Search

Many disorders of iron homeostasis (e.g., iron overload) are associated with the dynamic kinetic profiles of multiple non-transferrin bound iron (NTBI) species, chronic exposure to which is associated with deleterious end-organ effects. Here we discuss the chemical nature of NTBI species, challenges with measuring NTBI in plasma, and the clinical relevance of NTBI exposure based on source (iron overload disorder vs. intravenous iron-carbohydrate complex administration). NTBI is not a single entity but consists of multiple, often poorly characterized species, some of which are kinetically non-exchangeable while others are relatively exchangeable. Prolonged presence of plasma NTBI is associated with excessive tissue iron accumulation in susceptible tissues, with consequences, such as endocrinopathy and heart failure. In contrast, intravenous iron-carbohydrate nanomedicines administration leads only to transient NTBI appearance and lacks evidence for association with adverse clinical outcomes. Assays to measure plasma NTBI are typically technically complex and remain chiefly a research tool. There have been two general approaches to estimating NTBI: capture assays and redox-activity assays. Early assays could not avoid capturing some iron from transferrin, thus overestimating NTBI. By contrast, some later assays may have promoted the donation of NTBI species to transferrin during the assay procedure, potentially underestimating NTBI levels. The levels of transferrin saturation at which NTBI species have been detectable have varied between different methodologies and between patient populations studied.

In thalassemia major, iron overload is the joint outcome of multiple blood transfusions and an inappropriately increased iron absorption associated with ineffective erythropoiesis. Threshold values for iron toxicity are a liver iron concentration exceeding 440 mmoles/g dry weight, serum ferritin >2500 ng/mL, DFO urinary iron excretion >20 mg/day, and transferrin saturation >75%. The outpouring of catabolic iron that exceeds the iron-carrying capacity of transferrin results in the emergence of non-transferrin-bound iron (NTBI). NTBI is cleared preferentially by the liver and myocardium at a rate exceeding 200 times that of transferrin iron. NTBI catalyzes the formation of free radicals, resulting in oxidative stress and damage to mitochondria, lysosomes, lipid membranes, proteins, and DNA. The long-term consequences of iron toxicity, including cirrhosis, myocardiopathy, and endocrine disorders, are preventable and mostly reversible by effective iron chelation therapy. Recent technologic advances in the documentation of organ-specific siderosis and the improved efficiency of iron chelating programs resulted in a spectacular improvement in the prevention of iron-induced end-organ failure and improved survival in thalassemic patients.

Despite elegant regulatory mechanisms, iron deficiency anemia (IDA) remains one of the most common nutritional deficiencies of mankind. Iron deficiency is the result of an interplay between increased host requirements, limited external supply, and increased blood loss. When related to increased physiologic needs associated with normal development, iron deficiency is designated physiologic or nutritional. By contrast, pathological iron deficiency, with the exception of gross menorrhagia, is most often the result of gastrointestinal disease associated with abnormal blood loss or malabsorption. If gastroenterologic evaluation fails to disclose a likely cause of IDA, or in patients refractory to oral iron treatment, screening for celiac disease (anti-tissue transglutaminase antibodies), autoimmune gastritis (gastrin, anti-parietal or anti-intrinsic factor antibodies), and Helicobacter pylori (IgG antibodies and urease breath test) is recommended. Recent studies indicate that 20–27% of patients with unexplained IDA have autoimmune gastritis, about 50% have evidence of active H. pylori infection, and 4–6% have celiac disease. The implications for abnormal iron absorption of celiac disease or autoimmune gastritis are obvious. In patients with unexplained IDA and H. pylori infection, cure of refractory IDA by H. pylori eradication offers strong evidence for a cause-and-effect relation between H. pylori infection and unexplained IDA. Stratification by age cohorts in autoimmune gastritis implies a disease presenting as IDA many years before the establishment of clinical cobalamin deficiency. It is likely caused by an autoimmune process triggered by antigenic mimicry between H. pylori epitopes and major autoantigens of the gastric mucosa. Recognition of the respective roles of H. pylori and autoimmune gastritis in the pathogenesis of iron deficiency may have a strong impact on the diagnostic workup and management of unexplained, or refractory IDA.

