Assignment of complex species by affinity capillary electrophoresis: The case of Th(IV)-desferrioxamine B.

The electrophoretic mobility change of desferrioxamine B (DFO) was monitored by UV absorption spectrophotometry upon increasing the thorium(IV) concentration in the background electrolyte at two acidities ([HClO4 ]Tot = 0.0316 and 0.0100 M). These data enabled to assess the speciation model and to determine the equilibrium constant of [Th(DFO)H2 ]3+ at fixed ionic strength (I = 0.1 M (H,Na)ClO4 ). Affinity capillary electrophoresis (ACE) turned out to be most helpful in identifying the complexed species by ascertaining its charge and protonation state. The assignment of the correct stoichiometry relied on the reliable estimation of the electrophoretic mobility by assuming similar hydrodynamic radii for (DFO)H4+ and the chelate. The value of the apparent equilibrium constant (log ß112 = 38.7 ± 0.4) obtained by ACE compares favorably well with those reported in the literature for thorium and a range of other metal ions, according to a linear free-energy relationship. This method is useful for studying metal-ligand binding equilibria and provides valuable information for further modelling the behavior of tetravalent actinides under environmental conditions. Structural information about the prevalent solution species in acidic conditions was gained by DFT calculations, confirming the bishydroxamato coordination mode of Th4+ by the diprotonated ligand.

Quantifying Intranasally Administered Deferoxamine in Rat Brain Tissue with Mass Spectrometry.

Deferoxamine, a metal chelator, has been shown to be neuroprotective in animal models of ischemic stroke, traumatic brain injury and both subarachnoid and intracerebral hemorrhage. Intranasal deferoxamine (IN DFO) has also shown promise as a potential treatment for multiple neurodegenerative diseases, including Parkinson's and Alzheimer's. However, there have been no attempts to thoroughly understand the dynamics and pharmacokinetics of IN DFO. We developed a new high-performance liquid-chromatography electrospray-tandem mass spectrometry (HPLC/ESI-MS2) method to quantify the combined total levels of DFO, ferrioxamine (FO; DFO bound to iron), and aluminoxamine (AO; aluminum-bound DFO) in brain tissue using a custom-synthesized deuterated analogue (DFO-d7, Medical Isotopes Inc., Pelham NH) as an internal standard. We applied our method toward understanding the pharmacokinetics of IN DFO delivery to the brain and blood of rats from 15 min to 4 h after delivery. We found that IN delivery successfully targets DFO to the brain to achieve concentrations of 0.5-15 µM in various brain regions within 15 min, and decreasing though still detectable after 4 h. Systemic exposure was minimized as assessed by concentration in blood serum. Serum concentrations were 0.02 µM at 15 min and no more than 0.1 µM at later time points. Compared to blood serum, brain region-specific drug exposure (as measured by area under the curve) ranged from slightly under 10 times exposure in the hippocampus to almost 200 times exposure in the olfactory bulb with IN DFO delivery. These findings represent a major step toward future method development, pharmacokinetic studies, and clinical trials for this promising therapeutic.

A nanocellulose-based colorimetric assay kit for smartphone sensing of iron and iron-chelating deferoxamine drug in biofluids.

The current work describes the development of a "nanopaper-based analytical device (NAD)", through the embedding of curcumin in transparent bacterial cellulose (BC) nanopaper, as a colorimetric assay kit for monitoring of iron and deferoxamine (DFO) as iron-chelating drug in biological fluids such as serum blood, urine and saliva. The iron sensing strategy using the developed assay kit is based on the decrease of the absorbance/color intensity of curcumin-embedded in BC nanopaper (CEBC) in the presence of Fe(III), due to the formation of Fe(III)-curcumin complex. On the other hand, releasing of Fe(III) from Fe(III)-CEBC upon addition of DFO as an iron-chelating drug, due to the high affinity of this drug to Fe(III) in competition with curcumin, which leads to recovery of the decreased absorption/color intensity of Fe(III)-CEBC, is utilized for selective colorimetric monitoring of this drug. The absorption/color changes of the fabricated assay kit as output signal can be monitored by smartphone camera or by using a spectrophotometer. The results of our developed sensor agreed well with the results from a clinical reference method for determination of Fe(III) concentration in human serum blood samples, which revealed the clinical applicability of our developed assay kit. Taken together, regarding the advantageous features of the developed sensor as an easy-to-use, non-toxic, disposable, cost-effective and portable assay kit, along with those of smartphone-based sensing, it is anticipated that this sensing bioplatform, which we name lab-on-nanopaper, will find utility for sensitive, selective and easy diagnosis of iron-related diseases (iron deficiency and iron overload) and therapeutic drug monitoring (TDM) of iron-chelating drugs in clinical analysis as well.

Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method.

