Hepatic Steatosis and Fibrosis in Chronic Hepatitis C in Taiwan

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1 Jpn. J. Infect. Dis., 60, , 2007 Original Article Hepatic Steatosis and Fibrosis in Chronic Hepatitis C in Taiwan Meng-Hsuan Hsieh 1, Li-o Lee 2, Ming-Yen Hsieh 2, Kun-Bow Tsai 3, Jee-Fu Huang 4, Nai-Jen Hou 4, Shinn-Chern Chen 2,5, Zu-Yau Lin 2,5, Ming-Yuh Hsieh 2,5, Liang-Yen Wang 2,5, Chia-Yen Dai 1,2,5,6 *, Wan-Long Chuang 2,5 and Ming-Lung Yu 2,5 1 Department of Occupational and Environmental Medicine, 3 Department of athology, and 4 Department of Internal Medicine, Kaohsiung Municipal HsiaoKang Hospital; 2 Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital; and 5 Faculty of Internal Medicine and 6 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (Received May 21, Accepted September 5, 2007) SUMMARY: Hepatitis C virus (HCV) infection has been associated with hepatic steatosis. However, the role of hepatic steatosis in the pathogenesis of HCV infection remains controversial. In our study, 425 consecutive HCV-viremic patients with biopsy-proven chronic hepatitis C (male, 264; mean age, 49.0 years) were enrolled. Scoring of hepatic steatosis was based on the method described by Kleiner and on histopathology performed using the Knodell and Scheuer systems. HCV RNA level and genotypes were determined at the time of biopsy. Hepatic steatosis was observed in 30.8% of patients, including 113 mild, 16 moderate, and 3 with severe hepatic steatosis. atients with a body mass index (BMI) <23 kg/m 2 had a significantly lower rate (18.9%) of hepatic steatosis ( < 0.001). Hepatic steatosis did not correlate with the hepatic necroinflammatory activity, but was related to hepatic fibrosis ( = 0.035). Hepatic steatosis was also not associated with HCV RNA level, and the distribution was similar between patients with HCV genotype 1 and genotype 2 infection. According to multivariate analysis, BMI is the strongest risk factor associated with hepatic steatosis, followed by hepatic fibrosis and triglyceride level with odds ratios (95% confidence intervals) of 2.51 ( ), 2.06 ( ), and 1.02 ( ), respectively. Hepatic steatosis was associated with being overweight, hepatic fibrosis, and triglyceride level in chronic hepatitis C. INTRODUCTION Hepatic steatosis is a pathological phenomenon whereby fat is deposited within hepatocytes. Many factors are known to be risks for hepatic steatosis, including diabetes mellitus, hyperlipidaemia, certain drugs, and obesity (1,2). In recent years, some studies have shown that chronic hepatitis C (CHC) is also a risk factor, and indeed hepatic steatosis is associated with CHC (3,4). revious studies have shown that 30-70% of liver biopsies from patients with CHC include histological evidence of steatosis (5-7). Recent studies indicate that hepatitis C virus (HCV) core protein has a direct inhibitory effect on the function of microsomal triglyceride transfer proteins related to the secretion of very low density lipoproteins (VLDL) (8,9). Furthermore, HCV core protein is known to damage the mitochondria and therefore create oxidative stress within the hepatocytes, a process that disrupts the peroxidation of fats within hepatocytes and can lead to hepatic steatosis (5,8). In clinical observations, it remains controversial whether hepatic steatosis influences the progression of fibrosis in the liver histopathology of CHC patients. Steatosis has been reported to be independently associated with fibrosis in CHC (3,4); however, other studies have documented that steatosis is not associated with the presence of or subsequent progression to fibrosis in a cohort study group (10). Several HCV hyperendemic townships in southern Taiwan were found to have a prevalence of higher than 30% in previous studies *Corresponding author: Mailing address: Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 Tzyou 1st Rd, Kaohsiung City 807, Taiwan. Tel: ext. 7475, Fax: , d820195@yahoo.com.tw (11,12). Given that hepatic steatosis may therefore be an important histopathological factor in CHC, the