Background and Objectives: Since gastric acidity and ascorbate play a critical role in the solubilization and reduction of iron for subsequent absorption, the achlorhydria associated with autoimmune and Helicobacter pylori gastritis may explain the poor response of such patients to oral iron treatment. In order to circumvent this problem, we explored the therapeutic potential of a duodenal formulation of ferrous glycine sulfate consisting of micropellets that do not dissolve at the acid environment of the stomach but, owing to their solubility at a higher pH, discharge their content directly into the duodenum. Design and Methods: In a case-control study, the treatment results of 39 patients with iron deficiency anemia receiving a duodenal formulation of ferrous glycine sulfate (group A) were compared with the results of 39 patients receiving other oral iron compounds (group B). Autoimmune gastritis, H. pylori gastritis or both were present in over 75% of patients in each group. Results: After 1 and 3 months of treatment, mean hemoglobin in group A increased from 9.5 ± 1.2 to 11.2 ± 1.3 and 12.8 ± 1.3 g/dl, respectively. By comparison, in group B, the corresponding values increased from 9.3 ± 1.3 to 10.2 ± 1.5 (p = 0.019) and 11.1 ± 1.7 g/dl (p = 0.022). A favorable response, defined as a more than 2 g/dl increase in basal hemoglobin or hemoglobin exceeding 12 g/dl, was obtained in 33 of 39 patients in group A compared with only 18 of 39 in group B (p = 0.009). Because of treatment failure, 14 patients in group B were subsequently referred for intravenous ferric sucrose therapy versus only 3 in group A (p < 0.0001). Conversely, of 5 patients in group A managed by intravenous iron prior to referral, 4 became independent of parenteral iron after starting the duodenal formulation of ferrous glycine sulfate. Interpretation and Conclusions: In patients with iron deficiency anemia associated with autoimmune and H. pylori gastritis with a high rate of refractoriness to oral iron treatment, satisfactory response to a duodenal formulation of ferrous glycine sulfate can be elicited in the vast majority of cases, obviating the need for expensive, inconvenient and occasionally risky intravenous iron administration.

Background and objectives Helicobacter pylori infection with, or without coexisting autoimmune gastritis has been implicated in several recent studies as an important cause of IDA in patients with unexplained iron deficiency anemia (IDA). However, the role of H. pylori in the causation of IDA is still unsettled as the vast majority of reported patients were premenopausal women in whom menstrual blood loss was likely the dominant factor determining IDA. Design and methods Prospective study of 44 consecutive male IDA patients referred for hematologic evaluation. Following standard endoscopic studies, all patients were screened for non-bleeding GI conditions including celiac disease, autoimmune gastritis and H. pylori gastritis. All subject with H. pylori infection were offered triple therapy for H. pylori eradication. Results Only 15 patients had a likely source of blood loss identified. The 29 males with “unexplained” IDA were distinguished by their younger age (36 ± 20 vs. 57 ± 17 years p

Methods are now available to measure the magnitude of iron accumulation in the heart. Their validation currently relies on indirect evidence and not on chemical estimation in cardiac biopsies. All patients with symptomatic heart disease appear to have abnormal T2* values, but many patients without symptomatic heart disease also have evidence of increased myocardial iron. Although there is no proof to date that increased myocardial iron, as evidenced by abnormal magnetic resonance imaging, carries an adverse prognosis, it is likely that such new information will affect the chelating programme of patients. In these cases, there are a number of options available: (i) ongoing treatment with either desferrioxamine (DFO) or deferiprone may be intensified; (ii) the patient may be switched to the alternative chelator or (iii) combined chelation with both DFO and deferiprone may be started, which is more effective than using either chelator alone. For patients with symptomatic heart disease, continuous intravenous DFO with, or without deferiprone, remains the currently recommended treatment, in view of its documented ability to salvage these patients.

In chronic anemias associated with iron overload, iron chelation therapy is the only method available for preventing early death caused mainly by myocardial and hepatic iron toxicity. Although desferrioxamine (DFO) has been available for the treatment of transfusional iron overload since the early 1960s, the era of modern and effective iron chelation therapy started only 20 years ago with the introduction of subcutaneous DFO infusions by portable pumps. Today, long-term DFO therapy is an integral part of the management of thalassemia and other transfusion-dependent anemias, with a major impact on well-being and survival. However, the high cost and rigorous requirements of DFO therapy and the significant toxicity of deferiprone underline the need for the continued development of new and improved orally effective iron chelators. In recent years, more than 1000 candidate compounds have been screened in animal models. These efforts have led to the identification of several interesting compounds, a few of which may be of possible clinical usefulness. The present review covers some of the most outstanding of these compounds, including deferiprone (L1), pyridoxal isonicotinoyl hydrazone (PIH), the polyanionic amines, the substituted polyaza compounds and bishydroxyphenyl tiazole. The introduction of these promising new chelators and the evolution of improved strategies of iron chelation therapy require a better understanding of the pathophysiology of iron toxicity and of the mechanisms of action of iron chelating drugs.

