Clinical, Biochemical, virological and Histopathological Assessment of Hepatic Steatosis in Egyptian Patients with HCV genotype 4

and HCV Associated HCC

Mona M. Hassan*, Nihal M. El Assaly*, Naema I. El Ashry*, Mervat El Damarawy **,Shindy

M. Shindy***, and Tarek S Abou Shousha****

*Clinical Chemistry Department, ** I.C.U Department, *** Tropical Medicine Department,

**** Pathology Department at Theodor Bilharz Research Institute

Journal:-Egyptian Medical Journal Of The National Research Center; vol. 6 (1), June 2007:27-

37. ISSN 1687-1278

Abstract:

Liver steatosis is common in patients infected with hepatitis C virus (HCV), occurring in approximately 50% of biopsy specimens. This high prevalence suggests a specific interaction between HCV and lipid metabolism. The objective of this study was to assess some clinical, virological, histopathological and biochemical variables including serum adiponectin, leptin and beta-2- microglobulin (B2-MG) in relation to hepatic steatosis in Egyptian patients infected with HCV genotype 4 of early and late disease with cirrhosis and HCC.. Patients and Methodology: This study included a control group of 23 patients and 50 patients with HCV infection genotype 4 (group 1 of 25 HCV patients with early and active disease; and group 2 of 25 HCV cirrhotic patients associated with HCC) who were admitted to Theodor Bilharz Research Institute in Egypt. They were initially subjected to history; clinical examination, abdominal ultrasonography, liver function tests, HBs Ag, HCV-Ab and liver biopsy. Measurement of adiponectin, leptin, beta-2- microglobulin (done by ELISA) and lipid profile had been performed in all groups. Results: Steatosis of variable grades was detected in 80% of Egyptian patients with early active HCV genotype 4 infection and in 88% of those with cirrhosis and HCC. Steatosis was generally of higher grades in early active HCV than those with cirrhosis and HCC but the mean number of hepatocytes affected by steatosis were higher in cirrhotic patients but not significant (P>0.05). No correlation between body mass index (BMI) and the grade of steatosis in these patients (p = 0.236). No significant correlation between number of focal lesions, or their volumes and the degree of steatosis or histopathological grades; HAI (P > 0.05). Also, no significant correlation between number of focal lesions, or their volumes and all parameters studied including leptin, adiponectin and B2-MG. There was statistically significant higher viremia and HAI score in those with higher grades of steatosis than lower grades and in lower grades than those without steatosis (P < 0.05) and significant higher viremia in group 1 than group2 (P < 0.01). There were significantly lower serum triglycerides (P <0.05) and cholesterol (P < 0.01) (total, HDL, LDL) levels in both HCV groups than control group. Low triglycerides and cholesterol levels were significantly correlated with grade of steatosis (P < 0.05).There were significantly higher serum leptin (12.1± 7.35 & 11.75 ± 5..92 vs. 6.43 ± 4.13 ngm/ml) and beta 2 microglobulin (12.56 ± 3.44 & 4.73 ± 1.55 vs. 1.22) and lower adiponectin (10.40 ± 7.41 & 16.56 ±5.92 vs. 25.47 ± 12.38 micg/ml) in late and early HCV patients vs. control (P <0.001). Also, beta 2 microglobulin was significantly higher and adiponectin significantly lower in advanced patients with cirrhosis and HCC than early cases (P < 0.01). Significant correlations were found between beta 2-microglobulin concentration, adiponectin and grade of steatosis, HAI and fibrosis scores (P < 0.01). Leptin showed significant correlation only with grade of steatosis (P< 0.05). Age, level of alanine transaminase ALT (IU/L), low serum albumin, fibrosis, portal inflammation and lobular inflammation were significantly correlated with steatosis. In conclusion: In our Egyptian patients, HCV genotype 4 infection was associated with many factors that are highly linked to the development of steatosis such as high leptin and lower adiponectin serum levels. Also, beta 2 microglobulin which is a marker of more severe disease and poor outcome was significantly higher than control patients. Also steatosis is commonly seen in this genotype and its grade was associated with higher viral load, more severe inflammation and fibrosis. The steatosis in

genotype 4 is probably a genotype or viral-related process with all the biochemical changes secondary to it and these changes may re-perpetuate and exaggerate the process. The significant increase of leptin and

beta-2- microglobulin and decrease in adiponectin in HCV patients with cirrhosis could play a role in the pathogenesis of steatosis and progression of liver disease. Finally, steatosis should be considered a target for therapeutic interventions in addition to the viral and inflammatory process of the liver in such patients . Key words: Steatosis, adiponectin, B2-MG, liptin and HCV infection

Introduction:

Steatosis is defined as fat, largely triglyceride, exceeding 5% of the liver weight. It is

caused by failure of normal hepatic fat metabolism either due to a defect within the

hepatocyte or to delivery of excess fat, fatty acid or carbohydrate beyond the secretory

capacity for lipid of the liver cell. Liver biopsy and imaging procedures, such as ultrasound

and CT, are increasing the number of patients being identified with excess fat in the liver.

