Dynamics of the cytosolic chelatable iron pool of K562 cells

AbstractThe labile iron pool of cells (LIP) constitutes the primary source of metabolic and catalytically reactive iron in the cytosol. We studied LIP homeostasis in K562 cells using the fluorescent metal-sensitive probe calcein. Following brief exposure to iron(II) salts or to oxidative or reductive stress, LIP rose by up to 120% relative to the normal level of 350 nM. However, the rate of recovery to normal LIP level differed markedly with each treatment (respective t12s of 27, 65–88 and ≤17 min). We show that the capacity of K562 cells to adjust LIP levels is highly dependent on the origin of the LIP increase and on the pre-existing cellular iron status

Reduction in labile plasma iron during treatment with deferasirox, a once-daily oral iron chelator, in heavily iron-overloaded patients with β-thalassaemia

This subgroup analysis evaluated the effect of once-daily oral deferasirox on labile plasma iron (LPI) levels in patients from the prospective, 1-yr, multicentre ESCALATOR study. Mean baseline liver iron concentration and median serum ferritin levels were 28.6 ± 10.3 mg Fe/g dry weight and 6334 ng/mL respectively, indicating high iron burden despite prior chelation therapy. Baseline LPI levels (0.98 ± 0.82 μmol/L) decreased significantly to 0.12 ± 0.16 μmol/L, 2 h after first deferasirox dose (P=0.0006). Reductions from pre- to post-deferasirox administration were also observed at all other time points. Compared to baseline, there was a significant reduction in preadministration LPI that reached the normal range at week 4 and throughout the remainder of the study (P≤0.02). Pharmacokinetic analysis demonstrated an inverse relationship between preadministration LPI levels and trough deferasirox plasma concentrations. Once-daily dosing with deferasirox ≥20 mg/kg/d provided sustained reduction in LPI levels in these heavily iron-overloaded patients, suggesting 24-h protection from LPI. Deferasirox may therefore reduce unregulated tissue iron loading and prevent further end-organ damage

Objectives and Methods of Iron Chelation Therapy

Recent developments in the understanding of the molecular control of iron homeostasis provided novel insights into the mechanisms responsible for normal iron balance. However in chronic anemias associated with iron overload, such mechanisms are no longer sufficient to offer protection from iron toxicity, and iron chelating therapy is the only method available for preventing early death caused mainly by myocardial and hepatic damage. Today, long-term deferoxamine (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. Within recent years more than one thousand candidate compounds have been screened in animal models. The most outstanding of these compounds include deferiprone (L1); pyridoxal isonicotinoyl hydrazone (PIH) and; bishydroxy- phenyl thiazole. Deferiprone has been used extensively as a substitute for DFO in clinical trials involving hundreds of patients. However, L1 treatment alone fails to achieve a negative iron balance in a substantial proportion of subjects. Deferiprone is less effective than DFO and its potential hepatotoxicity is an issue of current controversy. A new orally effective iron chelator should not necessarily be regarded as one displacing the presently accepted and highly effective parenteral drug DFO. Rather, it could be employed to extend the scope of iron chelating strategies in a manner analogous with the combined use of medications in the management of other conditions such as hypertension or diabetes. Coadministration or alternating use of DFO and a suitable oral chelator may allow a decrease in dosage of both drugs and improve compliance by decreasing the demand on tedious parenteral drug administration. Combined use of DFO and L1 has already been shown to result in successful depletion of iron stores in patients previously failing to respond to single drug therapy, and to lead to improved compliance with treatment. It may also result in a “shuttle effect” between weak intracellular chelators and powerful extracellular chelators or exploit the entero-hepatic cycle to promote fecal iron excretion. All of these innovative ways of chelator usage are now awaiting evaluation in experimental models and in the clinical setting