The use of anthracycline antineoplastic drugs is limited by a cumulative, dose-dependent toxicity to the heart. Of the cellular organelles proposed as possible primary sites of anthracycline toxicity, the mitochondrial membrane appears to be most likely target. Cardiolipin, a major phospholipid component of the inner mitochondrial membrane is rich in polyunsaturated fatty acids and is particularly susceptible to peroxidative injury by harmful radicals produced by redox cycling of anthracyclines. This, in turn, leads to the inactivation of key enzymes in the mitochondrial respiratory chain. Since the formation of free radicals is catalyzed by iron through the Haber-Weiss reaction, it was hypothesized that iron depletion by deferoxamine (DFO) may limit anthracycline cardiotoxicity. Recent studies indicate that iron-loading aggravates doxorubicin cardiotoxicity by enhancing mitochondrial damage, and this can be prevented by prior DFO treatment. Although these observations are intriguing, further studies are required to show that the cardioprotective effects of DFO do not interfere with the therapeutic, antitumoral action of anthracyclines.

The redox cycling of anthracyclines promotes the formation of free radicals which are believed to play a central role in their cardiotoxicity. A number of observations indicate that the mechanism of the antineoplastic effect of anthracyclines is independent of their cardiotoxic effect and that it may be possible to prevent toxicity without interfering with therapeutic effect. Iron plays an important role in anthracycline toxicity by promoting the conversion of superoxide into highly toxic hydroxyl radicals through the Haber-Weiss reaction. Conversely, iron deprivation by its high-affinity binding to iron chelating compounds may inhibit anthracycline toxicity by interfering with free radical formation. ICRF-187, a bispiperazonedione which is hydrolyzed intracellularly into a bidentate chelator resembling EDTA, is able to decrease adriamycin-induced free hydroxyl radical formation and to prevent the development of clinical cardiac toxicity in patients receiving long-term anthracycline therapy. Our studies in rat heart cell cultures have shown that iron overload aggravates anthracycline toxicity and that this interaction can be prevented by prior iron chelating treatment. Since iron overload caused by multiple blood transfusions and bone marrow failure is a common condition in patients requiring anthracycline therapy, these observations may have significant clinical implications to the prevention of anthracycline cardiotoxicity.

Increased sensitivity to warfarin anticoagulation is usually attributed to liver disease, vitamin K deficiency, or drug interactions. We describe a patient with unexplained sensitivity to warfarin and mildly elevated prostate-specific antigen levels in whom subsequent developments indicated that warfarin sensitivity was the first manifestation of occult prostatic cancer. A review of all published cases of coagulopathy associated with cancer of the prostate shows that, unlike other solid tumors with secondary disseminated intravascular coagulation (DIC), in prostate cancer increased bleeding is more common than thrombotic phenomena. Chronic DIC due to occult prostate cancer should be included in the differential diagnosis of excessive prothrombin time prolongation in patients receiving anticoagulants.

Despite elegant regulatory mechanisms, iron deficiency anemia (IDA) remains one of the most common nutritional deficiencies of mankind. Iron deficiency is the result of an interplay between increased host requirements, limited external supply, and increased blood loss. When related to increased physiologic needs associated with normal development, iron deficiency is designated physiologic or nutritional. By contrast, pathological iron deficiency, with the exception of gross menorrhagia, is most often the result of gastrointestinal disease associated with abnormal blood loss or malabsorption. If gastroenterologic evaluation fails to disclose a likely cause of IDA, or in patients refractory to oral iron treatment, screening for celiac disease (anti-tissue transglutaminase antibodies), autoimmune gastritis (gastrin, anti-parietal or anti-intrinsic factor antibodies), and Helicobacter pylori (IgG antibodies and urease breath test) is recommended. Recent studies indicate that 20-27% of patients with unexplained IDA have autoimmune gastritis, about 50% have evidence of active H. pylori infection, and 4-6% have celiac disease. The implications for abnormal iron absorption of celiac disease or autoimmune gastritis are obvious. In patients with unexplained IDA and H. pylori infection, cure of refractory IDA by H. pylori eradication offers strong evidence for a cause-and-effect relation between H. pylori infection and unexplained IDA. Stratification by age cohorts in autoimmune gastritis implies a disease presenting as IDA many years before the establishment of clinical cobalamin deficiency. It is likely caused by an autoimmune process triggered by antigenic mimicry between H. pylori epitopes and major autoantigens of the gastric mucosa. Recognition of the respective roles of H. pylori and autoimmune gastritis in the pathogenesis of iron deficiency may have a strong impact on the diagnostic workup and management of unexplained, or refractory IDA.

