Saccharated Iron Oxide Research Articles

Some metabolic aspects of tolerance to bacterial endotoxin.

Berry, L. Joe (Bryn Mawr College, Bryn Mawr, Pa.), and Dorothy S. Smythe. Some metabolic aspects of tolerance to bacterial endotoxin. J. Bacteriol. 90:970-977. 1965.-The tolerance to bacterial endotoxins which is produced in mice given a series of daily injections of heat-killed Salmonella typhimurium failed to occur when actinomycin D was administered with the heat-killed cells. Neither ethionine nor 2-thiouracil, when given with endotoxin, altered the development of tolerance. An injection of endotoxin, actinomycin D, or ethionine lowered the activity of the liver enzyme tryptophan pyrrolase more significantly at either 4 or 17 hr postinjection in normal mice than in tolerant mice. Similarly, an injection of either saccharated iron oxide or Thorotrast lowered liver tryptophan pyrrolase activity more extensively in normal than in tolerant animals. Activation of the reticuloendothelial system (RES) of tolerant mice, as determined by an accelerated rate of carbon clearance from the blood, was observed, but this was prevented by the appropriate dose of actinomycin D. Similar results were obtained when saccharated iron oxide, rather than endotoxin, was used to activate the RES, but these animals were not resistant to endotoxin and their tryptophan pyrrolase was normally diminished after an injection of endotoxin. Thus, RES activation may occur without tolerance developing. A more nearly normal level of enzyme activity appears to be characteristic of the tolerant state.

ELECTRON MICROSCOPY OF ABSORPTION OF TRACER MATERIALS BY TOAD URINARY BLADDER EPITHELIUM.

The absorption of Thorotrast and saccharated iron oxide by the epithelium of the toad urinary bladder was studied by electron microscopy. Whether the toads were hydrated, dehydrated, or given Pitressin, no significant differences in transport of colloidal particles by epithelial cells were observed. This implies that these physiological factors had little effect on the transport of the tracer particles. Tracer particles were encountered in three types of epithelial cells which line the bladder lumen, but most frequently in the mitochondria-rich cells. Tracer materials were incorporated into the cytoplasm of epithelial cells after being adsorbed to the coating layer covering the luminal surface of the cells. In the intermediate stage (1 to 3 hours after introducing tracer) particles were present in small vesicles, tubules, and multivesicular bodies. In the later stages (up to 65 hours), the particles were more commonly seen to be densely packed within large membrane-bounded bodies which were often found near the Golgi region. These large bodies probably were formed by the fusion of small vesicles. Irrespective of the stages of absorption, no particles were found in the intercellular spaces or in the submucosa. Particles apparently did not penetrate the intercellular spaces of the epithelium beyond the level of the tight junction.

STUDIES ON THE MECHANISM OF EXPERIMENTAL PROTEINURIA INDUCED BY RENIN.

Renin-induced proteinuria in the rat was investigated, with special emphasis on the relationship between the enzymatic activity and the proteinuric effect of renin. The dependence of the proteinuric effect on the enzymatic activity was shown by using (a) renin preparations of widely varying purity and (b) chemically modified "active" and "inactive" renin derivatives. Angiotensin II, the pressor product of the enzymatic action of renin, also produced significant proteinuria. Adrenalectomy abolished the proteinuria induced by renin. Proteinuria, however, occurred as a result of pretreatment with DOCA, or aldosterone, or without treatment, 7 to 8 weeks after adrenalectomy. Electron microscopic studies of the kidney at the time of maximal proteinuria showed focal flattening and fusion of epithelial foot processes, as well as swelling and vesicle formation in endothelial and epithelial cells of the glomeruli. Studies with intravenously injected saccharated iron oxide showed increased permeability of the glomerular capillary basement membrane to these particles. These changes were transient and were not seen 24 hours after renin injection. Adrenalectomy prevented these changes. It is concluded that renin, acting through angiotensin, causes glomerular capillary damage with increased permeability of these structures to protein and resultant proteinuria. The adrenal glands participate in a permissive role in this phenomenon.

DISTRIBUTION OF INTRAVENOUSLY ADMINISTERED SACCHARATED IRON OXIDE IN THE POPLITEAL LYMPH NODE OF THE RABBIT.

LABELLED thoracic duct lymphocytes transfused into host rats have been shown to migrate across the walls of the post-capillary venules of the lymph node, through the cortex into the medullary lymph sinuses1,2. In the present investigation, the fate of non-cellular particles in the lymph node of rabbits has been examined using intravenous saccharated iron oxide (‘Ferrivenin’, Benger) as the marker.

