Liver steatosis correlates with iron overload but not with HFE gene mutations in chronic hepatitis C

Gdansk, Poland

Liver steatosis correlates with iron overload but not with HFE gene mutations in chronic hepatitis C

Katarzyna Sikorska, Piotr Stalke, Tomasz Romanowski, Robert Rzepko and Krzysztof Piotr Bielawski

Gdansk, Poland

BACKGROUND:Liver steatosis and iron overload, which are frequently observed in chronic hepatitis C (CHC), may contribute to the progression of liver injury. This study aimed to evaluate the correlation between liver steatosis and iron overload in Polish patients with CHC compared to nonalcoholic fatty liver disease (NAFLD) andHFE-hereditary hemochromatosis (HH) patients.

METHODS:A total of 191 CHC patients were compared with 67 NAFLD and 21 HH patients. Liver function tests, serum markers of iron metabolism, cholesterol and triglycerides were assayed. The inflammatory activity, fibrosis, iron deposits and steatosis stages were assessed in liver specimens.HFEgene polymorphisms were investigated by PCR-RFLP.

RESULTS:Liver steatosis was associated with obesity and diabetes mellitus. This disease was confirmed in 76/174 (44%) CHC patients, most of whom were infected with genotype 1. The average grade of steatosis was higher in NAFLD patients. CHC patients had significantly higher iron concentrations and transferrin saturations than NAFLD patients. Compared with CHC patients, HH patients had higher values of serum iron parameters and more intensive hepatocyte iron deposits without differences in the prevalence and intensity of liver steatosis. In the CHC group, lipids accumulation in hepatocytes was significantly associated with the presence of serummarkers of iron overload. No correlation between theHFEgene polymorphism and liver steatosis in CHC patients was found.

CONCLUSIONS:Liver steatosis was diagnosed in nearly half of CHC patients, most of whom were infected with genotype 1. The intensity of steatosis was lower in CHC patients than that in NAFLD patients because of a less frequent diagnosis of metabolic syndrome. Only in CHC patients were biochemical markers of iron accumulation positively correlated with liver steatosis; these findings were independent ofHFEgene mutations.

(Hepatobiliary Pancreat Dis Int 2013;12:377-384)

hepatitis C virus;iron overload; fatty liver; hemochromatosis; metabolic syndrome X

Introduction

Chronic HCV infection affects approximately 3% of the worldwide population and causes essential health problems due to difficulties in antiviral treatment and two serious consequences of chronic infection: liver cirrhosis and hepatocellular carcinoma.[1]Additionally, attempts to eradicate the virus with new directly acting antivirals may be ineffective as a result of enhanced side effects and drug resistance.[2]Liver steatosis and iron overload are observed in chronic hepatitis C (CHC) patients, likely as a result of host and viral factors, and may play an important role as cofactors of disease progression and hepatocellular carcinoma development.[3-5]

Fatty liver in CHC may be diagnosed as a comorbid disease entity, independent on HCV infection, associated with obvious host features of the coexisting metabolic syndrome (obesity, diabetes mellitus).[6]However, HCV itself may act as a strong inducerof complex syndrome of lipid and carbohydrate metabolism disorders. Experimental studies[7-11]have suggested that lipids accumulation in hepatocytes is a direct consequence of viral cytopathogenic effects. It was demonstrated that HCV core proteins may induce hepatocyte fat accumulation through at least the following mechanisms: (i) the activation of the proteasome activator 28γ (PA28γ) with the upregulation of sterol regulatory element binding protein 1 (SREBP-1) involved in liver fatty acid synthesis,[7]and (ii) the inhibition of microsomal transfer protein activity and very low-density lipoprotein secretion.[8]This model of virus-induced steatosis was reported to be particularly associated with HCV genotype 3 and viral load.[9-11]Moreover, insulin resistance and type 2 diabetes mellitus were confirmed to occur more frequently in CHC patients compared with healthy individuals.[12-14]A model of genotype-specific interference of HCV proteins with intracellular insulin signaling was proposed to explain this observation.[15-17]

Pathogenesis of liver iron overload observed in CHC is complicated. Very active process of necroinflammation may be followed by iron accumulation in liver tissue, especially in Kupffer cells. On the other hand, experimental studies[18,19]provided evidence on the impact of HCV on the dysregulation of iron homeostasis. It was suggested that iron overload in CHC may depend on the HCV-induced down-regulation of hepcidin expression through the reduced DNA binding activity of transcription factor C/EBPα, which results from oxidative stress in HCV infection.[18,19]However, the possible impact of host genetic factors can not be denied, especially mutations in theHFEgene and their potential causation of iron overload.[20,21]Iron hepatotoxicity is generally associated with oxidative stress. Products of oxidative stress and focal inflammatory reactions activate liver macrophages and profibrogenic cytokine release. The activation of hepatic stellate cells and myofibroblasts constitutes iron-induced liver injury and fibrogenesis.[22-24]The role of iron depletion therapy and its influence on the severity of CHC has been discussed, especially in non-responders to antiviral therapy.[25,26]