The main aspects of iron loading and the consequent clinical implications described in this series of articles are summarized in this final chapter. Despite mechanisms to maintain iron homeostasis, harmful iron accumulation can occur in patients with hereditary defects of regulatory proteins, such as hepcidin, or with transfusion-dependent anemias, such as thalassemia and myelodysplastic syndromes. Identifying the role of nontransferrin bound iron in the pathogenesis of disease allows for better treatment strategies to prevent and reverse iron toxicity. In addition, accurate noninvasive methods to reliably assess iron accumulation and chelation are now available. Continuous chelation coverage, which can be achieved with combination therapy (deferoxamine and deferiprone) or deferasirox, is expected to provide optimal protection from iron toxicity. As more long-term data on these drugs accumulate, the role of oral and combination chelation therapies in relation to blood transfusion, as well as other iron overload disorders, will become clearer.

Iron is one of the most common elements in nature. As a transition metal it is very efficient in electron transport and redox reactions. The proteins and enzymes in which iron is an essential component play a key role in respiration, energy production, detoxification of harmful oxygen species and cell replication. Despite the abundance of iron in nature, the solubility of its stable ferric form is extremely low. Hence, living organisms were compelled to develop efficient mechanisms for iron transport and storage.

Serum ferritin was assayed by an immunoradiometric method in 82 people above 60 years of age. For comparison purposes, the same assay was performed in 71 younger normal adults. The serum ferritin distribution in the elderly had a larger variance than in the younger adults and in addition, there was a clear shift to higher values in the elderly. The latter was more notable in females than in males but there was still a statistically lower mean in elderly females than in elderly males. Ten out of 55 elderly subjects with evidence of iron deficiency (response to oral iron therapy) had a normal or high serum ferritin which suggests that variables unrelated to iron status may operate in determining serum ferritin levels in the elderly. The shift to higher values appears to occur upon reaching grossly 70 years of age. Whether the shift is a physiologically normal event is at present an open question.

Iron binding in the sera of 35 patients with beta thalassaemia major and intermedia was studied. In patients receiving regular blood transfusions since infancy transferrin was completely saturated and about 2.7--7.1 mumol/l of the serum iron could be removed by dialysis or ultrafiltration in the presence of a chelating agent or by filtration on DEAE-Sephadex-catecholdisulphonic acid columns. In contrast, less than 1.0 mumol/l of transferrin bound iron was removed when subjected to the same procedures. The non-specific iron of thalassaemic sera could no longer be demonstrated after incubation with normal serum. These findings indicate that non-specific iron is a chelatable with normal serum. These findings indicate that non-specific iron is a chelatable compound which is readily available for transferrin binding. In view of the known toxicity of unbound iron, its identification in thalassaemic sera might be of relevance to the pathogenesis of tissue damage and the protective effect of iron chelating therapy in this disease.

Two young arab patients are described in whom malignant lymphoma developed within less than 1 year of the diagnosis of agnogenic myeloid metaplasia. Both patients showed a satisfactory response to combined chemotherapy. One of them died of hepatitis B at 10 months and the other is alive and in clinical remission 25 months after initial diagnosis. These observations demonstrate the close relation between myeloproliferative and lymphoproliferative syndromes and illustrate the diversity of malignant lymphoproliferative disorders into which agnogenic myeloid metaplasia may evolve in the course of disease. Our experience also demonstrates the ease with which some patients with an 'end stage' myeloproliferative disorder may respond to standard chemotherapy designed for the treatment of malignant lymphoma.

Summary Cell kinetics were studied by quantitative autoradiography in 29 patients with myelodysplastic syndromes (MDS) and in four with smouldering leukaemia (SML) in a prospective 6–year study. Cellular turnover in all FAB groups was characterized by reduced labelling index and prolonged DNA synthesis time indicating reduced proliferative activity. Two more parameters were derived from the measurements. (1) The relative cell production rate, which is increased in proerythroblasts and myeloblasts in most cases. This could be attributed to an increased number of divisions in early recognizable precursors in the absence of maturation, and (or) an increased influx from the progenitor compartments. (2) The maturation index of a cell lineage was determined by the ratio of relative cell production rate increase from the first to the last proliferative compartment over the corresponding normal value. It showed a high degree of abnormality in all FAB subgroups, indicating premature cell death in the bone marrow. None of the kinetic parameters proved to be of value in predicting the evolution of leukaemia. In contrast, an excellent correlation was found between the myeloid maturation index and survival (P

