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Nonalcoholic steatohepatitis (NASH) is a condition that progresses from nonalcoholic fatty liver disease (NAFLD) and is characterized by hepatic fat accumulation, inflammation, and fibrosis. It has the potential to develop into cirrhosis and liver cancer, and currently no effective pharmacological treatment is available. In this study, we investigate the therapeutic potential of targeting ceruloplasmin (Cp), a copper-containing protein predominantly secreted by hepatocytes, for treating NASH. Our result show that hepatic Cp is remarkedly upregulated in individuals with NASH and the mouse NASH model. Hepatocyte-specific Cp ablation effectively attenuates the onset of dietary-induced NASH by decreasing lipid accumulation, curbing inflammation, mitigating fibrosis, and ameliorating liver damage. By employing transcriptomics and metabolomics approaches, we have discovered that hepatic deletion of Cp brings about remarkable restoration of bile acid (BA) metabolism during NASH. Hepatic deletion of Cp effectively remodels BA metabolism by upregulating Cyp7a1 and Cyp8b1, which subsequently leads to enhanced BA synthesis and notable alterations in BA profiles. In conclusion, our studies elucidate the crucial involvement of Cp in NASH, highlighting its significance as a promising therapeutic target for the treatment of this disease., (© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Purpose: In diagnosing the pathogenesis of Graves' orbitopathy (GO), there is a growing interest in fibrosis generated by orbital fibroblasts (OFs); nevertheless, the involvement of ceruloplasmin (CP) in OFs remains unknown., Methods: Differentially expressed genes (DEGs) were identified through bioinformatic analysis. OFs were isolated from orbital tissue and identified with immunofluorescent staining. The levels of DEGs were validated in GO tissue samples and TGF-β-challenged OFs, and CP was selected for the following laboratory investigations. CP overexpression or knockdown was achieved, and cell viability and fibrosis-associated proteins were investigated to assess the cell phenotype and function. Signaling pathways were subsequently investigated to explore the mechanism of CP function in OFs., Results: CP and cathepsin C (CTSC) are two overlapped DEGs in GSE58331 and GSE105149. OFs were isolated and identified through fibrotic biomarkers. CP and CTSC were downregulated in GO tissue samples and TGF-β-challenged OFs. CP overexpression or knockdown was achieved in OFs by transducing a CP overexpression vector or small interfering RNA against CP (si1-CP or si2-CP) and verified using a qRT-PCR. CP overexpression inhibited cell viability and reduced the levels of α-SMA, vimentin, fibronectin, and collagen I, whereas CP knockdown exerted opposite effects on OFs. CP overexpression inhibited the phosphorylation of Smad3, Erk1/2, p38, JNK, and AKT; conversely, CP knockdown exerted opposite effects on the phosphorylation of factors mentioned above., Conclusion: CP was downregulated in GO and suppressed the expression of fibrosis-associated proteins in both GO and normal OFs. CP might serve as a promising therapeutic agent in the treatment regimens for GO., (© 2023. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Normal blood lipid levels have a crucial role in lowering cardiovascular mortality. This study was designed to investigate the effect of aqueous rhubarb extract on serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde, glutathione, and ceruloplasmin levels, as well as glutathione and malondialdehyde levels in the liver, kidney, and heart tissue in mice exposed to oxidative stress. 40 Balb/c mice were randomly allocated into 8 groups (n=5). Group 1: The control group was left eating feed and water without treatment for (15) days. Group 2: A group exposed to oxidative stress by giving hydrogen peroxide at a rate of (0.5%) with drinking water for 15 days. Group 3: A group exposed to oxidative stress induced by hydrogen peroxide at a rate of (0.5%) for 15 days with injecting on the seventh day, daily for a week, with insulin subcutaneously (15) units/kg. Group (4-8): the Groups were exposed to the oxidative stress induced by hydrogen peroxide (0.5%) for 15 days with injecting on the seventh day into the peritoneal cavity with both the cold aqueous and nonprotein extract, the extract of flavonoids at a dose of 400, 400, 0.4, 8.8, 1.96 mg/kg body weight, respectively. All animals were anesthetized on the last day of the experiment, blood samples were obtained for biochemical testing, and tissue samples from the livers were collected for research. The results revealed that the cold crude aqueous, non-proteinous extracts, flavonoids, proteinous precipitate, and proteinous compound caused a significant decrease ( P <0.05) in serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde levels in kidney, liver, and heart. The recorded data showed a significant increase ( P <0.05) in serum glutathione and ceruloplasmin in serum and glutathione levels in liver, kidney, and heart tissues in male mice exposed to oxidative stress. The results showed that all Rhubarb extracts have an antioxidant effect in mice exposed to oxidative stress., Competing Interests: The authors declare that they have no conflict of interest.

Background: Glioblastoma (GB) is the most common and most aggressive malignant brain tumor. In understanding its resistance to conventional treatments, iron metabolism and related pathways may represent a novel avenue. As for many cancer cells, GB cell growth is dependent on iron, which is tightly involved in red-ox reactions related to radiotherapy effectiveness. From new observations indicating an impact of RX radiations on the expression of ceruloplasmin (CP), an important regulator of iron metabolism, the aim of the present work was to study the functional effects of constitutive expression of CP within GB lines in response to beam radiation depending on the oxygen status (21% O 2 versus 3% O 2 )., Methods and Results: After analysis of radiation responses (Hoechst staining, LDH release, Caspase 3 activation) in U251-MG and U87-MG human GB cell lines, described as radiosensitive and radioresistant respectively, the expression of 9 iron partners (TFR1, DMT1, FTH1, FTL, MFRN1, MFRN2, FXN, FPN1, CP) were tested by RTqPCR and western blots at 3 and 8 days following 4 Gy irradiation. Among those, only CP was significantly downregulated, both at transcript and protein levels in the two lines, with however, a weaker effect in the U87-MG, observable at 3% O 2 . To investigate specific role of CP in GB radioresistance, U251-MG and U87-MG cells were modified genetically to obtain CP depleted and overexpressing cells, respectively. Manipulation of CP expression in GB lines demonstrated impact both on cell survival and on activation of DNA repair/damage machinery (γH2AX); specifically high levels of CP led to increased production of reactive oxygen species, as shown by elevated levels of superoxide anion, SOD1 synthesis and cellular Fe2 + ., Conclusions: Taken together, these in vitro results indicate for the first time that CP plays a positive role in the efficiency of radiotherapy on GB cells., (© 2022. The Author(s).)