ON THE FORMATION OF HAEMOSIDERIN AND ITS RELATION TO FERRITIN. II. A RADIOISOTOPIC STUDY.

In vivo biochemical studies on rabbits have shown that, with the intravenous injection of iron dextran, the rate of liver ferritin formation limits the uptake of iron by this organ. When ferritin production reaches 25‐30 mg. Fe per day, increased rates of iron dextran injection result only in increased serum concentrations of the compound. Regardless of the rate of injection, haemosiderin (insoluble iron) forms at a rate of approximately one‐tenth that of ferritin (soluble iron) until the total liver ferritin iron reaches 150‐200 mg. Therefore, when iron dextran is injected, the rate of haemosiderin formation is limited by the liver ferritin concentration. When this concentration reaches the ‘critical’level of 150‐200 mg. of iron and loading is continued, a process begins whereby ferritin production gradually decreases until the ferritin content of the liver reaches a maximum. Simultaneously the rate of haemosiderin production increases until all subsequent iron deposition is reflected in increased haemosiderin content (Shoden and Sturgeon, 1962a).Histochemical studies have shown that there is no appreciable deposition of stainable iron in the Kupffer cells of the liver when iron dextran is administered intravenously. When saccharated iron oxide is given, however, there is immediate and marked deposition of stainable iron in the Kupffer cells, even when relatively small amounts of iron are injected at slow rates. In contrast to the Kupffer cell insoluble iron, deposition of histochemically demonstrable iron in the parenchymal cells does not begin until the total liver ferritin iron reaches approximately the 150‐200 mg. level. At this level, regardless of the type of iron compound given, faint bluish areas or vacuoles, some of which may contain discrete blue granules, appear in the parenchymal cells. As the iron load is increased beyond this point, the number and size of the parenchymal cell granules increase. When iron dextran is given, the appearance of the blue granules is paralleled by a marked increase in the water‐insoluble iron fraction. Extraction with normal saline of small liver cubes, preceding the fixation and staining, shows that ferritin is removed, but not the haemosiderin granules (Shoden and Sturgeon, 1962b).From the above observations, the hypothesis was advanced that liver ferritin is a precursor of the haemosiderin granules found in the parenchymal liver cells (Shoden and Sturgeon, 1961). The present study was designed to test this hypothesis. It was planned to establish in the two iron storage ‘compartments’(ferritin and parenchymal haemosiderin) known specific activities (concentrations) of radioactive iron. Then, to study the pathway taken by new iron, non‐radioactive iron in form of iron dextran was administered. The sequence in the dilution of radioactive iron in the two compartments, and the shift of radioactivity between them,

Modification of cellular proliferation of the reticuloendothelial system in the rabbit

The intravenous injection of complete Freund's adjuvant results in a proliferation of RE cells, particularly in the liver, spleen and lungs. If saccharated iron oxide is administered prior to or with the adjuvant the proliferation of RE cells in the liver and spleen is impaired. This impairment persists despite repeated doses of adjuvant. Some of the cells which have phagocytized SIO respond to the adjuvant by proliferation. As the cells divide the iron is distributed to daughter cells. In the lung where there is no evidence of SIO in the RE cells, there is no alteration of the cellular reaction to the subsequent injection of adjuvant. The absence of iron in the newly proliferated histiocytes and macrophages of the lungs fails to support the concept that these cells are derived from the liver and spleen.

An electron microscopic study of the passage of colloidal particles from the blood vessels of the ciliary processes and choroid plexus of the rabbit.

The thinnest areas of the capillaries of the choroid plexus and ciliary processes in the eye of the rabbit are characterized by the presence of fenestrae. When various colloidal particles opaque to the electron beam (thorotrast, gold sol, and saccharated iron oxide) were injected into the blood stream, none were found in fenestrae or in areas that might suggest their having passed through fenestrae. The passage of marker particles from the lumen to the surrounding connective tissue does take place on occasion in the areas of thicker walls in the capillaries and venules rather than in the attenuated and fenestrated endothelial walls. The pathway taken by these markers may be either through the cytoplasm of the endothelial cells via membrane-bounded vesicles and vacuoles or through the intercellular spaces of the vessels. An altered aqueous humor (cloudy and plasmoid) was produced by endotoxin injection or by making a draining fistula in rabbit cornea. Both methods gave rise to the same changes in the blood vessels of the ciliary processes. Under such conditions of inflammation the passage of colloidal particles through the thicker walls of the capillaries and venules was greatly increased and occurred primarily as an intercellular passage between the endothelial cells. The attenuated and fenestrated areas of the endothelium of the small capillaries remained unchanged with no particles passing through them. These results on the altered vessels of the ciliary processes parallel those of Majno and Palade (26) on the rat cremaster muscle.