Liver biopsy is the best method of diagnosing steatosis, it is also the only method to

differentiate steatosis from non-alcoholic steato-hepatitis (NASH).1

Non-alcoholic fatty liver disease (NAFLD) is the term used to describe a spectrum of

disorders characterized by macrovesicular steatosis that occur in the absence of consumption

of alcohol in amounts considered to be harmful to the liver .2

Hepatitis C virus (HCV) and nonalcoholic fatty liver disease (NAFLD) are the two most

common causes of chronic liver disease.3 The spectrum of NAFLD consists of isolated

hepatic macrovesicular steatosis at one end and steatohepatitis at the other one.4, 5 Sustained

liver injury will lead to progressive fibrosis and cirrhosis in 10% to 25% of affected

individuals.2

Hepatic steatosis is frequently seen in subjects with HCV; occurring in approximately

50% of biopsy specimens. Also, the presence and severity of hepatic steatosis has emerged as

an important marker of progressive liver disease as well as virologic response to anti-HCV

therapy, it may be caused directly by the virus, as in genotype 3 infection, or be associated

with host metabolic factors.6,7 One study showed that genotype 3 was associated with greater

hepatic steatosis and inflammation than genotype 1. It was found specifically associated with

insulin resistance that was associated with a decrease in total and high molecular weight

(HMW) adiponectin levels.8 Another study found that infection with HCV genotype 3 was

associated with hepatic steatosis that appears to be a viral effect rather than a host effect, but

in most cases, with other genotypes, steatosis in hepatitis C represents the presence of 2

separate liver conditions.9

Obesity is a well-recognized risk factor for the development of steatosis in chronic

hepatitis C infection. The interaction of hepatitis C virus core protein with the lipoprotein

secretion pathways causes the characteristic alterations of lipid metabolism observed in

hepatitis C virus-related steatosis. Steatosis is a major determinant of the liver damage

progression in chronic hepatitis C (CHC), more severe or advanced hepatic fibrosis and

negatively affects the response rate to the anti-viral treatment with pegylated interferon and

ribavirin.10 Moreover, recent evidence suggests that steatosis may contribute to liver

carcinogenesis. Also, chronic hepatitis C is a recognized risk factor for type 2 diabetes and it

could be implicated into the pathogenesis of atherosclerosis. The role of hepatitis C virus

(HCV)-related steatosis in these epidemiological associations remains to be determined.5

Adiponectin is a key anti-inflammatory adipocytokine that may oppose TNF-alpha

activity in the liver, has been shown to have antifibrotic properties by reducing hepatic

stellate cell proliferation and increasing apoptosis.11 Plasma adiponectin concentrations are

reduced in patients with NASH and patients with increased insulin resistance.12 Also, HCV

genotype 3 was found specifically associated with insulin resistance that was associated with

a decrease in total and high molecular weight (HMW) adiponectin levels.8 Lack of

adiponectin enhanced the progression of NASH and exacerbated oxidative stress in mice.13

On the other hand, leptin was found to be higher in NAFLD or steatosis than in normal

controls.14 It may act as a mediator of hepatic inflammation and fibrosis, while low

adiponectin levels may contribute to the development of necroinflammation.15 Another

study in patients with both chronic HCV and steatosis showed a serum adiponectin/TNF-

alpha imbalance (with low adiponectin and high TNF-alpha) that was also associated with

insulin resistance.16 Thus, reduced adiponectin and increased leptin levels may be involved

in the pathogenesis of steatosis, which in turn accelerates progression of fibrosis in chronic

HCV.

The beta 2 microglobulin (b2-MG) concentration was also found to be elevated in HCV

in comparison to the normal values and IFN-alpha therapy caused an increase in the b2-MG.

The highest increase was observed among patients who did not eliminate the virus. A

significant increase in b2-MG during interferon therapy in patients with chronic HCV

infection is a predictor of poor outcome. Also, patients with HCC showed higher serum beta

2MG levels than did chronic hepatitis C patients or healthy subjects.17

The adipocyte-derived anti-obesity hormone leptin may prevent fat accumulation in the liver. It

was found that leptin deficiency in transgenic mice and in humans with lipodistrophies induced

severe liver steatosis (LS) and insulin resistance that are responsive to the administration of

exogenous leptin.18,19,20 In addition, hyperleptinemia has been observed to correlate with LS in

subjects with NASH and chronic hepatitis C, but the exact role of hyperleptinemia in the

pathogenesis of human NAFLD remains controversial.21,22,23,24

Finally, the presence of steatosis was found to impair the early reduction of viral load during

treatment in patients infected with HCV genotype 3 and non-3. Steatosis also, negatively affected

the final outcome of treatment mainly in patients infected with HCV genotype non-3 virus. Thus

interventions aiming at reducing hepatic steatosis prior to the onset of antiviral therapy may be of

benefit to patients infected with HCV.25 The steatosis in these patients was typically improved

following successful antiviral therapy.26,27,28

Aim of our study:

The aim of this work is to assess some clinical, virological, histopathological and biochemical

variables including serum adiponectin, leptin and beta-2- microglobulin in relation to hepatic

steatosis and liver disease severity, in Egyptian patients infected with HCV genotype 4 of early

and late disease with HCC.

Patients and Methodology:

This study included 73 individuals classified as following:

a) Control group: included 23 healthy individuals.

b) Group 1: early active HCV patients: included 25 patients with early HCV infection

genotype 4 who are not cirrhotic.

c) Group 2: cirrhotic with HCC: included 25 HCV cirrhotic patients associated with HCC

All were admitted to Theodor Bilharz Research Institute in Egypt. They were initially

subjected to history; clinical examination, abdominal ultrasonography, liver function tests,

HBs Ag and HCV Ab. Measurement of adiponectin, leptin, beta-2- microglobulin (done by

ELISA) and lipid profile had been performed in all groups.