Background: The relationship between the toxicity of cryoprotectants and their osmotic and/or oxidative stresses remains to be further investigated. OBJECTİVE: To investigate the toxic effects of different cryoprotectants and osmotic stress on Awassi ram sperm and to determine the relationship between oxidative and antioxidative status of the sperm., Materials and Methods: Pooled sperm samples were exposed to sucrose solutions of different concentrations (75 to 900 mOsm) and isosmotic condition (290-325 mOsm) was re-established by adding HEPES buffered Tyrode's lactate. Sperm samples were mixed with 0.5, 1.0 and 1.5 M of glycerol, methanol, 2-methoxyethanol, dimethylacetamide or 1,2-propanediol for 5 min and returned to isosmotic condition. Sperm samples were exposed to cryoprotectants at 4 degree C for 2 hours and isosmotic conditions were re-established. Motility, viability, acrosome integrity and oxidative or antioxidative parameters were determined., Results: Treatment with hypo- or hyperosmotic sucrose solution reduced motility and viability without affecting acrosome integrity. The addition and removal of glycerol and dimethylacetamide (1.0 or 1.5 M) decreased sperm motility, while cryoprotectants had no effect on viability except for 1.5 M glycerol. Chilling significantly reduced the motility and viability of the sperm, but not the acrosome integrity. Rapid addition or removal of cryoprotectants also did not affect the acrosome integrity. Cryoprotectants changed only the ceruloplasmin level, while there were significant post-chilling differences in lipid hydroperoxide, paraoxonase and ceruloplasmin levels., Conclusion: Cryoprotectants without other additives have limited protection and glycerol can be toxic to spermatozoa. The oxidative stress plays a role in cryoprotectant toxicity and chilling stress. doi.org/10.54680/fr22210110612.

Multiple System Atrophy (MSA) is a rare neurodegenerative synucleinopathy which leads to severe disability followed by death within 6-9 years of symptom onset. There is compelling evidence suggesting that biological trace metals like iron and copper play an important role in synucleinopathies like Parkinson's disease and removing excess brain iron using chelators could slow down the disease progression. In human MSA, there is evidence of increased iron in affected brain regions, but role of iron and therapeutic efficacy of iron-lowering drugs in pre-clinical models of MSA have not been studied. We studied age-related changes in iron metabolism in different brain regions of the PLP-αsyn mice and tested whether iron-lowering drugs could alleviate disease phenotype in aged PLP-αsyn mice. Iron content, iron-ferritin association, ferritin protein levels and copper-ceruloplasmin association were measured in prefrontal cortex, putamen, substantia nigra and cerebellum of 3, 8, and 20-month-old PLP-αsyn and age-matched non-transgenic mice. Moreover, 12-month-old PLP-αsyn mice were administered deferiprone or ceruloplasmin or vehicle for 2 months. At the end of treatment period, motor testing and stereological analyses were performed. We found iron accumulation and perturbed iron-ferritin interaction in substantia nigra, putamen and cerebellum of aged PLP-αsyn mice. Furthermore, we found significant reduction in ceruloplasmin-bound copper in substantia nigra and cerebellum of the PLP-αsyn mice. Both deferiprone and ceruloplasmin prevented decline in motor performance in aged PLP-αsyn mice and were associated with higher neuronal survival and reduced density of α-synuclein aggregates in substantia nigra. This is the first study to report brain iron accumulation in a mouse model of MSA. Our results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction and provide evidence that targeting iron in MSA could be a viable therapeutic option., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called "halogenating cycle". Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H 2 O 2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

Chronic submaximal physical load of aerobic-anaerobic charater leads to pro-atherogenic dyslipidemia, as manifested by increased levels of triglycerides, total cholesterol, and very-low-density lipoproteins. Ceruloplasmin (total dose 60 mg/kg body weight, triple intraperitoneal introduction on the 1st, 3rd, and 5th day of experiment) produced a partial correction of dyslipidemia, manifested by restoration of the normal levels of triglycerides, total cholesterol, and lipoproteins of very low, low, and high density. At the same time, ceruloplasmin administration did not influence the level of phospholipids.

Aim: To estimate the time course of clinical changes in patients with asthma during combination therapy using ceruloplasmin (CP)., Materials and Methods: A total of 92 asthmatic patients were examined. Their medical history data were collected; external lung function testing and clinical, laboratory, and instrumental examinations, involving the determination of the indicators of lipid peroxidation (LPO) (malonic dialdehyde (MDA), methemoglobin, carboxyhemoglobin) and the antioxidant system (superoxide dismutase (COD), sulfhydryl groups), were performed in all the patients over time. According to the therapy used, the patients were divided into 2 groups matched for gender, age, and clinical manifestations of the disease. A study group consisted of 45 patients who took CP in addition to conventional therapy. A comparison group included 47 patients receiving standard therapy., Results: During the combination therapy using CP, the asthmatic patients showed a reduction in the elevated concentrations of MDA, methemoglobin, and carboxyhemoglobin and increases in the activity of COD and in the levels of sulfhydryl groups, which was followed by a considerable clinical improvement. During the conventional therapy, the indicators of LPO remained high and those of the antioxidant system did low, suggesting permanent oxidative stress., Conclusion: CP incorporation into the combination therapy of asthmatic patients contributes to elimination of prooxidant-antioxidant imbalance, which is followed by a marked positive clinical effect.

The influence of administration of the antioxidant complexes consisting of nonenzymatic antioxidants (alpha-tocopherol acetate preparation) and enzymatic antioxidants (ceruloplasmin) has been studied in rabbits with experimental arthritis. The introduction of alpha-tocopherol acetate (at a daily dose of 4 mg) improved metabolic processes in the organism (decreased in the rate of erythrocyte precipitation, total leukocytes and their stub and segmental forms; increased in erythrocyte count; reduced the glycosaminoglycan content as determined from uronic acid and hexose level; decreased ceruloplasmin activity and malonic dialdehyde level ion blood serum, all at p < 0.05), thus favoring reduction in the total activity of the inflammatory process as judged from hematological and biochemical data. Intra-articular introduction of ceruloplasmin (1.5 mg/kg, once per week) positively influenced the state of joint structures in damaged knee joints of the animals: decreased the activity of ceruloplasmin (from 5.28 ± 0.06 to 3.94 ± 0.01 AU), and malonic dialdehyde level (0.18 ± 0.02 to 0.08 ± 0.01 μM) in the articular fluid (all at p < 0.05). These effects are probably related to the elimination of inefficiency of the antioxidant system in the synovial medium, thus preventing inflammatory destruction of articular tissues, hindering the development of pannus, and assisting the activation of reparative regeneration of connective tissue structures.

