Is Serum Angiotensin-Converting Enzyme Level Useful For Determining Necroinflammatory Activity In Chronic Hepatitis B Infection?

Abstract

Aim:

The purpose of this study was to investigate the relationship between the findings from liver biopsy and the serum angiotensin-converting enzyme (ACE) level to determine whether ACE might serve as a potential noninvasive sign of necroinflammatory activity in patients with Chronic Hepatitis B (CHB) infection.

Methods:

A total of 54 CHB patients referred for liver biopsy were enrolled in the study. Serum ACE levels were determined photometrically with a kinetic test.

Results:

The aspartate aminotransferase (AST), alanine aminotransferase (ALT), hepatitis B virus-deoxyribonucleic acid (HBV-DNA), histological activity index (HAI), and white blood cell counts were higher in patients with severe fibrosis, while albumin levels were low. The serum ACE levels showed a statistically significant correlation with HBV-DNA, HAI score, and ALT-AST levels.

Discussion:

In this study, a statistically significant relation between serum ACE levels and HAI scores was observed. This represents the first analysis to compare necroinflammation of the liver and serum ACE levels. There may be some explanations that the suppression of hepatocyte growth factor (HGF) by Angiotensin II and increased inflammatory damage might be a reason for the correlation between HAI and ACE. Serum ACE levels, HBV-DNA levels, and serum transaminase levels might be used together as noninvasive markers for the prediction of necroinflammation in CHB patients.

Introduction

Hepatitis B virus (HBV) is one of the main hepatotropic viruses that causes chronic hepatitis. It remains a serious health problem as it is communicable, common, and terminal since it leads to cirrhosis and hepatocellular adenoma (McMahon, 2004). Epidemiological data indicate that around 2 billion people have been infected with HBV, around 400 million people are diagnosed with a chronic HBV infection, and each year 500,000-700,000 people lose their lives globally due to diseases related to HBV (Thomas and Zoulim, 2012; Schweitzer et al., 2015).

Chronic hepatitis B (CHB) is unstable in its natural history, complicated, and poorly understood. Clinical data are vital for understanding the factors affecting the progression of the disease and for treatment planning. Determination of treatment and follow-up also requires histopathological evaluation of necroinflammation and fibrosis, in addition to all the other lab criteria. Specifying the grade and stage of chronic hepatitis by histology is of prognostic significance for assaying the severity and progression of the disease (Kamal et al., 1995). The histological activity index (HAI) is used to specify the degree of necroinflammatory activity (Prodromos et al., 1995). The histological degree sometimes shows a good correlation with clinical and biochemical evaluations, but it may, on occasion, be poorly correlated for many reasons. For example, histological changes occur relatively slowly compared to biological changes (Cooksley et al., 1986). This is why performing a liver biopsy, although a difficult and invasive method, is needed at least once in CHB cases (Park et al., 2007).

The renin-angiotensin-aldosterone (RAT) axis is a system comprising many essential regulators that maintain the balance of blood pressure, fluids, and electrolytes in the human body (Haznedaroglu and Beyazit, 2010). The importance of the RAT system in the pathogenesis of some diseases was increasingly recognized (Haznedaroğlu and Beyazit, 2010; Beyazit et al., 2011). Several studies have shown that angiotensin (Ang) plays a role in the pathogenesis of liver diseases (Anthuber et al., 1997; Beyazit et al., 2011). The angiotensin-converting enzyme (ACE), a vital part of RAT system, was viewed as the main molecule controlling systemic and portal circulation in some diseases (Beyazit et al., 2011).

The advancement of technology has led to a good number of noninvasive fibrotests for chronic viral hepatitis, and they are increasingly used in clinical practice (Albayrak et al., 2010; Lurie et al., 2015). However, few non-invasive methods are available for determining the HAI. They are vital for identifying necroinflammatory activity in liver biopsy. This study examines the relationship between liver biopsy and the serum ACE level to determine whether ACE might be a potential noninvasive sign of necroinflammatory activity in patients with CHB infection.

Patients and Methods

This study was approved by the Erzurum Region Training and Research Hospital (ERTRH) Ethics Committee on February 19, 2013. After Ethics Committee approvals a total of 54 CHB patients referred for liver biopsy were enrolled in the study.

