ERYTHROPOIETIN: FROM BENCH TO BEDSIDE**,* JERRY L. SPIVAKt BALTIMORE
Medical historians of a future generation surely will consider the development of the hematopoietic growth factors for clinical use as a major milestone in medical progress. They also will note that, owing to the power of recombinant DNA technology, the development of these growth factors was so rapid that their clinical application preceded a full understanding of their physiology. This is true even for erythropoietin, the first of the hematopoietic growth factors to be discovered, probably because it is the only one which behaves like a hormone. Produced in the kidneys and to a small extent in the liver, erythropoietin acts on erythroid progenitor cells in the bone marrow to promote their proliferation. Although erythropoietin was first described forty years ago (1), twentyseven years elapsed before its purification (2) and thirty-five years before the erythropoietin gene was molecularly cloned (3, 4) and the recombinant protein became available for clinical trials (5, 6). In spite of this long time interval, the physiology for erythropoietin is not widely appreciated. Presumably this is because for most of its known existence, erythropoietin was only of interest to experimental hematologists since it was not available for clinical use nor was there a clinically applicable assay for the protein. The development of sensitive immunoassays for erythropoietin with the use of either the purified protein (7) or recombinant-derived reagents (8) provided an opportunity to carefully scrutinize the behavior of erythropoietin in the circulation and correlate this with various clinical states. Such observations have provided new insights into the physiology of erythropoietin in both health and disease and are the subject of this report. For an assay of a circulating hormone to be useful clinically, a number of criteria must be met. First, the concentration of the hormone in the circulation should directly reflect its production. Second, hormone * From the Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD. ** Supported in part by grant DK 16702 from the National Institute of Diabetes and Digestive and Kidney Diseases. t Address correspondence and reprint requests to Jerry L. Spivak, M.D., Division of Hematology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205.
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production should reflect demand for the hormone. Third, the hormone should be biochemically unique. Fourth, only one form of the hormone should be present in the circulation. Fifth, the metabolism of the hormone should be independent of its plasma level and the size of its target cell population. Finally, immunoreactive hormone should be identical to biologically active hormone. Erythropoietin fulfills all of these criteria. First, erythropoietin production is regulated at the level of its gene and the stability of its mRNA (9, 10) and independently of its plasma concentration (11). Second, there are no significant preformed stores of erythropoietin in the kidneys or the liver (12). Demands for additional erythropoietin are met by de novo production; tissue hypoxia stimulates erythropoietin synthesis while erythrocytosis suppresses erythropoietin production but never completely. Third, erythropoietin is a highly conserved protein which has no homology with any known protein (3, 4). Fourth, posttranslational modification of erythropoietin is minimal [loss of only Argl66 (13)] and only one form of the hormone is present in the circulation. Fifth, the plasma clearance of erythropoietin is sluggish (14) and independent of its either concentration (15) or the size of the erythroid progenitor cell pool (16). Finally, immunoreactive erythropoietin appears to approximate biologically active erythropoietin (8). As a further convenience to clinical interpretation, neither age nor gender influence the plasma level of erythropoietin (17) and the diurnal variation in the level of the hormone is small (18) and not appreciably magnified by hypoxia (19). Using immunoassays, it has been possible to derive a normal range for erythropoietin in the circulation of 4-24 mU/ml. Although small with respect to the levels achieved with tissue hypoxia, this still represents a 6-fold variation which may reflect in part the variation observed in the plasma clearance of the hormone (4-12 hours) in normal individuals (20, 21, 22). Although erythropoietin levels vary from person to person, they vary very little from day to day in a given individual in the absence of hypoxia, bleeding or anemia (17). This is in keeping with known constancy of the hemoglobin or hematocrit in a given individual even though there is a 10-15% variation in these determinations between individuals of the same gender. The small variance in the circulating erythropoietin levels which is a feature of its normal physiology is, for unknown reasons, lost with the development of end-stage renal disease (23). Although it is widely accepted that there is a log-linear inverse correlation between circulating erythropoietin and the hemoglobin or hematocrit level, it is not widely appreciated that this relationship is
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tightly regulated and varies with the hemoglobin level. Indeed, the presence of a threshold with respect to erythropoietin production is central to understanding the behavior of erythropoietin in health and disease. Studies of erythropoietin mRNA expression have revealed that there is constitutive production of the hormone in certain cells in the kidney (24). With tissue hypoxia, these cells do not make more erythropoietin but rather additional cells are recruited to produce the hormone (24). Recruitment occurs in an exponential fashion, and with restoration of tissue oxygen delivery, there is an exponential down regulation of the number of erythropoietin-producing cells in the kidney. That production of the hormone is tightly regulated is evident from observations of humans or animals subjected to hypoxia. Unless the hypoxia is extreme, the initial increase in plasma erythropoietin is followed by a decline back to the normal range even in the presence of continued hypoxia (25). This down-regulation is not due to accelerated metabolism of the hormone since plasma erythropoietin levels parallel renal erythropoietin production (26) but rather to the other compensatory mechanisms available to the body for improving tissue oxygenation. Should these mechanisms fail, erythropoietin production will continue at an elevated level (27). Early evidence of a threshold for acceleration of erythropoietin production was provided by studies of erythropoietin excretion in the urine in normal individuals, patients with polycythemia vera and patients with secondary erythrocytosis (28). With repeated phlebotomy, there was a log-linear increase in urinary erythropoietin excretion. In patients with polycythemia vera, a greater degree of hematocrit reduction was required before a comparable increase in urine erythropoietin excretion was observed while in patients with secondary erythrocytosis, whose urinary excretion of erythropoietin was normal when their red cell mass was elevated, phlebotomy induced increments in urinary erythropoietin at a higher hematocrit than in the other two groups (28). Taken together, it appears that in polycythemia vera, erythrocytosis per se suppressed erythropoietin production and increased the threshold required for recruitment of erythropoietin-producing cells while in compensated secondary erythrocytosis due to hypoxia, the threshold for such recruitment was reduced. That erythrocytosis per se could inhibit erythropoietin production even in the presence of mild hypoxia was elegantly demonstrated in mice made plethoric with methemoglobincontaining erythrocytes (29). Importantly with regard to studies of plasma erythropoietin, as mentioned above, all evidence to date indicates that neither the plasma erythropoietin level (15) nor marrow cellularity
