Iron Ascorbate Research Articles

Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells.

Reactive oxygen species (ROS) induce HSCs activation, proliferation and collagen gene expression in vitro. Nitric oxide (NO) represents a reactive molecule that reacts with ROS, yielding peroxynitrite. We thus verified the effect of NO on ROS-induced HSCs proliferation in vitro and correlated iNOS expression and ROS formation to HSCs activation in the early phase of liver injury leading to hepatic fibrosis in vivo. HSCs were incubated with iron ascorbate (FeAsc) in vitro, which induced ROS production, ERK1/2 phosphorylation and increased cell proliferation. This effect was significantly reduced by the presence of the NO donor S-nitroso-N-acetylpenicillamine. Liver injury was induced in vivo in rats by dimethylnitrosamine administration. HSCs activation started 6 h after DMN administration and peaked at 1 week. ROS generation and neutrophil infiltration were evident for at least 48 h after DMN treatment, showing an identical distribution pattern. Only a few inflammatory cells expressed iNOS 6 h after DMN administration. we have shown that NO acts as a ROS scavenger in vitro, thus inhibiting HSCs proliferation. ROS production by infiltrating neutrophils occurs in the early phase of liver fibrosis and can represent a stimulus to HSCs activation in vivo. The reduced iNOS expression may account for the low NO levels and the inability to prevent the ROS-induced HSC activation in vivo.

Iron absorption from ferrous bisglycinate and ferric trisglycinate in whole maize is regulated by iron status

There is a need to determine whether iron absorption from iron amino acid chelates is protected from inhibition by dietary phytate and regulated normally by iron status. The objective of this study was to compare iron absorption from ferrous sulfate, ferrous bisglycinate, and ferric trisglycinate in whole-maize meal; to determine whether iron from ferrous bisglycinate and ferrous sulfate exchanges in the intestinal pool; and to assess iron absorption from ferrous bisglycinate and ferric trisglycinate over a range of iron statuses. In study 1A, 10 iron-sufficient men consumed ferrous sulfate-fortified whole-maize meal porridge equilibrated with (59)Fe-sulfate on day 1 and (55)Fe-bisglycinate on day 2. In study 1B, these volunteers consumed ferrous sulfate-fortified porridge equilibrated with (59)Fe-sulfate and (55)Fe-bisglycinate simultaneously. In studies 2A and 2B, iron absorption from 3 mg Fe as (59)Fe-ascorbate, (55)Fe-bisglycinate, or (59)Fe-trisglycinate in water and in porridge was compared in 23 subjects with a range of iron statuses. Iron absorption was determined from blood radioactivity on day 16. In study 1A, geometric mean iron absorption from ferrous bisglycinate was 6.0% (range: 2.6-13.6%), 4 times higher than that from ferrous sulfate (1. 7%; range: 1.0-3.3%; P < 0.05). In study 1B, absorption from neither source was different from that in study 1A. In studies 2A and 2B, absorption from all sources was strongly inversely related to serum ferritin, with geometric means of 32.5% (iron ascorbate), 9.1% (bisglycinate), and 15.3% (trisglycinate). Iron from ferric trisglycinate was poorly absorbed (2.3%; range: 0.5-9.2%) from maize. In whole-maize meal, iron from ferrous bisglycinate is better absorbed than is iron from ferrous sulfate and does not exchange with iron from maize or ferrous sulfate in the intestinal pool. Absorption of iron from bisglycinate and trisglycinate is regulated normally by iron status.

Myrica nagi attenuates cumene hydroperoxide-induced cutaneous oxidative stress and toxicity in Swiss albino mice.

