Clinical Aspects and Laboratory Iron Metabolism, Anemias

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3 Manfred Wick Wulf Pinggera Paul Lehmann Clinical Aspects and Laboratory Iron Metabolism, Anemias Concepts in the anemias of malignancies and renal and rheumatoid diseases Sixth, revised and updated edition With contributions by Volker Ehrhardt

4 Dr. Manfred Wick Institute of Clinical Chemistry, Klinikum Grosshadern, University of Munich, Germany Univ.-Prof. Dr. Wulf Pinggera Maria Taferl, Austria Dr. Paul Lehmann Mainz, Germany With contributions by Dr. Volker Ehrhardt Roche Diagnostics GmbH, Mannheim, Germany This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. 1991, 1995, 1996, 2000, 2003, 2011 Springer-Verlag/Wien Printed in Austria The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product Liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case, the respective user must check its accuracy by consulting other pharmaceutical literature. Typesetting: Thomson Press (India) Ltd., Chennai Printing: Holzhausen Druck GmbH, 1140 Wien, Austria Printed on acid-free and chlorine free bleached paper SPIN Library of Congress Control Number: With 57 Figures ISBN th edn. SpringerWienNewYork ISBN SpingerWienNewYork

5 Foreword Anemias are a worldwide problem. Severe anemia affects mainly older women and men. The WHO defines anemia as a hemoglobin concentration of less than 12 g/dl in women and less than 13 g/dl in men (World Health Organization. Nutritional Anemias. Technical Reports Series 1992; 503). According to these criteria, 10 20% of women and 6 30% of men above the age of 65 years are anemic. In this book, we place a new emphasis on the diagnosis and treatment of anemias of chronic disease (ACD) and renal anemias. Nevertheless, iron deficiency remains globally the most important cause of anemia. There have been so many advances in the diagnosis and, in particular, the therapy of the anemias in recent years that it appeared necessary to extend the spectrum of therapies and diagnostic methods described. Apart from renal and inflammatory anemias, new insights regarding the role of transferrin receptor, the physiology of erythropoietin production, and the genetic defect as well as the pathogenesis of hemochromatosis demanded a major update of the book. The authors are grateful to Annett Fahle and Ralf Röddiger of Roche Diagnostics GmbH for their committed cooperation and their support in the publication of this book. November 2010 M. Wick W. Pinggera P. Lehmann

6 Table of Contents Introduction Iron Metabolism Absorption of Iron Iron Transport in the Circulation Transferrin, Iron-Binding Capacity and Transferrin Saturation Iron Storage Ferritins, Isoferritins Distribution of Iron in the Body Iron Requirement and Iron Balance Transferrin Receptor Soluble Transferrin Receptor Hepcidin Erythropoiesis Physiological Cell Maturation Hemoglobin Synthesis Erythropoietin Erythrocyte Degradation Phagocytosis of Old Erythrocytes Hemoglobin Degradation Fundamentals Disturbances of Iron Metabolism/Disturbances of Erythropoiesis and Hemolysis Disturbances of Iron Balance Iron Deficiency Disturbances of Iron Distribution Anemias of Malignancies and Anemias of Chronic Diseases Differentiation between Shortage of Depot Iron and Functional Iron Deficiency

7 VIII Table of Contents Fundamentals Clinical Aspects Disturbances of Iron Utilization Renal Anemias Pathophysiology of Erythropoietin Synthesis Iron Overload Primary Hemochromatosis Other Hereditary States of Iron Overload Other Disturbances of Erythropoiesis Disturbances of Stem Cell Proliferation Vitamin B 12 and Folic Acid Deficiency Hemoglobinopathies Disturbances of Porphyrin Synthesis Pathologically Increased Hemolysis Haptoglobin Features of Severe Hemolysis Causes of Hemolysis (Corpuscular/Extracorpuscular) Diagnosis of Disturbances of Iron Metabolism Iron Balance Case History and Clinical Findings Ferritin, Transferrin, Transferrin Saturation, Soluble Transferrin Receptor Laboratory Diagnostics of Suspected Disturbances of Iron Metabolism Most Frequent Disturbances of Iron Metabolism and Erythropoiesis Hypochromic, Microcytic Anemias Iron Deficiency Diagnosis and Therapy Laboratory Diagnostics in Cases of Suspected Iron Deficiency Clinical Pictures of Iron Deficiency Oral Administration of Iron Parenteral Administration of Iron Side-Effects and Hazards of Iron Parenteral Therapy Disturbances of Iron Distribution and Hypochromic Anemias Iron and Cellular Immunity Activation of the Immunological and Inflammatory Systems Therapy with Erythropoietin and i.v. Iron Administration