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(16) appear to influence the plasma clearance of the hormone nor is there any effect of the plasma erythropoietin level on erythropoietin production (11). Tight regulation of erythropoietin production also occurs in chronic anemia as well as erythrocytosis. Thus, adult patients with sickle cell anemia ordinarily have high plasma erythropoietin levels but these are not as high as in patients with acquired hemolytic anemia (30). When patients with sickle cell anemia are exposed to a high oxygen tension, their erythropoietin level falls as does their reticulocyte count (31). When restored to ambient oxygen tension, there is a rapid rise in plasma erythropoietin but even though the patients are still anemic, this increase in erythropoietin synthesis is abruptly terminated and plasma erythropoietin falls rapidly (31). With the development of sensitive immunoassays, it was finally possible, for the first time, to quantitate the behavior of erythropoietin in normal individuals and several noteworthy observations have been made with respect to the regulation of erythropoietin production. First, within the normal range of hemoglobin (or hematocrit) for men and women (12-16 gm%), there is not an inverse correlation between hemoglobin and erythropoietin (17). Second, even though men have a higher red cell mass than women, their erythropoietin levels are identical (17). This could occur if some other factor was involved in the regulation of the red cell mass. The opportunity to test this hypothesis was provided in a group of men being given an LHRH agonist to suppress prostatic hypertrophy (32). With continued administration of this agonist, serum testosterone levels fell to castrate levels, and this was followed by a fall in the hemoglobin level of at least 1 gm% which was reversed when therapy with the LHRH agonist was discontinued. Throughout the six months of therapy and six months post therapy, while the hemoglobin was falling and recovering, there was no significant change in plasma erythropoietin (32). As shown in figures 1 and 2, there was an excellent correlation between hemoglobin and serum testosterone levels but no correlation of serum testosterone and erythropoietin levels. These data indicate that in men, within the normal range of hemoglobin testosterone is involved in regulating the red cell mass. To determine the effect of repeated phlebotomy on the circulating erythropoietin level, we examined erythropoietin levels in patients participating in an autologous blood donation program in preparation for elective surgery (33). As shown in Table 1, repeated weekly phlebotomies produced progressive anemia in spite of a small but significant increase in circulating erythropoietin within the normal range. This increase was associated with a small but significant increase in reticulocytes, but the
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JERRY L. SPIVAK
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TESTOSTERONE (ng/ml) FIG. 2. Correlation between testosterone and serum erythropoietin in men receiving the LHRH agonist nafarelin acetate. The data are from reference 32. response was insufficient to prevent the development of anemia as the phlebotomies were continued. The anemia was not a consequence of induced iron deficiency as there was no change in red cell protoporphyrin levels, and the patients were receiving supplemental oral iron. Similar observations have also been made by others in multiply phlebotomized individuals who were iron replete (34). Failure of mild anemia to induce increments in plasma erythropoietin outside the range of normal suggests that there is a threshold hemoglobin which must be reached before a significant increment in erythropoietin production occurs. That this is indeed the case has been demonstrated in several different ways. First, analysis of the hemoglobin level below
ERYTHROPOIETIN FROM BENCH TO BEDSIDE
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TABLE 1 Effect of Repeated Phlebotomies on Hematocrit, Reticulocyte Count and Serum Erythropoietin in Men Time
Hematocrit*
Reticulocyte* Count
(%o) (M) 42 ± 1.0 1.3 ± 0.1 Week 1 Week 2 1.7 ± 0.2t 39 ± 1.0t Week 3 38 ± 1.0t 2.3 ± 0.4t 3.2 ± 0.4t Week 4 35 ± 1.0t * Mean ± S.E.M. t Significantly different from Week 1 (p < 0.05) i: Significantly different from Week 1 (p < 0.025) The data are from reference 33.
Serum* Erythropoietin
(mU/ml) 13.2 ± 1.4 16.0 ± 1.3 17.6 ± 1.4t 22.4 ± 3.5t
which circulating erythropoietin levels were unequivocally elevated outside the normal range in patients with iron deficiency anemia yielded a value of 10.5 gm% (hematocrit 32%) (17). Second, in patients with sickle cell anemia, an unequivocal increment in serum erythropoietin only occurred below a hematocrit of 32% (35). Third, in patients postrenal transplantation, the circulating erythropoietin level fell to normal as the hematocrit exceeded 32% (36) and finally, in patients with a variety of anemias a significant increase in circulating erythropoietin was only seen when the hematocrit is below 32% (37). Thus, independent studies in different patient populations have yielded the same result i.e. a threshold exists below which the hemoglobin or hematocrit level must fall before the circulating erythropoietin level increases unequivocally outside the range of normal. This is not to imply that small increments in circualating erythropoietin do not occur with lesser degrees of anemia (or tissue hypoxia). They do (33, 38) but because increments are still within the range of normal, they would not be appreciated without serial measurements nor are such increments usually sufficient to prevent anemia if blood loss is repetitive over a short time interval. Thus, the tight regulation of erythropoietin production extends to the situation of mild anemia suggesting that the standard clinical definition of anemia is not synonymous with a degree of tissue hypoxia sufficient to elevate plasma erythropoietin above the range of normal. Several important predictions can be drawn from these observations. First, a random erythropoietin assay in a mildly anemic patient will probably not provide clinically useful information. Second, autologous blood donors should benefit from erythropoietin administration since their hemoglobin levels never reach the threshold for stimulating significant erythropoietin production (33). This prediction has been verified in a recent clinical trial (39).
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Although the inverse linear relationship between hemoglobin and circulating erythropoietin appears to hold true once the hemoglobin level falls below 10.5 gm% in uncomplicated situations such as iron deficiency anemia, certain disease states impact negatively on it. The inverse linear relationship is totally lost with renal disease. In other conditions such as rheumatoid arthritis (40, 41), AIDS (42) and cancer (43), erythropoietin production is inappropriately low with respect to the degree of anemia and in some patients with solid tumors, like patients with end-stage renal disease, the response is lost altogether (43). When viewed from the perspective of regression analysis, the slope of the erythropoietin:hemoglobin relationship in iron deficiency or anemia is -25.8 (R2 = 0.81) as compared with -12.6 (R2 = 0.32) for rheumatoid arthritis, -6.6 (R2 = 0.25) for AIDS and -4.9 (R2 = 0.09) for patients with solid tumors (17). The mechanisms involved in blunting the expected response to hypoxia are as yet unknown but the threshold concept may again be applicable. For example, although patients with end-stage renal disease lose the ability to maintain a sustained increase in erythropoietin production as they become anemic, they do not lose the ability to make substantial amounts of erythropoietin if they become sufficiently hypoxic (44). This is also true for patients with cancer (43) but in each case, the response is not sustained once the hypoxic stimulus is removed. Certain AIDS patients also appear to reset their threshold for erythropoietin production since with AZT administration, they produce more erythropoietin than other patients with comparable degrees of anemia (42). This type of analysis has a predictive value which is clinically useful since studies to date have confirmed that anemic patients with a blunted erythropoietin production will respond to administration of recombinant erythropoietin (45, 46, 47, 48). The tight regulation of erythropoietin production appears to provide a protective mechanism to avoid explosive increments in the erythroid progenitor cell pool since that pool expands exponentially with erythropoietin stimulation (49). There is also an apparent economy involved as well since erythroid progenitor cells at different stages of development have different requirements for erythropoietin. Thus, late erythroid progenitor cells are largely in cell cycle and appear only to require erythropoietin as a survival factor (50) while early erythroid progenitor cells appear to require erythropoietin as a mitogen as well as a survival factor (51) and probably in higher concentrations since they express fewer receptors for the hormone (52). Pharmacokinetic studies also indicate that sustained levels of circulating erythropoietin well above 100 mU/ml may not be efficient (53) since tissue receptors are probably saturated at that concentration.
