ascorbic-acid and ferric-citrate

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1-100 nM were rapidly iron-depleted when ferric citrate-but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.

Topics: Aquatic Organisms; Ascorbic Acid; Cells, Cultured; Edetic Acid; Ferric Compounds; Iron; Microalgae

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled (55)Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.

Topics: Arabidopsis; Arabidopsis Proteins; Ascorbic Acid; Biological Transport; Ferric Compounds; FMN Reductase; Iron; Iron Radioisotopes; Malates; Membrane Proteins; Nucleotidyltransferases; Phosphoric Monoester Hydrolases; Pisum sativum; Plant Proteins; Seeds

We studied mangiferin effects on the degradation of 2-deoxyribose induced by Fe(III)-EDTA/citrate plus ascorbate, in relation to ascorbate oxidation (measured at 265 nm). Results revealed that mangiferin was equally effective in preventing degradation of both 15 and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or citrate) caused a significant reduction in the protective effects of mangiferin. Interestingly, mangiferin strongly stimulated Fe(III)-EDTA ascorbate oxidation, but inhibited it when citrate was used as iron co-chelator. Mangiferin stimulated O2 consumption due to Fe(II) (formed by Fe(III) ascorbate reduction) autoxidation when the metal ligand was EDTA, but inhibited it when citrate was used. These results suggest that mangiferin removes iron from citrate, but not from EDTA, forming an iron-mangiferin complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. Taken together, these results indicate that mangiferin works mainly by a mechanism different from the classical hydroxyl radical scavengers, keeping iron in its ferric form, by complexing Fe(III), or stimulating Fe(II) autoxidation.

Topics: Antioxidants; Ascorbic Acid; Deoxyribose; Edetic Acid; Ferric Compounds; Iron Chelating Agents; Models, Chemical; Oxidation-Reduction; Xanthones

Dietary iron is present in the intestine as Fe(II) and Fe(III). Since enterocytes take up Fe(II) by the divalent metal transporter (DMT1), Fe(III) must be reduced. Whether other Fe(III) transport processes are present is unknown. Release of iron from the enterocyte into the plasma involves the iron-regulated transporter-1/metal transporter protein-1 (IREG-1/MTP-1, ferroportin) but ferroportin is also found on the apical membrane. We compared the uptake of iron from Fe(II):ascorbate and Fe(III):citrate using the rat intestinal enterocyte cell line-6 (IEC-6), in the presence of ferrous chelators, a blocking antibody to ferroportin, at different pH and during the over-expression of DMT1. Firstly, surface ferrireduction was absent. Secondly, blocking ferroportin partly and totally reduced Fe(II) and Fe(III) uptake, respectively. Thirdly, optimal Fe(II) uptake occurred at pH 5.5 but Fe(III) uptake was unaffected by pH and, fourthly, over-expression of DMT1 increased uptake of Fe(II) and Fe(III). This indicates that an increased extracellular H+ concentration facilitates DMT1-mediated Fe(II) uptake at the cell membrane. However, since Fe(III) uptake required DMT1, but not cell surface ferrireduction, and was independent of variations in extracellular pH, it appears that Fe(III) is internalised before ferrireduction and transport by DMT1. Ferroportin may function as a modulator of DMT1 activity and play a role in Fe(III) uptake, possibly by affecting the number or affinity of citrate binding sites.

Topics: Animals; Ascorbic Acid; Biological Transport; Cation Transport Proteins; Cell Line; Citric Acid; Ferric Compounds; Hydrogen-Ion Concentration; Intestines; Iron; Iron Chelating Agents; Iron-Binding Proteins; Rats

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Topics: Ascorbic Acid; Chelating Agents; Deoxyribose; Edetic Acid; Electron Spin Resonance Spectroscopy; Ferric Compounds; Free Radicals; Hydroxyl Radical; Isoniazid; Nitrilotriacetic Acid; Oxidation-Reduction; Oxidative Stress; Pyridoxal

