A simple and rapid method for the determination of "free" iron in biological fluids

We present a convenient method for determining "free" or non-protein-bound iron in biological fluids. The new method is based on the bathophenantroline method for determination of total serum iron, and comprises binding of iron by a chromogenic chelator (bathophenantroline-disulphonate, BPS), which is specific for ferrous iron. The ferrous complex of BPS absorbs strongly at 535 nm, and the detection limit is less than 1 μM in a sample size of 50 μl. The chelator does not liberate iron from either haemoglobin or transferrin. Interference from copper or zinc in concentrations up to 50 μM does not significantly disturb measurements. The main problem when measuring in blood plasma, the high and fluctuating background in the region around 535 nm, has been overcome through filtering techniques. Data from measurements of ferrous iron in microdialysate, cerebrospinal fluid, and blood plasma in different animal models and clinical conditions are presented as illustrative examples of the usefulness of the method. The method allows the determination of ferric, as well as ferrous, iron in the same sample.     

Kinetics and Mechanism of the Reaction of a Nitroxide Radical (Tempol) With a Phenolic Antioxidant

In the absence of redox-active transition metal ions, the removal of Tempol by Trolox occurs by a simple bimolecular reaction that, most probably, involves a hydrogen transfer from phenol to nitroxide. The specific rate constant of the process is small (0.1 M &#109 1 s &#109 1 ). Metals can catalyze the process, as evidenced by the decrease in rate observed in the presence of diethylenetriaminepentaacetic acid (DTPA). Furthermore, addition of Fe(II) (20 &#119 M ferrous sulfate and 40 &#119 M EDTA) produces a noticeable increase in the rate of Tempol consumption.     

Iron at the cell surface controls both DNA synthesis and plasma membrane redox system

Summary Addition of the impermeable iron II chelator bathophenanthroline disulfonate (BPS) to cultured Chinese hamster lung fibroblast (CCL 39 cells) inhibits DNA synthesis but not protein synthesis or cytoplasmic alkalinization, when cell growth is initiated with growth factors such as EGF plus insulin, thrombin, or ceruloplasmin. The BPS inhibition is reversed by addition of stoichiometric ferrous iron at stoichiometric concentration. BPS does not inhibit cell growth stimulated by fetal calf serum. The effect of the BPS differs from the inhibition of growth by hydroxyurea which acts on the ribonucleotide reductase. The BPS treatment leads to release of iron from the cells as determined by BPS iron II complex formation over 90 min. Cells treated with BPS just during starvation period cannot re-initiate DNA synthesis after mitogen stimulation even if BPS is removed from the medium and cells are previously washed. BPS treatment also inhibits transplasma membrane electron which is restored by incubation of cells with 10 M ferric ammonium citrate. Growth factor stimulation of DNA synthesis is restored by addition of 1 M ferrous ammonium sulfate or ferric ammonium citrate, or 0.1 M diferric transferrin. Copper, cobalt, nickel, zinc, gallium, aluminum, or apotransferrin cannot restore the activity. The BPS effect is consistent with removal of iron from a site on the cell surface which controls electron transport and DNA synthesis.Abbreviations BCS bathocuproine disulfonate - BPS bathophenan-throline disulfonate - CUP ceruloplasmin - FCS fetal calf serum - Fe₂Tf diferric transferrin - EGF epidermal growth factor - HU hydroxyurea - THR -thrombin     

Diferric transferrin reduction by K562 cells. A critical study

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.     

Effects of Iron Chelators,Iron Salts,and Iron Oxide Nanoparticles on the Proliferation and the Iron Content of Oligodendroglial OLN-93 Cells

The oligodendroglial cell line OLN-93 was used as model system to investigate the consequences of iron deprivation or iron excess on cell proliferation. Presence of ferric or ferrous iron chelators inhibited the proliferation of OLN-93 cells in a time and concentration dependent manner, while the application of a molar excess of ferric ammonium citrate (FAC) prevented the inhibition of proliferation by the chelator deferoxamine. Proliferation of OLN-93 cells was not affected by incubation with 300 μM iron that was applied in the form of FAC, FeCl₂, ferrous ammonium sulfate or iron oxide nanoparticles, although the cells efficiently accumulated iron during exposure to each of these iron sources. The highest specific iron content was observed for cells that were exposed to the nanoparticles. These data demonstrate that the proliferation of OLN-93 cells depends strongly on the availability of iron and that these cells efficiently accumulate iron from various extracellular iron sources.     

