Quantitative Measurement of the Iron‐Incorporating Enzyme in Relation to Marrow Cells and Liver Tissue in the Rabbit

THE incorporation of iron into haemoglobin appears to be deranged in several of the important anaemias those of infection, rheumatoid arthritis, uraemia, thalassaemia, cancer, lead poisoning and the more recently described refractory normoblastic (sideroblastic) anaemias (Dacie, Smith, White and Mollin, 1959; Heilmeyer, 1959). There have been several studies in recent years on the in-vitro incorporation of radioactive iron into protoporphyrine in the presence of blood, marrow and other tissues of man and experimental animals (Granick, 1954; Goldberg, Ashenbrucker, Cartwright and Wintrobe, 1956; Krueger, Melnick and Klein, 1956; Schwartz, Cartwright, Smith and Wintrobe, 1959; Kagawa, Minikami and Yoneyama, 1959; Lochhead and Goldberg, 1961), and there is good evidence that this invitro haem formation from iron and protoporphyrin is enzyme dependent. Grinstein, Bannerman and Moore (1959) have shown that [I"] protoporphyrin IX can be utilized for haem biosynthesis. The enzyme system which catalyses this incorporation has been fractionated and purified, and recently Schwartz, Goudsmit, Hill, Cartwright and Wintrobe (1961) have demonstrated that the incorporation is facilitated by the presence of globin, and have shown that the end-product of the reaction, as characterized by electrophoresis and chromatography, is haemoglobin. These studies have confirmed that the enzymic incorporation of iron into haemoglobin may be accurately assayed by the measurement of the in-vitro incorporation of 59Fe into protoporphyrin. It is not yet known whether the globin combines directly with newly formed haem or initially with protoporphyrin before the insertion of iron. The final elucidation of the exact mechanism of haemoglobin formation must probably await the isolation of the pure enzyme and subsequent lunetic studies. There are, however, several problems in the field of clinical and experimental haematology which may be profitably tackled at this stage, for example those which concern anaemias in which iron incorporation into haemoglobin appears to be deranged. Since a satisfactory assay method is already available it seemed worth while to quantitate the enzyme system in relation to a known number of erythrocyte precursors in the marrow. The mature mammalian erythrocyte is devoid of iron-incorporating enzyme activity; however, the reticulocyte and the erythrocyte precursors in the bone marrow are known to have such activity (London, Shemin and Rittenberg, 1948 ; Walsh, Thomas, Chow, Fluharty and Finch, 1949; Goldberg e l al., 1956; Lochhead and Goldberg, 1961). In these studies we have used rabbit bone marrow, prepared so that cell counts and measurement of enzyme activity could be carried out. The rabbits used were either normal or partially exsanguinated to mimic a haemorrhagic anaemia with marked marrow hyperplasia. In addition to the enzyme activity of marrow cells, that of hepatic cells was also measured in the normal and bled animals, in order to obtain a tissue control. The aim of these studies in experimental animals was to provide the basis of a technique for the measurement of the iron-incorporating enzyme system in human marrow cells in normal and diseased states. * McKenzie-McKinnon Research Fellow of the Royal Colleges of Physicians and Surgeons, England, 1960-61.