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SUMMARY Erythropoietin is unique amongst the hematopoietic growth factors since it is the only one which behaves like a hormone. The development of sensitive immunoassays for erythropoietin have provided an opportunity to examine its physiology more closely than ever before. Although the classical inverse-linear correlation between erythropoietin and hemoglobin has been amply confirmed, it has also become apparent that this relationship is tightly regulated and is only apparent below a threshold hemoglobin and not fully operative within the normal range of hemoglobin values. Certain disease states blunt the response of erythropoietin-producing cells to anemia, and in some cases this appears to be due to a resetting of the threshold for response while in others there may be a dichotomy between activation of the machinery for erythropoietin gene expression and net protein synthesis. The tight regulation of erythropoietin production may be directed in part at preventing explosive increases in the red cell mass and in part may conform to the actual demands of erythroid progenitor cells for the hormone, since, at least in vitro, erythropoietin effects these progenitor cells differently according to their stage of maturation and sustained high levels of the hormone are not necessary for certain of the desired effects. REFERENCES 1. Reissmann KR. Studies on the mechanism of erythropoietin stimulation in parabiotic rats during hypoxia. Blood 1956; 5: 372. 2. Miyaki T, Kung CK-H, Goldwasser E. Purification of human erythropoietin. J Biol Chem 1977; 252: 5558. 3. Jacobs K, Shoemaker C, Rudersdorf R, et al. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 1985; 313: 806. 4. Lin F-K, Suggs S, Lin C-H, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci 1985; 82: 7580. 5. Winearls CG, Pippard MJ, Downing MR, et al. Effect of human erythropoietin from recombinatn DNA on the anaemia of patients maintained on chronic haemodialysis. Lancet 1986; 2: 1175. 6. Eschbach JW, Egrie JC, Downing MR, et al. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med 1987; 316: 73. 7. Sherwood JB, Goldwasser E. A radioimmunoassay for erythropoietin. Blood 1979; 54: 885. 8. Egrie JC, Cotes PM, Lane J, et al. Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin tracer and immunogen. J Immunol Meth 1987; 99: 235. 9. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1988; 242: 1412. 10. Goldberg MA, Gaut CC, Bunn HF. Erythropoietin mRNA levels are regulated by both transcriptional events and by changes in RNA stability. Blood 1989; 74: 191a.
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11. Fried W, Barone-Varelas J. Regulation of the plasma erythropoietin level in hypoxic rats. Exp Hematol 1984; 12: 706. 12. Schooley JC, Mahlmann LJ. Evidence for the de novo synthesis of erythropoietin in hypoxic rats. Blood 1972; 40: 662. 13. Recny MA, Scoble HA, Kim Y. Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. J Biol Chem 1987; 262: 17156. 14. MacDougall IC, Neubert P, Coles GA, et al. Pharmacokinetics of recombinant human erythropoietin in patients on continuous ambulatory peritoneal dialysis. Lancet 1989; 1: 425. 15. Spivak JL, Hogans BB. The in vivo metabolism of recombinant human erythropoietin in the rat. Blood 1989; 73: 90. 16. Piroso E, Flaharty K, Caro J, et al. Erythropoietin half-life in rats with hypoplastic and hyperplastic bone marrows. Blood 1989; 74: 270a. 17. Spivak JL, Hogans BB. Clinical evaluation of a radioimmunoassay for serum erythropoietin using reagents derived from recombinant erythropoietin. Blood 1987; 70: 143a. 18. Wide L, Bengtsson C, Birgegard G. Circadian rhythm of erythropoietin in human serum. Brit J Haematol 1989; 72: 85. 19. McKeon JL, Saunders NA, Murree-Allen K, et al. Urinary uric acid: creatinine ratio, serum erythropoietin, and blood 2,3-diphosphoglycerate in patients with obstructive sleep apnea. Am Rev Respir Dis 1990; 142: 8. 20. Flaharty KK, Caro J, Erslev A, et al. Pharmacokinetics and erythropoietic response to human recombinant erythropoietin in healthy men. Clin Pharmacol Ther 1990; 47: 557. 21. Urabe A, Takaku F, Mizoguchi H, et al. Effect of recombinant human erythropoietin on the anemia of chronic renal failure. Intl J Cell Clon 1988; 6: 179. 22. McMahon FG, Vargas R, Ryan M, et al. Pharmacokinetics and effects of recombinant human erythropoietin after intravenous and subcutaneous injections in healthy volunteers. Blood, 1990 (in press). 23. Chandra M, Clemons GC, McVicar M, et al. Serum erythropoietin levels and hematocrit in end-stage renal disease: influence of the mode of dialysis. Am J Kidney Dis 1988; 12: 208. 24. Koury ST, Koury MJ, Bondurant MC, et al. Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration. Blood 1989; 74: 645. 25. Abbrecht PH, Littell JK. Plasma erythropoietin in men and mice during acclimatization to different altitudes. J Appl Physiol 1972; 32: 54. 26. Jelkmann W. Temporal pattern of erythropoietin titers in kidney tissue during hypoxic hypoxia. Pflugers Arch 1982; 393: 88. 27. Milledge JS, Cotes PM. Serum erythropoietin in humans at high altitude and relation to plasma renin. J Appl Physiol 1985; 59: 360. 28. Adamson JW. The erythropoietin/hematocrit relationship in normal and polycythemia man: implications of marrow regulation. Blood 1968; 32: 597. 29. Kilbridge TM, Fried W, Heller P. The mechanism by which plethora suppresses erythropoiesis. Blood 1969; 33: 104. 30. Dover GJ, Spivak JL, Hogans BB, et al. Erythropoietin levels in adults and children with sickle cell disease: relation to age, sex, hemoglobin, and fetal hemoglobin levels. Blood 1987; 70: 134a. 31. Embury SH, Barcia JF, Mohandas N, et al. Effects of oxygen inhalation on endogenous erythropoietin kinetics, erythropoiesis and properties of blood cells in sickle-cell anemia. N Engl J Med 1984; 311: 291. 32. Weber J, Walsh PC, Peters CA, et al. Effect of reversible androgen deprivation on
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34. 35.
36. 37.
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41. 42.
43. 44. 45. 46.
47. 48.
