METABOLIC FACTORS ASSOCIATED WITH HEPATIC STEATOSIS AND FIBROSIS IN CHRONIC HEPATITIS C PATIENTS

Ramy M ElSharkawy 1 , GhadaMostafa K 1 , Mahmoud Saif-Al-Islam AbdElfatah 1 , Eman Mohamed SalahEldein 2 , Tamer Elmokadem 3 ,Ahmed Hassan 3 andHydi Ahmed 4 . 1. Departments of Tropical Medicine and Gastroenterology, Faculty of Medicine, SohagUniversity. 2. Pathology, Faculty of Medicine, Sohag University. 3. Microbiology and Immunology, Faculty of Medicine, Sohag University. 4. Clinical Pathology,Faculty of Medicine, Sohag University and Ibn Sina National College, Jeddah,Saudi Arabia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

. The Egyptian Demographic Health Survey (EDHS), a cross sectional survey including hepatitis C virus (HCV) biomarkers, was conducted in 2008 on a large nationally representative sample (2) . It estimated HCV prevalence among the 15-59 years age group to be 14.7%. Accordingly, Egypt has the highest HCV prevalence in the world (3) . Liver steatosis is a common finding in patients infected with hepatitis C virus (HCV) (4) . HCV steatosis was recently identified as a risk factor for progression to extensive fibrosis (5) . Chronic HCV infection is closely related to the metabolic syndrome (MS). Accordingly, CHC should be classified into CHC with and CHC without MS. Insulin resistance (IR) is the main feature of the MS. In CHC, there is a close association between IR, hepatic steatosis (6) , and progression of fibrosis (7) .
The adipocytokine profile seems to play a distinct role, together with IR, in the pathogenesis of CHC (8) . Adiponectin modulates hepatic fat content and has an anti-steatotic effect on the liver (9) . Adiponectin is also a hepatic insulin sensitizer and has the opposite effect in comparison with tumor necrotic factor (TNF-α) on lipid metabolism, insulin sensitivity and inflammation (10) . In CHC, adiponectin levels are also associated with the degree of steatosis and insulin resistance (11) . Abnormalities of lipid metabolism such as the increase of serum triglyceride, cholesterol and LDL-cholesterol level and decrease in HDL-cholesterol may be the contributing factors in the development of NASH (12) .

Aim of the work:-
We aimed to evaluate metabolic factors associated with hepatic steatosis and fibrosis in patients infected with CHC, and to assess the impact of insulin resistance (as measured by HOMA-IR score) and serum adipocytokines levels on hepatic steatosis and fibrosis relative to other factors. Finally, to evaluate the relationship between CHC and metabolic syndrome.

Patients and Methods:-
The present study included 71 patients (49 males and 22 females) with chronic hepatitis C (CHC). Their ages ranged from 21-57 years. All patients were referred to the Department of Tropical Medicine and Gastroenterology, Sohag University Hospital and to Sohag Specialized Liver Institute (in the period from August 2011 to Septemper 2013) for doing liver biopsies and the complementary laboratory tests before starting treatment with pegylated interferon and ribavirin therapy at Sohag Specialized Liver Institute. Before inclusion in the study, all participants gave informed consents and the study protocol was approved by the Local Ethics Committee.
In addition, 12 age and sex matched healthy individuals were also included to serve as a control group. Patients were diagnosed as chronic hepatitis C based on clinical data, positive anti-HCV by ELISA test and HCV RNA by PCR for more than 6 months. Abdominal ultrasonography:-Histopathological assessment:-Seventy one liver biopsies were included in the study and submitted to histopathological examination. Hematoxylin and eosin (H&E) stained sections were done to assess both the grade and the stage of chronic viral hepatitis, in addition to the degree of steatosis. Metavir grading and staging systems were used.
Statistical Analysis:-Data were computed and analyzed using STATA intercooled version 9.2. Quantitative data were analyzed using ANOVA test and Post Hoc Bonferroni test for comparison of the means of three groups. When the data were not normally distributed Kruskal -Wallis rank test and Mann-Whitney test were used. Qualitative data were compared using Chi square test. P value was considered significant if it was less than 0.05. Both univariate and multivariate analyses were used to determine risk factors (predictors) of significant steatosis (moderate and severe), and the risk factors (predictors) of advanced fibrosis (stage 3 and 4).

