Hepatic Copper Concentrations Research Articles

Alleviation of Copper-Induced Hepatotoxicity by Bergenin: Diminution of Oxidative Stress, Inflammation, and Apoptosis via Targeting SIRT1/FOXO3a/NF-κB Axes and p38 MAPK Signaling.

Despite its biological importance, excess copper induces organ damage, especially to the liver. Disruption of critical signaling cascades that control redox status, inflammatory responses, and cellular apoptosis significantly contributes to the copper-induced hepatotoxicity. The present work explored the hepatoprotective ability of bergenin against the copper-induced hepatotoxicity using male Wistar rats as a mammalian model. The results revealed that bergenin suppressed the copper-evoked histopathological changes and hepatocellular necrosis as indicated by decreased activity of the liver enzymes ALT and AST in the sera of the copper-intoxicated rats. It decreased hepatic copper content and the copper-induced oxidative stress as revealed by reduced lipid peroxidation and improved activity of the antioxidant enzymes thioredoxin reductase, glutathione peroxidase, catalase, and superoxide dismutase. Bergenin downregulated the inflammatory cytokines TNF-α and IL-6, and the inflammatory cell infiltration to the liver tissues. Additionally, it inhibited the copper-induced apoptosis as indicated by significant reduction in caspase-3 activity. At the molecular level, bergenin activated the antioxidant transcription factor FOXO3a, inhibited the nuclear translocation of the inflammatory transcription factor NF-κB, and suppressed the inflammatory signaling molecules p38 MAPK and c-Fos. Interestingly, bergenin improved the expression of the anti-apoptotic protein Bcl2 and reduced the pro-apoptotic protein BAX. Bergenin markedly enhanced the expression of the histone deacetylase protein SIRT1 that regulates activity of NF-κB and FOXO3a. Collectively, these findings highlight the alleviating activity of bergenin against the copper-induced hepatotoxicity via controlling oxidative stress, inflammation, and apoptosis potentially through upregulation of SIRT1, activation of FOXO3a along with suppression of NF-κB and p38 MAPK signaling.

Exploring the impact of gut microbiota on liver health in mice and patients with Wilson disease.

Distinctive gut microbial profiles have been observed between patients with Wilson disease (WD) and healthy individuals. Despite this, the exact relationship and influence of gut microbiota on the advancement of WD-related liver damage remain ambiguous. This research seeks to clarify the gut microbiota characteristics in both human patients and mouse models of WD, as well as their impact on liver injury. Gut microbial features in healthy individuals, patients with WD, healthy mice and mice with early- and late-stage WD were analysed using 16S rRNA gene sequencing. Additionally, WD-afflicted mice underwent treatment with either an antibiotic cocktail (with normal saline as a control) or healthy microbiota (using disease microbiota as a control). The study assessed gut microbiota composition, hepatic transcriptome profiles, liver copper concentrations and hepatic pathological injuries. Patients with hepatic WD and mice with WD-related liver injury displayed altered gut microbiota composition, notably with a significant reduction in Lactobacillus abundance. Additionally, the abundances of several gut genera, including Lactobacillus, Veillonella and Eubacterium coprostanoligenes, showed significant correlations with the severity of liver injury in patients with WD. In WD mice, antibiotic treatment or transplantation of healthy microbiota altered the gut microbial structure, increased Lactobacillus abundance and modified the hepatic transcriptional profile. These interventions resulted in reduced hepatic copper concentration and alleviation of WD-related liver injury. Individuals and mice with pronounced WD-related liver injury exhibited shifts in gut microbial composition. Regulating gut microbiota through healthy microbiota transplantation emerges as a promising therapeutic approach for treating WD-related liver injury.

Notice of Retraction: Amundson et al. Sixteen years of canine hepatic copper concentrations within normal reference ranges in dogs fed a broad range of commercial diets. J Am Vet Med Assoc. Published online March 7, 2024.

