International Journal of Cell Cloning 8:211-226 Suppl 1 (1990)

Serum Immunoreactive Erythropoietin in Health and Disease Jerry L. Spivak Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Key Wrds. Erythropoietin

Radioimmunoassay

Anemia

Erythrocytosis

Abstract. The currently available radioimmunoassay for erythropoietin (Epo) using recombinant reagents is an accurate, reproducible, sensitive and specific assay which can be used to identlfy whether lack of Epo is contributingto anemia and,by extension, whether therapy with recombinantEpo might be appropriate. Elevation of the serum Epo level with anemia suggests that a marrow abnormality is the cause of the anemia, while a “high” Epo level in a non-anemic or plethoric patient suggests the presence of hypoxia or autonomous Epo production. Liver disease can elevate the serum Epo level, while modest degrees of anemia do not affect it appreciably. The lowest Epo levels occur in polycythemia Vera, but in a particular patient this finding is not completely diagnostic.

Introduction Erythropoietin (Epo) is the only hematopoieticgrowth factor which behaves like a hormone. An obligatory factor for erythroid cells, Epo is produced primarily in the kidneys, but also to a small extent in the liver, and interacts with erythroid progenitor cells in the bone marrow,stimulating them into cell cycle, supporting the survival of the cycling progenitor cells and permitting their differentiation [l] . Since Epo behaves like a hormone, its concentration in the blood should provide a useful guide to states of Epo insufficiency or excess. While this is indeed true, accurate interpretation of the Epo concentrationrequires an understanding of the biology of the hormone. In this review, the clinical significance of the circulating Epo concentration will be discussed with reference to the biology of Epo. Correspondence: Jerry L. Spivak, M.D., Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Room 1033, Baltimore, MD 21218, USA. Received September 29, 1989; accepted for publication September 29, 1989. 0737-1454/90/$2,00/0 @AlphaMedPress

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Molecular Biology and Biochemistry of Epo There is a single gene for Epo which is located on chromosome 7 [2,3]. The gene encodes a protein of 166 amino acids [4,5], while human urinary Epo, as well as its recombinant derivative produced in vitro by Chinese hamster ovary (CHO) cells, contains only 165 amino acids, having lost a single arginine (166) residue as a consequence of post-translational modification [6]. Thus, genomic Epo and native and recombinant human Epo (rhEpo) are virtually identical. This is in contrast to other hormones, such as insulin and ACTH, which must be processed after synthesis to smaller molecules to obtain the active form of these proteins. Furthermore, the Epo gene is highly conserved through evolution with over 80% homology between the rodent, primate and human genes [7]. This high degree of homology has important implications with respect to the assay for Epo, as well as its biological behavior. For example, as might be expected from such a well-conserved gene, the hormone is active in mammals across species bamers. Furthermore, human urinary Epo and rhEpo produced in CHO cells are identical biochemically, functionally and immunologicallyand, as a consequence, the recombinant protein is a satisfactory surrogate for the native protein for purposes of immunoassay. In addition to being highly conserved, the Epo gene is constructed more like a housekeeping gene than an inducible one [8]. This is also of importance with respect to Epo production and the blood Epo concentration, since it implies that Epo is constitutively produced and, by extension, that Epo is never absent from the blood. Additionally, one could anticipate that an elevation in circulating Epo in response to hypoxia might not be sustained unless the hypoxia was severe, and this has proved to be true [9-111.

Physiology of Epo Epo production in the kidneys appears to be restricted to peritubular inter131. Epo production can be trigstitial cells located mainly in the outer cortex [E, gered by the induction of anemia or a reduction in ambient oxygen tension. Since both protein and RNA synthesis are required for Epo synthesis [14] and Epo levels in the blood do not increase detectably for at least two hours after exposure to hypoxia [IS],laggingbehind renal Epo levels [lo, 151, it is clear that no preformed stores of the hormone exist in the kidneys or presumably, the liver. The signal transduction mechanism by which hypoxia stimulatesEpo production involves a heme protein [16], and this protein appears to reside in the cells which synthesize the hormone [17]. Since hepatoma cells can be induced by hypoxia to synthesize Epo [16], it seems likely that the same mechanisms for stimulating Epo production are operative in the liver as well as the kidney. However, liver Epo production in the adult is normally severely restricted [18], and its contribu-

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tion to total Epo production and to the blood Epo level is small even with hypoxia or the anephric state. Situations in which there is liver cell damage and repair, however, do stimulate Epo production [19], and this may be most obvious in the absence of the kidneys [20]. Normally, only a few cells in the kidney appear to be producing Epo as determined by in situ hybridization studies using a cDNA probe for the hormone [E,131. Indeed, by current techniques for mRNA expression, it is difficult to detect Epo mRNA in the kidneys in the absence of anemia or hypoxia [MI. With the induction of hypoxia, there is an increase in Epo production. This does not, however, appear to be due to an increase in Epo production in individual cells, but rather to the recruitment of additional cells to produce the hormone [21]. This occurs in an exponential and watershed fashion throughout the inner cortex of the kidney, and with the correction of the hypoxia or anemia, the pattern is reversed [21]. Thus, it is apparent that Epo production is regulated at the level of gene expression, and this implies that the circulatinglevel of Epo reflects renal Epo production. This is an important concept since, if true, the blood Epo level can serve as a useful measure of the endocrine activity of the kidneys, and a single Epo measurement has didactic value.

