Clinical research
Assessment of selected oxidative stress parameters in patients with Wilson’s disease

Introduction
Wilson’s disease is a recessive autosomal inherited disorder of copper metabolism that is caused by a mutation of the ATP7B gene which encodes an ATP-ase – the enzyme indispensable for biliary excretion of copper through the hepatocyte membrane [1]. The disease afflicts 1 in 40,000 individuals. Impaired transport of copper from the hepatocytes into bile leads to a toxic accumulation of copper in the liver followed by other tissues including the basal ganglia, cornea and erythrocytes. Damage to them is caused via generation of free radicals, lipid peroxidation and inhibition of protein synthesis [2]. Copper plays an important role in initiating the generation of reactive oxygen species. As a result of toxic activity of copper and creation of free oxygen radicals, a change in activity of antioxidative enzymes appears. The involvement of antioxidative factors including antioxidative enzymes from free radical scavengering groups in Wilson’s disease is not fully understood. The main enzymes of the antioxidative system are superoxide dismutase (SOD) and glutathione peroxidase (GPx). Both enzymes along with catalase, belong to the first-line of cellular defense mechanism against damage caused by reactive forms of oxygen. Antioxidative enzymes degrade organic superoxides to non-toxic hydroxylipids and in this way inhibit chain reaction of lipid peroxidation. Superoxide dismutase is a specific enzyme which is found in different cell organelles. It causes conversion of superoxide radical into elementary oxide and hydrogen superoxide which in turn undergoes further transformation into oxide and water by means of catalase. Glutathione peroxidase on the other hand, reacts with reduced glutathione and hydrogen superoxide which eventually leads to water and oxidized glutathione formation. Glutathione peroxidase is found both in the intracellular and in the extracellular space [3]. Activity of selenium-dependent GPx containing selenium in its active centers depends, along with other factors, on the bioavailability of this catalyst in the serum which in spite of being a trace element plays an important role in different metabolic processes [4-6]. Studies conducted so far show no selenium concentration disturbances in patients with Wilson’s disease [7]. Changes in erythrocytes selenium concentration and activities of antioxidative enzymes in this group of patients still remain unclear. Assessment of their activities could probably indirectly contribute to explanation of the mechanism of copper-dependent toxic liver injury.
The main aim of the study was the assessment of selected oxidative stress parameters: activity of GPx, SOD, serum antioxidative status (TAS), and erythrocyte selenium concentrations (SeE) in patients with Wilson’s disease.

Material and methods
The study involved 20 patients (12 men and 8 women, mean age 39.9 years) with Wilson’s disease who were treated at the Departments and Outpatient Clinics of Medical University of Gdańsk and in the Hepatological Outpatient Clinic of State Infectious Diseases Hospital in Gdańsk in Poland. Only patients with a confirmed diagnosis of Wilson’s disease were included in the study. The diagnosis had been made in patients with liver injury and/or neurological symptoms using the following criteria: decreased concentration of serum ceruloplasmin, increased 24-h urinary excretion of copper and presence of Kayser-Fleischer ring in the cornea. In some of the patients genetical tests were performed. Assessment of a mutation in ATP7B gene was performed in the Laboratory of the Department of Internal Medicine IV Gastroenterology and Hepatology of University of Vienna.
Twenty equally matched healthy controls also participated in the study (mean age 36.7 years).
Patients with coexisting hepatotoxic factors: hepatitis B virus (HBV) or hepatitis C virus (HCV) infection and those abusing alcohol or drugs were ruled out of the study.
During the study, the patients were in disease remission and did not present symptoms of active hepatic injury (oligosymptomatic group). Mild clinical symptoms still persisted in some patients with neurological form of Wilson’s disease although they were much less prominent. The patients had been treated with d-penicillamine and/or zinc sulphate for 2-9 years.
