A comparative study of serum proteins ability to scavenge active oxygen species: O2-. and OCl-

The ability of serum proteins (albumin, immunoglobulin G) and protein antioxidants (ceruloplasmin, superoxide dismutase and transferrin) to react with O2-. and OCl-, was studied. The interaction between serum proteins and OCl- was shown to be non-specific. Ceruloplasmin is the most effective OCl- trapping protein, and it reacts with O2-. with a considerable efficiency. Therefore, ceruloplasmin is supposed to be the main scavenger of toxic oxygen species generated by stimulated neutrophils.     

Erythrocyte oxidative damage by myeloperoxidase. The protective action of serum proteins

Influence of main serum proteins (albumin, immunoglobulin G) and proteins-antioxidants (ceruloplasmin, transferrin, superoxide dismutase) on the oxidative damage of erythrocytes by myeloperoxidase and hypochlorite was investigated. The proteins were determined to act as protectors and decrease the degree of hemoglobin oxidation, ceruloplasmin and albumin possessing the highest antioxidant activity.     

Serum protein degradation by hypochlorite

The structural integrity of serum proteins: albumin, immunoglobulin G, transferrin, ceruloplasmin and superoxide dismutase, and the functional activity of the latter two enzymes after their interaction with hypochlorite were studied. It was shown that the interaction between the proteins and hypochlorite resulted in protein injury and degradation of their native structure. In the case of ceruloplasmin and transferrin, a practically complete protein "dissipation" occurred, the albumin and superoxide dismutase structures being injured in a lesser degree. The inactivation of ceruloplasmin was slower than that of superoxide dismutase. The protein degradation by hypochlorite seems to be the main factor restricting the ability of the proteins to act as antiinflammatory drugs.     

Sertoli cells synthesize and secrete a ceruloplasmin-like protein

Sertoli cells synthesize and secrete a ceruloplasmin-like protein (testicular ceruloplasmin) that is immunologically similar to serum ceruloplasmin. Rat serum ceruloplasmin was purified and an antiserum was produced to the purified protein which specifically immunoprecipitated a 130,000 dalton protein from rat serum. This ceruloplasmin antiserum was found to also immunoprecipitate a 130,000 dalton protein synthesized and secreted by Sertoli cells. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was required during the immunoprecipitation procedure to prevent the proteolytic degradation of testicular ceruloplasmin. Immunoprecipitation of proteins secreted by Sertoli cells with an antiserum to rat serum proteins was found to precipitate two proteins, testicular ceruloplasmin and testicular transferrin.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998     

Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid

Lung epithelial lining fluid (ELF) is a thin layer of plasma ultrafiltrate and locally secreted substances that may provide antioxidant protection and serve as a "front-line" defense for the lower respiratory tract epithelium. To characterize the antioxidant properties of ELF, young, healthy, nonsmoking volunteers underwent bronchoalveolar lavage with determination of ELF volumes and ELF proteins. ELF (greater than 0.4 ml) is a potent inhibitor of lipid peroxidation as measured by malondialdehyde (MDA) production in an in vitro iron-dependent assay system. Two serum proteins, transferrin and ceruloplasmin, were quantitated in ELF and found to be potent inhibitors of lipid peroxidation. Other ELF components, including vitamin E, vitamin C, and albumin, did not function as antioxidants in this system. Several experimental observations suggest that ELF transferrin was more important than ceruloplasmin in inhibiting lipid peroxidation: 1) ELF concentrations of transferrin were 20-fold higher than those for ceruloplasmin; 2) ELF antioxidant activity was abolished by preincubation with Fe3+; 3) ELF antioxidant activity was minimally affected by sodium azide, which is known to inhibit ceruloplasmin ferroxidase activity; and 4) ELF ceruloplasmin ferroxidase activity was virtually nondetectable. ELF possesses a significant antioxidant activity that may be important in vivo in protecting the lung from oxidant injury.     

