High throughput fluorimetric assessment of iron traffic and chelation in iron-overloaded Caenorhabditis elegans.

The nematode Caenorhabditis elegans (C. elegans) is a convenient tool to evaluate iron metabolism as it shares great orthology with human proteins involved in iron transport, in addition to being transparent and readily available. In this work, we describe how wild-type (N2) C. elegans nematodes in the first larval stage can be loaded with acetomethoxycalcein (CAL-AM) and study it as a whole-organism model for both iron speciation and chelator permeability of the labile iron pool (LIP). This model may be relevant for high throughput assessment of molecules intended for chelation therapy of iron overload diseases.

Production of the natural iron chelator deferriferrichrysin from Aspergillus oryzae and evaluation as a novel food-grade antioxidant.

BACKGROUND: Deferriferrichrysin (Dfcy) is a siderophore found in foods fermented by Aspergillus oryzae and is a promising candidate for an antioxidant food additive because of its high binding constant toward iron. However, the Dfcy concentration is typically low in foods and cultures. RESULTS: We optimised culture conditions to improve Dfcy production to 2800 mg L(-1) from 22.5 mg L(-1) under typical conditions. Then, we evaluated the potential of Dfcy as a food additive by measuring its safety, stability, and antioxidant activity. Dfcy was sufficiently stable that over 90% remained after pasteurisation at 63 °C for 30 min at pH 3-11, or after sterilisation at 120 °C for 4 min at pH 4-6. Dfcy showed high antioxidant activity in an oil-in-water model, where inhibition of lipid oxidation was measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) assays. Dfcy decreased PV and TBARS by 83% and 75%, respectively. Antioxidant activity of Dfcy was equal to or higher than that of the synthetic chelator EDTA. CONCLUSION: Our study provides the first practical method for production of Dfcy. Dfcy can be a novel food-grade antioxidant and the first natural alternative to the synthesised iron chelator EDTA. © 2015 Society of Chemical Industry.

Assessment of iron chelates efficiency for photo-Fenton at neutral pH.

In this study, homogeneous photo-Fenton like at neutral pH was applied to remove sulfamethoxazole from water. The process was performed using different chelating agents in order to solubilize iron in a neutral water solution. The chelating agents tested were: ethylenediaminetetraacetic acid (EDTA); nitrilotriacetic acid (NTA); oxalic acid (OA) and tartaric acid (TA). The iron leaching was monitored over reaction time to evaluate the chelates stability and their resistance to HO· and UV-A radiation. Chelates of EDTA and NTA presented more stability than OA and TA, which also confirmed their higher efficiency. Total Organic Carbon (TOC) analyses were also performed to evaluate the contribution in terms of solution contamination related to the use of chelating agents. The better properties of biodegradability in respect of EDTA combined with better efficiency in terms of microcontaminant removal and the smallest TOC contribution indicate that NTA could represent a useful option to perform photo-Fenton processes at neutral pH.

Prospective randomized comparison of retroperitoneoscopic vs open pyeloplasty with minimal incision: subjective and objective assessment in adults.

OBJECTIVE: To determine the subjective and objective outcomes of retroperitoneoscopic vs open pyeloplasty with minimal incision in a prospective randomized comparison study. METHODS: In this study between August 2011 to July 2013, 30 patients underwent retroperitoneal laparoscopic pyeloplasty and 30 open pyeloplasty with minimal incision (incision length <10 cm) after randomization. The 2 groups were compared for the visual pain score on the first and second postoperative days as the primary end point of the study. Complications were recorded and graded using Dindo-modified Clavien classification of surgical complications. Success rates were evaluated by improvement in pain score and objectively by diethylene triamine penta-acetic acid renal scan and other parameters. Statistical analysis was performed with SPSS version 16.0 (IBM) with P <.05 considered statistically significant. RESULTS: The difference in the visual pain score (5.6 vs 3.2 on day 1; 3.8 vs 1.5 on day 2) and the diclofenac requirements (333.3 vs 178.75 mg) were statistically significant and more in the open pyeloplasty. The hospital stay and convalescence were significantly lower in retroperitoneoscopic group. Success rate was found to be 96.67% with 1 failure in each group. Two patients in retroperitoneoscopic group required conversion. Both groups showed significant improvement in pain score and drainage pattern on diethylene triamine penta-acetic acid scan with decrease in hydronephrosis on ultrasound evaluation. CONCLUSION: Although subjective and objective outcomes are equivalent in both the groups, the retroperitoneoscopic approach is associated with significantly less pain, less analgesic requirement, shorter hospital stay and short convalescence in comparison with open pyeloplasty.

