Vitamin B12 and the megaloblastic development.

In patients with vitamin B(12) deficiency, phytohemagglutinin-stimulated cultured lymphocytes had little or no thymidylate synthetase activity. Ample activity was found in such lymphocytes from normal individuals and patients with folic acid deficiency or pernicious anemia in remission. It therefore appears that the megaloblastosis that is associated with vitamin B(12) deficiency is related to low thymidylate synthetase activity.

Megaloblastic anemia as a result of an abnormal transcobalamin II (Cardeza).

A 34-year-old Black woman had severe megaloblastic anemia in childhood. Initially, and over the years, she responded well to massive doses of parenteral cobalamin (Cbl) or oral folic acid. Metabolic reactions involving Cbl and folate enzymes were normal during both relapse and remission except for the absence of thymidylate synthetase in relapse. Amino acid analyses of urine and plasma showed no significant abnormalities. Neither cystathionine, homocystine, formiminoglutamic acid, nor methylmalonic acid was detected in the urine. The serum Cbl level was repeatedly elevated even when the patient was receiving only folic acid therapy. The elevation of the vitamin in the serum was found to be a result of markedly increased levels of transcobalamin II (TC II), as identified by several physicochemical techniques. The patient's TC II-Cbl shared immunologic properties with normal TC II but did not facilitate or impede the uptake of Cbl or Cbl bound to normal TC II, respectively, by human cells.

Suggested educational changes and restructuring of medical schools.

The highlights of this commentary may be recapitulated as follows: First, an attempt has been made to emphasize the merits of having medical education as a continuous process after high school under the complete control of medical schools, i.e., incorporating pre-medical into medical education. Second, to restructure the departments of medical schools on the basis of human organs rather than disciplines as they presently exist. Third, to expand the Department of Family Medicine and have it as the principle department in the medical school with other departments serving as satellites. This commentary is quite ambitious, costly, and impractical if realized all at once, especially at existing schools. It might be easier and more practical for a new medical school, in this country or abroad, to adopt it in full or in part and/or in stages.

Clinical applicability and usefulness of ferritin measurements.

The accurate measurement of ferritin in the serum was first reported in 1972. Since then, the assay has become widely available to clinicians. However, the role of this assay in the diagnosis and treatment of various diseases is still poorly defined. Serum ferritin levels are clearly useful in the diagnosis of simple iron deficiency. Hepatic disease, malignancies, and other chronic diseases can cause an elevation in serum ferritin which does not represent an elevation in body iron stores. While markedly elevated in late hemochromatosis, the value of serum ferritin in the detection of early hemochromatosis or the carrier state is not certain.

The effects of free radicals on cobalamin and iron.

The aim of this communication is to show the means by which free radicals could deleteriously alter the metabolism of cobalamin (vitamin B12) and iron in their attempt to protect the body against neoplasia or inflammation and in doing so, create the anaemia of chronic disease.

Thirty-one-year survival following chemotherapy and autologous bone marrow in malignant lymphoma.

A 21-year-old woman with malignant lymphoma received a large dose of nitrogen mustard followed by autologous bone marrow. She achieved a complete remission that lasted 21 years. This was followed in a span of 10 years by two relapses that responded to chemotherapy and splenectomy. Death resulted from sepsis. At autopsy, there was minimal evidence of malignant lymphoma, but evidence for side effects of chemotherapy was present.

Inhibition of thymidylate synthetase of the human lymphocytes.

Previous studies had suggested that methotrexate (MTX) may have actions other than inhibition of dihydrofolic acid reductase. In this study MTX was added to the assay incubation mixture of the enzyme thymidylate synthetase. 5-fluorodeoxyuridine (FUdR) or folinic acid was added separately as controls. The three compounds inhibited thymidylate synthetase with MTX achieving the maximal inhibition. It is suggested that MTX could exert its antineoplastic effect through this mechanism especially if malignant cells have little or no activity of dihydrofolic acid reductase.

Tracing iron and transferrin in the macrophage by visual means.

The purpose of the study was to investigate the movement of iron and transferrin in the macrophage using light and electron microscopy. First, depicted here are the phagocytosis of antibody sensitized murine red cells by the murine bone marrow derived macrophage and the formation of red cell phagosomes. Second, we show the fusion of the lysosomes with the red cell phagosome to form a lysophagosome and the lysis of the red cell using acid phosphatase as a lysosome marker. Third by autoradiography, the presence of 55Fe silver grains in the phagocytosed red cells and its delivery to the organelles of the macrophage are demonstrated. Fourth a transferrin species is shown in red cells of all ages, in the phagocytosed as well as the non-phagocytosed, and in the phagocytosed as well as the non-phagocytosed, and in the macrophage itself. Transferrin was detected using immunogold and fluorescence labelling. These studies suggest that iron, using vesicles as means of transport, moves from the effete red cells inside the macrophage to the outside possibly bound to transferrin.

Modulators of macrophage transferrin or transferrin-like protein.

The effects of endotoxin or testosterone on the amount of transferrin 59Fe (or like protein) in the murine macrophages was investigated. The mouse peritoneal macrophages were laden with 59Fe tagged red cells following the injection of mice with either agent. After harvesting the cells, they were lysed and the transferrin iron was released with 40% trichloroacetic acid. The supernatant (extract transferrin iron) and the pellet (other iron proteins iron) were separated by centrifugation and their radioactivities counted. The results were expressed in percentage. The endotoxin group had a geometric mean of transferrin 59Fe of 0.14% compared to 0.28% for the control, p less than 0.001. The geometric mean for transferrin 59Fe of the testosterone treated group was 0.51% compared to 0.35% for the control, p less than 0.05. Therefore, the endotoxin seems to contract the transferrin pool whereas testosterone seems to expand it.

