Erythropoietin

Abstract

In 1890, Viault (1890) observed that 2 weeks after traveling in Peru from sea level (Lima) to the mountain area of Morococha (4200 m) his red blood cell count went from 5.0 million to 7.1 million/mm^{. Values of five other sojourners in his party ranged from 7.1 to 8.0 million. These simple observations provided the first convincing demonstration of the robust burst of erythropoiesis in man soon after exposure to high altitude hypoxia. During the transition into the next millennium the mechanism underlying this phenomenon became a topic of heated debate. Friedrich} Miescher (1893), well known for his discovery of DNA, proposed that a decrease in oxygen tension within the bone marrow provided a direct stimulus to erythroid cells. A half-century elapsed before this theory was disproven by carefully executed measurements of oxygen saturation in bone marrow specimens of patients with erythrocytosis, both primary (Berk et al. 1948) and secondary (Stohlman et al. 1954).

In 1906, Carnot and Deflandre (1906) proposed an alternate mechanism for hypoxic induction of erythropoiesis. They observed an increase in red blood cell counts following infusion of normal rabbits with serum from anemic animals and concluded that erythropoiesis is regulated by a humoral “factor” in the plasma. Attempts to reproduce this experiment over the ensuing decades yielded equivocal or negative results, thus casting doubt on this hypothesis. However, in the middle of the twentieth century, Krumdieck (1943) and Erslev (1953) modified the experimental design of Carnot and Deflandre by the addition of accurate measurements of reticulocytes and convincingly showed in rabbits the induction of new red cell production within 3–6 days following injection of anemic serum injected.

The notion that hypoxic stimulation of erythropoiesis involved an indirect humoral mechanism was strongly buttressed by experiments of Reissmann (1950) and Ruhenstroth-Bauer (1950). They used parabiotic pairs of rats whose circulations were connected at the capillary level by overlapping flaps of skin and elegantly showed that when one rat was exposed to low oxygen tension, whereas the other breathed room air and remained normoxic, both animals developed a surge of new red cell production and erythrocytosis.

Taken together, these studies led to the conception of a circulating erythroid-stimulating hormone, “erythropoietin” (Epo). Organ ablation studies in rats (Jacobson et al. 1957) and man (Nathan et al. 1964) firmly established that the kidney was the major site but not the sole site of Epo production. These findings led Eugene Goldwasser and his colleagues to undertake an intense and prolonged effort to isolate Epo. Initial attempts to obtain Epo from kidneys were unsuccessful owing to the release of proteolytic enzymes during tissue homogenization. In the search for a more tractable source of Epo, Goldwasser first turned to plasma of anemia sheep, then to urine from Argentinians with severe iron deficiency owing to hookworm infestation, and finally to urine from Japanese patients with aplastic anemia. This 15-year endeavor was greatly facilitated by the development of a sensitive and specific assay using incorporation of radio-labeled iron into newly produced red cells (Fried et al. 1956). By 1977, Goldwasser and his team were able to prepare 8 mg of highly purified human Epo (Miyake et al. 1977). Amino-terminal amino acid sequencing of this preparation enabled the synthesis of semidegenerate oligonucleotide probes that could then be used for the molecular cloning of the Epo gene (Jacobs et al. 1985; Lin et al. 1985). This advance opened up a new era in the exploration of the physiology and molecular biology of Epo and was exploited in the development of recombinant human Epo as a therapeutic agent for patients with various types of anemia.

For more detailed information on the history of Epo, see reviews by Grant and Root (1952), Erslev (1980, 1993), and Goldwasser (1996).

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