Comparison of the protective effects on the erythrocytes of ceruloplasmin from the blood of healthy donors and patients with hepatocerebral dystrophy

The binding of the ceruloplasmin (CP) from the healthy donor's blood and of ceruloplasmin--like protein (p-CP) isolated from the Wilson disease patient's blood with erythrocytes (RBC) of healthy donors and with RBC of Wilson's patients (p-RBC) was investigated. It was shown, that the CP number of binding sites both on the RBC and p-RBC was significantly lower than that for p-CP, but Kd value for p-CP binding of the both types of erythrocytes was approximately 10 times higher than Kd value for CP. The protective action of CP on copper stimulated hemolysis is almost 3 times higher than that of p-CP. The protective action of CP on ferrous ion stimulated hemolysis doesn't correlate with its ferroxidase activity. On the contrary the protective effect of p-CP which has no ferroxidase activity is more powerful than that of CP.     

Immunoenzyme determination of the total level of ceruloplasmin in the serum from patients with hepatocerebral dystrophy]

An immunoenzymatic method for ceruloplasmin analysis (IEA) based on the use of horseradish peroxidase-labelled monospecific antibodies as markers has been developed. IEA can be used for direct measurements of ceruloplasmin in blood serum, as can be evidenced from the coincidence of calibration plots obtained after the use of potassium-phosphate buffer and ceruloplasmin-free sera. The procedure allows the determination of the total content of ceruloplasmin present in the blood sera of patients with hepatocerebral dystrophies both in the active and inactive forms. The minimum ceruloplasmin concentration detectable by this method is 5 x 10(-9) g/ml. The method was used to determine ceruloplasmin levels in the blood of patients with various grades of hepatocerebral dystrophy. Analysis of blood sera from 6 patients revealed that the ceruloplasmin concentrations determined by IEA were very close, whereas the oxidase activities of this protein differed more than 7-fold. The amount of enzymatically active ceruloplasmin as determined from the oxidase activity made up to 10-68% of the total ceruloplasmin content in the sera, depending of the severity of the pathology.     

Protective effect of ceruloplasmin during human erythrocyte lysis induced by Fe2+ ions

In order to elucidate the nature of linkage between the oxidase activity and protective effect of ceruloplasmin during the Fe2(+)-induced lysis of erythrocytes, the both factors were identified in ceruloplasmin samples prepared from blood sera of healthy donors and patients with hepatocerebral dystrophy (HCD). It was found that the oxidase activity of healthy donor ceruloplasmin markedly exceeds that of HCD patients, whereas the protective effect of the HCD protein, contrariwise, markedly exceeds that of normal ceruloplasmin. The data obtained suggest that the protective effect of ceruloplasmin during Fe2(+)-induced erythrocyte lysis is not correlated with its oxidase (ferroxidase, in particular) activity.     

Protective effect of various forms of human ceruloplasmin in copper-induced erythrocyte lysis]

It is known that human ceruloplasmin (CP) is made up of several isoforms which differ by the structure of their carbohydrate fragment. One of these isoforms, CP1, which makes up to approximately 40% of the native CP molecule and which contains a carbohydrate fragment, [formula: see text] is specifically bound to human erythrocyte (ER) receptors. This isoform was isolated by using lectin affinity chromatography. It was found that CP1 produces a much stronger protective effect on ER during Cu(2+)-induced lysis as compared with CP. A kinetic analysis of Cu2+ accumulation and reduced glutathione (GSH) decline in ER revealed that the lack of correlation between these two processes. It was found that in the presence of CP and CP1 the GSH concentration is not critical for the hemolytic resistance of ER. In the presence of CP1 ER hemolysis occurs at a slower rate whereas the GSH decline at a much faster rate than in the presence of CP.     

Protective effect of capsaicin on the osmotic fragility of human erythrocytes

Capsaicin exerts a stabilizing effect on erythrocytes making them more resistant to lysis under hypotonic stress. The protective action of capsaicin on osmotic fragility (OF) was not receptor mediated since no dose responsive effect was observed. The results suggest that this protective effect of capsaicin on OF is due to a direct interaction of capsaicin with the erythrocyte membrane rather than due to any alteration in the intracellular metabolism of erythrocytes.     

Protective effect of placenta extracts against nitrite-induced oxidative stress in human erythrocytes

Aqueous-saline human placenta extract (HPE) is known to possess antioxidant activity due to the high concentration of bioactive substances. This fact allows its application in clinical practice in order to treat oxidation-induced diseases. Extract antioxidant activity is mainly conditioned by proteins. Freezing of extracts has been shown to lead to their antioxidant activity increasing due to protein conformation changes.     