The main inclusion criteria were patients age between 18 and 65 years, clinical history of CHB, and hepatitis B virus-deoxyribonucleic acid (HBV-DNA) level over 2000 IU/mL. Further inclusion and/or exclusion criteria were the absence of taking ACE inhibitor and the absence of liver cirrhosis. Patients with the following abnormal laboratory values were excluded: hemoglobin <13 g/dL for men or <12 g/dL for women; platelet count <150,000/mm³; serum creatinine ≥1.5 mg/dL; the international normalized ratio (INR) >1.2; serum albumin <3.5 g/dL.

To determine serum activity of ACE and other tests blood samples were collected in plain BD vacutainer tubes for serum chemistry (BD, New Jersey) and centrifuged at 4000 cycles for 8 min. The resulting serum was transferred to an Eppendorf tube and stored frozen at −80°C until used. Serums were brought to BD Vacutainer plastic serum tubes (BD, New Jersey) at room temperature, thawed, and serum ACE levels were determined photometrically with a kinetic test (Trinity Biotech, New Jersey) according to the manufacturer's instructions. Serum HBV-DNA levels were measured using a Roche Cobas TaqMan 48 system and the real-time polymerase chain reaction (Abbott; Architect i2000-Abbott) according to the manufacturer's instructions from blood samples collected in ethylenediamine tetraacetic acid (EDTA) tubes. Hepatitis B surface antigen HBsAg), hepatitis B surface antibody (Anti-HBs), hepatitis B early antigen (HBeAg), and hepatitis B early antibody (Anti-HBe) were determined by macro ELISA (enzyme-linked immunosorbent assay) (Architect i2000SR; Abbott Diagnostics, Chicago, IL). The INR was reported together with prothrombin time using a ThromborelR S (Diagnostica Siemens, Germany) at an International Sensitivity Index level of 1.07. Each parameter was measured in fully automated hemostasis analyzers (BCS XP System automatic coagulation analyzer; Siemens, Germany). Serum albumin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) values were determined from blood samples using a device (Roche PP800; Sweden) collected in EDTA tubes.

Liver fine-needle biopsies were performed by a standard procedure, on the same day as the blood samples were collected, on patients whose INR and thrombocyte counts were normal. The samples were obtained percutaneously using 17-G biopsy needles and ultrasonography. The biopsy samples were stained in the ERTRH pathology laboratory with hematoxylin/eosin, Masson trichome (MAS), silver reticulum, periodic acid Schiff, and Perls stains for evaluation of the necroinflammatory activity, fibrosis, and structural changes. All the samples were evaluated by the same pathologist. Fibrosis and necroinflammatory activity in the liver biopsy samples were determined using the Ishak scoring system. Biopsies with fibrosis scores 0-2 were defined as “mild” fibrosis and with scores 2-6 as “severe” fibrosis. Those with HAI scores 0-6 were defined as “low” and those with scores 7-18 were defined as “high” (Ishak et al., 1995).

The data were analyzed using the Statistical Packages for the Social Sciences (SPSS) 16 (SPSS, Inc., Chicago, IL) computer software. Digital data with normal distribution were presented as mean plus standard deviation, while digital data that were not normally distributed were presented as median (interquartile range), and categorical variables were shown as percentages. The Kolmogorov-Smirnov test was used to determine the normal distribution of the data. Numeric data with a normal group distribution were analyzed using the Student t-test and the Mann-Whitney U test was used for numeric variables that were not normally distributed. The Chi-square or Fisher test was used for analysis of the categorical variables. The Pearson or Spearmen correlation test was used for analysis of the relation between the variables. Variables with a significant correlation with serum ACE levels (p < 0.05) were further analyzed by multiple linear regression analysis. Those variables that were showing significant autocorrelation were not included in the regression analysis. In the analysis results, p < 0.05 was considered to be significant.

Results

In total, 54 patients were enrolled in the study [average age: 36.6 ± 10.6 years; 36 male (66.7%), 18 female (33.3%)]. Baseline data are as follows: the mean AST value for the entire group was 33 ± 10.9 IU/L and the ALT value was 47.5 ± 27.4 IU/L. The mean serum ACE level was 46.3 ± 29.7 U/L, the mean HAI was 7.2 ± 2.5, and the median HBV-DNA level was 49.8 × 10³ (6.7 × 10³-35,200 × 10³) IU/mL. A total of 15 (27.78%) patients were HbeAg positive and 38 (70.37%) were anti-HBe positive. The mean white blood cell value was 6.8 ± 1.5 × 10³/μL, and the mean platelet count was 217 ± 53.2 × 10³/μL. The average albumin level was 4.5 ± 0.28 g/dL (Table 1).