aim of this study was to establish the prevalence of hepatic steatosis in Taiwanese CHC patients and to evaluate whether hepatic steatosis was related to the virological characteristics of HCV and the histopathological features of liver biopsy. In addition, the association between hepatic steatosis and anthropometric and biometabolic markers in CHC patients was also explored. MATERIALS AND METHODS atients: A total of 425 consecutive CHC patients were enrolled at the Kaohsiung Municipal Hsiao-Kang Hospital (a regional core hospital) and Kaohsiung Medicine University Hospital (a medical center) from August 1998 to March They were all seropositive for antibody to HCV (anti- HCV) for more than 6 months, and positive for HCV RNA. All patients underwent liver biopsies and were proven to have CHC. The patients medical histories were taken and their body mass index (BMI) was measured on the date of their liver biopsy. Hypertension was determined according to the criteria defined by the National Committee on revention, Detection, Evaluation, and Treatment of High Blood ressure (13). atients with diabetes mellitus were classified according to the diagnostic criteria of the National Diabetes Data Group (14), or if the patient s past history mentioned taking oral hypoglycemic agents (OHA) or injecting insulin to control blood sugar. Hyperlipidaemia was defined as the fasting lipid criterion according to the third report of the National Cholesterol Education rogram (NCE) Expert anel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment anel III) final report (15). atients were classified as overweight if their BMI was greater 377

2 than or equal to 23 kg/m 2, according to the Asia-acific BMI criteria (16). atients were excluded if they were diagnosed with coexisting hepatocellular carcinoma, decompensated cirrhosis or hepatic failure, other hepatic diseases (such as Wilson s disease or 1-antitrypsin deficiency), biliary system disease, pancreatic disease or other severe systemic diseases (such as renal failure), or if concurrent hepatitis B or human immunodeficiency virus infection was present. atients with a history of alcohol consumption exceeding 40 g/day or those diagnosed with alcoholism, intravenous drug abuse, or exposure to hepatotoxic drugs were also excluded. Liver histopathology: Liver biopsy samples of at least 2 cm in length were taken and stored 4 C in a refrigerator for 8 h, before being reversed in a freezer under 70 C. Biopsy samples were stained with hematoxylin and eosin, and the results were then reported by one pathologist. The degree of hepatic necroinflammatory change was classified according to the histological activity index (HAI) score system (17), and the fibrosis stage was set according to the Scheuer scale (18). The extent of hepatic steatosis was assessed by light microscopy and graded as none (0-5%), mild (5-33%), moderate (33-66%), and severe (>66%), according to the area occupied by fatty hepatocytes (19). Laboratory tests: The presence of anti-hcv was tested by using third generation enzyme-linked immunosorbent assay kits (Abbott, North Chicago, Ill., USA). HCV RNA was detected using a standardized automated qualitative reverse transcription-polymerase chain reaction (CR) assay (COBAS AMLICOR Hepatitis C Virus Test, version 2.0; Roche, Branchburg, N.J., USA), with a detection limit of 50 IU/mL. HCV RNA levels were detected with a branched DNA assay (Versant HCV RNA 3.0; Bayer, Emeryville, Calif., USA), performed strictly in accordance with the manufacturer s instructions. Quantification was limited to 615 IU of HCV RNA per ml. HCV genotypes (1a, 1b, 2a, 2b, and 3a) were determined through amplification of the core region using genotype-specific primers (20). All data were collected at the time of the liver biopsies. Statistical analysis: The statistical tests used were Student s t test, the Mann-Whitney test, 2 square test, and Fisher exact test. All of these tests are two-sided, and the significance levels were set at = Logistic regression analysis was performed to determine the factors