In recent years, considerable efforts have been made to identify new chemopreventive agents which could be useful for man. Myrica nagi, a subtropical shrub, has been shown to possess significant activity against hepatotoxicity and other pharmacological and physiological disorders. We have shown a chemopreventive effect of Myrica nagi on cumene hydroperoxide-induced cutaneous oxidative stress and toxicity in mice. Cumene hydroperoxide treatment at a dose level of 30 mg/animal/0.2 ml acetone enhances susceptibility of cutaneous microsomal membrane for iron-ascorbate-induced lipid peroxidation and induction of xanthine oxidase activity which are accompanied by decrease in the activities of cutaneous antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and depletion in the level of cutaneous glutathione. Parallel to these changes a sharp decrease in the activities of phase II metabolizing enzymes such as glutathione S-transferase and quinone reductase has been observed. Application of Myrica nagi at doses of 2.0 mg and 4.0 mg/kg body weight in acetone prior to that of cumene hydroperoxide (30 mg/animal/0.2 ml acetone) treatment resulted in significant inhibition of cumene hydroperoxide-induced cutaneous oxidative stress and toxicity in a dose-dependent manner. Enhanced susceptibility of cutaneous microsomal membrane for lipid peroxidation induced by iron ascorbate and xanthine oxidase activities were significantly reduced (P<0.05). In addition the depleted level of glutathione, the inhibited activities of antioxidants, and phase II metabolizing enzymes were recovered to a significant level (P<0.05). The protective effect of Myrica nagi was dose-dependent. In summary our data suggest that Myrica nagi is an effective chemopreventive agent in skin and capable of ameliorating cumene hydroperoxide-induced cutaneous oxidative stress and toxicity.

Lipid oxidation in a chicken muscle model system: oxidative response of lipid classes to iron ascorbate or methemoglobin catalysis.

Catalysis by iron ascorbate and activated methemoglobin generated different oxidative responses in chicken muscle model systems. In iron ascorbate systems, large increases in hydroperoxides and thiobarbituric acid-reactive substances (TBARS) occurred during the initial stage of incubation. Thereafter, iron ascorbate catalysis led to a slow increase in the oxidation of triacylglcyerol (TG) and sarcoplasmic reticulum (SR) membrane lipids. By the end of incubation, 24, 36, and 32% of the initial content of n-3 fatty acids in free fatty acids, TG, and SR single-lipid model systems catalyzed by iron ascorbate had been lost. Reduced losses of n-3 fatty acids were observed in the SR and TG fractions (0 and 24%, respectively) when iron ascorbate model systems contained all three lipid fractions (mix). Hydroperoxides and TBARS in model systems catalyzed by activated methemoglobin were characterized by a lag phase during most of the incubation. Consistent with their role as antioxidants, losses of alpha-tocopherol (42-49%), gamma-tocopherol (36-42%), and protein sulfhydryls (41-52%) were observed in model systems catalyzed by activated methemoglobin. SR and mix model systems were 30-50% slower to oxidize than TG model systems when activated methemoglobin served as the catalytic agent.

Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis ofHIV-1 Tat-TAR complex.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans -activation responsive region (TAR) RNA, a 59 base stem-loop structure located at the 5'-end of all HIV transcripts. We have used an intramolecular RNA self-cleaving strategy to determine the folding of TAR RNA and its interactions with a Tat peptide. We incor-porated an EDTA analog at position 24 in the HIV-1 Tat binding site of the TAR RNA. After isolation and purification of the EDTA-TAR conjugate, RNA self-cleavage was initiated by the addition of an iron salt, ascorbate and hydrogen peroxide. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Sites of RNA cleavage were mapped by sequencing reactions. A Tat fragment, Tat(38-72), specifically inhibited RNA self-cleavage. To determine the structural changes caused by the Tat peptide, we performed Fe(II)-EDTA footprinting experiments on Tat-TAR complex. Our high-resolution footprinting results suggest that the inhibition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage and (ii) RNA goes through a structural change where EDTA-U24 is rigidly positioned out of the helix and cannot get access to other nucleotides in the loop of TAR RNA, which are not protected by the Tat peptide. Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures and RNA-protein interactions.

Peroxynitrite-Dependent Aromatic Hydroxylation and Nitration of Salicylate and Phenylalanine. Is Hydroxyl Radical Involved?

