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Erythropoietin and iron
Kidney International, Vol. 55, Suppl. 69 (1999), pp. S-49–S-56
Erythropoietin and iron JOACHIM P. KALTWASSER and RENE´ GOTTSCHALK Medizinische Klinik III, Zentrum der Inneren Medizin der J.W. Goethe-Universita¨t, Frankfurt am Main, Germany
Erythropoietin and iron. Serum ferritin concentration is most informative in estimating the amount of storage iron available for a particular individual. The serum transferrin receptor concentration, in contrast to serum ferritin, provides direct information about any deficit in the adequacy of iron supply to the erythropoiesis. The combination of serum transferrin receptor and serum ferritin provides complete information about storage and functional iron compartments. Using this combination along with the hemoglobin concentration, it is possible to define the iron nutritional status completely. Inflammatory conditions as well as parenteral iron administration interfere, however, with the direct and quantitative ferritin to storage iron relationship and, therefore, have to be considered carefully with respect to diagnostic purposes. The diagnostic use of the serum transferrin receptor is presently limited because of limitations in methodology and definition (standardization) of reference ranges.
aggregated form as hemosiderin. Ferritin is formed as an approximately spherical protein shell where, in its cavity, relatively large amounts of polynuclear hydrous Fe(III) oxide phosphate can be stored. Much of the body’s iron reserve is located in the cells of the macrophage-monocyte system, especially in the liver, spleen, bone marrow, and skeletal muscle. Iron stored in these sites can be mobilized and distributed via plasma transferrin to wherever it is needed. In the circulation, a tiny quantity of ferritin is found, which, in normal and iron-depleted subjects, is in close direct correlation with the total body iron reserve [1].
SERUM FERRITIN Ferritin and the transferrin receptor are important markers of two different compartments of iron metabolism. Ferritin is the major iron storage protein. In the physiology of iron metabolism ferritin has the important role of maintaining iron in a soluble, nontoxic, and biologically useful form. It has the capacity to sequester vast quantities of iron, and acts as a buffer against normal physiological variations in the iron requirements of tissues. The transferrin receptor is responsible for the mediation of cellular uptake of transferrin-bound iron. The receptor is predominantly present on the cell surface of erythroid progenitors. There is an increased synthesis of this receptor by erythroid cells under conditions of enhanced erythropoiesis, as well as during reduced iron supply to the bone marrow. In its truncated and in the circulation shedded form, the transferrin receptor may be regarded as a marker for the adequacy of iron supply to the erythroid marrow. FERRITIN Ferritin is a protein specialized for the storage of iron in tissues. Any iron that is surplus to immediate functional needs is stored in the tissue in ferritin or in its Key words: ferritin, iron storage, erythropoiesis, hemoglobin, transferrin receptor.
1999 by the International Society of Nephrology
There is a good correlation between the small amount of ferritin found in the serum (serum ferritin) and body iron stores [1, 2]. Serum ferritin concentrations are relatively stable in healthy persons. In patients with iron deficiency, serum ferritin concentrations are less than 12 mg/liter. A reduction of the level of reticuloendothelial stores is the only common cause of a low serum ferritin concentration. This is the key to the use of the serum ferritin assay in clinical practice [2, 3].
NORMAL VALUES The normal range for serum ferritin concentration in healthy subjects, based on hemoglobin as the selection criteria, was 15 to 300 mg/liter [2]. By using hemoglobin concentrations, histochemical bone marrow iron grading and/or measurement of intestinal iron absorption as indicators of a normal body iron status, however, normal values of 59 mg/liter (95% confidence range, 20 to 170 mg/liter) were observed in 44 healthy females and 91 mg/ liter (32 to 264 mg/liter) for 67 healthy males, respectively [1]. These normal ranges are in good accordance with findings of other groups [4, 5]. Significant differences in the ferritin concentration between men and women are consistent with the known difference in storage iron be-
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iron levels of approximately 20 g (Fig. 2). For clinical purposes, this variation must be considered when the amount of storage iron is calculated from serum ferritin measurements.
Fig. 1. Changes in serum ferritin concentration in healthy normal subjects as a function of age (according to Dallman et al [58]; used with permission).
tween the sexes (Fig. 1). In older men and women the mean concentrations are similar, while in children serum ferritin concentrations are generally lower than in adults.