Effects of a Direct Injection of Liposoluble Iron into Rat Striatum. Importance of the Rate of Iron Delivery to Cells

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical ( &#148 OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 &#119 M FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of &#148 OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced &#148 OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with &#148 OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular &#148 OH in damage associated with intracerebral iron injection.     

Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology

The ferrozine and phenanthroline colorimetric assays are commonly applied for the determination of ferrous and total iron concentrations in geomicrobiological studies. However, accuracy of both methods depends on slight changes in their protocols, on the investigated iron species, and on geochemical variations in sample conditions. Therefore, we tested the performance of both methods using Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, synthetic goethite, ferrihydrite, and pyrite, as well as microbially-formed magnetite and a mixture of goethite and magnetite. The results were compared to concentrations determined with aqua regia dissolution and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Iron dissolution prior to the photometric assays included dissolution in 1M or 6M HCl, at 21 or 60°C, and oxic or anoxic conditions. Results indicated a good reproducibility of quantitative total iron determinations by the ferrozine and phenanthroline assays for easily soluble iron forms such as Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, and ferrihydrite. The ferrozine test underestimated total iron contents of some of these samples after dissolution in 1M HCl by 10 to 13%, whereas phenanthroline matched the results determined by ICP-AES with a deviation of 5%. Total iron concentrations after dissolution in 1M HCl of highly crystalline oxides such as magnetite, a mixture of goethite and magnetite, and goethite were underestimated by up to 95% with both methods. When dissolving these minerals in 6M HCl at 60°C, the ferrozine method was more reliable for total iron content with an accuracy of ±5%, related to values determined with ICP-AES. Phenanthroline was more reliable for the determination of total pyritic iron as well as ferrous iron after incubation in 1M HCl at 21°C in the Fe(II)((aq)) sample with a recovery of 98%. Low ferrous iron concentrations of less than 0.5mM were overestimated in a Fe(III) background by up to 150% by both methods. Heating of mineral samples in 6M HCl increased their solubility and susceptibility for both photometric assays which is a need for total iron determination of highly crystalline minerals. However, heating also rendered a subsequent reliable determination of ferrous iron impossible due to fast abiotic oxidation. Due to the low solubility of highly crystalline samples, the determination of total iron is solely possible after dissolution in 6M HCl at 60°C which on the other hand makes determination of ferrous iron impossible. The recommended procedure for ferrous iron determination is therefore incubation at 21°C in 6M HCl, centrifugation, and subsequent measurement of ferrous iron in the supernatant. The different procedures were tested during growth of G. sulfurreducens on synthetic ferrihydrite. Here, the phenanthroline test was more accurate compared to the ferrozine test. However, the latter provided easy handling and seemed preferable for larger amounts of samples.     

Protective Activity of (−)-Epicatechin 3- O -gallate against Peroxynitrite-mediated Renal Damage

The protective effect of ( &#109 )-epicatechin 3- O -gallate (ECg) against peroxynitrite (ONOO &#109 )-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level, an indicator of ONOO &#109 production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 &#119 moles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO &#109 . In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 &#119 M) prior to 3-morpholinosydnonimine (SIN-1, 800 &#119 M) exposure reduced ONOO &#109 formation and increased the viability of cultured renal epithelial (LLC-PK 1 ) cells in a dose-dependent manner. In particular, ECg inhibited ONOO &#109 -mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G 2 /M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO &#109 in cellular and in vivo systems.     