associated with hepatic fibrosis based on the odds ratio and 95% confident intervals for these covariates. The covariates analyzed were age, sex, hypertension, diabetes mellitus, hyperlipidaemia, HCV genotype, HCV viral load, BMI, and hepatic necroinflammatory activity and fibrosis. The procedures were performed using the SSS 12.0 statistical package (SSS, Inc., Chicago, Ill., USA). Table 1. Basic demographics and factors associated with steatosis and non-steatosis patients in chronic hepatitis C patients atient Steatosis 1) ( ) Steatosis 1) (+) Fibrosis ( ) Fibrosis 2) (+) No. (%) No. (%) No. (%) No. (%) No. (%) No. of case 425 (100) 293 (68.2) 132 (30.8) 91 (21.4) 334 (78.6) Age (y) (mean ± SD) 49.0 ± ± ± ± ± ) Sex Male 264 (62.1) 177 (60.4) 87 (65.9) (70.3) 200 (59.9) 0.07 Female 161 (37.9) 116 (39.6) 45 (34.1) 27 (29.7) 134 (40.1) Hypertension 3) Without 357 (84.0) 250 (85.3) 107 (81.1) (91.2) 274 (82.0) ) With 68 (16.0) 43 (14.7) 25 (18.9) 8 (8.8) 60 (18.0) Diabetes mellitus 4) Without 384 (90.4) 267 (91.1) 117 (88.6) (93.4) 299 (89.5) 0.27 With 41 (9.6) 26 (8.9) 15 (11.4) 6 (6.6) 35 (10.5) Hyperlipidemia 5) Without 407 (95.8) 282 (96.2) 125 (94.7) (93.4) 322 (96.4) 0.21 With 18 (4.2) 11 (3.8) 7 (5.3) 6 (6.6) 12 (3.6) Triglyceride ± ± ± 36.6 < ) ± ± Cholesterol ± ± ± ± ± BMI (kg/m 2 ) (mean ± SD) 24.8 ± ± ± 3.20 < ) 24.0 ± ± < 23 kg/m (32.0) 111 (37.9) 25 (18.9) < ) 33 (36.3) 103 (30.8) kg/m (68.0) 182 (62.1) 107 (81.1) 58 (63.7) 231 (69.2) HCV genotype Type 1b 246 (57.9) 174 (59.4) 72 (54.5) (53.8) 197 (59.0) 0.74 Type 2a 140 (32.9) 96 (32.8) 44 (33.3) 32 (35.2) 108 (32.3) Type 2b 33 (7.8) 19 (6.5) 14 (10.6) 9 (9.9) 24 (7.2) Type unclassified 6 (1.4) 4 (1.4) 2 (1.5) 1 (1.1) 5 (1.5) Viral load log IU/ml (mean ± SD) 5.20 ± ± ± ± ± < IU/ml 233 (54.8) 155 (52.9) 78 (59.1) (52.7) 185 (55.4) IU/ml 192 (45.2) 138 (47.1) 54 (40.9) 43 (47.3) 149 (44.6) AST, IU/L (mean ± SD) 99.9 ± ± ± ± ± ) ALT, IU/L (mean ± SD) ± ± ± ± ± ) :Steatosis grade was according to criteria of Kleiner et al. (19). 2) :Fibrosis stage was according to criteria of Scheuer,.J. (18). 3) :Hypertension was determined according to the criteria defined by the National Committee on revention, Detection, Evaluation, and Treatment of High Blood ressure (13). 4) :atients with diabetes mellitus were classified if they met diagnostic criteria of the National Diabetes Data Group (14), or if patient past history mentioned taking oral hypoglycemic agents (OHA) or injecting insulin for controlling blood sugar. 5) : Hyperlipidemia was defined as the fasting lipid criteria according to the third report of the National Cholesterol Education rogram (NCE) Expert anel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment anel III) final report (15). 6) :In univariate analysis, triglyceride level and BMI were related to hepatic steatosis. 7) : In univariate analysis, age, hypertension history, and AST level were related to hepatic fibrosis. BMI, body mass index; AST, aspartate aminotransferase; ALT, alanine aminotransferase. 378

3 RESULTS Table 1 shows the basic demographics of 425 CHC patients, of whom 264 (62.1%) were male. The average age was 49.0 ± 12.2 years and the mean BMI was 24.8 ± 3.60 kg/ m 2. The prevalence of hypertension, diabetes mellitus, and hyperlipidaemia were 16.0, 9.6, and 4.2%, respectively. The HCV genotype distribution was as follows: 1b in 246 (57.9%) patients, 2a in 140 (32.9%) patients, 2b in 33 (7.8%) patients, and unclassified in 6 (1.4%) patients. The mean levels of HCV viral load, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were 5.20 ± 1.23 logs IU/ml, 99.9 ± 77.8 IU/L, and ± IU/L, respectively. One hundred and thirty-two (30.8%) patients had hepatic steatosis, of which 113 (85.7%), 16 (12.1%), and 3 (2.3%) were graded as mild, moderate, and severe steatosis, respectively. Factors associated with hepatic steatosis and fibrosis: A comparison of the results of covariates between patients with and without steatosis is shown in Table 1. The mean BMI and triglyceride level of the steatosis group were significantly higher than