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© Borgis - Postępy Nauk Medycznych 11/2014, s. 744-752
*Jarosław Kałuża1, Paweł Fijałkowski3, Daniela Dworniak2, Jan Błaszczyk3, Anna Skubała1, Anna Piekarska1
Ocena wybranych wskaźników metabolizmu tlenowego u pacjentów leczonych pegylowanym interferonem alfa i rybawiryną
Assessment of selected oxygen metabolism in patients with chronic hepatitis C during treatment with pegylated interferon alfa and ribavirin
1Infectious, Tropical Diseases and Parasitoses Department for Adults, Infectious Diseases and Hepatology Clinic, Medical University of Łódź
Head of Department: Anna Piekarska, MD, PhD, Associate Professor
2Infectious Diseases and Digestive Tract Diseases Department
Head of Department: prof. Daniela Dworniak, MD, PhD
3Physiology Department, Basic and Preclinical Sciences Chair, Medical University of Łódź
Head of Department: prof. Jan Błaszczyk, MD, PhD
Streszczenie
Wstęp. W 2012 roku nawet kilkaset milionów ludzi na świecie mogło być zakażonych wirusem zapalenia wątroby typu C (ang. hepatitis C virus – HCV). Duży odsetek chorych wykształcający przewlekłe zapalenie wątroby (pzw C) oraz progresja choroby ku marskości i pierwotnemu rakowi wątroby stanowią istotny problem medyczny. Etiopatogeneza schorzenia nie została do końca poznana. Podejrzewa się udział wolnych rodników w przebiegu choroby.
Cel pracy. Celem niniejszej pracy była ocena wybranych wskaźników metabolizmu tlenowego u chorych z pzw C leczonych pegylowanym interferonem (Peg-IFN) alfa i rybawiryną (RBV).
Materiał i metody. Badaniem objęto grupę 31 pacjentów (23 mężczyzn, 8 kobiet) o średniej wieku 44 +/- 11. W trakcie badania oznaczono czterokrotnie (przed leczeniem, po 12, 24 i 48 tygodniach) generowanie anionorodnika ponadtlenkowego (spoczynkowe, stymulowane), stężenie dialdehydu malonowego (MDA) oraz aktywność peroksydazy glutationowej (Gpx), katalazy (Cat) i dysmutazy ponadtlenkowej (SOD).
Wyniki. Uzyskano wyniki badań świadczące o podwyższonym generowaniu anionorodnika ponadtlenkowego u chorych z pzw C. Po zastosowanym leczeniu generowanie zmniejszało się, a w zakresie MDA uzyskano wyniki świadczące o redukcji stężeń. W zakresie wskaźników bariery antyoksydacyjnej uzyskano wyniki świadczące o podwyższonej aktywności enzymów wskaźników w pzw C przed leczeniem i systematyczne zmniejszanie się aktywności w trakcie i po zakończeniu terapii skojarzonej Peg-IFN i RBV.
Wnioski. 1. Przewlekłe zapalenie wątroby należy zaliczyć do chorób wolnorodnikowych. 2. Stres oksydacyjny występujący w pzw C stymuluje barierę antyoksydacyjną do wzmożonego generowania enzymów antyoksydacyjnych. 3. Skojarzone leczenie Peg-IFN i RBV wywiera korzystny wpływ na metabolizm tlenowy u chorych z pzw C, redukując generowanie reaktywnych form tlenu (RFT) oraz zmniejszając aktywność bariery antyoksydacyjnej.
Summary
Introduction. In the year 2012 there were several hundred millions patients with chronic hepatitis C (CHC). The etiopathogenesis of this disease is not clear. It was suspected that free radicals may play an important role in the process.
Aim. The aim of the research was to analyse of the indictors of oxygen metabolism among the patients with CHC treated with pegylated interferon alpha (Peg-IFN) and ribavirine (RBV).
Material and methods. The study was carried out on a group of 31 patients (23 males and 8 females) with an average age of 41 +/- 11. During the therapy results were controlled four times (before treatment, in the 12th, 24th and 48th week), measuring the level of superoxide anion radicals at rest and after stimulation, the malonic dialdehyde concetration (MDA), the activity of catalase (Cat), glutathione preoxidase (Gpx) and superoxide dismutase.