There is considerable dispute about whether the hydroxylating ability of peroxynitrite (ONOO-)-derived species involves hydroxyl radicals (OH*). This was investigated by using salicylate and phenylalanine, attack of OH* upon which leads to the formation of 2, 3– and 2, 5-dihydroxybenzoates, and o-, m- and p-tyrosines respectively. On addition of ONOO- to salicylate, characteristic products of hydroxylation (and nitration) were observed in decreasing amounts with rise in pH, although added products of hydroxylation of salicylate were not recovered quantitatively at pH 8.5, suggesting further oxidation of these products and underestimation of hydroxylation at alkaline pH. Hydroxylation products decreased in the presence of several OH* scavengers, especially formate, to extents similar to those obtained when hydroxylation was achieved by a mixture of iron salts, H2O2 and ascorbate. However, OH* scavengers also inhibited formation of salicylate nitration products. Ortho, p- and m-tyrosines as well as nitration products were also observed when ONOO- was added to phenylalanine. The amounts of these products again decreased at high pH and were decreased by addition of OH* scavengers. We conclude that although comparison with Fenton systems suggests OH* formation, simple homolytic fission of peroxynitrous acid (ONOOH) to OH* and NO2 would not explain why OH* scavengers inhibit formation of nitration products.

Previously unknown aldehydic lipid peroxidation compounds of arachidonic acid

Arachidonic acid was oxidized by iron ascorbate. Samples were withdrawn in time intervals. The aldehydic oxidation products were trapped by preparation of pentafluorbenzyloximes. Their trimethylsilylated derivatives were subjected to analysis by GC/MS. The main aldehydec lipid peroxidation product was found to be the well-known 4-hydroxy-2nonenal (HNE), but 2-hydroxy heptanal (HH) — a previously unknown lipid peroxidation product of arachidonic acid — was detected to be nearly equally abundant. Malondialdehyde (MDA), glyoxal and 2-hydroxy-4-decenal (HDE) were detected to be produced in up to 100 times lower amounts compared to HNE. The amounts of aldehydes increased steadily with time. In addition, n-l-hydroxy- n-oxo acids were detected. Similar aldehydes were obtained by iron ascorbate-induced oxidation of hydroxy acids derived by NaBH 4-reduction of 13-hydroperoxy-9- cis-11- trans-oc-tadecadienoic acid. Since this and analogous hydroxy acids (LOHs) are the main biological degradation products of hydroperoxides of unsaturated acids (LOOHs) their further peroxidation seems to be a main source of toxic aldehydes.

Ammonia Adsorption Properties and its Mechanism of Deodorant Filter Made of Iron Ascorbate on Porous Iron Frame.

通気性のある鉄多孔体 (比表面積 0.3m2/g, 気孔率 90%, 嵩比重 0.7) と L-アスコルビン酸を反応させて脱臭フィルターを製作し, アンモニア吸着容量とアンモニア吸着機構について活性炭と比較調査して, 次の結果を得た. (1) 鉄多孔体・アスコルビン酸フィルターの初期アンモニア吸着容量は活性炭のそれと同レベルであるが, 可逆的な脱着により吸着能が再生するため, 経時的に性能が劣化しない特徴がある. (2) 鉄多孔体・アスコルビン酸フィルターに対してアンモニアはFe (II) あるいはFe (III) 原子に置換配位することにより可逆的に吸脱着する. (3) このようなFe原子の活性はアスコルビン酸錯体を形成したためであり, それは鉄多孔体骨格を形成する金属鉄とアスコルビン酸とを反応させる製造過程で生成すると推定される。

In vitro duodenal iron uptake and serum and mucosal iron protein levels, with special reference to rheumatoid arthritis.

Duodenal biopsies from control subjects, patients with iron-deficiency anaemia and rheumatoid arthritic patients with anaemia of chronic disorders (ACD) were investigated for their ability to take up 59Fe from iron ascorbate. Additionally, duodenal tissues were analysed for iron and immunoreactive ferritin and transferrin. Biopsies from iron-deficient subjects showed a 2- to 3-fold increase in the apparent Vmax for 59Fe uptake, compared to control values. Uptake was inversely related to body iron stores. ACD patients showed similar rates of 59Fe uptake to controls; the rates were independent of the degree of anaemia or serum ferritin levels. Tissue analysis showed reductions in mucosal iron and ferritin levels in iron-deficient patients, whilst transferrin levels were within the normal range. ACD patients also exhibited lower mucosal iron levels, but had iron protein levels within the normal range. It is suggested that factors distant from the intestinal mucosa influence iron absorption in ACD.

