Circadian rhythm of serum erythropoietin in myelodysplastic syndromes

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1 European Review for Medical and Pharmacological Sciences 2000; 4: Circadian rhythm of serum erythropoietin in myelodysplastic syndromes P. PASQUALETTI, A. COLLACCIANI *, R. CASALE Department of Internal Medicine and Public Health, Faculty of Medicine and Surgery, University of L Aquila and Division of First Internal Medicine, S. Salvatore General Hospital of Coppito - L'Aquila, (Italy) * Division of General Medicine, Section of Hematology and Oncology, General Hospital of Tagliacozzo - L'Aquila (Italy) Abstract. Myelodysplastic syndromes (MSD) are a group of clonal disorders of the hemopoietic stem cell, often evolving in acute leukemia. They are characterized by anemia, and it has been attributed either to a deficiency in erythropoietin (EPO) secretion or to a resistance to EPO itself. Since in healthy subjects the serum circulating EPO levels fluctuate during the day, the aim of the study was to investigate the diurnal rhythm of EPO in MDS. Two groups of subjects were admitted to the study: (A) 20 adult clinically healthy subjects, and (B) 20 patients with MDS without renal failure. After standard life conditions in hospital lasting one week, venous blood samples were drawn during the span of a whole day and every four hours, starting from midnight, for the measurement of serum EPO levels by RIA. Statistical analysis was carried out by means of the cosinor method. The results show that the controls present a significant (p < 0.05) circadian rhythms in serum EPO levels with acrophase in the late afternoon; on the contrary, no significant (p > 0.05) rhythm was detected in patients with MDS. Patients with MDS presented significantly higher (p < 0.05) MESOR and lower (p < 0.001) amplitude of EPO circadian rhythm in respect to the controls; moreover, a significant (p < 0.005) difference was found between the two groups in overall EPO rhythm. These data confirm the existence of a physiological circadian rhythm in serum EPO concentrations with maximum in the afternoon. Because EPO levels are increased in the patient group, EPO deficiency does not seem to be the cause of anemia in MDS. Reduced EPO amplitude may be a compensatory mechanism for enhancing its activity in MDS. Finally, the stimulatory therapy in MDS with recombinant human EPO should be administered in the late afternoon hours, in order to respecting and simulating the physiological circadian rhythm of endogenous EPO. Key Words: Circadian rhythm, Erythropoietin, Myelodysplastic syndromes. Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hemopoietic disorders of the stem cell, characterized by abnormal hemopoietic differentiation and maturation; they manifest as peripheral cytopenias, with dysfunctional blood elements and have a high propensity for evolution into acute leukemia. The French-American- British (FAB) Cooperative Group 1 proposed the current morphological classification of MDS, based on the percentage of blast cells in blood and in bone marrow, degree of dyspoiesis, and additional features, such as ringed sideroblasts, monocytosis and Auer rods, into five subtypes: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess of blasts (RA.EB), refractory anemia with excess of blasts in transformation (RAEBt), and chronic myelomonocytic leukemia (CMML). More recently, the new World Health Organization classification of hematological malignancies 2 removed from the MDS classification RAEBt and CMML, and RAEB was split into two subgroups with medullary blasts belove or above 10%. Even if this new classification shows good reproducibility and reliability 3, the FAB classification is the most used and easily applicable 4. Anemia is a constant feature in MDS, ranging from moderate to severe and it is variable among FAB subtypes; anemia is usually characterized by macrocytosis and dual red cell populations on the peripheral blood film, whereas the bone marrow shows dyspoi- 111

