Free Ferritin Research Articles

Risk factors for coronary artery calcification in patients with end-stage renal disease

To explore risk factors for coronary artery calcification (CAC) inpatients with end-stage renal disease (ESRD). A total of 53 ESRD patients undergoing regular hemodialysis (3 times a week) from August 2014 to March 2015 in the Fourth Hospital of Hebei Medical University were enrolled in the study. The patients were divided into the negative control group (13 cases) and three positive groups (11 mild calcification cases, 12 moderate calcification cases and 17 severe calcification cases) based on coronary artery calcification score (CACs). Clinical data of all patients at study entry were collected. Arterial blood samples were also collected at the start of the first hemodialysis (HD) session of the week to measure the levels of serum albumin, uric acid, calcium (Ca), phosphorus (P), magnesium, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), C reactive protein (CRP), beta-2 microglobulin (β2-MG), free parathyroid hormone (iPTH), alkaline phosphatase, fibrinogen, hemoglobin (HGB) and ferritin. Meanwhile, levels of blood pH were detected after collecting pre- and post-HD blood samples to calculate ΔpH (post-HD pH subtracted pre-HD pH). Logistic regression analysis was performed to analyze the correlation of CACs with clinical data and previously-reported blood biochemical indicators, followed by analysis of the incidence of CAC and influential factors in ESRD patients. Severity of CAC was positively correlated with age (r=0.269), HD duration (r=0.341), serum calcium (r=0.358), serum phosphorus (r=0.186) and pre-HD pH (r=0.275), but negatively correlated with serum albumin (r=-0.192) and ΔpH (r=-0.302), all P<0.05. Logistic regression analysis revealed that age, HD duration, serum phosphorus level and ΔpH were independent risk factors for CAC in ESRD patients (P<0.05). In CAC positive groups, CAC was predominantly involved in the left anterior descending artery (P<0.05, P<0.01 and P<0.01 in mild, moderate and severe calcification group, respectively). CAC in ESRD patients seems to be affected by multiple factors, such as age, HD duration, serum phosphorus level and ΔpH. Moreover, ΔpH affects CAC mainly by pre-HD pH. Furthermore, left anterior descending artery is predominantly affected by CAC in ESRD patients.

SRAGE is associated with low waist circumference and Hb levels in NAFLD

Abstract Advanced glycation end products (AGEs) and the receptor RAGE interaction is involved in nonalcoholic fatty liver disease (NAFLD). Although exogenously administered soluble RAGE (sRAGE) has been shown to block the harmful effects of AGEs in animal models, there is still controversy about the role of sRAGE in humans. We examined here which anthropometric, metabolic and clinical variables were independent correlates of sRAGE levels in NAFLD patients. The study involved 77 biopsy-proven, unmedictaed NAFLD patients (44 male and 33 female) with a mean age of 43.4±13.0 years old. We examined which anthropometric, metabolic and clinical variables, including liver steatosis and fibrosis markers, are independently associated with serum levels of sRAGE. Mean serum levels of sRAGE were 710.7±290.2 pg/mL. Univariate analysis revealed that waist circumference (inversely), hemoglobin (inversely), number of white blood cells (inversely), total-bilirubin (inversely), free fatty acid (inversely), ferritin (inversely), and HbA1c (inversely) were significantly correlated with serum levels of sRAGE. In multiple stepwise regression analysis, waist circumference (p&lt;0.01, inversely) and hemoglobin (p&lt;0.01, inversely) were independently associated with serum levels of sRAGE (R2=0.176). The present study reveals that low serum levels of sRAGE are independently associated with waist circumference and hemoglobin in patients with NAFLD.