Cyanidin-3- O -β-glucopyranoside Protects Myocardium and Erythrocytes from Oxygen Radical-mediated Damages

The cyanidin-3- O - &#103 -glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 &#119 M C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 &#119 M C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 &#119 M C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 &#119 M C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 &#119 M was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 &#119 M). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Antioxidant α-Keto-carboxylate Pyruvate Protects Low-density Lipoprotein and Atherogenic Macrophages

Oxidative modification of low-density lipoprotein (oxLDL) plays a pathogenic role in atherogenesis. Classical antioxidants such as l -ascorbic acid can inhibit formation of oxLDL. &#102 -Keto-carboxylates such as pyruvate and congeners also display antioxidant properties in some cell-free and intact cell systems. We tested the hypothesis that pyruvate or &#102 -keto-glutarate may function as antioxidants with respect to LDL incubated with 5 or 10 &#119 M Cu 2+ alone or in combination with THP-1-derived macrophages. &#102 -Hydroxy-carboxylates ( l -lactate), linear aliphatic mono-carboxylates (acetate/caprylate) and l -ascorbic acid served as controls. The oxLDL formation was ascertained by electrophoretic mobility and oxLDL cytotoxicity was judged by macrophage viability and thiobarbituric acid reactive substances (TBARS) formation. Cu 2+ alone was not cytotoxic but increased electrophoretic mobility of cell-free LDL, stimulating TBARS. Millimolar pyruvate, &#102 -keto-glutarate, or micromolar l -ascorbic acid partially inhibited oxLDL formation, while &#102 -hydroxy-carboxylate or the aliphatic mono-carboxylates had no measurable antioxidant properties in cell-free LDL. Co-culture of LDL with macrophages and Cu 2+ augmented TBARS release and resulted in 95% macrophage death. Pyruvate improved macrophage viability with 5 &#119 M Cu 2+ up to 60%. l -Ascorbic acid ( &#83 100 &#119 M) protected macrophages up to 80%. When &#83 100 &#119 M l -ascorbic acid was combined with pyruvate, oxLDL formation and macrophage death were fully prevented. Thus, &#102 -keto-carboxylates, but not physiological &#102 -hydroxy-carboxylates or aliphatic mono-carboxylates qualify as antioxidants in LDL systems. Since &#102 -keto-carboxylates enhanced the antioxidant power of l -ascorbic acid, our findings may have implications for strategies attenuating atherosclerosis.     

Exposure to Malondialdehyde Induces an Early Redox Unbalance Preceding Membrane Toxicity in Human Erythrocytes

This work investigated the oxidative injury to human red blood cells (RBCs) by the exposure to exogenous malondialdehyde (MDA), in a physiological environment. When a 10% RBC suspension was incubated in autologous plasma, in the presence of 50 &#117 &#119 M MDA, 30% of MDA entered into the cells. A time-course study showed that MDA caused early (30-120 &#117 min) and delayed (3-18 &#117 h) effects. MDA caused a fast depletion of reduced glutathione, and loss of the glucose-6-phosphate dehydrogenase activity, followed by a decrease of HbO 2 . Accumulation of methemoglobin, and formation of small amounts of hemichrome were later evident. Also, an HbO 2 -derived fluorescent product was measured in the membrane. The redox unbalance was followed by structural and functional damage to the membrane, evident as the formation of conjugated diene lipid hydroperoxides, concurrent with a sharp accumulation of MDA, consumption of membrane vitamin E, and egress of K + ions. SDS--PAGE of membrane proteins showed formation of high molecular weight aggregates. In spite of the marked oxidative alterations, the incubation plasma prevented a substantial hemolysis, even after a 18 &#117 h incubation. On the contrary, the exposure of RBCs to 50 &#117 &#119 M MDA in glucose-containing phosphate saline buffer, resulted in a 16% hemolysis within 6 &#117 h. These results indicate that the exposure to MDA causes a rapid intracellular oxidative stress and potentiates oxidative cascades on RBCs, resulting in their dysfunction.     