Notice of Retraction: Amundson et al. Sixteen years of canine hepatic copper concentrations within normal reference ranges in dogs fed a broad range of commercial diets. J Am Vet Med Assoc. Published online March 7, 2024.

Marginal Zinc Deficiency Promotes Pancreatic Islet Enlargement While Zinc Supplementation Improves the Pancreatic Insulin Response in Zucker Diabetic Fatty Rats.

Zinc deficiency has been associated with the worsening of diabetes while zinc supplementation has been proposed to ameliorate diabetes. This study examined the effects of marginal zinc deficiency (MZD) and zinc supplementation (ZS) on obesity, glycemic control, pancreatic islets, hepatic steatosis and renal function of Zucker diabetic fatty (ZDF) rats. Male ZDF rats were fed an MZD, zinc control (ZC) or ZS diet (4, 30 and 300 mg Zn/kg diet, respectively), and lean Zucker rats were fed a ZC diet for 8 weeks. MZD and ZS did not alter body weight or whole-body composition in ZDF rats. MZD ZDF rats had reduced zinc concentrations in the femur and pancreas, a greater number of enlarged pancreatic islets and a diminished response to an oral glucose load based on a 1.8-fold greater incremental area-under-the-curve (AUC) for glucose compared to ZC ZDF. ZS ZDF rats had elevated serum, femur and pancreatic zinc concentrations, unchanged pancreatic parameters and a 50% reduction in the AUC for insulin compared to ZC ZDF rats, suggesting greater insulin sensitivity. Dietary zinc intake did not alter hepatic steatosis, creatinine clearance, or levels of proteins that contribute to insulin signaling, inflammation or zinc transport in epididymal fat. Potential adverse effects of ZS were suggested by reduced hepatic copper concentrations and elevated serum urea compared to ZC ZDF rats. In summary, ZS improved the pancreatic insulin response but not the glucose handling. In contrast, reduced zinc status in ZDF rats led to impaired glucose tolerance and a compensatory increase in the number and size of pancreatic islets which could lead to β-cell exhaustion.

WED-190 Elevated hepatic copper content in porto-sinusoidal-vascular disorder: leading down a wrong track

WED-190 Elevated hepatic copper content in porto-sinusoidal-vascular disorder: leading down a wrong track

RETRACTED: Sixteen years of canine hepatic copper concentrations within normal reference ranges in dogs fed a broad range of commercial diets.

To examine the effects of age, sex, breed, liver histopathology, and year of death/sample collection on liver copper concentrations in dogs fed various commercial dog foods throughout their lives. During necropsy, 336 samples were collected between the years 2006 and 2022 from dogs that were fed a variety of commercial dog foods on the market. This study utilized all liver samples available and did not require specific criteria for sample selection. Liver samples (n = 336) were analyzed as dry weight for copper concentration by inductively coupled plasma-optical emission spectrometry. The potential effects of animal age and year of death/collection (scatterplots and linear regression), sex, liver histopathology (t test), and breed (ANOVA) on liver copper concentration were assessed. Labrador Retrievers had lower liver copper concentrations than Beagles, but mixed breeds did not differ from Beagles or Labrador Retrievers. Analysis of year of death showed that liver copper concentrations decreased from 2006 through 2011, increased in 2012, decreased in 2013, and peaked in 2016, decreasing thereafter. Mean copper concentration of abnormal liver histopathology samples was lower than mean copper concentrations of normal liver histopathology samples. Age (12.9 ± 2.6 years) and sex had no effect on liver copper concentrations. Of note, some samples showed abnormal hepatic pathology. Liver copper concentrations varied significantly with breed and year of death; however, average liver copper concentrations of each year were within normal. However, this was a retrospective population study and diet histories of the dogs were unknown, requiring further investigation.