Epo Pharmacokinetics The metabolism of Epo is, of course, central to this issue since a change in the catabolismof the hormone could alter its concentration in the blood and render a single measurement of little value as an indicator of renal Epo synthesis. Epo, which has an MW of 30,400 [22], is a highly charged, heavily glycosylated hydrophobic protein. When administered i.v., its initial volume of distribution is the plasma volume and, thereafter, its plasma clearance is best defined by a twocompartment model [23,24] with an initial distribution phase of approximately 30 min and a slower elimination phase of approximately six to nine hours [25]. Its metabolism is, thus, similar to other glycoprotein hormones which circulate in the blood and is much more sluggishthan the hematopoietic growth factor, GMCSF,the bulk of which is rapidly cleared from the plasma within minutes of its introduction [26]. Approximately 10%of the Epo produced each day is excreted in the urine, and ureteral ligation does not influence the plasma clearance of the hormone substantially [24]. Although Epo is internalized and degraded by its target cells [27], expansion of the bone marrow Epo progenitor cell pool does not cause a substantial change in the plasma clearance rate of the hormone. The exact site of catabolism of circulating Epo is unknown, but if its behavior is similar to other plasma glycoproteins, the liver with its galactosyl receptors should be the major site of degradation [23]. This may account for the observation that in patients with hepatic disease, serum Epo is sometimes elevated out of proportion to the degree of anemia [28]. Unfor-

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tunately, since metabolic turnover studies have not been performed in such patients, it is not possible to distinguish between decreased catabolismand increased hepatic synthesis of the hormone in this situation. The latter could be a consequence of liver cell damage or possibly tissue hypoxia not recognized clinically and due to alterations in hemoglobin oxygen affinity [29]. Finally, recent studies also suggest that elevation of circulating Epo by exogenous administrationdoes not affect its clearance [24]. Therefore, there appears to be no evidence that changes in the plasma clearance of Epo are involved in regulating its concentration in the blood. Thus, measurement of the Epo in plasma or serum appears to be a valid method of assessing the renal response to tissue hypoxia. Regulation of Epo Production In order to interpret the clinical significance of a serum Epo measurement, it is necessary to understand the normal constraints on Epo production. These constraints are operative not only with respect to hypoxia, but also with respect to anemia. Tissue hypoxia stimulates Epo production, but unless the hypoxia is severe or the normal compensatory mechanisms are impaired, Epo production is downregulated even though the hypoxic stimulus continues [9-111. From a physiologic perspective, this provides protection against excess Epo production, since the hormone is active at picomolar levels and will potentiate its own effect [30]. Furthermore, because expansion of the erythroid progenitor cell pool is exponential [31], it is imperative that Epo excess be avoided. The clinical consequences of such an excess when endogenous regulatory control has been overridden by administration of recombinant Epo have been graphically described [32]. With respect to anemia, similar controls appear to apply. Thus, there appears to be a threshold below which the hemoglobin level or hematocrit must fall before a substantial elevation of Epo occurs outside the normal range (4-26 mU/ml) [33]. This is illustrated in Figure 1 for an autologous blood donor undergoing repeated phlebotomies. This is not meant to imply that small increments in serum Epo cannot be identified within the normal range by serial measurements: they can be. However, such small increments will not stimulate erythropoiesis sufficiently to rapidly restore the red cell mass. This presumably serves as an additional control against hormone excess. It should, however, be noted in this regard that if anemia is profound, substantial elevations in serum Epo will occur. Such elevations should not be considered with alarm, however, since obviously they would only be transient if the bone marrow was responsive to the hormone.

Assays For Epo The earliest assays for Epo in plasma, serum or urine were of necessity bio-

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Fig. 1. Effect of repeated phlebotomies on hematocrit and serum immunoreactive Epo in an autologous blood donor. Only when the hematocrit fell below 35% with phlebotomy was there a substantial increase in serum Epo.

assays, since a purified preparation of Epo was not available [reviewed in 34 and 351. In vivo bioassays, while cumbersome and insensitive, did have a satisfactory degree of specificity which was sometimes lacking in in vitro bioassays. Although bioassays were satisfactorywhen Epo levels were substantiallyelevated, they were not useful for detecting changes in serum Epo when anemia was modest nor were these assays widely available. With the purification of Epo, radioimmunoassay (RIA) of the hormone was finally possible [36], and this provided a more sensitive method for detecting Epo.With the development of rhEpo, there was a sufficient supply of the hormone to develop an RIA which was uniform in its characteristics because it used recombinant Epo as the labeled antigen and a polyvalent rabbit antiserum to the recombinant protein as the antibody [37]. Since rhEpo and human urine Epo are essentially identical biochemically, immunologically and biologically, the RIA using recombinantderived reagents has proven to be a sensitive, specific and reproducible assay for Epo in serum. Most importantly, since it is an immunoassay, it has been demonstrated by comparing the RIA with the standard in vivo Epo bioassay that the Epo detected immunologically is identical to biologically active Epo [37]. Furthermore, because genomic and circulating