Exclusion criteria: patients who underwent this study must not be having diabetes, alcohol

consumption, drugs causing steatosis such as NSAID’s, salicylate, corticoid, valproic acid,

amiodarone , perhexiline maleate, which are known to affect lipid metabolism at least for 6 weeks

and anti viral therapy. All HCV of genotypes other than genotype 4 were excluded from this

study. In addition, control group patients must be healthy and not obese.

Inclusion criteria were: increased serum amino- transferases levels, positive serum anti

HCV and HCV RNA, liver biopsy compatible with HCV diagnosis in all patients and HCC

on top of HCV in those patients with a focal lesion discovered by ultrasound. Also, 23

apparently healthy subjects, matched with patient’s age and sex, were enrolled in this study

as a reference or control group.

All the members of this study were subjected to clinical evaluation; including detailed

history and clinical examination; for all the patients with special emphasis on the possible

duration and stigmata of the HCV hepatic affection. Ultrasonography was done by an

ultrasound machine Hitachi EUB 515 A, using a convex linear transducer 3.5 MHz. Liver

steatosis (LS) was graded on a scale of 0–3 (0, absent; 1, mild; 2, moderate; 3, severe)

according to Saverymuttu et al.(1986).29 on the basis of abnormally bright echoes arising

from the hepatic parenchyma, difference in echo amplitude between liver, kidney and

pancreas, echo penetration into the deeper portion of the liver and clarity of the liver blood

vessel structure. Assessment of the size, surface, echogenicity and the presence or absence of

a focal lesion was done.

In chronic HCV patients, biopsy was done by a Hepafix needle 14 mm in diameter and

the biopsy was done according to the Menghini technique. In patients with liver cirrhosis

associated with focal lesion, two biopsies were performed. In addition to the biopsy taken by

the Menghini technique, an ultrasound guided biopsy was performed from the hepatic focal

lesion using 0.9 mm thin cutting edge needle (SBK – Angiomed – Germany). All biopsy

specimens were immersed in 10% formol and sent for histopathological analysis.

Pathological assessment was made on liver sections from formalin-fixed and paraffin

embedded liver biopsies stained with hematoxylin-eosin and Masson's trichrome stains.

According to Desmet et al. (1994)30, we considered the scores for necroinflammatory

changes and architectural alterations separately (12). The necroinflammatory activity was

scored according to the Histology Activity Index (HAI) by Knodell et al. (1981).31 The HAI

was determined by combining the scores for portal inflammation (0-4), lobular degeneration

and necrosis (0-4) and periportal necrosis (0-10). The stage was defined according to the

Scheuer fibrosis score (13, 14): 0: absence, 1: enlarged fibrotic portal tracts, 2: periportal or

portoportal septa but intact architecture, 3: fibrosis with architectural distortion, but not

obvious cirrhosis, 4: probable or definite cirrhosis. Steatosis was graded as follows: 0: absent,

1: 1-10% of hepatocytes affected, 2: 11-30% of hepatocytes affected, 3: 31-60% of

hepatocytes affected and 4: more than 60% of hepatocytes affected.

Blood samples were collected under aseptic conditions. All subjects were fasting

overnight for at least 10 hrs before sampling. Serum was separated from the other contents

and stored at –70 C until assayed. The following investigations had been done:-

- Liver function tests (Serum albumin, direct bilirubin, AST, ALT and protein) using

standard laboratory methods.

- Hepatitis markers HBsAg an HCV Ab by (ELISA) and HCV RNA by (PCR).

- Lipid profile: using standard laboratory methods to estimate serum (triglyceride, total

cholesterol, low density lipoprotein ((LDL)) and high density lipoprotein ((HDL)) by

dimension autoanalyser.

- Serum adiponectin by ELISA technique.

- Serum Beta -2 Microglobulin by ELISA technique.

- Serum lipitin by ELISA technique.

- Alph-feto protein by ELISA technique

Statistical analysis:

Medians were compared using the median test; continuous variables were expressed as

mean SD and were compared by using student’s t-test or correlated by using simple

regression. The degree of steatosis to the following parameters: age, disease duration, alanine

aminotransferase (ALT) activity, serum albumin, cholesterol, TG, adiponectin, beta-2

microglobulin, liptin, level of fibrosis, portal or lobular inflammation. Differences were

considered significant if P< 0.05.

Results:

This study included a reference group of 23 healthy subjects (reference group) and 50 patients

with chronic hepatitis C genotype 4 group of 25 were pure early active HCV (group1) and

cirrhotic with HCC group of 25 (group2). The clinical, sonographic and virological

characteristics of the studied groups compared to the reference group are shown in table 1 and

table 2. Table 3 shows the clinical, sonographic, biochemical and histopathological

characteristics features of the focal lesions in group 2. Table 4 and 5 show the liver function

tests and lipid profile in the two studied groups and the reference group. Table 6 shows the

serum levels of our non invasive new parameters for steatosis Serum leptin, adiponectin and