Introduction: Wilson's disease is an inherited autosomal recessive disorder of copper metabolism. The prevalence of Wilson's disease in most populations is approximately 1 in 30,000. The risk for offspring is 0.5%. The aim of this study was to establish the frequency of disease among offspring of a cohort of Wilson's disease patients., Materials and Methods: In February 2014, our registry included 760 cases of diagnosed Wilson's disease. We selected families in which Wilson's disease was diagnosed in the proband's offspring., Results: Between 1957 and 2014, 1,050 relatives of affected members were screened. Wilson's disease in subsequent generations was observed in nine non-consanguineous families, with 12 affected offspring from nine probands., Conclusion: We detected a higher (4.08%) than expected (0.5%) frequency of Wilson's disease among proband offspring, which is in accordance with a recent genetic study in the United Kingdom that suggested a higher WD prevalence in the European population., (© 2014 International Parkinson and Movement Disorder Society.)

In patients with active anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV), there are high levels of circulating C-reactive protein (CRP), which can inhibit the alternative complement pathway by binding factor H and triggering the classical complement pathway by binding C1q. However, the alternative, not the classical, complement pathway has been proven to play an important role in AAV. We found that both purified myeloperoxidase (MPO) and MPO released from ANCA-stimulated neutrophils could bind modified CRP (mCRP), but not pentameric CRP. Furthermore, MPO could block the binding between mCRP and factor H, as well as the binding between mCRP and C1q. Binding with mCRP did not influence the enzymatic activity of MPO. Binding with mCRP also did not influence the binding between MPO and its physical inhibitor, ceruloplasmin, as well as the binding between MPO and MPO-ANCA. The results indicated that MPO might be a complement regulator and inhibit the negative regulatory effect of CRP on the alternative complement pathway.

Objective: To investigate the roles of ceruloplasmin (Cp) in PI3K/PTEN cell signaling pathway change in human embryonic lung fibroblasts (HELFs) induced by silica., Methods: HELFs transfected with pGenesil1.1 plasmid and pGenesil1.1 with PTEN shRNA (PT) plasmid were successfully established. 100 µg/ml silica and different concentrations of Cp (10, 20, 30 µg/ml) were used in this experiment and Cp were treated cells after exposed to silica for 1h. Three different cell lines (including HELFs, PT and cells were transfected with p85 dominant negative mutant plasmid (DN-p85)) were divided into control groups, silica groups and silica+different concentrations of Cp groups. MTT assay was used to detect the effects of Cp on silica-induced cell proliferation after inhibiting PTEN and p85. When suppressing the expression of PTEN and p85, western blot assay was performed to detect the levels of p85, p110, AKT308, AKT473 and ERK, JNK and their phosphorylated levels., Results: After inhibition of PTEN, the high levels of p85 induced by 100 µg/ml silica with 30 µg/ml Cp were markedly decreased (P<0.05). When suppressing p85, the increased cell proliferation was not observed. And the high levels of AKT308, AKT473, ERK and phosphorylated JNK and ERK stimulated by 100 µg/ml silica with 30 µg/ml Cp were decrease (P < 0.05)., Conclusion: Cp could further strengthened silica-induced cell proliferation by PI3K/AKT/MAPK cell signaling pathway, of which the level of p85 was regulated by PTEN.

The role of commercially available lycopene (all-trans) from tomato in controlling arthritis has been reported. Even though many reports are available that the cis form of lycopene is more biologically active, no report seems to be available on lycopene (cis and trans) isolated from an easily available and culturable sources. In the present study, the anti-arthritic effect of lycopene (cis and trans) from the algae Chlorella marina (AL) has been compared with lycopene (all-trans) from tomato (TL) and indomethacin (Indo). Arthritis (CIA) was developed in male Sprague dawley rats by collagen and the following parameters were studied. The activities of inflammatory marker enzymes like cyclooxygenase (COX), lipoxygenase (LOX) and myeloperoxidase (MPO) were found to be decreased on treatment with AL when compared to TL and Indo. Changes in Erythrocyte sedimentation rate (ESR), white blood cell (WBC) count, red blood cells (RBC) count, hemoglobin (Hb), C-reactive protein (CRP), rheumatoid factor (RF), and ceruloplasmin levels observed in the blood of arthritic animals were brought back to normal by AL when compared to TL and Indo. Histopathology of paw and joint tissues showed marked reduction in edema on supplementation of AL. Thus these results indicate the potential beneficiary effect of algal lycopene on collagen induced arthritis in rats when compared to TL and even to the commonly used anti-inflammatory drug indomethacin. Therefore lycopene from C. marina would be recommended as a better natural source with increased activity and without side effects in the treatment of anti-inflammatory diseases.

Ceruloplasmin (Cp) is a ferroxidase important to the regulation of both systemic and intracellular iron levels. Cp has a critical role in iron metabolism in the brain and retina as shown in patients with aceruloplasminemia and in Cp-/-hep-/y mice where iron accumulates and neural and retinal degeneration ensue. We have previously shown that cultured lens epithelial cells (LEC) secrete Cp. The purpose of the current study was to determine if cultured retinal pigmented epithelial cells (RPE) also secrete Cp. In addition, the effects of exogenously added Cp on iron regulated proteins and pathways, ferritin, transferrin receptor, glutamate secretion and levels of hypoxia-inducible factor-1α in the nucleus were determined. Like LEC, RPE secrete Cp. Cp was found diffusely distributed within both cultured LEC and RPE, but the cell membranes had more intense staining. Exogenously added Cp caused an increase in ferritin levels in both cell types and increased secretion of glutamate. The Cp-induced increase in glutamate secretion was inhibited by both the aconitase inhibitor oxalomalic acid as well as iron chelators. As predicted by the canonical view of the iron regulatory protein (IRP) as the predominant controller of cellular iron status these results indicate that there is an increase in available iron (called the labile iron pool (LIP)) in the cytoplasm. However, both transferrin receptor (TfR) and nuclear levels of HIF-1α were increased and these results point to a decrease in available iron. Such confounding results have been found in other systems and indicate that there is a much more complex regulation of intracellularly available iron (LIP) and its downstream effects on cell metabolism. Importantly, the Cp increased production and secretion of the neurotransmitter, glutamate, is a substantive finding of clinical relevance because of the neural and retinal degeneration found in aceruloplasminemia patients. This finding and Cp-induced nuclear translocation of the hypoxia-inducible factor-1 (HIF1) subunit HIF-1α adds novel information to the list of critical pathways impacted by Cp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia-reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H(2)O(2)), the former activity by consuming H(2)O(2) and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO(+) species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H(2)O(2). In contrast to human CP, bovine CP consumed H(2)O(2) in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H(2)O(2) could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H(2)O(2)-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CP's putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The cytoarchitectural organization of the nervous system depends partly on extracellular serine proteases, including reelin. This 400K protein, which also exists as the N-terminally-derived 300K and 180K fragments, acts through binding to the lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR). Ceruloplasmin (CP), a multifunctional protein found in the circulation and also expressed on glial cells, was shown to bind to, and induce aggregation of neurons newly differentiated from P19 embryonic stem cells. This indicated a potential developmental role of CP in neuronal organization, possibly in relation with reelin and other extracellular serine proteases. Therefore, we analysed the effect of cell-impermeant, large spectrum, serine protease inhibitors on CP-induced neuroaggregation and studied reelin expression. Soybean trypsin inhibitor and aprotinin (SBTI+Apro) inhibited CP neuroaggregative action. Undifferentiated and neurally-differentiating cultures secreted the 400K reelin. The 180K fragment was present during and after differentiation whereas the 300K species was barely detectable. However, CP stimulated generation of the 300K in the differentiated neuronal cultures, and SBTI+Apro abolished this CP effect. Time course profiles and function-blocking antibody indicated that neuroaggregation does not depend on the generation of the 300K fragment or on reelin action. CP neuroaggregative action thus involves a pericellular serine protease, different from reelin. On the other hand, the CP stimulation of reelin cleavage is in line with a possible role of CP in nervous system development. Since P19 cells express ApoER2 and VLDLR, they can help understanding relationships existing between CP, reelin and intervening protease(s)., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Hemochromatosis is a known cause of osteoporosis, and iron overload has deleterious effects on bone. Although iron overload and its association with osteoporosis has long been recognized, the pathogenesis and exact role of iron have been undefined. Bone is an active tissue with constant remodeling capacity. Osteoblast (OB) development and maturation are under the influence of core binding factor alpha-1 (CBF-alpha1), which induces expression of OB-specific genes, including alkaline phosphatase, an important enzyme in early osteogenesis, and osteocalcin, a noncollagenous protein deposited within the osteoid. This study investigates the mechanism by which iron inhibits human OB activity, which in vivo may lead to decreased mineralization, osteopenia, and osteoporosis. We demonstrate that iron-provoked inhibition of OB activity is mediated by ferritin and its ferroxidase activity. We confirm this notion by using purified ferritin H-chain and ceruloplasmin, both known to possess ferroxidase activity that inhibited calcification, whereas a site-directed mutant of ferritin H-chain lacking ferroxidase activity failed to provide any inhibition. Furthermore, we are reporting that such suppression is not restricted to inhibition of calcification, but OB-specific genes such as alkaline phosphatase, osteocalcin, and CBF-alpha1 are all downregulated by ferritin in a dose-responsive manner. This study corroborates that iron decreases mineralization and demonstrates that this suppression is provided by iron-induced upregulation of ferritin. In addition, we conclude that inhibition of OB activity, mineralization, and specific gene expression is attributed to the ferroxidase activity of ferritin., (2010 American Society for Bone and Mineral Research)