Although potassium sorbate (PS), ascorbic acid and ferric or ferrous salts (Fe-salts) are used widely in combination as food additives, the strong reactivity of PS and oxidative potency of ascorbic acid in the presence of Fe-salts might form toxic compounds in food during its deposit and distribution. In the present paper, the reaction mixture of PS, ascorbic acid and Fe-salts was evaluated for mutagenicity and DNA-damaging activity by means of the Ames test and rec-assay. Effective lethality was observed in the rec-assay. No mutagenicity was induced in either Salmonella typhimurium strains TA98 (with or without S-9 mix) or TA100 (with S-9 mix). In contrast, a dose-dependent mutagenic effect was obtained when applied to strain TA100 without S-9 mix. The mutagenic activity became stronger increasing with the reaction period. Furthermore, the reaction products obtained in a nitrogen atmosphere did not show any mutagenic and DNA-damaging activity. PS, ascorbic acid and Fe-salts were inactive when they were used separately. Omission of one component from the mixture of PS, ascorbic acid and Fe-salt turned the reaction system inactive. These results demonstrate that ascorbic acid and Fe-salt oxidized PS and the oxidative products caused mutagenicity and DNA-damaging activity.

Topics: Ascorbic Acid; Diphosphates; DNA Damage; Edetic Acid; Ferric Compounds; Ferrous Compounds; Food Preservatives; Iron; Mutagenicity Tests; Ribosomal Proteins; Salmonella typhimurium; Sorbic Acid

In order to study the biological consequences of DNA damage induced by H2O2-mediated free radical reactions, DNA from bacteriophage PM2 was exposed to H2O2, Fe(3+)-citrate and ascorbate either alone or in combination. Induction of DNA lesions was determined as well as the biological activity of the phage DNA. Exposure to H2O2 alone resulted in max. 0.2 single-strand breaks per molecule; in the presence of Fe(3+)-citrate, the yield was approximately 4-fold higher. Under both conditions no double-strand breaks could be detected and the biological activity was not diminished. This indicates that low levels of single-strand breaks as generated by H2O2/Fe(3+)-citrate do not inactivate PM2 DNA. Exposure to ascorbate in the presence Fe(3+)-citrate resulted in extensive induction of single-strand breaks. However, at ascorbate concentration where approximately 3 single-strand breaks per molecule were induced, again no double-strand breaks could be detected and the biological activity of the DNA was not diminished. At 5 mM ascorbate, single-strand breaks were above the detection limit. Under these conditions, 0.02 double-strand breaks were induced and the biological activity was reduced to 50%. The contribution of double-strand breaks to biological inactivation was calculated to be approximately 3%. When PM2 DNA was exposed to H2O2 in the presence of ascorbate/Fe(3+)-citrate, a typical biphasic dose-effect relationship was observed both for the induction of double-strand breaks and biological inactivation, suggesting that one or more reactive species sensitive to H2O2 play a critical role. The .OH scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA, which may indicate that other reactive species than .OH are involved. Our data suggest that other reactive species than .OH, such as the ferryl ion, are involved in H2O2-mediated DNA damage induction and biological inactivation.

Topics: Ascorbic Acid; Bacteriophages; Butanols; DNA Damage; DNA, Viral; Ferric Compounds; Free Radicals; Hydrogen Peroxide; tert-Butyl Alcohol