2 P. PASQUALETTI, A. COLLACCIANI, R. CASALE etic megaloblastoid features with ineffective erythropoiesis 1,2,4,5. On the basis of these data, it has been hypothesized that the primary abnormality of the hemopoietic cells and their failure to grow adequately are due to a failure of response to cytokines 6, or that anemia is due directly to a deficiency in the renal EPO production 7. EPO is a glycoprotein produced mainly by the peritubular cells of the kidney and also by the liver. It has a predominant effect on the committed erythroid cells, colony-forming unit-erythroid, promoting their proliferation and differentiation into proerythroblasts. EPO may also stimulate the differentiation of a more primitive erythroid progenitor, the burst-forming unit-erythroid, in association with so-called burst-promoting activity; EPO also interacts with other hematopoietic growth factors to promote the production of megakaryocytes 8,9. EPO is detectable in the serum 10 and shows large fluctuations during the 24-hs period, with a well-marked circadian rhythm 11,12,13 with maximum levels in the afternoon, not influenced by the aging process 14. Therefore, the aim of the study was to investigate the circadian rhythms of the circulating serum levels of EPO in MDS, in order to verify whether these hematological disorders are associated with quantitative or qualitative changes in its circadian rhythm. After standard life conditions in hospital lasting one week, with sleep and/or rest period from 22:00 to 6:00, and meals at 8:00, 12:30 and 18:30 with free diet, venous blood samples were drawn from a peripheral vein of each subject during the span of a whole day and every four hours, starting from midnight. The serum levels of EPO were assayed in each sample by means of a commercial radioimmunoassay 10, with precipitation by the double antibody technique; the serum samples were assayed as a batch in two independent assays, and the EPO concentrations were recorded as the geometric mean values of these two estimates; the mean interassay coefficient of variation of estimates was 7.0%. The normal range using this technique for serum EPO concentration is U/L. The time related values of EPO were subjected to statistical analysis using chronograms (mean ± 1SD) and to inferential circadian statistical analysis by means of the mean-group cosinor method 15. This method is able to detect a significant (p < 0.05) circadian rhythm and the rhythm parameters: MESOR (average level of rhythm), amplitude (length from MESOR to acrophase), and acrophase (peak of rhythm). The circadian rhythms of EPO were compared among the two groups by means of the Hotelling s statistic test 15. Materials and Methods Two group of subjects were admitted to the study: Group A: 20 adult clinically healthy normocytemic subjects, 13 males and 7 females, aged years (mean age = 65.3 ± 6.4 yrs), with mean scrum hemoglobin = 13.5 ±1.7 gr/dl, and mean serum creatinine = 1.23 ± 0.14 mg/dl; Group B: 20 patients with MDS, 14 males and 6 females, aged years, (mean age = 73.2 ± 3.8), with mean hemoglobin = 8.2 ± 1.3 gr/dl, and mean serum creatinine = 1,15 ± All patients met the FAB diagnostic criteria for MDS 1 : there were 6 cases of RA, 4 of RARS, 5 of RAEB, 3 of RAEB-t, and 2 of CMML. The patients had not received drugs and/or blood transfusion in the month preceding the study. Results The serum levels of EPO fluctuated during the day in each group. When the data were analyzed by cosinor method, the controls (group A) presented a significant (p < 0.01) circadian rhythm for the circulating scrum EPO levels, with the acrophase in the late afternoon and the lowest values during nighttime; no rhythm (p > 0.05) was detected in MDS patients (group B). The MDS patients had significant (p < 0.01) higher MESOR and significant (p < 0.001) lower amplitude of EPO than the controls; significant difference (p < 0.005) was demonstrated between the overall rhythms of the two studied groups. Figure 1 illustrates in detail the results, both as chronograms and as derived by cosinor analysis. 112

3 Circadian rhythm of serum erythropoietin in myelodysplastic syndromes Figure 1. Diurnal fluctuation of serum erythropoietin in controls (group A) and in patients with myelodysplastic syndromes (group B), with the results of the cosinor analysis. The same results are plotted on polar coordinates: a rhythm is statistically significant (p < 0.05) when the ellipse region at 95% of probability does not overlap the center of the cosinor. Discussion This study extends to MDS our previous investigations on the circadian rhythm of EPO in course of multiple myeloma 16. Since the EPO circadian rhythm was preserved in patients without renal failure and lost in those with renal failure, we have hypothesized that the renal impairment in course of myeloma, and not the disease itself, is the main cause of anemia and of the loss of the EPO circadian rhythm in these patients. The present observations confirm that serum EPO presents in healthy subjects a definite circadian rhythm, with peak in the afternoon The mechanisms behind the observed circadian rhythm are still unclear. The accepted stimulus for EPO production is the tissue hypoxia 17,18, but this does not explain the rise in the afternoon hours of the hemopoietic growth factor. It is hypothesized that, since several of the hormones secreted or regulated by the pituitary gland show circadian rhythm 19, their rhythms may be involved in the control of the EPO circadian rhythm; moreover, changes in release rate and in metabolism of EPO and variations of the blood flow through the kidney during the day have to be considered 10-11, The diurnal rhythm of EPO is deranged in patients with MDS, suggesting, together with the observation of a higher MESOR, that EPO deficiency does not seems to have a fundamental role in the pathogenesis of anemia in the patients with MDS. Endogenous serum EPO levels in MDS are extremely variable; 113