Results. The results showed an elevated concentration of superoxide anion radicals for patients with chronic hepatitis C. The concentration decreased after the treatment. The MDA concentration was lower after the Peg-IFN + RBV treatment. The activity of antioxidant barrier was a proof of elevated rates of indicator enzymes in the case of patients with CHC and of continuous activity reduction during the treatment with Peg-INF and RBV.
Conclusions. 1. CHC should be described as an oxidative stress disease. 2. Oxidative stress CHC stimulate the antioxidant barrier to generate antioxidative enzymes. 3. Treatment with Peg-IFN and RBV has a positive impact on the oxygen metabolism among patients with CHC, reducing the production of free oxygen radicals and the activity of the antioxidative barrier.
Introduction
According to World Health Organization (WHO) estimation the number of people with confirmed diagnosis of HCV infection around the world is over 180 mln. Other assesments suggest that over 400 mln people is infected with HCV, which is about 6% of the population of the world (1).
HCV infection is characterized by low incidence of spontaneous eliminations. Between 50 and 80% of infected people will reveal chronic hepatitis, developing liver cirrhosis and primary hepatocellular carcinoma in the future (20% after 20 years) (2).
In Poland the number of infected people may reach up to 700 000 depending on an author (3). It is estimated that in the majority of cases the infection is connected with the contact with health service centres. Basic ways of transmission of the virus are: parenteral, sexual and vertical. Special risk groups include drug users (90% infected with the virus in some countries) and hemodialysis patients (according to different sources 30-60% infected) (4). What is important, about 40% of cases are „sporadic infections”, when the way of infection is impossible to determine.
Etiopathogenesis of HCV infection has not been clarified up to now. The receptor responsible for the virus penetration into the cell has not been defined unequivocally. The CD81 co-receptor is suspected of cooperation, binding viral E2 protein (envelope) and enabling virions to gather at the cell surface (5). SRBI (scavenger receptor class B type I) co-receptor binding viral HVR1 region (hypervariable region) is also responsible for HCV penetration into the cell. High mutation rate allows to avoid an organism immunologic response. Immunologic system pressure is also responsible for creating viral variants (subtypes and pseudotypes „quasispecies”). It is thought that viral proteins interfere with an organism natural and specific response.
The virus reveals tropism not only to hepatocytes. HCV-RNA was found in mononuclear cells as well. HCV-RNA was revealed in mononuclear cells of blood in 50-80% of the patients. These are the potential reservoirs of infection in further course of the disease. It has not been found whether and how the virus might influence their function (6).
The virus replicates in the cytoplasm of the cell. It is not the only intracellular localization. Viral protein presence has also been documented in mitochondria and nuclei of cells (7).
This localization is important because of the biochemical processes at the molecular level. HCV is considered to influence cell aerobic metabolism disturbing the homeostasis of free radicals production and impairing antioxidative mechanisms in the cell.
The cells of aerobic organisms physiologically produce necessary volumes of reactive oxygen forms – free radicals. One of the basic sites of their production is the inner mitochondrial wall, where the respiratory chain reactions generate superoxide anion radical among others (8).
Atoms or molecules with one or more unpaired electrons are considered as free radicals. Reactive oxygen forms (ROF) in the cell are all products of reactions of excitation and reduction of oxygen having higher biochemical activity than molecular oxygen (O2). ROF production of in the cell happen through enzymatic reactions (with oxidase from the oxidoreductase group), through xenobiotics oxygenation and through oxygenation of respiratory proteins.
Among the basic ROF we rate: singlet oxygen, ozone, superoxide anion radical, hydrogen peroxide, hydroxyl radical and hydrogen peroxide radical.
Physiologically free radicals production is controlled by a few mechanisms: antioxidant barrier enzymes (catalase, superoxide dysmutase, glutathione peroxidase), natural antioxidants – glutathione and microelements, and antioxidant vitamins (zinc, selenium, vitamins A, E, C). When inflammation begins at the tissue – regardless of its cause – those processes regulation fails, causing the oxidative stress to pathologically arise (9).
Because of their high reactivity free oxygen radicals react with all the basic cell components: lipids, proteins, nucleic acids, carbohydrates. The effects of those reactions are oxygenation of basic cell components, change of their structure and change or loss of their function.
