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(1) Introduction: Wilson's disease (WND) is an autosomal recessive disorder of copper (Cu) metabolism. Many tools are available to diagnose and monitor the clinical course of WND. Laboratory tests to determine disorders of Cu metabolism are of significant diagnostic importance. (2) Methods: A systematic review of the literature in the PubMed, Science Direct, and Wiley Online Library databases was conducted. (Results): For many years, Cu metabolism in WND was assessed with serum ceruloplasmin (CP) concentration, radioactive Cu test, total serum Cu concentration, urinary copper excretion, and Cu content in the liver. The results of these studies are not always unambiguous and easy to interpret. New methods have been developed to calculate non-CP Cu (NCC) directly. New parameters, such as relative Cu exchange (REC), reflecting the ratio of CuEXC to total serum Cu, as well as relative Cu exchange (REC), reflecting the ratio of CuEXC to total serum Cu, have been shown to be an accurate tool for the diagnosis of WND. Recently, a direct and fast LC-ICP-MS method for the study of CuEXC was presented. A new method to assess Cu metabolism during treatment with ALXN1840 (bis-choline tetrathiomolybdate [TTM]) has been developed. The assay enables the bioanalysis of CP and different types of Cu, including CP-Cu, direct NCC (dNCC), and labile bound copper (LBC) in human plasma. Conclusions: A few diagnostic and monitoring tools are available for patients with WND. While many patients are diagnosed and adequately assessed with currently available methods, diagnosis and monitoring is a real challenge in a group of patients who are stuck with borderline results, ambiguous genetic findings, and unclear clinical phenotypes. Technological progress and the characterization of new diagnostic parameters, including those related to Cu metabolism, may provide confidence in the more accurate diagnosis of WND in the future. [ABSTRACT FROM AUTHOR]

Introduction: The diagnosis of Wilson disease (WD) is plagued by biochemical and clinical uncertainties. Thus, calculated parameters have been proposed. This study aimed to: (a) compare the diagnostic values of non-caeruloplasmin copper (NCC), NCC percentage (NCC%), copper-caeruloplasmin ratio (CCR) and adjusted copper in WD; and (b) derive and evaluate a discriminant function in diagnosing WD. Methods: A total of 213 subjects across all ages who were investigated for WD were recruited. WD was confirmed in 55 patients, and the rest were WD free. Based on serum copper and caeruloplasmin values, NCC, NCC%, CCR and adjusted copper were calculated for each subject. A function was derived using discriminant analysis, and the cut-off value was determined through receiver operating characteristic analysis. Classification accuracy was found by cross-tabulation. Results: Caeruloplasmin, total copper, NCC, NCC%, CCR, adjusted copper and discriminant function were significantly lower in WD compared to non-WD. Discriminant function showed the best diagnostic specificity (99.4%), sensitivity (98.2%) and classification accuracy (99.1%). Caeruloplasmin levels <0.14 g/L showed higher accuracy than the recommended 0.20 g/L cut-off value (97.7% vs. 87.8%). Similarly, molar NCC below the European cut-off of 1.6 umol/L showed higher accuracy than the American cut-off of 3.9 umol/L (80.3% vs. 59.6%) (P < 0.001). NCC%, mass NCC, CCR and adjusted copper showed poorer performances. Conclusion: Discriminant function differentiates WD from non-WD with excellent specificity, sensitivity and accuracy. Performance of serum caeruloplasmin <0.14 g/L was better than that of <0.20 g/L. NCC, NCC%, CCR and adjusted copper are not helpful in diagnosing WD. [ABSTRACT FROM AUTHOR]