The effect of maturation on ferritin synthesis and iron uptake by erythroid precursor cells was studied in vitro in rabbit bone marrow cells synchronized by actinomycin D. Early mononuclear precursors were compared with basophilic normoblasts and reticulocytes. Apoferritin synthesis was measured by following the incorporation of 3H-amino acids into purified ferritin, and this was correlated with total protein synthesis and with radioactive iron uptake by whole cells and radioactive iron incorporation into heme and ferritin. With increasing cellular maturation, a progressive reduction in both apoferritin and total protein synthesis has been observed, undifferentiated cells synthesizing 12 times more protein and 20 times more ferritin than reticulocytes. The rate of ferritin synthesis was inversely related to the rate of radioactive iron uptake by whole cells and radioactive iron incorporation into heme; lowest rates of radioactive uptake and heme synthesis were found in undifferentiated cells, and highest rates in reticulocytes. Radioactive iron incorporation into ferritin was higher in basophilic normoblasts than in undifferentiated cells but was very low in reticulocytes. This sequence of events indicates that in developing erythroid cells the phase of maximal ferritin synthesis precedes the phase of maximal iron uptake. The accumulation of newly formed apoferritin is followed by an increase in cellular iron uptake and an increased retention of iron in ferritin. Finally, with continuing cellular maturation, ferritin iron and iron derived directly from transferrin are utilized for hemoglobin synthesis.

Patterns of erythropoiesis and granulopoiesis were studied in 15 patients with myelodysplastic syndromes and in one with smouldering leukaemia by correlating CFU-C, BFU-E and CFU-E cloning efficiency with erythroid and myeloid maturation indices derived from quantitative 14C-autoradiography and with ferrokinetics. Maturation index of a cell lineage was defined as the ratio of cell production rate increase from the first to the last proliferative compartment over the corresponding normal value. The myeloid maturation index was reduced in all cases, but CFU-C progenitor frequency was increased by a factor of 3. Erythroid maturation index was also reduced in most cases, and BFU-E progenitor frequency was reduced by a factor of 2. Similarly, CFU-E cloning efficiency corrected for the erythroid maturation index was 2.5-10-fold lower than normal. Comparison of the erythroid maturation index with ferrokinetics revealed a constant ratio of ineffective erythropoiesis with a maturation disturbance in the proliferative pool twice that in the non-proliferative pool. These findings indicate basic qualitative and quantitative abnormalities and a difference in the patterns of production of erythroid and myeloid cells in the myelodysplastic syndromes: Myeloid progenitors are increased in number but their maturation is grossly abnormal. On the other hand, the apparent reduction in erythroid progenitors and the reduced erythroid maturation index are attributable to both premature cell death of the more mature erythroblasts and an increased proerythroblast proliferation, i.e. extra divisions in the proerythroblast compartment at the expense of maturation.

Summary. Fifteen compounds with a high affinity to ferric iron have been screened for in vivo iron-chelating efficiency in a rat model. One of the most potent of these drugs was ethylenediamine-N,N′-bis(o-hydroxyphenylglycine) (EHPG). EHPG-induced iron excretion was up to 8 times higher than iron excretion induced by identical doses of desferrioxamine (DF). Studies employing selective radio-iron probes of reticuloendothelial and parenchymal iron stores showed that although EHPG is able to interact with both storage iron compartments, its effect on parenchymal iron is much more pronounced. Unlike DF which has two alternative routes of excretion, EHPG-induced iron excretion is restricted mainly to the gut. Although EHPG seems to be superior to DF in both its chelating efficiency and preferential interaction with hepatic parenchymal iron stores, information on its in vivo toxicity is at present insufficient and it cannot yet be recommended for clinical use.

We reported earlier an abnormal iron population in the serum of patients with transfusional hemosiderosis secondary to thalassemia. The iron is not bound to transferrin, ferritin, or hemoglobin and is referred to as nonspecific iron. This publication reports studies of the chemical reactivity and nature of the nonspecific iron and the development and validation of a method for its quantitative analysis. The quantitation depends on the mobilization of this iron from nonspecific macromolecular binding sites by ethylenediamine tetraacetic acid and subsequent separation from Fe3+-transferrin-CO⅔− and residual hemoglobin by ultrafiltration. Analyses of filtrate iron were carried out by atomic absorption and colorimetric methods. The accuracy of the test was established via an ultrafiltration titration of serum of known unsaturated iron-binding capacity. The method proved to be accurate and highly reproducible. Sera containing artificial introduced nonspecific iron were prepared. The reactivity of artificial and thalassemic nonspecific iron was examined with regard to reduction and chelation, and chelation by apotransferrin. Artificial and thalassemic nonspecific iron were found to be virtually identical in reactivity and to exhibit multiphasic kinetics consistent with nonspecific binding to several serum protein sites. The reaction of apotransferrin with nonspecific iron was complete within one minute. The reduction-chelation and apotransferrin reactivity studies offer independent analyses of nonspecific serum iron concentrations which compared closely with the chelation-ultrafiltration technique.