Table 1.

Demographic Characteristics of the 54 Chronic Hepatitis B Patients

Age, year^a	36.6 ± 10.6
Sex (male/female)^a, %	66.7/33.3
AST, U/L^a	33.0 (21.5-58.7)
ALT, U/L^a	47.5 (21-102)
INR^a	1.0 (0.95-1.00)
ACE, U/L^a	46.3 ± 29.7
WBC, 10³/μL^a	6.8 ± 1.5
Platelet, 10³/μL^a	217 ± 53.2
Albumin, g/dL^a	4.5 ± 0.3
HBV-DNA, × 10³, IU/mL^b	49.8 (6.7-35,200)
HbeAg, %	27.8
AntiHbe, %	72.2
HAI score^a	7.2 ± 2.5

Mean.

Median.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ACE, angiotensin-converting enzyme; HAI, histological activity index; HBV-DNA, hepatitis B virus-deoxyribonucleic acid; INR, international normalized ratio; WBC, white blood cell.

Six (11.11%) patients were rated as fibrosis 0, 32 (59.25%) as fibrosis 1, 6 (11.11%) as fibrosis 2, 9 (16.67%) as fibrosis 3, and 1 (1.85%) as fibrosis 5. No patients were rated as fibrosis 4 or 6. Based on these data patients were classified into 44 cases with mild fibrosis and 10 cases with severe fibrosis. Of these 17 patients had low HAI and 37 had high HAI.

Patient age, sex, AST, ALT, INR, serum ACE level, white blood cell count, platelet count, albumin level, HBV-DNA level, HBeAg positivity, and HAI variables were analyzed. The AST, ALT, HBV-DNA, HAI, and white blood cell counts were higher in patients with severe fibrosis, while albumin levels were low. However, no difference was noted in terms of age, sex, AST, ALT, INR, serum ACE level, HBeAg, and anti-HBe positivity ratio (Table 2).

Table 2.

Clinic Laboratory and Histopathological Differences Between Fibrosis Groups

	Mild fibrosis (n = 44)	Severe fibrosis (n = 10)	p
Age, year^a	36.4 ± 10.9	37.5 ± 9.4	0.781^b
Sex (male)^a, %	68	60	0.620^c
AST, U/L^a	28 (19-41)	143 (42-249)	<0.001^b
ALT, U/L^a	32 (19-73)	162 (63-425)	<0.001^b
INR^a	1 (1-1)	1 (1-1.1)	0.189^b
ACE, U/L^a	44.0 ± 27.9	56.2 ± 36.5	0.244^b
WBC, 10³/μL^a	6.6 ± 1.3	7.8 ± 2.1	0.028^b
Platelet, 10³/μL^a	219 ± 52.9	207 ± 55.9	0.520^b
Albumin, g/dL^a	4.6 ± 0.2	4.1 ± 0.2	<0.001^b
HBV-DNA, ×10³, IU/mL^d	17.1 (5.4-1463)	82800 (17810-170000)	0.005^b
HbeAg (%)	22.7	55.5	0.046^c
AntiHbe (%)	75	55.5	0.238^c
HAI score^a	6.7 ± 2.2	9.4 ± 2.5	0.002^b

Mean.

The Student t-test/the Mann-Whitney U test.

The Chi-square/Fisher test.

Median.

Among the groups, no difference was found in terms of age, gender, ALT, AST, platelet count, HBV-DNA level, fibrosis score, INR, serum ACE level, HBeAg, and anti-HBe values (Table 3), but a difference in white blood cell count was evident. The serum ACE levels showed a statistically significant correlation with HBV-DNA, HAI score, and ALT-AST levels (Table 4 and Fig. 1). Multiple linear regression analysis showed a significant auto-correlation between ALT and AST (r 0.934, p < 0.001), so only AST was studied in subsequent analyses. Multiple linear regression analysis revealed only the HAI score to be an independent predictor of the ACE serum level (Table 5).

FIG. 1.

(A) Correlation of the serum ACE level with AST; (B) Correlation of the serum ACE level with ALT; (C) Correlation of the serum ACE level with HBV-DNA; (D) Correlation of the serum ACE level with HAI scores. ACE, angiotensin-converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HAI, histological activity index; HBV-DNA, hepatitis B virus-deoxyribonucleic acid.

