Online immunocapture ICP-MS for the determination of the metalloprotein ceruloplasmin in human serum.

OBJECTIVE: The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity, precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32S mass trace. The ICP-MS signals were normalized on a 59Co signal. RESULTS: The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS.

Nanoconjugates of ferrocene and carbon-encapsulated iron nanoparticles as sensing platforms for voltammetric determination of ceruloplasmin in blood.

The nanoparticles comprising of iron core and carbon shell were decorated with ferrocene derivatives: ferrocenecarboxaldehyde (Fc-1) and ferrocenecarboxaldehyde oxime (Fc-2). A microdrop of suspension of the nanoconjugate was placed on a glassy-carbon electrode to prepare the recognition/sensing layer. Drying and purification of the sensing layer resulted in a well-defined and stable square-wave voltammogram of the ferrocene moiety. The height of the voltammetric peak increased in the presence of ceruloplasmin. That increase was linearly dependent on the logarithmic concentration of ceruloplasmin in blood. The applied external magnetic field was a factor which yielded better sensitivity and repeatability of the sensor response. The linearity of sensor response was found to be between 0.001 and 10µgdL-1 and 0.05-10µgdL-1 for both nanoconjugates: Fe@C-Fc-1 and Fe@C-Fc-2, in the presence and absence of the magnet, respectively. The obtained detection limit (LOD) for Fe@C-Fc-1 was found to be 0.60 and 0.10µgdL-1 in the absence and presence of magnetic field, respectively, whilst for Fe@C-Fc-2 was 0.4 and 0.07µgdL-1 in the absence and presence of a magnet, respectively. The proposed method is selective because the presence of common antioxidants in blood did not interfere significantly with the determination of the concentration of ceruloplasmin.

Evidence that ferritin is associated with light production in the mucus of the marine worm Chaetopterus.

The blue glow of the mucus from Chaetopterus involves a photoprotein, iron and flavins. Identity and respective role of these components remain, however, largely unresolved today, likely because of viscosity issues and inhibition of this system by oxidizers conventionally used to track bioluminescence activity. Here, we used gentle centrifugation to obtain a mucus supernatant showing no inhibition to oxidizers, allowing for further analysis. We applied conventional chromatographic techniques to isolate major proteins associated with light emission. Luminescence ability of elutriate fractions was tested with hydrogen peroxide to track photoprotein and/or protein-bound chromophore. Fractions producing light contained few major proteins, one with similarity to ferritin. Addition to the mucus of elements with inhibitory/potentiary effect on ferritin ferroxidase activity induced corresponding changes in light production, emphasizing the possible role of ferritin in the worm bioluminescence. DNA of the protein was cloned, sequenced, and expressed, confirming its identity to a Chaetopterus Ferritin (ChF). Both ferric and ferrous iron were found in the mucus, indicating the occurrence of both oxidase and reductase activity. Biochemical analysis showed ChF has strong ferroxidase activity, which could be a source of biological iron and catalytic energy for the worm bioluminescence when coupled to a reduction process with flavins.

Predictive value of ceruloplasmin for metabolic syndrome in adolescents.

The metabolic syndrome (MetS) is precisely defined and the cardiovascular risk associated with the clustering of its components has been demonstrated in adults. However, data on children and adolescents are still scarce, in part, because of difficulties in transposing the definition from adults. The identification of risk factors for the development of MetS at an early age is essential for prevention purposes with low-grade inflammation acting as a determinant for the association among the MetS components. The aim of this study was to investigate the associations of the MetS with systemic markers of inflammation and ceruloplasmin in a population of adolescents. The present is a cross-sectional study whose sample population consisted of 976 adolescents, 13.2 ± 1.2 years of age. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by ELISA. High-sensitivity C-reactive protein (hs-CRP) was determined by a solid-phase chemiluminiscent immunometric assay. Ceruloplasmin was measured by immunoturbidimetry. MetS adolescents exhibited higher levels of TNF-α, IL-6, CRP, and ceruloplasmin compared to non-MetS individuals. TNF-α, IL-6, and CRP showed strong correlations with the MetS components and insulin resistance but not relevant predictive values according to ROC curves (AUC values 0.544- 0.555). In contrast, ceruloplasmin only showed significant correlations in non-Mets individuals, but exhibited a very high predictive value (AUC=0.941, P < 0.001). The determination of serum ceruloplasmin in adolescents might be a useful tool to identify patients with the highest risk of future cardiovascular disease.