Assessment of labile plasma iron in patients who undergo hematopoietic stem cell transplantation.

Body iron disorders have been reported after myeloablative conditioning in patients undergoing hematopoietic stem cell transplantation (HSCT). There is a concern that labile plasma iron (LPI), the redox-active form of iron, can be involved in the occurrence of toxicity and other complications commonly observed in the early post-HSCT period. In order to better understand the LPI kinetics and its determinants and implications, we undertook sequential LPI determinations before and after conditioning until engraftment in 25 auto-HSCT patients. Increased LPI was present in only 5 patients before starting conditioning. Shortly after conditioning, LPI levels were increased in 23 patients, with peak at day 0, returning to normal range upon engraftment in 21 patients. Overall, LPI levels correlated weakly with serum ferritin and more strongly with transferrin saturation; however, both parameters were apparently not applicable as surrogate markers for increased LPI. Although this was a small cohort, logistic regression suggested that baseline LPI levels could predict occurrence of grade III or IV toxicity. In conclusion, LPI kinetics is influenced by aplasia following conditioning and engraftment. Measuring LPI before starting conditioning can offer an opportunity to predict toxicity and, perhaps, the need for chelation therapy.

Decontamination of spent iron-oxide coated sand from filters used in arsenic removal.

Sand filters devised with iron-rich adsorbents are extensively promoted and deployed in the arsenic-prone south and south-east Asian countries (e.g., Bangladesh). The approach offers superior performance in removing arsenic while the spent sludge from the sand filters is an issue of concern due to the possibility of toxic releases after being discarded. In this work, a new technique is proposed for the treatment of spent iron-oxide coated sand (IOCS) from filters used in arsenic removal. Chelant-washing of the arsenic-loaded IOCS is combined with the solid phase extraction treatment to accomplish the objective. The unique point of the proposed process is the cost-effective scheme, which includes the option of recycling of the washing solvent beside the decontamination of the spent arsenic-rich sludge.

Phytochemicals and antioxidant capacities in rice brans of different color.

Rice bran, a byproduct of the rice milling process, contains most of the phytochemicals. This study aimed at determining the concentrations of lipophilic, solvent-extractable (free), and cell wall-bound (bound) phytochemicals and their antioxidant capacities from brans of white, light brown, brown, purple, and red colors, and broccoli and blueberry for comparison. The concentrations of lipophilic antioxidants of vitamin E (tocopherol and tocotrienols) and γ-oryzanols were 319.67 to 443.73 and 3861.93 to 5911.12 µg/g bran dry weight (DW), respectively, and were not associated with bran color. The total phenolic, total flavonoid, and antioxidant capacities of ORAC (oxygen radical absorbance capacity), DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging, and iron-chelating in the free fraction were correlated with the intensity of bran color, while variations of these in the bound fraction were less than those in the free fraction among brans. Compounds in the bound fraction had higher antioxidant capacity of ORAC than DPPH, relative to those in the free fraction. The bound fraction of light-color brans contributed as much to its total ORAC as the free fraction. Total proanthocyanidin concentration was the highest in red rice bran, while total anthocyanin was highest in purple brans. The predominant anthocyanin was cyanidin-3-glucoside. Red and purple brans had several fold higher total phenolics and flavonoids as well as ORAC and DPPH, from both free and bound fractions, than freeze-dried blueberry and broccoli. These results indicate that rice brans are natural sources of hydrophilic and lipophilic phytochemicals for use in quality control of various food systems as well as for nutraceutical and functional food application.

