Determination of ultrafiltrable and exchangeable copper in plasma: stability and reference values in healthy subjects

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 Ultrafiltrable fraction of plasma copper is very instable in vitro. A 50% loss occurs within 6 hours after blood sampling     

Enriched copper isotope tracer for dietary copper incorporation into hair using neutron activation analysis in conjunction with direct-current argon plasma spectrometry

A method is described for labeling and measuring the incorporation of dietary copper into hair using the enriched (99.61 atomic-%) ⁶⁵Cu isotope. Natural isotope abundant copper exists as ⁶⁵Cu (30.86 atomic-%) and ⁶³Cu (69.17 atomic-%), but enriched (99.61 atomic-%) ⁶⁵Cu isotope may be obtained commercially and administered to change deliberately the natural isotope abundance of copper to “label” the copper source. Natural isotope abundant copper and the enriched (99.61 atomic-%) ⁶⁵Cu isotope were mixed into rat feed and administered daily by gavage. The isotope concentration was determined by neutron activation analysis (NAA). The copper concentration was determined by direct-current argon plasma (DCP) spectrometry.The measured copper isotope ratios (⁶⁵Cu/Cu_tot)_for the natural isotope abundant copper in National Bureau of Standards Standard Reference Materials (NBS SRM) 1571 Orchard Leaves and 1577a Bovine Liver were 0.3201 ± 0.0039 and 0.3187 ± 0.0089, respectively. Measured copper isotope ratios in rat hair were 0.3100 ± 0.0606, 0.4580 ± 0.2495 and 0.2712 ± 0.0645 from rats given a standard feed that contained natural isotope abundant copper, a daily supplement of natural isotope abundant copper sulfate mixed into the standard feed and a daily supplement of copper sulfate administered by gavage, respectively. The copper isotope ratio increased to 0.5251 ± 0.2860 and 0.7235 ± 0.0704 in hair from rats given enriched ⁶⁵Cu in their feed and by gavage, respectively. The statistically significant increase in the ⁶⁵Cu isotope when administered by gavage demonstrates strates successful labeling of dietary copper in the hair.     

Dialysis-Chelex method for determination of exchangeable copper in human plasma

A method based on dialysis in the presence of histidine, and subsequent copper adsorption and preconcentration using Chelex-100 resin, has been developed for selective extraction of exchangeable copper from blood plasma. The method was verified by comparative analysis using two different instrumental techniques, ICP–MS and AAS. The results obtained were in excellent agreement. Accurate quantification of the exchangeable copper and the firmly bound copper was achieved. The method was further validated by analysis of a lyophilised human serum certified reference material.     

Identification of two-dimensional copper signatures in human blood for bladder cancer with machine learning

Currently, almost all available cancer biomarkers are based on concentrations of compounds, often suffering from low sensitivity, poor specificity, and false positive or negative results. The stable isotopic composition of elements provides a different dimension from the concentration and has been widely used as a tracer in geochemistry. In health research, stable isotopic analysis has also shown potential as a new diagnostic/prognostic tool, which is still in the nascent stage. Here we discovered that bladder cancer (BCa) could induce a significant variation in the ratio of natural copper isotopes (⁶⁵Cu/⁶³Cu) in the blood of patients relative to benign and healthy controls. Such inherent copper isotopic signatures permitted new insights into molecular mechanisms of copper imbalance underlying the carcinogenic process. More importantly, to enhance the diagnostic capability, a machine learning model was developed to classify BCa and non-BCa subjects based on two-dimensional copper signatures (copper isotopic composition and concentration in plasma and red blood cells) with a high sensitivity, high true negative rate, and low false positive rate. Our results demonstrated the promise of blood copper signatures combined with machine learning as a versatile tool for cancer research and potential clinical application.

The blood Cu concentration and isotopic composition enable new insights into Cu imbalance and diagnosis of bladder cancer with machine learning.     

