Alberto Civolani

The relationship between copper and steatosis in Wilson's disease

BACKGROUND AND AIMS The histological similarities seen in Wilsons disease (WD) and non-alcoholic 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-five 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 Significant 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 significant correlation between liver copper content and steatosis scores (r=0.87; r(2)=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.

Non-invasive assessment of hepatic fibrosis in a series of patients with Wilson's Disease

BACKGROUND/AIMS Liver biopsy has always represented the standard of reference in hepatic fibrosis assessment. Recently, blood markers and instrumental methods have been proposed for non-invasive assessment. The aim of this study was to validate transient elastography and other non-invasive tests compared to liver histology in Wilsons Disease. METHODS Liver stiffness in 35 Wilsons Disease patients was evaluated by Fibroscan, serum fibrosis markers (AST-to-platelet-ratio index and FIB-4) and biopsy. RESULTS Compared to liver histology, the FibroScan values increased proportionally with progression of the histological fibrosis stage. Significant fibrosis could be predicted with a Fibroscan cut-off value of 6.6 kPa. Advanced fibrosis could be predicted with a FibroScan cut-off value of 8.4 kPa. Serum fibrosis marker values gave good correlation with hepatic stage. CONCLUSIONS A FibroScan value of 6.6 kPa was found to be a significant separation limit for differentiating significant fibrosis stages from milder stages and a fibroscan value of 8.4 kPa was found to be a significant separation limit for differentiating advanced fibrosis stages from milder stages. FibroScan values are clinically useful for predicting fibrosis stages and helpful in managing chronic therapy in Wilsons Disease patients.

Histologic evolution and long-term outcome of Wilson's disease: results of a single-center experience.

Background/aims Wilson’s disease (WD) is a rare inborn disease related to copper storage, leading to liver cirrhosis and neuropsychological deterioration. The aim of this study was to determine the clinical presentation and long-term outcome, and to examine the progression of hepatic histopathology in serial liver biopsies from WD patients. Materials and methods We carried out a retrospective analysis of 60 patients with WD treated with zinc and/or penicillamine. Demographic, clinical, and laboratory data were gathered and 40 patients underwent an initial biopsy and at least one repeat biopsy. Patients were divided into two groups: progressors (patients who presented worsening of at least one unit of fibrosis) and nonprogressors (patients who presented stable or improved fibrosis scores). Results A total of 33/40 (83%) patients (nonprogressors) showed stable hepatic histology or improvement. Seven of 40 (17%) patients (progressors) showed worsening of fibrosis. There was no significant correlation between the histological findings and clinical parameters or initial presentation. Conclusion In our study cohort, liver disease was stable or improving in most of the patients, and development of progressive hepatic symptoms while under treatment was a rare event. The development of new symptoms while under treatment or progression of pre-existing symptoms was more often recorded for neurological than for hepatic symptoms.

HFE gene mutations and Wilson's disease in Sardinia

BACKGROUND Hypocaeruloplasminaemia can lead to tissue iron storage in Wilsons disease and the possibility of iron overload in long-term overtreated patients should be considered. The HFE gene encodes a protein that is intimately involved in intestinal iron absorption. AIMS The aim of this study was to determine the prevalence of the HFE gene mutation, its role in iron metabolism of Wilsons disease patients and the interplay of therapy in copper and iron homeostasis. METHODS The records of 32 patients with Wilsons disease were reviewed for iron and copper indices, HFE gene mutations and liver biopsy. RESULTS Twenty-six patients were negative for HFE gene mutations and did not present significant alterations of iron metabolism. The HFE mutation was significantly associated with increased hepatic iron content (P<0.02) and transferrin saturation index (P<0.03). After treatment period, iron indices were significantly decreased only in HFE gene wild-type. CONCLUSIONS The HFE gene mutations may be an addictional factor in iron overload in Wilsons disease. Our results showed that an adjustment of dosage of drugs could prevent further iron overload induced by overtreatment only in patients HFE wild-type.

Clot wave analysis and thromboembolic score in liver cirrhosis: two opposing phenomena

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are abnormal but unreliable in cirrhotic patients to express their risk of bleeding. However, these patients may also suffer from thrombotic episodes. In order to investigate the dynamics of the formation of fibrin, the clot waveform analysis (CWA) of aPTT was studied together with a score for the evaluation of the thromboembolic risk.