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Epo differ by only one amino acid, the RIA provides an accurate measure of biologically active Epo. Only two exceptions to this contention have been identified. Sherwood et al. [38]identified by Epo RIA several different proteins in the serum of both normal individuals and patients with end-stage renal disease, and most recently, Cotes et al., using a similar assay, detected a larger MW species in two children [39].The significance of these observations and the frequency with which they occur remains to be determined. From a statistical perspective, the commercially available RIA (Smith Nine Bioscience, Van Nuys, CA)which we have employed has an interassay coefficient of variation of 14% at a mean serum Epo level of 9.6m U / d to 8.6% at a level of 99.3mU/ml. The lower limit of sensitivity of the assay is 1.9mU/ml with a suggested normal range of 4-26mU/ml. The correlation between repeat determinations in the same patient was 0.997,and as is the case with the red cell mass, the circulating Epo level in a given individual remained constant over time in the absence of interveninghypoxia or anemia. Non-anemic smokers do not as a group have different Epo levels than nonsmokers [40],and neither gender, age nor menstrual cycle appear to have any influence on the Epo assay [33,40-43].Furthermore, malnutrition does not appear to influence the production of Epo in response to anemia [44].Finally, with careful measurements, a diurnal variation in serum Epo has been detected with the highest levels occurring at 1O:OO p.m. and the lowest levels at 8:OOa.m. [45,46].All of these values are within the normal range, and thus, the circadian rhythm should not have a significant impact on the clinical usefulness of the assay, particularly if the blood samples are always obtained at the same time of day. Whether this rhythm is preserved or even measurable when Epo production is increased is unknown. It is of interest that a diurnal variation has also been detected in urine Epo excretion [47],and it is worth noting that while the RIA for Epo is highly reliable for circulating Epo, it does not appear to be reliable for measuring Epo in the urine [48]. For purposes of the discussion that follows, only data obtained with current RIAs will be reviewed, since these assays combine both sensitivity and specificity in contrast to the bioassays which they supercede.

Epo Levels in Normal Individuals Since tissue hypoxia stimulates Epo production while an increase in tissue oxygenation suppresses production of the hormone, it can be inferred that an inverse relationship should exist between hemoglobin or hematocrit and the serum Epo level. While this is true in the presence of anemia, as demonstrated for uncomplicated iron deficiency anemia (Fig. 2), it is not true when the hematocrit is normal. Thus, as shown in Table I, normal men and women differ with respect to their hemoglobin level, but they do not differ with respect to their serum Epo level. When large numbers of patients are studied, a very small but statistically

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Fig. 2. Relationship between hemoglobin and serum immunoreactive erythropoietin in patients with uncomplicated iron deficiency anemia (adapted from reference 56). 0 = women; 0 = men

significantdifference between men and women has been demonstrated [42], but the difference (1.6 mU/ml) is neither physiologically significant nor likely to be detectable except as part of a population study. It has also not been demonstrated that an inverse correlation exists between hemoglobin and serum Epo with respect to this difference. The lack of correlation between hemoglobin and serum Epo in non-anemic men and women within the normal hemoglobin range occurs after puberty [41]. It reflects the production of androgenic steroids in men, since following castration, male hemoglobin levels fall to the female level [49]. The lack of correlation also fits in well with the observations mentioned earlier regarding autologousblood donors: that a modest degree of anemia must be incurred to initiate an appreciable increase in serum Epo above normal PO]. As a corollary, in anemic post-renal transplantationpatients, serum Epo levels returned to normal when the hematocrit reached 32% [51], and a similar threshold has been observed in patients with sickle cell anemia [52].

Serum Epo Levels in Renal Disease The kidney is the major site of Epo production, and renal disease is com-

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lsible I. Hemoglobin and serum immunoreactive erythropoietin in normal men and women

Hemoglobin (gm96) Serum erythropoietin (mU/ml)

Men

Women

15.1 f 0.1’ 12.0 f 0.8

13.4 f 0.1 11.0 f 0.7

monly associated with the development of anemia when the creatinine clearance falls below 30 ml/min 1531. Inevitably, circulating Epo levels fail to increase in patients who become anemic as a consequence of their renal disease. Indeed, they present with the putative conundrumof a “normal” serum Epo level in the presence of anemia. However, because of the inverse relationshipbetween Epo production and the red cell mass, an Epo level can only be interpreted in conjunction with a simultaneously determined hemoglobin or hematocrit. Thus, a “normal” serum Epo level in an anemic patient is actually inappropriately low. Indeed, the normal inverse correlation between circulating Epo, and the red cell mass is lost in- renal disease when the serum creatinine is greater than 1.5 mg% [33]. This is not to say that high levels of serum Epo do not occur in patients with renal disease. Indeed, with careful measurements,a feedback relationship, albeit blunted, between hemoglobin and serum Epo can be detected [54], and in some patients, hemorrhage or hypoxia can be associated with increases in serum Epo [55]. In others, liver disease due to viral infection, drugs or toxins has been associated with an increase in serum Epo, and even a temporary amelioration of anemia and transfusion requirements during the period of hepatic cell regeneration [19]. This supports the contention that +e lack of Epo is a principal cause of anemia in patients with end-stage renal disease and also that liver Epo is biologically similar to renal Epo.