Application of gas chromatography/mass spectroscopy to the investigation of metabolic pathways of trace elements using stable isotopes as tracers

Summary Zinc and copper metabolism became easier to study after science provided two research ways [1]: Isotope tracer technique and atomic absorption spectroscopy. Using stable isotopes is advantageous in humans because exposure to radioactivity is avoided and therefore the method can be used safely in infants and pregnant women. According to Ehrenkranz et al. [2, 3] zinc absorption in premature infants is 60%. Ziegler and coworkers report a lower absorption of zinc in infants (20%–44%) [8]. The copper intestinal uptake has been reported to be 68% in adults [3, 4].We have investigated the bioavailability of zinc in a group of premature infants and one term infant weighing 1850 to 5630 g and the copper absorption in a man and an adolescent with M. Wilson (a copper storage disease) and discuss the accurate isotope analysis of human feces in relation to mineral bioavailability studies of premature infants. It is shown that for the determination of bioavailability of zinc and copper, the method of chelate-gas chromatography/mass spectrometry (GC/MS) is valuable for routine application to clinical experiments; the human fecal samples resulting from diet suitably enriched with isotopes⁶⁸Zn,⁷⁰Zn or⁶⁵Cu can be routinely analysed with a precision of about 1% for these stable isotopes [2]. The total zinc in feces was determined by atomic absorption spectroscopy and the⁷⁰Zn/⁶⁴Zn ratio by chelate-GC/MS (EI). We have found different absorption values between 3% to 70% for zinc in a group of different infants including twins, triplets and preterms. The copper uptake was determined to be 79.7% and 49.9%.

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Anwendung der Gas-Chromatographie/Massenspektrometrie für die Untersuchung der Stoffwechselwege von Spurenelementen mit Hilfe von stabilen Isotopen als Tracern

     

Determination of copper in coal fly ash in the presence of excess titanium by dynamic reaction cell inductively coupled plasma mass spectrometry

Inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) was used for the accurate determination of copper in coal fly ash samples in the presence of excess titanium, using the reaction of Cu(+) ions with NH(3) in the cell. The method eliminated the effect of polyatomic isobaric interferences at m/z 63 and 65 caused by the formation of (47)Ti(16)O(+), (49)Ti(16)O(+) and (47)Ti(18)O(+) on (63)Cu(+) and (65)Cu(+) by detecting Cu(+) as the product cluster ion Cu(NH(3))(2)(+). As the signal of (63)Cu(NH(3))(2)(+) overlapped with that of (97)Mo(+) which existed in the samples, (65)Cu(NH(3))(2)(+) was detected at m/z 99. The effect of the operating conditions of DRC system was studied in order to obtain the best signal to noise ratio for Cu(NH(3))(2)(+) at m/z 99. The formation of Cu(NH(3))(2)(+) was through the clustering reaction Cu(+)+2NH(3)-->Cu(NH(3))(2)(+) which resulted in the separation of analyte from the interfering oxide. The detection limit for Cu(NH(3))(2)(+) was 0.015 ng mL(-1) as Cu. The method was applied to the determination of copper in NIST SRM 1633a and 1633b coal fly ash reference materials. The precision between sample replicates was better than 2.0% and the analysis results were in good agreement with the certified values.     

The dual personality of ionic copper in biology

Biological copper is mainly involved in electron transport to catalyse essential oxido-reduction processes. It is an essential trace element which is extremely toxic because exchangeable intracellular copper is Cu(I) which generates reactive oxygen species. To handle this paradox the evolution has led to a fine homeostasis in which copper ions are never free. Intracellular Cu(I) instead is bound to numerous proteins forming specific cascades towards its targets.     

Spectroscopic and density functional studies of the red copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic structure

The electronic structure of the red copper site in nitrosocyanin is defined relative to that of the well understood blue copper site of plastocyanin by using low-temperature absorption, circular dichroism, magnetic circular dichroism, resonance Raman, EPR and X-ray absorption spectroscopies, combined with DFT calculations. These studies indicate that the principal electronic structure change in the red copper site is the sigma rather than the pi donor interaction of the cysteine sulfur with the Cu 3d(x2-y2) redox active molecular orbital (RAMO). Further, MCD data show that there is an increase in ligand field strength due to an increase in coordination number, whereas resonance Raman spectra indicate a weaker Cu-S bond. The latter is supported by the S K-edge data, which demonstrate a less covalent thiolate interaction with the RAMO of nitrosocyanin at 20% relative to plastocyanin at 38%. EXAFS results give a longer Cu-S(Cys) bond distance in nitrosocyanin (2.28 A) compared to plastocyanin (2.08 A) and also show a large change in structure with reduction of the red copper site. The red copper site is the only presently known blue copper-related site with an exogenous water coordinated to the copper. Density functional calculations reproduce the experimental properties and are used to determine the specific protein structure contributions to exogenous ligand binding in red copper. The relative orientation of the CuNNS and the CuSC(beta) planes (determined by the protein sequence) is found to be key in generating an exchangeable coordination position at the red copper active site. The exogenous water ligation at the red copper active site greatly increases the reorganization energy (by approximately 1.0 eV) relative to that of the blue copper protein site, making the red site unfavorable for fast outer-sphere electron transfer, while providing an exchangeable coordination position for inner-sphere electron transfer.     

