The effect of estrogens on serum ferritin levels in duck

Serum and tissue ferritin content is measured in duck by a RIA method before and after treatment with estrogens, as well as serum ferritin in laying and non-laying hen. Both serum ferritin and tissue ferritin decrease after treatment with estrogens, while serum iron increases. A relationship between serum ferritin and iron stores in duck is shown.     

Ultrasensitive detection of ferritin in human serum by Western blotting based on quantum dots-labeled avidin-biotin system

Various methods have been applied for serum ferritin detection, however, these methods still have some limitations. Over the last few years, quantum dots (QDs) have become very attractive for immunoassays because of their enormous potentials in ultrasensitive analysis. In this study, a Western blotting method combined with QDs‐labeled avidin–biotin system for detecting human serum ferritin was described. Meanwhile, the traditional diaminobenzidine (DAB)–horseradish peroxidase (HRP) method had been compared with the method. The linearity of this QDs‐based Western blotting method was from 0.27 to 1.1 ng, and the quantification limit was 0.27 ng, the sensitivity was up to pictogram values. Real serum samples such as hepatoma, thalassemia patient and normal individual sera were analyzed, the analysis results demonstrated that there was significant difference in the concentrations of ferritin between patients and normal individual serum. Furthermore, the recovery of ferritin from the serum samples of patients ranged from 98.15 to 119.67%, and the RSD (relative standard deviation) ranged from 8.73 to 11.61%, the repeatabilities were well within the acceptable range, which revealed that this method is a stable and reproducible method for detecting serum ferritin and have potential application prospect in clinical laboratory of serum ferritin detection.     

Turnover and tissue uptake of rabbit ferritin from rabbit plasma

Using a two-site immunoradiometric assay for rabbit liver ferritin normal NZW rabbits were found to have very low plasma ferritin concentrations (less than 4 micrograms/l). Purified preparations of rabbit liver and kidney ferritin were labelled with 125I and injected into rabbits. Clearance from plasma was extremely rapid with an initial half-life of 1-2 min as measured by immunoprecipitation of labelled ferritin. The rate of clearance was unaffected by the labelling procedure and by the method of ferritin purification. Autoradiography and organ uptake studies showed that 125I-rabbit liver ferritin was removed mainly by liver reticuloendothelial cells, although on a weight basis, spleen had the greatest radioactivity. These studies indicate that rabbit ferritin released into the circulation is promptly cleared by the RES.     

On the non-ferritin depot iron fraction in the rat liver

Liver depot iron can be divided into two fractions: ferritin iron and non-ferritin depot iron. Three methods intended to measure the non-ferritin depot iron in the rat liver were compared using livers of normal rats and livers of rats loaded with iron by transfusion of erythrocytes. Liver depot iron varied between 75 and 850 μg Fe/g liver. Non-ferritin depot iron, measured as the iron fraction sedimentable at 10 000 × g, was in the range 4–22 μg Fe/g liver. This fraction did contain ferritin. When measured as the difference between total liver depot iron and heat-stable iron (ferritin iron), the range was 10–270 μg Fe/g liver but this fraction also includes some ferritin iron.The values derived with both methods were linearly proportional to the total liver depot iron values.Non-ferritin depot iron, when measured as the difference between total liver depot iron and total ferritin iron, ranged from 0 to 190 μg Fe/g liver. In this last method no ferritin iron is included. This method provides the best estimate of the non-ferritin depot iron fraction. The concentrations obtained with this method were not always linearly proportional to the total liver depot iron concentration. Intravenous injection of rat liver ferritin resulted in a rapid accumulation of ferritin iron in the liver, together with an increase of the non-ferritin depot iron fraction from 18 μg Fe/g liver to 55 μg Ge/g liver. This confirms a relationship between ferritin catabolism and the non-ferritin depot iron fraction.     

