Research Article
Open Access
Songül Ünüvar*, Hamza Aslanhan, Zübeyde Tanrıverdi, Fuat Karakuş
The relationship between neopterin
and hepatitis B surface antigen positivity
https://doi.org/10.1515/pteridines-2018-0001
received November 14, 2017; accepted December 27, 2017.
Abstract: Hepatitis B is a life-threatening viral liver
infection caused by the hepatitis B virus. Neopterin is regarded as an immunologic biomarker of several diseases related to activation of the cellular immune system. Hepatitis B infection is associated with increased production of cellular immune system markers. We aimed to investigate whether there is a relationship between hepatitis B surface antigen-positivity (HBsAg +) and neopterin to determine the role of neopterin in the early diagnosis of hepatitis B infections. Seventy-two HBsAg (+) patients with normal liver function tests and forty-three controls were included in the study. Neopterin levels were 17.6 ± 0.13 nmol/L in HBsAg (+) patients; and 9.12 ± 0.09 nmol/L in infection-free controls, respectively. Compared to the control group, a statistically significant increase (p < 0.001) in the serum neopterin levels of the patients was observed. No significant relationship was determined between neopterin levels and age/sex (both, p > 0.05). With overstimulation of interferon-gamma, the production of neopterin increases by monocytes/macrophages. Likewise with other diseases associated with an activated cellular immune system, this study shows that neopterin can be a predictive biomarker for persistent carriers of hepatitis B infection.
Keywords: hepatitis B surface antigens; immune system;
interferon-gamma; liver diseases; neopterin.
Introduction
Each year, approximately 1.45 million people die worldwide as a result of viral hepatitis infections [1]. Hepatitis B virus (HBV) is the main cause of various liver diseases such as acute and chronic hepatitis B infections, liver cirrhosis and hepatocellular carcinoma. According to the data from World Health Organization (WHO), hepatitis C virus (HCV) and HBV are responsible for 90% of the deaths due to hepatitis infections. The infection may be chronic in about 5% of adults and 95% of newborns [2].
Hepatitis B virus, settling inside the liver, damages the liver cells. It has various viral surface antigens on the lipid envelope gained from the host cell surface. One of these
antigens is the HBsAg [3], which can be detected in the
serum approximately 6-8 weeks prior to the occurrence of the disease symptoms and 12 to 20 weeks before levels of
transaminases increase. HBsAg is the first indicator to be
evaluated in patients with acute hepatitis B. In recovering cases, it decreases in the serum and disappears within 6 months; its presence in the serum after this period indicates that either the disease has become chronic or the patient has become a carrier. In the serum, following the
disappearance of HBsAg, hepatitis B surface antibodies
(anti-HBs) appear and it can remain detectable for lifetime. The incubation period of the HBV may vary depending on the amount of the viruses exposed and the immunity of the patient. It is usually 60-90 days following the exposure to the virus, whereas it sometimes can be delayed as long as 45-180 days [4,5].
The vast majority of viruses that infect humans are effectively removed by the immune system; however, some viruses develop defense mechanisms against their elimination [6]. Natural killer cells, cytotoxic effectors, and cytokine production are important components of the natural immune system, which serves to eliminate viral infections. Natural killer cells have two primary functions in controlling viral infections. The first function is to attack the infected cell through direct cell-to-cell contact. The other is the secretion of antiviral cytokines
such as interferon-gamma (IFN-γ). Studies have suggested
that the immune response generated by the natural killer
*Corresponding author: Songül Ünüvar: Department of
Pharmaceutical Toxicology, Faculty of Pharmacy, İnönü University, Malatya, Turkey, E-mail: songul.unuvar@inonu.edu.tr
Hamza Aslanhan: Department of Family Medicine, Faculty of
Medicine, Dicle University, Diyarbakır, Turkey
Zübeyde Tanrıverdi, Fuat Karakuş: Department of Pharmaceutical
Toxicology, Faculty of Pharmacy, İnönü University, Malatya, Turkey
Fuat Karakuş: Department of Pharmaceutical Toxicology, Faculty of
Pharmacy, Ege University, İzmir, Turkey
Journal xyz 2017; 1 (2): 122–135
The First Decade (1964-1972)
Research Article
Max Musterman, Paul Placeholder
What Is So Different About
Neuroenhancement?
Was ist so anders am Neuroenhancement?
Pharmacological and Mental Self-transformation in Ethic Comparison
Pharmakologische und mentale Selbstveränderung im ethischen Vergleich
https://doi.org/10.1515/xyz-2017-0010
received February 9, 2013; accepted March 25, 2013; published online July 12, 2014 Abstract: In the concept of the aesthetic formation of knowledge and its as soon
as possible and success-oriented application, insights and profits without the reference to the arguments developed around 1900. The main investigation also includes the period between the entry into force and the presentation in its current version. Their function as part of the literary portrayal and narrative technique.
Keywords: Function, transmission, investigation, principal, period
Dedicated to Paul Placeholder
1 Studies and Investigations
The main investigation also includes the period between the entry into force and the presentation in its current version. Their function as part of the literary por-trayal and narrative technique.