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase.

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe(V) redox intermediate of myeloperoxidase, to Compound II, which has Fe(IV) in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin.

A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K(i) for neomycin (11 µM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A(610)/A(280) ~ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.

Electrophoretic separation and detection of metalloproteins by X-ray fluorescence mapping.

All living systems depend on metalloproteins. Yet, while tools for the separation and identification of apo-proteins are well developed, those enabling identification and quantitation of individual metalloproteins within complex mixtures are still nascent. Here, we describe the electrophoretic separation of a mixture of carbonic anhydrase, ceruloplasmin, urease, and hemoglobin using native 2D gel electrophoresis and X-ray fluorescence mapping-an approach we have developed to be broadly applicable, not require specialized equipment for sample preparation, and likely to be extensible in the future.

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes.

Hephaestin (Heph), a membrane-bound multicopper ferroxidase (FOX) expressed in duodenal enterocytes, is required for optimal iron absorption. However, sex-linked anemia (sla) mice harboring a 194-amino acid deletion in the Heph protein are able to absorb dietary iron despite reduced expression and mislocalization of the mutant protein. Thus Heph may not be essential, and mice are able to compensate for the loss of its activity. The current studies were undertaken to search for undiscovered FOXs in rodent enterocytes. An experimental approach was developed to investigate intestinal FOXs in which separate membrane and cytosolic fractions were prepared and FOX activity was measured by a spectrophotometric transferrin-coupled assay. Unexpectedly, FOX activity was noted in membrane and cytosolic fractions of rat enterocytes. Different experimental approaches demonstrated that cytosolic FOX activity was not caused by contamination with membrane Heph or a method-induced artifact. Cytosolic FOX activity was abolished by SDS and heat (78 °C), suggesting protein-mediated iron oxidation, and was also sensitive to Triton X-100. Furthermore, cytosolic FOX activity increased ∼30% in iron-deficient rats (compared with controls) but was unchanged in copper-deficient rats (in contrast to the reported dramatic reduction of Heph expression and activity during copper deficiency). Additional studies done in sla, Heph-knockout, and ceruloplasmin-knockout mice proved that cytosolic FOX activity could not be fully explained by Heph or ceruloplasmin. Therefore rodent enterocytes contain a previously undescribed soluble cytosolic FOX that may function in transepithelial iron transport and complement membrane-bound Heph.

Enfermedad de Wilson / Wilson´s disease

La enfermedad de Wilson es un trastorno del metabolismo del cobre que se hereda de forma autosómica recesiva. Está causada por mutaciones en el gen ATP7B que codifica para una ATPasa tipo P implicada en el transporte de cobre dentro del hepatocito, tanto al interior del aparato de Golgi para su incorporación a la apoceruloplasmina como en la excreción biliar del exceso de metal del organismo. El defecto en la función de esta proteína da lugar a la acumulación progresiva de cobre, primero en el hígado y posteriormente en el encéfalo y en otros tejidos, con manifestaciones clínicas principalmente hepáticas, neurológicas, psiquiátricas y oftalmológicas. Actualmente sigue representando un desafío diagnóstico, debido a que es una patología poco común, con manifestaciones clínicas inespecíficas y limitaciones en la exactitud de las diversas pruebas diagnósticas disponibles. El riesgo de que permanezca sin diagnosticar y progrese a muerte, junto con la existencia de un tratamiento eficaz, ponen de manifiesto la importancia de que se realice un diagnóstico correcto y temprano, siendo esencial para ello la aportación del laboratorio clínico (AU)