Cell division and phagocytic activity in liver reticulo-endothelial cells.

SummaryRadioautographs of liver sections from mice given tritiated thymidine were used to identify the cells which were preparing for division and colloidal saccharated iron oxide was used to identify the active phagocytic cells. In livers of mice whose reticulo-endothelial system was stimulated by estradiol, it was established that the cells preparing for division and those which had recently divided were actively phagocytic. In livers of mice whose reticulo-endothelial system had been “blockaded” with saccharated iron oxide, it was established that the cells which had phagocytized colloid were able to divide in the process of recovery from “blockade.” No evidence was found for a stem cell which proliferates and differentiates to provide the active phagocytic population.

The transport of particles across the walls of small blood vessels

Colloidal particles of thorium dioxide and saccharated iron oxide and molecules of ferritin enter the vesicles of the endothelium of the perfused rat heart. The first two substances have been shown to pass within the vesicles through the cells of the endothelium into the perivascular spaces.

Studies on the cornea. I. The fine structure of the rabbit cornea and the uptake and transport of colloidal particles by the cornea in vivo.

Physiological studies have demonstrated that ions, as well as large molecules such as hemoglobin or fluorescein, can diffuse across and within the cornea. Most of the substrates for corneal metabolism are obtained from aqueous humor filling the anterior chamber. In order to receive its nutrients and in order to maintain its normal conditions of hydration, the avascular cornea must transport relatively large amounts of solute and solvent across the cellular layers which cover this structure. It has been suggested in the past that there may be a morphological basis for the transport of large amounts of solvents and solutes by cells by the mechanism of pinocytosis. The use of electron-opaque markers to study fluid movements at the electron microscope magnification level was described by Wissig (29). The present study describes the fine structure of the normal rabbit cornea and the pathways of transport of colloidal particles by the cornea in vivo. Rabbit corneas were exposed in vivo to suspensions of saccharated iron oxide, thorium dioxide, or ferritin by injection of the material into the anterior chamber. In other experiments thorium dioxide or saccharated iron oxide was injected into the corneal stroma, producing a small bleb. Particles presented at the aqueous humor surface of the rabbit corneal endothelium are first attached to the cell surface and then pinocytosed. It appears that the particles are carried around the terminal bar by an intracellular pathway involving the pinocytosis of the particles and their subsequent transport in vesicles to the lateral cell margin basal to the terminal bar. Particles introduced at the basal surface of the endothelium (via blebs in the corneal stroma) are apparently carried through the endothelial cells in membrane-bounded vesicles without appearing in the intercellular space. There appears to be free diffusion of these particles through Descemet's membrane and the corneal stroma. The stromal cells take up large quantities of the particles when blebs are injected into the stroma.

Studies on the cornea. II. The uptake and transport of colloidal particles by the living rabbit cornea in vitro.

In vitro studies of the transport of colloidal particles by the cornea were carried out on intact corneas of adult rabbits in a chamber described by Donn, Maurice, and Mills (2) in which the epithelial or the endothelial surface of the cornea was exposed to thorium dioxide or saccharated iron oxide under various conditions. These studies confirmed the results of previous work in vivo and allowed modification of the experimental conditions. Particles are pinocytosed at the apical surface of the corneal endothelium and carried around the terminal bar in membrane-bounded vesicles. Basal to the terminal bar these vesicles fuse with the lateral cell margin and their contents are released into the intercellular space, in which they appear to be carried by a one-way flow down to Descemet's membrane and the corneal stroma. Indications that the endothelial transport is an active process are presented by the different pathways of transport into or out of the corneal stroma, as well as by the approximately 70 per cent reduction in transport activity at low temperatures.