Results:-
This study was conducted on 71 patients with chronic hepatitis C who fulfilled the study's inclusion criteria. The patients were predominantly males (49 males and 22 females), with ages ranging from 20 -57 years and a mean age of 41.86 ±9.70. Besides, 12 persons served as controls (7 males and 5 females), with ages ranging from 22-50 years and a mean age of 41.75 ± 7.66. According to the histopathology reports, the studied patients were classified according to the degree of steatosis into 2 groups: Steatosis group 1: includes 33 patients (46.5%) with chronic hepatitis C and steatosis< 33%. Steatosis group 2: includes 38 patients (53.5%) with chronic hepatitis C and steatosis> 33%.
The same patients were also categorized according to the fibrosis score into 3 groups: Fibrosis group 1: includes 40 patients (56.3%) with fibrosis score F 0/1. Fibrosis group 2: includes 16 patients (22.5%) with fibrosis score F2. Fibrosis group 3: includes 15 patients (21.1%) with fibrosis score F 3/4. 0.71 N=number When steatosis groups were compared regarding their laboratory data, we found that BMI, serum insulin and HOMA index were significantly higher in steatosis group 2 than group 1 (P= 0.0001, P= 0.0006 and P= 0.0001, respectively). Serum adiponectin was significantly higher in steatosis group 1 (P= 0.0001), while serum TNF-α was higher in steatosis group 2 (P= 0.01). Serum lipogram shows significantly higher triglycerides in steatosis group 2 (P= 0.003). While no significant difference was found between the two groups as regards age, gender, ALT, AST, serum cholesterol and viral load. Patients in steatosis group 1 have significantly higher serum ALT and AST levels (P= 0.001, P= 0.004 respectively), TNF-α (P= 0.0001), and serum triglycerides (P= 0.046), compared to the controls. Patients in steatosis group 2 have higher BMI (P= 0.0001), ALT, AST levels (P= 0.0002, P= 0.0001 respectively), serum triglycerides, serum TNF-α, HOMA index, (P= 0.0001 for each), and higher serum insulin (P= 0.02) compared to the controls. On the other hand, serum adiponectin was significantly lower in steatosis group 2 than the controls (P= 0.0001) ( Table 1). The clinical characteristics of studied population within each stage of fibrosis were shown in Table 2. We did not find any significant difference between the 3 groups of the studied population as regards serum ALT, AST, adiponectin, TNF-α and HOMA index. While a significant difference was found in BMI, and serum triglycerides being highest in stage 3/4 fibrosis (P= 0.04, P= 0.02 respectively).  Multivariate analysis of the factors mostly predicting higher degree of steatosis, showed that TNF-α (P= 0.03) and HOMA index (P= 0.001) are the factors mostly predicting higher degree of steatosis( Table 3). Multivariate analysis of the factors mostly predicting higher degree of fibrosis showed that BMI index (P= 0.01) and serum triglycerides (P= 0.03) are the factors mostly predicting higher stage of fibrosis (Table 4).  To assess whether hepatic steatosis in the current series is related to the presence of metabolic syndrome, patients were re-categorized into a group without MS (n=46) and a group with MS (n=25). We found 12 variables; patients with older age, higher BMI, elevated blood pressure more than 130/85 mmHg, higher serum triglycerides, total serum cholesterol, HDL-cholesterol in males, serum TNF-α, fasting blood glucose, fasting serum insulin, HOMA-IR, steatosis degree and lower serum adiponectinweresignificantly related to the MS (Table 5).

