Clinical and methodological factors affecting non-transferrin-bound iron values using a novel fluorescent bead assay.

Nontransferrin-bound iron (NTBI) is a heterogeneously speciated plasma iron, typically detectable when transferrin saturation (TfSat) exceeds 75%. Here, we examine factors affecting NTBI levels by a recently discovered direct chelator-based (CP851) fluorescent bead-linked flow-cytometric assay (bead-NTBI), compared with the established indirect nitrilotriacetate (NTA) assay in 122 iron-overloaded patients, including 64 on recent iron chelation therapy and 13 healthy volunteers. Both methods correlated (r = 0.57, P < 0.0001) but with low agreement, attributable to 2 major factors: (1) the NTA method, unlike the bead method, is highly dependent on TfSat, with NTBI under-estimation at low TfSat and over-estimation once Tf is saturated, (2) the bead method detects <3-fold higher values than the NTA assay in patients on recent deferiprone-containing chelation due to greater detection of chelate complexes but lower values for patients on deferasirox. The optimal timing of sample collection relative to chelation dosing requires further study. Patients with splenectomy, high-storage iron, and increased erythropoiesis had greater discrepancy between assays, consistent with differential access by both methods to the NTBI pools associated with these clinical variables. The bead-NTBI assay has advantages over the NTA assay, being less dependent on TfSat, hence of less tendency for false-negative or false-positive values at low and high TfSat, respectively.

Metabolism of rutin and poncirin by human intestinal microbiota and cloning of their metabolizing α-L-rhamnosidase from Bifidobacterium dentium.

To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-Lrhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni(2+)-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 µmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1-->6) bonds than (1-->2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.

A nanoparticulate ferritin-core mimetic is well taken up by HuTu 80 duodenal cells and its absorption in mice is regulated by body iron.

BACKGROUND: Iron (Fe) deficiency anemia remains the largest nutritional deficiency disorder worldwide. How the gut acquires iron from nano Fe(III), especially at the apical surface, is incompletely understood. OBJECTIVE: We developed a novel Fe supplement consisting of nanoparticulate tartrate-modified Fe(III) poly oxo-hydroxide [here termed nano Fe(III)], which mimics the Fe oxide core of ferritin and effectively treats iron deficiency anemia in rats. METHODS: We determined transfer to the systemic circulation of nano Fe(III) in iron-deficient and iron-sufficient outbread Swiss mouse strain (CD1) mice with use of (59)Fe-labeled material. Iron deficiency was induced before starting the Fe-supplementation period through reduction of Fe concentrations in the rodent diet. A control group of iron-sufficient mice were fed a diet with adequate Fe concentrations throughout the study. Furthermore, we conducted a hemoglobin repletion study in which iron-deficient CD1 mice were fed for 7 d a diet supplemented with ferrous sulfate (FeSO4) or nano Fe(III). Finally, we further probed the mechanism of cellular acquisition of nano Fe(III) by assessing ferritin formation, as a measure of Fe uptake and utilization, in HuTu 80 duodenal cancer cells with targeted inhibition of divalent metal transporter 1 (DMT1) and duodenal cytochrome b (DCYTB) before exposure to the supplemented iron sources. Differences in gene expression were assessed by quantitative polymerase chain reaction. RESULTS: Absorption (means ± SEMs) of nano Fe(III) was significantly increased in iron-deficient mice (58 ± 19%) compared to iron-sufficient mice (18 ± 17%) (P = 0.0001). Supplementation of the diet with nano Fe(III) or FeSO4 significantly increased hemoglobin concentrations in iron-deficient mice (170 ± 20 g/L, P = 0.01 and 180 ± 20 g/L, P = 0.002, respectively). Hepatic hepcidin mRNA expression reflected the nonheme-iron concentrations of the liver and was also comparable for both nano Fe(III)- and FeSO4-supplemented groups, as were iron concentrations in the spleen and duodenum. Silencing of the solute carrier family 11 (proton-coupled divalent metal ion transporter), member 2 (Slc11a2) gene (DMT1) significantly inhibited ferritin formation from FeSO4 (P = 0.005) but had no effect on uptake and utilization of nano Fe(III). Inhibiting DCYTB with an antibody also had no effect on uptake and utilization of nano Fe(III) but significantly inhibited ferritin formation from ferric nitrilotriacetate chelate (Fe-NTA) (P = 0.04). Similarly, cellular ferritin formation from nano Fe(III) was unaffected by the Fe(II) chelator ferrozine, which significantly inhibited uptake and utilization from FeSO4 (P = 0.009) and Fe-NTA (P = 0.005). CONCLUSIONS: Our data strongly support direct nano Fe(III) uptake by enterocytes as an efficient mechanism of dietary iron acquisition, which may complement the known Fe(II)/DMT1 uptake pathway.