Summary. Over 85% of ferritin 59Fe is recovered in the liver of rats within 24 hr of its intravenous injection. Autoradiographic studies have shown that this hepatic uptake is restricted entirely to the parenchymal cells. Following its hepatocellular uptake ferritin is catabolized and its iron moiety is recombined with locally produced apoferritin. The rate of hepatic uptake is reduced by bleeding and enhanced by inflammation. The electrophoretic properties and hepatic handling of ferritin preparations derived from the liver (parenchymal) or spleen (reticuloendothelial) are identical. These findings suggest that the increased amounts of circulating ferritin found in pathologic conditions such as transfusion haeniosiderosis and ineffective erythropoiesis, might play a significant role in the production of parenchymal iroll overload.

The causes of megaloblastic anemia were studied in a survey of patients admitted to six Israeli hospitals over a period of 15 yr. Among the 203 patients identified, 69% had pernicious anemia, 12% had gastrointestinal disease, 9% had primary nutritional deficiency of whom only 1% were associated with pregnancy, and 7% had selective vitamin B12 malabsorption with albuminuria. Comparison with previously published surveys showed, that in contrast with earlier studies where primary nutritional deficiency was the cause of megaloblastic anemia in about 70% of cases and pernicious anemia in only 20%, in more recent studies the proportion of cases with primary nutritional anemia in general and those associated with pregnancy in particular was much lower. This is most probably the result of improved standards of living and a national program of preventive folate supplementation at maternity clinics. A potential hazard of such preventive programs is the aggravation of neurological complications in patients with undiagnosed vitamin B12 deficiency. Early recognition of pernicious anemia and other forms of selective B12 malabsorption is a new challenge created by the changing pattern of megaloblastic anemias.

Summary. Iron exchange in the pregnant rat was quantitated by repeated determinations of plasma iron turnover (PIT), transferrin iron distribution and measurements of storage iron and food iron utilization employing selective radio-iron probes. Despite a sixfold increase in PIT, intestinal absorption of iron accounted for 40% of the PIT throughout pregnancy, with variations not exceeding ±5%. Increased fetal requirements were efficiently compensated for by the mobilization of iron from maternal tissue stores and by increased absorption, and there was no subsequent reduction in iron supply to maternal tissues. Enhancement of iron absorption occurred in the absence of a reduction in serum iron levels or the size of iron stores. In view of the close correlation between PIT and rates of absorption it is postulated that iron absorption in the pregnant rat is regulated by PIT which in turn is determined by the rate of plasma iron clearance by the placenta and the maternal erythroid marrow.

This study describes the effect of ferritin on lymphocyte function in vitro. Peripheral blood lymphocytes isolated from normal donors were incubated with purified human splenic ferritin, and the mitogenic effect of phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and mixed lymphocyte reaction (MLR) were assessed by the uptake of 3H-thymidine (3H-TdR). Ferritin (0.25--5.0 micrograms/ml culture) caused a marked suppression of PHA nad Con A blastogenesis but had no suppressive effect on PWM-induced transformation. Maximal suppression was obtained at a ferritin concentration of 1 microgram/ml and this was not enhanced by increasing ferritin concentrations. Ferritin also reduced the Con A capping phenomenon in normal lymphocytes from 22% to 6%, suppressed the MLR reaction but had no effect on the ability of normal lymphocytes to form E, EA and EAC rosettes or on in vitro lymphocyte cytoxicity against the K-562 cell line. Visual proof of the suppressive effect of ferritin on mitogen induced blastogenesis was provided by scanning electron microscopy, and direct evidence for the ability of lymphocytes to bind ferritin was obtained from studies with radioiodine labelled ferritin. The above findings indicate that ferritin suppresses certain parameters of T-lymphocyte function in vitro. The relation of the present findings to recognized abnormalities of T-cell function encountered in certain neoplastic disorders associated with high serum ferritin levels is at present unknown.

