G. E. Cartwright

The role of ceruloplasmin in iron metabolism

The importance of ceruloplasmin in iron metabolism was studied in swine made hypoceruloplasminemic by copper deprivation. When the plasma ceruloplasmin level fell below 1% of normal, cell-to-plasma iron flow became sufficiently impaired to cause hypoferremia, even though total body iron stores were normal. When ceruloplasmin was administered to such animals, plasma iron increased immediately and continued to rise at a rate proportional to the logarithm of the ceruloplasmin dose. The administration of inorganic copper induced increases in plasma iron only after ceruloplasmin appeared in the circulation. Thus, ceruloplasmin appeared to be essential to the normal movement of iron from cells to plasma. Studies designed to define the mechanism of action of ceruloplasmin were based on the in vitro observation that ceruloplasmin behaves as an enzyme (ferroxidase) that catalyzes oxidation of ferrous iron. Retention of injected ferrous iron in the plasma of ceruloplasmin-deficient swine was significantly less than that of ferric iron, reflecting impaired transferrin iron binding. Rat ceruloplasmin, which has little ferroxidase activity, was much less effective than porcine or human ceruloplasmin in inducing increases in plasma iron. These observations suggest that ceruloplasmin acts by virtue of its ferroxidase activity. Eight patients with Wilsons disease were evaluated in order to investigate iron metabolism in a disorder characterized by reduced ceruloplasmin levels. Evidence of iron deficiency was found in six of these, and in five of the six, plasma ceruloplasmin was less than 5% of normal. In comparison, the two patients without evidence of iron deficiency had ceruloplasmin levels of 11 and 18% of normal. It is suggested that iron deficiency tends to occur in those patients with Wilsons disease who have the severest degrees of hypoceruloplasminemia, possibly because of defective transfer of iron from intestinal mucosal cells to plasma.

Iron metabolism in copper-deficient swine

The way in which iron is handled by the duodenal mucosa, the reticuloendothelial system, the hepatic parenchymal cell, and the normoblast was investigated in copper-deficient swine.Copper-deficient swine failed to absorb dietary iron at the normal rate. Increased amounts of stainable iron were observed in fixed sections of duodenum from such animals. When (59)iron was administered orally, the mucosa of copper-deficient animals extracted iron from the duodenal lumen at the normal rate, but the subsequent transfer to plasma was impaired.When intramuscular iron supplements were given to copper-deficient pigs, increased amounts of iron were found in the reticuloendothelial system, the hepatic parenchymal cells, and in normoblasts (sideroblasts). Hypoferremia was observed in the early stages of copper deficiency, even though iron stores were normal or increased. When red cells that were damaged by prolonged storage were administered, the reticuloendothelial system failed to extract and transfer the erythrocyte iron to the plasma at the normal rate. Administration of copper to copper-deficient animals with normal iron stores resulted in a prompt increase in the plasma iron. The observed abnormalities in iron metabolism are best explained by an impaired ability of the duodenal mucosa, the reticuloendothelial system, and the hepatic parenchymal cell to release iron to the plasma. It is suggested that copper is essential to the normal release of iron from these tissues. This concept is compatible with the suggestion made by others that the transfer of iron from tissues to plasma requires the enzymatic oxidation of ferrous iron, and that the plasma copper protein, ceruloplasmin, is the enzyme (ferroxidase) which catalyzes the reaction. Because excessive amounts of iron were found in normoblasts, it is suggested that an additional defect in iron metabolism affects these cells and plays a major role in the development of anemia. As a result of the proposed defect, iron cannot be incorporated into hemoglobin and, instead, accumulates as nonhemoglobin iron.

STUDIES ON COPPER METABOLISM. IX. THE TRANSPORTATION OF COPPER IN BLOOD

The isolation and identification of the copper protein from serum have clarified the problem of the nature of serum copper (1, 2). This blue protein, ceruloplasmin, is an a-globulin with a molecular weight of approximately 151,000 and contains eight atoms of copper. Ceruloplasmin has true oxidase activity and since it acts on paraphenylene diamine and polyphenols but not on monophenols and monoamines, it has been classified as a laccase (3, 4). Most if not all of the copper in normal human or pig serum is stated to be present in the form of this blue protein. The copper in serum obtained from pregnant women and from patients with infections and other diseases, like ceruloplasmin, is precipitated by 50 per cent saturation with ammonium sulfate. Furthermore, the ability of serum to oxidize paraphenylene diamine is proportional to the copper content not only in normal serum but also in serum obtained from the umbilical vein, from pregnant womenand from patients with infections (5). Presumably then, in patients with hypercupremia the copper is in the form of ceruloplasmin. The problem still remains as to whether or not ceruloplasmin serves the function of transporting newly absorbed copper from the gastro-intestinal mucosa to the liver. Since the concentration of copper (ceruloplasmin) in normal human serum is relatively constant and is not influenced by meals or by the ingestion of copper (6-8), it seems unlikely that this protein is concerned in the active transport of copper. More important is the observation (1) that the copper in ceruloplasmin does not react directly with sodium diethyldithiocarbamate, whereas copper added to serum in sitro does react directly with this reagent (9). This would indicate that, unlike the ability of serum to

Neutrophil Kinetics in Acute Infection

Neutrophil kinetics of acute experimental infection were studied with diisopropylfluorophosphate-(32)P labeling in 31 dogs inoculated intrabronchially with pneumococci. In vitro neutrophil labeling indicated a rapid transit time through the blood in early infections, with an elevated marginal granulocyte pool sometimes preceding an elevation of the circulating granulocyte pool. 13 hr after infection, the circulating and total blood granulocyte pools were increased but the rate of neutrophil transit through the blood was normal. During the recovery from infection there was a marked prolongation of neutrophil blood transit time, suggesting virtually complete cessation of bone marrow release of neutrophils into the blood. Labeling of neutrophils in vivo indicated an increased rate of emptying of the bone marrow storage pool proportional to the severity of infection as measured by the fever index. The change in the blood ratio of nonsegmented to segmented neutrophils was a much more accurate index of the severity of infection than the blood granulocyte concentration, correlating significantly with the fever index.