Discussion:-
Chronic hepatitis C has many features which suggest that this disease must be viewed not only as a viral disease, but also as a metabolic liver disease which implies insulin resistance (13) , and high prevalence of steatosis (14) .Hourigan et al (15) found a significant relationship between hepatic fibrosis and steatosis, suggesting that in chronic HCV infection steatosis may play a role in disease progression.
Our results showed that significant hepatic steatosis (affecting >33% of cells) was present in 53.5% of studied CHC patients. Hepatic steatosis in our study was significantly higher than that reported in Greek patients with CHC genotype 4 where 26.4 % of them showed significant steatosis (16) .
In the current study, patients with different severity of steatosis did not show significant difference in their mean age. This was in agreement with El-Zayadi et al (17) who demonstrated that age is not significantly correlated with steatosis among HCV genotype 4 infected patients. In our study, we found no relation between hepatic fibrosis and age, unlike Hu et al (18) who declared that older patients had advanced stages of fibrosis.
Our study showed a significant association between BMI and the severity of steatosis. This agrees with Hu et al (18) and Negro and Sanyal (19) who found that BMI plays an important role in steatosis in patients with HCV, and disagrees with Adinolfi et al (8) who found that steatosis was not significantly associated with BMI in the overall cohort study of HCV infected patients.
In our study, we found a significant relation between BMI and stages of fibrosis. And this agrees with Hourigan et al (15) and Hu et al (18) .
We found that type 2 DM was present more in steatosis group 2 (47.4%) than in steatosis group 1 (18.19%), but with no statistically significant difference. El-Zayadi et al (17) also declared that DM was not significantly associated with hepatic steatosis in HCV genotype 4 patients. We as well as others, Castera et al (19) and Negro and Sanyal (20) also found a significant association between levels of serum triglycerides and the degree of steatosis in patients with CHC. This disagrees with Hu et al (21) who revealed in their cohort of patients with chronic hepatitis C, that hypertriglyceridemia was not significantly associated with hepatic steatosis.
In our study, we found a significant relationship between the serum triglycerides and fibrosis stage. This was in agreement with Hu et al (18) and in contrast to Solis-Herruzo et al (22) who failed to confirm this relation.
In the current study, mean serum adiponectin level was significantly lower in patients than controls. Also, it was significantly lower in steatosis group 2 than steatosis group 1. On the other hand, CHC patients showed significantly higher TNF-α level than the control. Also, TNF-α was significantly more raised in steatosis group 2 than 1069 steatosisgroup 1. The same result was also reported by Durante-Mangoni et al (23) who found that low level of adiponectin and elevated level of TNF-α were independently associated with grades of steatosis and HOMA-IR.
The elevated level of TNF-α had a direct relationship to the progression of fibrosis in CHC patients (24) . Lu et al (25) found that serum level of adiponectin did not differ significantly between healthy subjects and patients with HCV infection.
In the current study, higher serum insulin level was found in more advanced steatosis than in milder group and controls. Also, we found that insulin resistance (IR) measured by HOMA-IR was significantly higher in CHC patients than control, and it was significantly higher in steatosis group 2 than in steatosis group 1. This was in agreement with Cua et al (11) and Lawrence and Jacqueline (26) who demonstrated that patients with HCV have more insulin resistance than those without. This also agrees with Younossi et al (27) who demonstrated that patients with HCV have more insulin resistance and poor response to treatment. In contrast to our study, Muzzi et al (28) delineated in their cohort study that the level of insulin resistance was not correlated with hepatic steatosis in HCV infected patients.
In the present study, no correlation was found between insulin resistance as measured by HOMA-IR and hepatic fibrosis in CHC patients, and this conforms with Grigorscu et al (29) who revealed a lack of correlation between IR and fibrosis. In contrast to D'Souza et al (30) who reported that IR plays an important role in hepatic fibrosis in CHC patients, irrespective of the genotype.
Multivariate analysis of our data revealed that TNF-α (P= 0.03) and HOMA index (P= 0.001) are the most independent factors predicting hepatic steatosis in patients with CHC. Steatosis was not an independent factor associated with fibrosis, and only BMI (P= 0.01) and triglycerides (P= 0.03) were independently predict advanced fibrosis.
Our findings disagrees with Rubbia-Brandt et al (31) and Cholet et al (4) who reported a significant association between steatosis and high stage of fibrosis.
Our results showed that steatosis was significantly associated with the presence of MS in CHC patients. This was also reported by Grigorscu et al (29) in patients with HCV genotype 1.
This together with the absence of a significant association between viral load and degree of steatosis in our study strongly suggest that steatosis in the current series is due to metabolic origin.

Conclusion:-
In patients with CHC, higher BMI, HOMA-IR,higher serum TNF-α, triglycerides and lower serum adiponectin were associated with HCV hepatic steatosis and metabolic syndrome, while higher BMI and serum triglycerides were associated with more advanced fibrosis stage.