Rhodanine Stain Improves Utility of Leipzig Score to Diagnose Hepatic Wilson’s Disease in Noncholestatic Patients with Fibrosis

Abstract Introduction: Cholestasis may increase the difficulty of diagnosing Wilson’s disease (WD). We aimed to compare Leipzig score including hepatic parenchymal copper concentration or rhodanine stain, to diagnose hepatic WD, in noncholestatic patients with fibrosis. Materials and Methods: We defined cholestasis by biochemical and/or histological criteria. Of noncholestatic patients who had liver fibrosis and liver copper estimation, those with isolated hepatic WD comprised WD study group and those with liver disease other than WD, with either low serum ceruloplasmin, high urinary copper, or Kayser–Fleischer ring, were controls. WD diagnosis by Leipzig score was labeled highly likely, probable, or unlikely. Results: Twenty WD study group patients (12 males; 21 [6–52] years; median [range]) and 18 controls (13 males; 32 [10–69] years, all had high urinary copper) were recruited. Rhodanine stain was positive in 60% WD patients and in 33% of controls. With rhodanine stain, Leipzig score had sensitivity of 100% (95% confidence interval: 83.2%–100%), specificity of 66.7% (41%–86.7%), and positive likelihood ratio of 3 (1.56–5.77) to diagnose probable/highly likely WD. In contrast, on adding hepatic parenchymal copper concentration, Leipzig score had sensitivity of 100% (83.2%–100%), but specificity was 38.9% (17.3%–64.2%). In controls, Leipzig score (including rhodanine stain) of ≤ 2 ruled out WD. Conclusion: On excluding cholestatic patients, rhodanine stain performs better than hepatic parenchymal copper concentration to diagnose hepatic WD using Leipzig score in patients with fibrosis. Widespread availability is an added advantage of rhodanine stain.

Quantitative bioimaging of copper in frozen liver specimens from cats using laser ablation-inductively coupled plasma-mass spectrometry.

The aim of this study was to assess laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) as a tool for measuring concentrations and determining accumulation of copper in frozen liver specimens from cats. Six frozen liver specimens were evaluated by qualitative copper staining and quantitative flame atomic absorption spectroscopy. Tissue specimens were cryo-sectioned and quantitative bioimaging of copper was performed using LA-ICP-MS. Results were compared with those obtained using conventional methods. Of the six specimens, only one showed positive staining for copper with rhodanine. Using flame atomic absorption spectroscopy (FAAS), one specimen showed a deficient copper level (<100 µg/g dry weight), two specimens had copper within the reference interval (RI; 150-180 µg/g) and three specimens had copper concentrations above the RI. Bioimaging from LA-ICP-MS showed inhomogeneous distribution of hepatic copper. The areas with dense copper accumulation were represented as hotspots in the liver specimens. Hepatic copper quantification by LA-ICP-MS correlated well with copper quantified by FAAS (r = 0.96, P = 0.002). Our findings suggest that quantitative bioimaging by LA-ICP-MS could be used to demonstrate the distribution and concentration of copper in frozen liver specimens from cats. The distribution of copper in these specimens was inhomogeneous with dense accumulation represented as hotspots on tissue sections. A positive correlation of hepatic copper concentrations determined by LA-ICP-MS and FAAS was found. Further studies to establish an RI for hepatic copper using this technique and to further determine its clinical utility are warranted.

Comparison of copper concentration between non-alcoholic fatty liver disease patients and normal individuals: A meta-analysis.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Copper metabolism plays an important role in the pathogenesis of NAFLD. However, the relationship between serum/hepatic copper concentration and NAFLD is still debated. A literature search was performed using electronic databases to find publications up to September 2022, where the relationship between serum/hepatic copper or ceruloplasmin concentration and NAFLD was evaluated. Finally, 6 articles with 9 unique outcomes involving 2,607 NAFLD patients and 1,441 non-NAFLD normal individuals were included. The pooled results showed that hepatic copper concentration was significantly decreased in NAFLD patients (SMD = -0.98, 95% CI = [-1.21; -0.74], p < 0.0001), and the sensitivity analysis also confirmed this. Nevertheless, serum copper (SMD = -0.02, 95% CI = [-0.32; 0.28], p = 0.88) and ceruloplasmin (SMD = -0.03, 95% CI = [-0.69; 0.63], p = 0.93) were not associated with NAFLD. This meta-analysis revealed that low hepatic copper concentration was found in NAFLD patients and serum copper and ceruloplasmin were not associated with NAFLD. Larger cohort studies and related trials are needed to further validate the result of this meta-analysis in the future.

Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice.

Numerous studies have discovered that chronic stress induces metabolic disorders by affecting iron and zinc metabolism, but the relationship between chronic stress and copper metabolism remains unclear. Here, we explore the influence of chronic corticosterone (CORT) exposure on copper metabolism and its regulatory mechanism in mice. Mice were treated with 100 μg/mL CORT in drinking water for a 4-week trial. We found that CORT treatment resulted in a significant decrease in plasma copper level, plasma ceruloplasmin activity, plasma and liver Cu/Zn-SOD activity, hepatic copper content, and liver metallothionein content in mice. CORT treatment led to the reduction in duodenal expression of copper transporter 1 (CTR1), duodenal cytochrome b (DCYTB), and ATPase copper-transporting alpha (ATP7A) at the mRNA and protein level in mice. CORT treatment activated nuclear glucocorticoid receptor (GR) and down-regulated CRT1 expression in Caco-2 cells, whereas these phenotypes were reversible by an antagonist of GR, RU486. Chromatin immunoprecipitation analysis revealed that GR bound to the Ctr1 promoter in Caco-2 cells. Transient transfection assays in Caco-2 cells demonstrated that the Ctr1 promoter was responsive to the CORT-activated glucocorticoid receptor, whereas mutation/deletion of the glucocorticoid receptor element (GRE) markedly impaired activation of the Ctr1 promoter. In addition, CORT-induced downregulation of Ctr1 promoter activity was markedly attenuated in Caco-2 cells when RU486 was added. These findings present a novel molecular target for CORT that down-regulates intestinal CTR1 expression via GR-mediated trans-repression in mice.

Re-evaluation of the existing health-based guidance values for copper and exposure assessment from all sources.

Copper is an essential micronutrient and also a regulated product used in organic and in conventional farming pest management. Both deficiency and excessive exposure to copper can have adverse health effects. In this Scientific Opinion, the EFSA 2021 harmonised approach for establishing health-based guidance values (HBGVs) for substances that are regulated products and also nutrients was used to resolve the divergent existing HBGVs for copper. The tightly regulated homeostasis prevents toxicity manifestation in the short term, but the development of chronic copper toxicity is dependent on copper homeostasis and its tissue retention. Evidence from Wilson disease suggests that hepatic retention is indicative of potential future and possibly sudden onset of copper toxicity under conditions of continuous intake. Hence, emphasis was placed on copper retention as an early marker of potential adverse effects. The relationships between (a) chronic copper exposure and its retention in the body, particularly the liver, and (b) hepatic copper concentrations and evidence of toxicity were examined. The Scientific Committee (SC) concludes that no retention of copper is expected to occur with intake of 5mg/day and established an Acceptable Daily Intake (ADI) of 0.07mg/kg bw. A refined dietary exposure assessment was performed, assessing contribution from dietary and non-dietary sources. Background copper levels are a significant source of copper. The contribution of copper from its use as plant protection product (PPP), food and feed additives or fertilisers is negligible. The use of copper in fertilisers or PPPs contributes to copper accumulation in soil. Infant formula and follow-on formula are important contributors to dietary exposure of copper in infants and toddlers. Contribution from non-oral sources is negligible. Dietary exposure to total copper does not exceed the HBGV in adolescents, adults, elderly and the very elderly. Neither hepatic copper retention nor adverse effects are expected to occur from the estimated copper exposure in children due to higher nutrient requirements related to growth.