In all the subjects, TAS, erythrocyte GPx and SOD activities were assessed. The evaluation was carried out at the Department of Clinical Nutrition and Laboratory Diagnostics of Medical University of Gdańsk using Randox sets by precisely following the manufacturer’s instructions. Serum antioxidative status activity was assessed by means of colorimetric method [8] while SOD and GPx activities were evaluated using Ultraspect III spectrophotometer. In the examined groups, SeE was also determined by means of atomic absorption spectroscopy using hydride generation technique (HG-ASS). The tests were carried out at the Department of Physical Chemistry of Medical University of Gdańsk by the use of Cheman, Merck and Aldrich reagents. Moreover, serum copper and ceruloplasmin concentration, 24-h urinary copper excretion, biochemical markers of liver injury such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and g-glutamyltranspeptidase (GGTP) activities were determined. Furthermore parameters such as bilirubin, albumin and g-globulin concentration and values of normalized international prothrombin index (INR) were also assessed using standard methods. Serum used for the study was collected only after a consent of a patient to conduct the mentioned above tests.
The data were analyzed using nonparametric methods. The Mann-Whitney U test was used for between-group comparison. Spearmann method was applied for correlation assessment. The level of significance was set at p < 0.05. All calculations and figures were performed using the Statistica 8 software package (Statsoft Inc., Tulsa, USA).
The Local Ethics Committee consented to carry out the present study.

Results
Comparison of GPx, TAS and SOD activities in the patients and healthy volunteers indicated markedly higher activities of the tested enzymes and TAS in the sera of the patients when compared to the control group (Table I). Assessing erythrocyte selenium concentration, markedly higher values were also found in the group of the patients in comparison to the control group (Table I).
Comparison of the tested parameters in the patients presenting neurological symptoms of the disease and oligosymptomatic ones did not show statistically significant differences. However, a trend towards lower values of all tested parameters were noticed in patient with total remission of the disease (Table II).
The results obtained in the course of the study were also analyzed according to treatment types as shown in Table III. Enzyme activities remained unchanged in patients undergoing different treatments. However, TAS values were significantly higher in the patients treated with zinc preparations only or combination of zinc sulphate and d-penicyllamine as compared to the patients treated with d-penicyllamine alone.
Copper metabolism parameters in the group of the patients are shown in Table IV. Analysis of the relationship between the tested parameters and copper metabolism showed only a positive correlation between TAS and GPx activities and 24-h urinary copper excretion (p = 0.035, r = 0.47; p < 0.001, r = 0.7) (Figure 1, 2).
Biochemical markers of liver injury were within the normal range, so no correlation between them and the tested parameters were studied.

Discussion
Research concerning antioxidative system activity in patients with alcoholic or post-inflammatory liver injury has become quite popular [9-12]. In both groups, a significant reduction of the tested parameters in comparison to the healthy controls has been stated [9-15].
Our studies were concerned with the activity of antioxidative enzymes that are known to play a vital role in the processes involved in cell defense against toxic superoxides and oxidative radicals. Samuele et al. proved that the oxidative stress plays a crucial role in degradation of the hepatocytes and neurons of the central nervous system in the course of Wilson’s disease [16]. So far other antioxidative factors have been investigated in Wilson’s disease, however, few published papers address the activity of antioxidative enzymes in this group of patients [17, 18]. The papers mainly deal with decrease in vitamin E concentration in patients with chronic liver diseases including Wilson’s disease [19, 20]. Antioxidative activity of this vitamin is now quite often used in treatment of neoplastic disease [21, 22]. It has been proven that vitamin E analogues such as a-TOS stimulate apoptosis of proliferating endothelial cells and inhibit angiogenesis and tumor growth.
In the present work, we investigated a group of patients with Wilson’s disease in the remission stage which was initially confirmed by normal liver tests (no liver biopsy was performed at the time of the study). We found significantly higher activities of the tested enzymes and TAS in comparison to the control group. It may seem therefore that increased activity of the antioxidative enzymes is an adaptive mechanism in patients suffering from Wilson’s disease with intense generation of lipid peroxidation and free radicals. In our study, we did not find significant differences in the analyzed parameters between the patients presenting some neurological symptoms and oligosymptomatic patients. However, a tendency towards lower values of the tested parameters was noticed in the patient with total remission of the disease what may result from lower activity of the antioxidative system in this group of patients. And yet some authors have reported different results. Attri et al. stated decreased activities of erythrocyte antioxidative enzymes [23]. It should be underlined, however, that the study included 8 patients with Wilson’s disease in the stage of decompensation with acute haemolysis. Significantly higher values of the parameters mentioned above were observed by the same authors in the follow-up examination after treatment – during the clinical remission of the disease as in the present study. Nagasaka et al. stated a decrease in GPx activity but only in patients with fulminant liver failure due to Wilson’s disease [17]. In none of our patients, symptoms of acute or active liver injury appeared during the study. Only the patients with neurological symptoms were not in full remission.