Wilson's disease is an autosomal recessive disorder of copper metabolism. It is caused by mutations in the ATP7B gene, which encodes a P-type ATPase that functions in the transport of copper inside the hepatocyte, both into the trans-Golgi compartment for incorporation into apo-caeruloplasmin, and into the bile, for excretion of the excess metal. Defective function of this protein leads to progressive copper accumulation, first in the liver but ultimately in the brain and other tissues, with mainly hepatic, neurological, psychiatric and ophthalmologic signs and symptoms. Nowadays, it still represents a diagnostic challenge due to it being an uncommon disease, with unspecific clinical manifestations, and limitations in the accuracy of the available diagnostic tests. The risk that it remains undiagnosed together with the availability of effective treatments stresses the importance of an early and correct diagnosis, with the clinic laboratory playing an essential the role (AU)

Upregulation of a non-heme iron-containing ferritin with dual ferroxidase and DNA-binding activities in Helicobacter pylori under acid stress.

Helicobacter pylori is a spiral Gram-negative microaerophilic bacterium. It is unique and distinctive among various bacterial pathogens for its ability to persist in the extreme acidic environment of human stomachs. To address and identify changes in the proteome of H. pylori in response to low pH, we have used a proteomic approach to study the protein expression of H. pylori under neutral (pH 7) and acidic (pH 5) conditions. Global protein-expression profiles of H. pylori under acid stress were analysed by two-dimensional polyacrylamide gel electrophoresis (2-DE) followed by liquid chromatography (LC)-nanoESI-mass spectrometry (MS)/MS and bioinformatics database analysis. Among the proteins differentially expressed under acidic condition, a non-heme iron-containing ferritin of H. pylori (HP-ferritin) was found to be consistently upregulated at pH 5 as compared to pH 7. It was also found that HP-ferritin can switch from an iron-storage protein with ferroxidase activity to a DNA-binding/protection function under in vitro conditions upon exposure to acidic environment. Prokaryotic ferritins, such as non-heme iron-binding HP-ferritin with dual functionality reported herein, may play a significant urease-independent role in the acid adaptation of H. pylori under physiological conditions in vivo.

A novel and one-step purification of human ceruloplasmin by acharan sulfate affinity chromatography.

Human ceruloplasmin, a copper binding alpha(2)-glycoprotein, was purified by a single-step procedure using acharan sulfate affinity chromatography. Acharan sulfate was immobilized to amine-functionalized agarose matrix through carboxylic acids. Ceruloplasmin in human plasma was obtained from 0.4 M NaCl salt elution and characterized by SDS-PAGE (132 and 125 kDa), isoelectric focusing (pI 4.6), Western blotting, and MALDI-TOF-MS peptide mass fingerprinting. Ceruloplasmin was purified 106 fold with a specific oxidase activity of 0.53 U/mg protein.

Fortuitous structure determination of 'as-isolated' Escherichia coli bacterioferritin in a novel crystal form.

Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 A resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on 'as-isolated' E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.

Antioxidant and antibacterial genes are upregulated in early involution of the mouse mammary gland: sharp increase of ceruloplasmin and lactoferrin in accumulating breast milk.