Wilson’s Disease with Late Hepatic Involvement

Wilson’s Disease (WD) is an autosomal recessive condition that affects copper metabolism and manifests itself clinically in different ways. The diagnosis is indicated by low serum copper and ceruloplasmin concentrations, increased urine copper excretion and/ or increased hepatic copper concentrations. The present case report is about a 55-year-old male with chief complaints of loss of appetite, abdominal swelling, tremors in hand and head, slurring of speech for 10 days. Ultrasound (USG) findings were suggestive of liver cirrhosis with portal hypertension and liver function tests were also deranged. He was prescribed diuretics and asked to review after 10 days. Kayser-Fleischer ring was observed through slit lamp examination in both the eyes, which is a hallmark of WD. The Magnetic Resonance Imaging (MRI) of brain also revealed positive findings-hyperintensity throughout the mesencephalon for WD. Thereafter, the patient was treated with penicillamine and his symptoms improved after few days

Effect of oral zinc regimens on human hepatic copper content: a randomized intervention study

Zinc inhibits intestinal copper uptake, an effect utilized for treating Wilson’s disease (WD). We used copper-64 (64Cu) PET/CT to examine how much four weeks of treatment with different zinc regimens reduced the hepatic 64Cu content after oral 64Cu administration and test if alternative regimens were noninferior to the standard regimen of zinc acetate 50 mg × 3 daily. Forty healthy persons were randomized to four different zinc protocols. The WD standard treatment zinc acetate 50 mg × 3 reduced the hepatic 64Cu content from 26.9 ± 7.5% to 13.3 ± 5.6% of the administered 64Cu. Zinc gluconate 50 mg × 3 was noninferior (P = 0.02) (35.8 ± 9.0% to 17.4 ± 7.5%). Zinc acetate 150 mg × 1 (33.1 ± 9.9% to 17.4 ± 7.5%) and zinc gluconate 150 mg × 1 (28.1 ± 6.7% to 22.0 ± 6.7%) were less effective. These effects were intra- and inter-individually highly variable, and 14% had no effect of any zinc regimen, which may explain disparities in zinc treatment efficacy in WD patients.

Hepatic copper accumulates in ferrets with and without hepatobiliary disease.

To determine hepatic copper concentrations and zonal distribution in ferrets with and without hepatobiliary disease, validate rhodanine-based qualitative copper scoring and digital copper quantification in ferret hepatic samples, and ascertain whether clinical features predicted copper accumulation. 34 ferrets, including 7 with necroinflammatory disease, 5 with hepatocellular carcinoma, 13 with non-necroinflammatory disease, and 9 with no hepatobiliary disease. Rhodanine-based digital copper quantification was validated by use of liver dually measured by atomic absorption spectroscopy and digital scanning (R2 = 0.98). Clinical features and hepatic copper scores and concentrations (dry weight liver) were compared between groups. Zonal copper distribution was determined. Hepatic copper concentration was strongly correlated with copper scores (ρ = 0.88). Ferrets with hepatobiliary disease were significantly older and had significantly higher serum alkaline phosphatase and γ-glutamyltransferase activities and creatinine concentrations. Centrilobular copper accumulated in 23 of 34 (64%) ferrets with (n = 15) and without (8) hepatobiliary disease. Median copper concentrations were not significantly different between ferrets with and without hepatobiliary disease but were significantly higher within neoplastic hepatic tissue in ferrets with hepatocellular carcinoma. Hepatic copper concentrations exceeded feline (> 180 µg/g) and canine (> 400 µg/g) reference limits in 19 and 9 ferrets, respectively. Hepatic copper > 1,000 µg/g occurred in 5 ferrets with and 2 without hepatobiliary disease. Clinical features did not predict copper accumulation. Rhodanine-based digital copper quantification and qualitative copper scoring discerned liver copper accumulation in ferrets. Ferrets with and without hepatobiliary disease displayed a propensity for centrilobular hepatic copper accumulation of uncertain clinical importance. Clinical and clinicopathologic features could not exclusively implicate pathologic copper accumulation.