those of the non-steatosis group (26.0 ± 3.20 versus 24.3 ± 3.66, < 0.001; and ± 36.6 versus 96.2 ± 29.9, < 0.001). The prevalence of obesity (BMI > 23 kg/m 2 ) was significantly higher in the steatosis group than the non-steatosis group (81.1 versus 62.1%, < 0.001). The presence of hepatic steatosis was not related to sex, age, cholesterol level, HCV viral load and genotype, hepatic enzymes or a history of hypertension, diabetes, or hyperlipidemia. In univariate analysis, hepatic fibrosis was found to be associated with age, the presence of hypertension, and serum AST levels (Table 1). The relationship between hepatic histopathology and steatosis is shown in Table 2. There was no difference in hepatic necroinflammatory activity between patients with and without steatosis. atients with hepatic steatosis had significantly higher rates of hepatic fibrosis than those without hepatic steatosis (84.8 versus 75.8%, = 0.035). The independent predictive values of age, sex, HCV genotype and viral load, hepatic necroinflammatory activity and fibrosis, hypertension, diabetes, triglyceride and cholesterol levels, BMI, and liver enzymes were determined by using multivariate logistic regression analysis. BMI, fibrosis, and triglyceride levels were significant factors correlating with hepatic steatosis in CHC patients (Table 3). Hepatic steatosis, age, and periportal bridging necrosis were significant factors related to hepatic fibrosis in CHC patients (Table 4). Table 2. Histopathologic presence in chronic hepatitis C patients Steatosis Steatosis 1) ( ) Steatosis 1) (+) No. (%) No. (%) Intralobular degeneration and focal necrosis 3) Score (91.1) 119 (90.2) 0.75 Score (8.9) 13 (9.8) ortal inflammation 3) Score (35.8) 51 (36.8) 0.58 Score (64.2) 81 (61.4) eriportal ± bridging necrosis 3) Score (71.7) 90 (68.2) 0.47 Score (28.3) 42 (31.8) Total necroinflammatory score 3) Score (90.8) 117 (88.6) 0.49 Score > 8 27 (9.2) 15 (11.4) Fibrosis stage 2) Stage 0 71 (24.2) 20 (15.2) ) Stage (75.8) 112 (84.8) 1) : See Table 1, footnote 1). 2) : See Table 1, footnote 2). 3) :Hepatic necroinflammatory grade was according to the histological activity index (HAI) score system. 4) : In univariate analysis, fibrosis stage was related to hepatic steatosis. Table 3. Multivariates logistic regression analysis for factors associated with hepatic steatosis in chronic hepatitis C patients Factor Comparison Odds ratio 95% confidence interval BMI <23 kg/m 2 = 0, 23 kg/m 2 = Hepatic fibrosis F0 = 0, F1-4 = Triglyceride per 1 mg/dl increase <0.001 BMI, body mass index. Table 4. Multivariates logistic regression analysis for factors associated with hepatic fibrosis in chronic hepatitis C patients Factor Comparison Odds ratio 95% confidence interval eriportal ± bridging necrosis score 0-1 = 0, score 3-10 = <0.001 Hepatic steatosis absence = 0, presence = Age per 1 year increase

4 DISCUSSION In our study, 30.8% of Taiwanese CHC patients had hepatic steatosis. This figure is similar to that previously reported in western countries (5,6). BMI is the strongest risk factor associated with hepatic steatosis, followed by hepatic fibrosis and age. Hwang et al. (7) have reported that 52% of patients with CHC had hepatic steatosis in northern Taiwan, and that average BMI values (24.5 ± 3.9) were higher in patients with hepatic steatosis. Leandro et al. (21) have reported a metaanalysis involving 10 centers in Italy, Switzerland, France, Australia, and United States in which 93% of the subjects were Caucasian. Their results also showed a higher prevalence of steatosis (50.9%) than that found in our study. BMI value and age were related to hepatic steatosis, and hepatic steatosis and age were correlated with hepatic fibrosis in CHC patients. The inconsistency in the prevalence rate of hepatic steatosis in CHC may be due to the fact that factors such as case number, residual area, obesity definition, clinical features, patients recruitment biases, and varying the inclusion/ exclusion criteria were different between our study and the other two studies. revious anthropometric