Heme Synthesis Is Inhibited in Erythroid Cells Derived from Beta-Thalassemic Mice

Thalassemias are a heterogeneous group of red blood cell disorders characterized by ineffective erythropoiesis, peripheral hemolysis and anemia. Transfusion-dependent thalassemia (TDT) includes thalassemia major patients who require lifelong transfusions for survival. Non-transfusion dependent thalassemia (NTDT) patients do not need constant transfusions to survive, although their survival may depend on occasional or even frequent transfusions during defined periods of time. NTDT comprises three different forms of thalassemias: β-thalassemia intermedia, hemoglobin E/β-thalassemia (mild and moderate forms) and α-thalassemia intermedia (hemoglobin H disease). Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia.

The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. In β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation. The defective production of α-globin chains would be expected to cause the accumulation of uncommitted heme in thalassemic erythroblasts. However, we have observed that mice β-thalassemic erythroblasts have decreased heme levels when compared to wild type cells. This observation led us to hypothesize that heme synthesis pathway is inhibited in β-thalassemic erythroblasts.

To test this hypothesis, we used a β-thalassemic mice model, Th3,¹ which has a deletion of both β^major and β^minor genes. Mice homozygous for the deletion (Th3/Th3) die late in gestation and heterozygotes (Th3/⁺) survive and develop a phenotype similar to β-thalassemia intermedia in humans (anemia, tissue iron overload and ineffective erythropoiesis). We isolated reticulocytes from wild type (Wt) and Th3/⁺ mice and measured the rate of heme synthesis in them. Reticulocytes are an invaluable model to study heme synthesis and hemoglobin production in mice, since they are pure erythroid cells and responsible for a significant amount of all hemoglobin produced in mice during stress erythropoiesis.^2-4

We found that when Wt or Th3/⁺-derived reticulocytes were incubated with increasing amounts of ⁵⁹Fe-Tf (0.5-4 µM), the total iron uptake increased by the same percentage (51.9±5.4% and 63.3±6.4% [p>0.05; n=3] between 0.5 and 4.0 µM) in both Wt- and Th3/⁺-derived cells, while the amount of ⁵⁹Fe incorporated into heme was already maximal at 0.5 µM. Importantly, in Th3/⁺-derived reticulocytes the percentage of ⁵⁹Fe incorporated into heme was significantly lower than in Wt cells. Therefore, compared to Wt reticulocytes, Th3/⁺ reticulocytes are less efficient in utilizing iron for heme synthesis. We then examined whether heme synthesis could be stimulated in Th3/⁺ reticulocytes by supplying 5-aminolevulinic acid (ALA), which is the product of the first enzyme of heme biosynthesis. The incubation of Wt or Th3/⁺ reticulocytes with different amounts of ALA led to a significant increase in the ⁵⁹Fe incorporation into heme only in Th3/⁺ cells; there was a 48.31±4% increase in the presence of 2 mM ALA. Therefore, the deficiency of Th3/⁺ reticulocytes in synthesizing heme is not related to a lack of iron.

Our data suggest that there is a yet to be identified factor, which may, or not, be related to globin synthesis, which is responsible for decreased heme synthesis in Th3/⁺ erythroid cells. Additionally, the ALA-mediated increase in heme synthesis in Th3/⁺ reticulocytes also indicates that heme synthesis is inhibited in these cells prior to or at the step catalyzed by erythroid-specific ALA synthase.

¹Yang B, Kirby S, Lewis J, Detloff PJ, Maeda N, and Smithies O. A mouse model for beta 0-thalassemia. Proc Natl Acad Sci U S A. 1995;92(25):11608-12.

²Sassa A, and Bernstein SE. Levels of delta-aminolevulinate dehydratase, uroporphyrinogen-I synthase, and protoporphyrin IX in erythrocytes from anemic mutant mice. Proc Natl Acad Sci U S A. 1977;74(3): 1181-4.

³Sassa S, Kappas A, Bernstein SE and Alvares AP. Heme biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. J Biol Chem . 1979; 254(3):729-35.

⁴Abdulla M and Svensson S. Effect of phenylhydrazine on delta-aminolevulinic acid dehydratase in red blood cells. Enzyme . 1978;23(3):164-9.