The antigenic structure of ferritin. 1. Isolation of ferritin subunits and their immunochemical characteristics]

A method for the isolation of monomers of ferritin subunits has been developed. The procedure comprises dissociation of ferritin by treatment with thioglycolic acid in the presence of phosphate ions and subsequent gel-permeation chromatography. Ferritin and a number of its structural analogues (apoferritin, carboxymethylated ferritin, H- and L-subunits of ferritin) have been immunochemically characterized. The immunoreactivity of ferritin is shown to vary along with the degree of denaturation. Isolation of monomers of H- and L-subunits results in appearance of new antigenic sites. These "hidden" antigenic determinants are presumed to be localized in the regions of intersubunit contacts and intracapsular surface of the ferritin molecule and are responsible for the differences in immunochemical properties of its H- and L-subunits.     

Absorption of x-radiation by single crystals of proteins containing labile metal components: the determination of the number of iron atoms within the central core of ferritin

The number of metal atoms contained within a displaceable inorganic component of a metalloprotein was determined by considering X-ray absorption by single crystal samples of holo- and apo-proteins. Since this method is non-destructive, it can be used to determine the number of metal atoms associated with the molecules forming the crystal actually used for X-ray diffraction data collection and subsequent structure solution. The method has been applied to the iron storage protein ferritin, isolated from horse spleen, to give a reliable estimate of the average iron content of the ferritin molecules within the crystal. This value, of around 2000 iron atoms per molecule is consistent with that found for a typical ferritin preparation in solution and suggests non-selectivity of the crystallisation process for ferritin in terms of molecular iron content.     

单抗在人铁蛋白免疫亲和纯化中的应用

抗人铁蛋白单抗６Ｄ６和Ａ－ｈＦ－Ｃ与肝型铁蛋白和心型铁蛋白的反应性有所不同。６Ｄ６对两种铁蛋白的反应性相似;而Ａ－ｈＦ－Ｃ单抗与肝型铁蛋白的反应性较强。我们将６Ｄ６和Ａ－ｈＦ－Ｃ分别制成了亲和凝胶,用来纯化人肝脏和心脏粗抽提物中的铁蛋白。此法具有操作简便,产率和产品纯度高等优点。     

Isolation, purification and characterization of spleen ferritin of Gallus domesticus L

The ferritin from the spleen of the chickens has been isolated by a method of salt fractionation and by a pH change followed by purification in sephadex G-200. 2. The identification of the protein was carried out by acrylamide gel electrophoresis showing a single band. 3. The characterization of ferritin has been made by determination of molecular weight, amino acids analysis and the number of iron atoms (4520) which bound the ferritin. 4. The ferritin from the spleen of chicken is compared with the ferritin from the liver of pigeon.     

Musca domestica hemolymph ferritin

We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 ± 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae, decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage. © 1996 Wiley-Liss, Inc.     

Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm.

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.     

Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry.

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiff's bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.     

抗人铁蛋白单克隆抗体的制备及其在肝癌病人血清铁蛋白测定中的应用

铁蛋白是一种肿瘤相关蛋白质。我们从人肝癌组织中分离纯化了铁蛋白,并筛选到一株抗该铁蛋白的单克隆抗体杂交瘤细胞株（６Ｄ６）,它针对铁蛋白上的构象决定簇,并对人源铁蛋白高度专一。然后我们用此单抗建立了测定铁蛋白浓度的夹心ＥＬＩＳＡ法,并对方法的灵敏度,就确度及特异性作了研究。本法用于测定不同血清标本的结果表明：肝癌病人血清中的铁蛋白浓度明显高于正常人,这可能对临床诊断会有使用价值。     

Quantitative determination of ferritin by electroimmunoassay

A method for the quantitative determination of tissue ferritin protein is described. It is based on the electroimmunoassay of Laurell [Laurell, C. B. (1966) Anal. Biochem.15, 45–52] and uses the iron content of ferritin for its identification. It measures as little as 0.1 μg of ferritin protein, requires only a few milligrams of tissue, and is rapidly performed.     

A simple assay for determination of iron release from ferritin in neuroblastoma cells.