Acute erythroid leukaemias

Note Criteria for diagnosis of acute erythroid leukaemia. Erythroleukaemia Historically, AML with erythroid features has been designated M6 by the French-American-British (FAB) group. The FAB criteria for M6 diagnosis are: bone marrow erythroblasts equal to or greater than 50% and blasts equal to or greater than 30% of the non-erythroid cells. The World Health Organization (WHO) have recently recommended that the requisite blast percentage for a diagnosis of AML be 20% or greater, and this includes erythroid leukaemia. AML M6 would equate to the new WHO definition of erythroleukaemia (erythroid/ myeloid). If there are less than 20% blasts, the diagnosis is refractory anaemia with an excess of blasts (RAEB). Trilineage dysplasia is common but is not a prerequisite for diagnosis. Erythroid dysplasia may manifest as binuclearity, nucleo-cytoplasmic asynchrony and vacuolation. The morphological appearance of the myeloblasts is not characteristic and they may contain Auer rods. Myeloperoxidase and Sudan black B stains may be positive in the myeloblasts. The iron stain may show ringed sideroblasts and PAS may be positive in the erythroid precursors in a block or diffuse pattern. Pure erythoid leukaemia In addition to the typical AML M6 (erythroleukaemia), there is a second subtype of acute erythoid leukaemia where there is a neoplastic proliferation of immature cells entirely committed to the erythroid series (>80% of marrow cells) without evidence of a myeloid component. This is termed pure erythroid leukaemia by the WHO. Morphology is characterised by medium sized erythroblasts with fine nuclear chromatin, distinct nucleoli and deeply basophilic cytoplasm that often have vacuoles. Occasionally the blasts can resemble acute lymphoblastic leukaemia, distinction can be made by immunophenotyping. The erythroid nature of the blasts can be shown by electron microscopy demonstrating free ferritin particles. The blasts are negative for Sudan black B and myeloperoxidase (MPO), but positive for PAS in a block pattern.

Physiologische Einflussfaktoren im Alpinen Skirennlauf

Summary Introduction In the past several studies tried to identify parameters predicting success in elite alpine skiing. Most of these studies used cross-sectional designs. Therefore, the aim of this study was to determine influencing factors on alpine skiing performance in both a cross-sectional and a longitudinal design. Furthermore, the correlation between parameters of anthropometry, clinical chemistry, echocardiography and exercise testing and the success in alpine skiing (represented by FIS points) were investigated. Method We analyzed the parameters of 143 elite athletes (69 male, 74 female, age 18.8±3.9 years, BMI 23.7±2.2 kg*m -2 ) of the German National Ski Team during a term of 5 years. Results Athletes with higher fat free mass (FFM) and ferritin values were more successful than athletes with lower levels. In accordance, there were high correlation coefficients between FIS points and FFM (♀: r=-0.53, ♂: r=-0.73; all p Conclusion FFM and ferritin seems to be predicting factors for success in alpine ski racing. Therefore, training should be focused on the increase of FFM and iron status should be monitored carefully.

Iron Distribution in the Liver and Duodenum during Seasonal Iron Overload in Svalbard Reindeer

Seasonal iron overload in Svalbard reindeer was studied by light and electron microscopy and by X-ray microanalysis. The hepatic iron overload was of two types. The first type was characterized by massive siderosis of both parenchymal and non-parenchymal cells caused by a diet very rich in iron but low in energy and protein. Hepatocytes contained a moderate amount of free ferritin particles in the cytosol together with numerous siderosomes. This pattern is similar to that seen in primary haemochromatosis and thalassaemia. Kupffer cells contained large quantities of cytosolic ferritin, siderosomes and lysosomes with disintegrating red blood cells as seen in thalassaemia. The second type was characterized by massive non-parenchymal siderosis caused by an energy- and protein-poor diet with normal iron concentration. Hepatocytes contained little cytosolic ferritin and few siderosomes, but there were abundant electron-dense bodies without iron (i.e., autophagosomes). Kupffer cells were as described above. Ferritin was also present within the duodenal mucosa of these animals, located within enterocytes and lamina propria macrophages, as well as in the extracellular space and capillary and lacteal lumina. Ferritin was also present in the acinar cells of submucosal Brunner's glands. Changes consistent with exchange of ferritin particles between different cell types were observed. The role of ferritin as a possible iron transporter in this condition is discussed.

Protease-Resistant Human Prion Protein and Ferritin Are Cotransported across Caco-2 Epithelial Cells: Implications for Species Barrier in Prion Uptake from the Intestine

Foodborne transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob disease (CJD) has affected over 100 individuals, and probably millions of others have been exposed to BSE-contaminated food substances. Despite these obvious public health concerns, surprisingly little is known about the mechanism by which PrP-scrapie (PrP(Sc)), the most reliable surrogate marker of infection in BSE-contaminated food, crosses the human intestinal epithelial cell barrier. Here we show that digestive enzyme (DE) treatment of sporadic CJD brain homogenate generates a C-terminal fragment similar to the proteinase K-resistant PrP(Sc) core of 27-30 kDa implicated in prion disease transmission and pathogenesis. Notably, DE treatment results in a PrP(Sc)-protein complex that is avidly transcytosed in vesicular structures across an in vitro model of the human intestinal epithelial cell barrier, regardless of the amount of endogenous PrP(C) expression. Unexpectedly, PrP(Sc) is cotransported with ferritin, a prominent component of the DE-treated PrP(Sc)-protein complex. The transport of PrP(Sc)-ferritin is sensitive to low temperature, brefeldin-A, and nocodazole treatment and is inhibited by excess free ferritin, implicating a receptor- or transporter-mediated pathway. Because ferritin shares considerable homology across species, these data suggest that PrP(Sc)-associated proteins, in particular ferritin, may facilitate PrP(Sc) uptake in the intestine from distant species, leading to a carrier state in humans.