Desferrioxamine-caffeine (DFCAF) as a cell permeant moderator of the oxidative stress caused by iron overload.

Desferrioxamine (DFO) is a potent iron chelator used in the treatment of iron overload (IO) disorders. However, due to its low cell permeability and fast clearance, DFO administration is usually prolonged and of limited use for the treatment of IO in tissues such as the brain. Caffeine is a safe, rapidly absorbable molecule that can be linked to other compounds to improve their cell permeability. In this work, we successfully prepared and described DFO-caffeine, a conjugate with iron scavenging ability, antioxidant properties and enhanced permeation in the HeLa cell model.

Interleukin-6 and lipopolysaccharide modulate hepcidin mRNA expression by HepG2 cells.

Iron homeostasis is controlled by hepcidin (Hpc) as well as other ways. Hpc expression is regulated by iron (Fe) storage and by inflammation, but the joint effect of both stimuli remains unclear. We studied the modulatory role of inflammatory agents (IL6 and LPS) over Hpc and DMT1 mRNA expression in HepG2 cells preloaded with Fe. HepG2 cells were preloaded with different Fe concentrations (holo-Tf or Fe-NTA) and then incubated with IL6 or LPS. We measured intracellular Fe levels by AAS with graphite furnace, transferrin receptor (TfR) by ELISA and mRNA relative abundance of Hpc and DMT1 by qRT-PCR. The maximum effect on Fe uptake was observed in cells incubated with 30 ng/ml IL6 (p < 0.01) and 500 ng/ml LPS (p < 0.05). In HepG2 cells preloaded with holo-Tf or Fe-NTA and challenged with IL6 and LPS, we observed a decreased: (a) Hpc mRNA relative abundance (two-way ANOVA: p < 0.05 and p < 0.001, respectively), (b) DMT1 mRNA relative abundance and TfR1 protein levels (two-way ANOVA: p < 0.001), and (c) intracellular Fe concentration (two-way ANOVA: p < 0.001 and p < 0.01, respectively) compared to control cells incubated only with Fe (holo-Tf or Fe-NTA). Our results support the idea that Fe storage and inflammation act together to regulate Fe homeostasis and suggest a negative regulation in this hepatic cellular model to prevent excessive increases in Hpc.

Non-transferrin bound iron in Thalassemia: differential detection of redox active forms in children and older patients.

Non-transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron-binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly-transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox-active and readily chelatable fraction, which we have designated as "labile plasma iron" (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as "eLPI"), at concentrations that do not affect transferrin-bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly-transfused, iron overloaded thalassemia patients: non-chelated children (3-13 yrs, Gaza, Palestine), chelated adolescents-young adults (13-28 yrs, Israel), and chelated adults (27-61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r(2) = 0.61-0.75) with deferrioxamine-detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly-transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age-dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation.

Degradation of 2-fluorophenol by the brown-rot fungus Gloeophyllum striatum: evidence for the involvement of extracellular Fenton chemistry.

Iron-containing liquid cultures of the brown-rot basidiomycete Gloeophyllum striatum degraded 2-fluorophenol. Two simultaneously appearing degradation products, 3-fluorocatechol and catechol, were identified by gas chromatography and mass spectrometry (GC-MS). Concomitantly, fluoride was produced at approximately 50% of the amount that theoretically could be achieved upon complete dehalogenation. Defluorination was strongly inhibited in the presence of either the hydroxyl radical scavenger mannitol or superoxide dismutase, as well as in the absence of iron. The addition of the natural iron chelator oxalate caused a clear but less extensive inhibition, whereas supplementation with the artificial iron chelator nitrilotriacetic acid increased fluoride production. Extracellular 2-fluorophenol degradation was evidenced by defluorination, observed upon addition of 2-fluorophenol to cell-free culture supernatants derived from iron-containing fungal cultures. Ultrafiltered culture supernatants oxidized methanol to formaldehyde, known as a product of the reaction of methanol with hydroxyl radical. In addition, G. striatum was found to produce metabolites extractable with ethyl acetate that are capable of reducing Fe3+. GC-MS analysis of such extracts revealed the presence of several compounds. The mass spectrum of a prominent peak matched those previously reported for 2,5-dimethoxyhydroquinone and 4,5-dimethoxycatechol, fungal metabolites implicated to drive hydroxyl radical production in Gloeophyllum. Taken together, these findings further support an extracellular Fenton-type mechanism operative during halophenol degradation by G. striatum.

Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1.

Deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions, D. radiodurans R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO(2) and acetate but was unable to link this process to growth. D. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH(2)DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism. D. radiodurans reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml(-1)) but not in their absence. D. radiodurans also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that D. radiodurans can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also, D. radiodurans, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.

Ginseng extract scavenges hydroxyl radical and protects unsaturated fatty acids from decomposition caused by iron-mediated lipid peroxidation.

This study was conducted to investigate whether or not the antioxidation effect of ginseng extract directly inhibits decomposition of unsaturated fatty acid caused by iron and hydrogen peroxide-induced lipid peroxidation, and whether this effect involves a hydroxyl radical-scavenging mechanism. Thiobarbituric acid-reactive substances (TBARS), gas chromatography, and electron spin resonance (ESR) spectrometer were used to measure lipid peroxidation, unsaturated fatty acid, and hydroxyl radical. The results showed TBARS formed and the loss of arachidonic acid during lipid peroxidation, and that hydroxyl radical formed by the Fenton reaction were completely inhibited by ginseng extract. This antioxidant effect of ginseng may be responsible for its wide pharmacological actions in clinical practice by a free radical reaction-inhibition mechanism.

Iron release and uptake by plant ferritin: effects of pH, reduction and chelation.

Ferritins are iron-storage proteins that accumulate in plastids during seed formation, and also in leaves during senescence or iron overload. Iron release from ferritins occurs during growth of seedlings and greening of plastids. Depending on the concentration of the reducing agent ascorbate, either an overall iron release or uptake by ferritins from iron(III) citrate may occur. We have designed methods to measure these simultaneous and independent uptake and release fluxes. Each individual step of the exchange was studied using different iron chelates and an excess of ligand. It is shown that: (i) the chelated form of iron, and not ionic Fe3+, is the substrate for iron reduction, which controls the subsequent uptake by ferritin; (ii) iron uptake by ferritins is faster at pH 8.4 than at pH 7 or 6 and is inhibited by an excess of strongly binding free ligands; and (iii) strongly binding free ligands are inhibitory during iron release by ascorbate. When reactions are allowed to proceed simultaneously, the iron chelating power is shown to be a key factor in the overall exchange. The interactions of iron chelating power, reducing capacity and pH are discussed with regard to their influence on the biochemical mobilization of iron.

Kinetic evaluation of free malondialdehyde and enzyme leakage as indices of iron damage in rat hepatocyte cultures. Involvement of free radicals.

The present study relates to the effect of ferric iron supplementation on lipid peroxidation of adult rat hepatocyte pure cultures. Lipid peroxidation was evaluated by free malondialdehyde (MDA) using size exclusion chromatography (HPLC) as a specific and sensitive method. The ferric iron used under its complexed form with nitrilotriacetic acid (NTA) exhibited a prooxidant activity corresponding to an increase of free MDA recovery in the cells and in the culture medium. This enhancement of lipid peroxidation in the hepatocyte cultures supplemented with ferric iron was correlated with an intracellular enzyme leakage (lactate dehydrogenase and transaminase), suggesting that lipid peroxidation and enzyme release represented good parameters for cytotoxicity evaluation. The toxic effect of Fe-NTA on hepatocyte cultures was a function of the incubation time (from 0 to 48 hr) and of the concentration of ferric iron loading (i.e. 5, 20 and 100 microM). The mechanism by which Fe-NTA induced cellular damage involved free radical production, as increasing amounts of free radical scavengers corresponded to diminishing rates of both total free MDA and enzyme release. However, this reducing capacity varied from one scavenger to another, where they exhibited preferentially a decrease in lipid peroxidation or in enzyme leakage. This suggested a dissociation between the two parameters of cytotoxicity considered. Lipid peroxidation corresponding to alterations of both inner membranes and the plasma membrane, whereas enzyme release mainly corresponded to the damage of plasma membrane. Subsequently, some scavengers (superoxide dismutase, mannitol, alpha tocopherol, beta carotene) presented an intracellular activity, as they reduced mostly lipid peroxidation. Other ones (catalase, dimethylpyrroline N-oxide, thiourea) seemed essentially efficient in protecting the external plasma membrane, as shown an important decrease in enzyme leakage.