8 Table of Contents IX Anemias of Infection and Malignancy Biological Activity of Tumor Necrosis Factor Hemoglobin in Therapies with Cytostatic Drugs Erythropoietin and Iron Replacement in Tumor Anemias Anemias of Chronic Inflammatory Processes Anemias of Rheumatoid Arthritis Erythropoietin and Iron Therapy of Rheumatoid Arthritis Disturbances of Iron Utilization Uremic Anemia Therapy of Uremic Anemia Iron Overload Primary Hematochromatosis Secondary Hematochromatosis Non-Iron-Induced Disturbances of Erythropoiesis Macrocytic, Hyperchromic Anemia Folic Acid Vitamin B Normochromic, Normocytic Anemia Extracorpuscular Hemolytic Anemia Corpuscular Anemias Erythropoietin Therapy of Other Diseases Clinical Aspects Methods of Determination Serum/Plasma Parameters Iron Iron Saturation (Total Iron-Binding Capacity and Latent Iron-Binding Capacity) Iron-Binding Proteins Ferritin Transferrin Transferrin Saturation Soluble Transferrin Receptor Haptoglobin Laboratory

9 X Table of Contents Laboratory Ceruloplasmin Vitamin B Holotranscobalamin Folic Acid Homocysteine Erythropoietin Blood Count Automated Cell Counting Flow Cytometry Impedance-Based Cell Counters Hemoglobin Hematocrit Red Blood Cell Count Red Blood Cell Indices Reticulocyte Count Hemoglobin Content of Erythrocytes Erythrocyte Ferritin Zinc Protoporphyrin Tests for the Diagnosis of Chronic Inflammation Erythrocyte Sedimentation Rate C-Reactive Protein Interleukin Interleukin Tumor Necrosis Factor-α References Further Reading Subject Index

10 Introduction Disturbances of iron metabolism, particularly iron deficiency and iron redistribution are among the most commonly overlooked or misinterpreted diseases. This is due to the fact that the determination of transport iron in serum or plasma, which used to be the conventional diagnostic test, does not allow a representative estimate of the body s total iron reserves. In the past, a proper estimate was possible only by the costly and invasive determination of storage iron in the bone marrow. However, sensitive, well-standardized immunochemical methods for the precise determination of the iron storage protein ferritin in plasma are now available. Since the secretion of this protein correctly reflects the iron stores in the majority of cases, these methods permit fast and reliable diagnoses, particularly of iron deficiency status. In view of the high incidence of iron deficiency and its usual simple treatment, this fact should be common knowledge in the medical world. Even non-iron-related causes of anemia can now be identified rapidly by highly sensitive, well-standardized methods. We hope that this book will contribute to a better understanding of the main pathophysiologic relationships and diagnostic principles (Fig. 1) of iron metabo- Absorption Blood loss Hb synthesis Excretion Transport Storage Fig. 1: Physiological principles of iron metabolism

11 2 Introduction lism and anemias. However, the diagnosis of bone marrow diseases in the strict sense of the word, particularly if granulopoiesis or thrombopoiesis is involved, should remain the responsibility of hematologically experienced experts.

12 Iron Metabolism Absorption of Iron Iron, as a constituent of hemoglobin and cytochromes, is one of the most important biocatalysts in the human body. The absorption of iron by the human body is limited by the physico-chemical and physiologic properties of iron ions, and is possible only through protein binding of the Fe 2+ ion (Fig. 2). Fundamentals Fig. 2: Intestinal iron absorption DMT1: Divalent Metal Transporter; DCT1: Divalent Cation Transporter; Tf: Transferrin; HFE: Hemo-Iron; stfr: soluble Transferrin Receptor; Hep: Hepcidin

