Chaim Hershko

The importance of non-transferrin bound iron in disorders of iron metabolism.

The concept of non-transferrin bound iron (NTBI) was introduced 22 years ago by Hershko et al. (Brit. J. Haematol. 40 (1978) 255). It stemmed from a suspicion that, in iron overloaded patients, the large amounts of excess iron released into the circulation are likely to exceed the serum transferrin (Tf) iron-binding capacity (TIBC), leading to the appearance of various forms of iron not bound to Tf. In accordance with this assumption, NTBI was initially looked for and detected in patients with > or = 100% Tf-saturation. As techniques for its detection became more sophisticated and sensitive, NTBI was also found in conditions where Tf was not fully saturated, leading to a revision of the original view of NTBI as a simple spillover phenomenon. In this review, we will discuss some of the properties of NTBI, methods for its detection, its significance and potential value as an indicator for therapeutic regimens of iron chelation and supplementation.

Iron-chelating therapy

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.

NON-TRANSFERRIN PLASMA IRON

Transferrin, a glycoprotein of about 80000 molecular weight, has two high-ahity iron binding sites which, under normal conditions, prevent the existence of measurable amounts of unbound iron in the plasma. The main source of iron for transferrin is catabolism of non-viable red blood cells and its main destination is the erythroid marrow (Finch et al, 1970; Huebers & Finch, 1987). The high-afhity binding of iron to transferrin prevents its catalytic activity in oneelectron redox reactions, and thus protects the organism against the formation of harmful oxygen derivatives such as free hydroxyl radicals (Aisen, 1980).

Pathogenesis and management of iron toxicity in thalassemia

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.

Mitochondrial respiratory enzymes are a major target of iron toxicity in rat heart cells

Our previous studies in iron-loaded rat heart cells showed that in vitro iron loading results in peroxidative injury, manifested in a marked decrease in rate and amplitude of heart cell contractility and rhythmicity, which is correctable by treatment with deferoxamine (DF). In the present studies we explored the role of mitochondrial damage in myocardial iron toxicity. Iron loading by 24-hour incubation with 0.36 mmol/L ferric ammonium citrate resulted in a decrease in the activity of nicotinamide adenine dinucleotide (NADH)-cytochrome c oxidoreductase (complex I+III) to 35.3%+/-11.2% of the value in untreated controls; of succinate-cytochrome c oxidoreductase (complex II+III) to 57.4%+/-3.1%; and of succinate dehydrogenase to 63.5%+/-12.6% (p < 0.001 in all cases). The decrease in activity of other mitochondrial enzymes, including NADH-ferricyanide reductase, succinate ubiquinone oxidoreductase (complex II), cytochrome c oxidase (complex IV), and ubiquinol cytochrome c oxidoreductase (complex III), was less impressive and ranged from 71.5%+/-15.8% to 91.5%+/-14.6% of controls. That the observed loss of respiratory enzyme activity was a specific effect of iron toxicity was clearly demonstrated by the complete restoration of enzyme activities by in vitro iron chelation therapy. Sequential treatment with iron and doxorubicin caused a loss of complex I+III and complex II+III activity that was greater than that seen with either agent alone but was only partially correctable by DF treatment. Alterations in cellular adenosine triphosphate measurements paralleled very closely the changes observed in respiratory complex activity. These findings demonstrate for the first time the impairment of cardiac mitochondrial respiratory enzyme activity caused by iron loading at conditions formerly shown to produce severe abnormalities in contractility and rhythmicity.

Evaluation of Iron Status in Patients on Chronic Hemodialysis: Relative Usefulness of Bone Marrow Hemosiderin, Serum Ferritin, Transferrin Saturation, Mean Corpuscular Volume and Red Cell Protoporphyrin

The diagnostic usefulness of bone marrow hemosiderin, serum ferritin, transferrin saturation, mean corpuscular volume (MCV) and red cell protoporphyrin (EPP) in the evaluation of iron status in patients on chronic hemodialysis was studied in 39 subjects. The correlation between serum ferritin and the number of transfusions received per month was slightly higher (r = 0.717; p less than 0.001) than the correlation between bone marrow hemosiderin and transfusions (r = 0.685; p less than 0.01). Serum ferritin was useful in identifying subjects with both increased or reduced iron stores. In contrast, transferrin saturation could only be used for indicating iron overload. MCV for indicating iron deficiency, and EPP was not useful in either case. The abnormal increase of EPP in chronic uremia has not been previously described. It is unrelated to iron deficiency and is most probably explained by the known reduction in red cell ferrochelatase activity associated with chronic uremia. Serum ferritin is clearly the most useful diagnostic aid for assessing iron stores in patients on chronic hemodialysis. Whether ferritin is also the best predictor of response to iron therapy, cannot be determined on the basis of the present data.

Prevention of anthracycline cardiotoxicity by iron chelation.