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hemoglobin and serum immunoreactive erythropoietin in men. Accepted for publication, Am J Hematol. Kickler TS, Spivak JL. Effect of repeated whole blood donations on serum immunoreactive erythropoietin levels in autologous donors. JAMA 1988; 260: 65. Goodnough LT, Brittenham GM. Limitations of the erythropoietic response to serial phlebotomy: implications for autologous blood donor programs. J Lab Clin Med 1990; 115: 28. Sherwood JB, Goldwasser E, Chilcote R, et al. Sickle cell anemia patients have low erythropoietin levels for their degree of anemia. Blood 1986; 67: 46. Sun CH, Ward JH, Paul WL, et al. Serum erythropoietin levels after renal transplantation. N Engi J Med 1989; 321: 151. McGonigle RJS, Wallin JD, Shadduck RK, et al. Erythropoietin deficiency and inhibition of erythropoiesis in renal insufficiency. Kidney Intl 1984; 25: 437. Haga P, Cotes PM, Till JA, et al. Serum immunoreactive erythropoietin in children with cyanotic and acyanotic congenital heart disease. Blood 1987; 70: 822. Goodnough LT, Rudnick S, Price TH, et al. Increased preoperative collection of autologous blood with recombinant human erythropoietin therapy. N Engl J Med 1989; 321:1163. Baer AN, Dessypris EN, Goldwasser E, et al. Blunted erythropoietin response to anaemia in rheumatoid arthritis. Br J Haematol 1987; 66: 559. Hochberg MC, Arnold CM, Hogans BB, et al. Serum immunoreactive erythropoietin in rheumatoid arthritis: impaired response to anaemia. Arch Rheum 1988; 31: 1318. Spivak JL, Barnes DC, Fuchs E, et al. Serum immunoreactive erythropoietin in HIVinfected patients. JAMA 1989; 261: 3104. Miller CE, Jones RJ, Piantadosi S, et al. Decreased erythropoietin response in patients with anemia of malignancy. N Engl J Med 1990; 322: 1689. Chandra M, Clemons GK, McVicar MI. Relation of serum erythropoietin levels to renal excretory function: evidence for lowered set point for erythropoietin production in chronic renal failure. J Pediatr 1988; 113: 1015. Pincus T, Olsen NJ, Russell J, et al. Multicenter study of recombinant human erythropoietin in correction of anemia in rheumatoid arthritis. Am J Med 1990; 89: 161. Fischl M, Galpin JE, Levine JD, et al. Recombinant human erythropoietin for patients with AIDS treated with zidovudine. N Engl J Med 1990; 322: 1488. Ludwig H, Fritz E, Kotzmann H, et al. Erythropoietin treatment of anemia associated with multiple myeloma. N EngI J Med 1990; 322: 1693. Oster W, Herrmann F, Gamm H, et al. Erythropoietin for the treatment of anemia of malignancy associated with neoplastic bone marrow infiltration. J Clin Oncol 1990; 8:
956. 49. Wagemaker G, Visser TP. Erythropoietin-independent regeneration of erythroid progenitor cells following multiple injections of hydroxyurea. Cell Tissue Kinet 1980; 13: 505. 50. Koury M, Bondurant M. Erythropoietin retards DNA breakdown and prevents programmed cell death in erythroid progenitor cells. Science 1990; 248: 378. 51. Spivak JL, Pham T, Isaacs M, et al. Erythropoietin is both a mitogen and a survival factor. Blood 1991; 77: 1228. 52. Sawada K, Krantz SB, Dai C-H, et al. Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors. J Cell Physiol 1990; 142: 219. 53. MacDougall IC, Roberts DE, Neubert P, et al. Pharmacokinetics of recombinatn human
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erythropoietin in patients on continuous ambulatory peritoneal dialysis. Lancet 1989; 1: 425. DISCUSSION Braverman (Worcester): That was very interesting. The observations are reminescent of the changes in serum T4 and TSH concentrations, both well within the normal range, that occur with subtle decreases in T4. TSH rises, but only within the normal range, exactly the situation that you have reported with erythropoietin rising slightly with a drop in the hemoglobin as demonstrated in the men who were bled prior to surgery. It seems to be very similar to other endocrine systems. Spivak: I'm glad to hear that it's a more universal paradigm. I wanted to emphasize that because people think of this hormone as inherently going up the minute the hemoglobin changes, and that just isn't the case. Wheby (Charlottesville): Jerry, it was very nice. I think I've asked you this before and I still don't understand it. What is the situation of a compensated hemolytic state in which the bone marrow is sufficiently stimulated to compensate for the shortened red cell life span to prevent anemia? My question is, what keeps the marrow driven when there is no anemia? Spivak: The answer is, I think, in each one of us, the erythropoietin level is fixed just like the hemoglobin level is fixed, but amongst all of us it varies six fold. In someone with a compensated hemolytic anemia, what we don't know about is what normal was for them. If someone comes in with a compensated hemolytic anemia, and a hematocrit of 40% and reticulocytes of 7% and you did an erythropoietin level, I can tell you it will be normal. However, it might be normal at 24 whereas for them normal might have been 5. I think that's a conundrum. What I was trying to get at is that within that threshold, erythropoietin will go up. It is a very sensitive and very precise assay, but unless you know what the baseline is, within that six-fold range of normal those patients probably have an elevated erythropoietin level for them, but not necessarily outside the range of normal, because there is a threshold. Wheby (Charlottesville): That's what I was wondering when you showed the slide indicating that erythropoietin level didn't go up until hemoglobin reached about 10.5. Were those serial measurements in the same patient? Spivak: No. Wheby (Charlottesville): Does it go up for an individual patient as soon as the hemoglobin starts to fall? Spivak: Yes. I tried to indicate that in the men who were being phlebotomized. Within the normal range, for anyone of us if we get phlebotomized, or if we lower our oxygen saturation, you can see small changes, but unless you follow it serially in a given individual, you won't know that anything has changed because it's within normal, which as I say, has a six-fold range. Wheby (Charlottesville): Other factors can work, and you were showing testosterone. Do you know whether testosterone changes with hypoxia for instance. Spivak: That's a good question. I don't know if testosterone changes. To follow up on that, the erythropoietin receptor has analogies to the prolactin receptor and the growth hormone receptor, and the IL-3 receptor and IL-6 and IL-4 receptors. It is possible that there are other hormones which will stimulate erythroid progenitor cells in the presence or absence of erythropoietin. If you look at patients with Sickle Cell Anemia or you look at patients after renal transplant, in each case the erythropoietin level, until they get below a hematocrit of 32 or when they get up to a hematocrit of 32, erythropoietin switches down into the normal six-fold range, so that's the problem.
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Middleton (Buffalo): I was fascinated by the destruction of DNA in the cells in the absence of EPO and wondered whether or not this represents activation of an endonuclease in the absence of EPO. As I understand it, in other systems, programmed cell death is indeed dependent on activation of an endonuclease. Could you address that possibility? Spivak: Yes. This is not a finding alone of mine, it's a finding in other labs, for example, Koury at Vanderbilt in a different model, a late erythroid cell. As you suggest, programmed cell death is a commonality. If you take testosterone away from prostatic cells, they involute and they will show the same invariant, nonrandom DNA destruction. If you add cycloheximide back to these cells and block protein synthesis, but don't kill the cells, you can stop the programmed cell death. It is exactly as you say. There are some data that suggest that it's a calcium-activated endonuclease in the nucleus which is destroying the DNA. Middleton (Buffalo): Along the same lines, I wonder if EPO may have effects on other cell systems to prevent that kind of nuclease activation? Spivak: That's also an interesting question. There's been a bone of contention. Some geneticists at Hopkins gave mice an extra erythropoietin gene and the only thing that did was to make them very plethoric, but nothing happened to the white cells or the platelets. Erythropoeitin receptors have only been identified in erythroid progenitor cells. However, there has just been a recent article in PNAS, that very high levels of erythropoeitin will stimulate the proliferation of endothelial cells and there appears to be a receptor on endothelial cells. The levels have to be 20-50 times normal, so we're not talking about anything physiologic, but there may be some of the same mechanism involved, at least for endothelial cells, but not for any other cell. Bransome (Augusta): I'd like to refer to your six-fold variation in endogenous erythropoietin levels. This is erythropoietin measured by radioimmunoassay. Erythropoietin, of course, is inactive unless glycosylated. The glycosylation as with all other polypeptides I know of, I suspect varies from individual to individual. I have two questions. Has anyone looked at the variations in glycosylation with electrophoresis such as from an analytical point of view, can at least, begin to be approached? Is the glycosylation of this hormone regulated with changes in physiologic state? Spivak: Those are excellent questions. Erythropoietin is extremely heavily glycosylated. Some hematopoietic growth factors are hardly glycosylated at all. Erythropoeitin has to be glycosylated or it goes right out of the circulation and it loses biologic activity. The glycosylation is invariant in the sense that the recombinant molecule made in Chinese hamster ovary cells is identical in its glycosylation to native erythropoietin. We have done biologic assays and they correlate directly with the immunoassays. If you had variant glycosylation we would have a big problem. So far, with this assay, and all the other assays like it, the correlation of biologic activity and immunoactivity has been invariant. When we're measuring this range of levels, there is only biologically active erythropoietin. In reflection of the Metzger Lecture we heard earlier, unlike the POMC molecule, erythropoietin has virtually no post-translutional modification, and so what the gene makes and what you see are exactly the same. Braverman (Worcester): A comment in reference to the question about testosterone levels in anoxia that was asked previously. In a study that we participated in (I didn't climb) the Medical Expedition up Mt. Everest, we studied all sorts of hormonal changes at sea level and as they went up the mountain. The highest that we were able to get a sufficient number of climbers was at 22,000 feet. There were some changes in thyroid function. All the men complained that their libido had markedly decreased, and we measured serum testosterone, prolactin, and LH and compared them at 22,000 feet as compared to sea level. There was absolutely no change. Mohler (Charlottesville): I enjoyed that very nice review Jerry. I wonder what happens
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to the erythropoietin level in dilutional anemia when the hematocrit may drop as low as 30 or the high 20's and there is still normal red cell mass. Spivak: I have no data on dilutional anemias, but as I say, in every other situation that we or anyone else has ever looked at, there is a threshold. Once the hematocrit falls below 32 or the hemoglobin 10.5 gms. you can bet the erythropoietin levels will go up. Mohler (Charlottesville): Even though the red cell mass is normal? Spivak: Unfortunately we've never measured red cell mass under these circumstances, so I can't answer that.
