Iron overload and liver fibrosis

Abstract  The pathogenesis of liver fibrosis in genetic haemochromatosis and other iron overload states remains enigmatic. Recent advances in the cellular and molecular pathogenesis of liver fibrosis have determined a central role for hepatic stellate cells. These become activated to a myofibroblastic phenotype following most forms of liver injury and are the major cellular source of collagens and other matrix proteins laid down in fibrotic liver. Similar changes have now been reported in the liver in genetic haemochromatosis, with activation of stellate cells becoming more prominent with increasing hepatic iron concentration. In contrast to other liver diseases, this apparently occurs in the absence of significant necroinflammatory change. Unravelling the mechanism of liver fibrogenesis in iron overload states may, therefore, provide important general insights into the pathogenesis of liver fibrosis. The present article reviews current knowledge of this field with emphasis on the role of lipid peroxidation, sideronecrosis of hepatocytes and spillover of iron to Kupffer cells. An attempt is made to draw these observations together with previous studies of the mechanisms of stellate cell activation in other models and diseases. A unifying hypothesis emerges that helps to define some of the next research questions in the pathogenic mechanisms of liver fibrosis in iron overload.     

Serum Iron Levels and Hepatic Iron Overload in Nonalcoholic Steatohepatitis and Chronic Viral Hepatitis

Our objective was to determine the effect of serum iron levels and hepatic iron overload on hepatocellular damage in nonalcoholic steatohepatitis (NASH) and to compare this with chronic viral hepatitis. Twenty-five patients who had elevated transaminase levels on at least two occasions, without any evidence of viral and autoimmune hepatitis and diabetes, without a history of significant alcohol use, and with a liver biopsy consistent with NASH were enrolled in the study. Twenty-five patients with chronic viral hepatitis (13 patients with chronic hepatitis C and 12 with chronic hepatitis B) who were not under any antiviral treatment were taken as controls. Metabolic factors were studied in the NASH and chronic hepatitis groups. Biopsy specimens were stained with hematoxylin–eosin, and the grade of steatosis and the stage of fibrosis were evaluated as I, II, or III, I being mild and III being severe. Iron overload in the hepatic tissue was studied by Prussian blue staining. Serum ALT, AST, ALP, GGT, globulin, and ferritin levels were comparable in both steatohepatitis and chronic viral hepatitis groups. However, patients with chronic hepatitis had a lower albumin level and a higher serum iron level, with higher transferrin saturation. Among patients with NASH, mild, moderate, and severe steatosis was found in 7, 10, and 8 patients, respectively. Inflammatory infiltration was grade I in 24 patients and grade III in 1 patient. Fibrosis was mild in 12 patients and 13 patients had no fibrosis. Among patients with chronic viral hepatitis, inflammatory infiltration of grade I was seen in 11 patients, grade II in 11 patients, and grade III in 3 patients. Fibrosis was mild in 9 patients, moderate in 13 patients, and severe in 2 patients; 1 patient had no fibrosis. Compared to patients with NASH, those with chronic viral hepatitis cases had more severe inflammatory infiltration and fibrosis (P < 0.01). While five patients with chronic viral hepatitis had mild iron overload, patients with NASH had no hepatic paranchymal iron overload. Neither NASH nor chronic viral hepatitis revealed a relationship between hepatic iron overload and disease activity. This suggests that the iron overload actually may be a result of hemachromatosis gene mutation. The absence of hepatic parenchymal iron overload in the NASH group and only mild iron accumulation in the chronic hepatitis group may be explained by a lower frequency of the gene mutation in our country.     

Non-invasive assessment of tissue iron overload in the liver by magnetic resonance imaging

Summary. We investigated the clinical usefulness of a standard magnetic resonance imaging (MRI) system for non-invasive determination of the liver iron concentration in 38 patients with iron overload and 15 normal controls by measurement of the signal intensity ratio between liver and skeletal muscle (SIR). However, SIR was found dependent on the applied repetition time (TR) of the MRI system, which led us to investigate this relationship in autopsy material of liver and muscle tissue specimens with various iron content. Based on these results, adjustment of SIR measurements to a constant value of TR was achieved. By use of this technique we found a close correlation between MRI and chemically determined liver iron concentration ( r ²= 0.98) as well as the serum ferritin concentration ( r ²= 0.86). The reproducibility was sufficiently good for the use of MRI in the follow-up of iron reductive treatment. The use of iron store parameters in serum was found insufficient as indicators of endpoint for venesection therapy, if 20 μmol Fe/g dry weight was applied as the upper reference limit of the liver iron concentration.
It is concluded that MRI based on SIR measurements offers a precise and reproducible non-invasive method for the determination and follow-up of iron overload within a wide range of liver iron concentrations. Our findings may increase the clinical use of MRI in haematological patients with iron overload.     

