a(hypo)transferrinemia, transmitted as autosomal recessive trait is related to mutations in the transferrin gene, has early onset with severe iron deficiency anaemia and parenchymal iron-overload (11).

4. Mutations in the DMT1 (Divalent Metal Transporter) gene resulting in microcytic anaemia and hepatic iron excess (12).

Acquired iron-overload conditions included a heterogenous spectrum of health problems:

– Chronic excessive iron supply (13).

– Haematological disorders like thalassemia, myelodysplastic syndrome, congenital dyserythropoietic anemia, red cell enzymes deficiencies, sickle cell disease and other haemoglobinopathies that are responsible for hepatic iron excess both by downregulation of hepcidin production and multiple transfusions (14, 15).

– Chronic liver diseases, both by the liver damage and the cause of hepatic iron excess.

This last problem seems to be of particular importance due to a large number of affected patients. Generally a response to necro-inflammatory hepatocytic damage is a phagocytic process resulting redistribution of iron towards the Kupffer cells with slight iron-overload. End-stage liver disease may be associated with significant parenchymal iron excess, related to decreased transferrin and hepcidin synthesis secondary to impaired synthetic function of the liver (16). The pseudo-haemochromatotic cirrhosis pattern appears with increased transferrin saturation and ferritin level although without mutations in HFE gene, heterogenic distribution of iron throughout the liver and absence of iron deposits within fibrous septa at liver biopsy may also be seen.

Hepatitis C is associated with more specific mechanism of iron-overload. It is proposed that interaction between C virus and HFE gene might to some extend explain HCV-associated liver siderosis (3). In alcoholic liver disease (ALD) alcohol has been shown to inhibit hepcidin synthesis and could participate in alcohol-related liver siderosis (17).

A link between serum ferritin, insulin resistance, and non-alcoholic fatty liver disease (NAFLD) development has been suggested. Serum ferritin is postulated to be a potential marker of insulin resistance (18). Hepatic iron overload characterized by hyperferritinemia with normal or slightly increased transferrin saturation was described in non-C282Y homozygotes. Association between hepatic iron-overload with overweight, visceral distribution of fat, arterial hypertension, dyslipidemia, abnormal glucose metabolism was described and subsequently named as insulin-resistance hepatic iron-overload (IR-HIO) or dysmetabolic iron-overload syndrome (DIOS). On the other hand there are some suggestions that in subject with DIOS, hepcidin-resistance state can develop, because in morbid obesity low iron stores caused by hepcidin synthesis by the visceral adipose tissue were reported. Hyperhepcidinuria in patients with DIOS has also been shown (19, 20).

Alcohol and iron have been demonstrated to interact synergistically in the development of liver injury. Both acute and chronic alcohol exposure suppress hepcidin expression in the liver. The sera of patients with alcoholic liver disease, particularly those exhibiting higher serum iron indices, have also been reported to display reduced prohepcidin levels. Alcohol-mediated oxidative stress is involved in the inhibition of hepcidin promoter activity and transcription in the liver (17). This in turn leads to an increase in intestinal iron transport and liver iron storage. Alcohol acts within the parenchymal cells of the liver to suppress the synthesis of hepcidin. Due to its crucial role in the regulation of body iron stores, hepcidin may act as a secondary risk factor in the progression of alcoholic liver disease (21).

Homozygosity of C282Y of HFE gene is the commonest genotype associated with more than 90% of hereditary haemochromatosis in European populations but HFE gene has very low penetration ranging from 10% to 50% with clinical symptoms like fatigue and arthralgia, 11% when considering liver cirrhosis and 1% when the full blown disease is considered despite the 50-95% biochemical expressivity (22, 23, 24). These findings suggest environmental and genetic modifiers of HFE. Unfortunately, many studies found little evidence for correlation between environmental or dietetic factors and iron-overload (25, 26, 27).

Alcohol is responsible for a decrease of hepcidin transcription directly by decreasing expression/activity of the transcriptional factor (C/EBPa) as well as affecting ROS production and indirectly due to hepatic insufficiency. Haemochromatotic patients with excessive alcohol intake are prone to develop early and severe fibrosis when compared to moderate drinkers (28, 29).