Acute physical exercise was followed by a decrease in the osmotic resistance of erythrocytes, shortening of the time-to-onset of erythrocyte hemolysis, and increase in the sorption capacity of the cell membrane. Administration of ceruloplasmin 24 h before physical exercise normalizes membrane resistance in red blood cells.

CNS injury-induced hemorrhage and tissue damage leads to excess iron, which can cause secondary degeneration. The mechanisms that handle this excess iron are not fully understood. We report that spinal cord contusion injury (SCI) in mice induces an "iron homeostatic response" that partially limits iron-catalyzed oxidative damage. We show that ceruloplasmin (Cp), a ferroxidase that oxidizes toxic ferrous iron, is important for this process. SCI in Cp-deficient mice demonstrates that Cp detoxifies and mobilizes iron and reduces secondary tissue degeneration and functional loss. Our results provide new insights into how astrocytes and macrophages handle iron after SCI. Importantly, we show that iron chelator treatment has a delayed effect in improving locomotor recovery between 3 and 6 weeks after SCI. These data reveal important aspects of the molecular control of CNS iron homeostasis after SCI and suggest that iron chelator therapy may improve functional recovery after CNS trauma and hemorrhagic stroke.

Ceruloplasmin (Cp) is the major copper transport protein in plasma and catalyzes the conversion of toxic ferrous iron to the safer ferric iron. As an acute-phase protein, Cp is induced during inflammation. It is synthesized primarily in the liver and is expressed in several other tissues, including the brain. Elevated Cp levels have been observed in the brain of patients with neurodegenerative conditions, including Alzheimer's, Parkinson's, and Huntington's diseases. However, the exact role(s) of Cp in inflammatory and neuropathological conditions remains unclear. Microglia are the prime effector cells involved in immune and inflammatory responses in the central nervous system (CNS). They are activated during pathological conditions to restore CNS homeostasis, but chronic microglial activation endangers neuronal survival. Consequently, it is important to identify the regulators of microglial activation and the underlying mechanisms. We sought to examine whether Cp might modulate microglial activation. We observed that Cp induced nitric oxide (NO) release and inducible NO synthase mRNA expression in BV2 microglial cells and rat brain microglia. Cp also increased levels of mRNAs encoding tumor necrosis factor-alpha, interleukin-1beta, cyclooxygenase-2, and NADPH oxidase. Treatment of BV2 cells and primary microglia with Cp induced phosphorylation of p38 MAP kinase. Moreover, Cp induced nuclear factor (NF)-kappaB activation, showing a more sustained pattern than seen with bacterial lipopolysaccharide. Cp-stimulated NO induction was significantly attenuated by a p38 inhibitor, SB203580, and the NF-kappaB inhibitor SN50. Cp induced secretion of TNF-alpha and prostaglandin E(2) in primary microglial cultures. These results suggest that Cp may play an important role in neuropathological conditions by stimulating various proinflammatory and neurotoxic molecules in microglia.

Purpose: Ceruloplasmin (CP), an important serum antioxidant, was previously found to reduce the incidence of ventricular fibrillation (VF) induced by ischemia and reperfusion in isolated rat hearts. The present study investigated whether CP sterilized by gamma-irradiation maintains its antiarrhythmic capacity and in vitro antioxidant properties., Materials and Methods: Isolated rat hearts submitted to regional ischemia (15 min), were reperfused (10 min) with native CP or with CP irradiated at various doses (1-3 kGy) in the absence or presence of tyrosine (Tyr)., Results: All untreated hearts showed VF at reperfusion, which were all irreversible ventricular fibrillation (IVF). No IVF were found in hearts treated with native CP or gamma-irradiated CP. Cardioprotection afforded by irradiated CP (with or without Tyr) was slightly higher than that obtained with native CP. No VF at all (100% prevention) was found in hearts treated with CP irradiated alone or in the presence of tyrosine at 3 kGy. Tyrosine and irradiated tyrosine had no cardiotoxic or protective effects on reperfusion-induced arrhythmias. The Oxygen Radical Absorbing Capacity (ORAC), measured in vitro with beta-phycoerythrin (beta-PE) fluorescent indicator, was slightly higher for gamma-irradiated CP in the presence of Tyr., Conclusions: Ceruloplasmin sterilized by gamma-irradiation maintains antioxidant and antiarrhythmic effects in the post-ischemia reperfused isolated rat heart.