13 Fundamentals 4 Iron Metabolism Iron is absorbed as Fe 2+ in the duodenum and in the upper jejunum. Since iron in food occurs predominantly in the trivalent form it must (apart from the heme-bound Fe 2+ component) first be reduced e.g. by ascorbic acid (vitamin C). This explains why only about 10% of the iron in food, corresponding to about 1 mg per day, is generally absorbed. This daily iron intake represents only about 0.25 of the body s average total iron pool, which is approximately 4 g; this means that it takes some time to build up adequate reserves of iron. The actual iron uptake fluctuates considerably, depending on the absorption-inhibiting and absorptionpromoting influences in the upper part of the small intestine. The following factors inhibit absorption in clinically healthy individuals: reduced production of gastric acid, a low level of divalent iron as a result of an unbalanced diet (e.g., in vegetarians), a low level of reducing substances (e.g., ascorbic acid) in the food, or complex formation due to high consumption of coffee or tea. Conversely, absorption is promoted by a combination of a meat-rich diet with a plentiful supply of heme-bound iron and an acidic, reducing environment due to a high consumption of fruit and vegetables. Dietary iron is mostly present either as Fe 3+ or as heme. Whereas the absorption of heme is less completely characterized by the mechanism of Fe 3+ absorption has become clearer. It is assumed that it proceeds in two stages. At first, Fe 3+ is reduced by ferric reductase (duodenal cytochrome b) to Fe 2+ which is then transported across the cell membrane by a ferrous iron transporter (divalent metal transporter 1, DMT1 or DCT1). In the cells, Fe 2+ is stored bound to ferritin. Before export into the blood plasma by ferroportin, Fe 2+ is oxidized to Fe 3+ by an endooxidase (ceruloplasmin in macrophages, hephaestin in enterocytes). In the plasma, Fe 3+ is bound to transferrin [62]. The transport protein for iron, DTC1, is mainly localized in the duodenum within the enterocytic membrane and increases in concentration during alimentary iron deficiency. DTC1 is also expressed in the kidneys, liver, brain, and heart [62]. Within certain limits, the absorption of iron can be adjusted to meet the organism s iron requirement. Iron deficiency, anemia, and hypoxia lead via increased transferrin and DCT1 synthesis to an increase in the absorption and transport capacity. Two models were postulated for the regulation of iron absorption. In the so-called crypt programming model, immature crypt cells serve as iron sensors by uptake of Fe 3+ bound to transferrin via the basolateral

14 Iron Metabolism 5 membrane with the aid of the transferrin receptor. Essential for the normal function of the transferrin receptor is a protein encoded by the HFE gene. The amount of iron taken up by the immature enterocytes regulates the expression of the iron transporters described above. In case of iron deficiency, more transporters are exprimed; in case of iron overload, the expression of iron transporters is decreased. Consequently, after translocation of the matured enterocytes to the villi, iron absorption depends on the number of iron transporters exprimed in the immature cells. In the second model, the iron hormone hepcidin plays a central role in the regulation of iron recycling and iron balance. Hepcidin is a peptide sythesized by the liver. It inhibits iron uptake in the duodenum and the release of iron from intracellular storage pools. It is assumed that the inhibition of hepcidin is caused by binding to and inducing the degradation of ferroportin, the sole iron exporter in iron-transporting cells. The expression of hepcidin is regulated in response to anemia and hypoxia, and iron load. When oxygen delivery is inadequate, the hepcidin levels decrease and more iron is absorbed in the duodenum, and made available from intracellular iron stores. The opposite occurs with iron load. For the normal regulation of hepcidin levels, the intact HFE gene is required [10]. Fundamentals Iron Transport in the Circulation Iron is normally transported via the specific binding of Fe 3+ by transferrin in blood plasma [42]. The Fe 3+ -transferrin complex in turn is bound by transferrin receptors to cells of the target organs which allow specific iron uptake according to the individual needs of the various cells. Pronounced non-specific binding to other transport proteins, such as albumin, occurs in conditions of iron overload with high levels of transferrin saturation. When there is an excess supply of heme-bound iron, part of the Fe 2+ - heme complex may escape oxidation in the cells of the mucosa and be transported to the liver after being bound by haptoglobin or hemopexin. Transferrin, Iron-Binding Capacity, and Transferrin Saturation Transferrin (Fig. 3) is the most important and specific iron transport protein in the circulation. It is synthesized in the liver, and has a half-life

15 Fundamentals 6 Iron Metabolism Fig. 3: Transferrin crystals [64] of 8 12 days in the blood. Transferrin is a glycoprotein with a molecular weight of 79.6 kd, and β 1 electrophoretic mobility. Its synthesis in the liver may be increased as a corrective measure, depending on iron requirements and iron reserves. At present little is known about the details of the regulatory mechanisms involved. Transferrin is detectable not only in blood plasma, but also in many interstitial fluids, and a locally synthesized variant with a low neuraminic acid content (β 2 - or τ-transferrin) is found in the cerebrospinal fluid. The functional and immunologic properties of the many isoforms are substantially the same, the only important difference being the isoelectric point [42]. These forms are therefore of no practical interest with regard either to analytical technique or to the assessment of the iron metabolism (except CDT: diagnosis of alcoholism and β 2 -transferrin: CSF diagnostics). Each transferrin molecule can bind a maximum of 2 Fe 3+ ions, corresponding to about 1.41 μg of iron per mg of transferrin. By measurements of iron and transferrin concentrations, the total specific iron-binding capacity in plasma can be determined. Owing to its practicability, its low susceptibility to interference, and its high specificity, this method should be used to determine the iron transport by