22 research outputs found

    Urinary Copper Elevation in a Mouse Model of Wilson’s Disease Is a Regulated Process to Specifically Decrease the Hepatic Copper Load

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    Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson’s disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b‒/‒ mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated by distinct molecules. PET-CT and atomic absorption spectroscopy directly demonstrate an age-dependent decrease in the capacity of Atp7b‒/‒ livers to accumulate copper, concomitant with an increase in urinary copper. This reciprocal relationship is specific for copper, indicating that cell necrosis is not the primary cause for the initial phase of metal elevation in the urine. Instead, the urinary copper increase is associated with the down-regulation of the copper-transporter Ctr1 in the liver and appearance of a 2 kDa Small Copper Carrier, SCC, in the urine. SCC is also elevated in the urine of the liver-specific Ctr1‒/‒ knockouts, which have normal ATP7B function, suggesting that SCC is a normal metabolite carrying copper in the serum. In agreement with this hypothesis, partially purified SCC-Cu competes with free copper for uptake by Ctr1. Thus, hepatic down-regulation of Ctr1 allows switching to an SCC-mediated removal of copper via kidney when liver function is impaired. These results demonstrate that the body regulates copper export through more than one mechanism; better understanding of urinary copper excretion may contribute to an improved diagnosis and monitoring of WD

    Urinary Copper Elevation in a Mouse Model of Wilson’s Disease Is a Regulated Process to Specifically Decrease the Hepatic Copper Load

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    Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson’s disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b2/2 mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated by distinct molecules. PET-CT and atomic absorption spectroscopy directly demonstrate an age-dependent decrease in the capacity of Atp7b2/2 livers to accumulate copper, concomitant with an increase in urinary copper. This reciprocal relationship is specific for copper, indicating that cell necrosis is not the primary cause for the initial phase of metal elevation in the urine. Instead, the urinary copper increase is associated with the down-regulation of the copper-transporter Ctr1 in the liver and appearance of a 2 kDa Small Copper Carrier, SCC, in the urine. SCC is also elevated in the urine of the liver-specific Ctr12/2 knockouts, which have normal ATP7B function, suggesting that SCC is a normal metabolite carrying copper in the serum. In agreement with this hypothesis, partially purified SCC-Cu competes with free copper for uptake by Ctr1. Thus, hepatic down-regulation of Ctr1 allows switching to an SCC-mediated removal of copper via kidney when liver function is impaired. These results demonstrate that the body regulates copper export through more than one mechanism; better understanding of urinary copper excretion may contribute to an improved diagnosis and monitoring of WD

    Copper Capture in a Thioether-Functionalized Porous Polymer Applied to the Detection of Wilson’s Disease

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    Copper is an essential nutrient for life, but at the same time, hyperaccumulation of this redox-active metal in biological fluids and tissues is a hallmark of pathologies such as Wilson’s and Menkes diseases, various neurodegenerative diseases, and toxic environmental exposure. Diseases characterized by copper hyperaccumulation are currently challenging to identify due to costly diagnostic tools that involve extensive technical workup. Motivated to create simple yet highly selective and sensitive diagnostic tools, we have initiated a program to develop new materials that can enable monitoring of copper levels in biological fluid samples without complex and expensive instrumentation. Herein, we report the design, synthesis, and properties of PAF-1-SMe, a robust three-dimensional porous aromatic framework (PAF) densely functionalized with thioether groups for selective capture and concentration of copper from biofluids as well as aqueous samples. PAF-1-SMe exhibits a high selectivity for copper over other biologically relevant metals, with a saturation capacity reaching over 600 mg/g. Moreover, the combination of PAF-1-SMe as a material for capture and concentration of copper from biological samples with 8-hydroxyquinoline as a colorimetric indicator affords a method for identifying aberrant elevations of copper in urine samples from mice with Wilson’s disease and also tracing exogenously added copper in serum. This divide-and-conquer sensing strategy, where functional and robust porous materials serve as molecular recognition elements that can be used to capture and concentrate analytes in conjunction with molecular indicators for signal readouts, establishes a valuable starting point for the use of porous polymeric materials in noninvasive diagnostic applications

    PET-CT analysis of <sup>64</sup>Cu distribution in live mice at 24 hrs after oral administration of <sup>64</sup>CuCl<sub>2</sub>.

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    <p>Representative PET-CT images of (A) <i>Atp7b</i><sup>−/−</sup> and (B) wild-type mice at 24 hours post oral administration (PO) of <sup>64</sup>CuCl<sub>2</sub>. Orange-brown color denotes <sup>64</sup>Cu radioactivity. Red arrows and white arrows identify liver and gastrointestinal tract <sup>64</sup>Cu radioactivity, respectively. (C) Hepatic <sup>64</sup>Cu radioactivity in <i>Atp7b<sup>−/−</sup></i> and wild-type mice at 24 hrs PO (p<0.001). Age-dependent <sup>64</sup>Cu radioactivity in the (D) kidneys (p = 0.38 for group effect) and (E) urinary bladder of wild-type and <i>Atp7b</i><sup>−/−</sup> mice at 24 hr PO. %ID/g, percentage of administration dose per gram. Data presented as mean ± SD. N = 5, number of mice with same age and genotype. Additional data and details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s002" target="_blank">Figure S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s005" target="_blank">Information S1</a>.</p

    <i>Atp7b</i> inactivation induces age-dependent changes in urine copper content.

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    <p>(A) Urinary copper concentration and (B) total amount from wild-type (WT) and <i>Atp7b</i><sup>−/−</sup> (KO) mice at different ages. Urine collected over 24 hours and measured by atomic absorption. Data reported as mean concentration ± SD, n = 3 to 13, animals per age group. *P<0.05 versus age-matched wild type. Boxplot middle horizontal bar represents the median; the dotted horizontal line signifies the mean. The while upper and lower box values signify the 75<sup>th</sup> and 25<sup>th</sup> percentiles, whiskers represent the 10<sup>th</sup> and 90<sup>th</sup> percentiles. The black dots represent outliers (three SD from the mean).</p

    Elemental content of wild-type and <i>Atp7b</i><sup>−/−</sup> urine during disease progression.

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    <p>ICP-MS analysis of urine collected over 24 hours from age-matched wild-type and <i>Atp7b</i><sup>−/−</sup> mice. (A) Comparison of WT and KO copper amounts at different ages. (B) Urine amounts of Na, Mg, P, K, Ca, Se, and urine volume (relative to wild-type) at different ages. (C) Amounts of Fe and Zn at different ages (relative to wild type). N = 2 to 4 age-match WT/KO pairs for each time point. Data presented as mean ± SD, **P≤0.003. The data points (from left to right) in Panel A represent 6 weeks, 12 weeks, 15 to 20 weeks, and 60 to 65 weeks. Panels B and C data points (from left to right) represent 6 to 12 weeks, 15 to 20 weeks, and 60 to 65 weeks. Correlation coefficients found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s004" target="_blank">Table S1</a>.</p
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