Pathophysiological studies on ferric iron. Part 4. Bio­logical observation of serum iron colloid

The iron introduced into vein in the form of the serum iron colloid is rapidly incorporated into ferritin and hemoglobin in a markedly high level with the increase in their amounts, without showing any ill effects. Experiments also show that there is another course of iron metabolism for the incorporation into ferritin and hemoglobin than the physiologic course by the aid of metal combining protein. This is true, however, only in the case in which the normal function of RES is retained. The incorporation of iron into ferritin and hemoglobin is accelerated in anemic animals and delayed in those having RES whose function is disturbed. From these results the author would suggest that the anemic patients may be given a quantity of iron in the form of serum iron colloid directly into vein without causing any side effects.</p

Pathophysiological studies on ferric iron. Part 3. An electronmicroscopic studies on serum iron colloid and some iron preparates

Electron microscopic observations revealed that the serum ferric chloride mixture prepared in the range of pH 5.4 and 8.2 is of colloidal solution whose particles are composed of electron-dense iron colloid nuclei surrounded by protein cortex formed by the absorption around the iron colloid particles. The elevation of pH over 9.0 or the lowering below pH 3 in media results in the loss of the protein cortex enhancing the growth of each molecule to longer ones and to coagulate with each other. Ferritrat proved to have almost the same cortex surrounding the iron colloid as that of serum ferric chloride mixture, but the colloid particles were larger than those of the latter. Both Ferrobalt and the gelatin ferric chloride mixture are of colloid solution but the colloid particles are heterogenous both in size and shape and have no cortex part. Glufericon will be true solution of organic iron complex.</p

Pathophysiological studies on ferric iron. Part I. Chemi­cal reaction between ferric iron and serum

By mixing ferric iron with serum protein, 20 cc of serum and 1 cc of ferric chloride or ferric ammonium sulfate (l0 mg ferric iron/cc in each), in the range of pH 5.4 to 8.2, a transparent brownish red colored solution can be obtained. Paperelectrochromatography proved the iron can mainly be detected in &#946;-globulin fraction in bovine serum and in &#38;#945-globulin and albumin fractions in human and rabbit sera. But the absorption spectrum proved that there is no formation of any new compound, giving almost the same absorption curve as in the serum protein itself. And by lowering the pH of media below 5.4, the solution gives immediately the positive reaction of ferric iron. From these rerults it is suggested that iron will be maintained in a colloidal state keeping the stability of this state in the presence of protein molecules. Freezing and drying are the procedures quite useful for keeping this material for a long period of time without changing the chemical characteristics.</p

Pathophysiological studies on ferric iron. Part 2. Quan­titative observations on the reaction between ferric iron and the serum protein

According to the method presented in the first report the author mixed ferric iron solution and serum with the various proportions of serum and iron, limiting the pH level of the media wihin 5.4 to 8.3. It was found there was a certain level exceeding which the iron could no longer move with protein on the paperelectrochromatography. The maximum level was found to be81, 500&#947;% in the case of ferric chloride and 77, 200&#947;% in ferric ammonium sulfate, when the bovine serum was used as a protecting colloid. The iron added in excess of this level was found retarding at the starting line suggesting the formation of gross iron hydroxide colloid.</p

Histochemical Studies on the Nervous and Humoral Regulation of Lipids and Carbohydrate Metabolism

The purpose of the present study is to reveal the precise mechanism of nervous and humoral regulations of lipid and carbohydrate metabolisms in the adipose tissues. Histochemical and biochemical observations were made on the innervated and denervated interscapular brown adipose tissues and partly on the liver and adrenal cortex of male mice during starvation with or without carbohydrate introduction with special consideration to the changes of the lipid and glycogen contents and to the activities of several important enzymes as well as to pH values in the tissues. In a state of absolute starvation, the animals died in a few days showing a gradual discharge of stored lipids from the innervated brown adipose tissues, while in the denervated tissues the stored lipids increased gradually even in a state of slight or moderate starvation as well as in the cases of normally fed animals. The increase of lipids continued before the stage of severe starvation and the stored lipids being rapidly discharged became nil at the terminal stage of life. Introduction of glucose into starved animals caused also a more marked deposition of glycogen in the denervated than in the innervated tissues in proportion to the degree of starvation, although it did not cause the deposition in both tissues at the terminal stage of life. These facts represent that the nervous regulation is essential for the mobilization of lipids and carbohydrates from this tissue. Adrenalectomy also caused the death of animals within a few days with a gradual decrease of depot lipids. In this case denervation likewise caused a marked depositon of lipids in the brown adipose tissues, showing a sudden escape of lipids at the end of life. Experiments on transplanted adipose tissues taken from the animals at the terminal stage of starvation, proved that the tissue cells retain the ability to deposit lipids until the end of life. Chemical estimation elucidated that the serum glucose and lipids fall markedly at the terminal stage of life. The innervated tissues showed increased activities of succinic dehydrogenase, alkaline phosphatase, ATPase and lipase during starvation with a gradual discharge of lipids. Glucose injection increased the degree of the activities of all these enzymes, though in the terminal stage of starvation the ATPase activity declined again. The activity of total cholinesterase declined slightly in severe starvation. The pH value fell gradually with the progress of starvation. On the other hand, in the denervated tissues the activity of succinic dehydrogenase fell with an increased deposition of lipids, though in the final stage of starvation the activity rose with the discharge of lipids; while the activities of phosphatase, ATPase and lipase rose during starvation and total, unspecific and specific cholinesterase activities declined slightly. The pH value in the denervated tissues rose slightly during mild starvation and fell markedly in severe starvation. Observations proved that the activities Df these enzymes and pH, which are under the control of the autonomic nervous system, have close relationships to the deposition and the discharge of lipids and glycogen from the adipose tissues, and that the rapid discharge of lipids from the denervated tissue at the terminal stage of life is an expression of the onesided progress of oxidative process which may mean a complete loss of regulation of metabolism.</p