The mammary gland develops mainly after birth, and shows a repeated cycle of pregnancy-triggered proliferation, differentiation for lactation, and a regressive phase after weaning known as involution. Compared to the proliferation and differentiation phases, the molecular mechanisms of involution are largely unknown. In the present study we screened genes that could play a potential role in early involution of the mouse mammary gland using fluorescent differential display followed by gene-specific reverse transcription-polymerase chain reaction. We found that five genes were upregulated more than twofold 48 h after weaning: ceruloplasmin, chemokine (CXC motif) ligand 4, epoxide hydrolase 1, lactoferrin, and properdin P factor. The products of these genes can be linked to defense against oxidative stress and/or infectious bacteria. Electrophoretic analysis and mass spectrometry of milk proteins showed that the concentrations of ceruloplasmin and lactoferrin in milk were increased fivefold and more than 38-fold, respectively, within 48 h after weaning. These increases were in contrast to the constant presence of other major proteins including albumin, caseins, transferrin, and whey acidic protein. Ceruloplasmin and lactoferrin may cooperate in the defense of the mammary gland in the postlactation period.

Determination of ceruloplasmin in human serum by SEC-ICPMS.

This paper describes an analytical method for the determination of ceruloplasmin (Cp) in human serum. The method uses immunoaffinity chromatography and size-exclusion chromatography (SEC) to "purify" the serum sample prior to analysis of 63Cu and 65Cu by inductively-coupled plasma mass spectrometry (ICPMS). By removing the six most abundant proteins from serum with immunoaffinity chromatography and by using SEC to separate Cu bound by Cp from any free Cu that might be present in the serum sample, we demonstrated that SEC-ICPMS can accurately and reproducibly measure Cp in the ERM DA470 reference serum. Cp identification is based on retention time match of the unknown in the serum sample with the Cp external standard and the presence of 63Cu and 65Cu at a ratio of 2.2+/-0.1. This method was used to analyze a reference serum certified for Cp, 47 serum samples from four different diseases and a set of normal controls. The reference serum and a serum sample from a patient with myocardial infarction, as well as a Cp standard, were also analyzed by electrospray mass spectrometry to confirm the presence of Cp in the SEC fraction known to contain 63Cu.

Patients with nasopharyngeal carcinoma demonstrate enhanced serum and tissue ceruloplasmin expression.

The proteomics approach was adopted to study the simultaneous expression of serum proteins in patients with nasopharyngeal carcinoma (NPC). We have subjected unfractionated whole sera of ten newly diagnosed Malaysian Chinese patients with WHO type III NPC to two-dimensional gel electrophoresis (2-DE) and image analysis. The results obtained were then compared to that generated from sera of ten normal healthy controls of the same ethnic group and range of age. Our data demonstrated that the serum high abundance 2-DE protein profiles of NPC patients were generally similar to that of the controls, with exception of the ceruloplasmin (CPL) spots (identified by mass spectrometric analysis and MASCOT database search), which showed higher expression. The enhanced expression of CPL in the patients' sera was confirmed by competitive ELISA. Immunohistochemical analysis of nasopharyngeal lesions of NPC patients demonstrated moderate to strong positive CPL staining in the cytoplasm of cells at the regions of malignancy but only weak cytoplasmic staining at normal epithelial lining areas. When follow-up 2-DE and ELISA studies were performed on five of the NPC patients who responded positively to six months treatment, the difference in CPL expression was no longer significant.

Identification and isolation from breast milk of ceruloplasmin-lactoferrin complex.

The presence of a complex of the copper-containing protein ceruloplasmin (Cp) with lactoferrin (Lf) in breast milk (BM) is shown for the first time. In SDS-free polyacrylamide gel electrophoresis (PAGE), electrophoretic mobility of Cp in BM is lower than that of plasma Cp, coinciding with the mobility of the complex obtained upon mixing purified Cp and Lf. Affinity chromatography of delipidated BM on Cp-Sepharose resulted in retention of Lf. SDS-PAGE of the 0.3 M NaCl eluate revealed a single band with Mr approximately 78,000 that has the N-terminal amino acid sequence of Lf and reacts with antibodies to that protein. Synthetic peptides R-R-R-R (the N-terminal amino acid stretch 2-5 in Lf) and K-R-Y-K-Q-R-V-K-N-K (the C-terminal stretch 29-38 in PACAP 38) caused efficient elution of Lf from Cp-Sepharose. Cp-Lf complex from delipidated BM is not retained on the resins used for isolation of Cp (AE-agarose) and of Lf (CM-Sephadex). Anionic peptides from Cp--(586-597), (721-734), and (905-914)--provide an efficient elution of Cp from AE-agarose, but do not cause dissociation of Cp-Lf complex. When anti-Lf is added to BM flowed through CM-Sephadex, Cp co-precipitates with Lf. Cp-Lf complex can be isolated from BM by chromatography on CM-Sephadex, ethanol precipitation, and affinity chromatography on AE-agarose, yielding 98% pure complex. The resulting complex Cp-Lf (1 : 1) was separated into components by chromatography on heparin-Sepharose. Limited tryptic hydrolysis of Cp obtained from BM and from blood plasma revealed identical proteolytic fragments.