Beta 2 microglobulin in all studied groups. Table 7 shows the histological findings including

the percentage of steatosis of the studied groups and the reference group. Age, disease

duration, ALT, Albumin, Cholesterol, TG, Fibrosis, portal inflammation and lobular

inflammation are the parameters significantly related with steatosis in our analysis. TG was

inversely related with the percentage of steatosis. Fibrosis and lobular inflammation were

positively related with the percentage of steatosis showing a statistical significance where P <

0.05 on both groups compared to the reference group. Fibrosis was the factor independently

linked to the degree of steatosis. Tables 8 and 9 shows the histopathological parameters

related to steatosis of the studied groups compared to the reference. picture.1 showing the

ultrasonograghic echo of steatotic patient. Fig1 showing grade 2 hepatitis activity showing

mild steatosis activity, fig2 showing grade 3 hepatitis activity showing moderate steatosis

activity, fig3 showing high power of fig 2 showing micro and macrovesicular hepatocytes

steatosis and fig4 showing high severity of hepatocytic steatosis.

Table 1: Clinical and ultrasonographic data of the studied groups

Reference Group 1 Group 2

No of patients 23 25 25

Age 40.23 ± 14.54 39.81 ±12.22 45.14 ±11.93

Body mass index (BMI) 25.56 ± 3.25 24.71 ± 4.36 22.83 ± 5.16

Sex (Male : Female) 14 : 9 18 : 7 19 : 6

Clinical Examination

Hepatomegaly

Splenomegaly

Ascites

-

-

-

20*

4

0

11

19*

7

Ultrasound Hepatic Picture

-Bright (Fatty)

-Fibrofatty (bright coarse)

-Coarse

-Other US signs of cirrhosis #

-Focal lesions

2

-

-

-

-

17

7

1

3

-

5

13

7

25

25

HCV Genotype 4: - 25 25

# These include, among others, shrunken liver, hypertrophied caudate lobe, dilated portal vein, porto-systemic collaterals, attenuated hepatic veins, irregular surface and borders. * P <0.05.

Table 2: Ultrasonographic grading of steatosis:

Reference Group 1* Group 2

Absent (grade 0) 0 4 6

mild (grade 1) 1 4 13

Moderate (grade 2) 1 10 8

Severe ( grade 3) 0 7 0

All groups are comparable as regards to age and sex distribution. Hepatomegaly was significantly more in patients of group 1 with early active hepatitis than those with cirrhosis and HCC. Splenomegaly on the other hand was significantly more in cirrhotic group 2. Steatosis as detected by ultrasound examination was generally of higher grades in early active HCV than those with cirrhosis and HCC. No correlation between BMI and the grade of steatosis (p = 0.236).

Ultrasonograghic echo of steatotic patient

Table 3: Some ultrasonographic, biochemical and histopathological characteristics of HCC in group

2:

One focal lesion Two focal lesions >2 focal lesions Total

Number of patients 8 13 4

Range of diameters (cm)

Mean volume Of lesions (cm3)

1.1-8.5

31.54 14.34

1.0- 11.2*

42.32 16.85*

1.2-12.8

67.64 20.26*#

Echogenecity

Isoechoeic

Hypoechoeic

Hyperechoeic

2

4

2

3

8

2

1

3

0

6

15

4

Site of focal lesion in liver

o Rt. Lobe only

o Lt. lobe only

o Both lobes

6

2

0

6

4

3

1

0

3

14

8

6

Portal vein thrombosis 1 5 2 8

Ascites 1 3 3 7

Perihepatic lymph nodes 2 2 3 7

Distant metastasis 0 0 1 (lungs) 1

Alph-feto protein 526.34 205.25 985.18 282.69* 1234.65

274.14*#

915.39359.28

Histopathology:

o Well-differentiated

o Moderately differentiated

o Poorly differentiated

3

2

3

6

3

4

2

2

0

11

7

7

* Significant difference in comparison with patients with one focal lesion (P < 0.05) # Significant difference in comparison with patients with two focal lesions (P < 0.05)

There was significant direct correlation between the number of focal lesions, the total volume of the focal lesions in each patient and alpha fetoprotein level (P < 0.05) but not with other features. No significant correlation between number of focal lesions, or their volumes and the degree of steatosis or histopathological grades; HAI (P > 0.05). Also, no significant correlation between number of focal lesions, or their volumes and all parameters studied including leptin, adiponectin and B2-MG.

Table 4: Liver function tests in the controls and the studied groups:

reference group 1 group 2

Albumin ( g/L ) 42 3.52 32 4.9 * 30 4.3**

Protein ( g/ L ) 78 3.2 67 4.8 * 66 4.1**

ALT ( IU / dL ) 10 2.1 24.9 14.3* 13 8.1

AST ( IU /dL ) 8 2.5 26.8 8.7** 17.4 11.5

Direct bilirubin ( mg/dl ) 0.6 0.23 2.24 0.86*** 2.0 0.8**

*P< 0.05, ** P< 0.01, *** P < 0.005; compared to the reference group.

Normal values of ALT and AST: up to 12 IU/dl. There were significantly lower serum albumin and proteins levels in HCV patients (which are highly significant in group 2); significantly higher Liver enzymes in early active HCV patients and significant hyperbilirubinemia in both groups in comparison with control group.

Table 5 Serum lipid profile in the controls and the studied groups.