Table 3.

Clinic Laboratory and Histopathological Differences Between Histological Activity Index Groups

	Low HAI (n = 17)	High HAI (n = 37)	p
Age (year)^a	35.1 ± 11.0	37.3 ± 10.4	0.495^b
Sex (male)^a (%)	58.8	70.2	0.407^c
AST, U/L^a	33 (19-68)	33 (24-56)	0.941^b
ALT, U/L^a	46 (19-96)	48 (21-110)	0.582^b
INR^a	1 (1-1)	1 (1-1)	0.759^b
ACE, U/L^a	39.1 ± 15.3	49.6 ± 34.0	0.232^b
WBC, 10³/μL^a	6.22 ± 1.6	7.1 ± 1.5	0.041^b
Platelet, × 10³/μL^a	218 ± 53	216 ± 54	0.877^b
Albumin, g/dL^a	4.4 ± 0.3	4.6 ± 0.3	0.197^b
HBV-DNA, × 10³, IU/mL^d	894 (6.9-106593)	35.5 (5.4-11437)	0.496^b
HbeAg, %	35.2	25	0.437^c
AntiHbe, %	70.5	72.2	0.902^c

Mean.

The Student t-test/the Mann-Whitney U test.

The Chi-square/Fisher test.

Median.

Table 4.

Correlation of Serum Angiotensin-Converting Enzyme Levels with Clinical, Laboratory, and Histopathological Variables

	r^a	p^a
Age	−0.152	0.271
WBC, 10³/μL	0.109	0.432
Platelet, 10³/μL	−0.174	0.208
Albumin, g/dL	0.119	0.477
ALT, U/L	0.352	0.009
AST, U/L	0.369	0.006
HBV-DNA, IU/mL, × 10³	0.328	0.019
INR	0.237	0.084
Fibrosis score	0.223	0.105
HAI score	0.289	0.034

The Pearson/Spearmen correlation test.

Bold values indicate statistical significance.

Table 5.

Multiple Linear Regression Analysis for Determination of Independent Serum Angiotensin-Converting Enzyme Level Predictors

	Unstandardized β-coefficient	Standard error	Standardized β-coefficient	t	p^a
HAI score	3.274	1.633	0.272	2.023	0.050
AST, U/L	0.002	0.029	0.008	0.055	0.956
HBV-DNA, IU/mL × 10³	4.651	<0.001	0.169	1.192	0.239

Multiple linear regression analysis.

Discussion

In chronic hepatitis, the stage defines the degree of fibrosis and structural changes, whereas the HAI specifies the degree of necroinflammatory activity (Kamal et al., 1995; Prodromos et al., 1995). The basic premise of this activity is a lymphocytic moth-eaten appearance and lobular necrosis. This is more important than the severity of the portal inflammation, which parallels other symptoms in importance. Necroinflammation can occur as a result of viral and autoimmune hepatitis, hepatitis due to drugs, biliary diseases, and other types of hepatitis (Prodromos et al., 1995). The histological degree sometimes shows a good correlation with clinical and biochemical evaluations, but occasionally this correlation is poor for many reasons. One reason is that histological changes occur relatively slowly when compared to biological changes (Cooksley et al., 1986).

The liver needle biopsy is a difficult procedure in itself, but it is complicated by a common occurrence of coagulation incoherency failure in some cases, the heterogenic distribution of histopathologic findings in the tissue, and other different evaluations, which give rise to further concerns (Thampanitchawong and Piratvisuth, 1999). These reservations have prompted many studies aimed at identifying new index parameters for necrosis in liver biopsys, with particular emphasis on fibrosis and reagents that can detect increased fibrosis in serum (Albayrak et al., 2010; Lurie et al., 2015). Many studies have provided techniques for indirect detection of fibrosis in liver biopsy, but few of these are noninvasive methods for determining the HAI.

The RAT system is associated with pathogenic mechanisms, such as portal pressure increases due to vasospastic micro-occlusion, hepatic stellate proliferation, and inflammation of the liver (Rimola et al., 2004; Freise et al., 2006). Inflammatory processes are manifested by enhanced biosynthesis of inflammation mediators. Chronic inflammation consists of cellular infiltration, primarily by macrophages and lymphocytes (Shintani et al., 2011). These cells secrete various cytokines and proteases and form a complex network, which can maintain chronic inflammation for several years (Shintani et al., 2011). Evidence suggests that cytokines and tissue factors also regulate the pathophysiology of inflammation (Husain et al., 2015). One important biomarker of inflammation is C-reactive protein, which is generated by hepatic cells and is also modulated by interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF)-α (Yudkin et al., 1999).