CORRELATION TO IRON STORES A good correlation has been found between serum ferritin and storage iron, mobilized by phlebotomy, stainable iron in the bone marrow, and the concentration of both nonhem-iron and ferritin in the bone marrow [2]. There is also a significant inverse correlation between intestinal iron absorption and the logarithm of the serum ferritin concentration [4, 6]. This suggests that a close relationship exists between the total amount of storage iron and serum ferritin concentration in normal individuals. This correlation, however, does not exist in patients with iron overload caused by genetic hemochromatosis. Calculations derived from the correlation of serum ferritin with iron stores as determined by repeated phlebotomy suggest that in normal individuals, for each mg/liter ferritin there are 8 to 12 mg of storage iron [7, 8]. This ferritin-to-storage iron ratio, however, does not seem to be constant over the entire range from iron deficiency to heavy iron overload. Normal healthy volunteers with mobilizable iron stores ranging from 200 mg to 1.8 g were considered, together with patients with genetic hemochromatosis (iron stores 890 mg to 20.0 g). The ferritin-to-storage iron ratio decreased with increasing iron stores. At iron stores below 500 mg, the ratio averaged 15 mg storage iron per mg/liter of ferritin and decreased to a ratio of 6 mg per mg/liter of ferritin at storage
RED CELL FERRITIN The circulating erythrocytes contain a tiny residue of that ferritin present in its nucleated precursors in the bone marrow. The mean concentration in erythrocytes of the peripheral blood is approximately 10 ag/cell (10218 g/cell), measured with antibodies to H-type ferritin. The general red cell ferritin levels reflect the iron supply to the erythroid marrow and tend to vary inversely with the red cell protoporphyrin [9]. In untreated genetic hemochromatosis, red cell ferritin content is approximately 70 times the normal mean. Red cell ferritin was shown to reflect liver parenchymal cell iron concentrations [10]. High levels of red cell ferritin are also found in thalassemia, megaloblastic anemia, and myelodysplastic syndromes, presumably indicating a disturbance of erythroid iron metabolism in these conditions [2]. Despite these specific diagnostic advantages, the assay of red cell ferritin has little routine application thus far, mainly because it is necessary to have fresh blood in order to prepare red cells free of white cells, which have much higher ferritin levels [2]. SERUM FERRITIN AND INFLAMMATION Inflammation and neoplasia are associated with a significant rise in serum ferritin levels [11–16]. The response of serum ferritin levels to inflammation was found to parallel the acute phase plasma proteins, haptoglobin, and C-reactive protein [11, 14], suggesting that serum ferritin behaves as an acute phase reactant [17]. The degree of rise in serum ferritin concentration is, however, influenced by the iron status of the patient [14]. Serum ferritin most probably is a secretory protein because, in contrast to intracellular tissue ferritin, most of the circulating ferritin is glycosilated, representing the minor fraction of intracellular ferritin synthesized on membrane-bound polysomes [18–20]. Because of the behavior of ferritin as an acute phase reactant, inflammation can result in a false elevation of the ferritin values and can bring ferritin values for patients with true iron deficiency into the normal range [12]. With active inflammation, plasma levels of ferritin can rise sharply in the absence of any change in total body iron stores. These factors place limitations on the use of serum ferritin as a marker for iron deficiency, for example, in end-stage renal disease, but it remains the best available guide to the adequacy of iron stores prior to recombinant human erythropoietin (rHuEPO) therapy [21]. Once steady-state erythropoiesis and iron bal-
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Fig. 2. Correlation of storage iron mobilized by repeated phlebotomy, with serum ferritin concentration in 69 healthy male subjects (s) and 71 patients with genetic hemochromatosis (d) at various clinical stages. Note the direct and linear relationship in storage iron concentrations of less than 2.0 g and the remarkable changes of the ferritin to storage iron ratio in storage iron concentration of more than 2.0 g.
ance have been re-established, serum ferritin can again be used to assess iron stores. SERUM FERRITIN AND FUNCTIONAL IRON DEFICIENCY In otherwise healthy subjects, iron deficiency is considered “absolute” when iron stores are depleted or completely exhausted, as indicated by serum ferritin concentrations below the lower normal limits (mentioned earlier here). Complete exhaustion of iron stores normally is indicated by serum ferritin concentrations of less than 12 mg/liter [1] and inappropriate iron delivery to the erythroid marrow as evidenced by transferrin iron saturation (TISAT) levels below 15% [22]. In the specific situation of chronic renal failure (CRF), absolute iron deficiency has been defined as serum ferritin levels of less than 100 mg/liter and TISAT levels of less than 20% [23]. Besides the absolute iron deficiency, a different pattern of iron deficiency can occur when there is a need for a greater amount of iron to support hemoglobin synthesis than can be released from the reticuloendothelial iron stores. This situation, which can be caused by stimulation of erythropoiesis by erythropoietin or by limitation of storage iron release due to inflammatory processes, can occur in the presence of adequate or even increased iron stores. It has been called “functional” or “relative” iron deficiency [24]. Functional iron deficiency is a frequent issue associated with rHuEPO treatment in both renal anemia and the anemia of chronic disorders (ACD). It is also commonly observed when rHuEPO is used to increase autologous blood donation [25]. Patients with this condition do not meet traditional laboratory criteria for absolute