Serum Epo Levels in Patients with Inflammation, Infection or Cancer Patients with renal disease are not the only ones in whom Epo production is impaired. Studies at our institution, as well as at others, have indicated that anemic patients with rheumatoid arthritis have a blunted Epo response to anemia [55,56]. A similar blunted response was observed in anemic patients with AIDS [57] and also in anemic patients with solid tumors [58]. In each group, the mean Epo level in the anemic patients was less than the comparable levels in individuals with uncomplicated iron-deficiency anemia and the same degree of anemia. While the mechanism for this is unclear, what is apparent is that in each of the three situations for any given decline in hematocrit, there w a s less of an increment in serum Epo than in uncomplicated iron deficiency anemia. Thus, the nor-

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mal feedback relationship appears to be impaired, and anemia in these disorders

is in part a consequence of the lack of Epo. When other potential complicating factors such as liver disease or malnutrition (as determined by the serum albumin level) were considered, these did not correlate with the serum Epo level. Although an increase in catabolism cannot be excluded, it seems likely that Epo production is suppressed in patients with infection, inflammation or cancer. Indeed, when AIDS patients were treated with zidovudine, their serum Epo levels increased exponentially, as compared with pretreatment values [57]. The importance of the blunted Epo response in the pathogenesis of anemia in these disorders can be appreciated by the fact that the anemia can be ameliorated by administration of recombinant Epo [59, 601.

Serum Epo Levels in Hemolytic Anemias In autoimmune hemolytic anemia, like iron-deficiency anemia, the normal inverse relationship between serum Epo and hemoglobin or hematocrit is maintained [33]. This is not a trivial observation, since with persistent hemolysis there is marrow erythroid hyperplasia, while irondeficiency anemia represents a state of marrow hypoproliferationwith respect to erythropoiesis. Thus, since the Epo response in the two disorders is similar, marrow erythroid activity, per se, appears to have little role as a determinant of serum Epo levels. Sickle cell anemia is a life-long disorder characterized by a chronic, uncompensated hemolytic anemia with marrow hyperplasia and hypertrophy. In contrast, however, to uncomplicated autoimmune hemolytic anemia, Epo levels in adults with sickle cell anemia are not as high for a given hemoglobin level as might be predicted [52,61]. There are several possible reasons for this. First, renal function declines with age in patients with sickle cell anemia, and this could affect Epo production [62]. Second, recurrent tissue infatction and the attendant inflammation could influence Epo production. Third, tissue oxygenation might be more satisfactory for any given level of anemia in this disease than for other diseases, since hemoglobin oxygen affinity is reduced in this disorder [63] and cardiac output is higher in these patients than in other patients with the same degree of anemia [ a ] . Serum Epo Levels In Pregnancy and Prematurity There is a progressive increase in serum Epo during pregnancy after the first eight weeks, with the highest levels being reached at term [43]. This may be a consequence of the physiologic anemia that occurs during pregnancy. It is of interest in this regard that the placental receptors for Epo have been identified, suggesting that the fetus in some species may benefit from the increase in maternal Epo production [65, 661. The ability to produce Epo in response to anemia or hypoxia is certainly im-

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paired in premature infants [67, 681, and a period of several weeks may be required before the Epo response is appropriate [68].