Thymidylate synthetase of human lymphocytes augmented in vitro by methotrexate.

The addition of methotrexate to the assay system of thymidylate synthetase caused a reduction in the activity of the enzyme but addition of methotrexate to the culture of phytohemagglutinin stimulated normal human lymphocytes caused an increase in the activity of the enzyme which was abolished by the addition of actinomycin D or cycloheximide. These studies suggest that the antimetabolite augmented the enzyme activity by modulating the gene for the enzyme. This modulation of the gene could have been achieved by the thymineless state brought about by methotrexate or the antimetabolite could have affected gene reodont or brought about amplification of the gene. The results of the nucleoside incorporation were consistent with a thymidylate synthetase block; however, other explanations are offered.

Methylcobalamin corrects the deleterious in vitro effect of nitrous oxide on thymidylate synthetase.

In this study, cobalamin deficiency was produced in vitro by the use of nitrous oxide, known to inactivate the vitamin. In 14 sets of experiments, normal human lymphocytes stimulated with phytohemagglutinin on day 0 were exposed to nitrous oxide and oxygen on day 2. MeCbl was delivered later to half of the cells. Untreated cells served as a control. On day 3, the cells were harvested, the lymphocytes were lysed, and the obtained extracts were assayed for thymidylate synthetase. In 16 other experiments the same procedure was performed, and the incorporation of radioactive thymidine or deoxyuridine by the intact cells was measured. In additional experiments, a deoxyuridine suppression test of treated and untreated stimulated lymphocytes was also performed. The results indicate that nitrous oxide significantly reduces the activity of thymidylate synthetase and that this reduction is significantly corrected by MeCbl, suggesting a causative relation between the vitamin and the enzyme. However, there was no statistically significant effect of nitrous oxide demonstrated on the nucleoside incorporation nor on the deoxyuridine suppression test.

Treatment of primary defective iron-reutilization syndrome: revisited.

We encountered two patients who presented with hypochromic-microcytic anemia and were refractory to iron therapy. The symptoms were suggestive of anemia of chronic disease (ACD); however, there was no evidence of any such disease, either inflammatory or malignant. These patients were reminiscent of patients originally described as having primary defective iron reutilization. The hematologic picture consisted of hypochromic-microcytic anemia, low serum iron, low to normal iron binding capacity, high serum ferritin, and increased bone marrow iron in the absence of ringed sideroblasts. These patients had symptomatic anemia and received danazol (200 mg orally) three times per day to which they responded very well with an increase of approximately 3 g in the hemoglobin concentration over 1 year and amelioration of their symptoms. Danazol was well tolerated and did not cause any virilizing side effects. Doses were lowered in maintenance after 1 year to 200 mg once per week, and responses were sustained up to 36 months of follow-up duration. In the differential diagnosis of hypochromic-microcytic anemia, especially in postmenopausal women, one has to consider this type of treatable anemia when more common types such as iron deficiency, chronic inflammation, malignancy, sideroblastic anemia, or thalassemia have been ruled out.

Thymidylate synthetase and dihydrofolic acid reductase in the stimulated human lymphocyte.

The phytohemagglutinin (PHA)-stimulated human lymphocytes demonstrated trace or no activity (dihydro) folate acid reductase using three methods including a radioassay, but demonstrated ample activity of thymidylate synthetase. This was true regardless of the day of harvest, (first through seventh) of the stimulated lymphocyte. The lymphocyte extracts revealed no inhibitor to the reductase enzyme when these extracts were added before the liver extracts to the assay system. When methotrexate (MTX) was added to the culture media of the lymphocytes, there was, as expected, an increase in the synthetase activity, but the expected rise in the reductase activity did not occur, it remained nil. On the other hand, MTX did influence the incorporation of nucleosides by the stimulated lymphocytes in a fashion similar to its action on the incorporation of the same nucleosides by other cells.

[Transcobalamine-II-polymorphism: biochemical and clinical aspects of rare variants]. / Transcobalamin-II-Polymorphismus: Biochemische und klinische Aspekte seltener Varianten.

Transcobalamin II(TC II) is an essential transport protein for vitamin B12 in blood. TC II can be split up into isoproteins by polyacrylamide gel electrophoresis. Family studies are compatible with a genetic polymorphism of TC II and a five allele system. Screening of TC II isoprotein patterns in about 1000 individuals yielded two unusual TC II variants: The first case was a black female with severe megaloblastic anemia since infancy. Her TC II was elevated and bound B12, but displayed markedly diminished functional capacity to transfer radioactive cyanocobalamin in cellular systems. Comparison of this patient's TC II isoprotein pattern with known variants showed a distinct difference in electrophoretic mobility, indicating the presence of a sixth allele. The second patient, also presenting with pernicious anemia-like symptoms, was found to possess an unusual TC II variant with reduced TC II serum levels. Corresponding variants were also observed in the patient's asymptomatic children. Thus, abnormal TC II variants probably causing megaloblastic anemias both correlated with unusual isoprotein patterns.