M. M. Wintrobe

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

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.

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.

THE ANEMIA OF INFECTION. XIV. RESPONSE TO MASSIVE DOSES OF INTRAVENOUSLY ADMINISTERED SACCHARATED OXIDE OF IRON

The anemia of infection is associated with a number of alterations in iron metabolism. Previous studies (1-4) have shown that, with the development of inflammation, hypoferremia occurs and a decrease takes place in the concentration of the metal binding protein in the plasma. When iron is given by mouth, there is little change in the level of plasma iron and, following the intravenous injection of iron, only a transient rise in the plasma iron develops. These findings have suggested that diversion of iron from the plasma takes place in inflammation and infection. Studies in experimental animals have shown that iron is diverted primarily to the liver and spleen. Whether this iron is simply stored, since less can be used in the face of diminished hemoglobin production, or performs a special function in connection with the presence of inflammation, is unknown. These findings contrast with those associated

BLOOD GRANULOCYTE KINETICS IN CONDITIONS ASSOCIATED WITH GRANULOCYTOSIS

When granulocytes are labeled with DFP32 and then returned to the circulation of the donor, the labeled granulocytes are distributed in a pool of cells which is approximately two times larger than that calculated from the blood volume and the concentration of granulocytes in the circulating venous blood? This pool is referred to as the total blood granulocyte pool (TBGP) . Complete mixing of the labeled cells in the TBGP occurs rapidly and then the labeled cells are removed from the TBGP in an exponential f a s h i ~ n . ~ ~ The TBGP and the half-time disappearance (T % ) of the labeled cells from the TBGP have been measured in 45 normal subject^.^ The normal values along with the absolute values for the concentration of granulocytes (neutrophilic polymorphonuclear cells and metamyelocytes) in the blood are presented in TABLE 1.

Granulocytopoiesis in germfree mice.

Summary A comparison of the total number of neutrophils and neutrophil precursors within the humerus of germfree and conventional mice revealed no statistically significant difference between the two groups. The distribution of marrow neutrophils with respect to morphologic type and the number of neutrophils in mitosis were similar in the two groups. There was, however, a slight, but significantly, lower concentration of blood neutrophils in germfree mice as compared to controls. Removal of mice from the germfree environment was associated with a rapid decrease in the number of mature and relatively mature neutrophils in the marrow and with a subsequent increase in potentially mitotic, immature neutrophils, following which the marrow returned to normal. Germfree mice responded to endotoxm administration with an increase in rate of release of neutrophils from the marrow, as do normal mice. It was concluded that in this model system the presence or absence of microorganisms in the environment could not be assigned a major role in regulating granulocytopoiesis. We are indebted to Mrs. Vreni Bithell and Miss Lona Bindbeutel for technical assistance.

Studies on Copper Metabolism. XXV. Relationship Between Serum and Liver Copper.

Summary 1. Dietary restriction of copper in adult rats was followed by prompt decrease in serum copper and a somewhat slower and less extensive depletion of liver copper. 2. Feeding of diets high in copper was followed by increase in concentration of copper in both serum and liver. The increase occurred more rapidly in serum than in liver during the first 20 days. Thereafter, rate of increase was greater in liver than in serum. 3. It is concluded that concentration of copper in serum and liver is markedly influenced by dietary intake of copper and that there is a close correlation between serum and liver copper concentrations.