Solid-state NMR and EXAFS spectroscopic characterization of polycrystalline copper(I) O,O'-dialkyldithiophosphate cluster compounds: Formation of copper(I) O,O'-diisobutyldithiophosphate compounds on the surface of synthetic chalcocite

A number of polycrystalline copper(I) O,O'-dialkyldithiophosphate cluster compounds with Cu4, Cu6, and Cu8 cores were synthesized and characterized by using extended X-ray absorption fine-structure (EXAFS) spectroscopy. The structural relationship of these compounds is discussed. The polycrystalline copper(I) O,O'-diisobutyldithiophosphate cluster compounds, [Cu8{S2P(OiBu)2}6(S)] and [Cu6{S2P(OiBu)2}6], were also characterized by using 31P CP/MAS NMR (CP = cross polarization, MAS = magic-angle spinning) and static 65Cu NMR spectroscopies (at different magnetic fields) and powder X-ray diffraction (XRD) analysis. Comparative analyses of the 31P chemical-shift tensor, and the 65Cu chemical shift and quadrupolar-splitting parameters, estimated from the experimental NMR spectra of the polycrystalline copper(I) cluster compounds, are presented. The adsorption mechanism of the potassium O,O'-diisobutyldithiophosphate collector, K[S2P(OiBu)2], at the surface of synthetic chalcocite (Cu2S) was studied by means of solid-state 31P CP/MAS NMR spectroscopy and scanning electron microscopy (SEM). 31P NMR resonance lines from collector-treated chalcocite surfaces were assigned to a mixture of [Cu8{S2P(OiBu)2}6(S)] and [Cu6{S2P(OiBu)2}6] compounds.     

Determination of copper by electrothermal AAS after electrodeposition on a graphite disk electrode

The electrodeposition of copper on a graphite electrode at a constant potential with subsequent atomization in the graphite atomizer HGA-400 has been studied. A special graphite disk electrode is suitable for electrochemical enrichment at E = -0.7 V vs. SCE and the determination of copper by electrothermal-atomic absorption spectrometry (ET-AAS) if atomized at 2300 degrees C. In this way copper was determined in potable water and free Cu(2+) could be distinguished from that bound in chelate speciations after using a suitable deposition potential of the working electrode. This approach seems to be an alternative to the commonly used anodic stripping voltammetry (ASV) for the preconcentration and determination of free metal ions.     

Application of multiwalled carbon nanotubes as solid phase extraction sorbent for preconcentration of trace copper in water samples

The adsorption behavior of multiwalled carbon nanotubes (MWNTs) toward copper has been investigated systemically, and a new method has been developed for the determination of trace copper in water samples based on preconcentration with a microcolumn packed with MWNTs prior to its determination by flame atomic absorption spectrometry. The optimum experimental parameters for preconcentration of copper, such as pH of the sample, sample flow rate and volume, elution solution and interfering ions, have been investigated. Copper can be quantitatively retained by MWNTs in the pH range 5-8, and then eluted completely with 0.5 M HNO3. The detection limit of this method for Cu was 0.42 ng/mL, and the RSD was 3.5% at the 10 ng/mL Cu level. The method was validated using a certified reference material, and has been successfully applied for the determination of trace copper in water samples.     

Heteroleptic copper switches

Heteroleptic copper compounds have been designed and synthesized on solid supports. Chemical redox agents were used to change the oxidation state of the SiO(2)-immobilized heteroleptic copper compounds from Cu(I) to Cu(II) and then back to Cu(I). Optical spectroscopy of a dimethyl sulfoxide suspension demonstrated the reversibility of the Cu(I)/Cu(II) SiO(2)-immobilized compounds by monitoring the metal-to-ligand charge transfer peak at about 450 nm. Electron paramagnetic resonance spectroscopy was used to monitor the isomerization of Cu(I) tetrahedral to Cu(II) square planar. This conformational change corresponds to a 90° rotation of one ligand with respect to the other. Conductive atomic force microscopy and macroscopic gold electrodes were used to study the electrical properties of a p(+) Si-immobilized heteroleptic copper compound where switching between the Cu(I)/Cu(II) states occurred at -0.8 and +2.3 V.     