We investigated the effect of Fe deficiency on the nutritive utilization of Fe, Ca, P and Mg in rats. Aside from the well known depletion of Fe in liver, femur and sternum with low values of Hb, Fe deficiency impaired Ca, P and Mg metabolism at different degrees. Iron deficiency altered Mg absorption, lowered the concentration of Ca in the liver, femur and sternum, raised the concentration of P and Mg in the liver, and decreased P in the femur. The altered status was not completely rectified by iron supplementation as the animals were still slightly anemic at the end of the study. The second purpose of the study was to evaluate the ability of three iron compounds (ferric citrate, ferrous sulfate and ferrous ascorbate) to correct the undesirable effects of Fe deficiency. Ten days after treatment with these diets, Fe-deficient rats still had reduced Mg absorption, especially those fed ferric citrate. The concentrations of hemoglobin approached normal values in all groups; however, serum Fe remained low, indicating that Fe reserves were still depleted. Hepatic and femoral Fe concentrations were also lower in all Fe-deficient groups regardless of the diet given, compared with their respective controls, whereas Fe concentrations in the sternum increased significantly with all three diets, suggesting an increase in erythropoiesis. The concentration of Ca, P and Mg in liver approached normal values, and appeared to normalize in the femur, except that Ca and P concentrations remained low with the citrate diet. In the sternum, a site assumed to have higher requirements for these minerals, the concentrations of Ca, P and Mg also increased. These findings indicate that Fe is involved in the bone mineralization, and that in physiological terms, Fe interacts favorably with Ca, P and Mg metabolism, since Fe deficiency altered the status of these metals. These findings also suggest that ferrous ascorbate and ferrous sulfate were more effectively absorbed than was ferric citrate.

Topics: Analysis of Variance; Anemia, Iron-Deficiency; Animal Feed; Animals; Ascorbic Acid; Body Weight; Calcium; Eating; Ferric Compounds; Ferrous Compounds; Food, Fortified; Hemoglobins; Iron; Iron Deficiencies; Magnesium; Male; Phosphorus; Random Allocation; Rats; Rats, Wistar; Tissue Distribution

Mammalian cells accumulate iron from ferric citrate or ferric nitrilotriacetate through the activity of a transferrin-independent iron transport system [Sturrock, Alexander, Lamb, Craven and Kaplan (1990) J. Biol. Chem. 265, 3139-3145]. The uptake system might recognize and transport ferric-anion complexes, or cells may reduce ferric iron at the surface and then transport ferrous iron. To distinguish between these possibilities we exposed cells to either [59Fe]ferric citrate or ferric [14C]citrate and determined whether accumulation of iron was accompanied by the obligatory accumulation of citrate. In HeLa cells and human skin fibroblasts the rate of accumulation of iron was three to five times greater than that of citrate. Incubation of fibroblasts with ferric citrate or ferric ammonium citrate resulted in an enhanced accumulation of iron and citrate; the molar ratio of accumulation approaching unity. A similar rate of citrate accumulation, however, was observed when ferric citrate-incubated cells were exposed to [14C]citrate alone. Further studies demonstrated the independence of iron and citrate accumulation: addition of unlabelled citrate to cells decreased the uptake of labelled citrate without affecting the accumulation of 59Fe; iron uptake was decreased by the addition of ferrous chelators whereas the uptake of citrate was unaffected; reduction of ferric iron by ascorbate increased the uptake of iron but had no effect on the uptake of citrate. When HeLa cells were depleted of calcium, iron uptake decreased, but there was little effect on citrate uptake. These results indicate that transport of iron does not require the obligatory transport of citrate and vice versa. The mammalian transferrin-independent iron transport system appears functionally similar to iron transport systems in both the bacterial and plant kingdoms which require the activities of both a surface reductase and a ferrous metal transporter.

Topics: Ascorbic Acid; Calcium; Cell Membrane; Cells, Cultured; Citrates; Citric Acid; Ferric Compounds; Fibroblasts; FMN Reductase; HeLa Cells; Humans; Iron; Iron Chelating Agents; NADH, NADPH Oxidoreductases; Skin

Topics: Animals; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Female; Ferric Compounds; Free Radicals; Rats; Rats, Sprague-Dawley

The relative risk of primary hepatocellular carcinoma in genetic hemochromatosis (GH) is estimated at over 200 times as that of control populations. Recently, ferric ion chelated to citrate (Fe-citrate) was identified as the major non-transferrin-bound iron in the serum of GH patients. We investigated whether low concentration of Fe-citrate plus reductant could damage supercoiled plasmid DNA under physiological pH and ionic strength. Incubation of Fe-citrate with either H2O2, L-ascorbate, or L-cysteine induced single- and double-strand breaks in supercoiled plasmid pZ189 in a concentration- and time-dependent fashion. DNA strand breaks produced by Fe-citrate plus H2O2 increased at reduced pH (< or = 6.9). Catalase and free radical scavengers inhibited the DNA breakage produced by Fe-citrate in combination with each reductant, suggesting that H2O2 and finally .OH are responsible DNA damaging species. The catalytic ability of Fe-citrate to induce DNA strand breaks, particularly double-strand breaks (DSBs), may contribute to the carcinogenic processes observed in GH.