The assay in serum of non-caeruloplasmin copper, as exchangeable copper after complexation with EDTA (ExCu) and total copper has been evaluated and compared in patients with varying c-reactive protein(CRP). Measurement of ExCu and total copper, range 0.2–47.2 µmol/L, was developed using ICP-MS. The chelating agents EDTA and TEPA were compared over 0.0–10 g/L after incubation with serum for 60 mins followed by ultrafiltration with Amicon 10 kDa filter. The assay for ExCu was optimised with EDTA 3 g/L (8.1 mmol/L) maintained at pH 7.0–8.0 before ultrafiltration. TEPA was not as selective in chelation of copper. Patients n = 82 were studied in relation to changes in inflammatory marker CRP and a group of patients n = 37 with normal CRP. The ExCu assay gave excellent recoveries (94–102 % but poor recovery for free uncomplexed copper), good repeatability, limit of quantitation 0.19 µmol/l with a provisional reference range 0.48 to 1.63 µmol/L (n = 37 patients). The range for relative exchangeable copper (exchangeable copper divided by total serum copper) was 2.49 to 9.96 %. ExCu was elevated in conditions with increased CRP greater than 100 mg/L suggesting an effect of inflammation on the free copper fraction. A reliable and reproducible assay for ExCu and total copper has been developed. The upregulated inflammatory state increases the ExCu suggesting excess free copper. [ABSTRACT FROM AUTHOR]

Background: Defective copper regulation, primarily referred to as chelatable redox active Cu(II), has been involved in the etiology of diabetes, and Alzheimer's disease (AD).Objectives: However, no study has determined levels of labile copper non-bound to ceruloplasmin (non-Cp Cu, also known as 'free' copper) in the blood of subjects with diabetes compared with that of AD patients.Methods: To this aim, values of non-Cp Cu were measured in 25 Type 1 (T1D) and 31 Type 2 (T2D) subjects and in28 healthy controls, along with measurements of C-reactive protein, glycated hemoglobin A1c, cholesterol, and triglycerides. Non-Cp Cu levels were compared with those of an AD group previously studied.Results: T2D subjects had significantly higher non-Cp Cu levels than Controls and T1D subjects (both p < 0.001 after adjusting for age, sex, and body mass index). A multinomial logistic model revealed that a one unit standard deviation increase of non-Cp Cu increased the relative risk of having T2D by 9.64 with respect to Controls (95% CI: 2.86-32.47). The comparison of non-Cp Cu levels in T2D with those of an AD population previously studied shows rising blood non-Cp Cu copper levels from Controls to T2D and AD.Conclusion: These results suggest the involvement of catalytically-active Cu(II) and glucose dysregulation in oxidative stress reactions leading to tissue damage in both diseases. [ABSTRACT FROM AUTHOR]

Meta-analyses show that serum copper non-bound-to-ceruloplasmin (non-Cp-Cu) is higher in patients with Alzheimer's disease (AD). ATP7B gene variants associate with AD, modulating the size of non-Cp-Cu pool. However, a dedicated genetic study comparing AD patients after stratification for a copper biomarker to demonstrate the existence of a copper subtype of AD has not yet been carried out. An independent patient sample of 287 AD patients was assessed for non-Cp-Cu serum concentrations, rs1801243, rs1061472, and rs732774 ATP7B genetic variants and the APOE4 genotype. Patients were stratified into two groups based on a non-Cp-Cu cutoff (1.9 μM). Single-locus and haplotype-group analyses were performed to define their frequencies in dependence of the non-Cp-Cu group. The two AD subgroups did not differ regarding age, sex, MMSE score, or APOE4 frequency allele, while they did differ regarding non-Cp-Cu concentrations in serum, allele, genotype, and haplotype frequencies of rs1061472 A > G and rs732774 C > T after multiple testing corrections. AD patients with a GG genotype had a 1.76-fold higher risk of having a non-Cp-Cu higher than 1.9 μmol/L ( p = 0.029), and those with a TT genotype for rs732774 C > T of 1.8-fold ( p = 0.018). After 100,000 permutations for multiple testing corrections, the haplotype containing the AC alleles appeared more frequently in AD patients with normal non-Cp-Cu [43 vs. 33 %; Pm = 0.03], while the haplotype containing the GT risk alleles appeared more frequently in the higher non-Cp-Cu AD (66 vs. 55 %; Pm = 0.01). Genetic heterogeneity sustains a copper AD metabolic subtype; non-Cp-Cu is a marker of this copper AD. [ABSTRACT FROM AUTHOR]