Serum Epo Levels in Patients with Erythrocytosis Until the development of recombinant Epo for therapeutic purposes, the Epo assay was most widely employed to clarify the mechanism for erythrocytosis, and an understanding of the behavior of Epo production in response to hypoxia and hypertransfusion is obviously essential to the use of the assay for this purpose. As mentioned previously, unless a hypoxic stimulus is extreme, and thus uncompensated, increases in Epo production will not be sustained. However, intermittent hypoxia is sufficient for increasing the red cell mass [69]. This may account for the variations in serum Epo observed in some patients with “unexplained” erythrocytosis [70]. Furthermore, an’increasein red cell mass can, for unknown reasons, suppress Epo production [71]. However, it is not possible to completely suppress Epo production by increasing the red cell mass [72]. Finally, correlations between clinical estimates of oxygen transport and serum Epo can never be exact because there is no precise method for assessing tissue oxygen delivery, and some patients may actually tolerate a venous oxygen tension below that which should trigger Epo production (731. Studies of urine Epo excretion suggest that with compensatoryerythrocytosis, Epo production is suppressed as expected, but its production threshold is changed, and synthesis of the hormone increases at hematocrit levels above that at which Epo production would be increased in normal individuals [74]. As a general rule, the serum Epo level is normal in patients with cyanotic congenital heart disease in whom hypoxia is compensated by an increase in red cell mass [75,76]. As mentioned above, phlebotomy can trigger a large increase in serum Epo. The serum Epo level is variable in patients with tumor-induced erythrocytosis, but is usually above normal [70]. In patients with chronic, obstructive lung disease, serum Epo levels are variable, and some patients with hypoxic erythrocytosis may have a normal serum Epo level, while in others the level is elevated [69]. It appears that in patients with chronic pulmonary disease, cigarette smoking or nocturnal oxygen desaturation are the major determinants of whether erythrocytosis develops [69]. In polycythemia Vera patients as a group, serum Epo levels are lower than in any other group of patients with erythrocytosis [33,42,77,78]. However, because of some overlap within the normal range in patients with secondary erythrocytosis [70,77], a serum Epo determinationcannot be used by itself to distinguish autonomous erythrocytosis from secondary erythrocytosis. Indeed, since polycythemia Vera is a clonal disorder, only a marker of clonality will suffice for this purpose. However, other clinical findings will usually suggest the correct diagnosis. It is only in the patient with isolated erythrocytosis that a diagnosis may

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not be evident. This, however, cannot be considered a severe problem, since phlebotomy, not chemotherapy, is the only appropriate treatment for reducing the red cell mass.

References 1 Spivak JL. The mechanism of action of erythropoietin. Int J Cell Cloning 1986;4: 139-166. 2 Powell JS, Berkner KL, Lebo RV, Adamson JW.Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization. Proc Natl Acad Sci USA 1986;83:6465-6469. 3 Law ML, Cai G-Y, Lin F-K, et al. Chromosomal assignment ofthe human erythropoietin gene and its DNA polymorphism. Proc Natl Acad Sci USA 1986;83:6920-6924. 4 Jacobs K, Shoemaker C, Rudersdorf R, et al. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 1985;313:806-810. 5 Lin F-K, Suggs S, Lin C-H, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci USA 1985;82:7580-7584. 6 Recny MA, Scoble HA, Kim Y,et al. Structural characterization of natural human urinary and recombinant DNAderived erythropoietin. J Biol Chem l987,262:17156-17163. 7 McDonald JD, Lin F-K, Goldwasser E.Cloning, sequencing, and evolutionary analysis of the mouse erythropoietin gene. Mol Cell Biol l986;6:842-848. 8 Shoemaker CB, Mitsock LD. Murine erythropoietin gene: cloning, expression, and gene homology. Mol Cell Biol 1986;6:849-858. 9 Abbrecht PH, Littell JK. Plasma erythropoietin in men and mice during acclimatization to different altitudes. J Appl Physiol 1972;32:54-58. 10 Jelkmann W. Temporal pattern of erythropoietintiters in kidney tissue during hypoxic hypoxia. Pflugers Arch 1982;393:88-91. 11 Milledge JS, Cotes PM. Serum erythropoietin in humans at high altitude and its relation to plasma renin. J Appl Physiol l985;59:360-364. 12 b u r y ST,Bondurant MC, b u r y MJ. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood 1988;71:524-527. 13 Lacombe C, DaSilva L, Bmneval P, et al. Pentubularcells are the site of erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest 1988;81:620-623. 14 Schooley JC, Mahlmann LJ. Evidence for the de naw, synthesis of erythropoietin in hypoxic rats. Blood 1972;40:662-670. 15 Schuster SJ, Wilson JH, Erslev AJ, Can, J. Physiologic regulation and tissue localization of renal erythropoietin messenger RNA. Blood 1987,70:316-318. 16 Goldberg MA, Dunning SP, Bunn HE Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1988;242:1412-1414. 17 Schuster SJ,Badiavas EV, Costa-Giomi P, Weinmann R, Erslev AJ, Car0 J. Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure. Blood 1989;73:13-16. 18 Bondumnt MC, b u r y MJ. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver. Mol Cell Biol 1986;6:2731-2733. 19 Meyrier A, Simon P, Boffa G, Brissot P.Uremia and the liver. Nephron 1981;29:3-6. 20 Naughton BA, Kaplan SM, Roy M, Burdowski AJ, Gordon AS, Piliero SJ. Hepatic regeneration and erythropoietin production in the rat. Science 1977;196:301-302.