Liver steatosis and iron overload are important players in the pathogenesis of CHC, but the molecular mechanisms of their possibly reciprocal interactions and mutual influence on prognosis are still under investigation. Recently, on the basis of experiments with mice expressing HCV polyprotein, the iron-induced reactive oxygen species (ROS)-activation of the unfolded protein response has been recognized as one of the possible mechanisms of HCV-related liver steatosis.[27]This study was undertaken to investigate a possible correlation between liver steatosis and disturbances of iron homeostasis in Polish CHC patients compared with patients with non-alcoholic fatty liver disease (NAFLD) andHFE-hereditary hemochromatosis (HH).

Methods

One hundred ninety-one consecutive CHC patients who had been subjected to antiviral treatment in the Department of Infectious Diseases-Liver Unit, Medical University of Gdansk, were included in this study. The patients were compared with 67 consecutive NAFLD and 21 HH patients recruited in the same department. Patients using drugs or abusing alcohol (daily intake＞25 g) were excluded from the study. The study protocol conformed to the ethical guidelines of theHelsinki Declarationand was approved by the Local Independent Bioethics Committee at the Medical University of Gdansk (NKEB 270/2010). Informed consent for participation in this study was obtained from all enrolled subjects.

Diagnosis of liver damage

The results of blood morphology and some biochemical serum tests are presented in Table 1. Chronic HBV and HCV infections were detected by ELISA (HBsAg and anti-HCV, respectively) and confirmed by PCR assays of serum samples.[28]HCV genotyping was carried out by RT-PCR LINEAR ARRAY HCV (Roche), and HCV viral load was measured by Roche Taqman according to the manufacturer's instructions. HCV genotyping was performed in 114 patients who were qualified for treatment with pegylated interferon and ribavirin. Other patients included in the study were recruited during qualification for treatment with classical interferon and ribavirin, and HCV genotyping was not carried out in these patients. In the present group of patients with CHC, 96 patients (84%) were infected with genotype 1, 14 (12%) with genotype 3, and 4 (4%) with genotype 4.

The diagnosis of NAFLD was based on liver ultrasound examination, increased blood fatty acids concentration and the exclusion of other liver damaging factors. Liver steatosis was confirmed by liver biopsy. Hyperlipidemia was diagnosed in cases with increased serum cholesterol (＞180 mg/dL).

Serum markers of iron overload were defined as increased serum concentration of iron (＞28.3 μmol/L) and increased transferrin saturation (＞50% in men;＞45% in women) and/or increased serum ferritin concentration (＞300 ng/mL in men; ＞200 ng/mL in women). The diagnosis of HH was based on thepresence of biochemical markers of iron overload and detection of homozygous C282YHFEgene mutation.[29]

Table 1.Selected demographic, laboratory and histologic features in CHC, NAFLD and HH patients

Interpretation of liver biopsy

Liver biopsy was performed using the Menghini method[30]in 174/191 CHC and 61/67 NAFLD patients. In 17 CHC and 6 NAFLD patients, liver biopsy was not carried out because of contraindications. The preparation of liver biopsy specimen and the assessment of inflammatory activity and fibrosis were described previously.[28]Pathologist was unaware of the patients' clinical findings. Perls' Prussian blue staining for the detection of iron deposits was performed. Semiquantitative grading was used on a scale of 0-4 for the assessment of iron in hepatocytes.[31]Liver steatosis was graded on a four-grade scale: grade 0: no steatosis, grade I: steatosis in ＜10% of hepatocytes, grade II: steatosis in 10%-30% of hepatocytes, and grade III: steatosis in more than 30% of hepatocytes. This study was focused on the investigation of the correlation between liver steatosis and iron accumulation. Iron accumulation in hepatocytes, but not in the reticuloendothelial cells, refers to an early phase of iron overload caused by down-regulation of hepcidin. For this reason only deposits found in hepatocytes were scored. Sideronecrosis in liver tissue, with iron accumulation in Kupffer cells, is observed in cases of heavy iron overload, in the advanced phase of HH.[32,33]