Ethical issues and risk/benefit assessment of iron chelation therapy: advances with deferiprone/deferoxamine combinations and concerns about the safety, efficacy and costs of deferasirox.

New developments in the area of iron and other metal metabolism and toxicity and the effects and uses of chelators have been presented at the 16th International Conference on Chelation (ICOC), Limassol, Cyprus in October 2006. Marketing practices by pharmaceutical companies, contradictory policies by regulatory authorities and ineffective policies by health authorities deprive thousands of thalassemia and other transfused patients of life saving iron chelating drugs and of efficacious chelation treatments. Thousands of patients were using deferasirox (DFRA) worldwide a few months after the European Union (EU) authorities, and about 1 year after the Food and Drugs Administration (FDA), proceeded to its accelerated approval with no sufficient evidence that the drug was efficacious, especially for clearing excess cardiac iron, and also safe. Cases of fatal, acute, irreversible renal and liver failure, fatal agranulocytosis and other toxicities have recently been reported with DFRA. The FDA has not yet approved deferiprone (L1) depriving thousands of patients of potentially life saving treatment. The high cost of DFRA at 60 euros/g, L1 at 5.5 euros/g and deferoxamine (DFO) at 8.3 euros/g, diminishes the prospects of universal chelation therapy, especially for patients in developing countries. The safety and efficacy record of L1, DFO, and their combination in particular, appear to provide universal solutions in the treatment of transfusional iron overload, and also in reducing mortality because of their ability to clear rapidly and effectively excess cardiac iron.

Assessment of chelatable mitochondrial iron by using mitochondrion-selective fluorescent iron indicators with different iron-binding affinities.

Chelatable cellular iron, and chelatable mitochondrial iron in particular, has yet to be well characterized, so the overall strength with which these "loosely bound" iron ions (presumably mainly Fe(II)) are intracellularly/intramitochondrially bound is unclear. We have previously reported the first selective mitochondrial iron indicator: rhodamine B 4-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). With this compound as a model, we have now developed two additional mitochondrial iron indicators with very different iron-binding affinities and have applied these to the study of the chelatable iron pool in the mitochondria of isolated rat liver cells. With the new indicator rhodamine B 4-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester (RDA), with 2,2'-bipyridine as chelating unit (log beta(3)=17.5), essentially the same iron concentration (16.0+/-1.9 microM) was determined as with RPA (log beta(3)=21.1), despite the four orders of magnitude difference in Fe(II)-binding affinity. This not only demonstrates the reliability of the procedure, but also confirms that iron complexation by these indicators does not induce any significant release of iron from the iron-storage proteins on the timescale of the experiment. In contrast, the indicator rhodamine B 4-[bis(pyridin-2-ylmethyl)aminomethyl]benzyl ester (PIRO), with an N,N-bis(pyridin-2-ylmethyl)amine group as chelating component (log beta(2)=12.2), could not compete against the array of endogenous ligands. The intramitochondrial concentrations of the three indicators were determined to be in the range of 100 microM: that is, about three orders of magnitude lower than the total concentration of endogenous compounds that might chelate iron ions. It is therefore estimated that chelatable mitochondrial iron ions are bound by endogenous ligands with apparent stability constants (log K(app)) of between 9 and 14.

Synthesis and physicochemical assessment of novel 2-substituted 3-hydroxypyridin-4-ones, novel iron chelators.

Novel 3-hydroxypyridin-4-one containing tridentate ligands were synthesised and their physicochemical properties characterised, including ionisation constants and stoichiometric titration with Fe(III). There is an urgent demand for orally active iron chelators with potential for the treatment of thalassaemia. In principle, tridentate ligands are likely to be more kinetically stable than bidentate molecules, but to date no satisfactory molecules have been identified. Fe(III) stability constants were assessed by competition with the hexadentate ligand EDTA. In all cases no evidence was found for a tridentate mode of iron chelation; instead the ligands behaved as bidentate hydroxypyridinones. As a consequence they provide no advantage over the more simple alkyl hydroxypyridinones.

Assessment of structural features of the pseudomonas siderophore pyochelin required for its ability to promote oxidant-mediated endothelial cell injury.