Lipid parameter Reference group 1 group 2

TG ( mg / dl ) 120 16.5 100 17.9* 100 15*

Cholesterol ( mg / dl ) 163 13.3 145 12.3** 139 13.9**

HDL ( mg /dl ) 45.9 4.2 35 4.5* 34 4.2*

LDL (mg /dl ) 84.9 13 72.9 1.5** 73 13**

*P< 0.01 and ** P< 0.05; compared to the reference group. There were significantly lower serum triglycerides and cholesterol (total, HDL, LDL) levels in Both HCV groups than control group. Low triglycerides and cholesterol levels were significantly correlated with grade of steatosis (P < 0.05).

Table 6: serum levels of leptin, adiponectin and Beta 2 microglobulins in all groups.

Leptin (ng/ml) Adiponectin (microg/ml) Beta 2 Microglobulin

control Early HCV

Cirrhosis & HCC

control Early HCV

Cirrhosis & HCC

control Early HCV

Cirrhosis & HCC

Mean 6.43 11.75 12.1 25.47 16.56 10.40 1.22 4.73 12.56

SD 4.13 5.92* 7.35* 12.38 5.92* 7.41*# .076 1.55* 3.44*#

* Significant difference in comparison to control group (P< 0.001). # Significant difference in comparison to early HCV patients (P < 0.01). Significant correlations were found between beta 2-

microglobulin concentration, adiponectin and grade of steatosis, HAI and fibrosis scores (P < 0.01). Leptin showed significant correlation only with grade of steatosis (P< 0.05).

Age, level of ALT (IU/L), low serum albumin, serum cholesterol, low TG and chlesterol, high serum leptin, low serum adiponectin, high B2-MG, fibrosis, portal inflammation and lobular inflammation were significantly correlated with steatosis in our analysis.

Table 7: The histopathological parameters of the studied groups.

score Reference group 1 Group 2

Portal inflammation 0.33 ± 0.41 2.4 ± 0.73* 2.21 ± 0.85*

Lobular degeneration & necrosis 0.11 ± 0.05 1.3 ± 0.62* 1.83 ± 0.37*

Periportal necrosis 0 5.36 ± 2.81* 6.14 ± 2.67*

Fibrosis 0 3.1 ± 0.45* 4.55 ± 1.64*+

Hepatocytic Steatosis (mean % )

Grade 1

Grade 2

Grade 3

grade 4

Number of patients with steatosis /total number

3.4±2.18

2 (100%)

0 (0%)

0 (0%)

0 (0%)

2/23

37.7±14.32*

3(15%)

8(40%)

7(35%)

2(10%)

20/25 (80%)

40.3±10.25*

4 (18.18%)

9 (40.9%)

7 (31.8%)

2 (11%)

22/25 (88%)

*Significant. difference from the control group (P < 0.002). The mean number of hepatocytes affected by steatosis was higher in cirrhotic patients but not significant (P>0.05)

Table 8: Relation of HAI and viremia to the grades of steatosis in group 1 patients:

Grade of Hepatocyte Steatosis

0 1-2 3-4

Number of patients

HAI total score (Mean ± S.D)

Portal inflammation

Lobular degeneration

Periportal necrosis

Fibrosis score

Viremia (copies/ml)

0

3.25 ± 0.62

2.52 ± 0.35

0.19 ± 0.12

1.05 ± 0.27

1.21 ± 0.32

651500

±1075928.53

9

5.08 ± 0.34*

2.86 ± 0.24*

1.13 ± 0.32*

1.83 ± 0.58*

1.93 ± 0.62*

1321500

±1173465.71*

13

5.94 ± 0.77*#

3.14 ± 0.97*

1.52 ± 0.81*

2.64 ± 0.59*#

2.87 ± 0.68*#

2550200

±1345645.60*#

*Significant difference with grade 0 (P<0.05). # Significant difference with grade 1-2 (P<0.05).

There was statistically significant higher viremia in those with higher grades of steatosis than lower grades and in lower grades than those without steatosis (P < 0.05).

Table 9: Relation of HAI and viremia to the grades of steatosis in group 2 patients:

Grade of Hepatocyte Steatosis

0 1-2 3-4

Number of patients 5 13 7

HAI total score (Mean ± S.D)

Portal inflammation

Lobular degeneration

Periportal necrosis

Fibrosis score

Viremia (copies/ml)

3.87 ± 0.25

2.45 ± 0.87

0.7 ± 0.13

1.82 ± 0.44

3.02 ± 0.12

412500

± 536780.55

5.76 ± 0.10*

3.13 ± 0.74*

1.25 ± 0.18*

2.66 ± 0.73*

3.37 ± 0.83*

449400

± 596733.61

6.49±0.18*#

3.74±0.38*#

1.56±0.37*#

2.72±0.95*

3.65±0.54*

956300

± 715661.61#

*Significant difference with grade 0 (P<0.05). # Significant difference with grades 1-2 (P<0.05). There was statistically significant higher viremia in group 1 than group2 (P = 0.01). There was statistically significant higher viremia in those with steatosis grade 3-4 than lower grades 2-1 (P < 0.05) but insignificant statistically between lower grades than those without steatosis (P = 0.062).

Steatosis was mild in 30 % of patients, moderate in 32 % and severe in 12 % of patients.