Carotenoids Induce Apoptosis in the T-lymphoblast Cell Line Jurkat E6.1

Epidemiologically, a high-carotenoid intake via a fruit- and vegetable-rich diet is associated with a decreased risk of various forms of cancer. The mechanisms by which carotenoids exert this protective effect are controversial. In this study, we examined the potency of a range of carotenoids commonly found in human plasma to induce apoptosis in Jurkat E6.1 malignant T-lymphoblast cells. At a concentration of 20 &#119 M, the order of potency to induce apoptosis after 24 h was: &#103 -carotene > lycopene > lutein> &#103 -cryptoxanthin=zeaxanthin. Canthaxanthin failed to induce apoptosis under these conditions. &#103 -Carotene induced apoptosis in a time- and concentration-dependent manner with a lowest effective concentration of about 3 &#119 M. Pre-conditioning of &#103 -carotene for 72 h destroyed its pro-apoptotic activity almost completely, whereas degradation for 6 h or less did not, indicating that either &#103 -carotene itself and/or an early degradation product of &#103 -carotene are the death-inducing compounds. Apoptosis induced by &#103 -carotene was characterized by chromatin condensation and nuclear fragmentation, DNA degradation, PARP cleavage and caspase-3 activation. The antioxidant BO-653 inhibited the degradation of &#103 -carotene in vitro and significantly increased its cytotoxicity, indicating that a pro-oxidant effect of &#103 -carotene is unlikely to cause its pro-apoptotic activity. The induction of apoptosis in transformed cells by carotenoids may explain their protective effect against cancer formation in humans. Possible pathways for induction of apoptosis by carotenoids are discussed.     

Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection

This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH + GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20–45 years old) and 55 patients with chronic hepatitis C (20–49 years old) and the resulting values were in a good agreement with results published earlier. The calibration curves were linear over a concentration range of 5–100 μM in plasma (r ² = 0.985−0.996). The intraday and interday coefficients of variation were 3–6% and 4–7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2–0.5 ng per 10 μl of the injection volume (signal-to-noise ratio of 3).     

The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E

Common methods for plant iron determination are based on atomic absorption spectroscopy, radioactive measurements or extraction with subsequent spectrophotometry. However, accuracy is often a problem due to background, contamination and interfering compounds. We here describe a novel method for the easy determination of ferric iron in plants by chelation with a highly effective microbial siderophore and separation by high performance liquid chromatography (HPLC). After addition of colourless desferrioxamine E (DFE) to plant fluids, the soluble iron is trapped as a brown-red ferrioxamine E (FoxE) complex which is subsequently separated by HPLC on a reversed phase column. The formed FoxE complex can be identified due to its ligand-to-metal charge transfer band at 435 nm. Alternatively, elution of both, DFE and FoxE can be followed as separate peaks at 220 nm wavelength with characteristic retention times. The extraordinarily high stability constant of DFE with ferric iron of K=10³² enables extraction of iron from a variety of ferrous and ferric iron compounds and allows quantitation after separation by HPLC without interference by coloured by-products. Thus, iron bound to protein, amino acids, citrate and other organic acid ligands and even insoluble ferric hydroxides and phosphates can be solubilized in the presence desferrioxamine E. The “Ferrioxamine E method” can be applied to all kinds of plant fluids (apoplasmic, xylem, phloem, intracellular) either at physiological pH or even at acid pH values. The FoxE complex is stable down to pH 1 allowing protein removal by perchloric acid treatment and HPLC separation in the presence of trifluoroacetic acid containing eluents. Published online December 2004     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 &#45 28 nerve fibers consisting of 63 &#45 24 unmyelinated and 12 &#45 6 myelinated fibers. The PAN was composed of 195 &#45 50 nerve fibers, namely 129 &#45 28 unmyelinated and 66 &#45 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 &#119 m with a maximum between 0.3 and 0.6 &#119 m. The myelinated nerve fibers in the MAN ranged from 1 to 8 &#119 m with a peak between 2 and 5 &#119 m. In the PAN, their diameters ranged from 1 to 12 &#119 m with a clearly visible peak at 4-5 &#119 m and a plateau at 8-9 &#119 m that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Synthesis of Aspartame Precursor Using Protease Suspended in Microaqueous Molten Amino Acids Mixture