Hemin-induced lysis of rat erythrocytes. Protective action of ceruloplasmin and different serum albumins

Hemin-induced lysis of rat erythrocytes is markedly reduced by ceruloplasmin (human) and serum albumins from different species, the order of effectiveness beings: bovine albumin approximately equal to ceruloplasmin greater than human albumin approximately equal to dog albumin greater than apotransferrin (human). Although the proteins studied had hemin binding capacity, the best protective agents, ceruloplasmin and bovine albumin, did bind hemin less strongly than human and dog albumin. The results suggest the existence of another protective mechanism, possibly involving an interaction between erythrocyte membranes and serum proteins.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin

Photosensitized hemolysis of human erythrocytes by hematoporphyrin was suppressed by flavonols such as quercetin and rutin at submillimolar concentrations. The suppression of photohemolysis was accompanied by inhibition of lipid peroxidation by the reagents. Quercetin and rutin were photooxidized in the presence of hematoporphyrin and the photooxidation was partially suppressed by 1 mM NaN3, a quencher of singlet molecular oxygen. Flavonols were also oxidized by radicals formed during degradation of lauroyl peroxide. These results indicate that flavonols can function as antioxidants in biological systems by terminating radical chain reactions and removing singlet molecular oxygen. A pharmacological function of flavonols, decrease of the increased permeability and fragility of capillary, was discussed in relation to their antioxidative functions.     

The effect of temperature on lysis of human erythrocytes by palmitic acid]

An analysis of kinetic curves of erythrocyte hemolysis induced by palmitic acid has shown the existence of some stages of this process. The activation energy of hemolysis, as determined by the temperature dependence of the hemolysis rate constant, was 210 +/- 30 kJ/mol. It was shown by the method of stepwise thermoinactivation of erythrocytes proteins that at temperature of 49 degrees C which corresponded to the framework protein spectrin denaturation temperature, the erythrocyte membrane stability sharply decreased. On the contrary, changes of the cell shape induced by the hyperosmotic medium (0.5 M sucrose) inhibited the palmitic acid-induced erythrocytes hemolysis.     

Antihemolytic effect of various benzodiazepines on human erythrocytes]

The benzodiazepines, medazepam, chlorodiazepoxyde, and oxazepam, act antihemolytically just as chloropromazine in hypotonic NaCl solution on human erythrocytes. The effect depends on concentration and is biphasic with the exception of oxazepam. The maximal antihemolytic concentrations are for medazepam 9.1-10-5 M, for chlorodiazepoxyde 4.6-10-4 M and, respectively, 9.1-10-4 M for diazepam and oxazepam. No linear correlation exists between the concentration at maximal hemolytic protection and the lipid solubility.     

Oxidative modification of human ceruloplasmin by peroxyl radicals

Ceruloplasmin (CP), the blue oxidase present in all vertebrates, is the major copper-containing protein of plasma. We investigated oxidative modification of human CP by peroxyl radicals generated in a solution containing 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). When CP was incubated with AAPH, the aggregation of proteins was increased in a time- and dose-dependent manner. Incubation of CP with AAPH resulted in a loss of ferroxidase activity. Superoxide dismutase and catalase did not protect the aggregation of CP, whereas hydroxyl radical scavengers such as ethanol and mannitol protected the protein aggregation. The aggregation of proteins was significantly inhibited by the copper chelators, diethyldithiocarbamate and penicillamine. Exposure of CP to AAPH led to the release of copper ions from the enzyme and the generation of protein carbonyl derivatives. Subsequently, when the amino acid composition of CP reacted with AAPH was analyzed, cysteine, tryptophan, methionine, histidine, tyrosine, and lysine residues were particularly sensitive.     

Protective effect of selenium compounds in the freeze preservation of erythrocytes]

The influence of sodium selenite, selenomethionine and sodium selenite with tocopherols in combination on the survival of cryopreserved erythrocytes was investigated. Percent hemolysis is marked decreased after a three-hour incubation of the whole blood with addition of selenomethionine as well as sodium selenite with tocopherole in combination before cryopreservation. The protective effect is attributed to the antioxydative and membrane stabilizing effectiveness of selenium and tocopherol.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Electron microscope study on human ceruloplasmin.

Electron microscopy of human ceruloplasmin (CP) molecules revealed a few distinctive types of particle images. Analysis of these images allows to propose a tentative model for CP: six "subunits" (which we call domains) not much different in size are arranged with 32 point group pseudosymmetry. The determination of the number of polypeptides arising at the spontaneous specific proteolytic fragmentation of CP and their molecular weights conform with this assumption. The electrophoretic studies of the CP samples prepared both with and without potent proteolytic inhibitor, PMSF, revealed that CP is a single-chain protein with molecular weight of 130 000. Isolated and stored without PMSF the polypeptide chain of CP undergoes specific proteolytic cleavage which results in the appearance of polypeptides with molecular weights of 16 000, 48 000, and 64 000. The latter two polypeptides degradate to about two- and three-fold decreased molecular weights fragments, respectively. Therefore, the single polypeptide chain of CP contains at least five peptide bonds which are particularly susceptible to proteolytic attack and which connect six principal segments of the chain. The hydrolysis of these bonds results in liberation of the six fragments which were integrated in the enzymatically active globule of CP.     

Lack of effect of synthetic atrial natriuretic factor on rubidium uptake by human erythrocytes

The effect of synthetic atrial natriuretic factor on the ouabain-sensitive and the furosemide-sensitive rubidium uptake by human erythrocytes has been studied. This peptide with potent diuretic and natriuretic effects did not affect any rubidium uptake system at concentrations of 10(-7) and 10(-9) M. These results do not support that the natriuretic effect is based on the inhibition of active transport systems in the renal tubules.