AnalyzingHFEgene polymorphisms

The genomic DNA from peripheral blood leukocytes was extracted by a High Pure PCR Template Preparation Kit (Roche Diagnostics) according to the manufacturer's instructions. The followingHFEmutations were detected by the PCR-RFLP assay: C282Y, H63D, and S65C.[34]

Statistical analysis

Statistical analysis was carried out using data analysis software STATISTICA version 8.0 (StatSoft Inc., USA). All statistical data were presented as a mean ± standard error (SE) or median value (histopathological data). SE was used since the distributions of data were skewed. An analysis of differences between variations was performed using nonparametric statistics: the Mann-WhitneyUtest, the Chi-square test and Spearman's rank-order correlation coefficient test. Relationshipsbetween variables were estimated by multivariate linear and logistic regressions. APvalue of less than 0.05 was considered statistically significant.

Results

Characteristics of CHC patients

The majority of CHC patients were infected with HCV genotype 1, and more than 50% of these patients had a BMI above 25 kg/m2. Diabetes mellitus was diagnosed in 25% and hyperlipidemia in 9% of CHC patients. HCV 3 genotype-infected patients had significantly lower BMIs (r=-0.199;P＜0.05). The median value of inflammation activity and fibrosis, in patients subjected to liver biopsy, corresponded to chronic hepatitis of moderate intensity. Eighty-two patients presented elevated serum parameters of iron overload. The mean concentrations of serum iron, ferritin and transferrin saturations exceeded normal values. Hepatocyte steatosis was present in 76 and hepatocyte iron deposits were detected in 74 CHC patients. In most of CHC patients, both steatosis and iron deposition assessed in histopathological examination were mild. Elevated biochemical iron parameters correlated with increased fibrotic severity (P=0.00059) but not with inflammation activity. Mean HCV viral load was (4.344±1.212)×106IU/mL. Load was not associated with liver steatosis or serum and liver iron overload. DifferentHFEgene mutations were detected in 78 CHC patients. Coexistence of CHC with HH was confirmed in 3 patients.

Differences between patients with and without liver steatosis in the CHC group are presented in Table 2. Univariate analysis confirmed the presence of liver steatosis more frequently in CHC patients with serum markers of iron accumulation than those without (OR=2.076;P=0.02; 95% CI: 1.118-3.856). Multivariate analysis in the CHC group revealed that elevated serum iron parameters were associated only with higher BMI and more frequent occurrence of diabetes mellitus (χ2=19.2;P=0.00007). Moreover, liver steatosis was diagnosed more frequently in patients with arterial hypertension and higher BMIs (χ2=13.6;P=0.001). In the CHC patients with liver steatosis, tissue iron accumulation without biochemical iron disturbances occurred more rarely than in hepatocytes with iron deposits accompanied by elevated serum iron indices (χ2=15.73;P=0.0001).

Comparison of CHC with NAFLD and HH patients

Differences between CHC patients and NAFLD or HH patients are presented in Table 1. Gender and BMI distributions were different in the CHC and NAFLD patients, but the mean age was similar. CHC and HH patients were not different in relation to these features (Table 2). The presence of serum and tissue markers of iron overload was similar in CHC and NAFLD patients. Iron overload, confirmed by both serum iron parameters and liver iron deposits, wasmore severe in HH than that in CHC patients. In all patients with genetic hemochromatosis subjected to liver biopsy, hepatocyte iron deposition of very high intensity was confirmed. Among patients with liver steatosis, the higher biochemical activities of liver enzymes related to liver injury and higher serum iron concentrations with higher transferrin saturations were found more frequently in CHC patients than in NAFLD patients (Table 2). NAFLD patients presented stronger associations of BMI and hyperlipidemia with liver steatosis than CHC patients did (Table 2). Higher liver inflammation activities and fibrosis stages were confirmed in CHC patients compared with NAFLD patients, and they were independent of liver steatosis (Table 2).

Table 2.Selected demographic, laboratory and histologic features in study subjects with hepatocyte steatosis

In the NAFLD group liver steatosis was not correlated with elevated biochemical iron parameters. However, higher serum ferritin concentration was significantly associated with the presence of hepatocytes steatosis in NAFLD patients (729±76.6 vs 543±101 ng/ mL;P=0.018). No significant relationship was found between hepatocyte iron deposits and liver steatosis. In the CHC and NAFLD groups, elevated biochemical iron parameters correlated with increased fibrotic severity (P=0.00022) but not with increased inflammatory severity. In the CHC group significantly more active liver inflammation and more intense liver fibrosis were observed. In the CHC group, however, multivariate logistic regression modeling showed that only serum markers of iron overload were independent factors associated with liver steatosis (P=0.021; OR=2.18; 95% CI: 1.12-4.22).

