Gabriela Link

Pathophysiology of Iron Overloada

Abstract: In thalassemia, iron overload is the joint outcome of excessive iron absorption and transfusional siderosis. While iron absorption is limited by a physiologic ceiling of about 3 mg/d, plasma iron turnover in thalassemia may be 10 to 15 times normal, caused by the wasteful, ineffective erythropoiesis of an enormously expanded erythroid marrow. This outpouring of catabolic iron exceeds the iron‐binding capacity of transferrin and appears in plasma as non‐transferrin‐plasma iron (NTPI). The toxicity of NTPI is much higher than of transferrin‐iron as judged by its ability to promote hydroxyl radical formation resulting in peroxidative damage to membrane lipids and proteins. In the heart, this results in impaired function of the mitochrondrial respiratory chain and abnormal energy metabolism manifested clinically in fatal hemosiderotic cardiomyopathy. Ascorbate increases the efficacy of iron chelators by expanding the intracellular chelatable iron pool, but, at suboptimal concentrations is a pro‐oxidant, enhancing the catalytic effect of iron in free radical formation. NTPI is removed by i.v. DFO in a biphasic manner and reappears rapidly upon cessation of DFO, lending support to the continuous, rather than intermittent, use of chelators. Unlike DFO and other hexadentate chelators, bidentate chelators such as L1 may produce incomplete intermediate iron complexes at suboptimal drug concentrations.

Fructose-Induced Fatty Liver Disease: Hepatic Effects of Blood Pressure and Plasma Triglyceride Reduction

The most known risk factor for nonalcoholic fatty liver disease (NAFLD) is the metabolic syndrome. In this study, we characterized changes in liver pathology, hepatic lipid composition, and hepatic iron concentration (HIC) occurring in rats given fructose-enriched diet (FED), with and without therapeutic maneuvers to reduce blood pressure and plasma triglycerides. Rats were given FED or standard rat chow for 5 weeks. Rats on FED were divided into 4 groups: receiving amlodipine (15 mg/kg per day), captopril (90 mg/kg per day), bezafibrate (10 mg/kg per day) in the last 2 weeks, or a control group that received FED only. FED rats had hepatic macrovesicular and microvesicular fat deposits develop, with increase in hepatic triglycerides (+198%) and hepatic cholesterol (+89%), but a decrease in hepatic phospholipids (−36%), hypertriglyceridemia (+223%), and hypertension (+15%), without increase in HIC. Amlodipine reduced blood pressure (−18%), plasma triglycerides (−12%), but there was no change in hepatic triglycerides and phospholipids concentrations. Captopril reduced blood pressure (−24%), plasma triglycerides (−36%), hepatic triglycerides (−51%), and hepatic macrovesicular fat (−51%), but increased HIC (+23%), with a borderline increase in hepatic fibrosis. Bezafibrate reduced plasma triglycerides (−49%), hepatic triglycerides (−78%), hepatic macrovesicular fat (−90%), and blood pressure (−11%). We conclude that FED rats can be a suitable model for human NAFLD. Drugs administered to treat various aspects of the metabolic syndrome could have hepatic effects. An increase in HIC in rats with NAFLD could be associated with increased hepatic fibrosis.

Iron Chelators for Thalassaemia

The iron-chelating compound desferrioxamine (DFO) was discovered accidentally, as a byproduct of antibiotic research by scientists in the Swiss Federal Institute of Technology in Zurich and Ciba in Basel. Its development into a useful clinical drug was not by design, but thanks to natural curiosity and ingenuity, as described by Keberle (1992). Although DFO has been available for treating transfusional iron overload from the early 1960s, the era of modern and effective iron-chelating 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 thalassaemia and other transfusion-dependent anaemias, with a major impact on wellbeing and survival. In this Review we will discuss the abnormalities in iron metabolism associated with thalassaemia major and the chelatable iron pools representing the targets of iron-chelating therapy, examine the results of longterm DFO therapy in thalassaemia, and, finally, review the development of new, orally effective, iron chelators which may be considered for present or future use.

Objectives and Mechanism of Iron Chelation Therapy

Abstract: Prevention of cardiac mortality is the most important beneficial effect of iron chelation therapy. Unfortunately, compliance with the rigorous requirements of daily subcutaneous deferoxamine (DFO) infusions is still a serious limiting factor in treatment success. The development of orally effective iron chelators such as deferiprone and ICL670 is intended to improve compliance. Although total iron excretion with deferiprone is somewhat less than with DFO, deferiprone may have a better cardioprotective effect than DFO due to deferiprones ability to penetrate cell membranes. Recent clinical studies indicate that oral ICL670 treatment is well tolerated and is as effective as parenteral DFO used at the standard dose of 40 mg/kg of body weight/day. Thus, for the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available to avoid serious organ damage. Finally, combined chelation treatment is emerging as a reasonable alternative to chelator monotherapy. Combining a weak chelator that has a better ability to penetrate cells with a stronger chelator that penetrates cells poorly but has a more efficient urinary excretion may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.