Plasma iron turnover (PIT) and ferritin synthesis in the liver and spleen were studied in rats within the first 24 h of inflammation produced by turpentine injection. Comparison of the sequential changes in PIT and ferritin synthesis showed that alterations in ferritin synthesis preceded the changes in plasma iron exchange throughout the study. Thus, after 4 h inflammation ferritin synthesis was twice normal whereas plasma iron and PIT were still unchanged. Conversely, maximal reduction in plasma iron occurred after 12 h inflammation, at a time when ferritin synthesis had already declined to normal rates. These correlations seem to indicate that, in analogy with other acute phase reacting proteins, increased ferritin synthesis is a primary nonspecific response which is part of a general pattern of the systemic effects of inflammation. This increase in ferritin synthesis is assumed to be responsible for the diversion of labile iron into ferritin stores, and its reduced availability for release from tissues.

The effect of iron deficiency on tumor growth and on host survival was studied in BALB/c mice with transplanted Merwin Plasma Cell‐II tumors. Iron deficiency was induced by maintaining the animals on an iron‐free diet consisting of milk‐cornflour supplemented with CuCl2 and vitamins A and D. Iron deficiency resulted in retardation of both body and tumor growth in weanling BALB/c mice. Their survival, however, was not significantly different from that of iron supplemented controls. The inhibitory effect of iron deficiency on host and tumor growth could not be reproduced in adult BALB/c mice. The survival of tumor‐bearing hosts maintained on a milk‐cornflour diet (whether supplemented with iron or not) was significantly longer than that of animals maintained on a purina diet.

Summary. The mechanism of increased ferritin synthesis in inflammation was studied in rat livers 0—48 h after turpentine injection. A subcellular protein synthesizing system was employed in which the respective roles of cell sap factors and polysomes from normal and treated animals could be studied. Two waves of increased ferritin synthesis were found, an early wave with peak activity at 6 h of inflammation, and a second wave starting at about 24 h. The early wave of enhanced ferritin synthesis was associated with increased activity of cell sap factors. In contrast, the late enhancement of ferritin synthesis was characterized by increased polysomal activity as well as increased cell sap activity. These observations suggest a post-transcriptional control mechanism for the early phase of enhanced ferritin synthesis in inflammation, and a transcriptional as well as post-transcriptional control for the late phase of enhanced ferritin synthesis.

The value of serum ferritin and mean corpuscular volume (MCV) measurement in distinguishing between iron deficient, beta-thalassaemia trait and normal subjects has been studied. Normal subjects had normal ferritin and MCV, iron-deficient ones had low ferritin and low or normal MCV, and thalassaemics had normal ferritin and low MCV. By the combined use of these two measurements it was possible to identify individuals belonging to one of the three categories with an accuracy of over 95%. Although definitive diagnosis of beta-thalassaemia trait requires the demonstration of abnormal haemoglobin A2 levels or reduced beta-chain synthesis, serum ferritin and MCV measurements are useful screening procedures for the initial diagnosis of beta-thalassaemia trait and iron deficiency. Because of the very small amounts of blood required for both of these measurements, they are particularly suitable for surveying large numbers of subjects in populations with a high prevalence of hypochromic-microcytic anaemias.

SUMMARY. The mechanism of desferrioxamine (DF)-induced iron excretion was investigated in 16 thalassaemic patients. Tran~ferrin-~~Fe has been injected intravenously and the fraction of radioiron located to iron stores was determined as the difference between total injected S9Fe and YO RBC utilization. Following the intravenous infusion of 4 g DF, the specific acitivity of excreted urinary iron has been measured in I I patients and the total chelatable pool was calculated as (100- % RBC utilization)/specific activity of urinary iron. Despite the wide variation in DF induced urinary iron excretion, ranging from 3 to 86 mg/d, an unexpected uniformity in the specific activity of chelated urinary iron was found in all patients, and the calculated chelatable pool was 4.5k1.2 (meanfSD) g of iron. These findings suggested that iron chelated by DF was probably not obtained directly from the total storage iron pool but rather from a smaller high specific activity iron compartment derived from the catabolism of labelled non-viable erythrocytes. This postulate was tested by the direct measurement of specific activities in the liver and spleen in five patients undergoing surgery. In all patients, the specific activity of splenic non-haem iron representing RE iron stores (3 I k 8% SyFe/g Fe), was much higher than that of hepatic iron (2 k I %), and the specific activity of iron excreted in the urine (302 8%) was identical with the specific activity of splenic iron. These findings indicate that DF-induced urinary iron excretion is derived from a chelatable pool located in the RE system. Iron overload is a major cause of mortality in patients with homozygous /I-thalassaemia or refractory anaemia receiving multiple blood transfusions. A potential solution for this severe complication is the use ofiron chelating agents such as desferrioxamine (DF). Renewed interest in DF within the last few years (Modell & Beck, 1974; Seshadri et al, 1974; Propper et al, 1976, 1977; Pippard ef al, 1977; Hussain et al, 1976, 1977) has been largely the result of the work of Barry et a1 (1974) who have demonstrated in a prospective controlled study that it is possible to prevent the accumulation of iron and the progression of hepatic fibrosis in thalassaemic patients by prolonged DF administration. Clinical correlations indicate that the damaging effect of iron