Background and purpose The severity of Wilson's disease ( WD) is linked to free copper accumulating in the liver and brain. Exchangeable copper (Cu EXC) is a new technique to determine plasmatic copper and is useful in the diagnosis of WD. It is hypothesized that it may also enable a good evaluation of extra-hepatic involvement and its severity. Methods Forty-eight newly diagnosed WD patients were prospectively evaluated using hepatic, neurological, ophthalmological and brain magnetic resonance imaging ( MRI) scores. Three phenotypic presentations were distinguished: pre-symptomatic, hepatic and extra-hepatic. Cu EXC was determined in addition to standard copper assays before decoppering therapy. Correlations between biological parameters and the different scores were determined and compared in the hepatic and extra-hepatic groups. Results Extra-hepatic patients had significantly higher Cu EXC values than those with the hepatic form ( P < 0.0001). The overall ability of Cu EXC to separate the two forms was satisfactory, with an area under the curve of 0.883 (95% confidence interval 0.771-0.996) and an optimal threshold for extra-hepatic diagnosis of 2.08 μmol/l (sensitivity 85.7%; specificity 94.1%). In extra-hepatic patients, Cu EXC was the only biological marker to be positively correlated with the Unified Wilson Disease Rating Score ( r = 0.45, P = 0.016), the Kayser−Fleischer ring score ( r = 0.46, P = 0.014) and the brain MRI score ( r = 0.38, P = 0.048), but it was not correlated with the hepatic score. Conclusions Exchangeable copper determination is useful when diagnosing WD as a value >2.08 μmol/l is indicative of the severity of the extra-hepatic involvement. In the case of purely hepatic presentation, atypical or mild neurological signs, it should encourage physicians to search for lesions in the brain and eyes. [ABSTRACT FROM AUTHOR]

In a recent meta-analysis by Ventriglia and colleagues studying the association of zinc levels with Alzheimer's disease (AD), serum zinc has been found significantly decreased in AD patients compared with healthy controls. However, such a finding does not necessarily propose the causal role of low zinc in the pathophysiology of this neurodegenerative disease. On the basis of available evidence, free copper toxicosis may play a causal role in age-related AD, and zinc therapy can be a rational causal treatment. Nevertheless, a randomized controlled clinical trial testing a definite hypothesis is needed before conclusions can be drawn about the value of zinc supplements in the treatment of AD. [ABSTRACT FROM AUTHOR]

For acceptance of any chemical agent as an endogenous chemical mediator of inflammation, the agent in question must fulfill some biological requirements which are (a) it should be ubiquitously present in tissues in inactive form, (b) it should be activated during process of inflammation whose increase should be identifiable, (c) it should induce or amplify some events of inflammation, (d) there must be some natural inhibitor of such active form in tissues, (e) it should be able to induce inflammatory reaction after exogenous injection, (f) such reaction should be inhibited by exogenous use of their antagonists, and (g) it should be amplified by use of agonists. Copper in its protein free or protein bound form are reported to act as pathogenic factor in inflammatory processes due to oxidative stress. But their role as endogenous chemical mediator of inflammation does not appear to be investigated thoroughly in light of abovementioned biological criterion of mediator. Present study aims at thorough exploration on role of free copper as endogenous chemical mediator of inflammation in light of above facts. It was done by estimation of total copper, protein-bound copper, and free copper along with estimation of free radical generation, increase in vascular permeability, and cellular infiltration during acute inflammatory reaction induced by carrageenan and concanavalin using chicken skin as test model. It was further evaluated by use of exogenous free copper in experimental model and their subsequent inhibition and amplification by chemical chelators of copper. Present study confirms that free copper fulfilled all the biological requirements for accepting it as an endogenous chemical mediator of inflammation. [ABSTRACT FROM AUTHOR]