Storage of Iron in Bone Marrow Plasma Cells

The physiological site of iron storage in the human bone marrow is the macrophage (orthotopic iron store). Iron-storing plasma cells, as bone marrow indicators of iron overload, and/or chronic alcoholism represent a pathological (heterotopic) iron store. They were demonstrated by light and electron microscopy in 23 iron-overloaded patients: transfusional siderosis (n = 8), genetic haemochromatosis (GH; n = 11), iron-loading anaemias (n = 4) and in patients with bone marow impairment due to chronic alcoholism (n = 6). The pattern of iron storage in bone marrow plasma cells was monitored during iron loading in patients receiving continuous transfusional therapy (n = 7) and also upon reversal of iron overload by repeated phlebotomies in GH (n = 9) and in iron-loading anaemias (n = 4). The first ultrastructural evidence of iron storage in plasma cells was the appearance of free ferritin molecules in the cytosol, thus indicating endogenous ferritin synthesis. Accumulation of iron proceeded with the additional formation of ferritin- and haemosiderin-containing lysosomes (siderosomes) in these cells. Lysosomal ferritin partially showed a paracrystalline arrray. The siderosomes originated from autophagocytosis of cytoplasmic areas containing free ferritin molecules. In addition, the formation of ferritin-containing vesicles in the proximity of the Golgi apparatus was observed. The heterotopic iron store of bone marrow plasma cells was less readily mobilizable by blood-letting than the orthotopic store of macrophages.

Mucosal IgA response to rectally administered antigen formulated in IgA-coated liposomes

Ferritin, a soluble model antigen, was used to test whether liposomes can provide an effective delivery vehicle for mucosal immunization via the rectum, and whether the local coloniclrectal secretory immune response to antigen in liposomes can be enhanced by immunoadjuvants. The coloniclrectal IgA response to liposomal ferritin was significantly enhanced over the response to free ferritin but only when cholera toxin (CT) was present as adjuvant. The presence of IgA on the liposome surface increased the uptake of liposomes into Peyer's patch mucosa, and the local rectallcolonic immune response to ferritin about 5-fold over uncoated liposomes. These results show that (1) liposomes coadministered with immunoadjuvants can be used for mucosal immunization via the rectum. (2) Cholera toxin is an effective immunoadjuvant in the rectallcolonic mucosa. (3) IgA can enhance the local secretory immune response to antigen in liposomes, apparently by increasing liposome uptake via M cells.

Pattern of Iron Storage in the Rat Heart Following Iron Overloading with Trimethylhexanoyl-Ferrocene

Female Wistar rats with slight iron deficiency anemia were kept on a diet containing 0.5% trimethylhexanoyl (TMH)-ferrocene for up to 79 weeks. In the state of iron deficiency, the heart was free of light-microscopically detectable iron. After 7 weeks of the TMH-ferrocene diet, the first iron-positive granules appeared in perivascular macrophages. Further oral administration caused a progression of iron deposition in these cells, visible in the form of a granular staining but also as a diffuse iron staining of the cytoplasm. Accordingly, at the electron-microscopical level, the iron was stored partly as free ferritin molecules in the cytosol, and partly in lysosomes in the form of ferritin and/or hemosiderin. After 11 weeks, further iron-positive cells with relatively small dark-blue granules were found in the vicinity of capillaries, which could be identified as fibrocytes by means of electron microscopy. In addition, slight iron deposition occurred in the endothelial cells of the cardiac capillaries, likewise mainly in the form of small, uniform siderosomes. The myocytes showed no product of Perls' Prussian blue reaction during the whole period of investigation. From the 11th week onwards, discrete ferritin molecules were detected electron microscopically within lysosomes of these cells. Their amount increased slowly with progression of the TMH-ferrocene feeding period. Free ferritin molecules could be observed in the cytosol of fibrocytes, endothelial cells and myocytes in only very slight concentrations, whilst they were more plentiful in macrophages. In hereditary hemochromatosis and posttransfusional siderosis, the iron is found predominantly in myocytes and appears to cause cell damage, whilst this is not the case in experimental iron overload in rats.