iron deficiency, but may demonstrate an increase in hemoglobin when i.v. iron is administered. Distinguishing between functional iron deficiency and an inflammatory iron block is a common clinical problem. The percentage of hypochromic red cells does appear to be a reliable and sensitive indicator for functional iron deficiency and has been shown to be helpful in the diagnosis of functional iron deficiency (Fig. 3) [26, 27]. Normally, there are less than 2.5% of red cells with individual cell hemoglobin of less than 28 pg/dl. Values exceeding 10% are compatible with functional iron deficiency in rHuEPOtreated patients. This measurement is, however, presently performed as part of routine full blood samples that requires a Technicont H-1, H-2, or H-3 automated cell counter (Bayer Diagnostics, Mu¨nchen, Germany), which may be not available in many medical centers in Europe or the United States [23]. Because of these limitations, currently the best tests of iron status are the percentage TISAT and the serum ferritin. Although no single value of TISAT or serum ferritin accurately discriminates between CRF patients who are or are not functionally iron deficient, available data demonstrate that the lower the TISAT and serum ferritin, the higher the likelihood that a patient is iron deficient, and the higher the TISAT and the serum ferritin, the lower the likelihood that a patient is iron deficient [23]. SERUM FERRITIN AND IRON TREATMENT Serum ferritin concentrations below the lower levels of normal ranges always indicate the presence of absolute iron deficiency and therefore are a clear indication for iron supplementation. Because the distinction between absolute and functional iron deficiency in CRF
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Fig. 3. Course of hemoglobin and percentage of hypochromic red cells (A) as well as serum ferritin and saturation of TIBC (B) in a female patient with severe inflammatory active rheumatoid arthritis treated with recombinant human erythropoietin (150 IU/kg s.c. twice a week) together with an intravenous iron supply (125 to 200 mg twice a week). Note the development of functional iron deficiency after initiation of EPO treatment as shown by the inappropriate hemoglobin increase and rise of hypochromic red cells above 10% (left hand side), whereas serum ferritin increased continuously and the saturation of TIBC remained below 20%. Abbreviations are: OP, start of surgical treatment (knee joint TEP); Recormont, recombinant human erythropoietin; Ferrlecitt, Fe(III) gluconate; Venofert, iron sucrose complex.
patients, specifically before or during EPO treatment, is more crucial, several recent consensus reports have recommended that serum ferritin should be kept at more than 100 mg/liter in order to maintain a sufficient iron reserve [21, 23, 28]. Most EPO-treated patients require intravenous iron to build up their respective iron stores. A small percentage of hemodialysis patients as well as many peritoneal dialysis and predialysis patients are also able to maintain adequate iron stores using only oral iron supplements [21, 23, 29]. Patients with initial ferritin levels of less than 100 mg/liter, however, will almost certainly require intravenous iron to support the requirements of the marrow during rHuEPO treatment [30]. During oral iron administration in therapeutic doses (100 to 200 mg/day) in absolute iron-deficient patients with unimpaired intestinal iron absorption, the serum ferritin concentration within the first 5 to 10 days increases up to normal levels and decreases again when increasing
hemoglobin regeneration takes place. A relevant stable increase in storage iron, as indicated by increasing ferritin levels, is observed only when hemoglobin regeneration reaches a steady state. In contrast, after i.v. iron administration (100 to 200 mg/day), serum ferritin increases disproportionally to the amount of storage iron present [31, 32]. Therefore, for serum ferritin to be used as an indicator of the iron stores rebuilt by i.v. iron supplementation, it should not be determined earlier than two weeks after the last i.v. iron dose. There is little information available that clearly establishes the upper limit of safety for serum ferritin in patients receiving i.v iron therapy. Serum ferritin levels between 300 and 800 mg/liter have been common in dialysis patients, and there is no evidence that such levels are associated with adverse iron-mediated effects [23]. Iron therapy should, however, be used or continued with extreme caution in patients whose serum ferritin is greater
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than 1000 mg/liter or where the TISAT level is above 50% [21].
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These findings were confirmed in subsequent studies [35, 36, 45]. In controlled phlebotomy studies, the receptor does not increase until iron stores are completely exhausted (Fig. 4) [47]. When serum ferritin fell below 12 mg/liter, the serum receptor increased above normal levels, roughly in proportion to the deficit in functional iron.
SERUM TRANSFERRIN RECEPTOR On actively growing human tumor cells, Sutherland et al have found a specific receptor for the iron transport protein transferrin [33]. By somatic hybrid cell studies, Goodfellow et al assigned the transferrin receptor (TRFR) locus to chromosome 3 [34]. The number of transferrin receptors on the cell surface is directly related to the erythroid activity [35–37]. The transferrin receptor molecule consists of two identical domains. On each domain, the binding of two molecules of transferrin is possible. Transferrin receptors are found on cell membranes and allow iron-bound transferrin to enter the cell. In the case of high saturation with iron, transferrin is bound with a significantly higher affinity to the binding sites of the receptor [38]. When the iron supply is inadequate, there is an up-regulation of transferrin receptors to enable the cell to compete more effectively for iron. As known with other membrane proteins, a shaded form of the transferrin receptor is known. This sTRF-R is detectable in the blood serum, and it is found as a truncated fragment of the transmembrane receptor. The number of membrane receptors is in proportion to the receptors found in the circulating blood plasma. In comparison to the complete molecule, sTRF-R lacks the cytoplasmic and the transmembrane domains [39]. Like serum iron and transferrin, sTRF-R shows a diurnal and seasonal pattern in its peak trough differences, but many authors confirmed that measurement of sTRF-R in serum is a sensitive indicator for both the total erythropoiesis and iron deficiency [35–38, 40–43].
