Hepatic Steatosis : Prevalence and Host / Viral Risk Factors in Iranian Patients with Chronic Hepatitis B Infection

Hepatitis B virus (HBV) chronic infection frequently leads to undesirable complications like cirrhosis and hepatocellular carcinoma (HCC). One of the potential risk factors for the progression to cirrhosis includes hepatic steatosis (HS). HS is characterized by the deposition of lipid droplets, mainly triglycerides, in hepatocytes that exceed 5% of the total weight of liver, or excessive fat accumulation in more than 5% of hepatocytes cytoplasm under light microscopic examination (Schiff et al., 1999; Powell et al., 2005). The accumulation of lipids within the hepatocytes exposes the liver to injury from a variety of causes (Yang et al., 1997; 2001; Wan et al., 2000), which make them vulnerable to factors associated with further hepatic injury by their increased sensitivity to oxidative stress and to cytokine-mediated hepatic damage (Ates et al., 2011). HS is also shown to be associated with higher programmed cell death by apoptosis with stellate cell activation (Walsh et al., 2004; Basaranoglu and Basaranoglu, 2011). Moreover, HS can influence the


Introduction
Hepatitis B virus (HBV) chronic infection frequently leads to undesirable complications like cirrhosis and hepatocellular carcinoma (HCC).One of the potential risk factors for the progression to cirrhosis includes hepatic steatosis (HS).HS is characterized by the deposition of lipid droplets, mainly triglycerides, in hepatocytes that exceed 5% of the total weight of liver, or excessive fat accumulation in more than 5% of hepatocytes cytoplasm under light microscopic examination (Schiff et al., 1999;Powell et al., 2005).The accumulation of lipids within the hepatocytes exposes the liver to injury from a variety of causes (Yang et al., 1997;2001;Wan et al., 2000), which make them vulnerable to factors associated with further hepatic injury by their increased sensitivity to oxidative stress and to cytokine-mediated hepatic damage (Ates et al., 2011).HS is also shown to be associated with higher programmed cell death by apoptosis with stellate cell activation (Walsh et al., 2004;Basaranoglu and Basaranoglu, 2011).Moreover, HS can influence the

Hepatic Steatosis: Prevalence and Host/Viral Risk Factors in Iranian Patients with Chronic Hepatitis B Infection
Vahdat Poortahmasebi 1 , Seyed Moayed Alavian 2 , Hossein Keyvani 3 , Mehdi Norouzi 1 , Mahmood Mahmoodi 4 , Seyed Mohammad Jazayeri 1 * progression of chronic liver diseases to non-alcoholic steatohepatitis (NASH) with development and acceleration of fibrosis, which is associated with an increased risk of cirrhosis or even a cause for hepatocellular carcinoma (Matteoni et al., 1999;Tsochatzis et al., 2007;Starley et al., 2010).Concerning the latter, lines of evidence support the notion that HS may account for a large proportion of cirrhosis (and also idiopathic or cryptogenic cirrhosis), which predisposes these patients to the development of HCC (Bugianesi, 2007;Kim et al., 2010).Moreover, HS could influence the response to given therapy (Jiang et al., 2006;Ates et al., 2011).
However, preliminary data from HBV patients also suggest that hepatic steatosis in these patients may be related to host factors rather than viral factors (Altlparmak et al., 2005;Gordon et al., 2005;Thomopoulos et al., 2006;Bondini et al., 2007).Accumulating data show that while the association between steatosis and HCV is specific, this not the case in HBV-infected patients (Ates et al., 2011).
The aim of this study was to compare CHB patients with steatosis in terms of demographic, virological, biochemistry and histological parameters with control group (CHB patients without steatosis) and to determine the factors that could be potentially associated with steatosis in CHB patients.

Patients
160 HBsAg-positive chronic carrier patients who were referred to the Iranian Hepatitis Network and Digestive Disease center for GI, Tehran during (2010)(2011) were enrolled in a cross-sectional study.They had no evident of co-infection with hepatitis C virus, human immunodeficiency virus (HIV), and they were treatment-naive.None had history of alcohol consumption, drug-induced hepatitis or other known diseases.The diagnosis of chronic CHB disease was made by clinical, biochemical, radiological and endoscopic criteria.The stages and scores of liver pathology and fibrosis were based on biopsy results.All patients gave their informed consent prior to liver biopsy.The clinical data of all patients were retrospectively reviewed in order to collect information about previous laboratory tests and demographic (gender and age) data.
Demographic information including age, gender, weight, height, and body mass index (BMI, calculated the body weight in kilograms divided by the height in meter squared) were obtained from all patients.The study was approved by the Local Committee of Ethics and conformed to the ethical guidelines of Tehran Hepatitis Center.