Although an essential nutrient, iron can catalyze damaging free radical reactions. Therefore elaborate mechanisms have evolved to carefully regulate iron metabolism. Ceruloplasmin, a protein with ferroxidase activity, and transferrin, an iron binding protein have important roles in maintaining iron homeostasis in cells. Since oxidative damage is a hallmark of cataractogenesis, it is essential to determine iron's role in lenticular physiology and pathology. In the current study of lens epithelial cells, the effects of ceruloplasmin and transferrin on intracellular distribution and efflux of iron were determined. Both ceruloplasmin and transferrin increased iron efflux from these cells and their effects were additive. Ceruloplasmin had significant effects on extracellular iron distribution only in cases of iron overload. Surprisingly, both transferrin and ceruloplasmin had significant effects on intracellular iron distribution. Under physiological conditions, ceruloplasmin increased iron incorporation into the storage protein, ferritin. Under conditions of iron overload, it decreased iron incorporation into ferritin, which is consistent with increased efflux of iron. Measurements of an intracellular chelatable iron pool indicated that both transferrin and ceruloplasmin increased the size of this pool at 24 h, but these increases had different downstream effects. Finally, lens epithelial cells made and secreted transferrin and ceruloplasmin. These results indicate an important role for these proteins in iron metabolism in the lens.

Ceruloplasmin (CP) is essential for brain iron homeostasis. However, its precise function in brain iron transport has not been definitely determined. In this study, we investigated the effects of soluble CP on iron influx and efflux in primary neuronal culture from the midbrain (the substantia nigra and striatum) and the hippocampus. Our data showed that low concentrations of CP (2, 4, 8 microg/ml) can promote iron influx into iron-deficient neurons, but not the neurons with normal iron status. The same concentrations of CP had no effect on iron efflux from iron-sufficient and normal-iron neurons. Contrary to our expectation, we did not find any regional difference in the effects of CP on iron influx as well as efflux in neurons. The changes in quenching (iron influx) and also dequenching (iron efflux) of intracellular fluorescence, induced by the addition of CP with iron, in the midbrain neurons were no different from those in the hippocampus neurons. The data showed that soluble CP has a role in iron uptake by iron-deficient brain neurons under our experimental conditions. The physiological significance of the results forms the focus for future work., (2006 Wiley-Liss, Inc.)

In 81.25% patients with an acute necrotic pancreatitis and in 50% of healthy persons the blood neutrophils are sensitive to cerulloplasmin in metabolic activity. In 62.5% of patients the phagocytic index have had reduced in addition of cerulloplasmin. Phagocytic number in addition of cerulloplasmin have constituted over 1,5 conditional units, in 75% of patients, without such addition - in 84%. The conclusion was made about necessity of individual selection of immunomodulators in patients with an acute necrotic pancreatitis.

Previous studies indicated that sex might influence the response to copper exposure. Ceruloplasmin (Cp) is an indicator of Cu status, but it is not clear whether and how it reflects changes of Cu status among healthy individuals. In this study, 82 apparently healthy women and men were chosen from 800 individuals because their Cp values belonged to the higher and lower 10% of the group Cp distribution curve. Before and after receiving a supplement of 10mg Cu/day (upper limit of daily intake) for 2 months, we performed blood and urinary biochemical measurement of potential Cu markers. We used principal component analysis and linear discriminant analysis to identify blood and/or urinary Cu indicators that showed a differential response to copper. Results showed that Cp values in serum represent a reliable indicator to differentiate subgroups within the normal population in their response to Cu exposure. The response depends on Cp values and on sex, such that women with higher and men with lower Cp values exhibit the greatest response.

Natural killer (NK) cells are functionally suppressed and induced to apoptosis by reactive oxygen species (ROS) produced by mononuclear phagocytes (MPs). These inhibitory events are reversed by the biogenic amine serotonin. MPs generate hydrogen peroxide (H(2)O(2)), which is processed further by myeloperoxidase (MPO) to even more toxic compounds. Earlier studies suggest that serotonin scavenges MP-derived oxygen radicals generated by the MPO-H(2)O(2) system. These findings led us to explore the capability of MPO-deficient MPs to induce NK cell dysfunction. We show that MPs recovered from subjects with MPO deficiency trigger inhibition of NK cells. In addition, MPs recovered from healthy subjects conveyed suppression of NK cells in the presence of the MPO inhibitor ceruloplasmin. We conclude that ROS-dependent inhibition of NK cell function is unrestricted by the availability of MPO-derived oxygen radicals and that the protecting properties of serotonin may operate in the absence of functional MPO. Our data suggest a complex mechanism of MP-induced NK cell inhibition, which comprises the generation of interchangeable oxygen radicals.

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid beta peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid beta peptides (Abeta) can stimulate copper-mediated free radical formation. We found that neurotoxic Abeta (1-42), Abeta (1-40), and Abeta (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Abeta (40-1) did not. Formation of ascorbate free radical was significantly increased by Abeta (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric-amyloid beta peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid beta peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Abeta peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric-amyloid beta peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.

The reticuloendothelial system has a central role in erythropoiesis and iron homeostasis. An important function of reticuloendothelial macrophages is phagocytosis of senescent red blood cells. The iron liberated from heme is recycled for delivery to erythrocyte precursors for a new round of hemoglobin synthesis. The molecular mechanism by which recycled iron is released from macrophages remains unresolved. We have investigated the mechanism of macrophage iron efflux, focusing on the role of ceruloplasmin (Cp), a copper protein with a potent ferroxidase activity that converts Fe2+ to Fe3+ in the presence of molecular oxygen. As shown by others, Cp markedly increased iron binding to apotransferrin at acidic pH; however, the physiological significance of this finding is uncertain because little stimulation was observed at neutral pH. Introduction of a hypoxic atmosphere resulted in marked Cp-stimulated binding of iron to apotransferrin at physiological pH. The role of Cp in cellular iron release was examined in U937 monocytic cells induced to differentiate to the macrophage lineage. Cp added at its normal plasma concentration increased the rate of 55Fe release from U937 cells by about 250%. The stimulation was absolutely dependent on the presence of apotransferrin and hypoxia. Cp-stimulated iron release was confirmed in mouse peritoneal macrophages. Stimulation of iron release required an intracellular "labile iron pool" that was rapidly depleted in the presence of Cp and apotransferrin. Ferroxidase-mediated loading of iron into apotransferrin was critical for iron release because ferroxidase-deficient Cp was inactive and because holotransferrin could not substitute for apotransferrin. The extracellular iron concentration was critical as shown by inhibition of iron release by exogenous free iron, and marked enhancement of release by an iron chelator. Together these data show that Cp stimulates iron release from macrophages under hypoxic conditions by a ferroxidase-dependent mechanism, possibly involving generation of a negative iron gradient.