Synthesis, characterization, and antifouling potential of functionalized copper nanoparticles

The synthesis, characterization, and antimicrobial properties of functionalized copper nanoparticle/polymer composites are reported. Copper nanoparticles (Cu NPs) are stabilized by surface attachment of the acrylic functionality that can be copolymerized with other acrylic monomers, thus, becoming an integral part of the polymer backbone. Biological experiments show that Cu NP/polymer composites exhibit antimicrobial activity similar to that of conventional copper-based biocides. Atomic absorption spectroscopy shows the smallest amount of copper ions leaching from chemically bound acrylated Cu NPs compared to the nonfunctionalized biocides. These composites have a strong potential for use in antibacterial or marine antifouling coatings.     

Probing the influence of X-rays on aqueous copper solutions using time-resolved in situ combined video/X-ray absorption near-edge/ultraviolet-visible spectroscopy

Time-resolved in situ video monitoring and ultraviolet-visible spectroscopy in combination with X-ray absorption near-edge spectroscopy (XANES) have been used for the first time in a combined manner to study the effect of synchrotron radiation on a series of homogeneous aqueous copper solutions in a microreactor. This series included both non biologically relevant (pyridine, bipyridine, neocuproine, terpyridine, dimethylpyridine, ammonia, ethylenediamine, and 1,10-phenanthroline) and biologically relevant (histidine, glycine, and imidazole) ligands. It was found that when water is present as solvent, gas bubbles are formed under the influence of the X-ray beam. At the liquid-gas interface of these bubbles, in particular cases colloidal copper nanoparticles are formed. This reduction process was found to be influenced by the type of copper precursor salt (SO(4)(2-), NO(3)(-), and Cl(-)), the ligands surrounding the copper cation, and the redox potential of the copper complexes (ranging between +594 and -360 mV). In other words, in some cases, no reduction was encountered (e.g., ammonia in the presence of SO(4)(2-) and NO(3)(-)), whereas in other cases reduction to either Cu(+) (neocuproine with SO(4)(2-)) or Cu(0) (e.g., histidine and imidazole both with SO(4)(2-), NO(3)(-), and Cl(-)) was observed. These results illustrate the added value of video spectroscopy for the interpretation of in situ XANES studies. Not only do the results give an illustration of the parameters that are important in the redox processes that occur in biological systems, they also show the potential problems associated with studying catalytic processes in aqueous solutions by XANES spectroscopy.     

A targetable fluorescent sensor reveals that copper-deficient SCO1 and SCO2 patient cells prioritize mitochondrial copper homeostasis

We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu(+)-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu(+) in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu(+) pools in SCO1 and SCO2 patient fibroblasts are largely unaltered relative to wild-type controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.     

Tuning the excitonic and plasmonic properties of copper chalcogenide nanocrystals

The optical properties of stoichiometric copper chalcogenide nanocrystals (NCs) are characterized by strong interband transitions in the blue part of the spectral range and a weaker absorption onset up to ~1000 nm, with negligible absorption in the near-infrared (NIR). Oxygen exposure leads to a gradual transformation of stoichiometric copper chalcogenide NCs (namely, Cu(2-x)S and Cu(2-x)Se, x = 0) into their nonstoichiometric counterparts (Cu(2-x)S and Cu(2-x)Se, x > 0), entailing the appearance and evolution of an intense localized surface plasmon (LSP) band in the NIR. We also show that well-defined copper telluride NCs (Cu(2-x)Te, x > 0) display a NIR LSP, in analogy to nonstoichiometric copper sulfide and selenide NCs. The LSP band in copper chalcogenide NCs can be tuned by actively controlling their degree of copper deficiency via oxidation and reduction experiments. We show that this controlled LSP tuning affects the excitonic transitions in the NCs, resulting in photoluminescence (PL) quenching upon oxidation and PL recovery upon subsequent reduction. Time-resolved PL spectroscopy reveals a decrease in exciton lifetime correlated to the PL quenching upon LSP evolution. Finally, we report on the dynamics of LSPs in nonstoichiometric copper chalcogenide NCs. Through pump-probe experiments, we determined the time constants for carrier-phonon scattering involved in LSP cooling. Our results demonstrate that copper chalcogenide NCs offer the unique property of holding excitons and highly tunable LSPs on demand, and hence they are envisaged as a unique platform for the evaluation of exciton/LSP interactions.     