CORRELATION TO IRON STORES Distinguishing between IDA and ACD remains unsatisfactory in some cases. Serum ferritin, serum iron, and TISAT are sometimes too low in value to classify the different forms of anemias. In particular, the combination of ACD and iron deficiency anemia, which is often seen, for example, in patients suffering from CRF or rheumatoid arthritis, could remain a diagnostic problem because serum ferritin levels are higher in patients with both IDA and ACD compared with those with absolute iron deficiency alone. sTRF-R in these special cases can distinguish between both forms because elevated sTRFR levels are characteristic for IDA regardless of whether there is chronic inflammation [48]. In contrast, sTRF-R remains unaffected in patients with anemia caused by chronic disorders [49–52]. In patients with anemia of CRF, Beguin et al demonstrated that sTRF-R could predict the response to rHuEPO therapy [53]. In this group of patients, the best response rates were shown when the sTRF-R (and fibrinogen) level was low [53]. This could be because responders to treatment with rHuEPO and intravenous iron administration probably show higher levels of sTRF-R during the therapy interval [54]. It was also shown that sTRF-R increases progressively for a six-week period and reaches a plateau when patients with CRF are treated with rHuEPO [55].
SERUM TRANSFERRIN RECEPTOR ASSAY
CONCLUSION
Measurement of sTRF-R is based on enzyme-linked immunosorbent essays (ELISA) with monoclonal antibodies against parts of the receptor molecule, and by latex agglutination nephelometric immunoassay [37, 38, 43, 44]. The normal range for serum transferrin receptor depends strongly on the method used for the determination of the protein [45]. In a study using a monoclonal ELISA technique, the mean value for 82 healthy males and females was 5.6 mg/liter, and for iron deficiency anemia it was 18 mg/liter [45]. However, validation of the different methods is missing thus far, and normal values independent from the underlying method are not available [46]. This is caused by the fact that purified transferrin receptors were not available, and standardization of the test systems was not possible. In patients suffering from iron-deficiency anemia (IDA), strictly elevated levels of sTRF-R were reported [37].
One of the major diagnostic problems is to distinguish iron deficiency from other causes of anemia, that is, anemia of chronic diseases. Because most of the tissue receptors have been found on the erythroid precursor cell, the level of sTRF-R measured in serum is directly proportional to erythroid activity and reflects the total body mass of tissue receptor [36, 38, 41, 56]. The serum ferritin level and serum transferrin receptor level can portray the entire spectrum of iron deficiency from normal to severe ID. Unlike serum ferritin, serum transferrin receptor remains normal in patients with acute or chronic inflammation or liver disease [47] and appears to be effective in distinguishing iron deficiency anemia from anemia of chronic disease [49]. This new parameter provides a quantitative measure of increased erythropoietic activity, for example, absolute or functional iron deficiency, and could be a useful
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alternative in distinguishing the associated anemia from that of chronic disease [56, 57]. Additionally, this parameter leads to a more stable measurement than transferrin saturation and is affected earlier in the development of functional iron deficiency than traditional hematological indices such as the erythrocyte protoporphyrin or mean corpuscular volume. On the other hand, it must be noted that determination of sTRF-R using different techniques leads to uncomparable results. Furthermore, the measurement of serum transferrin receptor is not routinely used in clinical practice because it is still expensive; it should only carried out in patients in whom the quantitation of hypochromic red cells is not available or when the determination of serum ferritin, serum iron, transferrin, and saturation of transferrin does not lead to an accurate classification of the type of anemia. Reprint requests to Dr. Joachim P. Kaltwasser, Medizinische Klinik III, Zentrum der Inneren Medizin, J.W. Goethe-Universita¨t, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: [email protected]
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Fig. 4. Course of serum ferritin and soluble transferrin receptor (sTRFR) during consecutive weekly phlebotomies (500 ml) of healthy male subjects (own data; P 0–P 5: phlebotomy week 0 to week 5; number of subjects in brackets). (A) The solid horizontal line indicates serum ferritin values in case of completely exhausted body iron stores. (B)
The solid horizontal line indicates the upper normal value of sTRF-R levels. The bars represent the upper and lower two-second interval. There is a continuous increase within the normal range of sTRF-R levels in relation to decreasing iron stores. sTRF-R values cross the upper normal value before the iron stores are completely depleted.