Overweight and steatosis in C282Y patients are associated with increased risk of fibrosis. On the other hand obesity is associated with lower iron burden due to visceral adipose tissue production of hepcidin (20).

Acute inflammation is associated with decreased transferrin saturation in C282Y homozygotes whereas chronic inflammation does not influence iron burden in these subjects (30).

Proton-pump inhibitors therapy results in decreasing phlebotomy requirements in C282Y homozygotes probably by suppressing gastric acidity necessary to iron absorbtion (31). Nifedipine – L-type calcium channel blocker – modulates iron mobilization from the liver by its effect on DMT1 and increased urinary iron excretion (32).

Among other modifiers, female gender was considered by its association with menses. Number of pregnancies was not found to play a role (33). Coinheritance of mutation in the hepcidin gene or in the hemojuvelin gene and C282Y homozygosity is associated with more severe iron burden and of earlier onset. Common variants of BMP (Bone Morphogenic Protein) – the member of TGF-β superfamily, involved in hepcidin synthesis (BMP 1 and 2) and transcription (BMP 2,4 and 9) – are associated with iron burden, which suggests that full expression of HFE haemochromatosis is linked to abnormal hepatic expression of hepcidin via impairment in the HFE function and functional modulation in the BMP pathway (4). Most recent study utilizing BMP6-null mice showed BMP6 to be a crucial regulator of hepcidin expression and iron metabolism (34). Endoplasmic reticulum stress has also been found as a potent inductor of hepcidin expression (35). The other considering genes are superoxide dismutase, increasing the risk of cardiomyopathy and TGF-β1 codon 25 gene polymorphism associated with cirrhosis in haemochromatotic patients (4).

Evaluation of transferrin saturation together with ferritin level is crucial for diagnosis of iron-overload. Elevation of ferritin alone can be caused by hepatitis independently of origin due to cell necrosis or increased synthesis in acute or chronic inflammation, chronic alcohol consumption or insulin resistance. Causes of elevated ferritin level are shown in table 2. Normal transferrin saturation rules out haemochromatosis except in case of coexistent inflammatory syndrome or obesity. Genetic finding of homozygotic mutation C282Y allows to establish the diagnosis of hereditary haemochromatosis. The other HFE genotypes should be interpreted with caution: C282Y/H63D heterozygosity does not result in clinically relevant iron-overload, although 10% of compound heterozygotes present with slight hepatic iron excess. H63D homozygosity, C282Y and H63D heterozygosity are associated with increased iron burden only together with additional factors influenced iron metabolism (4).

Magnetic Resonance Imagine (MRI) is a modality able to assess liver iron concentration. Liver biopsy has both diagnostic and prognostic value especially in non-C282Y homozygotes. In C282Y homozygotes liver fibrosis can regress with venesection therapy. Predictive index for fibrosis regression was proposed based upon gammaglobulins, platelet count and prothrombin activity (4).

Venesection therapy remains the standard treatment for HFE, non-HFE haemochromatosis and acquired iron-overload conditions unrelated to haematological diseases. Nutritional advices include low alcohol intake, no excessive vitamin C supplementation and drinking tea. Reducing iron intake is not useful.

In other forms of iron-overload, phlebotomies are not effective and chelating agents are drugs of choice. Subcutaneous injections of desferrioxamine are efficient in aceruloplasminemia (36) and together with deferiprone is effective in reducing iron burden in iron-loading anemias. A new oral chelator – deferasirox – seems to be of choice in iron-loading anemias due to its good tolerance (4). Perhaps it may found a place of adjunct therapy to or replace venesection therapy in haemochromatosis in future.

Haemochromatosis was described in the 19 th century, its HLA linkage defined in 1978 and the gene responsible for its classical form discovered in 1996. From 2000 the other genes involved in iron metabolism were detected and finally hepcidin gene was described as playing a critical role in regulating systemic iron homeostasis. As a result our understanding, classification and management of human disorders associated with iron-overload have been greatly enhanced. Efforts which have been focused on the development of drugs interacting with hepcidin production may in future help in treating hepcidin deficiency in haemochromatosis and hepcidin excess in inflammatory anaemia.