Oxidative stress in autism: increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin--the antioxidant proteins

Autism is a neurological disorder of childhood with poorly understood etiology and pathology. We compared lipid peroxidation status in the plasma of children with autism, and their developmentally normal non-autistic siblings by quantifying the levels of malonyldialdehyde, an end product of fatty acid oxidation. Lipid peroxidation was found to be elevated in autism indicating that oxidative stress is increased in this disease. Levels of major antioxidant proteins namely, transferrin (iron-binding protein) and ceruloplasmin (copper-binding protein) in the serum, were significantly reduced in autistic children as compared to their developmentally normal non-autistic siblings. A striking correlation was observed between reduced levels of these proteins and loss of previously acquired language skills in children with autism. These results indicate altered regulation of transferrin and ceruloplasmin in autistic children who lose acquired language skills. It is suggested that such changes may lead to abnormal iron and copper metabolism in autism, and that increased oxidative stress may have pathological role in autism.     

Age-related changes in plasma proteins of analbuminemic rats

A mutant strain, Nagase analbuminemia rats (NAR), was established from Sprague-Dawley rats. Age-related changes in plasma proteins of NAR were investigated to obtain information of their abnormalities of protein metabolism. The total protein concentration in the serum of NAR of various ages was almost the same as that of normal rats of the same age. The albumin level of NAR was less than 0.05 mg/ml at all ages examined. The concentrations of serum alpha 1-antitrypsin, alpha-X protein, alpha 2-macroglobulin, transferrin, ceruloplasmin, IgG, IgA and IgM were higher in NAR than in normal rats except for the perinatal stage, but alpha 1-acid glycoprotein level in NAR was normal. The serum transferrin and ceruloplasmin levels were especially high in female adult NAR. The plasma fibrinogen concentration was also increased in NAR. These findings indicate that the normal total serum protein level of NAR was maintained by increase in the globulin concentration.     

Blood protein polymorphism in the one-humped camel (Camelus dromedarius) in the Sudan

The blood protein polymorphism of serum albumin, haptoglobin, transferrin, ceruloplasmin and haemoglobin have been studied in 135 samples from one-humped Arabian camel (Camelus dromedarius) of the Sudan by starch gel electrophoresis. Only the serum albumin and haptoglobin systems exhibited polymorphism with the estimated frequencies of 0.0222, 0.2227 and 0.7773 for Alb^v, Hp¹Hp⁰respectively. The frequency of Hp was 0.0325. No electrophoretic variant was observed at transferrin, ceruloplasmin and haemoglobin loci in the camel. The activity of the ceruloplasmin of the camel sera was weak.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

Role of ceruloplasmin, transferrin and lipid peroxidation in bacterial infections of the CNS

Ceruloplasmin and transferrin blood serum levels were measured during bacterial infection of the CNS. A decrease in ceruloplasmin level and a gradual increase in transferrin level were observed on the 5th day of the disease. The minimum ceruloplasmin level was observed simultaneously with the maximum MDA and GOT levels. There was a close correlation between the time course of ceruloplasmin, transferrin, MDA and GOT changes and the clinical condition of the patient.     

The effect of synovial fluid proteins in the degradation of hyaluronic acid induced by ascorbic acid

The degradation of hyaluronic acid induced by ascorbic acid and the effect of synovial fluid proteins, such as ceruloplasmin, transferrin, and albumin, were investigated on the basis of the elution volume and the molecular weight of hyaluronic acid using high-performance gel permeation chromatography. Hyaluronic acid was degraded to less than one-third of the original molecular weight in the range of the physiological concentrations of ascorbic acid. Synovial fluid proteins protected against the ascorbate-dependent degradation of hyaluronic acid at their physiological concentrations. It is suggested that the inhibitory activity of ceruloplasmin mainly depends on the ferroxidase activity and that of transferrin is probably due to iron binding property.     

Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.     

Linkage relationship among loci determining genetic variants of serum and milk proteins in cattle

Special methods allowing usage of inadequate pedigrees were employed to examine linkage among the milk protein loci alpha s1-casein, beta-casein, chi-casein and beta-lactoglobulin, and the loci for serum amylase, ceruloplasmin and transferrin. Linkage was evident between the alpha s1-casein and beta-casein loci, the alpha s1 and chi-casein loci, the beta-casein and chi-casein loci, also amylase and transferrin loci. Recombination fractions for these corresponding combinations were 0.00; 0.00; 0.00 and 0.30. Weak linkage (recombination fraction being 0.46; 0.44 and 0.42) between the beta-lactoglobulin and beta-casein loci, the amylase and ceruloplasmin loci, ceruloplasmin and transferrin loci is supposed.     