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56. Beguin Y, Clemons GK, Pootrakul P, Fillet G: Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin. Blood 81:1067–1076, 1993 57. Cook JD, Skikne BS, Baynes RD: Serum transferrin receptor. Ann Rev Med 44:63–74, 1993 58. Dallman PR, Siimes MA, Steckel A: Iron deficiency in infancy and childhood. Am J Clin Nutr 33:86–118, 1980
Erythropoietin and iron JOACHIM P. KALTWASSER and RENE´ GOTTSCHALK Medizinische Klinik III, Zentrum der Inneren Medizin der J.W. Goethe-Universita¨t, Frankfurt am Main, Germany
Erythropoietin and iron. Serum ferritin concentration is most informative in estimating the amount of storage iron available for a particular individual. The serum transferrin receptor concentration, in contrast to serum ferritin, provides direct information about any deficit in the adequacy of iron supply to the erythropoiesis. The combination of serum transferrin receptor and serum ferritin provides complete information about storage and functional iron compartments. Using this combination along with the hemoglobin concentration, it is possible to define the iron nutritional status completely. Inflammatory conditions as well as parenteral iron administration interfere, however, with the direct and quantitative ferritin to storage iron relationship and, therefore, have to be considered carefully with respect to diagnostic purposes. The diagnostic use of the serum transferrin receptor is presently limited because of limitations in methodology and definition (standardization) of reference ranges.
aggregated form as hemosiderin. Ferritin is formed as an approximately spherical protein shell where, in its cavity, relatively large amounts of polynuclear hydrous Fe(III) oxide phosphate can be stored. Much of the body’s iron reserve is located in the cells of the macrophage-monocyte system, especially in the liver, spleen, bone marrow, and skeletal muscle. Iron stored in these sites can be mobilized and distributed via plasma transferrin to wherever it is needed. In the circulation, a tiny quantity of ferritin is found, which, in normal and iron-depleted subjects, is in close direct correlation with the total body iron reserve [1].
SERUM FERRITIN Ferritin and the transferrin receptor are important markers of two different compartments of iron metabolism. Ferritin is the major iron storage protein. In the physiology of iron metabolism ferritin has the important role of maintaining iron in a soluble, nontoxic, and biologically useful form. It has the capacity to sequester vast quantities of iron, and acts as a buffer against normal physiological variations in the iron requirements of tissues. The transferrin receptor is responsible for the mediation of cellular uptake of transferrin-bound iron. The receptor is predominantly present on the cell surface of erythroid progenitors. There is an increased synthesis of this receptor by erythroid cells under conditions of enhanced erythropoiesis, as well as during reduced iron supply to the bone marrow. In its truncated and in the circulation shedded form, the transferrin receptor may be regarded as a marker for the adequacy of iron supply to the erythroid marrow. FERRITIN Ferritin is a protein specialized for the storage of iron in tissues. Any iron that is surplus to immediate functional needs is stored in the tissue in ferritin or in its Key words: ferritin, iron storage, erythropoiesis, hemoglobin, transferrin receptor.
1999 by the International Society of Nephrology
There is a good correlation between the small amount of ferritin found in the serum (serum ferritin) and body iron stores [1, 2]. Serum ferritin concentrations are relatively stable in healthy persons. In patients with iron deficiency, serum ferritin concentrations are less than 12 mg/liter. A reduction of the level of reticuloendothelial stores is the only common cause of a low serum ferritin concentration. This is the key to the use of the serum ferritin assay in clinical practice [2, 3].
NORMAL VALUES The normal range for serum ferritin concentration in healthy subjects, based on hemoglobin as the selection criteria, was 15 to 300 mg/liter [2]. By using hemoglobin concentrations, histochemical bone marrow iron grading and/or measurement of intestinal iron absorption as indicators of a normal body iron status, however, normal values of 59 mg/liter (95% confidence range, 20 to 170 mg/liter) were observed in 44 healthy females and 91 mg/ liter (32 to 264 mg/liter) for 67 healthy males, respectively [1]. These normal ranges are in good accordance with findings of other groups [4, 5]. Significant differences in the ferritin concentration between men and women are consistent with the known difference in storage iron be-
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iron levels of approximately 20 g (Fig. 2). For clinical purposes, this variation must be considered when the amount of storage iron is calculated from serum ferritin measurements.
Fig. 1. Changes in serum ferritin concentration in healthy normal subjects as a function of age (according to Dallman et al [58]; used with permission).
tween the sexes (Fig. 1). In older men and women the mean concentrations are similar, while in children serum ferritin concentrations are generally lower than in adults.