Iron overload of the bone marrow by trimethylhexanoyl-ferrocene in rats.

Iron-deficient female Wistar rats were fed a diet which contained 0.5% 3,5,5-trimethylhexanoyl (TMH)-ferrocene over a 57-week period. The state of iron deficiency was characterized by means of the absence of stainable iron in the bone marrow. After the first days on the iron-enriched diet, ferritin-containing siderosomes were found, in numerous erythroblasts up to orthochromatic normoblasts and in reticulocytes, i.e. the dispensed iron was used for haemoglobin synthesis. After 1 week the first macrophages showed a positive Perls' Prussian blue reaction. In the cytoplasm they stored the iron in the form of free ferritin molecules and lysosomally as aggregated ferritin and/or haemosiderin. The iron loading of the macrophages increased in both of the storage qualities proportionally with duration of the feeding period and reached a maximum after 38 weeks. Final stages showed extremely iron-loaded macrophages with high concentrations of free ferritin molecules and large siderosomes, partially flowing together to still greater units. Iron deposits within endothelial cells of bone marrow sinusoids can be observed for the first time after 4 weeks. In these cells the iron is stored as ferritin in siderosomes of relatively small and uniform size; free ferritin molecules in the cytosol were of only slight concentration. The TMH-ferrocene model of iron overload shows in the bone marrow: (1) an unimpeded utilization of the iron component for erythropoiesis, (2) development of excessive iron overload of the bone marrow in macrophages and endothelial cells of sinusoids and (3) a pattern of distribution of iron as seen in secondary haemochromatosis.

Transport of siderotic Kupffer cells to the lung and bronchial excretion of iron in experimental iron-overload.

In 3,5,5-trimethylhexanoylferrocene-induced iron overload of rats, three different types of iron-loaded macrophages and derivatives thereof were found in the lungs. On the basis of their localization and of their pattern of iron load it was possible to distinguish: (1) Resident macrophages, showing an alveolar localization and a moderate iron content represented by lysosomal ferritin and haemosiderin. (2) Liver-derived macrophages and giant cells, as well as fragments of them. They showed an exclusive localization in capillaries and alveolar septa, and high concentrations of free ferritin molecules in addition to polymorphous ferritin- and haemosiderin-containing siderosomes. (3) Monocyte-derived intravascular pulmonary macrophages. Initially, they contained iron only as lysosomal aggregates of ferritin and haemosiderin, as a result of phagocytosis of liver-derived macrophageal cell fragments. Later in iron overload, they also showed free ferritin molecules in the cytosol and fused intrapulmonarily to giant cells. The resident as well as the liver-derived siderotic pulmonary macrophages provide a way for iron excretion through the airways.

Free radicals in the pathogenesis of kwashiorkor.

The skeletal spectre of the marasmic child clearly results from a lack of intake of the bulk nutrients. It can be easily and consistently reproduced in any convenient species of experimental animal. In stark contrast, the other major form of malnutrition, kwashiorkor, has only once been convincingly reproduced in experimental animals. Coward & Whitehead (1972) produced oedema, fatty liver, skin lesions, hair discoloration and friability, hypoproteinaemia and mental changes in baboons by feeding the animals exactly the same diets that children with kwashiorkor received in Uganda. The authors demonstrated that the syndrome could be reproduced experimentally but made no attempt to discover the factor or factors in the diet responsible for the disease. In other studies the individual features of kwashiorkor had been singly and inconsistently reproduced in animals using a whole variety of, often extreme, experimental manipulations. The conditions necessary to give a ‘model’ for kwashiorkor oedema, for instance, are quite different from those used to give experimental fatty liver or hair discoloration. Why should the kwashiorkor syndrome be so difficult to reproduce experimentally I Why have the applied programmes designed to prevent kwashiorkor been so uniformly unsuccessful? The obvious first answer to be considered is that all the hypotheses that have been proposed, tested experimentally and used as the conceptual framework for the applied programmes, are incorrect. Certainly the extant hypotheses of protein deficiency (Williams, I 935)’ niacin deficiency (Gillman & Gillman, 195 I), antidiurectic-hormone-like action of free ferritin (Srikantia, 1958), dysadaptation to protein deficient stress (Gopalan, 1968), hormonal dysadaptation (Whitehead, 1979), or aflatoxin intoxication (Hendrickse, 1984), do not adequately explain more than a few of the features of kwashiorkor listed in Table I. These features must all be reconciled in any unifying hypothesis. Recently we have proposed that kwashiorkor results from an imbalance between the production of free radicals and their safe disposal (Golden, 1985). It is the purpose of the present paper to examine the evidence for such a hypothesis. The hypothesis, illustrated in Fig. I, states that in kwashiorkor, various noxae are imposed on the subject. These noxae produce free-radical-mediated lipid peroxides and toxic carbonyls. Under normal metabolic and nutritional