Ferritin was not correlated with inflammation activity, and it was a good predictor of liver iron deposition in the CHC and NAFLD groups (P＜0.000001). But its concentration was dependent on the severity of fibrosis (P=0.009 for CHC andP=0.019 for NAFLD patients).

HFEgene mutations in the CHC and NAFLD groups

HFEgene mutations were detected in NAFLD at a similar frequency in CHC (31/67, 46%) patients (Table 3). In CHC and NAFLD patients, C282Y mutation was associated with elevated serum iron parameters (allP=0.002) and liver iron accumulation (P=0.0295 andP=0.0002, respectively). OtherHFEgene mutations were detected more frequently in HCV-infected patients with liver iron deposits (P=0.04) but without statistical significance in patients with elevated serum iron parameters (P=0.08). In the NAFLD group, the association ofHFEmutations with elevated biochemical iron parameters and liver iron overload was significant (P=0.024 andP=0.003, respectively). Additionally, inthe NAFLD group, but not the CHC group, H63DHFEpolymorphism was positively correlated with higher serum iron concentrations (P=0.04) and liver iron deposits (P=0.006). No correlation of liver steatosis withHFEpolymorphism was found in CHC or NAFLD patients. HH patients compared with CHC patients presented similar frequency and intensity of hepatocyte steatosis despite more severe serum and liver iron overload.

Discussion

Liver accumulation of iron or fat deposits in CHC patients have been reported previously. However, the potential impact of excess iron, induced by HCV infection on steatosis development has not been a frequent subject of studies. Fatty changes in liver biopsy specimens were found in 44% of HCV-infected patients.[35-37]Clinical observations revealed that liver steatosis was dependent on the following: viral factors in HCV genotype 3 or metabolic syndrome in HCV genotype 1 infection.[37-39]We did not find the correlation of liver steatosis with HCV genotype 3 because only few patients who had been infected with this genotype were included in the study. Genotype 1b is dominant in the Polish population. The prevalence of HCV genotype 3 has been increasing since 2000, especially among intravenous drug users.[40,41]In our series, only 3 patients reported intravenous use of drugs, and 81/191 (42%) individuals had been infected before 2000. Moreover the frequency of metabolic syndrome symptoms influenced the above results as patients infected with genotype 1b were more frequently obese. An overrepresentation of males in our study corresponded to results from previous studies on liver disease morbidity.[42]

Obesity was the strongest factor associated with liver steatosis in all subjects; however, lipid metabolism disorders were associated with steatosis only in NAFLD patients. Most patients with NAFLD had simple liver steatosis explaining the lower ALT and AST activitiesand lower liver inflammation and fibrosis grades in this group. The prevalence of serum markers of iron accumulation was similar in the CHC and NAFLD groups. However, higher serum iron concentration and transferrin saturation in the CHC group compared with NAFLD group reflected different pathogenic mechanisms leading to iron overload. In HCV-infected patients, it was partially due to HCV-induced liver injury and down-regulation of hepcidin.[18,19]

Our study confirmed the association of ferritin concentration with fibrotic staging, and found that ferritin concentration was a good predictor of liver iron deposition in the study groups. Liver steatosis was associated with higher ferritin concentration in NAFLD patients and could be related to insulin resistance syndrome with iron overload.[43,44]This syndrome, frequently associated with NAFLD, was characterized by hyperferritinemia, normal transferrin saturation and mild to moderate iron overload. In NAFLD patients with insulin resistance syndrome, increased ferritin concentration was an indicator of growing tissue iron deposition; such deposition may be resulted from the insulin induced stimulation of cellular iron uptake and a redistribution of intracellular transferrin receptors to the plasma membrane.[45]In our study, we did not diagnose insulin resistance syndrome. In the CHC group, the type of disturbances of iron homeostasis was different from that described for this syndrome. Hepatocyte steatosis in CHC patients correlated both with higher serum iron concentration and transferrin saturation.