Tunable diode laser spectroscopy of copper,cadmium, and indium at 325 nm

A tunable frequency-doubled external-cavity diode laser has been recently developed for atomic absorption spectroscopy in the wavelength range from 320 to 327 nm. The line width of the laser is 0.003 pm. In this work the light source has been applied to the laser absorption spectroscopy of copper at 324.754 nm, cadmium at 326.106 nm, and indium at 325.609 nm and 325.856 nm. The Cu transition was measured in three different environments: in a direct current plasma, a diffusion flame, and in a low-pressure hollow-cathode discharge. Both the plasma and the flame were at atmospheric pressure. The Cd and In transitions were measured in the diffusion flame. A Voigt profile was fit to the measured spectra taking into account the hyperfine structure. From the measured absorption lines spectral reference data for Cu is extracted.     

Electropolymerized coatings of polyaniline on copper by using the galvanostatic method and their corrosion protection performance in HCl medium

The synthesis of polyaniline coatings on the copper (Cu) surface has been investigated by using the galvanostatic method. The synthesized coatings were characterized by Fourier transform infrared spectroscopy, UV–visible absorption spectrometry and scanning electron microscopy. The anticorrosion performances of polyaniline coatings were investigated in 0.5 M HCl medium by the potentiodynamic polarization technique and electrochemical impedance spectroscopy. The corrosion rate of polyaniline‐coated Cu was found to be ～27 times lower than bare Cu, and potential corrosion increased from ?0.21 V versus Ag/AgCl for uncoated Cu to ?0.19 V versus Ag/AgCl for polyaniline‐coated Cu electrodes. Electrochemical measurements indicate that polyaniline coating has good inhibiting properties with a mean efficiency of ~96% at 10 mAcm^?2 current density applied on Cu corrosion in acid media. The results of this study clearly ascertain that the polyaniline has an outstanding potential to protect Cu against corrosion in an acidic environment. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of adsorptive cathodic differential pulse stripping method for simultaneous determination of copper and molybdenum using pyrogallol red

A reliable and sensitive procedure for the simultaneous determination of trace levels of copper and molybdenum is proposed. The complexing of copper(II) and molybdenum(VI) with pyrogallol red (PGR) is analyzed by cathodic stripping differential pulse voltammetry based on the adsorption collection of the complexes onto a hanging mercury drop electrode (HMDE). The effect of chemical and instrumental parameters on the sensitivity and selectivity were studied. Copper and molybdenum peaks potential were observed at about +0.13 and −0.22 V versus Ag/AgCl electrode, respectively. A standard addition method was utilized for the analysis of voltammogram data, under the optimum conditions and with accumulation time of 90 s. The measured peak current at about +0.14 and −0.22 V is proportional to the concentration of Cu(II) and Mo(VI) over the range of 2-70 and 0.8-80 ng ml⁻¹, respectively. The limit of detection are 0.3 and 0.1 ng ml⁻¹ for Cu(II) and Mo(VI), respectively. The capability of the method for the analysis of real samples was evaluated by determination of copper and molybdenum in river water, tap water and alloy. Atomic absorption spectrometry was applied as a reference method for determination of copper and molybdenum in water samples.     

Dependence of copper species on the nature of the support for dispersed CuO catalysts

Copper catalysts prepared by chemisorption-hydrolysis technique over silica (Cu/Si) and silica-alumina (Cu/SiAl) supports were studied to understand the role of the support on the nature and surface properties of the copper species stabilized on their surfaces. The morphological and surface properties of the copper phases have been characterized by complementary techniques, such as HRTEM, EXAFS-XANES, EPR, XPS, and FTIR. For the FTIR investigation, molecular probes (CO and NO) were also adsorbed on the surfaces to test the reactivity of the copper species. Moreover, the catalytic performances of the two catalysts have been compared in the HC-SCR reaction (NO reduction by C(2)H(4)) performed in highly oxidant conditions. The superior activity and selectivity of the supported silica-alumina catalyst with respect to that supported on silica could be related with the different nature of the copper species stabilized on the two supports, as emerged from the results obtained from the spectroscopic investigations. Small and well-dispersed CuO particles were present on silica, whereas isolated copper ions predominated on silica-alumina, likely in regions rich in alumina that made some exchangeable sites available, as indicated by FTIR spectra of adsorbed CO. The less positive global charge of copper species on Cu/SiAl than in Cu/Si has been confirmed by EPR, XPS, and EXAFS-XANES analyses.