The antioxidant and antihypoxant effects of the drug ceruloplasmin were studied on an experimental model of hypobaric hypoxia in rats. Two forms of the drug characterized by low and high enzymatic activity were administered (intramuscularly or intraperitoneally) 20 min or 1 day before hypobaric hypoxia test. The drug effect was independent of the enzymatic activity level. The maximum activity of ceruloplasmin was observed when the drug was administered one day before test. Analyses showed that the drug activated superoxide dismutase, while not changing the catalase activity.

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl-alpha-D-glucoside was ineffective on Cu2+-induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu+; this is more efficient in decomposing lipid peroxide than Cu2+, accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.

A 34 year old diabetic man with a complete deficiency of serum ferroxidase activity, regardless of the presence of serum ceruloplasmin (Cp), a multicopper ferroxidase protein, is described. The patient had had diabetes mellitus for 13 years, and was also found to have retinal degeneration accompanied by the development of a hearing disturbance of unknown aetiology. Laboratory examination showed markedly increased serum ferritin and low serum iron. Magnetic resonance imaging showed a pronounced hypointensity in the putamen, caudate, cerebellar dentate, and thalamus on T2 weighted images, and also disclosed a low level signal in the liver, suggesting the accumulation of some magnetic substances in the brain and liver. Liver biopsies histochemically identified iron deposition in the hepatocytes. Most of these findings were consistent with the newly established autosomal recessive disease "aceruloplasminaemia", except for the presence of serum Cp and the lack of apparent neurological symptoms. Interestingly, no ferroxidase activity was detected in the patient's serum, whereas suppressed ferroxidase activity was found in his mother's serum. A nucleotide sequence analysis of the Cp gene showed two mutations; a C to T substitution at nucleotide 2701 in exon 16, resulting in a nonsense mutation at amino acid 882 (Arg882ter), and a T to G substitution at nucleotide 2991 in exon 17, resulting in an amino acid alternation at amino acid 978 (His978Gln). The second mutation was also found in the patient's mother. The absence of serum ferroxidase activity despite the presence of serum Cp protein in this compound heterozygote was considered to be due to the production of a non-functional Cp harbouring no ferroxidase activity.

Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood-derived CD34+ cells were cultured in liquid medium supplemented with Kit- ligand, FLt3, interleukin 6 (IL-6), thrombopoietin and IL-3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/10(7) cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/10(7) cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/10(7) cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony-forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34+ cells in low Cu was extended (12-16 weeks), and in high Cu was shortened (2-4 weeks), compared with control cultures (6-8 weeks). These effects required modulation of intracellular Cu only during the first 1-3 weeks of the culture; the long-term effects persisted thereafter, suggesting that the decision process for either self-renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.

In a previous study, we found that human ceruloplasmin (hCP) promotes iron uptake rather than release in BT325 cells, a human glioma cell line. In this study, we investigated the effect of ferroxidase activity of hCP and different species of ceruloplasmins on CP-mediated iron uptake by the cells. The cells were incubated for 30 min at 37 degrees C with 1 microM 59Fe2+ with or without 150 microg/ml of the untreated and the ferroxidase-defective hCPs (apohCP and heat-inactivated hCP) or different species of ceruloplasmins (human CP, rabbit CP and bovine CP). The untreated hCP induced a significant increase in iron uptake by BT325 cells, while ferroxidase-defective hCPs with (heat-inactivated hCP) or without cooper ions (apohCP) had no such role. The untreated hCP increases significantly internalized iron but not membrane-bound iron, implying that hCP stimulated iron entry into the cell rather than increased extracellular binding of iron to the cell surface. All species of ceruloplasmins could promote iron uptake by the cells and the difference in degree of stimulatory effect among them was insignificant. These results suggested that ferroxidase activity of hCP is essential for the hCP-mediated iron uptake process and also that CP-stimulated iron uptake is not associated with copper ions in the protein, and that the effect of ceruloplasmin on iron uptake by BT325 cells is not species specific.

It has been shown, that the total X-ray irradiation in the dozes of 0.5 and 1 Gy influences on the content of lipid peroxidation products and enzymatic activity of antioxidant system in rat spleen and thymus cells. The influence of preparations "AMMIVIT" and "Ceruloplasmin" on these processes is investigated also. So, the animals feeding by the vitamin concentrate "AMMIVIT" have lead to increase of MDA level (a final product of lipid peroxidation) and the overactivity of some antioxidant enzymes in rat spleen and thymus cells. Injection of the preparation "Ceruloplasmin" to experimental animals up 1 hour before the irradiation has normalized LPO intensity and activity of AO enzymes.

Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Büdy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.

The red color of muscle is principally due to the presence of oxymyoglobin. Oxidation of heme iron from the ferrous to the ferric state produces a brownish color, which consumers find undesirable. The aim of this study was to use enzymic and nonenzymic antioxidants to simulate in situ muscle antioxidation reactions in order to understand better the mechanism by which the iron redox cycle catalyzes membrane lipid peroxidation and oxymyoglobin oxidation. The inclusion of superoxide dismutase (SOD) in the model system decreased oxymyoglobin oxidation by 10% without affecting lipid peroxidation. Addition of catalase decreased oxymyoglobin oxidation by approximately 40% but not lipid peroxidation. Increasing the ceruloplasmin concentration inhibited lipid peroxidation but increased oxymyoglobin oxidation, which was inhibited by SOD and catalase. Conalbumin (50 microM), a specific iron chelator, inhibited peroxidation and oxymyoglobin oxidation by almost 50%. The addition of the antioxidant catechin (500 microM) decreased lipid peroxidation by 90% but oxymyoglobin oxidation by only 50%. Feeding turkeys with vitamin E at several levels significantly increased the alpha-tocopherol level of membranes, thus preventing oxymyoglobin and lipid oxidation. In conclusion, oxymyoglobin stability in the model system was affected by two pathways: (a) oxygen active species, such as O(2)*(-), H(2)O(2), HO*, and ferryl, generated during autoxidation of myoglobin and oxidation of ferrous ions and ascorbic acid; and (b) lipid radicals, such as ROO*, RO*, and hydroperoxides, generated during lipid peroxidation. Maximum inhibition could be achieved only by introducing inhibitors of both pathways into the system.