Accelerated hepatic fibrosis in patients with combined hereditary hemochromatosis and chronic hepatitis C infection

BACKGROUND/AIMS: Hereditary hemochromatosis (HH) and chronic hepatitis C virus (HCV) infection can both result in hepatic fibrosis and cirrhosis. It has been proposed that iron overload and HCV may have potentiating effects on hepatic fibrogenesis. This study determined if HH patients with HCV would present with hepatic fibrosis/cirrhosis at a younger age and at a lower hepatic iron concentration compared to patients with HH or HCV alone. METHODS: Ten patients with combined HCV and HH were compared to 13 patients who had HH alone and 24 patients who had HCV alone. All patients had advanced fibrosis/cirrhosis on liver biopsy. All HH patients were homozygous for the C282Y mutation. RESULTS: At presentation with advanced fibrosis/cirrhosis, the mean age of the HH/HCV group was significantly lower than that of the HH group and the HCV group. The mean hepatic iron concentration was lower in the combined HH/HCV group compared to that of the HH group. CONCLUSIONS: HH patients with HCV present with advanced fibrosis/cirrhosis at a younger age and at a lower hepatic iron concentration compared to HH patients without HCV. These findings support the concept that the combination of HH-induced iron overload and HCV has a potentiating effect on hepatic fibrogenesis.     

Haemochromatosis: a clinical update for the practising physician

Haemochromatosis is most commonly due to the autosomal recessive inheritance of a C282Y substitution in the HFE protein, whereby both alleles of the corresponding gene are affected. The disease is characterised by an inappropriate increase in intestinal iron absorption due to reduced expression of the iron regulatory protein, hepcidin. Progressive iron deposition in parenchymal tissues may ultimately lead to liver and other organ toxicity. The characteristic biochemical abnormalities are raised serum ferritin and transferrin saturation, which can be used in conjunction with genetic tests and emerging magnetic resonance imaging‐based techniques to diagnose patients with the disorder. Progressive iron overload can manifest clinically as advanced fibrosis, cirrhosis and hepatocellular carcinoma. Enigmatically, the penetrance of both raised iron indices and clinically significant disease is incomplete in patients with hereditary haemochromatosis. Regardless, advanced clinical presentations of the disease have become less common due to increased awareness and earlier diagnosis. On the other hand, obesity and alcohol have been identified as major risk factors that can compound the risk of liver injury in people with hereditary (HFE) haemochromatosis. The prospect of modifying genes that may contribute to the clinical expression of the disease is the subject of ongoing research. Treatment with phlebotomy remains the first‐line therapy, and if instigated early leads to a normal life expectancy. A healthy, well‐balanced diet is recommended to be incorporated as part of the ongoing management of the disease.     

Liver fibrosis in genetic hemochromatosis. Respective roles of iron and non-iron-related factors in 127 homozygous patients.

A retrospective study of 127 patients with untreated homozygous genetic hemochromatosis (HGH) was conducted to evaluate the respective roles of iron overload and non-iron-related factors in the development of hepatic fibrosis in HGH. Twenty-seven percent of the patients had cirrhosis, 21% had liver fibrosis and 52% had no fibrosis (prefibrotic group). The mean value of liver iron concentration was increased significantly (p < 0.001) in cirrhotic (378 +/- 144 mumol/g dry wt.) and in fibrotic (331 +/- 168) subjects compared to prefibrotic (237 +/- 108) patients. Of 13 patients with liver iron concentration > or = 500, 12 had liver fibrosis or cirrhosis, versus 48/134 with liver iron concentration < 500. Chronic alcoholic men exhibited hepatic fibrosis or cirrhosis more frequently than non-alcoholic men (p < 0.001). Non-alcoholic men had hepatic fibrosis or cirrhosis more often than non-alcoholic women (p < 0.05). Cirrhotic and fibrotic patients were significantly older than prefibrotic patients whilst a significant correlation between age and liver iron concentration was found in younger patients only. These results suggest that the iron overload threshold necessary to induce fibrosis is modulated by non-iron-related factors such as alcoholism, sex and age. The development of fibrosis in HGH with liver iron concentration < 500 mumol/g is frequent and must lead to a search for associated non-iron-related fibrogenic factors.     