Age, disease duration, ALT (IU/L), Albumin, Cholesterol, TG, adiponectin, liptin , Beta-2

microglobulin, Fibrosis, portal inflammation and lobular inflammation are the parameters

significantly related with steatosis in our analysis. TGs were inversely related with the

percentage of steatosis. Fibrosis and lobular inflammation were positively related with the

percentage of steatosis showing a statistical significance where P<0.05 on both groups

compared to the reference group. Adiponectin, liptin, and beta-2 microglobulin

concentrations and fibrosis were the factors independently linked to the degree of steatosis.

Adiponectin and beta-2 microglobulin show a positive correlation with the degree of

steatosis, portal inflammation, periportal necrosis and fibrosis with a high significant increase

of there level in both groups.

Discussion:

Study of liver steatosis with chronic hepatitis C is confused by the probable

coexistence of two distinct entities: some patients may have steatosis independently of HCV

infection, secondary to common causes such as obesity, diabetes or alcohol consumption,

while others may have steatosis as a direct consequence of HCV infection.32

This study was performed on Egyptian patients with early active HCV infection and those

with late cirrhosis and having hepatocellular ca1rcinoma to assess the clinical, virological,

histopathological and biochemical parameters that may have some relation to hepatic steatosis.

Those with Genotype 4 only were selected to find the reflection of this genotype on the

pathogenesis, degree of steatosis and liver disease severity. Many studies had been performed

in the same manner but in different genotypes and found important relation between steatosis,

severity of liver disease, viral load and response to treatment.33, 34, 35, 36, 37, 38 The steatosis in

these patients was found to be improved following successful antiviral therapy.39, 40, 41

Histopathological examination of liver biopsy was the most sensitive and specific tool to

diagnose the extent and degree of steatosis in addition to the necro-inflammatory and fibrotic

effect of the virus. It appears from this study that the grade or the severity of steatosis

decreased with advancement of the liver disease despite the more number of cells affected as

detected by both ultrasound examination and histopathology. This was also associated with

more enlargement of the liver due to inflammation, more lipid accumulation in the liver and

less fibrosis in early active disease.

Also, it was found in this study that there was statistically significant higher viremia in

those with higher grades of steatosis than lower grades and in lower grades than those without

steatosis (P < 0.05). These findings were also detected in previous studies particularly with

genotype 3.33 This might mean that steatosis can be directly related to the pathogenesis of

these viruses. Thus, viral steatosis may be more commonly seen in subjects infected with HCV

genotype 3 and 4 than other genotypes and the extent of steatosis in these patients is associated

with high viral load. In contrast, steatosis in patients infected with genotypes other than 3 and 4

may be linked to obesity and metabolic disorders including peripheral insulin resistance which

resembles nonalcoholic steato-hepatitis.33, 35, 36, 37

In group 2 patients with cirrhosis and HCC, more than half of patients had two focal

lesions. In general, the number and total volume of lesions showed significant direct

correlation with serum alpha fetoprotein but not with the grades of steatosis and HAI score.

Also, with more and larger focal lesions, more complications were detected such as ascites,

portal vein thrombosis and perihepatic lymph nodes. However, because of small number of

patients, these differences did not reach statistical significance. Of course, there were

significant hypoalbuminemia and hyperbilirubinemia in patients with chronic HCV in relation

to reference group.

In this study also, it was found that the body weight as indicated by body mass index was

not correlated with the degree of steatosis in chronic HCV patients. Thus, in these patients,

obesity might have no role in the pathogenesis of steatosis. Also, it was found that there were

significantly lower serum triglycerides and cholesterol (total, HDL, LDL) levels in Both HCV

groups than the reference group. Low triglycerides and cholesterol levels were significantly

correlated with grade of steatosis (P < 0.05). We think that these findings were probably due to

dietary habit and to a less extent due to liver dysfunction itself. It is well known habit that most

Egyptian patients with chronic liver diseases restrict fat intake and lose weight. Also, it could

be due to decrease in the levels of plasma lipoproteins as it is well-known that most plasma

apolipoproteins, endogenous lipids and lipoproteins are synthesized in the liver which depends

on the integrity of cellular functions of liver. 42, 43, 44 It was also found that hepatic cellular

damage and HCC impaired these processes, leading to alterations in plasma lipid and

lipoprotein pattern. Also, HDL and its major apolipoproteins, apoAI and apoAII, are frequently

reduced in the patients suffering from cirrhosis or HCC.45 Also, the decrease in the level of

serum LDL cholesterol in patients with liver disease was significantly correlated to the

increasing severity of the disease.46, 47, 48 and 49 Also, decreased serum levels of cholesterol and

apoAI may indicate a poor prognosis.49, 50, 51

One of our main findings in this study was that the HAI and fibrosis scores were

significantly higher in those with higher grades of steatosis. Thus, steatosis was associated with

more severe liver disease. It is assumed from this study that steatosis should be considered as a

risk factor in our patients with HCV genotype 4 and should be considered as a target for

therapeutic interventions in addition to the viral and inflammatory process of the liver. This is

supported also by the second most important finding of higher viremia present in those with

higher grades of steatosis. Thus, this study and previous studies showed that steatosis is a

major determinant of the liver damage progression in chronic hepatitis C, more severe or

advanced hepatic fibrosis and negatively affects the response rate to the anti-viral treatment

with pegylated interferon and ribavirin.10 Moreover, recent evidence suggests that steatosis

may contribute to liver carcinogenesis.52

In this study, serum beta 2 microglobulin levels showed statistically significant higher

levels in those with advanced cirrhotic liver disease and HCC than those with early disease and

in both HCV groups than the reference group. In our study also, there was significant

correlation between B2-MB serum levels and both HAI and fibrosis scores (P = 0.0051 and