CORRELATION TO IRON STORES A good correlation has been found between serum ferritin and storage iron, mobilized by phlebotomy, stainable iron in the bone marrow, and the concentration of both nonhem-iron and ferritin in the bone marrow [2]. There is also a significant inverse correlation between intestinal iron absorption and the logarithm of the serum ferritin concentration [4, 6]. This suggests that a close relationship exists between the total amount of storage iron and serum ferritin concentration in normal individuals. This correlation, however, does not exist in patients with iron overload caused by genetic hemochromatosis. Calculations derived from the correlation of serum ferritin with iron stores as determined by repeated phlebotomy suggest that in normal individuals, for each mg/liter ferritin there are 8 to 12 mg of storage iron [7, 8]. This ferritin-to-storage iron ratio, however, does not seem to be constant over the entire range from iron deficiency to heavy iron overload. Normal healthy volunteers with mobilizable iron stores ranging from 200 mg to 1.8 g were considered, together with patients with genetic hemochromatosis (iron stores 890 mg to 20.0 g). The ferritin-to-storage iron ratio decreased with increasing iron stores. At iron stores below 500 mg, the ratio averaged 15 mg storage iron per mg/liter of ferritin and decreased to a ratio of 6 mg per mg/liter of ferritin at storage
RED CELL FERRITIN The circulating erythrocytes contain a tiny residue of that ferritin present in its nucleated precursors in the bone marrow. The mean concentration in erythrocytes of the peripheral blood is approximately 10 ag/cell (10218 g/cell), measured with antibodies to H-type ferritin. The general red cell ferritin levels reflect the iron supply to the erythroid marrow and tend to vary inversely with the red cell protoporphyrin [9]. In untreated genetic hemochromatosis, red cell ferritin content is approximately 70 times the normal mean. Red cell ferritin was shown to reflect liver parenchymal cell iron concentrations [10]. High levels of red cell ferritin are also found in thalassemia, megaloblastic anemia, and myelodysplastic syndromes, presumably indicating a disturbance of erythroid iron metabolism in these conditions [2]. Despite these specific diagnostic advantages, the assay of red cell ferritin has little routine application thus far, mainly because it is necessary to have fresh blood in order to prepare red cells free of white cells, which have much higher ferritin levels [2]. SERUM FERRITIN AND INFLAMMATION Inflammation and neoplasia are associated with a significant rise in serum ferritin levels [11–16]. The response of serum ferritin levels to inflammation was found to parallel the acute phase plasma proteins, haptoglobin, and C-reactive protein [11, 14], suggesting that serum ferritin behaves as an acute phase reactant [17]. The degree of rise in serum ferritin concentration is, however, influenced by the iron status of the patient [14]. Serum ferritin most probably is a secretory protein because, in contrast to intracellular tissue ferritin, most of the circulating ferritin is glycosilated, representing the minor fraction of intracellular ferritin synthesized on membrane-bound polysomes [18–20]. Because of the behavior of ferritin as an acute phase reactant, inflammation can result in a false elevation of the ferritin values and can bring ferritin values for patients with true iron deficiency into the normal range [12]. With active inflammation, plasma levels of ferritin can rise sharply in the absence of any change in total body iron stores. These factors place limitations on the use of serum ferritin as a marker for iron deficiency, for example, in end-stage renal disease, but it remains the best available guide to the adequacy of iron stores prior to recombinant human erythropoietin (rHuEPO) therapy [21]. Once steady-state erythropoiesis and iron bal-
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Fig. 2. Correlation of storage iron mobilized by repeated phlebotomy, with serum ferritin concentration in 69 healthy male subjects (s) and 71 patients with genetic hemochromatosis (d) at various clinical stages. Note the direct and linear relationship in storage iron concentrations of less than 2.0 g and the remarkable changes of the ferritin to storage iron ratio in storage iron concentration of more than 2.0 g.