Evaluation of donor hepatic iron concentration as a factor of early fibrotic progression after liver transplantation

BACKGROUND/AIMS: Hepatic iron may act as an important co-morbid factor in non-hemochromatotic liver diseases, but whether it may favour fibrogenesis after liver transplantation is not known. To verify whether the hepatic iron concentration of the graft might play a role in the rapid fibrotic progression frequently observed after liver transplantation for chronic hepatitis C. METHODS: The hepatic iron concentration, measured at the time of the donor operation, was retrospectively related to the histological follow-up data of 68 recipients (49 males, 19 females), of whom 38 were hepatitis C virus positive. RESULTS: The hepatic iron concentration in donor liver biopsies ranged from 25 to 7,100 microg/gdw. After a median follow-up of 19 months, nine patients (five HCV positive) had a staging score >3. There was a significant association between a higher frequency of increasing staging and donor age >50 years. In female HCV-positive recipients, a graft hepatic iron concentration >1,200 microg/gdw was associated with fibrosis progression >0.15 fibrosis units per month (4/4 vs. 1/7, p<0.01). CONCLUSIONS: The graft hepatic iron concentration may be one of the factors involved in early fibrosis progression due to recurrent hepatitis C in female recipients.     

Hepatic iron overload in aceruloplasminaemia

We report the case of a 52 year old male with diabetes mellitus and long standing evidence of hepatic iron excess. Initially considered to have haemochromatosis, this patient was reevaluated when hepatic iron stores were found to be unaffected by a prolonged course of weekly phlebotomy. The development of neurological disease prompted diagnostic consideration of aceruloplasminaemia, which we confirmed by demonstration of a novel frameshift mutation in the ceruloplasmin gene. Our inability to resolve the patient's iron overload by regular phlebotomy is consistent with recent animal studies indicating an essential role for ceruloplasmin in cellular iron efflux. Evaluation of this case underscores the clinical relevance of aceruloplasminaemia in the differential diagnosis of hepatic iron overload and provides insight into the pathogenetic mechanisms of hepatocellular iron storage and efflux.     

Hepatotoxicity of iron overload: mechanisms of iron-induced hepatic fibrogenesis

While iron is a vital requirement for normal cellular physiology, excessive intestinal absorption of iron as seen in hemochromatosis leads to its deposition in parenchymal cells of various organs such as the liver, heart, and pancreas, resulting in cellular toxicity, tissue injury, and organ fibrosis. Cellular injury is induced by iron-generated oxyradicals and peroxidation of lipid membranes. In the liver, lipid peroxidation results in damage to hepatocellular organelles, such as mitochondria and lysosomes, which is thought to contribute to hepatocyte necrosis and apoptosis, and ultimately lead to the development of hepatic fibrogenesis. Hepatic stellate cells are central to the development of hepatic fibrosis, as they can be activated into collagen-producing myofibroblasts. Numerous potential stimuli associated with hepatic iron overload and iron-induced hepatocellular injury have been assessed in an attempt to explain stellate cell transformation in hemochromatosis. Stellate cell activation and fibrosis appear to be regulated by a series of events involving cellular interaction between resident and nonresident cells of the liver, the sequestration of free iron versus the transport and storage of mobilizable iron, and extracellular matrix remodeling as well as intracellular signaling events associated with inflammatory and fibrogenic cytokines.     

Effects of iron overload in a rat nutritional model of non-alcoholic fatty liver disease.