Among harmful effects of ROF influence on a cell we can find: collagen degradation, lipids peroxidation, enzymes inactivation, DNA chain brakes (impairments of the helix causing mutations and neoplasms as a result), erythrocytes’ lysis, oxidative phosphorylation inhibition.
The result of abovementioned reactions is impairment of function and structure of the cell, including the cell death.
There are three basic mechanisms of defense for the organism against free radicals:
1) prevention – of the ROF reactions with biologically active agents,
2) intervention – interruption of free radicals reactions,
3) elimination – of ROF reactions effects.
Among the enzymes decomposing the precursors of hydroxyl radical – superoxide anion radical and hydrogen peroxide, we find enzymes using dismutation properties (disproportionate) of ROF. This group comprises enzymes of an antioxidant barrier – Cat, Gpx, SOD (10).
Currently the most effective method of treatment for chronic hepatitis C is a combined therapy with pegylated interferon alpha (Peg-IFN) and ribavirine (RBV).
Interferons are species specific pleiotropic cytokines released by cells as a reaction to viral infection. Currently two subtypes of alpha interferon are used in the therapy: 2a and 2b. Effectiveness of interferon monotherapy was low (15-20%). Adding of poliethylene glycol molecule (pegylation) to the interferon molecule caused a change in its physical and chemical properties, increasing the drug effectiveness. Pegylated interferon is more water soluble, has a better bioavailability, pegylated particle is chemically neutral and protects the drug against opsonisation and phagocytosis. Also it elongates the half-life (less frequent drug injections) (11).
Currently the effectiveness of the treatment with Peg-IFN alpha with ribavirine allows to reach satisfactory effect in 50% of the patients (12).
The virus eradication not always is possible to achieve. Positive is to achieve lasting suppression of viral replication and as a result inhibition of necrotico-inflammatory processes in the liver. It prevents the progression to liver cirrhosis and primary hepatocellular carcinoma.
Aim
The aim of this work is to assess an aerobic metabolism rates in patients with chronic hepatitis C and evaluation of the influence of the treatment with pegylated interferon alpha 2a and ribavirine to chosen rates of aerobic metabolism.
Material and methods
The study comprised 31 patients (8 women and 23 men) aged 19 to 58 (on average 44 +/- 11). The group consisted of the patients hospitalised at the Infectious Diseases Clinic of Military Medical Division and at City Units in Biegański’s Provincial Specialised Hospital in Łódź (Wojewódzki Specjalistyczny Szpital im. dr. Wł. Biegańskiego w Łodzi).
Control group comprised 22 healthy adults, adjusted according to the sex and age.
The patients qualified to the study were diagnosed with chronic hepatitis C (CHC) according to the serological tests (presence of antibodies anti-HCV), biochemical test (increased ALT, AST activity, minimum 2 times higher than normal), presence of HCV-RNA and histopathology. Liver tissue sampled during liver biopsy was assessed with 5-grade Batts and Ludwig scale has minimum S2 (inflammation).
Disqualifying criteria were autoimmunologic diseases, haematologic disorders, unstable liver failure, heart failure, unstable ischaemic heart disease, pregnancy, breast feeding, other severe devastative diseases.
Treatment
All the patients were treated with 180 ug of Peg-IFN alpha 2 subcutaneously once weekly for 48 weeks. Daily they were treated with 1000 or 1200 mg of RBV (depending on body weight).
The samples for the study were collected in the quantity of 2 ml from the ulnar vein to the vaccutainers with K3 EDTA. Blood samples were taken during the tests essential to the monitoring of the treatment. Assessment of each parameter was performed before start of the treatment, after 12, 24 and 48 weeks of treatment, which is after completion of the combined therapy.
In the subsequent samples we tested:
– in neutrophiles of blood – production of superoxide anion radical at rest and after stimulation,
– in erythrocytes of blood – concentration of malonic dialdehyde (MDA), catalase activity (Cat), glutathione peroxidase activity (Gpx), superoxide dismutase activity (SOD).
Photocolorimetric method was implemented to the tests. Superoxide anion radical activity was tested with Bellavita method, MDA concentration – with Placer method, SOD activity – with Misdry and Fridroviha method, Gpx activity – with Lindsay method in Little and O’Brien modification, Cat activity – with Beers and Sizer method.