We previously showed that iron chelated to the Pseudomonas aeruginosa siderophore pyochelin enhances oxidant-mediated injury to pulmonary artery endothelial cells by catalyzing hydroxyl radical (HO(*)) formation. Therefore, we examined pyochelin structural/chemical features that may be important in this process. Five pyochelin analogues were examined for (i) capacity to accentuate oxidant-mediated endothelial cell injury, (ii) HO(*) catalytic ability, (iii) iron transfer to endothelial cells, and (iv) hydrophobicity. All compounds catalyzed similar HO(*) production, but only the hydrophobic ones containing a thiazolidine ring enhanced cell injury. Transfer of iron to endothelial cells did not correlate with cytotoxicity. Finally, binding of Fe(3+) by pyochelin led to Fe(2+) formation, perhaps explaining how Fe(3+)-pyochelin augments H(2)O(2)-mediated cell injury via HO(*) formation. The ability to bind iron in a catalytic form and the molecule's thiazolidine ring, which increases its hydrophobicity, are key to pyochelin's cytotoxicity. Reduction of Fe(3+) to Fe(2+) may also be important.

Improving the oral bioavailability of the iron chelator HBED by breaking the symmetry of the intramolecular H-bond network.

Physicochemical analysis and Monte Carlo simulations were used to identify structural features which prevent oral absorption of HBED, a potent iron chelator. In water the dominant conformations of HBED involve the hydrophobic collapse of the two aromatic rings. These conformations are favored in polar media because they expose the polar phenolic hydroxy groups to the solvent and partially shield the nonpolar aromatic rings. In a less polar solvent such as chloroform, a symmetrical H-bond network between the carboxylates and the amines dominates the conformational space. This leads to the exposure of the phenolic hydroxy groups to the solvent, which is unfavorable for solvation. The low solubility of HBED in nonpolar solvents was confirmed experimentally by determination of the partition coefficients in octanol, chloroform, and cyclohexane and may explain the poor membrane permeability of this compound. The high conformational stability which disfavors partitioning into phospholipids is mainly due to the symmetrical H-bond network. Potentiometric titrations of a monoester of HBED in MeOH/water indicate that the protonation sequence was changed compared to that of the parent compound, suggesting that the symmetrical H-bond network was disrupted. Conformational analysis in chloroform confirmed that, in contrast to HBED, no symmetric interaction between the carboxylate and the nitrogen amines is possible in the half-ester and a variety of conformations which allow partial shielding of the polar phenolic OH groups are energetically possible. This theoretical model predicting a better solubility of the half-esters in nonpolar solvents was supported by the large increase in the partition coefficients in octanol, chloroform, and cyclohexane measured experimentally. The high absorbability predicted by physicochemical and computer simulation methods was corroborated by in vivo experiments in marmoset monkeys where the monoethyl ester derivative of HBED was well-absorbed orally while the parent compound was nearly ineffective in the same model.

[A new property of aspirin and other salicylates. Their capacity for radical generation at the expense of chelating-oxidizing action on iron cations]. / Novoe svoistvo aspirina i drugikh salitsilatov. Ikh sposobnost' k generatsii radikalov za schet khelatiruiushche-okisliaiushchego deistviia na kationy zheleza.

It was demonstrated that salicylic acid, by binding ions of ferrous iron, induces its oxidation to the oxide form by molecular oxygen with generation of active forms of oxygen. Salicylic acid may therefore form stable ferrosalicylic complexes only with iron oxide, in which case the affinity of salicylic acid for iron oxide is higher than that of o-phenanthroline and lower than that of EDTA. In the presence of ascorbate in the system with salicylic acid and iron ions continuous radical formation may take place due to alternating cycles of oxidation-reduction iron ions through the combined effect of salicylic acid and ascorbate on them. The initial salicylic acid content in commercial aspirin preparations and its intensive formation at pH values close to the physiological parameters were determined by colorimetry and the inner standard method. After incubation of aspirin preparations at pH close to the physiological values, accelerated oxidation of ferrous iron with formation of active oxygen forms was recorded, which was evidently due to salicylic acid produced as the result of aspirin breakdown under such conditions.