BACKGROUND/AIMS: This study sought to determine whether excess hepatic iron potentiates liver injury in the methionine choline-deficient (MCD) model of non-alcoholic fatty liver disease (NAFLD). METHODS: Iron-loaded rats were fed either MCD or control diets [MCD diet plus choline bitartrate (2 g/kg) and DL-methionine (3 g/kg)] for 4 and 12 weeks, after which liver pathology, hepatic iron, triglyceride, lipid peroxidation products and hydroxyproline (HYP) levels and serum alanine aminotransferase (ALT) levels were evaluated. RESULTS: Iron supplementation in MCD animals resulted in histologic evidence of hepatic iron overload at 4 and 12 weeks and a 14-fold increase in hepatic iron concentration at 12 weeks (P < 0.001). Iron supplementation in these animals was associated with increased lobular necroinflammation at 4 weeks (P < 0.02) and decreased hepatic steatosis (P < 0.01), hepatic triglyceride levels (P < 0.01), hepatic-conjugated dienes (CD; P < 0.02) and serum ALT levels (P < 0.002) at 12 weeks. Reduced hepatic steatosis (P < 0.005) and CD (P < 0.01) were apparent by 4 weeks. Iron supplementation was associated with a trend towards increased perivenular fibrosis not hepatic HYP content. CONCLUSION: Hepatic iron overload in the MCD model of NAFLD is associated with decreased hepatic lipid, decreased early lipid peroxidation products, increased necroinflammation and a trend towards increased perivenular fibrosis.     

Evaluation of cardiac and hepatic iron overload in thalassemia major patients with T2* magnetic resonance imaging

Objectives: Recent advancements have promoted the use of T2* magnetic resonance imaging (MRI) in the non-invasive detection of iron overload in various organs for thalassemia major patients. This study aims to determine the iron load in the heart and liver of patients with thalassemia major using T2* MRI and to evaluate its correlation with serum ferritin level and iron chelation therapy.

Methods: This cross-sectional study included 162 subjects diagnosed with thalassemia major, who were classified into acceptable, mild, moderate, or severe cardiac and hepatic iron overload following their T2* MRI results, respectively, and these were correlated to their serum ferritin levels and iron chelation therapy.

Results: The study found that 85.2% of the subjects had normal cardiac iron stores. In contrast, 70.4% of the subjects had severe liver iron overload. A significant but weak correlation (r?=??0.28) was found between cardiac T2* MRI and serum ferritin, and a slightly more significant correlation (r?=?0.37) was found between liver iron concentration (LIC) and serum ferritin.

Discussion: The findings of this study are consistent with several other studies, which show that patients generally manifest with liver iron overload prior to cardiac iron overload. Moreover, iron accumulation demonstrated by T2* MRI results also show a significant correlation to serum ferritin levels.

Conclusion: This is the first study of its kind conducted in Indonesia, which supports the fact that T2* MRI is undoubtedly valuable in the early detection of cardiac and hepatic iron overload in thalassemia major patients.     

Glucose abnormalities in non‐alcoholic fatty liver disease and chronic hepatitis C virus infection: the role of iron overload

Non‐alcoholic fatty liver disease (NAFLD) and chronic hepatitis C virus (HCV) infection are major causes of liver disease frequently described in outpatient patients with glucose abnormalities. Hyperferritinemia, which suggests that iron overload plays a decisive role in the pathophysiology of insulin resistance and hyperglycemia, is a common finding in both disorders. However, the role of the hepatic iron deposition differs from one to the other. In NAFLD, a moderate liver iron accumulation has been observed and molecular mechanisms, including the downregulation of the liver iron exporter ferroportin‐1, have been described. Iron overload will enhance intrahepatic oxidative stress that promotes hepatic fibrosis, interfere with insulin signalling at various levels and may hamper hepatic insulin extraction. Therefore, liver fibrosis, hyperglycemia and hyperinsulinemia will lead to increased levels of insulin resistance and the development of glucose abnormalities. Furthermore, iron depletion by phlebotomy removes liver iron content and reduces serum glucose and insulin resistance in NAFLD patients. Therefore, it seems that iron overload participates in those glucose abnormalities associated with NAFLD. Concerning chronic HCV infection, it has been classically assumed that iron overload contributes to insulin resistance associated with virus infection. However, recent evidence argues against the presence of iron overload in these patients and points to inflammation associated with diabetes as the main contributor to the elevated ferritin levels. Therefore, glucose abnormalities, and specially type 2 diabetes, should be taken into account when evaluating serum ferritin levels in patients with HCV infection. Copyright © 2009 John Wiley & Sons, Ltd.     