0.011). Previous studies showed similar findings in HCV patients in comparison to the normal

values. Also, IFN-alpha therapy caused more increase in the B2-MG. The highest increase was

observed among patients who did not eliminate the virus.17 It was found that significant

increase in B2-MG during interferon therapy in patients with chronic HCV infection was a

predictor of poor outcome. Similar to our results also, previous study showed that patients with

HCC had higher serum B2-MG levels than did chronic hepatitis C patients or healthy

subjects.17 It was assumed in this study that the increase in the beta2MG serum level reflects

the tumor size and seems to be a consequence of the stimulation of hepatocytes by humoral

components of immunological response, such as IL-6. Weakening of the immune system, due

to IL-6, may be responsible for a more severe progression of HCC and the hyperexpression of

beta2-MG.17

Adiponectin was found in this study to decrease significantly in HCV patients either early

or late and complicated and this decrease was correlated with the degree of liver affection

whether by steatosis, inflammation or even fibrosis. Many studies demonstrated low

adiponectin levels in patients with steatosis. The severity of steatosis was inversely correlated

with adiponectin levels. HCV genotype 3-infected patients also, showed lower adiponectin

levels than those with other genotypes. An independent predictor of low adiponectin levels in

genotype 3 infection was the severity of steatosis. It was concluded that reduced adiponectin

levels may be involved in the pathogenesis of steatosis, which in turn accelerates progression

of fibrosis in chronic HCV. In chronic HCV patients, hypoadiponectinemia is significantly

associated with the development of liver steatosis. The fact that the plasma levels of

adiponectin inversely correlate with steatosis in HCV-infected subjects suggests that

hypoadiponectinemia may contribute to hepatic steatosis progression and liver injury in this

population. Other data which supports such assumption are that therapy to increase circulating

adiponectin concentration, such as overweight reduction or thiazolidinediones, provides the

potential to improve steatosis in chronic hepatitis C infection, 14,16 and that the logarithmic

serum adiponectin level was inversely correlated with the logarithmic levels of serum AST,

ALT and gamma GT.54 Also, the change of adiponectin levels still significantly related to the

response to IFN-alpha. In one more study, high HCV load and genotype 2 were significantly

associated with a lower serum adiponectin level.53

On the other hand, serum leptin level was found in this study to increase significantly with

the increase of steatosis and the progression of liver disease. However, its level showed

correlation only with the grade and severity of steatosis but not with inflammation or fibrosis.

In patients with chronic hepatitis C and steatosis, results of leptin serum levels in different

studies were controversial. One study showed values in these

. patients similar to healthy individuals and no relationship was found between leptin levels

and severity of steatosis.55 Other studies found that hepatic steatosis was related to genotype,

fibrosis degree, and serum leptin levels. Genotype 3 seems to have a viral specific steatogenic

effect. Leptin seems to be a link between obesity and steatosis development in HCV genotype

1–infected patients as it was found that there were correlations between HCV, leptin, body

mass index, percentage of body fat, and visceral obesity.56 In other studies, it was found that

chronic hepatitis C patients with and without steatosis were similar with respect to their serum

glucose, lipid and leptin levels and serum leptin levels were correlated with obesity and with

liver transaminases only in patients with steatosis. Also, it was found that chronic hepatitis C

patients with or without steatosis had similar leptin levels. Thus it was concluded from this

study that leptin levels were well correlated with anthropometrical parameters in chronic

hepatitis C patients and leptin levels were associated with evidence of impaired hepatic

function and may be a prognostic marker in patients with chronic HCV related steatosis.57 also,

it can have a role in the regulation of hepatic fibrosis.56

Conclusion: In our Egyptian patients, HCV genotype 4 infection was associated with many

factors that are highly linked to the development of steatosis such as high leptin and lower

adiponectin serum levels, and beta 2 microglobulin which is a marker of more severe disease

and poor outcome. Steatosis is commonly seen in genotype 4 and its extent or grade was

associated with high viral load, more severe inflammation and fibrosis. The pathogenesis of

HCV-related steatosis in general is likely to be multifactorial; probably genotype or viral-

related process with all the biochemical changes secondary to it. These factors may re-

perpetuate and exaggerate the pathological conditions. It is assumed from this study that

steatosis should be considered as a risk factor in our patients with HCV genotype 4 and should

be considered as a target for therapeutic interventions in addition to the viral and inflammatory

process of the liver. Further investigations are needed to prove the effect of such therapeutic

intervention on the prognosis of such patients.

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التقييم السريرى والكيمائى و الفيروسى والنسيجى لمرض التشمع الكبدى فى المرضى المصريين طان الكبدوكذلك المصاحب لسر ٤المصابين بالفيروس الكبدى ج النوع الوراثى

منى حسن و نهال العسالى ونعيمة العشرى و ميرفت الدمراوى وشندى محمد شندى وطارق أبو شوشة.