The Use of FE59and CR51for Estimating Red Cell Production and Destruction: An Interpretive Review

Abstract In surveying some of the limitations of studies with Cr51 and Fe59 my purpose has been not to deny their usefulness but to put them in perspective. These technics have made possible many extensions of the fundamental understanding of red cell physiology and iron metabolism. They will continue to be valuable experimental tools. It is hoped that if some of the limitations of isotope technics are considered, the non-experimental use of these isotopes will be confined to situations in which otherwise unavailable information of diagnostic or therapeutic importance can be obtained. Unfortunately isotope technics are used when more conventional means would be adequate or even preferable. An extreme example is the suggestion24 that repeated Fe59 turnover studies might be used to determine the total dose of parenteral iron (as Imferon or saccharated iron oxide) to be given in iron deficiency anemia, pointing out that in so doing the possibility of iatrogenic hemochromatosis could be avoided. The usual calculations for determining dose25 however are not only safer but more accurate. The use of Fe59 and Cr51 entails some risk, the main hazards being hepatitis, with the use of donor plasma or cells, and the possibility of untoward effects from radiation. An estimate of the risk of hepatitis can be gained from its incidence after transfusion. The radiation hazard is more difficult to assess. Leukemia has occurred after large doses of radiation but the extent of the radiation hazard is unknown from the much smaller doses of radiation employed in the usual isotopes studies. Certainly the risk is not such as to preclude the use of isotopes to obtain information essential for diagnosis. However, when such information can be obtained by other means or when results cannot be adequately interpreted, the use of isotopes in clinical medicine appears unwarranted. In considering the use of isotopes in the doubtful case, the dose of radiation to be delivered should not be thought of as an isolated event but rather as adding to a total radiation dose, which as shown by the British survey28 may be appreciable.

Sensitivity of mice to bacterial lipopolysaccharide following alteration of activity of the reticulo-endothelial system.

It has generally been assumed that resistance of animals to the pyrogenic and lethal effects of bacterial endotoxin is largely dependent on the phagocytic capacity of the reticulo-endothelial system. Thus it has been shown that stimulation of the reticulo-endothelial system with non-lethal doses of bacterial lipopolysaccharide or endotoxin results in protection of the animals against a subsequent lethal dose of endotoxin1. Again, numerous workers have demonstrated that a ‘blockade’ of the reticulo-endothelial system by colloidal particles, for example saccharated iron oxide, thorotrast and trypan blue, increases the sensitivity of the animals to the lethal effects of these substances2. Recently, however, it has been shown that in certain circumstances it is possible for mice to possess an increased phagocytic activity, as measured by their ability to clear colloidal carbon, and yet be highly sensitive to the effects of endotoxin. This can be seen during the course of an intracellular infection3, or following the administration of zymosan4. The apparent contradiction of these results has yet to be resolved, and it is not unlikely that such resolution may lead to an elucidation of the mode of action of the endotoxins.

The transport and distribution of colloidal iron and its relation to the ultrastructure of the cell

Rabbits were given saccharated iron oxide intravenously. The iron accumulated in the cytoplasm of a variety of cells, but particularly in the Kupffer's cells and the cells lining the sinusoids of the spleen. The distribution of iron-containing particles in the cytoplasm and nucleus is described. Pinocytosis did not appear to be important in the uptake of the colloidal iron. The passage of these particles across the nuclear membranes was not related to the nuclear pores. It is suggested that fairly large molecules may be diffusible from the cytoplasm through the nuclear envelope, and the system of nuclear pores may be limited to the exchange of ribonucleoprotein between the nucleus and cytoplasm.

Intranuclear aggregates of ferritin in liver cells of mice treated with saccharated iron oxide. Their possible relation to nuclear protein synthesis.

Several months following parenteral injections of saccharated iron oxide into DBA/2J mice, granules rich in iron were found in nuclei of scattered parenchymal liver cells as well as in the cytoplasm. As seen in the light microscope, the intranuclear granules were brown; most of them measured between 0.5 micro and 1 micro in cross-section. They gave positive Prussian blue tests, and were not selectively stainable with pyronine. Electron micrographs of the granules showed closely packed aggregates of ferritin molecules, occasionally in paracrystalline order. The intranuclear collections were often surrounded by bands of material of moderate opacity. Scattered ferritin molecules and collections of such molecules were also present in the cytoplasm of many liver cells, but there seemed to be no quantitative relationship between intranuclear and cytoplasmic ferritin. Liver cells from untreated control mice failed to reveal intranuclear deposits of ferritin. Although the site of origin of the intranuclear aggregates of ferritin is unknown, the findings suggest the possibility that under suitable circumstances ferritin synthesis may take place within nuclei of liver cells-perhaps induced by the presence of colloidal iron.

Experimental retinitis proliferans in the rabbit.