Medical historians of a future generation surely will consider the development of the hematopoietic growth factors for clinical use as a major milestone in medical progress. They also will note that, owing to the power of recombinant DNA technology, the development of these growth factors was so rapid that their clinical application preceded a full understanding of their physiology. This is true even for erythropoietin, the first of the hematopoietic growth factors to be discovered, probably because it is the only one which behaves like a hormone. Produced in the kidneys and to a small extent in the liver, erythropoietin acts on erythroid progenitor cells in the bone marrow to promote their proliferation. Although erythropoietin was first described forty years ago (1), twentyseven years elapsed before its purification (2) and thirty-five years before the erythropoietin gene was molecularly cloned (3, 4) and the recombinant protein became available for clinical trials (5, 6). In spite of this long time interval, the physiology for erythropoietin is not widely appreciated. Presumably this is because for most of its known existence, erythropoietin was only of interest to experimental hematologists since it was not available for clinical use nor was there a clinically applicable assay for the protein. The development of sensitive immunoassays for erythropoietin with the use of either the purified protein (7) or recombinant-derived reagents (8) provided an opportunity to carefully scrutinize the behavior of erythropoietin in the circulation and correlate this with various clinical states. Such observations have provided new insights into the physiology of erythropoietin in both health and disease and are the subject of this report. For an assay of a circulating hormone to be useful clinically, a number of criteria must be met. First, the concentration of the hormone in the circulation should directly reflect its production. Second, hormone * From the Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD. ** Supported in part by grant DK 16702 from the National Institute of Diabetes and Digestive and Kidney Diseases. t Address correspondence and reprint requests to Jerry L. Spivak, M.D., Division of Hematology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205.
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production should reflect demand for the hormone. Third, the hormone should be biochemically unique. Fourth, only one form of the hormone should be present in the circulation. Fifth, the metabolism of the hormone should be independent of its plasma level and the size of its target cell population. Finally, immunoreactive hormone should be identical to biologically active hormone. Erythropoietin fulfills all of these criteria. First, erythropoietin production is regulated at the level of its gene and the stability of its mRNA (9, 10) and independently of its plasma concentration (11). Second, there are no significant preformed stores of erythropoietin in the kidneys or the liver (12). Demands for additional erythropoietin are met by de novo production; tissue hypoxia stimulates erythropoietin synthesis while erythrocytosis suppresses erythropoietin production but never completely. Third, erythropoietin is a highly conserved protein which has no homology with any known protein (3, 4). Fourth, posttranslational modification of erythropoietin is minimal [loss of only Argl66 (13)] and only one form of the hormone is present in the circulation. Fifth, the plasma clearance of erythropoietin is sluggish (14) and independent of its either concentration (15) or the size of the erythroid progenitor cell pool (16). Finally, immunoreactive erythropoietin appears to approximate biologically active erythropoietin (8). As a further convenience to clinical interpretation, neither age nor gender influence the plasma level of erythropoietin (17) and the diurnal variation in the level of the hormone is small (18) and not appreciably magnified by hypoxia (19). Using immunoassays, it has been possible to derive a normal range for erythropoietin in the circulation of 4-24 mU/ml. Although small with respect to the levels achieved with tissue hypoxia, this still represents a 6-fold variation which may reflect in part the variation observed in the plasma clearance of the hormone (4-12 hours) in normal individuals (20, 21, 22). Although erythropoietin levels vary from person to person, they vary very little from day to day in a given individual in the absence of hypoxia, bleeding or anemia (17). This is in keeping with known constancy of the hemoglobin or hematocrit in a given individual even though there is a 10-15% variation in these determinations between individuals of the same gender. The small variance in the circulating erythropoietin levels which is a feature of its normal physiology is, for unknown reasons, lost with the development of end-stage renal disease (23). Although it is widely accepted that there is a log-linear inverse correlation between circulating erythropoietin and the hemoglobin or hematocrit level, it is not widely appreciated that this relationship is
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tightly regulated and varies with the hemoglobin level. Indeed, the presence of a threshold with respect to erythropoietin production is central to understanding the behavior of erythropoietin in health and disease. Studies of erythropoietin mRNA expression have revealed that there is constitutive production of the hormone in certain cells in the kidney (24). With tissue hypoxia, these cells do not make more erythropoietin but rather additional cells are recruited to produce the hormone (24). Recruitment occurs in an exponential fashion, and with restoration of tissue oxygen delivery, there is an exponential down regulation of the number of erythropoietin-producing cells in the kidney. That production of the hormone is tightly regulated is evident from observations of humans or animals subjected to hypoxia. Unless the hypoxia is extreme, the initial increase in plasma erythropoietin is followed by a decline back to the normal range even in the presence of continued hypoxia (25). This down-regulation is not due to accelerated metabolism of the hormone since plasma erythropoietin levels parallel renal erythropoietin production (26) but rather to the other compensatory mechanisms available to the body for improving tissue oxygenation. Should these mechanisms fail, erythropoietin production will continue at an elevated level (27). Early evidence of a threshold for acceleration of erythropoietin production was provided by studies of erythropoietin excretion in the urine in normal individuals, patients with polycythemia vera and patients with secondary erythrocytosis (28). With repeated phlebotomy, there was a log-linear increase in urinary erythropoietin excretion. In patients with polycythemia vera, a greater degree of hematocrit reduction was required before a comparable increase in urine erythropoietin excretion was observed while in patients with secondary erythrocytosis, whose urinary excretion of erythropoietin was normal when their red cell mass was elevated, phlebotomy induced increments in urinary erythropoietin at a higher hematocrit than in the other two groups (28). Taken together, it appears that in polycythemia vera, erythrocytosis per se suppressed erythropoietin production and increased the threshold required for recruitment of erythropoietin-producing cells while in compensated secondary erythrocytosis due to hypoxia, the threshold for such recruitment was reduced. That erythrocytosis per se could inhibit erythropoietin production even in the presence of mild hypoxia was elegantly demonstrated in mice made plethoric with methemoglobincontaining erythrocytes (29). Importantly with regard to studies of plasma erythropoietin, as mentioned above, all evidence to date indicates that neither the plasma erythropoietin level (15) nor marrow cellularity
