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The anemia of ageing is not associated with increased plasma hepcidin levels
Blood Cells, Molecules, and Diseases 41 (2008) 252–254
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y b c m d
The anemia of ageing is not associated with increased plasma hepcidin levels Pauline Lee, Terri Gelbart, Jill Waalen, Ernest Beutler ⁎ Department of Molecular and Experimental Medicine The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e
i n f o
Article history: Submitted 25 June 2008 Available online 3 August 2008 (Communicated by E. Beutler, M.D., 25 June 2008) Keywords: Hemoglobin Inflammation Ferritin Immunoassay
a b s t r a c t It has been proposed that the anemia of ageing may be caused, at least in part, by elevated hepcidin levels, representing a response to increased IL-6 levels. Using a recently developed immunoassay, we have measured the plasma hepcidin levels of eight patients with the anemia of ageing and sex- and age-matched controls, and found that the levels of hepcidin were not increased in patients with the anemia of ageing. In contrast, patients with the anemia of inflammation have higher hepcidin levels than sex- and age-matched controls. In the overall group there was a strong correlation between serum ferritin levels and hepcidin levels, as has been found previously. © 2008 Elsevier Inc. All rights reserved.
Introduction Numerous studies show that there is a distinct decline in the hemoglobin levels with increasing age. In many elderly patients anemia is a consequence of diseases such as cancer, chronic inflammatory disorders, bleeding from benign lesions, or vitamin B12 or folate deficiency. However, even excluding such patients, the hemoglobin levels of the elderly are lower, particularly in males [1–4]. The modest decline in hemoglobin levels that occurs during ageing has gained potential clinical importance with the publication of a number of studies indicating that the outcomes of a variety of disorders, including heart failure [5], percutaneous coronary intervention [6], and acute myocardial infarction [7,8] are related to hemoglobin levels, even when confounding factors are taken into account. A prospective study of 5888 community-dwelling individuals with median 11.2 year follow-up (53,866 person years) led to the conclusion that overall mortality was increased in persons with lower hemoglobin levels, even when adjustments were made for comorbidities [9]. A “reverse J-shaped” relationship was found between hemoglobin levels and mortality [9], a pattern that has also been noted in patients with the acute coronary syndromes. The cause(s) of the anemia of ageing are not known. It has been suggested that increased levels of interleukin-6 that have been reported to occur in the course of ageing may stimulate hepcidin production. The consequent dysregulation of iron homeostasis could result in decreased erythropoiesis and anemia [4]. Accurate measurement of plasma hepcidin levels has proven to be technically difficult.
⁎ Corresponding author. Fax: +1 858 784 2083. E-mail address: [email protected] (E. Beutler). 1079-9796/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.06.005
They have been measured previously only using a mass spectroscopybased technique [10,11] which has a fairly large coefficient of variation [10], and one immunoassay [12], which has not been generally validated. A more reproducible immunoassay [13] has recently become commercially available, which has made it possible for us to measure plasma hepcidin levels in a group of patients with the anemia of ageing. Plasma levels of hepcidin in a few patients with the anemia of chronic inflammation were also determined, to validate the technique, since it is well known that chronic inflammation increases hepcidin levels. Materials and methods To study the anemia of ageing 20 white patients from the Scripps/ Kaiser study [14], aged 63–83 years of age were selected for study. Ten patients were anemic, with hemoglobin levels b12.4 g/dl for males and b11.4 g/dl for females. None of these patients had a known cause of anemia, although, as noted below, 2 patients were subsequently found not to meet the criteria of the anemia of ageing, and were removed from the analysis, one because the mean corpuscular volume (MCV) was increased to 105.1 fl, and one because of a diagnosis of autoimmune hemolytic anemia. All subjects had serum ferritin levels N18 ng/ml, serum transferrin saturations N15%, and serum creatinine levels b1.4 mg%. None had a history of inflammatory disease or of active neoplasia. Each of the anemic patients were age- and sexmatched with males with hemoglobin levels of N14.5 g/dl or females with hemoglobin levels N13.7 g/dl. Seven additional patients with inflammatory disease were studied, including some patients with active rheumatoid arthritis, some with lupus erythematosis, and others with an elevated erythrocyte sedimentation rate of unknown origin. Seven age-, race- and sexmatched controls served as the additional control group.