HBV serological tests
Serological markers for HBV including HBsAg, HBeAg, and anti-HBe, as well as anti-HDV were confirmed in stored serum samples from all patients.All serological markers were measured by enzyme linked immunosoebent assay (ELISA) using a commercial kit (Enzygenost ® HBS Ag, USA).

HBV DNA quantification
HBV DNA was extracted from 200-µL serum samples using the QIAamp DNA mini-kit (Qiagen, Hilden, Germany) and eluted in 100-µL according to the manufacturer's instructions.Total HBV DNA was determined by the COBAS-TaqMan real-time PCR (Roche Molecular Diagnostics, NJ, USA) and was expressed in copy/ml according to the manufacturer's instructions.

Statistical analysis
Data were analyzed with the Statistical Program for Social Sciences (SPSS-16, SPSS Inc., Chicago, Illinois, USA).Continuous variables were expressed as the mean±standard deviation (SD) or median and were compared using independent t-test or Mann-Whitney U-test.Categorical variables were expressed as percentages, and differences between groups were judged for significance using the chi-squared test or Fisher's exact test.Multivariate logistic regression was performed to establish independent predictors of steatosis and high fibrosis stage.For all comparisons, p value <0.05 was considered as statistically significant.
The total BMI (kg/m 2 ) values for groups I and II were 26.67±4.06 and 22.92±3.14,respectively (p>0.001) (Table 1), therefore, patients with increased weight showed a more chance for acquiring liver steatosis.

Virological features
Of all patients, 113 (70.6%) and 47 (29.4%) were positive for anti-HBe and HBeAg, respectively.13 (18.3%)and 34 (38.2%) were positive for HBeAg in group I and II, respectively, on the other hand, 58 (81.7%) and 55 (61.8%) were positive for anti-HBe in group I and II, respectively.Results of chi-square tests showed that there was a significant associations between both groups in terms of HBeAg negativity (p=0.006),indicating the presence of steatosis with anti-HBe positivity in CHB patients.On the other hand, when HBeAg/anti-HBe positivity was compared with the age classification: <25, 25-29, 30-34 and >35 years old, the same statistical methodology showed no strong associations between HBeAg/anti-HBe status of patients and steatosis (results not shown).
Five patients in each of both groups (7% and 5.6% in groups I and II, respectively) were positive for HDV antibody.There was no significant association between HDV positivity and the presence of steatosis (Table 1).

Biochemistry features
The mean levels for liver enzyme tests (including: ALT, AST, Alkaline phosphatase, bilirubin (total and direct) and prothrombin time (PT) were not significantly different between both groups (Table 1).However, the fasting blood glucose and serum lipids including cholesterol and triglycerides levels were higher in group I (with steatosis) compared with control group with strong associations (p values for all three ≤0.001).