Many authors underline a possible connection between activity of the antioxidative enzymes and use of zinc preparations [24-26]. Santon et al. stated an increase in activity of hepatic GPx and concentration of metallothioneins in the liver and intestines of patients treated with zinc preparations in comparison to the ones not receiving this element and a control group [26]. However, we observed significantly higher values of the tested parameters even in the patients who did not get zinc preparations. So activation of the antioxidative system is due not only to the type of treatment but may also result from the disease.
In the present study, we also assessed concentration of erythrocytic selenium which is an indispensable component of GPx. We found significantly higher concentrations of selenium in the group of the patients comparing to the healthy controls. Comparison of our own results with any data from the literature is impossible because of lack of papers regarding this matter. Compounds containing selenium play an important role in the organism removing not only toxic oxygenic substances but also metals. However, reactions between selenium and metals result in generation of poorly soluble metal selenides what in turn may lead to accumulation of metals in the parenchymatous organs. A protein called AE1 takes part in the transport of oxyanions – selenates, phosphatic, sulphatic and magnesic oxyanions – through the erythrocyte membranes, so mutual changes in the concentrations of other elements are responsible for maintenance of selenium concentration in the erythrocytes [27].
In our study, we also analyzed correlation between GPx, SOD and TAS activities, SeE concentration and copper metabolism parameters. A positive correlation between 24-h urinary copper excretion and both TAS and GPx activities was found. It should be stressed that these significant correlations were also observed in the group of patients who did not received d-penicyllamine – a drug increasing urinary copper excretion. Unfortunately no paper assessing correlation between TAS and activity of the antioxidative enzymes and copper metabolism in patients with Wilson’s disease has been found in the available literature. Dalgiç et al. found decreased values of other antioxidative stress parameters (vitamin C and b-carotene) in patients with cirrhosis due to different chronic liver diseases including Wilson’s disease [19]. In our paper, we did not assess a correlation between the tested enzymes and selenium and the liver parameters because they were within the normal range of values.
The results of the present study leave many problems unexplained but they deal with the problem of the antioxidative system activity in Wilson’s disease. Activities of the antioxidative enzymes and TAS in treated patients with Wilson’s disease are high what differs from other liver pathologies and what may result from the stimulation of the antioxidative system in this group of patients.
In conclusion, the antioxidative status expressed by means of activities of the anti-oxidative enzymes is high in patients with Wilson’s disease who are in good clinical condition.
Higher concentration of selenium in the erythrocytes requires further research – it may be due to accumulation of this element in case of oxidative stress threat.

Acknowledgments
The authors would like to thank professor Peter Ferenci from the Department of Internal Medicine, Gastroenterology and Hepatology of University of Vienna for making it possible to perform genetical tests in our patients.

References
1. Brewer GJ. Recognition, diagnosis, and management of Wilson’s disease. Proc Exp Biol Med 2000; 223: 39-46.
2. Britton RS. Metal-induced hepatotoxicity. Semin Liver Dis 1996; 16: 3-12.
3. Murray RK, Granner DK, Mayes PA, Rodwell VW. Harper’s Biochemistry. PZWL, Warszawa 1995.
4. Bettger WJ. Zinc and selenium, site-specific versus general antioxidation. Can J Physiol Pharmacol 1993; 71: 721-4.
5. Turan B, Dalay N, Afrasyap L, et al. The effects of selenium supplementation on antioxidative enzyme activities and plasma and erythrocyte selenium levels. Acta Physiol Hung 1993; 81: 87-93.
6. Feher J, Nemeth E, Nagy V, Lengyel G, Feher J. The preventive role of coenzyme Q10 and other antioxidants in injuries caused by oxidative stress. Arch Med Sci 2007; 3: 305-14.
7. Świątkowska-Stodulska R, Dejneka W, Jabłońska-Kaszewska I, et al. Serum selenium concentration in patients with Wilson’s disease. Hepatogastroenterology 2007; 54: 1788-90.
8. Katsoulis K, Kontakiotis T, Baltopoulos G, Kotsovili A, Legakis IN. Total antioxidant status and severity of community-acquired pneumonia: are they correlated? Respiration 2005; 72: 381-7.