ance have been re-established, serum ferritin can again be used to assess iron stores. SERUM FERRITIN AND FUNCTIONAL IRON DEFICIENCY In otherwise healthy subjects, iron deficiency is considered “absolute” when iron stores are depleted or completely exhausted, as indicated by serum ferritin concentrations below the lower normal limits (mentioned earlier here). Complete exhaustion of iron stores normally is indicated by serum ferritin concentrations of less than 12 mg/liter [1] and inappropriate iron delivery to the erythroid marrow as evidenced by transferrin iron saturation (TISAT) levels below 15% [22]. In the specific situation of chronic renal failure (CRF), absolute iron deficiency has been defined as serum ferritin levels of less than 100 mg/liter and TISAT levels of less than 20% [23]. Besides the absolute iron deficiency, a different pattern of iron deficiency can occur when there is a need for a greater amount of iron to support hemoglobin synthesis than can be released from the reticuloendothelial iron stores. This situation, which can be caused by stimulation of erythropoiesis by erythropoietin or by limitation of storage iron release due to inflammatory processes, can occur in the presence of adequate or even increased iron stores. It has been called “functional” or “relative” iron deficiency [24]. Functional iron deficiency is a frequent issue associated with rHuEPO treatment in both renal anemia and the anemia of chronic disorders (ACD). It is also commonly observed when rHuEPO is used to increase autologous blood donation [25]. Patients with this condition do not meet traditional laboratory criteria for absolute
iron deficiency, but may demonstrate an increase in hemoglobin when i.v. iron is administered. Distinguishing between functional iron deficiency and an inflammatory iron block is a common clinical problem. The percentage of hypochromic red cells does appear to be a reliable and sensitive indicator for functional iron deficiency and has been shown to be helpful in the diagnosis of functional iron deficiency (Fig. 3) [26, 27]. Normally, there are less than 2.5% of red cells with individual cell hemoglobin of less than 28 pg/dl. Values exceeding 10% are compatible with functional iron deficiency in rHuEPOtreated patients. This measurement is, however, presently performed as part of routine full blood samples that requires a Technicont H-1, H-2, or H-3 automated cell counter (Bayer Diagnostics, Mu¨nchen, Germany), which may be not available in many medical centers in Europe or the United States [23]. Because of these limitations, currently the best tests of iron status are the percentage TISAT and the serum ferritin. Although no single value of TISAT or serum ferritin accurately discriminates between CRF patients who are or are not functionally iron deficient, available data demonstrate that the lower the TISAT and serum ferritin, the higher the likelihood that a patient is iron deficient, and the higher the TISAT and the serum ferritin, the lower the likelihood that a patient is iron deficient [23]. SERUM FERRITIN AND IRON TREATMENT Serum ferritin concentrations below the lower levels of normal ranges always indicate the presence of absolute iron deficiency and therefore are a clear indication for iron supplementation. Because the distinction between absolute and functional iron deficiency in CRF
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Fig. 3. Course of hemoglobin and percentage of hypochromic red cells (A) as well as serum ferritin and saturation of TIBC (B) in a female patient with severe inflammatory active rheumatoid arthritis treated with recombinant human erythropoietin (150 IU/kg s.c. twice a week) together with an intravenous iron supply (125 to 200 mg twice a week). Note the development of functional iron deficiency after initiation of EPO treatment as shown by the inappropriate hemoglobin increase and rise of hypochromic red cells above 10% (left hand side), whereas serum ferritin increased continuously and the saturation of TIBC remained below 20%. Abbreviations are: OP, start of surgical treatment (knee joint TEP); Recormont, recombinant human erythropoietin; Ferrlecitt, Fe(III) gluconate; Venofert, iron sucrose complex.
patients, specifically before or during EPO treatment, is more crucial, several recent consensus reports have recommended that serum ferritin should be kept at more than 100 mg/liter in order to maintain a sufficient iron reserve [21, 23, 28]. Most EPO-treated patients require intravenous iron to build up their respective iron stores. A small percentage of hemodialysis patients as well as many peritoneal dialysis and predialysis patients are also able to maintain adequate iron stores using only oral iron supplements [21, 23, 29]. Patients with initial ferritin levels of less than 100 mg/liter, however, will almost certainly require intravenous iron to support the requirements of the marrow during rHuEPO treatment [30]. During oral iron administration in therapeutic doses (100 to 200 mg/day) in absolute iron-deficient patients with unimpaired intestinal iron absorption, the serum ferritin concentration within the first 5 to 10 days increases up to normal levels and decreases again when increasing
hemoglobin regeneration takes place. A relevant stable increase in storage iron, as indicated by increasing ferritin levels, is observed only when hemoglobin regeneration reaches a steady state. In contrast, after i.v. iron administration (100 to 200 mg/day), serum ferritin increases disproportionally to the amount of storage iron present [31, 32]. Therefore, for serum ferritin to be used as an indicator of the iron stores rebuilt by i.v. iron supplementation, it should not be determined earlier than two weeks after the last i.v. iron dose. There is little information available that clearly establishes the upper limit of safety for serum ferritin in patients receiving i.v iron therapy. Serum ferritin levels between 300 and 800 mg/liter have been common in dialysis patients, and there is no evidence that such levels are associated with adverse iron-mediated effects [23]. Iron therapy should, however, be used or continued with extreme caution in patients whose serum ferritin is greater
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than 1000 mg/liter or where the TISAT level is above 50% [21].
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These findings were confirmed in subsequent studies [35, 36, 45]. In controlled phlebotomy studies, the receptor does not increase until iron stores are completely exhausted (Fig. 4) [47]. When serum ferritin fell below 12 mg/liter, the serum receptor increased above normal levels, roughly in proportion to the deficit in functional iron.
