Clinics and Research in Hepatology and Gastroenterology (2013) 37, 3640

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ORIGINAL ARTICLE

The relationship between copper and steatosis in Wilsons disease

Mauro Liggi , Debora Murgia , Alberto Civolani , Enrico Demelia , Orazio Sorbello , Luigi Demelia
UOC of Gastroenterology, Azienda Ospedaliero Universitaria, Cagliari, Italy Available online 7 May 2012

Summary Background and aims: The histological similarities seen in Wilsons disease (WD) and nonalcoholic steatohepatitis (NASH) led us to verify possible correlations between glucose and/or lipid and/or iron metabolism alterations and hepatic steatosis in WD patients. Methods: Thirty-ve WD patients (20 females, 15 males, mean age 40.1 5.4 years), and 44 NASH patients (25 females, 19 males, mean age 42.8 6.7 years) were enrolled in the study. BMI, total/HDL/LDL-cholesterol, triglycerides and glucose serum levels were established in all subjects. HOMA index was calculated. Percutaneous liver biopsy with quantitative evaluation of steatosis and copper tissue content was performed in all WD patients and in NASH control group. Results: Signicant difference was seen in baseline serum levels of glucose, HOMA index, total cholesterol, triglycerides, and ferritin between the WD group and NASH group (P < 0.05) but steatosis scored was similar between two groups. No correlation between the level of steatosis and metabolic factors studied was highlighted. In WD, hepatic parenchymal copper concentration was 753 65.3 mcg/g dry weight against 54.5 16.9 mcg/g dry weight in NASH patients (P < 0.05). Higher liver copper concentrations were seen in patients with severe steatosis compared to those with mild (P = 0.004) and moderate, (P = 0.038) steatosis. Positive signicant correlation between liver copper content and steatosis scores (r = 0.87; r2 = 0.76) was observed. Conclusions: The hepatic steatosis in WD is not induced by metabolic comorbidities but by the accumulation of copper in the liver tissue. The hypothesise that the metabolic alterations could be co-factors in the pathogenesis of steatosis in these patients cannot be excluded. 2012 Elsevier Masson SAS. All rights reserved.

Introduction

Corresponding author. Via Giudice Chiano 31, 09131 Cagliari, Italy. Tel.: +347 865 9513; fax: +078 163 998. E-mail address: macnol@tiscali.it (M. Liggi).

Wilsons disease (WD) is an autosomal recessive hereditary disease, caused by the mutation of the gene that codies ATP7B, a cationic transporter with cationic adenosin-

2210-7401/$ see front matter 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.clinre.2012.03.038

Copper and steatosis triphosphatasic function, essential in the metabolism of copper [1]. This mutation alters the hepatic excretion of copper with consequent accumulation in diverse tissues of the organism, particularly in the liver, central nervous system, cornea and kidney [2]. The most frequently observed hepatic lesions in WD are steatosis [3,4], Mallory bodies, glycogenic nuclei, portal, periportal and pericentral brosis [5]. These alterations are also observed in nonalcoholic steatohepatitis (NASH) [6,7]. It is known that obesity, insulin-resistance [8] and hyperlipidemia [9] are associated with NASH, while controversy exists concerning the data on the effects of iron overload [10,11] the limited studies on copper accumulation [12] and the pathogenesis of NASH. Alterations of the metabolism of glucose [13,14] and lipid [15] have only been reported in single cases in WD patients. The presence of alterations has never been documented systematically in patients with hepatic forms of WD or before the start of therapy with copper chelators D-penicillamine and trientine, or zinc. Histological similarity between WD and NASH led us to verify whether hepatic steatosis in WD is correlated to the presence of changes in glucose and/or lipid and/or iron metabolism or whether it can be attributed exclusively to copper metabolism changes.

37 with hepatic decompensated cirrhosis, pregnancy or breastfeeding were excluded from the study. Body mass index (BMI) BMI = weight (kg)/height(m2 ) [29], total cholesterol (n.v. < 200 mg/dl), HDL-cholesterol, LDLcholesterol, triglycerides (n.v. > 170 mg/dl), UIBC, ferritin and glucose serum levels, HOMA index were performed for each subject [30]. Results were expressed as means standard error of the mean (SEM). Variance analysis was used to evaluate the signicant differences between the average values of the continuous variables of data from the different groups. The Student T test was used to compare continuous variables between the two groups, accepting the normal distribution of these data. Values of P < 0.05 were considered statistically signicant. Correlation between two qualitative variables was quantied using the regression coefcient (r) and the determination coefcient (r2 ). Informed consent was obtained from all the study participants.

