Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796515
Title: Haem biosynthesis in isolated human erythroblasts
Author: Houston, Tracey
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1990
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Abstract:
Hepatic haem biosynthesis has been well characterised. The first enzyme of the haem biosynthetic pathway, 5-aminolaevulinic acid (ALA) synthase, is rate-limiting and under negative feedback control by haem. Liver is, however, a relatively minor site of haem synthesis as 80% of haem is formed in the erythroid cells of the bone marrow. Within developing erythroid cells haem is required both for its specific complexing with globin and for the coordinate regulation of erythroblast metabolism and differentiation. The study of erythroid haem synthesis has been hampered by the heterogeneous nature of the marrow cell population, the various differentiation stages of the erythroblast population, the small sample sizes available and the lack of sensitive enzyme assays. A variety of models and techniques have been used to study erythroid haem synthesis. Accordingly, the results have been inconclusive. This work describes human bone marrow fractionation to provide four purified age-matched erythroblast cell populations. Myeloid (white) cells were removed from the sample by monoclonal antibody mediated cell lysis. The remaining cells were then fractionated by equilibrium density gradient centrifugation. These 'in vivo' human erythroblasts were used to examine the effects of normal and abnormal erythroid differentiation on the first and last enzymes of the haem pathway, ALA synthase and ferrochelatase. A sensitive radiochemical assay for ALA synthase was adopted and improved. This was then used to examine the temperature-dependent inactivation of erythroid ALA synthase. A novel radiochemical assay for ferrochelatase was developed. Both assays utilise HPLC for isolation of the radioactive product and are capable of detecting picomoles of activity. The pattern of ALA synthase and ferrochelatase activities during normoblastic erythropoiesis was established. ALA synthase activity was maximal in the most immature erythroid cells and diminished as the cells matured. Ferrochelatase activity was maximal in the intermediate erythroid cells. Hence, the development of enzyme activity appears to be sequential rather than simultaneous. On a quantitative basis, however, it is uncertain whether the development of ferrochelatase activity is limiting for haem formation. The effects of iron and haem deficiency on haem enzyme activity were studied. In iron deficient erythroblasts ALA synthase activity was significantly reduced, particularly in the most immature erythroid cells. This enzyme reduction is likely to result from consequent haem deficiency rather than iron deficiency per se as an iron replete patient with haem deficiency associated with hepatoerythropoietic porphyria also showed reduced enzyme activity. This suggests that erythroid haem synthesis is controlled in a different manner to hepatic haem synthesis - a view compatible with the recent identification of specific genes and tissue-specific isoenzymes for ALA synthase. ALA synthase and ferrochelatase activities were measured in patients with sideroblastic anaemia (SA). Haem synthesis is impaired in SA and ALA synthase is generally believed to be the site of the primary defect. ALA synthase activity was markedly impaired in 3 patients with congenital SA (CSA) and 7 patients with primary acquired SA (PASA). One patient with secondary SA (SSA) demonstrated normal activity. Ferrochelatase activity was measured in six patients (1 CSA, 5 PASA) and was found to be significantly reduced. The latter 6 patients (1 CSA, 5 PASA) were treated with haem arginate in an attempt to correct the haem deficiency and to stimulate haem enzyme activity. Haem arginate was clinically ineffective (Hb levels did not change in any of the patients). However, the study demonstrated that exogenous haem is able to enter erythroblasts and influence haem enzyme activity. In two patients (1 CSA, 1 PASA), both ALA synthase and ferrochelatase activities returned to normal. This indicates a potential role for haem arginate in the treatment of haematological disorders other than SA which may respond to stimulated haem biosynthesis. The results also suggest that the primary abnormality in these cases of CSA and PASA is not ALA synthase (or ferrochelatase) deficiency. Furthermore, the stimulatory effect of exogenous haem on enzyme activity and the reduced ALA synthase activity in haem deficiency provide strong evidence of independent regulatory mechanisms for haem biosynthesis in erythroid and hepatic tissue.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796515  DOI: Not available
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