SERUM TRANSFERRIN RECEPTOR On actively growing human tumor cells, Sutherland et al have found a specific receptor for the iron transport protein transferrin [33]. By somatic hybrid cell studies, Goodfellow et al assigned the transferrin receptor (TRFR) locus to chromosome 3 [34]. The number of transferrin receptors on the cell surface is directly related to the erythroid activity [35–37]. The transferrin receptor molecule consists of two identical domains. On each domain, the binding of two molecules of transferrin is possible. Transferrin receptors are found on cell membranes and allow iron-bound transferrin to enter the cell. In the case of high saturation with iron, transferrin is bound with a significantly higher affinity to the binding sites of the receptor [38]. When the iron supply is inadequate, there is an up-regulation of transferrin receptors to enable the cell to compete more effectively for iron. As known with other membrane proteins, a shaded form of the transferrin receptor is known. This sTRF-R is detectable in the blood serum, and it is found as a truncated fragment of the transmembrane receptor. The number of membrane receptors is in proportion to the receptors found in the circulating blood plasma. In comparison to the complete molecule, sTRF-R lacks the cytoplasmic and the transmembrane domains [39]. Like serum iron and transferrin, sTRF-R shows a diurnal and seasonal pattern in its peak trough differences, but many authors confirmed that measurement of sTRF-R in serum is a sensitive indicator for both the total erythropoiesis and iron deficiency [35–38, 40–43].
CORRELATION TO IRON STORES Distinguishing between IDA and ACD remains unsatisfactory in some cases. Serum ferritin, serum iron, and TISAT are sometimes too low in value to classify the different forms of anemias. In particular, the combination of ACD and iron deficiency anemia, which is often seen, for example, in patients suffering from CRF or rheumatoid arthritis, could remain a diagnostic problem because serum ferritin levels are higher in patients with both IDA and ACD compared with those with absolute iron deficiency alone. sTRF-R in these special cases can distinguish between both forms because elevated sTRFR levels are characteristic for IDA regardless of whether there is chronic inflammation [48]. In contrast, sTRF-R remains unaffected in patients with anemia caused by chronic disorders [49–52]. In patients with anemia of CRF, Beguin et al demonstrated that sTRF-R could predict the response to rHuEPO therapy [53]. In this group of patients, the best response rates were shown when the sTRF-R (and fibrinogen) level was low [53]. This could be because responders to treatment with rHuEPO and intravenous iron administration probably show higher levels of sTRF-R during the therapy interval [54]. It was also shown that sTRF-R increases progressively for a six-week period and reaches a plateau when patients with CRF are treated with rHuEPO [55].
SERUM TRANSFERRIN RECEPTOR ASSAY
CONCLUSION
Measurement of sTRF-R is based on enzyme-linked immunosorbent essays (ELISA) with monoclonal antibodies against parts of the receptor molecule, and by latex agglutination nephelometric immunoassay [37, 38, 43, 44]. The normal range for serum transferrin receptor depends strongly on the method used for the determination of the protein [45]. In a study using a monoclonal ELISA technique, the mean value for 82 healthy males and females was 5.6 mg/liter, and for iron deficiency anemia it was 18 mg/liter [45]. However, validation of the different methods is missing thus far, and normal values independent from the underlying method are not available [46]. This is caused by the fact that purified transferrin receptors were not available, and standardization of the test systems was not possible. In patients suffering from iron-deficiency anemia (IDA), strictly elevated levels of sTRF-R were reported [37].
One of the major diagnostic problems is to distinguish iron deficiency from other causes of anemia, that is, anemia of chronic diseases. Because most of the tissue receptors have been found on the erythroid precursor cell, the level of sTRF-R measured in serum is directly proportional to erythroid activity and reflects the total body mass of tissue receptor [36, 38, 41, 56]. The serum ferritin level and serum transferrin receptor level can portray the entire spectrum of iron deficiency from normal to severe ID. Unlike serum ferritin, serum transferrin receptor remains normal in patients with acute or chronic inflammation or liver disease [47] and appears to be effective in distinguishing iron deficiency anemia from anemia of chronic disease [49]. This new parameter provides a quantitative measure of increased erythropoietic activity, for example, absolute or functional iron deficiency, and could be a useful
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alternative in distinguishing the associated anemia from that of chronic disease [56, 57]. Additionally, this parameter leads to a more stable measurement than transferrin saturation and is affected earlier in the development of functional iron deficiency than traditional hematological indices such as the erythrocyte protoporphyrin or mean corpuscular volume. On the other hand, it must be noted that determination of sTRF-R using different techniques leads to uncomparable results. Furthermore, the measurement of serum transferrin receptor is not routinely used in clinical practice because it is still expensive; it should only carried out in patients in whom the quantitation of hypochromic red cells is not available or when the determination of serum ferritin, serum iron, transferrin, and saturation of transferrin does not lead to an accurate classification of the type of anemia. Reprint requests to Dr. Joachim P. Kaltwasser, Medizinische Klinik III, Zentrum der Inneren Medizin, J.W. Goethe-Universita¨t, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: [email protected]
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Fig. 4. Course of serum ferritin and soluble transferrin receptor (sTRFR) during consecutive weekly phlebotomies (500 ml) of healthy male subjects (own data; P 0–P 5: phlebotomy week 0 to week 5; number of subjects in brackets). (A) The solid horizontal line indicates serum ferritin values in case of completely exhausted body iron stores. (B)
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