Topics: Ascorbic Acid; Catalase; Cysteine; Deferoxamine; DNA Damage; DNA, Superhelical; Ferric Compounds; Free Radical Scavengers; Hydrogen Peroxide; Hydrogen-Ion Concentration; Osmolar Concentration; Plasmids; Superoxides

We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.

Topics: Animals; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Ferric Compounds; Free Radicals; Lipid Peroxidation; Male; Rats; Rats, Inbred Strains

Pea seed ferritin is able to incorporate ferrous iron into the mineral core. Fe2+ may be formed by reduction of exogenous Fe3+ with ascorbate or by photoreduction by ferritin and by ferric citrate. In our experimental conditions the bulk of the photoreduction is carried out by ferritin, which is able to photoreduce its endogenous iron. Citrate does not enhance the photoreduction capacity of ferritin, and exogenous ferric citrate improves the yield of the reaction by about 30%. The mineral core of the ferritin is shown to photoreduce actively, and the protein shell does not participate directly in the photoreduction. Low light intensities and low concentration of reducing agents do not allow a release of iron from ferritins, but induce a 'redox mill' of photoreduction and simultaneous ferroxidase-mediated incorporation. High ascorbate concentrations induce the release of ferritin iron. These reactions are accompanied by the correlated occurrence of damage caused by radicals arising from Fenton reactions, leading to specific cleavages in the 28 kDa phytoferritin subunit. This damage caused by radicals occurs during the oxidative incorporation into the mineral core and is prevented by o-phenanthroline or by keeping the samples in the dark.

Topics: Ascorbic Acid; Chelating Agents; Citrates; Citric Acid; Fabaceae; Ferric Compounds; Ferritins; Ferrous Compounds; Free Radicals; Iron; Iron Radioisotopes; Oxidation-Reduction; Oxygen; Phenanthrolines; Photochemistry; Plants, Medicinal; Seeds

Since iron is essential for the multiplication of microorganisms, the effect of the iron chelator deferoxamine, with or without ascorbic acid, on the growth of 43 strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Alcaligenes faecalis, Neisseria meningitidis and species of Salmonella, Enterobacter, Pseudomonas and Providencia, was investigated with the use of an automated turbidimeter. Addition of deferoxamine (25-400 micrograms/ml) to the incubation medium was inhibitory in a dose-dependent fashion. At concentrations between 200-400 micrograms/ml, growth was about 25% lower than control values. However, when ascorbic acid (100 micrograms/ml) was added to the culture medium, this antimicrobial activity of deferoxamine was significantly increased to on average 75% of the control value (p less than 0.05). Ascorbic acid alone had no bacteriostatic properties. Growth in the presence of 200 micrograms/ml deferoxamine combined with 100 micrograms/ml ascorbic acid was significantly lower than that in control media without additions (p less than 0.001). Addition of ferric citrate to the culture medium at a concentration sufficient to saturate all of the deferoxamine with iron, abolished the growth inhibiting effect of deferoxamine. The results provide evidence that deferoxamine is bacteriostatic due to its capacity to deplete iron which would otherwise be used for bacterial multiplication, and that ascorbic acid enhances this antibacterial property of deferoxamine.

Topics: Alcaligenes; Ascorbic Acid; Bacteria; Deferoxamine; Dose-Response Relationship, Drug; Drug Synergism; Enterobacteriaceae; Ferric Compounds; Neisseria meningitidis; Pseudomonas; Staphylococcus