Introduction In 1931, Chuta Oguchi 1 produced experimental retinitis proliferans in the eyes of rabbits by intravitreal injection of autogenous blood. Ashton 2 recently succeeded in producing vascular proliferation into the vitreous body of kittens by raising them in a chamber containing highly oxygenated air. In this study, we induced fibrous and vascular tissue proliferation into the vitreous of rabbits by injecting autogenous blood or saccharated iron oxide. Materials and Methods One-tenth of a milliliter of blood was withdrawn from the ear artery of 18 rabbits and injected into the vitreous of the same animal. The right eyes were used for the experiment whereas left eyes were injected with saline or sucrose solution for control. After 2 weeks to 1 year clinical observation, the animals were killed and the eyes were enucleated, fixed in formalin solution, and embedded in paraffin. The sections were stained with Prussian blue 3,4 and counter-stained

EFFECTS OF BACTERIAL ENDOTOXINS ON METABOLISM

There exists an inverse proportionality between number of heat-killed cells of Salmonella typhimurium injected intraperitoneally into mice and the quantity of urinary nitrogen the animals excrete during a 17 hour period following the subcutaneous administration of 2 units of ACTH. This relationship has been developed into an assay for bacterial endotoxin. Mice immunized against S. typhimurium require 10 to 20 times the number of cells needed by control animals to suppress urinary nitrogen excretion to the same extent. Intravenous saccharated iron oxide sensitizes animals so that fewer heat-killed salmonellae can be detected. Heat-killed cells of Staphylococcus aureus are without effect in the assay. Several lipopolysaccharides derived from Gram-negative bacteria are effective in preventing the rise of urinary nitrogen excreted in response to ACTH and the amount required, compared to the LD(50), is in the same ratio for all of them. Citrated mouse serum partially inactivates the endotoxin during in vitro incubation for 1 hour at 37 degrees C. while normal serum does not. Dichloroisoproterenol protects mice against the lethal effects of lipopolysaccharide and it lowers its effectiveness in the assay. The minimum amount of endotoxin reliably determined by the test is 0.25 microg. of an E. coli preparation that was given intravenously in mice in which the reticuloendothelial system had been "blocked" with saccharated iron oxide.

The use of intramuscular iron in the prophylaxis of the iron-deficiency anemia of prematurity.

Iron-deficiency anemia is still the most prevalent anemia occurring in infancy, despite modern knowledge concerning iron metabolism and the causation of this deficiency state. The premature infant is extremely vulnerable to iron deficiency because his total body hemoglobin available for use is smaller and his growth rate is usually greater than that of the full-term infant. Recently an iron-dextran complex has been found to be effective in supplying the human organism parenterally with large quantities of iron for hemoglobin synthesis. The preparation 1 is a third as toxic in mice as saccharated iron oxide solutions. It has been used widely for the treatment of iron deficiency of children 2-4 and adults. 5,6 It was felt worth while to evaluate its use in the prevention of the late (iron-deficiency) anemia of prematurity. This report concerns observations of two groups of infants born prematurely who were examined hematologically until one year of

Proliferation of the reticuloendothelial system in the liver

The rate of removal from blood of intravenously injected colloidal carbon was used to measure the functional capacity of liver phagocytes. The carbon disappearance rate constant was initially reduced and gradually returned to normal following injection into mice of saccharated iron oxide or colloidal carbon. This index of phagocytic activity increased above normal after zymosan, estradiol or bacterial endotoxin administration. To determine whether these increases were associated with cell proliferation, liver DNA synthesis was measured. Incorporation of radioactive phosphate into DNA of mouse livers rose markedly after administration of these substances. To characterize the cell type responsible for the large increases in DNA synthesis, radioautographs of liver sections were prepared from mice previously given tritium-labeled thymidine. The label was rarely seen in parenchymal cells, whereas many littoral cells were labeled.

Comparison of intravenous saccharated iron oxide and whole blood in treatment of hypochromic anemia of pregnancy

1.1. A study is presented comparing two commonly used methods of treatment of hypochromic anemia in the pregnant woman.2.2. A general discussion of the metabolism of iron is outlined.3.3. A brief résumé of the present methods of iron replacement in iron-deficiency anemia is presented.4.4. Alternating cases, 50 patients were treated with transfusions of whole blood, and 54 patients with a total of 279 injections treated by administration of intravenous saccharated iron oxide. Hematologic response was comparable but varied with the initial severity of the anemia, being more pronounced in those patients with a more severe iron depletion.5.5. It is noted that the comparison between these two methods of therapy is not a strictly fair one since the actual amount of iron given per patient is less when the patient is treated by transfusion than that given by intravenous elemental iron replacement.6.6. The occurrence of possible side effects and their management are presented.7.7. In view of the possibility of transfusion reaction and the difficulties surrounding the procurement and administration of whole blood, the method of choice of iron replacement in patients refractory to oral therapy was felt to be intravenous iron in divided doses.