The statistical analysis comprised 24 patients who completed 48-week therapy. 7 patients were disqualified from treatment because of side effects or unsatisfactory treatment results. The analysis used dependent sample, which is the same group at different subsequent points of time. For the statistical significance level 0.05 (p < 0.05) was assumed. Program Statistica 7 was used for the statistical analysis.
Results
All the patients were infected with HCV genotype 1. In 28 cases genotype 1b was found, and in 2 patients – genotype 1a (tab. 1).
Table 1. HCV genotypes in the tested group.
 Viral genotype
1a1b3a
Number of patients2281
Histological advancement in the liver sample obtained during the liver biopsy was assessed with Batts and Ludwig scale was G1S2 to G4S4. At the majority of cases (19 patients) medium intensity of changes was found (G2S2). The histological results are presented in table 2.
Table 2. Liver biopsy results at the tested group.
Inflammation (G)
Fibrosis (S)
(Batts and Ludwigs scale)
Number of patients
(n = 31)
G1S2
G1S3
G2S2
G2S2/3
G3S2
G3S3
G3S3/4
G4S3
2
1
19
1
3
1
3
1
After 48 weeks of Peg-IFN and RBV treatment 21 patients achieved ETR (end of treatment response). 17 patients (56% of the patients who started treatment) achieved SVR (sustained virological response) after 6 months.
Three patients were disqualified because of side effects (leucopaenia) induced by interferon. Three patients were disqualified from further treatment after 12 weeks because of lack of effects of treatment EVR-negative (early virological response). The results are presented in table 3.
Table 3. HCV-RNA test results in a tested group of patients with CHC in three periods of time during pegylated interferon alpha 2a and ribavirine therapy (IU/ml = 7.8 viral copies/ml).
 GroupNumber of patients
(n)
(%)
AGroup of the patients with no HCV-RNA after 48 weeks of treatment
(ETR-positive)
2170
BGroup of patients with HCV-RNA present after 48 weeks of treatment
(ETR-negative)
310
CGroup of patients in which the treatment was ceased due to the lack of positive treatment prognosis
(EVR-negative)
310
DPatients who has the treatment ceased due to side effects310
EPatients who achieved SVR1756
ETR (end of treatment reaction) – RNA-HCV after the treatment completion:
ETR-positive – RNA HCV elimination
ETR-negative – lack of RNA-HCV elimination
EVR (early virological responce) – RNA-HCV test in 12th week of treatment:
EVR-positive – no RNA-HCV or decrease in viral load by 2 log
EVR-negative – no minimal 2 lgo RNA-HCV decrease
SVR (sustained virological responce) – RNA-HCV test after 24 weeks after the treatment completion:
SVR-positive – no RNA-HCV
SVR-negative – RNA-HCV present
Aerobic metabolizm rates
Superoxide anion radical production at rest, before treatment was 9.24 to 23.160 (nmolO*2/min/kom); on average 13.621 +/- 3.188 (nmolO*2/min/kom). These results were higher than in a control group 6.239 to 16.670 (nmolO*2/min/kom); on average 6.76 +/- 0.520 (nmolO*2/min/kom). After 24 weeks lower production of superoxide anion radical was found (4.290 to 11.3 (nmolO*2/min/kom); on average 7.988 +/- 1.933 (nmolO*2/min/kom). The lowest levels were fund after 48 weeks of treatment (4.340 to 11.09 (nmolO*2/min/kom); on average 7.988 +/- (nmolO*2/min/kom). These results were higher than in a control group (p < 0.05), and lower than the results before treatment (p < 0.05). Superoxide anion radical production at rest in the patients with CHC treated with Peg-IFN and RBV are presented in table 3 and figure 1.
Fig. 1. Superoxide anion radical level – production at rest in the patients with CHC treated with Peg-IFN alpha and ribibavirine.

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otrzymano: 2014-09-10
zaakceptowano do druku: 2014-10-14

Adres do korespondencji:
*Jarosław Kałuża
Infectious, Tropical Diseases and Parasitoses Department for Adults Infectious Diseases and Hepatology Clinic Medical University of Łódź
ul. Kniaziewicza 1/5, 91-347 Łódź
tel. +48 (42) 251-62-65
karandil@o2.pl

Postępy Nauk Medycznych 11/2014
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