data showed that for a given BMI, Asians (mostly Chinese) have more body fat in a more prominent central distribution than Caucasians (22-24). No similar data were studied in CHC patients, and it remains unclear whether ethnicity is associated with a different prevalence rate of hepatic steatosis in CHC. This topic requires further study. Day and James (25) proposed the two hits theory. The first hit is peripheral insulin resistance, which leads to increased lipolysis and delivery of free fatty acids (FFA) to the liver. These events may in turn result in triglyceride accumulation (26) and may lead to hepatic steatosis. The second hit is the accumulation of triglycerides in hepatocytes, which will increase fatty acid -oxidation which, in the presence of mitochondrial abnormalities, would result in free radical formation, consequent cell injury, inflammation, and fibrosis (27). The results of previous studies (7,10) and of ours indicate that triglyceride levels were correlated with hepatic steatosis, a finding that is consistent with Day and James theory (25). Furthermore, the increased risk of hepatic steatosis with increasing BMI may reflect increasing insulin resistance, which also causes intracellular -oxidation, mitochondria damage, and fat deposition within the hepatocyte (28). Our study and previous studies (7,21,29) showed that raised BMI in patients with CHC could be used as an indicator of risk for concurrent hepatic steatosis, even though the threshold of BMI values differed in different studies. It therefore appears that metabolic factors, especially higher BMI values, really do reflect an increased risk for hepatic steatosis in CHC patients, irrespective of whether or not they are Taiwanese or Caucasians. The presence of a history of diabetes mellitus, hypertension or hyperlipidemia was not associated with hepatic steatosis. A possible explanation for this is that these patients are on medication for these conditions, which alters their blood pressure, blood sugar, and lipid profile. In addition, the only a small number of such patients have been studied, especially patients with hyperlipidemia history. Our results showed that there was a relationship between age and hepatic steatosis. revious research has suggested that patients with both CHC and hepatic steatosis are more likely to have had CHC for a longer period than patients without hepatic steatosis. It therefore follows that the length of time of exposure to HCV can affect the incidence of hepatic steatosis (30,31). However, our results showed that patients with hepatic steatosis were younger than those without steatosis, and that age was not related to hepatic steatosis. These results differ from the report of Leandro et al. (21), which indicated that older age was associated with a higher risk of steatosis. We did not find evidence for possible causes that correlated with steatosis incidence, and further investigation is required to address this issue. In contrast, the mean age of patients with fibrosis was higher in our study. This result is similar to that found by Leandro et al. (21). We suggest that patients with longer periods of HCV infection have a higher rate of developing hepatic fibrosis, because slow and steady histopathologic progression (such as necroinflammation or fibrosis) is found in most untreated patients with chronic HCV infection. CHC may cause virus-induced inflammatory changes that lead to increased oxidative stress, increased peroxidation, increased production of cytokines, and ultimately, cell death (28,32). It has been noted that HCV genotype type 3 creates greater DNA oxidative damage, and therefore we thought that the genotype of hepatitis C might also be a possible factor influencing the progression to hepatic steatosis (6). Oxidative stress can induce the peroxidation of fat, which can lead to hepatic steatosis (5,8). HCV core protein can directly inhibit the function of microsomal triglyceride transfer proteins and the secretion of VLDL from hepatocytes (8,9). In this way, HCV core protein itself can induce