4 P. PASQUALETTI, A. COLLACCIANI, R. CASALE approximately one-third of patients have levels that are inappropriately low for the degree of anemia 20, and an inverse relationship between the level of EPO and the degree of anemia was found 7. Moreover, no correlation bas been demonstrated between serum EPO concentration and total erythroid production 21. On the other hand, the lower amplitude supports the hypothesis of a resistance to the activity of the growth factor. In fact, the reduced amplitude, with constant daily serum levels, represents, from a chronobiological point of view, a compensatory mechanism: constant levels of any biological active substance have the same effectiveness of higher, but fluctuating, levels. A circadian difference exists in the susceptibility of the marrow to the effect of radiation, myelotoxic drugs and growth factor, and in vivo EPO administration enhances daily rhythms in erythroid colony numbers by increasing their amplitudes while leaving their circadian shapes virtually unchanged 22. The observation that the increment in erythroid colony numbers after EPO administration varies up to 16-fold with the time of day of treatment 22, and the present demonstrations that EPO presents its higher values in the afternoon and that this rhythm is lost in patients with MDS suggest that the therapy with recombinant human EPO, that actually represent a supportive therapy for the MDS patients, with a response in the 20-30% of cases 23,24, should be administered in this time of the day. This time of administration, in fact, should resemble its own physiological circadian rhythm, simulating and respecting the endogenous hemopoietic growth factor 25. In effect a study on patients with renal failure undergone to hemodialysis has demonstrated that the evening intravenous administration of recombinant human EPO seems to have a quicker therapeutic success 26. References 1) BENNETT JM, CATOVSKY D, FLANDRIN G, GALTON DAG, GRALNICK HR, SULTAN C. Proposal for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 51: ) HARRIS NL, JAFFE ES, DIEBOLD J et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-airlie House, Virginia, November J Clin Oncol 1999; 17: ) GERMING U, GATTERMANN N, STRUPP C, AIVADO M, AUL C. Validation of the WHO proposals for a new classification of primary myelodysplastic syndromes: a retrospective analysis of 1600 patients. Leuk Res 2000; 24: ) TEFFERI A. Chronic myeloid disorders: classification and treatment review. Semin Hematol 2001; 38: ) JACOBS A, CLARK RE. Pathogenesis and clinical variations in the myelodysplastic syndromes. Clin Hematol 1986; 15: ) MAY SJ, SMITH SA, JACOBS A, WILLIAMS A, BAILEY- WOOD R. The myelodysplastic syndromes: analysis of laboratory characteristics in relation to the FAB classification. Br J Haematol 1985; 59: ) JACOBS A, JANOWSKA-WIECZOREK A, CARO J, BOWEN DT, LEWIS T. Circulating erythropoietin in patients with myelodysplastic syndromes. Br J Haematol 1989; 73: ) ERSLEY AJ. Erythropoietin. N Engl J Med 1991; 324: ) KRANTZ SB. Erythropoietin. Blood 1991, 77: ) COTES PM. Immunoreactive erythropoietin in serum. Br J Haematol 1982; 50: ) WIDE L, BENGTSSON C, BIRGEGÅRD G. Circadian rhythm of erythropoietin in human serum. Br J Haematol 1989; 72: ) ALLEGRA A, BUEMI M, CORICA F, GIACOBBE MS, FRISINA N, CERUSO D. Erythropoietin: present and future. Rec Progr Med 1989; 90: ) PASQUALETTI P, CASALE R. Circadian rhythm of serum erythropoietin in healthy subjects. Eur Rev Med Pharmacol Sci 1996; 18: ) PASQUALETTI P, C ASALE R. No influence of aging on the circadian rhythm of erythropoietin in healthy subjects. Gerontology, 1997, 43: ) NELSON W, TONG YL, LEE JK, HALBERG F. Method for cosinor-rhythmometry. Chronobiologia 1979; 6: ) PASQUALETTI P, C OLLACCIANI A, CASALE R. Circadian rhythm of erythropoietin in multiple myeloma. Am J Hematol 1996; 53: ) FITZPATRICK F, MAC KAY T, WHYTE KF et al. Nocturnal saturation and serum erythropoietin: a study in patients with chronic obstructive pulmonary disease and in normal subjects. Clin Sci 1993; 84: ) CASALE R, PASQUALETTI P. Diurnal rhythm of serum erythropoietin circulating levels in chronic obstructive pulmonary disease. Panminerva Med 1997; 47:

5 Circadian rhythm of serum erythropoietin in myelodysplastic syndromes 19) NATALI GF, ACITELLI P, C ASALE R, COLANTONIO D, FESTUCCIA V, PASQUALETTI P. Circadian time structure of antero-pituitary, adrenocortical, gonadal and thyroidal hormones in healthy man. Rass Med Sper 1984; 31: ) STEIN RS, ABELS RI, KRANTZ SB. Pharmacologic doses of recombinant human erythropoietin in the treatment of mylodyspalstic syndromes. 1991; 78: ) BOWEN DT, JACOBS A, COTES PM, LEWIS TC. Serum erythropoietin and erythropoiesis in patients with myelodysplastic syndromes. Eur J Haematol 1990; 44: ) WOOD PA, HRUSHESKY WJ, KLEVECZ R. Distinct circadian time structures characterize myeloid and erythroid progenitor and multipotential cell clonogenicity as well as marrow precursor proliferation dynamics. Exp Hematol 1998; 26: ) ROSE EH, ABELS RI, NELSON RA, MC CULLOUGH DM, LESSIN L. The use of r-huepo in the treatment of anaemia related to myelodysplasia (MDS). Br J Haematol 1995; 89: ) SEIPELT G, OTTMANN OG, HOELZER D. Cytokine therapy in myelodysplastic syndromes. Curr Opin Hematol 2000; 7: ) HAYASHI N, KINOSHITA H, YUKAWA E, HIGUCHI S. Pharmacological analysis of subcutaneous erythropoietin administration with non linear mixed effect model including endogenous production. Br J Clin Pharmacol 1998; 46: ) BUEMI L. ALLEGRA A, LAGANÀ A, et al. Effects of the evening IV administration of erythropoietin in haemodialyzed patients. Eur Rev Med Pharmacol Sci 1993; 15: Acknowledgements This work was supported by grants of Ministero dell Università e della Ricerca Scientifica e Tecnologica (ex M.U.R.S.T. 60%). 115