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21 b u r y ST, b u r y MJ, Bondurant MC, Car0 J, Graber SE. Quantitation of erythropoietin-producingcells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietinmRNA, and serum erythropoietinconcentration. Blood 1989;74:645-651. 22 Davis JM, Strickland TW,Yphantis DA. Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells. Biochemistry 1987,26: 2633-2638. 23 Spivak JL, Hogans BB. The in vivo metabolism of recombinant human erythropoietin in the rat. Blood 1989;73:90-99. 24 Emmanouel DS, Goldwasser E, Katz AI. Metabolism of pure human erythropoietin in the rat. Am J Physiol l984;247Fl68-176. 25 MacDougall IC, Roberts DE, Neubert P, et al. Pharmacokinetics of recombinant human erythropoietin in patients on continuous ambulatory peritoneal dialysis. Lancet 1989;i:425-427. 26 Cebon J, Dempsey P, Fox R, et al. Pharmacokinetics of human granulocyte-macrophage colony-stimulatingfactor using a sensitive immunoassay. Blood 1988;72:l340-l347. 27 Sawyer ST, Krantz SB, Goldwasser E. Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells. J Biol Chem 1987,262: 5554-5562. 28 Harris ML, Spivak JL. Serum immunoreactiveerythropoietinlevels in patients with liver disease or inflammatory bowel disease. Gastroenterology l988;94:172a. 29 Farber MO, Carlone S,Serra P, et al. The oxygen affinity of hemoglobin in hepatic encephalopathy. J Lab Clin Med 1981;98:135-144. 30 Gurney CW,Wackman N, Filmanowicz E. Studies on erythropoiesis. XVII. Some quantitativeaspects of the erythropoietic response to erythropoietin. Blood 1961;17 531-546. 3 1 Wagemaker G, Visser TP. Erythropoietin-independentregeneration of erythroid progenitor cells following multiple injections of hydroxyurea. Cell Tissue Kinet 1980; l3:505-517. 32 Tomson CRV, Venning MC, Ward MK. Blood pressure and erythropoietin. Lancet 1988;i:351-352. 33 Spivak JL, Hogans BB. Clinical evaluation of a radioimmunoassay for serum erythropoietin using reagents derived from recombinanterythropoietin. Blood 1987; 70:143a. 34 Popovic WJ,Adamson JW.Erythropoietin assay: present status of methods, pitfalls, and results in polycythemic disorders. CRC Critical Rev Clin Lab Sci 1978;10:57-87. 35 Spivak JL, Sieber F. Erythropoietin. In: Fotherby K, Pal SB, eds. Hormones in Normal and Abnormal Human Tissues, Volume m. New York:Walter de Gruyter & Co., 1983~63-96. 36 Sherwood JB,Goldwasser E. A radioimmunoassay for erythropoietin. Blood 1979; 54~885-893. 37 Egrie JC, Cotes PM, Lane J. Gaines Das RE, Tam RC. Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin as tracer and immunogen. J Immunol Meth l987,99:235-241. 38 Sherwood JB, Carmichael D, Goldwasser E. The heterogeneity of circulating human serum erythropoietin. Endocrinology l988;122:1472-1477. 39 Cotes PM, Hellesbostad M, Tam RC, et al. Abnormal “big” immunoreactive erythropoietin in serum. Exp Hematol 1989;17590a. 40 Miller ME, Chandra J, Garcia JF. Clinical applications of measurement of serum immunoreactive levels of erythropoietin. AM NY Acad Sci 1985;459:375-381.

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41 Hellebostad M, HAgA P, Cotes PM. Serum immunoreactive erythropoietin in healthy normal children. Br J Haematol 1988;70:247-250. 42 Garcia JF, Ebbe SN, Hollander L, Cutting HO, Miller ME, Cronkite EP. Radioimmunoassay of erythropoietin: circulating levels in normal and polycythemic human beings. J Lab Clin Med 1982;99:624-635. 43 Cotes PM, Canning CE, Lind T. Changes in serum immunoreactive erythropoietin during the menstrual cycle and normal pregnancy. Br J Obstet Gyn 1983;90:304-311. 44 Wickramasinghe SN. Cotes PM, Gill DS, Tam RC, Grange A, Akinyanju 00. Serum immunoreactive erythmpoietin and erythmpoiesis in protein-energy malnutrition. Br J Haematol l985;60:515-524. 45 Cotes PM, Brozovic B. Diurnal variation of serum immunoreactive erythropoietin in a normal subject. Clin Endocrinol l982;17:419-422. 46 Wide L, Bengtsson C,Birgegard G. Circadian rhythm of erythropoietin in human serum. Br J Haematol 1989;72:85-90. Alexanian R, Martinez C, Finch CA.Erythropoietin excretion in nor47 Adamson JW, mal man. Blood 1966;28:354-364, 48 Cohen RA, Clemons G, Ebbe S. Correlation between bioassay and radioimmunoassay for erythropoietin in human serum and urine concentrates. Proc Soc Exp Biol Med 1985;179:296-299. 49 McCullagh EP, Jones R. Effects of androgens on blood count of men. J Clin Endocrinol Metab 1942;2:243-251. 50 Kickler TS,Spivak JL. Effect of repeated whole blood donations on serum immunoreactive erythropoietin levels in autologous donors. JAMA 1988;260:65-67. 51 Sun CH, Ward HJ, Pad WL, €kyle MA, Yanagrnva N, Lee DBN. Serum erybpoietin levels after renal transplantation. N Engl J Med 1989;321:151-157. 52 Sherwood JB,Goldwasser E, Chilcote R, Carmichael D, Nagel RL. Sickle cell anemia patients have low erythropoietin levels for their d e p of anemia. Blood l986;6746-49. 53 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;113:1015-1021. 54 Walle AJ, Wong, GY, Clemons GK, et al. Erythropoietin-hematocritfeedback circuit in the anemia of end-stage renal disease. Kidney Int 1987;31:1205-1209. 55 Baer AN, Dessypris EN, GoldwasserE, Krantz SB. Blunted erythropoietinresponse to anaemia in rheumatoid arthritis. Br J Haematol 198766559-564. 56 Hochberg MC, Arnold CM, Hogans BB, Spivak JL. Serum immunoreactive erythropoietin in rheumatoid arthritis: impaired response to anemia. Arth Rheum 1988;31: 13184321. 57 Spivak JL, Barnes CD, Fuchs E, Quinn TC.Serum immunoreactive erythropoietin in HIV-infected patients. JAMA 1989;261:3104-3107. 58 Miller CB, Jones RJ, Piantadosi S, Abeloff MD, Spivak JL. Decreased erythropoietin (EPO) response associated with the anemia of malignancy. Proc Am Soc Clin Oncol 1989;8:182a. 59 Rudnick SA.Human recombinant erythropoietin (r-HuEPO): a double-blind, placebocontrolled study in acquired immunodeficiency syndrome (AIDS) patients with anemia induced by disease and AZT. Proc Am Soc Clin Oncol 1989;8:2a. 60 Means W Jr, Olsen NJ, Krantz SB, et al. Treatment of the anemia of rheumatoid arthritis with recombinant human erythropoietin:clinical and in vitro studies. Arth Rheum 1989;32 :638-642. 61 Dover GJ, Spivak JL, Hogans BB, Sejeant GR. Erythropoietin levels in adults and children with sickle cell disease: relation to age, sex, hemoglobin, and fetalhemoglobin levels. Blood 1987,70:134a.