The idea that copper may play a role in the pathogenesis of Alzheimer's disease is gaining momentum. Serum copper and ceruloplasmin were measured by both enzymatic (eCp) and immunologic (iCp) methods in 28 patients with Alzheimer's disease and 29 age-matched controls. "Free copper" was determined by subtracting copper accounted for in the eCp assay from total serum copper. Percentage free copper, that is the proportion of serum copper not bound to ceruloplasmin, was significantly elevated in patients with Alzheimer's compared to controls. There was significantly more "defective" ceruloplasmin, which is apoceruloplamin lacking its copper, in Alzheimer's disease than in normal controls. This abnormality may precede the clinical onset of the disease and help predict risk of disease onset. Increased exposure to environmental copper (eg, the spread of copper plumbing and the use of copper in supplements) and/or defective ceruloplasmin function may play a role in the current epidemic of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The knowledge of copper (Cu) distribution in blood contributes to a better understanding of copper metabolism and to a better approach and follow up of related diseases such as Wilson’s disease (WD). Many tests can be used to investigate patients who may have WD but they show many drawbacks and do not allow real patient monitoring. Knowing that the Cu overload can result from the free and easily exchangeable form of copper in plasma, a two-step method (ultrafiltration–determination by ETAAS) was carried out to determine these two fractions. The ultrafiltration procedure and the instrumental determination showed good repeatability, and a very low limit of detection was obtained (0.7 nmol/L). In vitro stability of both ultrafiltrable copper (CuUF) and exchangeable copper (CuEXC) was studied. Plasma was ultrafiltered in 44 presumably healthy subjects to determine CuUF and CuEXC and to set reference values ranges. The method was applied on a few patients showing good correlation between both parameters and the clinical and biological features of the patients. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Changes in life cycle parameters (survival, growth, reproduction) and feeding rate of the tropical cladoceran Ceriodaphnia silvestrii as affected by Cu contaminated algae Pseudokirchneriella subcapitata were investigated. The dietary copper exposure ranged from 3 × 10-15 to 68 g 10-15 g Cu algal cell-1. Low waterborne copper exposure (around 10-10 mol l-1 free Cu2+ ions) was kept in the experiments. The results show an increasing toxic effect on C. silvestrii with copper increase in algal cells; at the highest copper exposure, all life cycle parameters were significantly affected. A concentration of 38 × 10-15 g Cu algal cell-1 reduced egg hatching percentile and the number of neonates produced per female, but did not cause any statistically significant effect on animals survival nor to the number of eggs produced per female. The following sequence of events was observed from the lowest to the highest copper contamination: reproduction, feeding rate, body length and, at last, survival was affected. We conclude that algal cells are an important route of copper exposure and toxicity to cladocerans. [ABSTRACT FROM AUTHOR]

Metal ecotoxicity testing with microalgae is subject to interferences such as change in bioavailable metal fraction by excreted metabolites. Moreover, high metal loads are usually necessary for obtaining 50% effect concentration (EC50) values based on cell number or chlorophyll a. Thus, sensitive parameters, which promptly respond to low dissolved metal variations at environmentally relevant concentrations, are much needed. The genus Scenedesmus, a coenobium-forming freshwater microalga, is cosmopolitan and inhabits pristine and contaminated waters. The present investigation addressed cellular phenotypic alterations in Scenedesmus acuminatus (Largerheim) Chodat 1902 caused by the presence of copper. Copper toxicity to the alga was investigated under laboratory-controlled conditions using a concentration range of 10-8 mol 1-1 to 10-3 mol 1-1 total dissolved Cu2+, thus including environmentally relevant concentrations. Free Cu2+ concentrations were determined using an ion-selective electrode calibrated with a metal ion buffer. The phenotypic plasticity of S. acuminatus was measured as the number of cells per coenobia and found to be readily affected at low copper concentrations. Under healthy conditions (10-8 mol 1-1 total dissolved Cu2+, 10-11 mol 1-1 free Cu2+) S. acuminatus formed eight-celled coenobia, whereas at 10-7 mol 1-1 total dissolved Cu2+ (3 × 10-10 mol 1-1 free Cu2+) these were absent and at 10-5 mol 1-1 total dissolved Cu2+ (5 × 10-8 mol 1-1 free Cu2+) free cells were predominant. Chlorophyll a synthesis was affected by 5 × 10-8 mol 1-1 free Cu-2+ or higher. EC50 (3.3 × 10-5 mol 1-1 free Cu2+) was obtained using chlorophyll a values after 96-h exposure to the toxicant. The number of cells per coenobia was a more sensitive parameter than chlorophyll a to assess Cu toxicity, and at concentrations that may be encountered in the environment. [ABSTRACT FROM AUTHOR]