10 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
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
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>.
<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
Copper Capture in a Thioether-Functionalized Porous Polymer Applied to the Detection of Wilson’s Disease
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
<i>Atp7b</i> inactivation induces age-dependent changes in urine copper content.
<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
Renal function is altered in <i>Atp7b</i><sup>−/−</sup> mice older than 20 weeks.
<p>Water intake (A), Food intake (B) and urine volume output (C) relative to age-matched wild-type mice were measured over a 24 hr period. Double asterisks denote p≤0.003. N = 3 to 7 age-matched pairs; data presented as mean ± SD; additional supporting data and details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s001" target="_blank">Figure S1</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
Elemental content of wild-type and <i>Atp7b</i><sup>−/−</sup> urine during disease progression.
<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
Ctr1 and Atp7A levels in the liver and kidney of Atp7b<sup>−/−</sup> and wild-type mice.
<p>(A) Real-time PCR analysis of Atp7A mRNA levels in the liver of wild-type and Atp7b<sup>−/−</sup> mice at different ages. Data presented as mean ± SD, n = 3 to 4 different samples of each genotype and age. (B) Representative Western blot illustrating ATP7A protein expression in the livers of wild-type and <i>Atp7b</i><sup>−/−</sup> mice at 7 and 20 weeks. Twenty weeks old <i>Atp7b</i><sup>−/−</sup> kidneys were used as a positive control for ATP7A protein expression. “ST” denotes molecular weight standards. Black arrow points to ATP7A band. Weak bands in the liver samples are due to non-specific staining (C, D) Quantitation of CTR1 protein levels by Western blot analysis and densitometry (relative to wild type) in liver and kidneys of wild-type and <i>Atp7b</i><sup>−/−</sup> mice at different ages. Band intensity in each sample was normalized to a β-actin loading control. Data presented as mean ± SD. n = 2 for each genotype per age group.</p
Characterization of urine copper component(s).
<p>(A) Representative graph of gel filtration analysis on 20 µl of <i>Atp7b</i><sup>−/−</sup> urine along with copper levels in resulting fractions. (B) % of total copper found in the filtrate or retentate of wild-type and <i>Atp7b</i><sup>−/−</sup> urine after application of whole urine to a filter with 3 kDa cutoff. Ages varied from 6 to 65 weeks. Data presented as % of total copper ± SD, n = 21 age-matched wild-type/<i>Atp7b</i><sup>−/−</sup> pairs. (C) Representative graph of copper profile from gel filtration of 3 kDa filtrate from wild-type and <i>Atp7b</i><sup>−/−</sup> urine. Arrows point to the elution time of indicated compounds.</p
Functional interaction of SCC and CTR1.
<p>(A) Copper concentrations in fractions obtained from gel filtration of wild-type, <i>Atp7b</i><sup>−/−</sup>, and liver-specific <i>Ctr1</i><sup>−/−</sup> urine. (B) 64Cu uptake assay by HEK293 cells stably overexpressing hCtr1 in the presence of increasing concentrations of gel filtration purified SCC from urine of <i>Atp7b</i><sup>−/−</sup> and wild-type mice. Radioactive copper-64 was kept constant at 0.25 µM. Sodium phosphate buffer pH 7.4 was used as the buffer control. 1× is equal to a copper concentration of 0.25 µM for <i>Atp7b<sup>−/−</sup></i> SCC and 2× to 20× are multiples of the original 1× concentration. The volumes used for the assays were set to achieve the desired 1× to 20× concentrations specifically for <i>Atp7b<sup>−/−</sup></i> SCC. The wild-type SCC 1× to 20× assays are done with the same volumes of SCC utilized for corresponding <i>Atp7b<sup>−/−</sup></i> SCC assays, thus wild-type concentrations used are low due to inherent lower copper levels in WT SCC. Data presented as mean ± SD, performed in triplicates. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s003" target="_blank">Figure S3</a>.</p
Copper Capture in a Thioether-Functionalized Porous Polymer Applied to the Detection of Wilson’s Disease
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