Results
Baseline characteristics of the two groups are shown in Table 1. In the WD group, four patients (11%) had a BMI of > 25 and 10 of < 20 (27.7%) with average values SEM of 21.1 0.5. Signicant difference was seen in baseline serum levels of glucose, HOMA index, total cholesterol, triglycerides, and ferritin between the WD group and NASH group (P < 0.05). Alterations of the glucidic and lipidic metabolisms (total cholesterol, LDL-cholesterol, triglycerides glucose serum levels) were reported in 20 out of 35 WD patients (56%). Twelve of the 35 patients showed only one metabolic alteration whereas eight of 35 patients showed multiple alterations. All WD and NASH patients underwent liver biopsy (Table 2): in WD group mild steatosis was observed in 19/35 (> 5% < = 33%), moderate steatosis in 10/35 (> 33% < = 66%)

Materials and methods

Between 1995 and 2011, 35 WD patients, (20 females/15 males, mean age 40.1 5.4 years) were studied; none had undergone therapy with chelants or zinc before being submitted to the investigations performed in the present study. A control group of 44 subjects affected by NASH was also included in the study. The diagnosis of NASH is established by the presence of a characteristic pattern of steatosis, inammation and hepatocellular ballooning on liver biopsies in the absence of signicant alcohol consumption [16]. WD diagnosis was based on the presence of clinical abnormalities observed in liver disease and/or on the presence of Kayser Fleisher (slit lamp examination) and/or abnormal neurological features and low serum ceruloplasmin (< 20 mg/dl). Diagnosis was conrmed by the determination of total serum copper (< 70 g/dl), of cupruria baseline (<100 g/24 h) and hepatic parenchymal copper concentration (> 250 mcg/g dry weight) following hepatic agobiopsy [17,18]. The copper excretion in 24 hours urine following oral administration of d-penicillamine (n.v. < 300 g/24 h), and gene mutations of ATP7B using molecular biology tests were also determined [19]. WD patients underwent percutaneous liver biopsy [20] with histological ndings, copper and iron tissue content with hepatic iron index calculation (HII) [21] (n.v. < 1.9). Histological evaluation of grading and staging of brosis was carried out using the criteria proposed by Metavir and Ishak [22,23]. Quantitative evaluation of hepatic steatosis was also carried out in all the patients [24,25]. Steatosis was scored as mild (</ = 33%), moderate (> 33% < = 66%) or severe (> 66%) of parenchymal involvement [26]. The NAFLD Activity Score (NAS) can range from 0 to 8 and is calculated by the sum of scores of steatosis (03), lobular inammation (03) and hepatocyte ballooning (02) [27,28]. WD patients

Table 1 Baseline Wilsons disease (WD) and non-alcoholic steatohepatitis (NASH) patient characteristics. Values expressed as mean SEM. WD n = 35 Age Sex Male Female BMI >25 20/25 < 20 Basaline glucose serum level Homa index Total Cholesterol HDL Triglycerides Ferritin UIBC 40.1 5.4 15 20 21.1 0.5 7 22 6 84.2 1.6 1.1 0.3 194.1 7.0 61.4 3.4 100.8 9.4 131 24.4 155.3 12.1 NASH n = 44 42.8 6.7 19 25 27.5 2.5 28 12 4 107.4 43.0 5.5 0.9 223.5 58.0 58.6 19.2 131.1 12.5 215.6 158.5 187.5 45.6

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Table 2 Histology in Wilsons disease (WD) and nonalcoholic steatohepatitis (NASH) patient. WD Grading 1 2 3 Staging I II III IV Steatosis Mild Moderate Severe Cu 24 8 3 8 16 7 4 21 9 10 735.6 67.3 NASH 31 11 2 12 19 9 4 15 12 17 54.5 16.9