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 antimalarial effect of iron chelators: Studies in animal models and in humans with mild falciparum malaria

In this study we explore the antimalarial effects of 3-hydroxypyridin-4-ones (CP compounds), a family of bidentate orally effective iron chelators in experimental animal systems in vivo and in vitro, and examine whether the iron chelator deferoxamine (DF) is active against human infection with P. falciparum. There was direct relation between lipid solubility of the CP compounds, which would facilitate membrane transit, and their in vivo antimalarial action, suggesting direct intracellular iron chelation as the most likely explantation for the antimalarial effect of iron chelators. Results of the double-blind, placebo controlled trial of DF in humans with asymptomatic parasitemia provided unequivocal evidence that this iron-chelating agent has antimalarial activity. Depriving the parasite of a metabolically important source of iron may represent a novel approach to antimalarial drug development. DF is a relatively ineffective intraerythrocytic chelator, and our data indicate that other orally effective iron chelators may have superior antimalarial activity in vivo. A systematic screening of available iron chelating drugs may result in the identification of potentially useful antimalarial compounds.

CONTROL OF STEROID‐RESISTANT AUTOIMMUNE HAEMOLYTIC ANAEMIA BY CYCLOSPORINE

adriamycin, vinblastine and procarbazine. He has remained well and euglycaemic since prednisone therapy was withdrawn 5 months after the commencement of chemotherapy. The presence of circulating antibodies against the insulin receptor usually leads to insulin resistance though occasionally hypoglycaemia occurs (Taylor et al, 1982). Recently two other reports have shown the presence of such antibodies, in association with Hodgkin’s disease (Braund et al, 1987: Walters et al, 1987), capable of completely occupying or down regulating insulin binding receptors in vitro, and also possessing a n agonistic effect accounting for the hypoglycaemia. Hypoglycaemia in both of these patients occurred when Hodgkin’s disease was clinically not evident. Autoimmune phenomenon such as haemolytic anaemia, erythema nodosum and thrombocytopenia are uncommon but well recognized in Hodgkin’s disease and may precede all other clinical manifestations. It seems conceivable that as a prodromal feature of the illness the polyclonal lymphocytes in Hodgkin’s disease are capable of production of insulin receptor antibodies possibly expressed against abnormal receptors on tumour cells. An anti-idiotype response to insulin antibody complexes resulting in the formation of insulin-receptor antibodies is a recognized phenomenon (Reeves, 198 5): however, we were unable to demonstrate the presence of insulin antibodies in our patient. Patients with autoantibodies to insulin receptor may exhibit either fasting hypoglycaemia or hyperglycaemia and extreme insulin resistance. Hypoglycaemia results from the direct insulin-like agonistic effect of the antibodies, whereas with prolonged exposure the competitive inhibition of insulin binding together with desensitization effects can result in insulin resistant hyperglycaemia. The net metabolic response is not determined by the heterogeneity of the anti-insulin receptor antibodies but is determined by the host tissue response and the caloric intake (Dons et al. 1983). The rapid reversal of hypoglycaemia with steroid therapy in this case appears to have resulted from suppression of such activity as shown by the changes of in vitro binding and biological activity before and after treatment. Our case represents the third in the literature showing the association of autoimmune hypoglycaemia with Hodgkin’s disease and the first in which the diagnosis was made during life, leading directly to the discovery of relapse of the disease. In all three cases hypoglycaemia occurred at a time when there was no other definite clinical evidence of disease activity.

Iron, infection and immune function

A decrease in circulating Fe, or hypoferraemia, is one of the most constant features of infectious disease. Since Fe deprivation in bacterial cultures is regularly associated with inhibition of growth, it has been suggested that Fe deficiency may represent an important defence mechanism (Weinberg, 1990). The term ‘nutritional immunity’ has been introduced by Kochan (1973) to underline the importance of Fe deprivation as a key mechanism limiting the growth of invading organisms. Interleukin-1 (IL-l), a protein released by mononuclear phagocytes in response to microbial invasion, is a key mediator in the inflammatory reaction and is directly responsible for the hypoferraemia of inflammation (Dinarello, 1984). It enhances the synthesis of a number of acute-phase proteins such as fibrinogen, haptoglobin, ceruloplasmin, amyloid A protein and ferritin (Fig. 1). The result of increased ferritin synthesis is a block in Fe release resulting in reduced serum Fe levels. Because of the paucity of clinical information supporting the significance of Fe deficiency or overload in determining the severity of infectious disease in man, the nutritional immunity hypothesis has remained a topic of continued controversy (Hershko & Peto, 1988). This controversy is of more than academic interest, since both Fe deficiency and infectious diseases are common conditions, and Fe supplementation in some populations may resolve one problem while aggravating the other. In the text that follows, I shall discuss briefly the importance of microbial Fe requirements, the role of Fe