This study was initiated to investigate the mechanism of ceruloplasmin (CP)-mediated iron release from brain cells using BT325 cells (a glioblastoma cell line); however, negative results were obtained. The BT325 cells were preloaded with 1 microM 59Fe2+ in sucrose (pH 5.8) for 60 min, and then with CP (0-300 microg/ml) for 30 min at 37 degrees C. The addition of CP, either at low (25 microg/ml) or high (300 microg/ml) concentrations, did not lead to a significant change in iron release from iron-loaded BT325 cells. No significant difference in total iron of cells was found between all CP treated groups and the control (P>0.05). Although apotransferrin (apoTf) was shown to have a role in iron release from the cells, the effect of apoTf was not significantly affected by the addition of different concentrations of CP. When the cells were incubated with 1 microM 59Fe2+ in the presence of varying amounts of CP for 30 min at 37 degrees C, it was found that CP increased iron uptake. The total iron uptake by BT325 cells in CP treatment groups (25, 75, 150, 300 microg/ml) was significantly higher than that in the control (no CP addition) (all P<0.01). Furthermore, in contrast to our expectation, CP was shown to promote significantly iron uptake in not only iron-sufficient but also iron-deficient cells. These results showed that CP had a role in iron uptake rather than release in BT325 cells.

Aim: To study generation of active oxygen forms in the whole blood of patients with chronic bronchitis and correction of the detected disorders., Material and Methods: By means of registration of spontaneous and staphylococcus-induced luminol-dependent chemiluminescence (LDCL) of the whole blood, generation of active oxygen forms in 73 patients with chronic bronchitis (CB) has been studied., Results: By the intensity of luminescence, all the examinees were divided into two groups. In group 1 LDCL was high, in group 2 LDCL was under the control level. Combined treatment of group 1 patients included inhalations of ceruloplasmin antioxidant. Patients of group 2 were administered sodium hypochlorite that possesses prooxidation properties. Compared to conventional treatment, the above two treatments produced marked positive changes in the studied indices and clinical symptoms., Conclusion: The method of chemiluminescence registration of the whole blood allows validation of pathogenetic therapy of patients with CB and improves outcomes of the treatment.

The mechanism of iron release from the placenta into the fetal circulation is not well understood. Ceruloplasmin, a plasma ferroxidase, has been implicated in iron efflux from a variety of cell types. The hypothesis is that circulating ceruloplasmin facilitates iron efflux by oxidizing the released Fe(II) to Fe(III) for incorporation into transferrin. We tested whether this mechanism mediates iron release from placental cells into the fetal circulation, using the BeWo cell line, a choriocarcinoma which can differentiate into a syncytium.(59)Fe release from undifferentiated or differentiated cells and from cells grown on porous filters was not stimulated by extracellular ceruloplasmin. Instead, we found that BeWo cells express an endogenous ferroxidase. The protein is membrane bound and cross-reacts with an anti-ceruloplasmin antibody, but has a different size; 100 and 140 kDa. Similar immunoreactivity was identified in first- and third-trimester human placentae. In BeWo cells, the protein has a perinuclear localization but does not entirely co-localize with markers for the endoplasmic reticulum or Golgi apparatus. We propose that this oxidase performs the same function as serum ceruloplasmin and is involved in iron release into the fetal circulation., (Copyright 2000 Harcourt Publishers Ltd.)

The aim of the work was to examine immunotropic features of celluroplasmine++ (Cp) and estimate its capacity as radioprotector. The pulse radiolysis method was used to set the mechanism of elementary reactions, which were responsible for antioxidant activity, and to demonstrate a particular role of reversible oxycomplexis, Cp...O2. Within in vitro test-systems the effects of Cp were examined on the model of interaction between lymphocytes from Shelter staff which constantly contacts with ionizing irradiation, and autologous erythrocytes in rosset-forming phenomenon (223 Shelter employees in comparison with 253 donors of Kyiv blood transfucion station). Expression of this Index in the presence of antigens with known poly-specificity was determined (antigens were taken from cortical and pyramid sections of kidney, liver, lungs, myocard, pancreatic gland, grey matter and aorta). Simultaneously the presence of analogous autoantibodies was determined (ELISA). It has been shown that Cp can reprogram the level of expression of immunovaluable receptors towards to norm. It has been also defined that Cp presence in blood irradiated in vitro (1-15 rem) promotes the masking of active centers of autoantibodies of different tissue specificity.

Ceruloplasmin (CP) is a major multicopper-containing plasma protein that is not only involved in iron metabolism through its ferroxidase activity but also functions as an antioxidant. However, physiological substrates for CP have not been fully identified nor has the role of CP been fully understood. The reaction of nitric oxide (NO) with CP was investigated in view of nitrosothiol (RS-NO) formation. First, formation of heavy metal- or CP-catalyzed RS-NO was examined with physiologically relevant concentrations of NO and various thiol compounds (RSH) such as glutathione (GSH). Among the various heavy metal ions and copper-containing enzymes and proteins examined, only copper ion (Cu(2+)) and CP showed potent RS-NO (S-nitrosoglutathione)-producing activity. Also, RS-NO-forming catalytic activity was evident for CP added exogenously to RAW264 cells expressing inducible NO synthase in culture, but this was not the case for copper ion. Similarly, CP produced endogenously by HepG2 cells showed potent RS-NO-forming activity in the cell culture. One-electron oxidation of NO appears to be operative for RS-NO production via electron transfer from type 1 copper to a cluster of types 2 and 3 copper in CP. Neurological disorders are associated with aceruloplasminemia; besides RS-NO, S-nitrosoglutathione particularly has been shown to have neuroprotective effect against oxidative stress induced by iron overload. Thus, we suggest that CP plays an important catalytic role in RS-NO formation, which may contribute to its potent antioxidant and cytoprotective activities in vivo in mammalian biological systems.

The death of macrophage-derived foam cells contributes to the formation of the lipid core in atherosclerotic lesions. Although the underlying mechanism is not yet clear, apoptosis has been shown to be responsible, at least in part, for the cell death of lipid-laden macrophages in atherosclerotic plaques. In the present study, we demonstrated that copper, in the presence of 8-hydroxyquinoline, was able to induce apoptosis of murine J774.A1 cells in culture. Ceruloplasmin exerts similar a effect, but not iron or hemin. Further experiments demonstrated that the expression of immediate early genes, including c-jun, c-fos and egr-1, was also induced by copper treatment in these cells, although only egr-1 mRNA was induced in a time- and dose-dependent manner. The antioxidant, N-acetylcysteine, exhibited remarkable inhibitory effect on the copper-induced apoptosis dose-dependently. Time course experiment revealed that prior treatment of cells with N-acetylcysteine is essential for the anti-apoptotic effect of this compound. Results also demonstrated that under the condition; in which N-acetylcysteine inhibited the copper-induced apoptosis, this antioxidant also abolished the gene expression of egr-1. Collectively, these results suggest that egr-1 gene expression is closely associated with the apoptosis induced by copper in macrophages.