*Max Musterman: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning
Road Keelung 20224, Taiwan (R.O.C), e-mail: email@mail.com
Paul Placeholder: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning
Road Keelung 20224, Taiwan (R.O.C), e-mail: email@mail.com
Open Access. © ���� Mustermann and Placeholder, published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives �.� License.
Open Access. © 2018 Songül Ünüvar et al., published by De Gruyter.
Journal xyz 2017; 1 (2): 122–135
The First Decade (1964-1972)
Research Article
Max Musterman, Paul Placeholder
What Is So Different About
Neuroenhancement?
Was ist so anders am Neuroenhancement?
Pharmacological and Mental Self-transformation in Ethic
Comparison
Pharmakologische und mentale Selbstveränderung im
ethischen Vergleich
https://doi.org/10.1515/xyz-2017-0010
received February 9, 2013; accepted March 25, 2013; published online July 12, 2014
Abstract: In the concept of the aesthetic formation of knowledge and its as soon
as possible and success-oriented application, insights and profits without the reference to the arguments developed around 1900. The main investigation also includes the period between the entry into force and the presentation in its current version. Their function as part of the literary portrayal and narrative technique.
Keywords: Function, transmission, investigation, principal, period
Dedicated to Paul Placeholder
1 Studies and Investigations
The main investigation also includes the period between the entry into force and the presentation in its current version. Their function as part of the literary por-trayal and narrative technique.
*Max Musterman: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning
Road Keelung 20224, Taiwan (R.O.C), e-mail: email@mail.com
Paul Placeholder: Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning
Road Keelung 20224, Taiwan (R.O.C), e-mail: email@mail.com
Open Access. © ���� Mustermann and Placeholder, published by De Gruyter. This work is
licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives �.� License. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.
cells may provide the possibility to control the disease in the early stages of acute HBV infections. Additionally, in HBV infections, the evaluation of the parameters, which are involved in the host defense system also provides information on whether the infection is in its active phase or not [7]. Although HBV does not lead to a significant natural immune response in the liver, it may lead to HBV-specific adaptive T-cell responses. Despite the advancements gained in the studies on HBV, the important aspects of HBV immunology and pathogenesis, particularly the immune mechanisms that the persistent virus-related effects are responsible for, could not yet be adequately identified [8].
The clinical features of the hepatitis-related disorders varies widely. Approximately half of the patients are asymptomatic [9]. At the end of the incubation period, before clinical symptoms occur, neopterin levels increase in body fluids. The highest neopterin levels are observed just before the detection of virus-specific antibodies [10,11].
It is known that immune system activation can be observed following the development of a viral infection. Change in concentration of neopterin in viral infections, which is one of the indicators of immune system activation, provides insight into the relationship between infectious diseases and the immune system [12]. Neopterin, which is secreted by macrophages that are activated by inflammation, can be used as an indicator of liver diseases such as acute hepatitis, chronic hepatitis, and cirrhosis. A positive correlation has been found between the elevated serum neopterin levels and the disease severity in many disorders [12-16].
The aim of our study was to evaluate the serum neopterin levels, which is an indicator of cellular immune
system activation in persistent carriers of HBsAg (+)
patients.
Materials and methods
Subjects and samples
A total of 115 serum samples were collected. The study
group comprised of 72 HBsAg carriers (male: 44, female:
28) and 43 healthy individuals (male: 26, female: 17). The average age of the patient group was 40.49 ± 0.20 years, whereas control group’s average age was 33.12 ± 0.22 years.
Blood samples were collected from the patients and healthy individuals who applied to Family Medicine Polyclinic. Diagnostic routine hepatitis tests for acute or
chronic HBV infections were requested from patients with suspected hepatitis B infection. The patients who had
positive HBsAg test results for more than four months were
formed patient group. The health status was thoroughly questioned in all of the individuals in the control group. Individuals who applied to Family Medicine Polyclinic without an infectious disease, chronic disease, malignancy and immune system-related disorders were selected for control group. Additional blood was collected from both control group and patient group when the physician requested blood for routine biochemical tests and for the evaluation of hematological parameters.
Informed consent: Informed consent has been
obtained from all individuals included in this study.
Ethical approval: The research related to human
use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the İnönü University’s local ethics committee.
Measurement of neopterin
During collection of the blood samples required for routine biochemical and hematological tests, additional blood samples were also taken in biochemistry tubes. Then, the samples were centrifuged at 3.500 rpm for 10 minutes. The serum samples were collected and stored at -20°C until the time of measurement.
Serum neopterin levels were measured with enzyme linked immunoassay (ELISA) method by using commercially available DRG Diagnostics GmbH (Marburg, Germany). Results were expressed as nmol/L.
Statistics
All results are presented as means ± standard error of the mean (SEM) using the Statistical Package for the Social Sciences (SPSS) 11.5 program. Mann-Whitney U test was used for the comparison between the two independent groups and Pearson correlation test was used in the evaluation of the correlations. p values < 0.05 were considered statistically significant.