Leukokinetic Studies. XI. Blood Granulocyte Kinetics in Polycythemia Vera, Infection, and Myelofibrosis

Although it seems evident that the neutrophilic leukocytosis commonly encountered in patients with purulent infections, polycythemia rubra vera, and a variety of other clinical disorders probably indicates an increased mass of neutrophils in the blood and increased neutrophil production, turnover, and utilization, it has not been possible to quantify these processes directly until recently. In normal subjects it was demonstrated that approximately one-half of the neutrophilic granulocytes in the blood are circulating freely [circulating granulocyte pool (CGP)], whereas the remainder adhere to the walls of small venules [marginal granulocyte pool (MGP)] (1). Since these two pools were shown to be in rapid equilibrium with each other they may be considered to form a single total blood granulocyte pool (TBGP) for kinetic purposes. These facts together with the finding that neutrophilic granulocytes disappear from the blood in a random manner (2) have made it possible to approximate the rate of production and destruction of neutrophils in normal man. In the present study the size of the TBGP, the distribution of cells in the two subcompartments, the CGP and the MGP, the blood granulocyte half disappearance time (tj), and the granulocyte turnover rate (GTR) were measured in patients with polycythemia vera, myelofibrosis, chronic infections, and diseases of other kinds. Studies in patients with chronic myelocytic leukemia are the

Superoxide dismutase activity in copper-deficient swine.

Summary These experiments demonstrate the dependency of cuprozinc superoxide dismutase activity in red cells and liver on an adequate dietary intake of copper. The superoxide dismutase activity in red cells decreased to 15% of control values and, therefore, these cells may be used as a convenient model for studying the physiologic consequences of free radicals. The authors are grateful for the technical assistance of Mr. George Trappett, Mr. Dale Chlarson, Ms. Jacqueline E. Thomas, and Mrs. Alice W. Tustison.

Neutrophil Releasing Activity in Plasma of Normal Human Subjects Injected with Endotoxin

Summary Normal human subjects were injected with endotoxin and their plasma was harvested at various times thereafter. This plasma was later infused into the same subject, to determine if neutrophilia inducing activity was demonstrable in such plasma. Infusion of normal control plasma induced no significant change in total neutrophil concentration and nonsegmented neutrophils tended to decline after such infusions. Plasma collected after injection of endotoxin did not induce a significant change in blood neutrophils as compared to control plasma if obtained at a time when no significant increase in the rate of release had developed in the donor. Postendotoxin plasma, collected when the donor was releasing neutrophils at an abnormally rapid rate, induced a significant increase in both total and nonsegmented neutrophils. Infusion of active postendotoxin plasma induced a more rapid onset of neutrophilia than did endotoxin injection. This observation and the inactivity of certain plasmas which were collected after the same dose of endotoxin yielding active plasma suggested that the activity of postendotoxin plasma was not representative of persistence of the injected endotoxin. This study, in conjunction with similar studies in other species, suggests that the rate of release of neutrophils from the marrow to the blood may be controlled by a humoral factor.

The role of superoxide anion radical in the reduction of ferritin iron by xanthine oxidase.

Superoxide dismutase exerted a pronounced inhibitory effect upon xanthine oxidase-mediated reduction of iron in ferritin, ferric chloride, or ferric ADP. Maximal inhibition was observed when the superoxide dismutase concentration was only about 1% of that found in normal porcine liver. These observations indicate that superoxide anion radical is an intermediate in the reduction of iron by xanthine oxidase in vitro but not in vivo.

Heme biosynthesis in copper deficient swine.

Summary The steps in the heme biosynthetic pathway were evaluated in normal and in copper deficient swine. As anemia developed, the activity of heme biosynthetic enzymes increased. These data suggest that the anemia of copper deficiency is not the result of defective heme biosynthesis and, therefore, that copper is not a cofactor in any of the reactions studied. Since the morphologic characteristics of the anemia of copper deficiency suggest defective hemoglobin synthesis, it is concluded that copper is essential either for the normal metabolism of iron or for the synthesis of globin.

The Blood and Bone Marrow Neutrophil Response to Graded Doses of Endotoxin in Mice.

Summary Studies were undertaken to characterize more definitively the effect of endotoxin upon neutrophil kinetics. The data presented herein indicate that the neutrophil response to endotoxin in the mouse is dose related. With relatively small doses of endotoxin, neutrophilia appeared earlier, was of greater magnitude and was not preceded by neutropenia. After larger doses, neutropenia persisted for several hours and was later followed by neutrophilia. The response in marrow neutrophils was determined at 6 and 16 hours following the injection of endotoxin in order to correlate changes occurring in the marrow with those observed in the blood. Smaller doses of endotoxin resulted in an earlier and greater degree of blood neutrophilia than did larger doses but fewer cells were released from the marrow after small doses than after large doses. At least 2 dose related kinetic events explain the neutrophil changes occurring after the administration of endotoxin. With small doses of endotoxin, the acceleration of release rate from the marrow exceeds the rate of loss from the blood. Larger doses have a more profound effect than small doses in accelerating the rate of release of neutrophils from the marrow but they have an even greater effect on acceleration of the outflow of neutrophils from the blood.