Immunohistochemical Expression of Oxidative Stress and Apoptosis Markers in Archived Liver Specimens from Dogs with Chronic Hepatitis

Chronic hepatitis (CH) in dogs is histologically characterized by an inflammatory infiltration of the liver accompanied by hepatocellular apoptosis or necrosis, varying degrees of fibrosis and regeneration. Oxidative stress has been suggested to play a role in the pathogenesis of various liver diseases, including CH. This study assessed the immunohistochemical expression of markers of oxidative stress (4-hydroxynonenal [4-HNE] and malondialdehyde [MDA]) and apoptosis (active caspase-3 [casp-3]) in 35 surplus archived formalin-fixed paraffin-embedded liver biopsies from 25 dogs with CH and 10 control dogs that had no significant hepatic changes. Correlations between immunohistochemical markers and necroinflammatory, fibrosis and histological copper scores, and hepatic copper concentrations were also determined. There were no significant differences in 4-HNE expression between the two groups. Control dogs had lower hepatic MDA scores than dogs with CH. MDA scores were positively correlated with copper scores as well as hepatic copper concentrations. There was no significant difference in casp-3-positive hepatocytes between groups. However, a positive correlation between casp-3 immunoreactivity and copper scores, as well as hepatic copper concentrations, was identified. Necroinflammatory and fibrosis scores were positively correlated with immunoreactivity for MDA and casp-3. MDA and casp-3 are expressed in canine liver and both markers are correlated with necroinflammatory scores, fibrosis scores and hepatic copper accumulation. Our results suggest the utility of immunolabelling for MDA and casp-3 for assessment of hepatic oxidative stress and apoptosis, respectively, in dogs with CH.

Tratamento de búfalos deficientes em cobre, por intermédio da aplicação de cobre parenteral

ABSTRACT The objective of this study was to investigate the effectiveness of the treatment of copper deficiency, by parenteral route, in buffalo calves raised on the Marajó Island (PA). Fourteen animals were divided into two groups. Group 1 received three doses of 120 mg of copper glycinate (20mgCu/mL), intramuscularly, on days 1, 45 and 90; in the group 2 (control) the animals were Not supplemented with copper. In both groups liver biopsies were performed on days 1 and 135. The diagnosis of copper deficiency was established through history, clinical signs, and by the analysis of hepatic copper content. In group 1, the mean hepatic copper content increased from 17.4±5.84mg/kgDM (before administration) to 311.2±133.5mg/kgDM (after 135 days) (P&lt;0.05). In the control group, the mean of hepatic copper contents decreased from 17.1±3.4 to 9.11±1.1mg/kgDM at the end of the experiment (P&lt;0.05). Based on the results, it was demonstrated that the administration of copper, by parenteral route, was efficient in the treatment of copper deficiency in buffalo calves and could become a very promising option for the reality of buffalo producers on the Island of Marajó.

Assessment of the intracellular distribution of copper in liver specimens from cats.

The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 μg/g dry weight, eight had copper concentrations between 100 and 180 μg/g, 14 had copper concentrations between 181 and 700 μg/g, and two had copper concentrations >700 μg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.

Effects of Sodium Monensin on Copper Metabolism of Brazilian Santa Inês Sheep Submitted to Different Dietary Copper.