Animal models are reliably mimicking human diseases? A morphological study that compares animal with human NAFLD

Non‐alcoholic fatty liver disease (NAFLD) is a clinical‐pathological syndrome that includes a wide spectrum of morphological alterations. In research, animal models are crucial in evaluating not only the pathogenesis of NAFLD and its progression, but also the therapeutic effects of various agents. Investigations on the ultrastructural features of NAFLD in humans are not copious, due to the difficulty to obtain human samples and to the long time of NAFLD to evolve. Translational comparative studies on the reliability of animal models in representing the histopathologic picture as seen in humans are missing. To overcome this lack of investigations, we compared the ultrastructural NAFLD features of an animal model versus human. Sprague‐Dawley rats were fed with a high fat diet (HFD) for 1–4 weeks, while control rats were fed with a standard diet. Human specimens were collected from patients with diagnosed fatty liver disease, undergoing liver biopsies or surgery. Rat and human samples were examined by light microscopy and by transmission and high resolution scanning electron microscopy. The present work demonstrated that NAFLD in animal model and in human, share overlapping ultrastructural features. In conclusion, animal HFD represent an appropriate tool in studying the pathogenesis of NAFLD. Microsc. Res. Tech. 77:790–796, 2014.

Uneven distribution of hepatic copper concentration and diagnostic value of double-sample biopsy in Wilson's disease.

Abstract Background and aims. Determination of hepatic copper (Cu) concentration is important in Wilsons disease (WD) diagnosis. The aim of this study was to evaluate uneven distribution of liver Cu concentration and the utility of double-sample biopsy in WD diagnosis. Methods. Thirty-five WD patients (20 male; mean age 41 ± 9 years) were enrolled in the study and double-liver samples for biopsy were obtained. A further 30 WD patients, in whom Cu determination was performed using single-liver samples, were also enrolled as controls. Results. A marked difference in hepatic Cu concentration was observed between the two sample groups (p < 0.0001). This difference is statistically significant for all levels of liver fibrosis (p < 0.001) and for the comparison of hepatic and neurological phenotypes (p < 0.01). The sensitivity of the Cu concentrations obtained from the double-sample biopsies for the conventional cut-off value of 250 mg/g dry weight of tissue was 85.7% compared to 80% in the single-sample biopsies. By lowering the cut-off value from 250 to 50 µg/g of dry weight of tissue, the sensitivity of Cu content to diagnose WD increased to 97% for double-sample liver biopsy compared to 93% for single-sample liver biopsy. Conclusions. Liver Cu content was unevenly distributed in the WD subjects, irrespective of fibrosis levels and disease phenotypes; hence WD can be misdiagnosed using single-sample liver Cu measurement. Double-sample biopsy sensitivity is greater than that obtained with single-sample biopsy and should therefore be considered to evaluate liver Cu concentration at initial diagnosis in all patients.

Are prothrombin time and clot waveform analysis useful in detecting a bleeding risk in liver cirrhosis

Prothrombin time is thought to be unreliable in cirrhotic patients to predict the risk of bleeding. We investigated whether prothrombin time ratio was an independent risk factor for bleeding alongside its clot waveform analysis.

Animal models are reliably mimicking human diseases? A morphological study that compares animal and human NAFLD

Nonalcoholic fatty liver disease (NAFLD) affects up to 20% of western population and, when untreated, it can progress from simple fatty liver or steatosis to a more severe condition, such as NASH (non alcoholic steatohepatitis) and cirrhosis (1). NAFLD is a clinical-pathological syndrome that include a wide spectrum of morphological alteration but such studies on humans are not copious, as human samples are difficult to obtain because of ethical limitations. Experimental models are crucial to study steatosis’ progression, not only for elucidating the pathogenesis of NAFLD but also in examining therapeutic effects of various agents. Animal models may be developed on genetic or nutritional basis, or a combination of both. It is important to select the best model fitted to the aim of the study. But the question that arises is: can the animal model reflect hepatic histopathology and pathophysiology of human NAFLD? This question is always neglected as well as the evaluation of ultrastructural features of NAFLD. In order to overcome this lack of investigations we compared ultrastructural features of NAFLD in an animal model and in human samples of NAFLD patients. NAFLD animal model was obtained using Sprague Dawley rats fed by a high fat diet (HFD) (71% of energy from fat), while control rats were fed by a standard diet (35% of energy from fat). Diets were given ad libitum and rats were killed after 1, 2, 3, and 4 weeks. Human specimens were obtained from patients with fatty liver disease undergoing to liver biopsies. Normal liver was taken from patients undergoing surgery for other pathologies. Hepatic steatosis and normality of the liver were assessed by parallel examinations at light microscopy, transmission and high resolution scanning electron microscopy. Light microscopy results showed that different degrees of NAFLD observed in human samples corresponded to similar morphological changes in treated rats. Ultrastructural examination revealed that in the HFD model the histopathology closely reflected that of human NAFLD, although the first did not replicate the full spectrum of the disease in humans. In summary, we showed that, at least morphologically, HFD model overlays to human NAFLD. This could point out for reliability in evaluating other pathological features in animal models. Moreover, animal HFD mimics human nutritional dysregulation that may induce the same biochemical and molecular modifications observed in human patients and might represent a more appropriate tool for studying the pathogenesis of NAFLD over genetic models (2).