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(16) appear to influence the plasma clearance of the hormone nor is there any effect of the plasma erythropoietin level on erythropoietin production (11). Tight regulation of erythropoietin production also occurs in chronic anemia as well as erythrocytosis. Thus, adult patients with sickle cell anemia ordinarily have high plasma erythropoietin levels but these are not as high as in patients with acquired hemolytic anemia (30). When patients with sickle cell anemia are exposed to a high oxygen tension, their erythropoietin level falls as does their reticulocyte count (31). When restored to ambient oxygen tension, there is a rapid rise in plasma erythropoietin but even though the patients are still anemic, this increase in erythropoietin synthesis is abruptly terminated and plasma erythropoietin falls rapidly (31). With the development of sensitive immunoassays, it was finally possible, for the first time, to quantitate the behavior of erythropoietin in normal individuals and several noteworthy observations have been made with respect to the regulation of erythropoietin production. First, within the normal range of hemoglobin (or hematocrit) for men and women (12-16 gm%), there is not an inverse correlation between hemoglobin and erythropoietin (17). Second, even though men have a higher red cell mass than women, their erythropoietin levels are identical (17). This could occur if some other factor was involved in the regulation of the red cell mass. The opportunity to test this hypothesis was provided in a group of men being given an LHRH agonist to suppress prostatic hypertrophy (32). With continued administration of this agonist, serum testosterone levels fell to castrate levels, and this was followed by a fall in the hemoglobin level of at least 1 gm% which was reversed when therapy with the LHRH agonist was discontinued. Throughout the six months of therapy and six months post therapy, while the hemoglobin was falling and recovering, there was no significant change in plasma erythropoietin (32). As shown in figures 1 and 2, there was an excellent correlation between hemoglobin and serum testosterone levels but no correlation of serum testosterone and erythropoietin levels. These data indicate that in men, within the normal range of hemoglobin testosterone is involved in regulating the red cell mass. To determine the effect of repeated phlebotomy on the circulating erythropoietin level, we examined erythropoietin levels in patients participating in an autologous blood donation program in preparation for elective surgery (33). As shown in Table 1, repeated weekly phlebotomies produced progressive anemia in spite of a small but significant increase in circulating erythropoietin within the normal range. This increase was associated with a small but significant increase in reticulocytes, but the
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JERRY L. SPIVAK
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TESTOSTERONE (ng/ml) FIG. 2. Correlation between testosterone and serum erythropoietin in men receiving the LHRH agonist nafarelin acetate. The data are from reference 32. response was insufficient to prevent the development of anemia as the phlebotomies were continued. The anemia was not a consequence of induced iron deficiency as there was no change in red cell protoporphyrin levels, and the patients were receiving supplemental oral iron. Similar observations have also been made by others in multiply phlebotomized individuals who were iron replete (34). Failure of mild anemia to induce increments in plasma erythropoietin outside the range of normal suggests that there is a threshold hemoglobin which must be reached before a significant increment in erythropoietin production occurs. That this is indeed the case has been demonstrated in several different ways. First, analysis of the hemoglobin level below
ERYTHROPOIETIN FROM BENCH TO BEDSIDE
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TABLE 1 Effect of Repeated Phlebotomies on Hematocrit, Reticulocyte Count and Serum Erythropoietin in Men Time
Hematocrit*
Reticulocyte* Count
(%o) (M) 42 ± 1.0 1.3 ± 0.1 Week 1 Week 2 1.7 ± 0.2t 39 ± 1.0t Week 3 38 ± 1.0t 2.3 ± 0.4t 3.2 ± 0.4t Week 4 35 ± 1.0t * Mean ± S.E.M. t Significantly different from Week 1 (p < 0.05) i: Significantly different from Week 1 (p < 0.025) The data are from reference 33.
Serum* Erythropoietin
(mU/ml) 13.2 ± 1.4 16.0 ± 1.3 17.6 ± 1.4t 22.4 ± 3.5t
which circulating erythropoietin levels were unequivocally elevated outside the normal range in patients with iron deficiency anemia yielded a value of 10.5 gm% (hematocrit 32%) (17). Second, in patients with sickle cell anemia, an unequivocal increment in serum erythropoietin only occurred below a hematocrit of 32% (35). Third, in patients postrenal transplantation, the circulating erythropoietin level fell to normal as the hematocrit exceeded 32% (36) and finally, in patients with a variety of anemias a significant increase in circulating erythropoietin was only seen when the hematocrit is below 32% (37). Thus, independent studies in different patient populations have yielded the same result i.e. a threshold exists below which the hemoglobin or hematocrit level must fall before the circulating erythropoietin level increases unequivocally outside the range of normal. This is not to imply that small increments in circualating erythropoietin do not occur with lesser degrees of anemia (or tissue hypoxia). They do (33, 38) but because increments are still within the range of normal, they would not be appreciated without serial measurements nor are such increments usually sufficient to prevent anemia if blood loss is repetitive over a short time interval. Thus, the tight regulation of erythropoietin production extends to the situation of mild anemia suggesting that the standard clinical definition of anemia is not synonymous with a degree of tissue hypoxia sufficient to elevate plasma erythropoietin above the range of normal. Several important predictions can be drawn from these observations. First, a random erythropoietin assay in a mildly anemic patient will probably not provide clinically useful information. Second, autologous blood donors should benefit from erythropoietin administration since their hemoglobin levels never reach the threshold for stimulating significant erythropoietin production (33). This prediction has been verified in a recent clinical trial (39).
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Although the inverse linear relationship between hemoglobin and circulating erythropoietin appears to hold true once the hemoglobin level falls below 10.5 gm% in uncomplicated situations such as iron deficiency anemia, certain disease states impact negatively on it. The inverse linear relationship is totally lost with renal disease. In other conditions such as rheumatoid arthritis (40, 41), AIDS (42) and cancer (43), erythropoietin production is inappropriately low with respect to the degree of anemia and in some patients with solid tumors, like patients with end-stage renal disease, the response is lost altogether (43). When viewed from the perspective of regression analysis, the slope of the erythropoietin:hemoglobin relationship in iron deficiency or anemia is -25.8 (R2 = 0.81) as compared with -12.6 (R2 = 0.32) for rheumatoid arthritis, -6.6 (R2 = 0.25) for AIDS and -4.9 (R2 = 0.09) for patients with solid tumors (17). The mechanisms involved in blunting the expected response to hypoxia are as yet unknown but the threshold concept may again be applicable. For example, although patients with end-stage renal disease lose the ability to maintain a sustained increase in erythropoietin production as they become anemic, they do not lose the ability to make substantial amounts of erythropoietin if they become sufficiently hypoxic (44). This is also true for patients with cancer (43) but in each case, the response is not sustained once the hypoxic stimulus is removed. Certain AIDS patients also appear to reset their threshold for erythropoietin production since with AZT administration, they produce more erythropoietin than other patients with comparable degrees of anemia (42). This type of analysis has a predictive value which is clinically useful since studies to date have confirmed that anemic patients with a blunted erythropoietin production will respond to administration of recombinant erythropoietin (45, 46, 47, 48). The tight regulation of erythropoietin production appears to provide a protective mechanism to avoid explosive increments in the erythroid progenitor cell pool since that pool expands exponentially with erythropoietin stimulation (49). There is also an apparent economy involved as well since erythroid progenitor cells at different stages of development have different requirements for erythropoietin. Thus, late erythroid progenitor cells are largely in cell cycle and appear only to require erythropoietin as a survival factor (50) while early erythroid progenitor cells appear to require erythropoietin as a mitogen as well as a survival factor (51) and probably in higher concentrations since they express fewer receptors for the hormone (52). Pharmacokinetic studies also indicate that sustained levels of circulating erythropoietin well above 100 mU/ml may not be efficient (53) since tissue receptors are probably saturated at that concentration.