Angiotensin II (Ang II) is a potent suppressor of hepatocyte growth factor (HGF) production in various organs, such as the myocardium, kidney, and vessels (Nakano et al., 1998; Taniyama et al., 2000). ACE inhibitors are known to induce the expression of HGF in the liver, and HGF causes a shift in the cytokine profile toward a less active inflammatory state (Yayama et al., 2007; Shintani et al., 2011). HGF is protective for these tissues against inflammatory damage (Kaido et al., 1997; Okada et al., 2004; Homsi et al., 2009). ACE therefore plays a role in inflammation of the liver in association with HGF (Kaido et al., 1997; Nakano et al., 1998; Taniyama et al., 2000; Okada et al., 2004; Yayama et al., 2007; Homsi et al., 2009). The purpose of our study was to investigate whether serum ACE levels could be used as a noninvasive marker for determination of the liver inflammation levels.

We found a statistically significant relation between serum ACE levels and HAI scores. To date, no study has been undertaken in the literature to compare necroinflammation of the liver and serum ACE levels, although serum ACE and inflammation levels have been investigated in other diseases. For example, Morell et al. (2002) found the ACE level of 40 sarcoidosis patients to be a significant marker for inflammation, in agreement with our findings. Kavala et al. (2005), examined the ACE levels of 132 psoriasis vulgaris patients before and after treatment and observed a statistically significant decrease in the serum ACE levels following the treatment. They reported higher levels of ACE in damaged tissues and also pointed out a destructive role for ACE regarding substance P, bradykinin, and other kininogens that cause inflammation during psoriasis (Kavala et al., 2005). Even if the study of Kavala et al. and our study are on two different diseases and have different pathogenesis, it may explain the relationship between serum ACE levels and HAI because both of the diseases are associated with inflammation. This effect might be one reason for the significant relation between serum ACE level and HAI observed in our study. Additionally, the suppression of HGF by Ang II and increased inflammatory damage might be another reason for the correlation between HAI and ACE. In our study was also observed correlation between serum ACE levels, HBV-DNA, and ALT-AST values. This suggests that serum ACE levels, HBV-DNA levels, and serum transaminase levels might be used together as noninvasive markers for the prediction of necroinflammation in CHB patients.

In our study, no statistically significant difference was found between the high and low fibrosis groups and ACE levels. By contrast, Purnak et al. (2012) reported a relationship between serum ACE levels and fibrosis in their study conducted on 50 CHB patients. In that study, 28 (56%) patients had a fibrosis score 2 and higher, whereas in our study, only 10 (18.5%) patients had a fibrosis score 2 and higher. Therefore, the previous researchers might have found a relationship between serum ACE levels and fibrosis only because the majority of their patients had high fibrosis values. Conversely, our study was conducted on a patient group with low fibrosis scores, which might be the reason for the absence of a relationship between serum ACE levels and fibrosis. Therefore, future studies should be conducted on patients with high fibrosis scores and more homogenous fibrosis distribution to provide a more accurate assessment of the relationship between serum ACE level and fibrosis.

A significant relationship was observed between the serum ACE level and HAI score in patients newly diagnosed with CHB, indicating that the serum ACE level in CHB patients might be a useful inflammation indicator. We suggest that serum ACE levels, together with HBV-DNA and serum transaminase levels, might be used as noninvasive indicators for necroinflammation, allowing for an early detection of activations in CHB patients. In our study no significant relationship was observed between the serum ACE levels and fibrosis in patients newly diagnosed with CHB. Our study has some limitations. The number of cases in our study was only 56, so the results are not suitable for generalization. Additionally, the fact that no patient with a higher HAI score than 12 was present in our study might be preventing more accurate results. The majority of our patients had low fibrosis levels, so the relationship between the fibrosis of the liver and serum ACE levels could not be sufficiently established. Our opinion is that presented associations between serum ACE levels, HAI score, and fibrosis score should be more accurately defined with further prospective-randomized studies that use a larger number of patients with more homogeneously distributed HAI and fibrosis scores.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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