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62 Buckalew VM Jr, Someren A. Renal manifestations of sickle cell disease. Arch Intern Med 1974;133:660-669. 63 Milner PF. Oxygen transport in sickle cell anemia. Arch Intern Med 1974;133:565-572. 64 Varat MA, Adolph RJ, Fowler NO. Cardiovascular effects of anemia. Am Heart J 1972;83:415-426. 65 b u r y MJ, Bondurant MC, Graber SE, et al. Erythropoietin messenger RNA levels in developing mice and transfer of *2sI-erythropoietinby the placenta. J Clin Invest 1988;82:154-159. 66 Sawyer ST, Krantz SB, Sawada K. Receptors for erythropoietin in mouse and human erythroid cells and placenta. Blood 1989;74:103-109. 67 Shannon KM, Naylor GS, Torkildson JC, et al. Circulating erythroid progenitors in the anemia of prematurity. N Engl J Med 1987;317728-733. 68 Keyes WG, Donohue PK, Spivak JL, Jones MD Jr, Oski FA. Assessing the need for transfusion of premature infants and role of hematocrit, clinical signs, and erythropoietin level. Pediatrics 1989;84:412-417. 69 Wedzicha JA, Cotes PM, Empey DW, Newland AC, Royston JP, Tam RC. Serum immunoreactive erythropoietin in hypoxic lung disease with and without polycythaemia. Clin Sci 1985;69:413-422. 70 Cotes PM, Dore CJ, Liu Ym JA, et al. Determination of serum immunoreactive erythropoietin in the investigation of erythrocytosis. N Engl J Med 1986;315:283-287. 71 Kilbridge TM, Fried W, Heller P. The mechanism by which plethora suppresses erythropoiesis. Blood 1969;33:104-113. 72 Moccia G, Miller ME, Garcia JF, Cronkite EP. The effect of plethora on erythropoietin levels. Proc Soc Exp Biol Med 1980;163:36-38. 73 Charache S, Achuff S, Winslow R, Adamson J, Chervenick P. Variability of the homeostatic response to altered pso. Blood 1978;52:1156-1162. 74 Adamson JW.The erythropoietin/hematocritrelationship in normal and polycythemia man: implications of marrow regulation. Blood 1968;32:597-609. 75 Haga P, Cotes PM, Till JA, Minty BD, Shinebourne EA. Serum immunoreactive erythropoietin in children with cyanotic and acyanotic congenital heart disease. Blood 198770~822-826. 76 ”)mW MR, Teitel DF, Lutin WA, Clemons GK,Dallman PR. Serum erythropoietin levels in patients with congenital heart disease. J Pediatr 1987,110:538-544. 77 Koeffler HP, Goldwasser E. Erythropoietin radioimmunoassay in evaluating patients with polycythemia. Ann Intern Med 1981;94:44-47. 78 Birgegard G, Miller 0, Car0 J, Erslev A. Serum erythropoietin levels by radioimmunoassay in polycythaemia. Scand J Haematol 1982;29:161-167.