oxidative stress and mitochondrial damage. Hepatic steatosis is thought to precede hepatic fibrosis irrespective of the HCV genotype causing hepatitis, but it is particularly common following genotype 3 CHC (21,33). Leandro et al. (21) showed that genotype 3 CHC was related to hepatic steatosis; however, HCV genotype 3 is very rare in Taiwan (12), and our results showed that the rate at which hepatic steatosis is present is similar between patients with genotype 1 and genotype 2. In addition, our study and Hwang et al. s study (7) pointed out that HCV viral load was not related to hepatic steatosis. Our study documents that virological factors play little role in hepatic steatosis in Taiwanese CHC patients, and that metabolic factors are important for this condition. revious studies have also indicated that patients with HCV infection and histological evidence of steatosis are at greater risk of progressing to hepatic fibrosis (34). Hwang et al. (7) have pointed out that necroinflammation is not specifically related to hepatic steatosis, but that the stage of hepatic fibrosis is generally more advanced in patients with hepatic steatosis compared to those without steatosis. Leandro et al. (21) and Yoon and Hu (33) believed that hepatic steatosis is an important and independent risk factor for developing hepatic fibrosis in CHC infection. However, one study performed by erumalswami et al. (10) suggested that hepatic steatosis was not related to hepatic fibrosis in CHC infection. In our study, hepatic steatosis was found to be related to fibrosis, and vice versa. Necroinflammatory conditions, such as intralobular degeneration and focal necrosis, portal inflammation, and periportal and/or bridging necrosis, were not significantly related to hepatic steatosis. However, we did not observe a positive correlation between the grade of hepatic steatosis and the severity of hepatic fibrosis in our CHC patients. This might be due to the fact that the number of cases of moderate and severe hepatic steatosis was too limited in our series for us to make a meaningful comparison between the severity of hepatic fibrosis and steatosis. The possible causes for patients with hepatic fibrosis having a 380

5 higher risk for steatosis are still unclear. We propose that hepatic fibrosis is multifactorial and is associated with older age, possibly due to the likelihood of a longer period of CHC infection and higher probable contact with factors that cause hepatic steatosis. The reasons for the different results concerning hepatic steatosis and fibrosis among many studies are not well understood. Further studies are needed to clarify the controversy. Furthermore, in our study, hypertension was found to correlate with hepatic fibrosis in univariate analysis, but not in multivariate analysis. We hypothesized that this may be due to an increased average age in the hepatic fibrotic group and a greater prevalence of hypertension in this group. In addition, hypertension was also not related to hepatic steatosis, which was related to hepatic fibrosis in our study. However, the fact that no association was found using multivariate analysis suggests that hypertension is not a risk factor for hepatic fibrosis in CHC patients. This issue should be studied further in the future. In conclusion, we demonstrated that 30.8% of Taiwanese patients with CHC had hepatic steatosis, with most of these cases graded as mild. BMI was the strongest risk factor associated with hepatic steatosis, followed by hepatic fibrosis and triglyceride level for CHC patients in Taiwan. ACKNOWLEDGMENTS This study was supported by grants from Taiwan Liver Research Foundation, Taiwan. REFERENCES 1. Angulo,. and Lindor, K.D. (2002): Non-alcoholic fatty liver disease. J. Gastroenterol. Hepatol., 17, Suppl., S Angulo,. (2002): Nonalcoholic fatty liver disease. N. Engl. J. Med., 346, Hourigan, L.F., Macdonald, G.A., urdie, D., et al. (1999): Fibrosis in chronic hepatitis C correlates significantly with body mass index and steatosis. 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