Lactate-proton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots

Exposure of nervous tissue to hypoxia results in interstitial acidification. There is evidence for concomitant decrease in extracellular pH to the increase in tissue lactate. In the present study, we used double-barrelled pH-sensitive microelectrodes to investigate the link between lactate transport and acid-base homeostasis in isolated rat spinal roots. Addition of different organic anions to the bathing solution at constant bath pH caused transient alkaline shifts in extracellular pH; withdrawal of these compounds resulted in transient acid shifts in extracellular pH. With high anion concentrations (30 mM), the largest changes in extracellular pH were observed with propionate >l-lactate ≈ pyruvate >62; 2-hydroxy-2-methylpropionate. Changes in extracellular pH induced by 10 mMl- andd-lactate were of similar size. Lactate transport inhibitors α-cyano-4-hydroxycinnamic acid and 4,4′-dibenzamidostilbene-2,2′-disulphonic acid significantly reducedl-lactate-induced extracellular pH shifts without affecting propionate-induced changes in extracellular pH. Hypoxia produced an extracellular acidification that was strongly reduced in the presence of α-cyano-4-hydroxycinnamic acid and 4,4′-dibenzamidostilbene-2,2′-disulphonic acid. In contrast, amiloride and 4,4′-di-isothiocyanostilbene-2,2′-disulphonate were without effect on hypoxia-induced acid shifts. The results indicate the presence of a lactate-proton co-transporter in rat peripheral nerves. This transport system and not Na+/H+ or C1−/HCO−3 exchange seems to be the dominant mechanism responsible for interstitial acidification during nerve hypoxia

Performance of Redox Active and Chelatable Iron Assays to Determine Labile Iron Release From Intravenous Iron Formulations

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137448/1/cts12443_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137448/2/cts12443.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137448/3/cts12443-sup-0003-figureS2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137448/4/cts12443-sup-0002-figureS1.pd

Response of iron overload to deferasirox in rare transfusion-dependent anaemias: equivalent effects on serum ferritin and labile plasma iron for haemolytic or production anaemias

Objectives: It is widely assumed that, at matched transfusional iron-loading rates, responses to chelation therapy are similar, irrespective of the underlying condition. However, data are limited for rare transfusion-dependent anaemias, and it remains to be elucidated if response differs, depending on whether the anaemia has a primary haemolytic or production mechanism. Methods: The efficacy and safety of deferasirox (Exjade (R)) in rare transfusion-dependent anaemias were evaluated over 1 yr, with change in serum ferritin as the primary efficacy endpoint. Initial deferasirox doses were 10-30 mg/kg/d, depending on transfusion requirements; 34 patients had production anaemias, and 23 had haemolytic anaemias. Results: Patients with production anaemias or haemolytic anaemias had comparable transfusional iron-loading rates (0.31 vs. 0.30 mL red blood cells/kg/d), mean deferasirox dosing (19.3 vs. 19.0 mg/kg/d) and baseline median serum ferritin (2926 vs. 2682 ng/mL). Baseline labile plasma iron (LPI) levels correlated significantly with the transfusional iron-loading rates and with serum ferritin levels in both cohorts. Reductions in median serum ferritin levels were initially faster in the production than the haemolytic anaemias, but at 1 yr, similar significant reductions of 940 and 617 ng/mL were attained, respectively (-26.0% overall). Mean LPI decreased significantly in patients with production (P < 0.0001) and haemolytic (P = 0.037) anaemias after the first dose and was maintained at normal mean levels (< 0.4 mu m) subsequently. The most common drug-related, investigator-assessed adverse events were diarrhoea (n = 16) and nausea (n = 12). Conclusions: At matched transfusional iron-loading rates, the responses of rare transfusion-dependent anaemias to deferasirox are similar at 1 yr, irrespective of the underlying pathogenic mechanism

Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders

Non-transferrin-bound iron and its labile (redox active) plasma iron component are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. We compared ten worldwide leading assays (6 for non-transferrin-bound iron and 4 for labile plasma iron) as part of an international inter-laboratory study. Serum samples from 60 patients with four different iron-overload disorders in various treatment phases were coded and sent in duplicate for analysis to five different laboratories worldwide. Some laboratories provided multiple assays. Overall, highest assay levels were observed for patients with untreated hereditary hemochromatosis and beta-thalassemia intermedia, patients with transfusion-dependent myelodysplastic syndromes and patients with transfusion-dependent and chelated beta-thalassemia major. Absolute levels differed considerably between assays and were lower for labile plasma iron than for non-transferrin-bound iron. Four assays also reported negative values. Assays were reproducible with high between-sample and low within-sample variation. Assays correlated and correlations were highest within the group of non-transferrin-bound iron assays and within that of labile plasma iron assays. Increased transferrin saturation, but not ferritin, was a good indicator of the presence of forms of circulating non-transferrin-bound iron. The possibility of using non-transferrin-bound iron and labile plasma iron measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of assay