P. Lee et al. / Blood Cells, Molecules, and Diseases 41 (2008) 252–254
Fig. 1. Plasma hepcidin levels of five elderly anemic female and four elderly male patients with “the anemia of ageing” and age- and sex-matched non-anemic controls. One anemic male subject was excluded because he was found to have an elevated erythrocyte mean corpuscular volume (MCV) (plasma hepcidin = 90.7 ng/ml). One anemic female was excluded because she was found to have a diagnosis of autoimmune hemolytic anemia (plasma hepcidin = 41.9 ng/ml).
Frozen plasma from each patient was thawed and immunoassayed for hepcidin at Intrinsic LifeSciences, LLC, La Jolla, California, 92037 [13]. Results The results of the hepcidin assays of elderly patients with anemia are summarized in Fig. 1. The hepcidin levels of the anemic patients were actually lower, albeit not statistically significantly so, than the levels in the controls. Pooling the data from the eight anemic patients and 10 controls the mean hepcidin level (±1 S.D.) was 165.8 ±119.7 ng/ml and 195.6 ± 141.3 ng/ml, respectively. The corresponding median values were 146.4 and 164.7. There was no relationship between hemoglobin levels and plasma hepcidin levels. Fig. 2 presents a comparison of the hepcidin assays of elderly patients, as shown in Fig. 1, with patients with the anemia of chronic inflammation (ACI). In contrast to the patients with the anemia of ageing, the patients with chronic inflammation tend to have higher plasma hepcidin levels. Although the number patients studied was small, the pooled data from both sexes was statistically significant at p b 0.05 (Mann–Whitney test; one-tail).
253
Fig. 3. The relationship between serum ferritin and plasma hepcidin levels in all patients. The line is a linear regression with log transformation (p b 0.001; r2 = 0.41).
The serum ferritin concentration of all of the control, anemia of ageing, and anemia of chronic inflammation patients are compared with the plasma hepcidin levels in Fig. 3. Discussion It has been proposed that the anemia of ageing might be due to increased IL-6 levels leading to increased secretion of hepcidin [4]. The current studies fail to show such an effect. Although the number of patients studied was very small, the fact that both the mean and median hepcidin level was lower in the anemic patients rather than higher, the hypothesis that was being examined, it is unlikely that statistically significant higher values for plasma hepcidin concentrations would have been achieved even with vast expansion of the number of cases examined. The method used for estimating plasma hepcidin levels using this approach is validated, in part, by showing that the hepcidin levels measured correlate with serum ferritin concentrations and are increased in patients with chronic inflammation. Such changes have been documented previously using urinebased immunoassays [15–17] and mass spectroscopy-based serum assays [10,18]. Acknowledgments This is manuscript 19606-MEM from The Scripps Research Institute. This work was supported by grants from the National Institutes of Health grant AG029135–02 and the Stein Endowment Fund. References
Fig. 2. Hepcidin assays performed on patients with the anemia of ageing and age- and sex-matched controls (same data shown in Fig. 1) and patients with the anemia of chronic inflammation (ACI) and age/sex matched controls. While there is no increase in hepcidin levels in elderly patients with anemia, hepcidin levels are increased, on average, in patients with the anemia of chronic inflammation.