Peroxidation reactions in plant membranes: effects of free fatty acids.

Free fatty acids accumulate in plant membranes after exposure of plants to environmental stress, such as freezing and desiccation. Fatty acid accumulation has been linked to various biophysical changes and to the occurrence of lipid peroxidation, but the relationships appear complex and inconsistent. The interactions between oxygen free radicals, free fatty acids and lipid peroxidation in plant membranes were examined further by studying peroxidation reactions in a model membrane system composed of a complex mixture of plant phospholipids, including various free fatty acids. Multilamellar liposomes were treated with oxygen free radicals generated from iron ascorbate. Increased concentrations of free palmitic acid up to 10 mol% (fatty acid/phospholipid) reduced the production of aldehydes detected by the thiobarbituric acid assay, but enhanced the production of fluorescent products. By contrast, increased concentrations of free linolenic acid increased aldehyde production and reduced the formation of fluorescent products. The two free fatty acids both enhanced the susceptibility of phospholipids to degradation as shown by the reduced recovery of esterified polyunsaturated fatty acids (linoleic and linolenic). The free radical reactions with or without free fatty acid additions catalyzed the selective degradation of phospholipids in the order phosphatidylethanolamine > phosphatidylcholine > phosphatidylinositol > phosphatidylglycerol. Selective degradation of phospholipids is often observed after periods of environmental stress or during senescence of plants, and has been cited as evidence for the involvement of phospholipases in these degenerative processes. The results indicate that selectivity is not a criterion for eliminating the involvement of oxygen free radicals in these degenerative processes. Furthermore, the results suggest that modifications of lipid composition during a plant's acclimation to adverse environments may determine the types of free radical reactions that occur due to stress.

Activated THP-1 cells depress mitochondrial respiration in HEP G2 cells infected with influenza B virus

Influenza B virus has been aetiologically linked to Reye Syndrome (RS), but the mechanism(s) by which this pathogen could disrupt liver metabolism and produce the hepatic mitochondrial injury characteristic of the syndrome are unknown. In this study, two mechanisms by which infection of hepatocytes with influenza B virus could disrupt cellular metabolism were investigated. (1) virus-induced increase in pro-oxidant iron with subsequent iron-induced lipid peroxidation (LP) and (2) increased membrane permeability. Hep G2 cells, a well-differentiated continuous human liver cell line derived from a hepatoblastoma, were infected with allantoic-fluid derived influenza B Lee/40 virus (AFDV) at a multiplicity of infection of 10 for 24 h; productive infection was confirmed by both haemagglutination of chick erythrocytes and by plaque assay. Infection of Hep G2 cells preloaded with 59Fe-transferrin resulted in increased release of 59Fe (153 ± 17% of controls, P <0·03). However, the iron released did not result in increased LP (assessed by thiobarituric acid reactive substances; TBARS). To confirm that this lack of increase in TBARS was not due to insensitivity of the cell line to pro-oxidant iron, cells were exposed to 15 μM iron ascorbate for 60 min. Production of TBARS was increased (122 ± 4%) of controls, P <0·0003). Release of 51Cr from infected cells was also increased (128 ± 12% of controls, P<0·05); thus the infected cells exhibited a generalized increase in membrane permeability. However, infection did not depress mitochondrial respiration (as assessed by the formation of MTT-f3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan. To determine if the combination of viral infection and soluble products of activated macrophages would affect mitochondrial respiration, infected hepatocytes were expose to the supernatant fluid from THP-1 cells which had previously been incubated with lipopolysaccharide at 100 ng ml -1 for 18 h. This supernate did depress the formation of MTT-f (81 ± 5% of controls, P<0·03). We conclude that influenza B virus does productively infect Hep G2 cells, and does increase hepatocyte membrane permeability. This effect does not impair mitochondrial respiration directly. However, infection does act in concert with soluble products of activated macrophages to depress hepatic mitochondrial respiration. Whether this interaction can be explained by virus-induced permeability changes and/or other effects of infection deserves further investigation.