A new method for counting the reticulocyte number

The counting of reticulocyte number by the routine method on the dye fillms often leads to a poor result. This can be avoided by counting them on the collodion dye film on which the almost equal distribution of reticulocytes can be attained.</p

The Role of Marrow Architecture and Stromal Cells in the Recovery Process of Aplastic Marrow of Lethally Irradiated Rats Parabiosed with Healthy Litter Mates

Bone marrow aplasia was induced in rats by whole body lethal irradiation (1,000 rads by x-ray), and rats died of irradiation injury within 7 days. Correlative studies at light (LM), transmission (TEM) and scanning electron microscopy (SEM) demonstrated swelling of endothelial and reticular cells and hemorrhage due to detachment of sinus endothelial cells on days 1 and 2. With time, structural recovery occurred without hemopoietic recovery. Reticular cells developed small intracytoplasmic lipid droplets on days 3 and 4. This resulted in fatty a plastic marrow within 7 days. On the other hand, in the marrow of irradiated rats parabiosed with healthy mates by aortic anastomosis, hemopoiesis was initiated by adhesion of nucleated blood cells to fine cytoplasmic pseudopods of fat-stored cells on days 1 and 2 after parabiosis. On days 3 to 5, reticular cells with large lipid droplets and fine pseudopods increased, then hemopoietic foci became clear and extensive. On day 8 after parabiosis, the a plastic bone marrow recovered completely both its structure and hemopoietic activity. Thus, hemopoietic recovery in lethally irradiated marrow begins with recovery of vascular endothelial cells, re-establishment of sinusoidal structure, and morphological and functional recoveries of reticular cells from fat-storage cells by releasing intracytoplasmic lipid droplets. Marrow stromal cells, namely reticular, fat-storage and fibroblastoid cells, share a common cellular origin, and regain their structure and function when fat-storage cells and fibroid cells are placed in contact with hemopoietic precursor cells

Generation of active oxygen species by iron nitrilotriacetate (Fe-NTA).

Ferric nitrilotriacetate (Fe3+-NTA) solution showed maximum absorbance at pH 7.5. The iron was in ferric high-spin state and coordinated octahedrally with a relatively symmetric structure and also probably pentagonally. A spin trapping technique employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) yielded a DMPO spin adduct of unknown radical with three doublets (DMPO-Z) and a simple nitroxide radical (Y-NO.) in serum from rats injected intraperitoneally with Fe3+-NTA. When the Fe3+-NTA solution was diluted 500-fold with 50 mM NTA solution, DMPO-Z, Y-NO. and an additional signal, DMPO-OH were observed. The DMPO-Z signal was suppressed by a decrease in oxygen tension, alpha-tocopherol and 3-tert-butyl-4-hydroxy-anisole (BHA). The DMPO-OH signal was suppressed in the presence of ethanol and catalase. Fe2+-NTA solution hardly produced DMPO spin adducts. The Fe3+-NTA solution produced a strong DMPO-OH signal in the presence of H2O2. Rose Bengal solution, a singlet oxygen generating system, produced the same DMPO adducts. Fe3+-NTA reacted with oxygen in solution. The oxygen was activated and might be similar to singlet molecular oxygen. In the presence of H2O2, the Fe3+-NTA solution generated a hydroxyl radical. Fe3+-NTA itself generated free radicals, but Fe2+-NTA did not.</p

Enhancement of DNA synthesis of mouse myelogenous cells by cyclic adenosine 3',5'-monophosphate (cAMP)

To observe the possible role of cAMP on the DNA synthesis during specialization-division of myelogenous precursor cells, the authors observed the DNA and RNA synthesis of the cells by in vitro autoradiography. And it is concluded that cAMP or its dibutyryl derivative added to the media penetrated into myelogenous precursor cells and metamyelocytes of mice and enhanced the DNA synthetic capacity of them. cAMP hardly enhanced RNA synthesis. Discussion is made on relation between enhancement of DNA synthesis of metamyelocytes and their possible rejuvenation.</p

Distribution of ferritin and hemosiderin in the liver, spleen and bone marrow of normal, phlebotomized and iron overloaded rats.

The distribution of ferritin has been studied in many tissues, but has not yet been established on the cellular level. We investigated the cellular distribution of ferritin in the liver, spleen and bone marrow using the immunoperoxidase method, and compared it with that of hemosiderin. We also examined changes in the distribution of these proteins after phlebotomy and iron overload. In normal rats, ferritin was seen in centrilobular hepatocytes, Kupffer cells, macrophages in the red and white pulp of the spleen and central macrophages in bone marrow. Hemosiderin was observed almost exclusively in the red pulp and partly in tangible body macrophages of the white pulp. After phlebotomy, neither ferritin nor hemosiderin were detectable in these cells except for ferritin-positive cells in the white pulp, which showed little change after either phlebotomy or iron overload. In iron overloaded rats, both ferritin and hemosiderin increased in hepatocytes and reticulo-endothelial (RE) cells. Ferritin-positive cells in the liver were mainly located in the periportal area. These results indicated that hepatocytes and RE cells except for those in the white pulp may play an important role in iron storage, and that ferritin-positive cells in the white pulp may have a function other than iron reserve. They also suggested that the zonal distribution of ferritin-positive hepatocytes may be due to microcirculation in the hepatic lobules.</p