[1] D.A. Lipschitz, C.O. Mitchell, C. Thompson, The anemia of senescence, Am. J. Hematol. 11 (1981) 47–54. [2] E. Beutler, J. Waalen, The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration? Blood 107 (2006) 1747–1750. [3] M. Shayne, M.A. Lichtman, Hematology in older persons, in: M.A. Lichtman, E. Beutler, T.J. Kipps, U. Seligsohn, K. Kaushansky, J. Prchal (Eds.), Williams Hematology, McGraw-Hill, New York, 2006, pp. 111–121. [4] R. Eisenstaedt, B.W. Penninx, R.C. Woodman, Anemia in the elderly: current understanding and emerging concepts, Blood Rev. 20 (2006) 213–226. [5] G.M. Felker, K.F. Adams, W.A. Gattis, C.M. O'Connor, Anemia as a risk factor and therapeutic target in heart failure, J. Am. Coll. Cardiol. 44 (2004) 959–966. [6] P.C. Lee, A.S. Kini, C. Ahsan, E. Fisher, S.K. Sharma, Anemia is an independent predictor of mortality after percutaneous coronary intervention, J. Am. Coll. Cardiol. 44 (2004) 541–546. [7] E. Nikolsky, E.D. Aymong, A. Halkin, C.L. Grines, D.A. Cox, E. Garcia, R. Mehran, J.E. Tcheng, J.J. Griffin, G. Guagliumi, T. Stuckey, M. Turco, D.A. Cohen, M. Negoita, A.J. Lansky, G.W. Stone, Impact of anemia in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention — analysis from the Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications (CADILLAC) trial, J. Am. Coll. Cardiol. 44 (2004) 547–553. [8] M.S. Sabatine, D.A. Morrow, R.P. Giugliano, P.B.J. Burton, S.A. Murphy, C.H. McCabe, C.M. Gibson, E. Braunwald, Association of hemoglobin levels with clinical outcomes in acute coronary syndromes, Circulation 111 (2005) 2042–2049.
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P. Lee et al. / Blood Cells, Molecules, and Diseases 41 (2008) 252–254
[9] N.A. Zakai, R. Katz, C. Hirsch, M.G. Shlipak, P.H. Chaves, A.B. Newman, M. Cushman, A prospective study of anemia status, hemoglobin concentration, and mortality in an elderly cohort: the Cardiovascular Health Study, Arch. Intern. Med. 165 (2005) 2214–2220. [10] E.H. Kemna, H. Tjalsma, V.N. Podust, D.W. Swinkels, Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications, Clin. Chem. 53 (2007) 620–628. [11] A.T. Murphy, D.R. Witcher, P. Luan, V.J. Wroblewski, Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry, Blood 110 (2007) 1048–1054. [12] G. Dallalio, T. Fleury, R.T. Means, Serum hepcidin in clinical specimens, Br. J. Haematol. 122 (2003) 996–1000. [13] T. Ganz, G. Olbina, D. Girelli, E. Nemeth, M. Westerman, Immunoassay for human serum hepcidin. Submitted for publication.
[14] E. Beutler, V.J. Felitti, J.A. Koziol, N.J. Ho, T. Gelbart, Penetrance of the 845G–NA (C282Y) HFE hereditary haemochromatosis mutation in the USA, Lancet 359 (2002) 211–218. [15] S.L. Kearney, E. Nemeth, E.J. Neufeld, D. Thapa, T. Ganz, D.A. Weinstein, M.J. Cunningham, Urinary hepcidin in congenital chronic anemias, Pediatr. Blood Cancer 48 (2007) 57–63. [16] E. Nemeth, E.V. Valore, M. Territo, G. Schiller, A. Lichtenstein, T. Ganz, Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein, Blood 101 (2003) 2461–2463. [17] E. Nemeth, A. Roetto, G. Garozzo, T. Ganz, C. Camaschella, Hepcidin is decreased in TFR2 hemochromatosis, Blood 105 (2005) 1803–1806. [18] N. Tomosugi, H. Kawabata, R. Wakatabe, M. Higuchi, H. Yamaya, H. Umehara, I. Ishikawa, Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System(R), Blood 108 (2006) 1381–1387.