Screening for Active Small Molecules in Mitochondrial Complex I Deficient Patient's Fibroblasts, Reveals AICAR as the Most Beneficial Compound

Congenital deficiency of the mitochondrial respiratory chain complex I (CI) is a common defect of oxidative phosphorylation (OXPHOS). Despite major advances in the biochemical and molecular diagnostics and the deciphering of CI structure, function assembly and pathomechanism, there is currently no satisfactory cure for patients with mitochondrial complex I defects. Small molecules provide one feasible therapeutic option, however their use has not been systematically evaluated using a standardized experimental system. In order to evaluate potentially therapeutic compounds, we set up a relatively simple system measuring different parameters using only a small amount of patients fibroblasts, in glucose free medium, where growth is highly OXPOS dependent. Ten different compounds were screened using fibroblasts derived from seven CI patients, harboring different mutations. 5-Aminoimidazole-4-carboxamide ribotide (AICAR) was found to be the most beneficial compound improving growth and ATP content while decreasing ROS production. AICAR also increased mitochondrial biogenesis without altering mitochondrial membrane potential (Δψ). Fluorescence microscopy data supported increased mitochondrial biogenesis and activation of the AMP activated protein kinase (AMPK). Other compounds such as; bezafibrate and oltipraz were rated as favorable while polyphenolic phytochemicals (resverastrol, grape seed extract, genistein and epigallocatechin gallate) were found not significant or detrimental. Although the results have to be verified by more thorough investigation of additional OXPHOS parameters, preliminary rapid screening of potential therapeutic compounds in individual patients fibroblasts could direct and advance personalized medical treatment.

A rapid and sensitive ELISA for serum ferritin employing a fluorogenic substrate

A fluorescent enzyme-linked immunosorbent assay is described for the rapid measurement of serum ferritin. Increased sensitivity was achieved by using 4-methyl-umbelliferyl-beta-D-galactopyranoside as the substrate for beta-galactosidase coupled to the purified antiferritin antibody. Further enhancement of the specific antigen-antibody reaction was attained by the addition of 4% polyethylene glycol 6000 to the antiferritin-beta-galactosidase conjugate. The procedure is performed in microELISA plates. These modifications of the method permit the measurement of serum ferritin at concentrations ranging from 0.25 to 50 microgram/liter with a coefficient of variation of 8% or less. The entire procedure is performed at ambient temperature and is completed within one working day. The cost of the assay is less than 10% of the immunoradiometric assay for serum ferritin.

Cardioprotective effect of α-tocopherol, ascorbate, deferoxamine, and deferiprone: Mitochondrial function in cultured, iron-loaded heart cells

Because mitochondrial inner membrane respiratory complexes are important targets of iron toxicity, we used iron-loaded rat heart cells in culture to study the beneficial effect on mitochondrial enzymes of the iron chelators deferoxamine (DFO) and deferiprone (L1) and of antioxidants and reducing agents (ascorbate and alpha-tocopherol). Reduced nicotinamide adenine dinucleotide-cytochrome c oxidoreductase (complex I-III) and succinate dehydrogenase were the most-sensitive indicators of iron toxicity and cardioprotective effect. Although at concentrations below 0.3 mmol/L the iron-mobilizing effect of L1 was less than that of DFO, both were equally effective in protecting or restoring mitochondrial respiratory enzyme activity. At 1.0 mmol/L, L1 toxicity was manifested in respiratory enzyme inhibition, whereas DFO had no such effect. Ascorbate (0.057 to 5.7 mmol/L) had a mild cardioprotective effect at the highest concentration only, in association with decreased cellular iron uptake. By contrast, alpha-tocopherol (0.023 mmol/L) completely inhibited mitochondrial iron toxicity without affecting iron uptake or release, and irrespective of whether it was used before, during, or after in vitro iron loading. These observations illustrate the usefulness and limitations of iron chelators and other agents used for preventing iron toxicity to the heart and other vital organs, and they underline the need for exploring in more detail the effects of these agents in the clinical setting.

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 Role of Iron and Iron Chelators in Anthracycline Cardiotoxicity

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.

Effects of Amlodipine, Captopril, and Bezafibrate on Oxidative Milieu in Rats with Fatty Liver

Oxidative stress may initiate significant hepatocyte injury in subjects with fatty liver. We characterized changes in hepatic oxidative anti-oxidative parameters in rats given a fructose-enriched diet (FED) with and without medications to reduce blood pressure or plasma triglycerides. FED rats had an increase in malondialdehyde (MDA) concentration, a reduction in α-tocopherol concentration, a reduction in paraoxonase (PON) activity, an increase in glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R) activity. Amlodipine increased PON and GSH-Px, but decreased GSSG-R activity and α-tocopherol concentration. Captopril decreased MDA concentration and the activity of both GSH-Px and GSSG-R, but increased α-tocopherol concentration and PON activity. Bezafibrate increased α-tocopherol concentration and PON activity, but decreased the activity of GSSG-R. Animals with fatty liver exhibit an increase in peroxidative stress but also a defect in anti-oxidative pathways. Drugs administered to treat hypertension and hypertriglyceridemia could lead to a variety of changes in the hepatic oxidative, anti-oxidative milieu.