Model studies of the iron-catalysed Haber-Weiss cycle and the ascorbate-driven Fenton reaction.

Complementary hydroxylation assays and stopped-flow e.s.r. techniques have been employed in the investigation of the effect of various iron chelators (of chemical, biological and clinical importance) on hydroxyl-radical generation via the Haber-Weiss cycle and the ascorbate-driven Fenton reaction. Chelators have been identified which selectively promote or inhibit various reactions involved in hydroxyl-radical generation (for example, NTA and EDTA promote all the reactions of both the Haber-Weiss cycle and the ascorbate-driven Fenton reaction, whereas DTPA and phytate inhibit the recycling of iron in these reactions). The biological chelators succinate and citrate are shown to be relatively poor catalysts of the Haber-Weiss cycle, whereas they are found to be effective catalysts of .OH generation in the ascorbate-driven Fenton reaction. It is also suggested that continuous redox-cycling reactions between iron, oxygen and ascorbate may represent an important mechanism of cell death in biological systems.

Effect of milk and casein on the absorption of supplemental iron in the mouse and chick.

Milk is an attractive vehicle for introducing iron supplements into iron-deficient infants and children. This study compares the effects of milk and caseins on the whole-body absorption of radioactive iron complexes in an attempt to resolve the controversy over whether milk and its constituent phosphoproteins seriously impair iron absorption. Evidence is presented to clarify the role of the calcium-casein micelles of cow's milk in binding iron donated by the ferric-nitrilotriacetate (NTA) complex. The absorption of iron from isolated Fe(III)-casein complexes was studied in mice as a function of the casein--to--Fe ratio and was compared with the absorption of Fe(III)-NTA at equivalent levels. Even at casein--to--Fe ratios higher than those found in conventional iron-supplemented cow's milk (10-15 mg Fe/qt; casein P:Fe congruent to 34), absorption of iron(III) from the casein or NTA complex was not significantly different. There was no significant difference in the absorption of iron administered to mice and chicks as ferrous ion, ferric-NTA, or ferric fructose; nonfat cow's milk did not inhibit the absorption of these iron compounds. For the chick, in fact, milk significantly enhanced the absorption of iron from the ferric-NTA chelate. In order to affect iron absorption significantly casein would have to be present considerably in excess of that found in conventionally supplemented cow's milk.

The metabolism of nitrilotriacetate by a pseudomonad.

1. An organism that grows on nitrilotriacetate as sole source of carbon and energy was isolated in pure culture and was identified as a pseudomonad. 2. Cell-free extracts of the nitrilotriacetate-grown pseudomonad contain an enzyme that catalyses the NADH-and O(2)-dependent oxidation of nitrilotriacetate to iminodiacetate and glyoxalate. This enzyme is absent from extracts of glucose-grown cells. 3. Compared with growth on glucose, growth on nitrilotriacetate results in increased activities of enzymes of glycine and serine metabolism, namely serine hydroxymethyltransferase, glycine decarboxylase, serine-oxaloacetate aminotransferase and hydroxypyruvate reductase. 4. Cell-free extracts of the nitrilotriacetate-grown organism contain the enzyme glyoxalate carboligase and, when supplemented with NADH, Mg(2+) and thiamin pyrophosphate, can catalyse the anaerobic conversion of glyoxalate into glycerate. 5. These results are incorporated in a scheme which shows the oxidative metabolism of nitrilotriacetate by the successive removal of C(2) units to form 2mol of glyoxalate and 1mol of glycine per mol of nitrilotriacetate degraded. The glyoxalate and glycine are then both metabolized to glycerate by separate pathways, via tartronic semialdehyde and serine respectively. The role of this scheme in the growth of the organism on nitrilotriacetate is discussed.