Discussion
In patients with chronic hepatitis C, steatosis of the liver increases the severity of fibrosis and adversely influences the response to given therapy; however, the association of liver steatosis and CHB is less clear.
Our study evaluated the frequency of hepatic steatosis in liver biopsies of 160 adult CHB patients to determine whether the presence of steatosis was associated with viral and host metabolic factors and its potential impact on the severity of necroinflammation and stage of fibrosis was also evaluated.Previous studies showed that hepatic steatosis is a common phenomenon in CHB patients.In the present study, the prevalence of hepatic steatosis was 44.4% in CHB patients, in agreement with most published reports and higher than that in Iranian general population of 27-51% (Sass et al., 2005;Jin et al., 2012), hinting its potential effects in CHB.
On univariate analysis we found that the presence of steatosis was significantly associated with male gender, older age, BMI index, high serum glucose (FBS) and lipid levels and HBeAg negativity, similar to other studies studies (Malhotra et al., 2000;Bondini et al., 2007;Minakari et al., 2009).Indeed, sex, BMI, Cholesterol and FBS levels was found on multivariate analysis to be the parameters to independently were associated with the presence of steatosis.On the other hand, we did not find any significant association between steatosis and HBV DNA and ALT levels and the HAI scores of necroinflammation.In accordance to previous reports, we found that hepatic steatosis was significantly associated with higher body mass index and waist circumference.
There was no significant association between patients with and without steatosis in terms of liver grade scores.Likewise, in other reports, no significant association was found between the stage of fibrosis and the severity of steatosis (Bondini et al., 2007;Peng et al., 2008;Rastogi et al., 2011).However, by univariate analysis, when the cut off of >3 was used as criterion decision for severe fibrosis, a strong relationship between fibrosis and HS was achieved (p=0.026), a finding similar to two reports (Shi et al., 2008;Petta et al., 2011).Moreover, on multivariate analysis when we compared the HIA scores with the stages of fibrosis in both groups of patients, higher stages of fibrosis (>3) were associated with HAI scores of necroinflammation (P value<0.001),consistent with other reports of patients with CHC and CHB (Altlparmak et al., 2005;Peng et al., 2008).Serum triglyceride level was found on multivariate analysis to be the only parameter to independently influence HS.The blood sugar, serum leptin, c-peptide levels, and the waist circumference did not reveal any statistically significant association.These parameters associated with superimposed NASH in patients with CHB are known components of metabolic syndrome.
In terms of viral factors, a majority of CHB patients who had HS were HBeAg-negative (81.7%, p value 00.6) with approximately 16.7% of them having at least moderate HS.However, other studies did not confirm a strong association between either the presence or the severity of HS and HBeAg status of CHB patients (Bondini et al., 2007;Tsochatzis et al., 2007;Ates et al., 2011).Further, no significant association was found between HBV-DNA and HS.In fact, previous reports showing that HBV-DNA titre, HBeAg status and liver enzymes did not associate with the presence of hepatic  (Altlparmak et al., 2005;Bondini et al., 2007;Tsochatzis et al., 2007;Rastogi et al., 2011).A possible explanation for the lack of association between HBeAg negativity (and also HBV DNA levels) in all studies so far, could be the fluctuation of HBV replication in HBeAg-negative CHB patients together with bias on patients selection (as a majority of CHB patients included in investigations were HBeAg negative).However, this hypothesis does need the ability to differentiate active CHB status from non-active CHB status in longitudinally or perhaps cohort studies to elucidate the relationship between HBV-DNA levels and the severity of fibrosis.
On the other hand, a role of steatogenic effect for HBx protein in the pathogenesis of HCC has been described (Kim et al., 2001;2007).In a study by Na et al, this role has been attributed to the expression and transcriptional activity of liver X receptor α (LXRα), a regulatory protein in the expression of genes involved in the metabolism of lipids and cholesterol which was increased in HBVassociated HCC in transgenic mice (Na et al., 2009).
The cause and clinical importance of hepatosteatosis accompanying CHB are not well defined.Therefore, we cannot draw any conclusions about the ability of HBV to cause steatosis directly or indirectly.As a result, the progression of steatosis in CHB may be primarily due to the host metabolic status of the host, not directly affect the development of steatosis.These findings probably imply that HBV does not induce accumulation of hepatic steatosis and we believe that steatosis in HBeAg-negative CHB is due to host metabolic risk factors and is not related to the virus per se.
In the present study, HBV genotyping could not be performed due to lack of enough materials for further molecular studies.Regarding the association between HS and HBV genotypes, in one report, authors found that hepatic steatosis tended to present more among patients with viral genotype C (37.9%) compared to genotype B (24.0%) (Lesmana et al., 2012), while other study did not reveal statistically significant differences between genotype A and D (Rastogi et al., 2011).Similarly, two studies from Turkey, a region with HBV genotype D predominance, showed no association between HS and viral genotypic factors (Altlparmak et al., 2005;Ates et al., 2011).Hepatitis B infection in Iran is accepted to be virtually all genotype D (almost 100%); hence, a genotype effect is not expected to influence HS in our CHB patients.It seems that these genotypes do not seem to be steatogenic per se.Nonetheless, there is little data in the literature addressing this relationship.This is in contrast to CHC, in which genotype 3 directly involved in the development of steatosis (Gordon et al., 2005;Lonardo et al., 2006;Yoon and Hu, 2006).
In conclusion, the presence of steatosis associate with various host factors andHBeAg negativity, but not with viral load.Additional studies on molecular aspects and prospective epidemiological studies are needed to explore the real impact of HBV infection on the pathogenesis of steatosis in CHB patients.

Table 2 . Multivariate Analysis of the Association of Steatosis with Clinical and Virologic Parameters in Patients with CHB
Prevalence and Host/Viral Risk Factors in Iranian Patients with Chronic Hepatitis B Infection steatosis or NASH