Hepatic iron overload is common in chronic hepatitis B and is more severe in patients coinfected with hepatitis D virus

Hepatic iron overload has been described in chronic hepatitis C as a cofactor affecting fibrosis progression. Data in patients with chronic hepatitis B infection are scarce. We investigated hepatic iron deposits and serum iron indices in 205 consecutive patients with hepatitis B and compensated liver disease. Mean age of the patients was 42.4 ± 12.4 years and 72.5% were males. Coinfection with hepatitis delta virus (HDV) was present in 8.8%. At least one of the serum iron indices was elevated in 41.5% of cases. Hepatic iron deposits were detected in 35.1% of patients, most of them being minimal (grade I) (59.7%) or mild (grade II) (27.8%). Variables significantly associated with hepatic iron deposits were male gender (P = 0.001), serum ferritin (P = 0.008), γGT (P = 0.05) and alkaline phosphatase (P = 0.05) levels. By multivariate analysis hepatic iron deposits correlated with serum ferritin [odds ratio (OR) 1.2, 95% confidence interval (CI) 1.05-1.4, P = 0.002]. Presence of mild-moderate (grades II and III) hepatic iron deposits could be excluded with high negative predictive value (90%) when serum ferritin was within normal values. A significant correlation between coinfection with HDV and hepatic iron deposits was also found (OR 4.23, 95% CI 1.52-11.82, P = 0.003). When compared to monoinfected cases, HDV positive patients had more elevated γGT (P = 0.03), more advanced fibrosis and more severe iron deposits (P < 0.0001). In conclusion, in well-compensated chronic hepatitis B infection, hepatic iron deposits and elevation of serum iron indices are common, especially in male gender and in patients coinfected with HDV. As HBV/HDV liver disease is generally more rapidly progressive than that caused by HBV monoinfection, we speculate that iron overload may be one of the factors contributing to the severity of liver disease.     

Hepatic iron accumulation may be associated with insulin resistance in patients with chronic hepatitis C

Background/Aim: Insulin resistance and hepatic iron overload are frequently demonstrated in hepatitis C virus (HCV)-related liver diseases. We investigated the relationship between insulin resistance and hepatic iron deposition in patients with chronic HCV infection. Methods: Insulin resistance was evaluated using the homeostasis model assessments for insulin resistance (HOMA-IR) in 56 non-diabetic non-obese patients with biopsy proven chronic hepatitis C. The relationship between insulin resistance and serum ferritin levels or the grade of hepatic iron deposition was assessed. Results: The levels of plasma immunoreactive insulin (IRI) and HOMA-IR were significantly correlated with serum ferritin levels and the grade of hepatic iron deposition (P = 0.003).Although IRI and HOMA-IR increased in parallel with the development of hepatic fibrosis, insulin resistance (HOMA-IR > 2) was observed in 11 (26.2%) of 42 patients even without severe fibrosis (F0-2). Among patients without severe fibrosis, IRI and HOMA-IR were significantly higher in patients with iron deposits than in those without iron deposits. Conclusion: Hepatic iron overload may be associated with insulin resistance in patients with chronic hepatitis C, especially in patients with mild to moderate fibrosis.     

Liver Affection in Iron Overload Studied with Serum Ferritin and Serum Aminotransferases

ABSTRACT Liver dysfunction as measured by S-ALAT activity was present in 72% of patients over 40 years of age with HLA-related iron overload, mainly detected by laboratory screening. Liver dysfunction was correlated to the amount of iron stored (r=0.54, p<0.001). When iron was removed by phlebotomy, liver function returned to normal. S-ALAT activity was closely correlated to serum ferritin concentration (r=0.73, p<0.001). Even a mild iron excess can affect hepatocytes and result in increased levels of ferritin and aminotransferases in serum. Patients with “transaminitis” should be investigated for iron overload.     