The plasma copper protein ceruloplasmin (CP) was found to inhibit endothelial nitric-oxide synthase activation in cultured endothelial cells, in line with previous evidence showing that the endothelium-dependent vasorelaxation of the aorta is impaired by physiological concentrations of ceruloplasmin. The data presented here indicate a direct relationship between the extent of inhibition of agonist-triggered endothelial nitric oxide synthase activation and CP-induced enrichment of the copper content of endothelial cells. Copper discharged by CP was mainly localized in the soluble fraction of cells. The subcellular distribution of the metal seems to be of relevance to the inhibitory effect of CP, because it was mimicked by copper chelates, like copper-histidine, able to selectively enrich the cytosolic fraction of cells, but not by copper salts, which preferentially located the metal to the particulate fraction.

Background: The purpose of this study was to characterize the recently reported inhibition of myeloperoxidase (MPO) by ceruloplasmin and to determine whether this may be disturbed in the presence of anti-MPO antibodies., Methods: Specificity of the binding between ceruloplasmin and MPO was confirmed by Western blotting and enzyme-linked immunosorbent assay (ELISA), and the enzymatic activity of MPO was measured in the presence of ceruloplasmin, affinity-purified anti-MPO antibodies, or both. The affinity of the binding between MPO and ceruloplasmin and MPO and the anti-MPO antibodies was measured using a biosensor, with the results confirmed by chaotrope ELISA., Results: Affinity-purified anti-MPO antibodies from patients with microscopic polyangiitis and florid renal vasculitis inhibited the binding between ceruloplasmin and MPO to a maximum of 72.9 +/- 12.8%, whereas those from patients with Wegener's granulomatosis and only minimal renal involvement inhibited the binding to a maximum of only 36.8 +/- 10.9% (P < 0. 001), with comparable reversal of the ceruloplasmin-mediated inhibition of MPO activity. Measurement of the affinity of the interactions demonstrated that binding between MPO and the anti-MPO antibodies is stronger than that between MPO and ceruloplasmin (1.61 x 107 to 1.33 x 108 vs. 7.46 x 106 m-1), indicating that binding to the autoantibody would be favored in vivo., Conclusions: This study confirms a role for ceruloplasmin as a physiological inhibitor of MPO, and demonstrates how the inhibition may be disrupted in the presence of anti-MPO antibodies. Because a majority (16 of 21) of the antibodies did not themselves inhibit MPO activity, their interference with the inhibition mediated by ceruloplasmin may be brought about by steric hindrance consequent upon the binding of the antibody to a dominant epitope at or near the active site.

The chain-breaking antioxidant potential of caeruloplasmin and bovine serum albumin (BSA) has been investigated in comparison with other well-established antioxidants. Their Oxygen Radical Absorbing Capacity (ORAC), was measured by using beta-phycocyanin (beta-PC) as a fluorescent indicator protein, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) as a peroxyl radical generator and the water soluble vitamin E analogue, Trolox, as a reference standard. The relative peroxyl absorbing capacities/mole for Trolox, caeruloplasmin, heat-denatured caeruloplasmin (hCP), catalase, bovine serum albumin (BSA), superoxide dismutase (SOD), and deferoxamine were 1; 2.6; 3.3; 3.7; 1.2; 0.1; 0.2, respectively. Caeruloplasmin was far more effective as a peroxyl radical scavenger than SOD, deferoxamine and BSA, but slightly less effective than catalase. The peroxyl radical absorbing capacity of caeruloplasmin was enhanced by heat-denaturation of the protein. Electron paramagnetic resonance (EPR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap, was applied in order to measure the scavenger abilities of caeruloplasmin on superoxide radical and hydroxyl radical production and the concentration required to inhibit by 50% oxygen free radical formation (IC50) was determined. The IC50 values of caeruloplasmin, hCP, and BSA for the superoxide radical were 12, 2, 260 microM and for the hydroxyl radical 15, 2, 200 microM. These results show that caeruloplasmin is an effective chain-breaking antioxidant for a variety of radicals, independently of its catalytic ferroxidase activity.

Particulate air pollution causes increased cardiopulmonary morbidity and mortality, but the chemical determinants responsible for its biologic effects are not understood. We studied the effect of total suspended particulates collected in Provo, Utah, an area where an increase in respiratory symptoms in relation to levels of particulate pollution has been well documented. Provo particulates caused cytokine-induced neutrophil chemoattractant-dependent inflammation of rat lungs. Provo particulates stimulated interleukin-6 (IL-6) and IL-8 production, increased IL-8 messenger RNA (mRNA) and enhanced expression of intercellular adhesion molecule-1 (ICAM-1) in cultured BEAS-2B cells, and stimulated IL-8 secretion in primary cultures of human bronchial epithelium. Cytokine secretion was preceded by activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and was reduced by treatment of cultures with superoxide dismutase, deferoxamine, or N-acetylcysteine. These biologic effects were replicated by culturing BEAS cells with quantities of Cu2+ found in Provo extract. IL-8 secretion by BEAS cells could be modified by addition of normal constituents of airway lining fluid to the culture medium. Mucin significantly reduced IL-8 secretion, and ceruloplasmin significantly increased IL-8 secretion and activation of NF-kappaB. These findings suggest that copper ions may cause some of the biologic effects of inhaled particulate air pollution in the Provo region of the United States, and may provide an explanation for the sensitivity of asthmatic individuals to Provo particulates that has been observed in epidemiologic studies.

Aim: To study the effects of epinephrine, homocysteine, and other complexing agents on the cytotoxicity of copper sulfate., Methods: In vitro suspensions of human red cells incubated with cupric sulfate were used, and hemolysis was determined by extracellular hemoglobin., Results: The hemolytic activity of CuSO4 (0.3 mmol.L-1) was enhanced by the presence of epinephrine and to a lesser extent by homocysteine, whereas D-penicillamine, succimer, and mercaptodextran reduced the copper-induced hemolysis. The latter 3 chelating thiols also reduced the copper-epinephrine-induced hemolysis. The plasma protein ceruloplasmin reduced markedly the copper-epinephrine-induced hemolysis, even upon concentrations < 20% of that of copper. Chromic chloride, as well, acted anti-hemolytically., Conclusion: The latter protectors may interact with the production or activity of toxic oxygen, while classical copper chelators sequester cupric ions from interaction with epinephrine or homocysteine.

Individuals with hereditary ceruloplasmin (Cp) deficiency have profound iron accumulation in most tissues, which suggests that Cp is important for normal release of cellular iron. Here, in contrast to expectations, Cp was shown to increase iron uptake by HepG2 cells, increasing the apparent affinity for the substrate by three times. Consistent with its role in iron uptake, Cp synthesis was regulated by iron supply and was increased four- to fivefold after iron depletion. Unlike other iron controllers that are posttranscriptionally regulated, Cp synthesis was transcriptionally regulated. Thus, iron-deficient cells could increase Cp synthesis to maintain intracellular iron homeostasis, so that defects would lead to global accumulation of iron in tissues.