Discussion O’ReiUy: Do you have any data with regard to the effects of the cytoreductive regimen, for example, the Hiroshima-type doses of chemotherapy and radiation, which are used in terms of the cortical cells that generate erythropoietin? Spivak: I think that’s an excellent question, and we don’t have any data on that. There is the question of whether maintenance doses of therapy could have an affect, and we are looking at that right now. We have 70+ transplant patients, followed for over six months to see what happens to erythropoietin levels. The most recent data published in the British Journal of Haemtology suggests that it didn’t make a difference what cytoreductive regimen was used-they saw the same type of effect.

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Eridani: Have you observed any case of polycythemia due to inappropriate erythropoietin production? Spivak: I think I have, but sometimes it’s hard to prove. For example, we’ve had patients with renal carcinoma, and their erythropoietin levels were higher than they ought to have been, and sometimes the patient had erythrocytosis. I would say that in secondary forms of erythrocytosis you can generally expect the erythropoietin level to be higher than normal. Some patients with liver disease also have higher erythropoietin levels than normal. And I would say also this: if you take a cancer patient who’s anemic and has a low erythropoietin level, and that patient gets pulmonary metastasis and hypoxia, they can raise their erythropoietin levels. Therefore, the capacity is there, but the threshold for response has changed. I don’t understand the mechanism, but we have something to evaluate.

O’ReWy:You have described situations where there were clear non-relationshipsbetween hypoxia and erythropoietin production, and the query that comes up where you’re looking at the ultimate metastasis is: is it red cell mass or is it really going to be hypoxia alterations? Spivak: When we looked at, for example, AIDS patients, I wanted to be very careful because they a l l have opportunistic infections. We did blood gases in any patient we suspected might have a problem, and there was no correlation between blood gas measurements and erythropoietin levels in those patients. What I am trying to say is that all those who had inappropriately high erythropoietin levels were not hypoxic by blood gas measurements. We also did not find anyone with an inappropriately low level who was hypoxic. So, there was no correlation when we looked at blood gases. In cancer patients, however, when they got severely hypoxic, their erythropoietinlevels went up. Perhaps the AIDS patients were very suppressed.

Anonymous: Can you comment on the patients with pure red cell aplasia. I’m referring to papers discussing the possibility of autoantibodies against erythropoietinor against the erythropoietin receptors?

Spivak: I think that’s an interesting question. There is an article on a patient from Italy where there is a description of an antibody against erythropoietin, but any red cell aplasia patient that we’ve looked at has had a high erythropoietin level. I can’t say that I’ve looked at 100, because they’re hard to come by, but erythropoietin has been given to a lot of patients, over the past few years, and no one has an made antibody to it. I have a suspicion that it’s such a well-conserved protein that I doubt that we will see an anemia due to an erythropoietin antibody. Receptor antibodies are another story. The hormone is always present in the plasma. It’s constitutively made because it is vital for the maintenance of erythroid progenitor cell survival, and I have a feeling that it’s not in the body’s best interest to make an antibody against it.

Mitsuyasu: I have a question regarding those patients who restored their erythropoietin response after AZT. Did you find that those patients had become resistant to AZT or were they less likely to have a restoration of response? Is there any change in therapeutic effect here that may in fact be correlated with the erythropoietin response? Spivak: When we went back and looked at those who had higher erythropoietin levels and looked at viral titers before and after AZT, they hadn’t changed. And, when we looked at the titer of a number of HIV antibodies, they hadn’t changed. When we looked at their lymphocyte counts, they hadn’t changed. So, we couldn’t show that there was a change

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in the infection by those criteria, nor did their reticulocyte counts go up. These are probably the patients with the worst bone marrow failure, and they are now responding like other types of bone marrow failure patients. On the other hand, AZT may be doing something in the kidney, just like the bone marrow transplant-conditioning regimens.

O’Reilly: In conditions where you might have other controls over red cell mass, such as in the WW’ mouse, do you find compensatory elevations of erythropietin-or are they like the myelodysplastic patients, inappropriate in their response? Spivak: I don’t know if the WW’ mouse has been transfused and then studied, but WW‘

mice do have high erythropoietin levels. I don’t know if you transfuse them whether their erythropoietin levels will remain evaluated.

Peschle: I should like to comment about the antibody to erythropoietin in pure red cell aplasia. We observed only one out of 30 or 40 cases examined in a multicenter trial, which was done in the seventies, so I would agree that this is an extremely rare type of situation. However, at the experimental level, a similar situation has been observed-a situation in which an autoantibody against erythropoietin has been observed by a number of investigators, including ourselves. This was observed first by Schooley and then by others. This is the case where you inject a rabbit with human erythropoietin to raise an antibody. In some rabbits, it’s very clear that you get an autoimmune type of response, and the antibody neutralizes the human erythropoietin that was injected. But at a lower level, it’s also going to neutralize endogenous rabbit erythropoietin and there is actually going to be a very clear-cut relationship between injections of the exogenous erythropoietin, and the resulting antibodies will make the animal anemic. I think that it’s really a very interesting question which needs further study.

Serum immunoreactive erythropoietin in health and disease.

The currently available radioimmunoassay for erythropoietin (Epo) using recombinant reagents is an accurate, reproducible, sensitive and specific assa...
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