Natural antioxidants in grapes and wines

The concentrations of phenolics of three grape varieties and two red wines were determined. The red grape variety and the red wines contain phenolics at concentrations of 920 mg/kg and 1800 and 3200 mg/L, respectively. The antioxidative effects of wine phenolics on the catalysis of lipid peroxidation by biological catalysts such as myoglobin, cytochrome c, iron ascorbate, and copper ions were estimated. Lipid peroxidation catalyzed by myoglobin, cytochrome c, and iron ascorbate was inhibited (I 50 ) by wine phenolics at concentrations of 0.2, 0.35, and 0.9 μg of phenolics/mL

Suppressive Effects of Lactoferrin on Bleomycin-dependent DNA Damage by the Iron Ion and Ascorbate.

The effects of the iron ion and reductones on bleomycin (BLM)-induced DNA degradation were observed by measuring the amount of malondialdehyde (MDA) formed. The formation of MDA was detected after adding Fe(2+) or Fe(3+) + Asc combined to a solution consisting of DNA and BLM, but not after adding Fe(3+) alone, catecholamines or phenolic compounds. Marked suppressive effects of lactoferrin (Lf) and ovotransferrin on MDA formation were observed in the solution with Fe(2+) or Asc. However, serum albumin, β-lactoglobulin, α-lactalbumin, and ovoalbumin had no effect on suppressing MDA formation. The suppression by bovine Lf (bLf) of MDA formation decreased with increasing iron saturation at 1.0 mm Fe(2+), but at 100% iron saturation, 51.2% of apo-bLf remained suppression. In the solution with 0.25 mm Asc, 100% iron saturated bLf markedly enhanced MDA formation.

Iron speciation at physiological pH in media containing ascorbate and oxygen.

The stability of iron ascorbate solutions was investigated, under both anaerobic and aerobic conditions, with the Fe2+ and Fe3+ indicators, respectively ferrozine and mimosine, at different pH values. The species present under the differing conditions were investigated by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy and by gel-filtration chromatography. At physiological pH (6.8-7.4) iron ascorbate solutions rapidly form mononuclear chelatable Fe3+ species as reflected by indicator studies and EPR. Mössbauer spectroscopy fails to detect any Fe2+ species. EPR studies show a time-dependent decrease in rhombic Fe3+, particularly in oxygenated buffers, consistent with a conversion to polynuclear Fe. O2 uptake studies show that the conversion of Fe2+ to Fe3+ in Fe-ascorbate solutions at pH > 7.0 was accompanied by rapid O2 consumption but preceded depletion of ascorbate. Addition of high concentrations of mannitol (50-200 mM) reduces the O2 consumption and partly stabilizes the rapidly chelatable Fe form. Gel filtration studies show that the oxidation of Fe-ascorbate solutions at pH 7.4 is accompanied by an increase in the apparent relative molecular mass of the Fe, presumably due to Fe polymer formation. These studies indicate that inherent instability of Fe-ascorbate solutions above neutral pH and clearly have important implications in the use of ascorbate in studies of Fe physiology.

Studies of the reactions of iron(II) ascorbate mixtures with molecular oxygen in solution

Mössbauer and electronic absorbance spectroscopic studies on the reactions of iron(II): ascorbic acid with molecular oxygen in aqueous and methanolic solutions are reported. Both spectroscopic techniques show that in the starting mixtures there are no iron(II): ascorbate complexes. On mixing the iron(II)/ascorbate solution with solutions containing molecular oxygen at pH 6–7 high spin iron(III) is observed in the Mössbauer spectrum. Coloured intermediates, the lifetimes of which are solvent dependent, are seen by stopped-flow spectrophotometry. We assign these coloured intermediates as iron(III) ascorbate complexes. The stoichiometry of the initial reaction between iron(II) and oxygen is shown to be 2Fe(II):O 2 by stopped-flow methods. A scheme for the overall reactions is discussed.