We confirmed the association between elevated biochemical iron parameters and liver steatosis by univariate regression analysis in CHC patients. However, these parameters did not serve as an independent factor correlated with steatosis in the multivariate analysis, perhaps because of the strong effects of metabolic features (＞50% of patients with BMIs above the normal value and, ＞25% of patients with coexisting diabetes mellitus). The above-mentioned correlation which was found only in CHC patients was intriguing. This correlation indicates an effect of iron overload on steatosis development and corresponds to the results of experiments, suggesting the impact of HCV on hepatic lipogenesis induction in the presence of iron.[27]

The association of hepatocyte iron deposits with liver steatosis was not shown in this study. In an Italian study of 242 CHC patients, liver iron deposits correlated with liver steatosis grade. The authors confirmed this association in patients infected with HCV genotype 3 and assessed not only the presence of iron in hepatocytes but also the reticuloendothelial iron pattern.[39]In our study we did not analyze the correlation of iron deposits in macrophages with fat accumulation in the liver. We focused on the investigation of the possible influence of serum or liver iron concentrations on the development of liver steatosis. Most of the patients presented with mild or moderate liver iron deposits in their hepatocytes. Heavy iron loading and severe necroinflammatory activity in the liver was observed in a minority of patients. The hepatocyte pattern of iron accumulation is related to hepcidin inhibition, which is proposed to play an essential role in the pathogenesis of most common syndromes of iron overload. Experiments on mice furnished evidence for the down-regulation of hepcidin during HCV infection.[18,19]Controlling plasma iron levels is the main function of hepcidin, the peptide regulator of iron homeostasis. Decreased level of hepcidin results in free, unlimited efflux of iron out of macrophages and enterocytes with increased plasma iron and transferrin saturation. Binding and oxidation of Fe2+within the shell of ferritin protects cells against the toxicity of a labile iron pool (nonprotein-bound iron), and liver alone serves in this protective role as the main storage location for iron in the body. When the physiological capacity of liver iron storage is not sufficient for increasing body iron content (transferrin saturation ＞75%), it leads to the appearance of toxic non-transferrin bound iron (NTBI), which has a high potential for generating reactive oxygen radicals.[32,46]The activity of highly toxic NTBI is suggested to be mainly responsible for tissue damage due to iron toxicity.[47]Perhaps the impact of iron and HCV cooperation on liver steatosis is dependent more on the presence of toxic NTBI or free iron than the presence of hepatocyte iron deposits in terms of iron's association with moderate increases in serum ferritin.[27]CHC patients with liver steatosis presented significantly more frequently with serum and tissue markers of iron accumulation, not simple hepatocyte iron deposits. This unique model of interaction between disturbed iron homeostasis and liver steatosis in CHC patients can also be confirmed by observing HH patients. In the HH group, no association of severe serum or tissue iron overload with liver steatosis was observed.

Contrary to observations by Valenti et al,[37]HFEgene mutations may lead to iron disorders in chronic liver diseases, but their prevalence was not correlated with liver steatosis in a population of Polish patients. In CHC patients, a significant association of onlyHFEgene mutations, excluding the C282Y mutation, with the presence of hepatic iron overload indicates that chronic HCV infection itself disrupts most strongly the normal regulation of plasma iron levels. Nevertheless,the interaction between fat accumulation in hepatocytes and iron disorders in CHC patients needs further clinical validation. If a probable synergistic influence in the course of this disease was proved, the issue of iron removal, as an option for CHC treatment, especially in difficult-to-treat HCV-infected patients, would require reconsideration.

In conclusion, in this series liver steatosis was diagnosed in nearly half of patients with CHC, most of whom were infected with genotype 1. The intensity of steatosis was lower in CHC patients compared with NAFLD patients possibly because of a less frequent diagnosis of metabolic syndrome. In the CHC group only biochemical markers of iron accumulation positively were correlated with liver steatosis. In CHC and NAFLD patients, the presence ofHFEgene mutations was not associated with liver steatosis. Therefore we postulate that in CHC patients virusinduced disruption of iron homeostasis may act as a cofactor for liver steatosis.

Contributors:SK proposed the study. SK and SP collected and analyzed the data, and prepared the manuscript. RT performed genetic tests. RR performed histopathological examinations of liver biopsy specimens. BKP coordinated the research team and reviewed the manuscript.

Funding:The study was supported by a grant from Medical University of Gdansk (W-175).

Ethical approval:The study protocol was approved by the Local Ethics Committee (NKEB 270/2010).

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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April 3, 2012

Accepted after revision December 25, 2012

Author Affiliations: Department of Infectious Diseases, Medical University of Gdansk, Smoluchowskiego 18, 80-214 Gdansk, Poland (Sikorska K and Stalke P); Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland (Romanowski T and Bielawski KP); Department of Pathology, Medical University of Gdansk, Debinki 7, 80-952 Gdansk, Poland (Rzepko R)

Krzysztof Piotr Bielawski, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland (Tel: 48-58-3412887; Email: bielawski@biotech.ug.gda.pl)

? 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(13)60059-4