Results
The serum level of neopterin in HBsAg (+) group was 17.6
± 0.13 nmol/L (range 8.25-68.40 nmol/L), whereas it was 9.12 ± 0.09 nmol/L (range 3.66-20.30 nmol/L) in the control
group. The mean neopterin level of the HBsAg (+) patients increased when compared to the control group, and this increase was found statistically significant (p < 0.001) (Figure 1).
Both in the control group and the patient group, there was no significant relationship between gender and serum neopterin level (both, p > 0.05) (Figure 2).
There were positive correlations between the serum neopterin level and age in both the control group (r = 0.1, p > 0.05) and the patient group (r = 0.09, p > 0.05). However, these correlations were not found to be significant (Figure 3).
Discussion
The pathogenesis of the liver diseases caused by hepatitis B virus is mostly based on the immune system-mediated mechanisms. However, it can rarely be caused by direct hepatotoxic damage. The principal mechanism of immune system-mediated hepatotoxicity is the destruction of the infected hepatocytes through cytotoxic T cells [4].
In hepatitis B infection, IFN-g, which is released from
cytotoxic T cells, activates the macrophages in the liver, stimulating the release of neopterin from these
macrophages [17]. IFN-g production increases due to the
stimulation of T lymphocytes by various specific antigens, mainly viral antigens [15,18,19].
Neopterin is regarded as an early valuable marker for monitoring of infectious disease activity [20]. Serum neopterin levels were not only correlated with activity of disease but also, they are useful and informative for an early differentiationbetween infectious and non-infectious patients [21]. It has been suggested that serum neopterin level may be an important indicator for differential diagnosis of viral hepatitis and non-infectious hepatitis [15,22].
Serum neopterin levels in patients with acute and chronic hepatitis B were found to be higher than those of healthy individuals. The relationship between neopterin level and the severity of the disease has been proved, and it has been suggested that neopterin level can be used as a prognostic indicator of the disease progression in combination with clinical data [12,15,23]. High neopterin levels have been found in hepatitis B e antigen positive (HbeAg +) chronic hepatitis B patients. It can be concluded that there is an association between elevated neopterin levels and HbeAg-positivity [23]. The results of a pediatric study suggested that serum neopterin levels can be used as an inflammatory marker in children with
hepatitis-B-related chronic liver disease [15]. In acute hepatitis, chronic inactive hepatitis, chronic active hepatitis, liver cirrhosis, hepatocellular carcinoma, alcoholic liver
diseases and in asymptomatic HBsAg carriers, serum
and urine neopterin levels were found to be higher than those of the control group. The highest levels of neopterin
Figure 1: Neopterin levels (mean +/- SEM) in patients with HBsAg (+)
and control.
Figure 2: The comparison of neopterin levels (mean +/- SEM) according to the gender.
0
5
10
15
20
Control
HBsAg (+)
N
eopte
rin
(nm
ol
/L)
Male FemaleFigure 2: The comparison of neopterin levels (mean +/- SEM)
according to the gender.
Figure 3: The distribution of neopterin concentrations according to the age. 0 10 20 30 40 50
0 10 20 30 40 50 60 70
N
eopte
rin
(nm
ol
/L)
Age (Year)
Control HBsAg (+)Figure 3: The distribution of neopterin concentrations according to
were detected in patients with acute hepatitis [15,24]. Our results are in agreement with previous studies [12,15,22-24]. We found a significant increase in serum neopterin levels
in the HBsAg carriers due to the activation of the cellular
immune system compared to the healthy individuals. Neopterin levels are reduced when the immune system is suppressed, due to infectious diseases such as chronic hepatitis B and chronic hepatitis C infections. Conversely, when the chronic hepatitis is treated with an IFN therapy, neopterin concentrations increase due to stimulation of the immune system [25]. The development of necroinflammation and fibrosis in chronic hepatitis B infection is caused by the immune response in which T lymphocytes and macrophages play roles. It has been considered that neopterin, which is secreted by macrophages during this immune response, reflects the presence and the severity of fibrosis and necroinflammation in the liver. Neopterin measurement in the body fluids not only informs about the level of the cellular immune response but also helps to predict the progression of the disease [15,26].
Elevated neopterin levels in body fluids can be detected immediately before the occurrence of the clinical symptoms at the end of the incubation period, and it show a significant increase with the emergence of clinical symptoms. High neopterin concentrations are more pronounced in diseases in which monocyte and macrophage activities are intense. Neopterin release begins three days before T lymphocyte proliferation reaches its peak and neopterin can be used as an early indicator of inflammation since an increase in neopterin production is observed approximately one week before the specific antibodies become positive. After the emergence of neutralizing antibodies in the convalescence period, however, neopterin secretion is reduced to its normal level [17,27].
As it is known that cellular immune system activation is observed following cardiovascular diseases, infectious diseases, autoimmune disorders, malignancies, and organ
transplantations. HBsAg can be detected in the serum from
several weeks before occurrence of the clinical symptoms. We aimed to evaluate the serum neopterin level, which is one of the early indicators of the cellular immune system activation.
In conclusion, we suggest that measurement of serum neopterin level can be used for assessment of the immune system activity in combination with other parameters in hepatitis B-related diseases.
Conflict of interest: Authors state no conflict of interest
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