قسم الكيمياء االكلينيكية و قسم الرعاية المركزة و قسم األمراض المتوطنة وقسم الباثولوجى معهد تيودور بلهارس لألبحاث

ملخص البحث:

فى عينات الكبد. وهذا إنما يدل %05المصابين بالفيروس الكبدى ج وقد يصل إلى إن التشمع الكبدى شائع فى المرضى

على تداخل خاص بين هذا الفيروس والتمثيل الغذائى للدهون. وقد كان الهدف من هذا البحث هو تقييم بعض العوامل

وعالقتها بالتشمع ٢كروجلوبيولين بالسريرية والكيمائية و الفيروسية والنسيجية بما فى ذلك االديبونكتين واللبتن والمي

المبكر وكذلك المصاحب للتليف ولسرطان ٤الكبدى فى المرضى المصريين المصابين بالفيروس الكبدى ج النوع الوراثى

الكبد.

٤مريضا مصابين بالفيروس الكبدى ج النوع الوراثى ٠٥شخصا و ٢٢وقد شمل هذا البحث مجموعة مقارنة مكونة من

مريضا بالتليف والمصاحب ٢٠مريضا بااللتهاب الكبدى المبكر النشط والثانية ٢٠لى مجموعتين: األولى ومقسمين ا

تم فحص المرضى وعمل الفحوص ومتابعة و قدلسرطان الكبد والذين تم حجزهم بمعهد تيودور بلهارس لألبحاث بالقاهرة.

ى الدم وأثاره على الكبد بما فى ذلك وظائف الكبد وخزعة المرضى بالتحاليل الالزمة للفيروس بجميع دالالته ومستواه ف

وجميع أنواع الدهون فى الدم. ٢االديبونكتين واللبتن والميكروجلوبيولين بالكبد والموجات فوق الصوتية وقياس مستوى

ن بالفيروس من المرضى المصريين المصابي ٪٠٥التشمع الكبدى بدرجاته المختلفة يوجد بنسبة وقد أثبت هذا البحث أن

وان كان عدد الخاليا من المصابين بالتليف والمصاحب لسرطان الكبد ٪٠٠المبكر النشط وفى ٤الكبدى ج النوع الوراثى

المتأثرة كان أكثر فى حاالت التليف عنها فى المرض المبكر النشط ولكن بدون داللة إحصائية. كما كان ال يوجد عالقة بين

وال بين عدد األورام أو حجمها وبين جميع العوامل التى تم شمع أو درجة االلتهاب النسيجيمعدل كتلة الجسم ودرجة الت

. وكان عدد الفيروس فى الدم أعلى علوا ذى داللة ٢دراستها بما فى ذلك االديبونكتين واللبتن والميكروجلوبيولين ب

رجات أقل إحصائية فى المرضى الذين عندهم درجات أعلى من التشمع عن الذين عندهم درجات أقل وعند الذين عندهم د

عن الذين ليس لديهم تشمع وفى المرض المبكر النشط عن مرضى التليف والسرطان. وكان مستوى الدهون الثالثية

والكولسترول أقل بدرجة ذى داللة إحصائية فى مرضى الفيروس الكبدى ج عن مجموعة المقارنة. وكان معدل انخفاض

. وكان هناك زيادة ذو داللة إحصائية فى كل من اللبتن الدهون الثالثية والكولسترول مرتبطا بدرجة التشمع

وانخفاض ذو داللة إحصائية فى االديبونكتين فى مرضى الفيروس الكبدى ج عن مجموعة ٢والميكروجلوبيولين ب

ى وانخفاض ذو داللة إحصائية فى االديبونكتين ف ٢المقارنة. وكان هناك زيادة ذو داللة إحصائية فى الميكروجلوبيولين ب

وقد وجد أن مستوى اللبتن مرضى الفيروس الكبدى ج المتليف والمصاحب بسرطان الكبد عن الحاالت المبكرة النشطة.

وكذلك كال من العمر ومستوى خمائل الكبد األالنين ودرجة التليف وااللتهاب الكبدى كان متناسبا تناسبا طرديا مع درجة

التشمع.

يوجد ارتباط وثيق بين ٤المصريين المصابين بالفيروس الكبدى ج النوع الوراثىيستنتج من هذا البحث أن فى المرضى

حدوث التشمع الكبدى وعدة عوامل مثل ارتفاع مستوى اللبتن وانخفاض االديبونكتين فى الدم وكذلك ارتفاع مستوى

شائعا فى هذا النوع من والذى هو دليل على شدة المرض وسوء تطوره. وقد كان التشمع الكبدى ٢الميكروجلوبيولين ب

الفيروس وكانت درجته مرتبطة بمستوى الفيروس فى الدم وشدة االلتهاب واأللياف فى الكبد. ومن هنا نعتقد أن الفيروس

بطريقة قد تدل على أن هذا التشمع الكبدى عملية لها صلة بالفيروس ٢مشابها للنوع الوراثى ٤الكبدى ج النوع الوراثى

لوراثى وأن جميع التغيرات الكيمائية هى نتيجة لذلك وفى نفس الوقت قد تؤدى الى زيادة و اطراد في نفسه أو نوعه ا

وانخفاض االديبونكتين فى مرضى ٢عملية التشمع. ومن هنا يستنتج أن الزيادة فى كل من اللبتن والميكروجلوبيولين ب

الكبدى وتطور العلة الكبدية وقد يفترض أيضا من خالل الفيروس الكبدى ج المتليف قد يكون لها دورا فى عملية التشمع

االلتهاب الكبدى أيضا. هذه الدراسة أن التشمع الكبدى يجب أن يكون هدفا هاما للتدخل العالجى باإلضافة إلى الفيروس وعملية