A comparison between concentrations of free erythrocyte protoporphyrin and serum ferritin during development of iron deficiency by phlebotomy in polycythaemia vera patients

A comparison between concentrations of free erythrocyte protoporphyrin and serum ferritin during development of iron deficiency by phlebotomy in polycythaemia vera patients

Enhancement of antigenic potency in vitro and immunogenicity in vivo by coupling the antigen to anti-immunoglobulin.

We have examined the effect of targeting an antigen to the immune system, by covalently coupling it to anti-immunoglobulin (Ig), on its efficacy for T cell stimulation in vitro and its immunogenicity for antibody production in vivo. In vitro, we compared the potency (for stimulation of a ferritin-specific T cell line) of free ferritin, ferritin coupled to goat antimouse IgM (heavy (H) chain specific), ferritin coupled to anti-IgG (H and light (L) chain specific), or ferritin coupled to anti-IgA (H chain specific), as well as a mixture of free ferritin plus goat anti-IgG. The ferritin coupled to anti-IgM or to anti-IgG (H + L), which could bind to surface Ig of B cells, stimulated T cell proliferation at concentrations of ferritin at least 10-fold lower than those required for the other forms of the antigen over the entire time course of the response, with 1000 rad-irradiated spleen cells as presenting cells. Because the goat antibodies were all of the same IgG isotype and coupling ratio, the failure of goat anti-IgA to enhance potency served as a control to exclude Fc receptor binding as the mechanism. The effect was not due to the nonspecific activation of B cells to become more efficient antigen-presenting cells, because mixtures of ferritin plus anti-IgG (H + L) had no effect, and the anti-IgG coupled to ferritin did not enhance presentation of myoglobin to a myoglobin-specific T cell line. The enhanced presentation of ferritin conjugated to goat anti-IgG (H + L) or to anti-IgM was sensitive to radiation doses greater than 2000 R, and was effective at less than one-tenth the number of spleen cells, consistent with the predominance of B cells as antigen-presenting cells for this form of the antigen rather than macrophages and dendritic cells only. When B cells and accessory cells were purified from T-depleted spleen cells, only the B cell preparation but not the accessory cell population manifested enhanced presentation of ferritin coupled to anti-IgG compared with free ferritin, and it was radiosensitive. Finally, allogeneic B cells could not mediate the enhancement in the presence of syngeneic splenic accessory cells (SAC); therefore, the enhancement was not due to shedding of immune complexes from B cells and subsequent presentation by SAC. We conclude that targeting the antigen to B cells as presenting cells greatly enhances its efficacy in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaemia and iron status of symptom-free adult males in northern Nigeria.

The prevalence of anaemia and iron deficiency have been evaluated in 276 symptom-free male inhabitants of northern Nigeria. One hundred and eighteen were classified as non-elite and 158 as elite. Twenty-six of the elite had a high consumption of alcohol, and have been considered as a separate sub-group. Measurements included haemoglobin (Hb), serum iron (SI), total iron-binding capacity (TIBC), free erythrocyte protoporphyrin (FEP) and serum ferritin (SF). The prevalence of anaemia (Hb less than 13.0 g dl-1) was considerably higher in non-elite (35.6%) than in 132 non-alcohol drinking elites (7.0%), while none of the alcohol drinkers was anaemic. The geometric mean SF was 54.6 micrograms l-1 in the non-elites, 100.6 micrograms l-1 in the non-drinking elites and 149.3 micrograms l-1 in the alcohol-drinkers. Iron deficiency [at least two of transferrin saturation (TS) less than or equal to 20%, FEP greater than or equal to 70 micrograms dl-1 and SF less than or equal to 30 micrograms l-1] was diagnosed in 15.3% of the non-elites and in only 1.3% of the elites. Iron deficiency in the non-elites probably resulted from poor availability of iron from the local staples guinea corn (Sorghum spp.) and millet (Pennisetum spp.), with chronic haemorrhage due to helminthic infestations contributing in some. Evidence of iron overload (TS greater than 90%, SF greater than 600 micrograms l-1 was found in two of the drinkers, one of whom had a history of prolonged consumption of burukutu, a beer locally brewed from cereals.