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y b c m d
The anemia of ageing is not associated with increased plasma hepcidin levels Pauline Lee, Terri Gelbart, Jill Waalen, Ernest Beutler ⁎ Department of Molecular and Experimental Medicine The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e
i n f o
Article history: Submitted 25 June 2008 Available online 3 August 2008 (Communicated by E. Beutler, M.D., 25 June 2008) Keywords: Hemoglobin Inflammation Ferritin Immunoassay
a b s t r a c t It has been proposed that the anemia of ageing may be caused, at least in part, by elevated hepcidin levels, representing a response to increased IL-6 levels. Using a recently developed immunoassay, we have measured the plasma hepcidin levels of eight patients with the anemia of ageing and sex- and age-matched controls, and found that the levels of hepcidin were not increased in patients with the anemia of ageing. In contrast, patients with the anemia of inflammation have higher hepcidin levels than sex- and age-matched controls. In the overall group there was a strong correlation between serum ferritin levels and hepcidin levels, as has been found previously. © 2008 Elsevier Inc. All rights reserved.
Introduction Numerous studies show that there is a distinct decline in the hemoglobin levels with increasing age. In many elderly patients anemia is a consequence of diseases such as cancer, chronic inflammatory disorders, bleeding from benign lesions, or vitamin B12 or folate deficiency. However, even excluding such patients, the hemoglobin levels of the elderly are lower, particularly in males [1–4]. The modest decline in hemoglobin levels that occurs during ageing has gained potential clinical importance with the publication of a number of studies indicating that the outcomes of a variety of disorders, including heart failure [5], percutaneous coronary intervention [6], and acute myocardial infarction [7,8] are related to hemoglobin levels, even when confounding factors are taken into account. A prospective study of 5888 community-dwelling individuals with median 11.2 year follow-up (53,866 person years) led to the conclusion that overall mortality was increased in persons with lower hemoglobin levels, even when adjustments were made for comorbidities [9]. A “reverse J-shaped” relationship was found between hemoglobin levels and mortality [9], a pattern that has also been noted in patients with the acute coronary syndromes. The cause(s) of the anemia of ageing are not known. It has been suggested that increased levels of interleukin-6 that have been reported to occur in the course of ageing may stimulate hepcidin production. The consequent dysregulation of iron homeostasis could result in decreased erythropoiesis and anemia [4]. Accurate measurement of plasma hepcidin levels has proven to be technically difficult.
⁎ Corresponding author. Fax: +1 858 784 2083. E-mail address: [email protected] (E. Beutler). 1079-9796/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.06.005
They have been measured previously only using a mass spectroscopybased technique [10,11] which has a fairly large coefficient of variation [10], and one immunoassay [12], which has not been generally validated. A more reproducible immunoassay [13] has recently become commercially available, which has made it possible for us to measure plasma hepcidin levels in a group of patients with the anemia of ageing. Plasma levels of hepcidin in a few patients with the anemia of chronic inflammation were also determined, to validate the technique, since it is well known that chronic inflammation increases hepcidin levels. Materials and methods To study the anemia of ageing 20 white patients from the Scripps/ Kaiser study [14], aged 63–83 years of age were selected for study. Ten patients were anemic, with hemoglobin levels b12.4 g/dl for males and b11.4 g/dl for females. None of these patients had a known cause of anemia, although, as noted below, 2 patients were subsequently found not to meet the criteria of the anemia of ageing, and were removed from the analysis, one because the mean corpuscular volume (MCV) was increased to 105.1 fl, and one because of a diagnosis of autoimmune hemolytic anemia. All subjects had serum ferritin levels N18 ng/ml, serum transferrin saturations N15%, and serum creatinine levels b1.4 mg%. None had a history of inflammatory disease or of active neoplasia. Each of the anemic patients were age- and sexmatched with males with hemoglobin levels of N14.5 g/dl or females with hemoglobin levels N13.7 g/dl. Seven additional patients with inflammatory disease were studied, including some patients with active rheumatoid arthritis, some with lupus erythematosis, and others with an elevated erythrocyte sedimentation rate of unknown origin. Seven age-, race- and sexmatched controls served as the additional control group.