9. Koch OR, Pani G, Borrello S, et al. Oxidative stress and antioxidant defenses in ethanol-induced cell injury. Mol Aspects Med 2004; 25: 191-8.
10. Hoek JB, Pastorino JG. Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol 2002; 27: 63-8.
11. Cemek M, Dede S, Bayiroglu F, Caksen H, Cemek F, Mert N. Relationship between antioxidant capacity and oxidative stress in children with acute hepatitis A. World J Gastroenterol 2006; 12: 6212-5.
12. Popadiuk S, Liberek A, Korzon M, Renke J, Woźniak M. Free radical reactions and activity of antioxidant barrier in children with chronic hepatitis B [Polish]. Med Wieku Rozwoj 2004; 8: 395-402.
13. Czuczejko J, Zachara BA, Staubach-Topczewska E, Halota W, Kędziora J. Selenium, glutathione and glutathione peroxidases in blood of patients with chronic liver diseases. Acta Biochim Pol 2003; 50: 1147-54.
14. Dworkin BM, Rosenthal WS, Stahl RE, Panesar NK. Decreased hepatic selenium content in alcoholic cirrhosis. Dig Dis Sci 1988; 33: 1213-7.
15. Jabłońska-Kaszewska I, Świątkowska-Stodulska R, Łukasiak J, et al. Serum selenium levels in alcoholic liver disease. Med Sci Monit 2003; 9 (Suppl 3): 15-8.
16. Samuele A, Mangiagalli A, Armentero MT, et al. Oxidative stress and pro-apoptotic conditions in rodent model of Wilson’s disease. Biochim Biophys Acta 2005; 1741: 325-30.
17. Nagasaka H, Inoue I, Inui A, et al. Relationship between oxidative stress and antioxidant systems in the liver of patients with Wilson’s disease: hepatic manifestation in Wilson’s disease as a consequence of augmented oxidative stress. Pediatr Res 2006; 60: 472-7.
18. Dastych M. Serum levels of zinc, copper and selenium in patients with Wilson’s disease treated with zinc [Czech]. Vnitr Lek 1999; 45: 217-9.
19. Dalgiç B, Sönmez N, Biberog˘lu G, Hasanog˘lu A, Erbas¸ş D. Evaluation of oxidant stress in Wilson’s disease and non-Wilsonian chronic liver disease in childhood. Turk J Gastroenterol 2005; 16: 7-11.
20. Rodo M, Czonkowska A, Pulawska M, Swiderska M, Tarnacka B, Wehr H. The level of serum lipids, vitamin E and low density lipoprotein oxidation in Wilson’s disease patients. Eur J Neurol 2000; 7: 491-4.
21. Yu W, Jia L, Wang P, et al. In vitro and in vivo evaluation of anticancer actions of natural and synthetic vitamin E forms. Mol Nutr Food Res 2008; 52: 447-56.
22. Dong LF, Swettenham E, Eliasson J, et al. Vitamin E analogues inhibit angiogenesis by selective induction of apoptosis in proliferating endothelial cells: the role of oxidative stress. Cancer Res 2007; 67: 11906-13.
23. Attri S, Sharma N, Jahagirdar S, Thapa BR, Prasad R. Erythrocyte metabolism and antioxidant status of patients with Wilson’s disease with haemolytic anaemia. Pediatr Res 2006; 59: 593-7.
24. Farinati F, Cardin R, D’Inca R, Naccarato R, Sturniolo GC. Zinc treatment prevents lipid peroxidation and increases glutathione availability in Wilson’s disease. J Lab Clin Med 2003; 141: 372-7.
25. Sturniolo GC, Mestriner C, Irato P, Albergoni V, Longo G, D’Inca R. Zinc therapy increases duodenal concentrations of metallothionein and iron in Wilson’s disease patients. Am J Gastroenterol 1999; 94: 334-8.
26. Santon A, Irato P, Medici V, D’Inca R, Albergoni V, Sturniolo GC. Effect and possible role of Zn treatment in LEC rats, an animal model of Wilson’s disease. Biochim Biophys Acta 2003; 1637: 91-7.
27. Galanter WL, Hakimian M, Labotka RJ. Structural determinants of substrate specificity of the erythrocyte anion transporter. Am J Physiol 1993; 265: C918-26.