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.     

Annotations on the measurement of liver ferritin, especially in the rat.

A radial immunodiffusion method (RID) to measure liver ferritin protein has been reevaluated. The RID has been compared with an electroimmunoassay (EIA) and with an enzyme-linked immunoassay (ELISA). The RID and ELISA provided similar results; the RID measured more ferritin protein in liver homogenates as compared with the EIA. After incubation of liver homogenate with deoxycholate (30 mg/ml) a slight increase of the ferritin concentration is detectable.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

Uptake of Ferritin-labeled Antibodies by Tetrahymena1

Experiments with Tetrahymena thermophila using ferritin probes revealed that these cells take up ferritin conjugated to antibodies (not directed against Tetrahymena) much more readily than they do ferritin or cationized ferritin. The massive and rapid uptake of antibody-ferritin offers certain advantages for studies of endocytosis and membrane flow in cells of this type, and the method may be applicable to other types of protozoa as well.     

Inflammatory macrophages in the dog contain high amounts of intravesicular ferritin and are associated with pouches of connective tissue fibers

We have studied the subcellular distribution of ferritin in inflammatory macrophages present in regional lymph nodes from dogs subjected to a pulmonary inflammatory reaction. The inflammatory reaction was induced by intrabronchial instillation of calcium tungstate (CaWO4), a water-insoluble powder. Ferritin was identified by electron microscopy, and its electron density was enhanced by the use of a modified Perls method. From day 14 on after the CaWO4 deposition, tungsten-positive lymph node macrophages showed a massive accumulation of ferritin. Most of the ferritin was stored in membrane-bounded vesicles that showed heterogeneous concentrations of the protein. A significant complement of ferritin was also detected in the cytoplasmic ground substance of phagocytes. The cell surface of the ferritin-rich, tungsten-positive macrophages showed deep infoldings that encompassed small pockets of connective tissue fibers. These features were not observed in control samples or in lymph nodes from dogs subjected to CaWO4-induced inflammation for periods shorter than 1 week. Our data indicate that inflammatory macrophages greatly increase their content of ferritin macrophages greatly increased their content of ferritin and that ferritin is stored predominantly by a membrane-bounded vesicular compartment. This is in contrast with suggestions that the inflammation-induced increase in macrophage iron is restricted to the labile pool of iron and it does not involve the iron bound to ferritin molecules. Our observation of nodules of connective-tissue fibers in intimate topographical association with ferritin-rich macrophages may indicate that the increase in intracellular ferritin in the macrophage is in some way related to the secretion of factors by the phagocyte that will stimulate fibrillogenesis by neighboring fibroblats.     

A simple two-step labeling procedure for ultrastructural localization of cell surface anionic sites

We propose a new method for ultrastructural localization of cell surface anionic sites. The method consists of sequential interaction of aldehyde-fixed cells with a polycationic reagent, poly-L-lysine (PL), followed by secondary interaction with a negatively charged marker, ferritin. By use of PL of low molecular weight (4000) on aldehyde-pre-fixed red blood cells and macrophages, the reaction resulted in binding of ferritin particles to cell surface anionic sites with a density distribution resembling that of cationized ferritin (CF). The density of the attached ferritin molecules increased in direct correlation with the MW of PL used. The primary PL interaction can be carried out at low pH (less than 2), thus restricting the labeling mainly to membrane-bound sialyl residues.     

Dendritic translocation of the rat ferritin H chain mRNA

To elucidate the mechanism regulating the selective transport of mRNAs to synaptic sites, we compared the synaptosomal mRNAs with those from the forebrain using the differential display method. The ferritin H chain mRNA was found to be highly enriched in the synaptosomes. In situ hybridization for the ferritin H chain mRNA in the cultured dissociated neurons and in the hippocampal brain slices demonstrated its existence in the dendritic region. These data clearly indicate the selective translocation of the ferritin H chain mRNA into the dendrites and suggested the local expression of ferritin at the synapse.