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SUMMARY Erythropoietin is unique amongst the hematopoietic growth factors since it is the only one which behaves like a hormone. The development of sensitive immunoassays for erythropoietin have provided an opportunity to examine its physiology more closely than ever before. Although the classical inverse-linear correlation between erythropoietin and hemoglobin has been amply confirmed, it has also become apparent that this relationship is tightly regulated and is only apparent below a threshold hemoglobin and not fully operative within the normal range of hemoglobin values. Certain disease states blunt the response of erythropoietin-producing cells to anemia, and in some cases this appears to be due to a resetting of the threshold for response while in others there may be a dichotomy between activation of the machinery for erythropoietin gene expression and net protein synthesis. The tight regulation of erythropoietin production may be directed in part at preventing explosive increases in the red cell mass and in part may conform to the actual demands of erythroid progenitor cells for the hormone, since, at least in vitro, erythropoietin effects these progenitor cells differently according to their stage of maturation and sustained high levels of the hormone are not necessary for certain of the desired effects. REFERENCES 1. Reissmann KR. Studies on the mechanism of erythropoietin stimulation in parabiotic rats during hypoxia. Blood 1956; 5: 372. 2. Miyaki T, Kung CK-H, Goldwasser E. Purification of human erythropoietin. J Biol Chem 1977; 252: 5558. 3. Jacobs K, Shoemaker C, Rudersdorf R, et al. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 1985; 313: 806. 4. Lin F-K, Suggs S, Lin C-H, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci 1985; 82: 7580. 5. Winearls CG, Pippard MJ, Downing MR, et al. Effect of human erythropoietin from recombinatn DNA on the anaemia of patients maintained on chronic haemodialysis. Lancet 1986; 2: 1175. 6. Eschbach JW, Egrie JC, Downing MR, et al. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med 1987; 316: 73. 7. Sherwood JB, Goldwasser E. A radioimmunoassay for erythropoietin. Blood 1979; 54: 885. 8. Egrie JC, Cotes PM, Lane J, et al. Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin tracer and immunogen. J Immunol Meth 1987; 99: 235. 9. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1988; 242: 1412. 10. Goldberg MA, Gaut CC, Bunn HF. Erythropoietin mRNA levels are regulated by both transcriptional events and by changes in RNA stability. Blood 1989; 74: 191a.
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11. Fried W, Barone-Varelas J. Regulation of the plasma erythropoietin level in hypoxic rats. Exp Hematol 1984; 12: 706. 12. Schooley JC, Mahlmann LJ. Evidence for the de novo synthesis of erythropoietin in hypoxic rats. Blood 1972; 40: 662. 13. Recny MA, Scoble HA, Kim Y. Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. J Biol Chem 1987; 262: 17156. 14. MacDougall IC, Neubert P, Coles GA, et al. Pharmacokinetics of recombinant human erythropoietin in patients on continuous ambulatory peritoneal dialysis. Lancet 1989; 1: 425. 15. Spivak JL, Hogans BB. The in vivo metabolism of recombinant human erythropoietin in the rat. Blood 1989; 73: 90. 16. Piroso E, Flaharty K, Caro J, et al. Erythropoietin half-life in rats with hypoplastic and hyperplastic bone marrows. Blood 1989; 74: 270a. 17. Spivak JL, Hogans BB. Clinical evaluation of a radioimmunoassay for serum erythropoietin using reagents derived from recombinant erythropoietin. Blood 1987; 70: 143a. 18. Wide L, Bengtsson C, Birgegard G. Circadian rhythm of erythropoietin in human serum. Brit J Haematol 1989; 72: 85. 19. McKeon JL, Saunders NA, Murree-Allen K, et al. Urinary uric acid: creatinine ratio, serum erythropoietin, and blood 2,3-diphosphoglycerate in patients with obstructive sleep apnea. Am Rev Respir Dis 1990; 142: 8. 20. Flaharty KK, Caro J, Erslev A, et al. Pharmacokinetics and erythropoietic response to human recombinant erythropoietin in healthy men. Clin Pharmacol Ther 1990; 47: 557. 21. Urabe A, Takaku F, Mizoguchi H, et al. Effect of recombinant human erythropoietin on the anemia of chronic renal failure. Intl J Cell Clon 1988; 6: 179. 22. McMahon FG, Vargas R, Ryan M, et al. Pharmacokinetics and effects of recombinant human erythropoietin after intravenous and subcutaneous injections in healthy volunteers. Blood, 1990 (in press). 23. Chandra M, Clemons GC, McVicar M, et al. Serum erythropoietin levels and hematocrit in end-stage renal disease: influence of the mode of dialysis. Am J Kidney Dis 1988; 12: 208. 24. Koury ST, Koury MJ, Bondurant MC, et al. Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration. Blood 1989; 74: 645. 25. Abbrecht PH, Littell JK. Plasma erythropoietin in men and mice during acclimatization to different altitudes. J Appl Physiol 1972; 32: 54. 26. Jelkmann W. Temporal pattern of erythropoietin titers in kidney tissue during hypoxic hypoxia. Pflugers Arch 1982; 393: 88. 27. Milledge JS, Cotes PM. Serum erythropoietin in humans at high altitude and relation to plasma renin. J Appl Physiol 1985; 59: 360. 28. Adamson JW. The erythropoietin/hematocrit relationship in normal and polycythemia man: implications of marrow regulation. Blood 1968; 32: 597. 29. Kilbridge TM, Fried W, Heller P. The mechanism by which plethora suppresses erythropoiesis. Blood 1969; 33: 104. 30. Dover GJ, Spivak JL, Hogans BB, et al. Erythropoietin levels in adults and children with sickle cell disease: relation to age, sex, hemoglobin, and fetal hemoglobin levels. Blood 1987; 70: 134a. 31. Embury SH, Barcia JF, Mohandas N, et al. Effects of oxygen inhalation on endogenous erythropoietin kinetics, erythropoiesis and properties of blood cells in sickle-cell anemia. N Engl J Med 1984; 311: 291. 32. Weber J, Walsh PC, Peters CA, et al. Effect of reversible androgen deprivation on
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956. 49. Wagemaker G, Visser TP. Erythropoietin-independent regeneration of erythroid progenitor cells following multiple injections of hydroxyurea. Cell Tissue Kinet 1980; 13: 505. 50. Koury M, Bondurant M. Erythropoietin retards DNA breakdown and prevents programmed cell death in erythroid progenitor cells. Science 1990; 248: 378. 51. Spivak JL, Pham T, Isaacs M, et al. Erythropoietin is both a mitogen and a survival factor. Blood 1991; 77: 1228. 52. Sawada K, Krantz SB, Dai C-H, et al. Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors. J Cell Physiol 1990; 142: 219. 53. MacDougall IC, Roberts DE, Neubert P, et al. Pharmacokinetics of recombinatn human