Iron species in iron ascorbate solutions at physiological pH

Iron/ascorbate solutions are widely used as a source of Fe2+ for biochemical studies. Ascorbate is regarded as a good reducing agent for maintaining iron in a soluble form at physiological pH (Conrad & Schade, 1968). We have been investigating Fe2+ uptake by mouse intestinal brush-border membrane vesicles using 20-fold excess ascorbate to maintain Fe2+ in the reduced state (Simpson & Peters, 1986). Evidence was found for the ageing of these solutions at pH greater than 7.0. We therefore set out to study Fe/ascorbate solutions using U.V. and visible spectroscopy, gel filtration,

The effect of raw wheat bran, alfalfa meal and alpha-cellulose on iron ascorbate chelate and ferric chloride in three binding solutions

Iron ascorbate chelate was prepared and purified with iron and ascorbate combined in a 1:1 molar ratio. Some iron dissociated from the chelate and bound to raw wheat bran and alfalfa meal, indicating something in these fiber sources produced the dissociation. The chelate bound intact to alpha-cellulose in bicarbonate and phosphate buffer. More iron as ferric chloride bound to the three fiber sources than did iron as chelate. Iron became more soluble in bicarbonate buffer in the presence of raw wheat bran and alfalfa meal, but became less soluble in phosphate buffer in the presence of these fiber sources.

Discrimination of ascorbate-dependent nonenzymatic and enzymatic, membrane-bound reduction of nitric oxide in denitrifying Pseudomonas perfectomarinus

The marine nitrite-respiring (denitrifying) bacterium, Pseudomonas perfectomarinus, catalyzes by a membrane-bound enzyme the reduction of nitric oxide to nitrous oxide with ascorbate-reduced phenazine methosulfate as electron donor. The entire nitric oxide-reducing capability of a cell-free system was membrane bound and this process was studied with respect to pH and substrate dependency. The enzymatic process was perturbed by an identical nonenzymatic reduction by iron(II) ascorbate in neutral to alkaline aqueous solution. 2 mol nitric oxide and 1 mol ascorbate were consumed per mol nitrous oxide formed. Enzymatic and nonenzymatic processes were discriminated by their differential behavior towards pH and metal-chelating agents. The pH optimum for the enzymatic and nonenzymatic reaction was 5.2 and greater than 7.0, respectively. EDTA (10 mM) inhibited the nonenzymatic reduction completely without interfering with the membrane-bound activity. The nonenzymatic system mimics the reaction of nitric oxide reductase and could serve as a model to study the formation of the N-N bond in denitrification. Enzymatic generation of nitric oxide by cytochrome cd and subsequent nonenzymatic reduction to nitrous oxide simulate an overall quasi-enzymatic nitrous oxide formation by cytochrome cd. The nonenzymatic reduction of nitric oxide might have occurred in previous work due to the ubiquitous use of ascorbate in studies on nitrite respiration and the likelihood of adventitious iron in biological samples.

Lipid peroxidation in liver: Hydroxy dimethyl carbazole a new potent inhibitor

The results of our structure-activity relationship study on the anticancer drug 9-hydroxyellipticine for its antioxydant properties show that 6-hydroxy-1,4-dimethyl carbazole has a remarkable inhibitory action in several In vitro peroxidative systems. The effectiveness of this carbazole is one order of magnitude larger than that of promethazine in the iron ascorbate or cysteine initiated systems.

Determination of Filtration Fraction Using the Gamma Scintillation Camera

A simple technique for determining effective renal plasma flow, filtration fraction and filtration rate is described. The determination depends upon the use of 131iodine labeled hippuran and 99mtechnetium labeled iron ascorbate or Sn diethylenetriaminepentaacetic acid. The filtration fraction is determined by dividing the corrected uptake for the technetium compound by the corrected uptake for radiohippuran. The uptake is simply determined by the 1 to 2-minute count over the kidneys with background subtraction and taking into account the amount injected. The technique is simple, quick and appears to be a useful clinical tool.