Serum amino-terminal propeptide of type III procollagen and 7S domain of type IV collagen correlate with hepatic iron concentration in patients with chronic hepatitis C following alpha-interferon therapy

BACKGROUND: It has been reported that chronic infection with hepatitis C virus is associated with excess iron deposits in the liver of subjects who are neither alcoholics nor recipients of blood transfusions. However, little is known about the relationship between hepatic iron concentration (HIC) and the serum levels of hepatic fibrogenesis markers, which were caused by interferon therapy for chronic hepatitis C. Therefore, changes in the serum amino-terminal propeptide of type III procollagen (P-III-P) and the 7S domain of type IV collagen (7S-IV) in 16 patients treated with alpha-interferon (IFN-alpha) were studied, and their HIC and histological assessment evaluated. Hepatic iron concentrations were measured by using liver biopsy specimens obtained before and 6 months after the cessation of treatment. METHODS AND RESULTS: Eight subjects (50%) who had normal alanine transaminase levels at 6 months after therapy showed significantly lowered HIC, and attenuated hepatic iron staining with decreased serum levels of P-III-P and 7S-IV compared to the remaining subjects. The HIC was significantly correlated with the serum levels of P-III-P and 7S-IV in all subjects. CONCLUSIONS: These findings suggest that IFN-alpha treatment may decrease stimuli for fibrogenesis, at least in part, by reducing the hepatic iron deposition in patients with chronic hepatitis C.     

Iron Through the Prism of Haematology

Since the inception of the British Society for Haematology (BSH) 60 years ago, our increased scientific understanding of iron metabolism, together with clinical developments, have changed the way we diagnose and treat its disorders. In the UK, perhaps the most notable contributions relate to iron overload, some of which I will outline from personal experience. Diagnostically, this began with the identification of serum ferritin as a marker of iron overload and continued later with the application of MRI-based imaging techniques for iron and its distribution. Therapeutically, the first trials of both parenteral and oral chelation, which have radically changed the outcomes of transfusional iron-overloaded patients, took place in the UK and are now part of standard clinical practice. During this time, our scientific understanding of iron metabolism at a cellular and systemic level have advanced the diagnosis and treatment of inherited disorders of iron metabolism. There are potential novel applications related to our recent understanding of hepcidin metabolism and manipulation.     

Cellular expression and regulation of iron transport and storage proteins in genetic haemochromatosis

: Genetic haemochromatosis is a common iron overload disorder of unknown aetiology. To characterize the defect of iron metabolism responsible for this disease, this study localized and semiquantified the mRNA and protein expression of transferrin, transferrin receptor and ferritin in the liver and duodenum of patients with genetic haemochromatosis. Biopsies were obtained from iron-loaded non-cirrhotic patients with genetic haemochromatotic and control patients with normal iron stores. Additional duodenal biopsies were obtained from patients with iron deficiency. Immunohistochemical and in situ hybridization analysis for transferrin, transferrin receptor and ferritin was performed. Hepatic transferrin, transferrin receptor and ferritin protein expression was localized predominantly to hepatocytes and was increased in patients with genetic haemochromatosis when compared with normal controls. Interestingly, hepatic ferritin mRNA expression was not increased in these same patients. In the genetic haemochromatotic duodenum, ferritin mRNA and protein was localized mainly to crypt and villus epithelial cells and the level of expression was decreased compared with normal controls, but similar to iron deficiency. Duodenal transferrin receptor mRNA and protein levels colocalized to epithelial cells of the crypt and villus were similar to normal controls. Early in the course of genetic haemochromatosis and before the onset of hepatic fibrosis, transferrin receptor-mediated iron uptake by hepatocytes contributes to hepatic iron overload. Increased hepatic ferritin expression suggests this is the major iron storage protein. While persisting duodenal transferrin receptor expression may be a normal response to increased body iron stores in patients with genetic haemochromatosis, decreased duodenal ferritin levels suggest that duodenal mucosa is regulated as if the patient were iron deficient.     

The haemochromatosis gene: A co-factor for chronic liver diseases?

There is increasing evidence that hepatotoxins, such as alcohol and the hepatitis viruses, act as co-factors in causing hepatic fibrosis and cirrhosis. For example, alcohol aggravates the hepatic damage produced by iron in hereditary haemochromatosis. We present evidence that the reverse is also true, that is, that iron loading of mild to moderate degree due to heterozygosity or homozygosity for the haemochromatosis genetic mutations acts as a significant hepatotoxin aggravating hepatic damage from other causes of liver disease. These include non-alcoholic steatohepatitis, chronic hepatitis C, porphyria cutanea tarda and possibly primary liver cell cancer. However, any additional hepatotoxic effect is due to the hepatic iron concentration and not the mutations in the haemochromatosis genes.