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erythropoietin in patients on continuous ambulatory peritoneal dialysis. Lancet 1989; 1: 425. DISCUSSION Braverman (Worcester): That was very interesting. The observations are reminescent of the changes in serum T4 and TSH concentrations, both well within the normal range, that occur with subtle decreases in T4. TSH rises, but only within the normal range, exactly the situation that you have reported with erythropoietin rising slightly with a drop in the hemoglobin as demonstrated in the men who were bled prior to surgery. It seems to be very similar to other endocrine systems. Spivak: I'm glad to hear that it's a more universal paradigm. I wanted to emphasize that because people think of this hormone as inherently going up the minute the hemoglobin changes, and that just isn't the case. Wheby (Charlottesville): Jerry, it was very nice. I think I've asked you this before and I still don't understand it. What is the situation of a compensated hemolytic state in which the bone marrow is sufficiently stimulated to compensate for the shortened red cell life span to prevent anemia? My question is, what keeps the marrow driven when there is no anemia? Spivak: The answer is, I think, in each one of us, the erythropoietin level is fixed just like the hemoglobin level is fixed, but amongst all of us it varies six fold. In someone with a compensated hemolytic anemia, what we don't know about is what normal was for them. If someone comes in with a compensated hemolytic anemia, and a hematocrit of 40% and reticulocytes of 7% and you did an erythropoietin level, I can tell you it will be normal. However, it might be normal at 24 whereas for them normal might have been 5. I think that's a conundrum. What I was trying to get at is that within that threshold, erythropoietin will go up. It is a very sensitive and very precise assay, but unless you know what the baseline is, within that six-fold range of normal those patients probably have an elevated erythropoietin level for them, but not necessarily outside the range of normal, because there is a threshold. Wheby (Charlottesville): That's what I was wondering when you showed the slide indicating that erythropoietin level didn't go up until hemoglobin reached about 10.5. Were those serial measurements in the same patient? Spivak: No. Wheby (Charlottesville): Does it go up for an individual patient as soon as the hemoglobin starts to fall? Spivak: Yes. I tried to indicate that in the men who were being phlebotomized. Within the normal range, for anyone of us if we get phlebotomized, or if we lower our oxygen saturation, you can see small changes, but unless you follow it serially in a given individual, you won't know that anything has changed because it's within normal, which as I say, has a six-fold range. Wheby (Charlottesville): Other factors can work, and you were showing testosterone. Do you know whether testosterone changes with hypoxia for instance. Spivak: That's a good question. I don't know if testosterone changes. To follow up on that, the erythropoietin receptor has analogies to the prolactin receptor and the growth hormone receptor, and the IL-3 receptor and IL-6 and IL-4 receptors. It is possible that there are other hormones which will stimulate erythroid progenitor cells in the presence or absence of erythropoietin. If you look at patients with Sickle Cell Anemia or you look at patients after renal transplant, in each case the erythropoietin level, until they get below a hematocrit of 32 or when they get up to a hematocrit of 32, erythropoietin switches down into the normal six-fold range, so that's the problem.
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Middleton (Buffalo): I was fascinated by the destruction of DNA in the cells in the absence of EPO and wondered whether or not this represents activation of an endonuclease in the absence of EPO. As I understand it, in other systems, programmed cell death is indeed dependent on activation of an endonuclease. Could you address that possibility? Spivak: Yes. This is not a finding alone of mine, it's a finding in other labs, for example, Koury at Vanderbilt in a different model, a late erythroid cell. As you suggest, programmed cell death is a commonality. If you take testosterone away from prostatic cells, they involute and they will show the same invariant, nonrandom DNA destruction. If you add cycloheximide back to these cells and block protein synthesis, but don't kill the cells, you can stop the programmed cell death. It is exactly as you say. There are some data that suggest that it's a calcium-activated endonuclease in the nucleus which is destroying the DNA. Middleton (Buffalo): Along the same lines, I wonder if EPO may have effects on other cell systems to prevent that kind of nuclease activation? Spivak: That's also an interesting question. There's been a bone of contention. Some geneticists at Hopkins gave mice an extra erythropoietin gene and the only thing that did was to make them very plethoric, but nothing happened to the white cells or the platelets. Erythropoeitin receptors have only been identified in erythroid progenitor cells. However, there has just been a recent article in PNAS, that very high levels of erythropoeitin will stimulate the proliferation of endothelial cells and there appears to be a receptor on endothelial cells. The levels have to be 20-50 times normal, so we're not talking about anything physiologic, but there may be some of the same mechanism involved, at least for endothelial cells, but not for any other cell. Bransome (Augusta): I'd like to refer to your six-fold variation in endogenous erythropoietin levels. This is erythropoietin measured by radioimmunoassay. Erythropoietin, of course, is inactive unless glycosylated. The glycosylation as with all other polypeptides I know of, I suspect varies from individual to individual. I have two questions. Has anyone looked at the variations in glycosylation with electrophoresis such as from an analytical point of view, can at least, begin to be approached? Is the glycosylation of this hormone regulated with changes in physiologic state? Spivak: Those are excellent questions. Erythropoietin is extremely heavily glycosylated. Some hematopoietic growth factors are hardly glycosylated at all. Erythropoeitin has to be glycosylated or it goes right out of the circulation and it loses biologic activity. The glycosylation is invariant in the sense that the recombinant molecule made in Chinese hamster ovary cells is identical in its glycosylation to native erythropoietin. We have done biologic assays and they correlate directly with the immunoassays. If you had variant glycosylation we would have a big problem. So far, with this assay, and all the other assays like it, the correlation of biologic activity and immunoactivity has been invariant. When we're measuring this range of levels, there is only biologically active erythropoietin. In reflection of the Metzger Lecture we heard earlier, unlike the POMC molecule, erythropoietin has virtually no post-translutional modification, and so what the gene makes and what you see are exactly the same. Braverman (Worcester): A comment in reference to the question about testosterone levels in anoxia that was asked previously. In a study that we participated in (I didn't climb) the Medical Expedition up Mt. Everest, we studied all sorts of hormonal changes at sea level and as they went up the mountain. The highest that we were able to get a sufficient number of climbers was at 22,000 feet. There were some changes in thyroid function. All the men complained that their libido had markedly decreased, and we measured serum testosterone, prolactin, and LH and compared them at 22,000 feet as compared to sea level. There was absolutely no change. Mohler (Charlottesville): I enjoyed that very nice review Jerry. I wonder what happens
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to the erythropoietin level in dilutional anemia when the hematocrit may drop as low as 30 or the high 20's and there is still normal red cell mass. Spivak: I have no data on dilutional anemias, but as I say, in every other situation that we or anyone else has ever looked at, there is a threshold. Once the hematocrit falls below 32 or the hemoglobin 10.5 gms. you can bet the erythropoietin levels will go up. Mohler (Charlottesville): Even though the red cell mass is normal? Spivak: Unfortunately we've never measured red cell mass under these circumstances, so I can't answer that.