Preparation of monoclonal antibody-ferritin conjugates of high specific activity.

125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.

A simplified method of producing biologically active monomeric ferritin-insulin for use as a high resolution ultrastructural marker for occupied insulin receptors.

A new, simplified, and highly reproducible method for preparing biologically active monomeric ferritin-insulin that can be used as a high-resolution ultrastructural marker for occupied insulin receptors is described. The ferritin-insulin conjugate was stable, and contained negligible free insulin and low levels of free ferritin. The hormone conjugate behaved identically to native insulin in both binding assays and biological activity (glucose oxidation) assays. The immunological and biological activities of the insulin in the conjugate were equal. Biogel 1.5 column purification was used to yield a product monomeric in ferritin.

Postembedding staining of Brunner's gland with lectin-ferritin conjugates.

Postembedding staining of intracellular carbohydrates of rat Brunner's gland cells embedded in Epon and acrylamide was carried out with Ricinus communis agglutinin-ferritin, concanavalin A-ferritin, and wheat germ agglutinin-ferritin conjugates. Th Golgi vacuoles and mucous granules were stained with these conjugates. In each staining, the tissues embedded in acrylamide were stained more strongly than those embedded in Epon. The staining intensity of wheat germ agglutinin-ferritin was the strongest among the three conjugates and the staining intensity of Ricinus communis agglutinin-ferritin was stronger than that of concanavalin A-ferritin in both embedding methods. Free ferritin showed almost no binding to these structures and staining with the conjugates was inhibited by the addition of appropriate competitive sugars to the staining solutions. Osmium-postfixed tissues were not stained well with the conjugates. Washing of the sections with bovine serum albumin solution after staining was an essential step in the present method to reduce the nonspecific adsorption of the conjugates. The present method was very simple and had good reproducibility.

Autophagy of ferritin incorporated into the cytosol of Hela cells by liposomes.

Ferritin was trapped in negatively charged liposomes consisting of phosphatidyl choline and phosphatidyl serine in a molar ratio of 9:1. Unilamellar vesicles were prepared by ultrasonic treatment. HeLa cells were incubated with these liposomes for 2h and chased for 0, 2, 14 and 20 h. The migration pathway of ferritin in the cells was followed by quantitative electron microscopy. Ferritin was detected exclusively in cytosol and lysosomes. Evidence is presented that free ferritin in cytosol enters lysosomes.

Hormone receptor topology and dynamics: Morphological analysis using ferritin-labeled epidermal growth factor

Previous studies using a biologically active 1:1 conjugate of EGF and ferritin (F-EGF) have traced the binding and internalization of the hormone molecules. In the present report, we develop ultrastructural criteria for identification of the F-EGF.receptor complex, and, thereby, enable utilization of the F-EGF as an indirect marker to localize the receptor for this peptide hormone. The ferritin cores of bound F-EGF are situated 4-6 nm from the extracellular surface of the membrane. When cells were incubated for up to 30 min at 37 degrees C, this characteristic spatial relationship was observed in all uptake stages (surface clustering, endocytosis, and incorporation into multivesicular bodies), indicating that the hormone.receptor complex remains intact through these steps. However, when incubation was continued for periods sufficient to allow hormone degradation (30-60 min), pools of free ferritin were observed in lysosomes. In the presence of various amine inhibitors of hormone degradation, internalization and multivesicular body incorporation proceeded, but hormone.receptor degradation was blocked as evidenced by preservation of the ferritin-membrane relationship; i.e., no pools of free ferritin were seen after 60 min. These data provide morphological support for the hypothesis that down-regulation of surface receptors involves internalization of intact hormone.receptor complexes. In addition, we have developed a method for viewing the surface of intact cells en face, allowing closer scrutiny of the clustering of F-EGF.receptor complexes in the plane of the membrane prior to internalization. The particles in the F-EGF clusters observed by this method are spaced at 12 nm center-to-center, serving to set upper limits on the packing dimensions of the EGF.receptor complex.