[Isolation of stable human ceruloplasmin and its interaction with salmon protamine].

An interaction was discovered between ceruloplasmin (CP, a ferro-O2-oxidoreductase, EC 1.16.3.1), a copper-containing protein of human blood plasma, and salmon protamine (PR), a cationic polypeptide of vertebrates that provides a compact structure of spermatozoid DNA. Addition of PR to CP at a molar ratio of 2: 1 decreases the CP electrophoretic mobility. Two types of CP binding centers for PR were determined: two centers with a high (Kd1 of 5.31 x 10(-7) M) and four centers with a low affinity (Kd2 of 1.56 x 10(-5) M). PR was shown to form complexes with CPs of various animal species. The CP-PR complex dissociates at an increased ionic strength (0.3 M NaCl), at pH decreased below 4.7, or in the presence of added polyanions (DNA, lipopolysaccharides, or heparin) and/or polylysine, which indicates the electrostatic nature of the interaction. The CP-PR interaction increased 1.5-fold the rate of CP-catalyzed oxidation of Fe2+. The preliminary treatment of blood plasma with arginine-Sepharose and heparin-Sepharose (to remove the blood coagulation factors) and affinity chromatography on PR-Sepharose helped isolate the practically unproteolyzed monomeric CP in 90% yield; it remained stable for more than two months at 37 degrees C. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

Purification and partial characterization of camel (Camelus Dromedarius) ceruloplasmin.

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8+/-0.3 atoms per molecule. The spectroscopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K(m)=0.42 mM and V(max)=0.93 microM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.

High levels of ceruloplasmin in the serum of transgenic mice developing hepatocellular carcinoma.

Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver-specific human antithrombin-III promoter all develop well-differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of the Cp gene as well as to the increase in Cp mRNA stability in the livers of the transgenic mice. One-half of the overproduced Cp is charged with copper and Cp-associated serum oxidase activity increases in parallel with the holo-Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour-bearing animals and which increased in parallel with Cp concentration; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased.

Purification of human ceruloplasmin as a by-product of C1-inhibitor.

Human ceruloplasmin (Cp) has been purified from cryoprecipitate-poor plasma as a by-product of the C1-inhibitor production chain. Highly purified Cp was obtained by subsequent ion-exchange chromatography on sulfate-Fractogel EMD and TMAE-Fractogel EMD. Treatments for viral safety included application of the solvent-detergent method and two nanofiltration steps using 35- and 15-nm pore size filters at the end of the process. Overall antigen yield was 95 (+/-5) %. Purified human ceruloplasmin was studied by electron spin resonance (ESR) to characterize its different types of copper complexes and to check its antioxidant properties. We distinguished three types of complexes: one type-2 Cu(II) with g// = 2.25 and A// = 180 G and two type-I Cu(II) exhibiting different narrow hyperfine splitting (A// = 72 G and A// = 90 G) with close g// (2.20 and 2.21). Purified Cp has a specific activity of 24.5+/-0.2 mU/mg of proteins. This process provides a method for Cp purification that could be easily integrated into modern plasma fractionation.