P. Lee et al. / Blood Cells, Molecules, and Diseases 41 (2008) 252–254
Fig. 1. Plasma hepcidin levels of five elderly anemic female and four elderly male patients with “the anemia of ageing” and age- and sex-matched non-anemic controls. One anemic male subject was excluded because he was found to have an elevated erythrocyte mean corpuscular volume (MCV) (plasma hepcidin = 90.7 ng/ml). One anemic female was excluded because she was found to have a diagnosis of autoimmune hemolytic anemia (plasma hepcidin = 41.9 ng/ml).
Frozen plasma from each patient was thawed and immunoassayed for hepcidin at Intrinsic LifeSciences, LLC, La Jolla, California, 92037 [13]. Results The results of the hepcidin assays of elderly patients with anemia are summarized in Fig. 1. The hepcidin levels of the anemic patients were actually lower, albeit not statistically significantly so, than the levels in the controls. Pooling the data from the eight anemic patients and 10 controls the mean hepcidin level (±1 S.D.) was 165.8 ±119.7 ng/ml and 195.6 ± 141.3 ng/ml, respectively. The corresponding median values were 146.4 and 164.7. There was no relationship between hemoglobin levels and plasma hepcidin levels. Fig. 2 presents a comparison of the hepcidin assays of elderly patients, as shown in Fig. 1, with patients with the anemia of chronic inflammation (ACI). In contrast to the patients with the anemia of ageing, the patients with chronic inflammation tend to have higher plasma hepcidin levels. Although the number patients studied was small, the pooled data from both sexes was statistically significant at p b 0.05 (Mann–Whitney test; one-tail).
253
Fig. 3. The relationship between serum ferritin and plasma hepcidin levels in all patients. The line is a linear regression with log transformation (p b 0.001; r2 = 0.41).
The serum ferritin concentration of all of the control, anemia of ageing, and anemia of chronic inflammation patients are compared with the plasma hepcidin levels in Fig. 3. Discussion It has been proposed that the anemia of ageing might be due to increased IL-6 levels leading to increased secretion of hepcidin [4]. The current studies fail to show such an effect. Although the number of patients studied was very small, the fact that both the mean and median hepcidin level was lower in the anemic patients rather than higher, the hypothesis that was being examined, it is unlikely that statistically significant higher values for plasma hepcidin concentrations would have been achieved even with vast expansion of the number of cases examined. The method used for estimating plasma hepcidin levels using this approach is validated, in part, by showing that the hepcidin levels measured correlate with serum ferritin concentrations and are increased in patients with chronic inflammation. Such changes have been documented previously using urinebased immunoassays [15–17] and mass spectroscopy-based serum assays [10,18]. Acknowledgments This is manuscript 19606-MEM from The Scripps Research Institute. This work was supported by grants from the National Institutes of Health grant AG029135–02 and the Stein Endowment Fund. References
Fig. 2. Hepcidin assays performed on patients with the anemia of ageing and age- and sex-matched controls (same data shown in Fig. 1) and patients with the anemia of chronic inflammation (ACI) and age/sex matched controls. While there is no increase in hepcidin levels in elderly patients with anemia, hepcidin levels are increased, on average, in patients with the anemia of chronic inflammation.