This study aimed to evaluate the influence of sodium monensin on the hepatic accumulation of copper in sheep. Twenty-four Santa Inês crossbred sheep were used and allocated in a 2 × 2 factorial experiment with six repetitions and considering the factors dietary copper (basal and high) and supplementation (with and without sodium monensin). Thus, four homogeneous groups were formed: control (basal diet); monensin (Mon), 30ppm of monensin; copper (Cu), 10 10mg/kg BW per day of copper; monensin + copper (MonCu). The experimental period lasted 14weeks. Liver and bile samples were collected at the beginning and end of the experiment to determine mineral element concentrations, and weekly blood samples for biochemical, hematological, and mineral evaluation. Liver copper concentrations at the beginning of the experiment did not vary between groups, while mean liver copper concentrations at the end of the experiment were higher in the MonCu, Cu, and Mon groups when compared to the control. At the end of the study, hepatic copper concentration was influenced by copper (p = 0.0001) and monensin (p = 0.0003) supplementation. Copper-supplemented groups had reduced liver iron contents (p = 0.0287) and increased copper concentrations in bile. The biochemical evaluation showed increased serum GGT and AST activity (p < 0.05) in the Cu and MonCu groups from the eleventh week on compared to the control and Mon groups. The increase in activity of these enzymes was influenced by copper supplementation (p = 0.0340). Monensin interferes positively with the hepatic accumulation of copper and the supplementation of this additive may predispose sheep to copper poisoning.

Analyzing the Therapeutic Efficacy of Bis-Choline-Tetrathiomolybdate in the Atp7b-/- Copper Overload Mouse Model.

Bis-choline-tetrathiomolybdate, introduced as WTX101 (now known as ALXN1840), is a first-in-class copper-protein-binding agent for oral therapy of Wilson’s disease. In contrast to other decoppering agents such as trientine or D-penicillamine it acts by forming a tripartite complex with copper and albumin, thereby detoxifying excess liver and blood copper through biliary excretion. Preclinical animal experimentation with this drug was typically done with the alternative ammonium salt of tetrathiomolybdate, which is expected to have identical properties in terms of copper binding. Here, we comparatively analyzed the therapeutic efficacy of ALXN1840, D-penicillamine and trientine in lowering hepatic copper content in Atp7b−/− mouse. Liver specimens were subjected to laser ablation inductively conductively plasma mass spectrometry and electron microscopic analysis. We found that ALXN1840 caused a massive increase of hepatic copper and molybdenum during early stages of therapy. Prolonged treatment with ALXN1840 reduced hepatic copper to an extent that was similar to that observed after administration of D-penicillamine and trientine. Electron microscopic analysis showed a significant increase of lysosomal electron-dense particles in the liver confirming the proposed excretory pathway of ALXN1840. Ultrastructural analysis of mice treated with dosages comparable to the bis-choline-tetrathiomolybdate dosage used in an ongoing phase III trial in Wilson’s disease patients, as well as D-penicillamine and trientine, did not show relevant mitochondrial damage. In contrast, a high dose of ALXN1840 applied for four weeks triggered dramatic structural changes in mitochondria, which were notably characterized by the formation of holes with variable sizes. Although these experimental results may not be applicable to patients with Wilson’s disease, the data suggests that ALXN1840 should be administered at low concentrations to prevent mitochondrial dysfunction and overload of hepatic excretory pathways.

Diagnosis of Wilson Disease and Its Phenotypes by Using Artificial Intelligence.

WD is caused by ATP7B variants disrupting copper efflux resulting in excessive copper accumulation mainly in liver and brain. The diagnosis of WD is challenged by its variable clinical course, onset, morbidity, and ATP7B variant type. Currently it is diagnosed by a combination of clinical symptoms/signs, aberrant copper metabolism parameters (e.g., low ceruloplasmin serum levels and high urinary and hepatic copper concentrations), and genetic evidence of ATP7B mutations when available. As early diagnosis and treatment are key to favorable outcomes, it is critical to identify subjects before the onset of overtly detrimental clinical manifestations. To this end, we sought to improve WD diagnosis using artificial neural network algorithms (part of artificial intelligence) by integrating available clinical and molecular parameters. Surprisingly, WD diagnosis was based on plasma levels of glutamate, asparagine, taurine, and Fischer’s ratio. As these amino acids are linked to the urea–Krebs’ cycles, our study not only underscores the central role of hepatic mitochondria in WD pathology but also that most WD patients have underlying hepatic dysfunction. Our study provides novel evidence that artificial intelligence utilized for integrated analysis for WD may result in earlier diagnosis and mechanistically relevant treatments for patients with WD.