M. Liggi et al.

Discussion
Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of histological alterations characterized mainly by hepatic steatosis that affects subjects who do not consume alcohol in quantities dangerous for the liver [31,32]. The progression of NAFLD to NASH is characterized by the periportal and lobular inammatory inltration [33,34]. Numerous studies claim that NAFLD represents the hepatic component of the metabolic syndrome characterized by obesity, insulin-resistance and/or type 2 diabetes [35,36], hypertriglyceridemia and arterial hypertension [3740]. The histological picture of WD is various and is not specic to the disease; levels of evolution from steatosis with minimal lesions (typical in children and adolescents) to chronic active hepatitis (young adults) and nally cirrhosis can be identied [41]. Ultra-structural analysis of the liver with steatosis reveals signicant mitochondrial anomalies (megamitochondria, dilation of the crest points) [42], which in the absence of colestasis, are considered pathognomonic of WD. Steatosis, Mallory bodies, glycogenic nuclei, portal, periportal and pericentral brosis with associated lobular inammation are the most frequently observed basic lesions in WD that are also seen in NASH. Studies in vivo show that copper overload at hepatic levels causes lipidic peroxidization producing free radicals and subsequent oxidization with damage to the membrane functions dependant on the mitochondria (oxidative metabolism) and to the lysosomes (membrane integrity, uidity and pH) [43,44]. This lipoperoxidation should be modulated with zinc salt therapy [45]. Our data highlight signicant correlation between copper concentration and hepatic steatosis with an increase in severity as tissue copper content increases; signicantly higher copper concentrations were observed in patients with severe steatosis compared to those with moderate steatosis. It is possible, therefore, that steatosis in WD patients is due to the inhibition of mitochondrial function following damage caused by copper accumulation at hepatic levels; in fact even if copper overload determines damage to the mitochondrial respiratory chain by inhibiting cytocromo c oxidasis activity [46], it is possible that the mitochondrial damage is involved in beta oxidization inhibition. Peroxidative damage with the production of free radicals, due to further accumulation (overload) of tissue copper, determines the inammatory process from steatosis with minimal lesions to steatohepatitis [2]. UIBC, ferritin levels and HII in the WD patients studied were all within range; we can therefore exclude a role of iron overload in steatosis in WD. Furthermore, 56% of the WD patients reported at least one metabolic alteration that was observed in 51% of the patients with moderate or mild steatosis. In our study, we observed that the prevalence of metabolic risk factors is statistically signicant between WD and NASH patients. Nevertheless, hepatic steatosis score are similar. In conclusion, we can say that hepatic steatosis in Wilsons disease is not induced by metabolic comorbidities but by the accumulation of copper in the liver tissue. The hypothesise that the metabolic alterations could be

and severe steatosis in 6/35 (> 66%) while in NASH patients mild steatosis was observed in 15/44 (> 5% < = 33%), moderate steatosis in 12/44 (> 33% < = 66%) and severe steatosis in 17/44 (> 66%). No correlation between the level of steatosis and the metabolic factors studied singularly or globally was highlighted. One or more metabolic alterations were revealed in nine of 19 patients (56,2%) with mild or moderate steatosis. Iron tissue content dosage was 248.3 53.1 mcg/g dry weight e HII revealed values between 0.1 and 0.84 with an average of 0.22 0.04 (n.v. < 1.9). In WD average values SEM of Cu tissue content were 735.6 67.3 mcg/g dry weight against 54.5 16.9 mcg/g dry weight in NASH patients (P < 0,05). WD patients with mild steatosis showed lower liver copper concentrations (500.3 87.6 mcg/g dry weight) compared to patients with moderate (859.2 104.9 mcg/g dry weight; p = 0.038) and severe steatosis (1053.7 83.4 mcg/g dry weight; P = 0.004). Signicant correlation was observed between liver copper content and steatosis scores; regression analysis between hepatic copper and steatosis showed a positive signicant correlation (r = 0.87 and r2 = 0.76; Fig. 1).

Figure 1 Regression line between steatosis and copper in Wilsons disease (WD) patients.

Copper and steatosis co-factors in the pathogenesis of steatosis in these patients cannot be excluded.

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Disclosure of interest
The authors declare that they have no conicts of interest concerning this article.

Acknowledgements
We would like to thank Barry Mark Wheaton for his invaluable linguistic assistance.

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