Enzymatic synthesis of the aspartame precursor, N -(benzyloxycarbonyl)- l -aspartyl- l -phenylalanine methyl ester (Z-AspPheOMe) was performed with highly concentrated molten substrates. A mixture composed of molten N -(benzyloxycarbonyl)- l -aspartic acid (Z-Asp) and l -phenylalanine methyl ester (PheOMe) mixtures of 20 M could be prepared at 50°C. This Z-Asp/PheOMe mixture was applied to the enzymatic synthesis of Z-AspPheOMe using free thermolysin. Synthesis of Z-AspPheOMe was observed in the range of 100-150 &#119 l of NaOH solution (12.5 M) addition to a reaction mixture consisting of 1.0 mmol Z-Asp and 1.0 mmol PheOMe at 50°C. The enzymatic activity increased with increasing water addition, and reached a maximum at 100 &#119 l in addition to the reaction mixture of 1.0 mmol Z-Asp, 1.0 mmol PheOMe and 125 &#119 l of the NaOH solution. In this reaction system, the conversion at the reaction equilibrium was about 60%, the initial reaction rate calculated on the basis of the enzyme weight was 2.2 &#119 mol/g s, and the productivity calculated on the basis of the reaction mixture volume was 300 mol/m 3 h.     

HPLC determination of a novel aroylhydrazone iron chelator (o-108) in rabbit plasma and its application to a pilot pharmacokinetic study

A high performance liquid chromatographic method for the determination of a biocompatible iron chelator, pyridoxal 2-chlorobenzoyl hydrazone (o-108), in rabbit plasma was developed and validated. The separation was achieved on a C18 column with the mobile phase composed of a mixture of 0.01 M phosphate buffer (pH 6) with the addition of EDTA (2 mM), methanol and acetonitrile (42:24:14; v/v/v). The method was validated with respect to selectivity, linearity (0.8-150 microg/mL), intra- and inter-day variability and stability. This method was successfully applied to the analysis of the samples obtained from a pilot pharmacokinetic experiment, in which the chelator was administered intravenously to rabbits.     

The plant-endogenous Fe(II)-chelator nicotianamine restricts the ferrochelatase activity of tomato chloroplasts

Stripped chloroplasts were prepared from young leaves of a tomatowild type (Lycopersicon esculentum Mill.) and its mutant chloronerva.Several morphological and biochemical abnormalities of thismutant are caused by the total lack of the plant-endogenousFe²⁺ chelator nicotianamine (NA). The ferrochelatase activitywas estimated by determination of ⁵⁹Fe incorporated into haem.A mercaptoethanol concentration of 250 mM was necessary to maintainfull enzyme activity. The reducing agent supported the reducedstate of the active site of the enzyme more than that of theiron as revealed by use of ferrous and ferric ionproviding compoundsas substrates. Chloroplasts of both genotypes exhibited a similar enzyme activity.NA inhibited this activity by nearly 100% depending on the concentrationapplied. On the basis of the formation constant of the Fe(ll)–NAcomplex and the concentrations of iron and NA in the enzymeassay as well as in the tomato shoot apex region it is proposedthat ferrochelatase acts in vivo with an iron level at the attomolarrange which is provided by NA. Key words: Ferrochelatase activity, ferrous ion concentration, nicotianamine, tomato chloroplasts, substrate limitation     

Rapid and direct determination of selenium, copper, and zinc in blood plasma by flow injection-inductively coupled plasma-mass spectrometry

A flow injection-inductively coupled plasma-mass spectrometry (FI-ICP-MS) with a simple sample preparation procedure was developed for the determination of selenium, copper, and zinc in blood serum/plasma. A serum/plasma sample was filtered through a 0.45-μm membrane filter and diluted with a mixture of trace elements in a standard solution (9∶1, v/v). Measurement of the reference serum sample confirmed the accuracy of our method for selenium, copper, and zinc concentration. In the case of blood plasma samples obtained from six healthy adult males, the selenium, copper, and zinc concentrations were similar to those of a typical healthy male in Japan. These results suggest that the sample prepartive procedure coupled with FI-ICP-MS can be used for the routine determination of selenium, copper, and zinc in human blood serum/plasma.