Because of the catalytic action of iron in one-electron redox reactions, it has a key role in the formation of harmful oxygen derivatives and production of peroxidative damage to vital cellular structures. The clinical manifestations of iron overload may be prevented and even reversed by the effective administration of the iron-chelating drug deferoxamine (DF). Recent experimental evidence suggests that DF may also be useful in modifying disease conditions unrelated to iron overload by preventing the formation of free radicals, the powerful final effectors of tissue damage resulting from the respiratory burst of granulocytes and macrophages participating in the inflammatory response. Although much experimental work is still needed, this novel approach in iron-chelating therapy may have far-reaching implications in the management of autoimmune disease, adult respiratory distress syndrome, and organ transplantation. The poor intestinal absorption of DF, its almost prohibitive price, and short duration of action underline the need for new, orally effective iron chelators. A number of very promising orally effective drugs have been identified in recent years, such as the polyanionic amines, aryl hydrazones, and hydroxypyridones. Further development for clinical use of this new generation of iron-chelating drugs is a major challenge for future research.

A patient with methimazole-induced aplastic anemia is described. Despite severe pancytopenia and the complete disappearance of hemopoietic elements from the bone marrow, recovery of hemopoiesis has been observed within 14 days of discontinuing methimazole therapy. In vitro studies of CFU-C inhibition of peripheral mononuclear cells harvested in remission, were performed by incubation with autologous sera collected at earlier phases of the disease. These studies provide evidence in favor of a humoral, and most probably autoimmune mechanism as the cause of transient bone marrow aplasia.

SummaryThe influence of vitamin E on the mobilization and excretion of storage iron induced by DF was studied in normal and iron-overloaded rats. Vitamin E administration in pharmacologic doses resulted in complete inhibition of the effect of DF on storage iron in iron-overloaded rats while no such effect could be demonstrated in rats with normal iron stores. The mechanism of the observed inhibition of DF action by vitamin E is at present unknown. Nevertheless this drug interaction has to be considered in view of ongoing therapeutic trials where both antioxidants and iron chelating drugs are administered simultaneously to thalassemic patients with transfusion induced iron overload.The skillful technical assistance of Ludmilla Eilon, Luda Gelfand, and Aliza Shifter is gratefully acknowledged.

Serum ferritin was measured in a variety of hematologic malignancies at presentation, in remission following therapy, and in relapse. Ferritin was strikingly increased in all acute leukemias at presentation and in relapse, in the blastic crisis of CML, and in smouldering leukemia. Remission in both ALL and ANLL was associated with a reduction of serum ferritin, and this normalization was a function of remission duration. In the malignant lymphomas serum ferritin was related to tumor histology. Highest levels were found in Hodgkin disease and histiocytic lymphoma, normal levels in lymphocytic lymphoma, and intermediate levels in mixed histiocytic-lymphocytic lymphoma. In all cases, remission was associated with normalization of serum ferritin. These correlations suggest that serum ferritin measurements may be of clinical usefulness in the initial evaluation and in the assessment of response to therapy in patients with acute leukemia and malignant lymphoma.

Summary. We studied the role of ABH antigens in determining graft outcome in 104 patients who received HLA-identical bone marrow transplants for aplastic anaemia and acute leukaemia. ABH compatibility had no significant effect on incidence of graft rejection or graft-versus-host disease. Fifteen recipients had pre-transplant antibodies against donor ABH antigens. In 14, large volume plasma exchange and transfusion of donor-type erythrocytes was successful in reducing the antibody titre to low or undetectable levels. In one patient, plasma exchange was unsuccessful and red cells were removed from the marrow inoculum by unit gravity sedimentation. This approach prevented transfusion reaction, and permitted engraftment of all haematopoietic cell lines despite persistently elevated antibody titres. Parallel in vitro studies revealed that antibodies to ABH antigens failed to inhibit the growth of progenitor cells committed to both granulocyte-macrophage (CFU-C) and erythroid (BFU-E) development. These findings indicate that ABH-antigens are not clinically important transplantation antigens and suggest that ABH antigens are not operationally present on hematopoietic stem cells.