[1] D.A. Lipschitz, C.O. Mitchell, C. Thompson, The anemia of senescence, Am. J. Hematol. 11 (1981) 47–54. [2] E. Beutler, J. Waalen, The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration? Blood 107 (2006) 1747–1750. [3] M. Shayne, M.A. Lichtman, Hematology in older persons, in: M.A. Lichtman, E. Beutler, T.J. Kipps, U. Seligsohn, K. Kaushansky, J. Prchal (Eds.), Williams Hematology, McGraw-Hill, New York, 2006, pp. 111–121. [4] R. Eisenstaedt, B.W. Penninx, R.C. Woodman, Anemia in the elderly: current understanding and emerging concepts, Blood Rev. 20 (2006) 213–226. [5] G.M. Felker, K.F. Adams, W.A. Gattis, C.M. O'Connor, Anemia as a risk factor and therapeutic target in heart failure, J. Am. Coll. Cardiol. 44 (2004) 959–966. [6] P.C. Lee, A.S. Kini, C. Ahsan, E. Fisher, S.K. Sharma, Anemia is an independent predictor of mortality after percutaneous coronary intervention, J. Am. Coll. Cardiol. 44 (2004) 541–546. [7] E. Nikolsky, E.D. Aymong, A. Halkin, C.L. Grines, D.A. Cox, E. Garcia, R. Mehran, J.E. Tcheng, J.J. Griffin, G. Guagliumi, T. Stuckey, M. Turco, D.A. Cohen, M. Negoita, A.J. Lansky, G.W. Stone, Impact of anemia in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention — analysis from the Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications (CADILLAC) trial, J. Am. Coll. Cardiol. 44 (2004) 547–553. [8] M.S. Sabatine, D.A. Morrow, R.P. Giugliano, P.B.J. Burton, S.A. Murphy, C.H. McCabe, C.M. Gibson, E. Braunwald, Association of hemoglobin levels with clinical outcomes in acute coronary syndromes, Circulation 111 (2005) 2042–2049.
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P. Lee et al. / Blood Cells, Molecules, and Diseases 41 (2008) 252–254
[9] N.A. Zakai, R. Katz, C. Hirsch, M.G. Shlipak, P.H. Chaves, A.B. Newman, M. Cushman, A prospective study of anemia status, hemoglobin concentration, and mortality in an elderly cohort: the Cardiovascular Health Study, Arch. Intern. Med. 165 (2005) 2214–2220. [10] E.H. Kemna, H. Tjalsma, V.N. Podust, D.W. Swinkels, Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications, Clin. Chem. 53 (2007) 620–628. [11] A.T. Murphy, D.R. Witcher, P. Luan, V.J. Wroblewski, Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry, Blood 110 (2007) 1048–1054. [12] G. Dallalio, T. Fleury, R.T. Means, Serum hepcidin in clinical specimens, Br. J. Haematol. 122 (2003) 996–1000. [13] T. Ganz, G. Olbina, D. Girelli, E. Nemeth, M. Westerman, Immunoassay for human serum hepcidin. Submitted for publication.
[14] E. Beutler, V.J. Felitti, J.A. Koziol, N.J. Ho, T. Gelbart, Penetrance of the 845G–NA (C282Y) HFE hereditary haemochromatosis mutation in the USA, Lancet 359 (2002) 211–218. [15] S.L. Kearney, E. Nemeth, E.J. Neufeld, D. Thapa, T. Ganz, D.A. Weinstein, M.J. Cunningham, Urinary hepcidin in congenital chronic anemias, Pediatr. Blood Cancer 48 (2007) 57–63. [16] E. Nemeth, E.V. Valore, M. Territo, G. Schiller, A. Lichtenstein, T. Ganz, Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein, Blood 101 (2003) 2461–2463. [17] E. Nemeth, A. Roetto, G. Garozzo, T. Ganz, C. Camaschella, Hepcidin is decreased in TFR2 hemochromatosis, Blood 105 (2005) 1803–1806. [18] N. Tomosugi, H. Kawabata, R. Wakatabe, M. Higuchi, H. Yamaya, H. Umehara, I. Ishikawa, Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System(R), Blood 108 (2006) 1381–1387.