Glycosyl phosphatidylinositol-anchored ceruloplasmin is expressed by rat Sertoli cells and is concentrated in detergent-insoluble membrane fractions.

The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule.     

Relationships between the copper and iron systems in hemodialysis patients and variables affecting these systems

The copper-binding protein ceruloplasmin oxidizes ferrous iron to ferric iron, an action that is critical for the binding of iron to transferrin in plasma. Ceruloplasmin, in common with ferritin and transferrin, is an acute-phase protein that is altered by inflammation. We sought to identify interrelationships between the copper and iron systems by measuring copper, ceruloplasmin, ferroxidase, ferritin, transferrin, iron, and iron-binding capacity in a group of hemodialysis patients. We looked for evidence of inflammation and free-radical injury by assaying for protein carbonyl groups, protein pyrrolation, di-tyrosine, and advanced oxidation protein products. Our findings were compatible with an active inflammatory state that affected both iron and copper metabolism. Transferrin levels were low, whereas ceruloplasmin levels were elevated compared to normal. Copper concentration was increased proportional to ceruloplasmin. Several variables including ceruloplasmin and transferrin were observed to correlate significantly with the level of pyrrolated protein. The data suggest that posttranslational modification of circulating proteins may affect their structural, enzymatic, and ligand-binding properties. Abnormalities in copper metabolism and their influence on iron handling in renal failure are complex and will require additional study before their importance can be defined.     

Purification and characterization of testicular transferrin secreted by rat Sertoli cells.

Sertoli cells synthesize and secrete a transferrin-like protein (testicular transferrin) [Skinner & Griswold (1980) J. Biol. Chem. 255, 1923-1925]. The purpose of the present study was to purify and characterize testicular transferrin and to compare it with serum transferrin. Testicular transferrin was obtained from the medium of cultured rat Sertoli cells, whereas serum transferrin was obtained from rat serum. Both proteins were purified with the use of phenyl-Sepharose hydrophobic chromatography and transferrin immunoaffinity chromatography. The purified proteins were shown to have similar molecular masses (75 000 Da) and amino acid compositions. The pattern of tryptic peptides from testicular and serum transferrin were found to be essentially the same when analysed by reverse-phase high-pressure liquid chromatography. The carbohydrate composition of both transferrins was determined by several colorimetric assays and g.l.c. Testicular transferrin, isolated from cell culture medium, had increased amounts of glucose, galactose and glucosamine. Serum transferrin that was incubated with cell culture medium also had a large amount of associated glucose. The results show that testicular transferrin and serum transferrin are structurally very similar and are possibly products of the same gene expressed in two different tissues, the testis and liver. However, the amount of carbohydrate associated with these two proteins is different.     

Serum protein polymorphisms in four populations of Afghanistan.

Gene frequencies of the serum proteins third component of complement (C3) transferrin (Tf), haptoglobin (Hp), group specific component (Gc), serum cholinesterase (E1), alpha1-antitrypsin (Pi), beta2-glycoprotein I (Bg), and ceruloplasmin (Cp) in the Tajiks, Pushtoons, Hazaras, and Usbeks in Afghanistan were reported. Rare variants were observed in the C3, Tf, and Pi systems.     

Interrelations between ceruloplasmin and Fe status during human pregnancy

It is well established that Fe and ceruloplasmin interact in animals and in in vitro models. However, Fe-mediated regulation of ceruloplasmin has never been investigated in humans. In an observational study, 53 pregnant women aged 19–39 yr (29.8±0.7 yr, mean ± SEM) were recruited at the Aberdeen Antenatal Clinic, Aberdeen Maternity Hospital, UK. All requirements for local ethical committees were followed. Venous blood samples were taken from each woman at 34 wk gestation for measurement of Fe status and ceruloplasmin. Various parameters were used to test for Fe status. The most sensitive one appeared to be soluble transferrin receptor, which increased with parity. In the population studied, there was no relationship between hemoglobin or ferritin and serum ceruloplasmin. However, using soluble transferrin receptor (sTfR) levels, we were able to demonstrate an inverse linear relationship (r=0.37, p=0.021, n=41) between Fe status and ceruloplasmin. Fe supplementation, number of previous pregnancies, and smoking habits did not affect this relationship. Our data support in vitro results showing regulation of ceruloplasmin by Fe and also suggest that the interactions between Fe and ceruloplasmin should be considered when Fe supplementation is given.