The Actinobacterium Gordonia rubripertincta CWB2 (DSM 46758) produces hydroxamate-type siderophores (188 mg L-1) under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth which was confirmed by the Fe-CAS assay. Elution profile and its absorbance spectrum were similar to those of commercial (des)ferrioxamine B which was used as reference compound. This confirms previously made assumptions and shows for the first time that the genus Gordonia produces desferrioxamine-like siderophores. The reversed-phase HPLC protocol was optimized to separate metal-free and -loaded oxamines. This allowed to determine siderophore concentrations in solutions as well as metal affinity. The metal loading of oxamines was confirmed by ICP-MS. As a result, it was demonstrated that desferrioxamine prefers trivalent metal ions (Fe3+ > Ga3+ > V3+ > Al3+) over divalent ones. In addition, we aimed to show the applicability of the newly established reversed-phase HPLC protocol and to increase the re-usability of desferrioxamines as metal chelators by immobilization on mesocellular silica foam carriers. The siderophores obtained from strain CWB2 and commercial desferrioxamine B were successfully linked to the carrier with a high yield (up to 95%) which was verified by the HPLC method. Metal binding studies demonstrated that metals can be bound to non-immobilized and to the covalently linked desferrioxamines, but also to the carrier material itself. The latter was found to be unspecific and, therefore, the effect of the carrier material remains a field of future research. By means of a reversed CAS assay for various elements (Nd, Gd, La, Er, Al, Ga, V, Au, Fe, As) it was possible to demonstrate improved Ga3+- and Nd3+-binding to desferrioxamine loaded mesoporous silica carriers. The combination of the robust reversed-phase HPLC method and various CAS assays provides new avenues to screen for siderophore producing strains, and to control purification and immobilization of siderophores.

Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function.

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies.

Accurate mass MS/MS/MS analysis of siderophores ferrioxamine B and E1 by collision-induced dissociation electrospray mass spectrometry.

Siderophores are bacterially secreted, small molecule iron chelators that facilitate the binding of insoluble iron (III) for reuptake and use in various biological processes. These compounds are classified by their iron (III) binding geometry, as dictated by subunit composition and include groups such as the trihydroxamates (hexadentate ligand) and catecholates (bidentate). Small modifications to the core structure such as acetylation, lipid tail addition, or cyclization, make facile characterization of new siderophores difficult by molecular ion detection alone (MS(1)). We have expanded upon previous fragmentation-directed studies using electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS/MS) and identified diagnostic MS(3) features from the trihydroxamate siderophore class for ferrioxamine B and E1 by accurate mass. Diagnostic features for MS(3) include C-C, C-N, amide, and oxime cleavage events with proposed losses of water and -CO from the iron (III) coordination sites. These insights will facilitate the discovery of novel trihydroxamate siderophores from complex sample matrices. Graphical Abstract ᅟ.

Plutonium(IV) sorption to montmorillonite in the presence of organic matter.

The effect of altering the order of addition in a ternary system of plutonium(IV), organic matter (fulvic acid, humic acid and desferrioxamine B), and montmorillonite was investigated. A decrease in Pu(IV) sorption to montmorillonite in the presence of fulvic and humic acid relative to the binary Pu-montmorillonite system, is attributed to strong organic aqueous complex formation with aqueous Pu(IV). No dependence on the order of addition was observed. In contrast, in the system where Pu(IV) was equilibrated with desferrioxamine B (DFOB) prior to addition of montmorillonite, an increase in Pu(IV) sorption was observed relative to the binary system. When DFOB was equilibrated with montmorillonite prior to addition of Pu(IV), Pu(IV) sorption was equivalent to the binary system. X-ray diffraction and transmission electron microscopy revealed that DFOB accumulated in the interlayer of montmorillonite. The order of DFOB addition plays an important role in the observed sorption/desorption behavior of Pu. The irreversible nature of DFOB accumulation in the montmorillonite interlayer leads to an apparent dependence of Pu sorption on the order of addition in the ternary system. This work demonstrates that the order of addition will be relevant in ternary systems in which at least one component exhibits irreversible sorption behavior.

A simple small size and low cost sensor based on surface plasmon resonance for selective detection of Fe(III).

A simple, small size, and low cost sensor based on a Deferoxamine Self Assembled Monolayer (DFO-SAM) and Surface Plasmon Resonance (SPR) transduction, in connection with a Plastic Optical Fiber (POF), has been developed for the selective detection of Fe(III). DFO-SAM sensors based on appropriate electrochemical techniques can be frequently found in the scientific literature. In this work, we present the first example of a DFO-SAM sensor based on SPR in an optical fiber. The SPR sensing platform was realized by removing the cladding of a plastic optical fiber along half the circumference, spin coating a buffer of Microposit S1813 photoresist on the exposed core, and finally sputtering a thin gold film. The hydroxamate siderophore deferoxamine (DFO), having high binding affinity for Fe(III), is then used in its immobilized form, as self-assembled monolayer on the gold layer surface of the POF sensor. The results showed that the DFO-SAM-POF-sensor was able to sense the formation of the Fe(III)/DFO complex in the range of concentrations between 1 µm and 50 µm with a linearity range from 0 to 30 µm of Fe(III). The selectivity of the sensor was also proved by interference tests.

Characterisation of iron binding ligands in seawater by reverse titration.

Here we demonstrate the use of reverse titration - competitive ligand exchange-adsorptive cathodic stripping voltammetry (RT-CLE-ACSV) for the analysis of iron (Fe) binding ligands in seawater. In contrast to the forward titration, which examines excess ligands in solution, RT-CLE-ACSV examines the existing Fe-ligand complexes by increasing the concentration of added (electroactive) ligand (1-nitroso-2-naphthol) and analysis of the proportion of Fe bound to the added ligand. The data manipulation allows the accurate characterisation of ligands at equal or lower concentrations than Fe in seawater, and disregards electrochemically inert dissolved Fe such as some colloidal phases. The method is thus superior to the forward titration in environments with high Fe and low ligand concentrations or high concentrations of inert Fe. We validated the technique using the siderophore ligand ferrioxamine B, and observed a stability constant [Formula: see text] of 0.74-4.37×10(21) mol(-1), in agreement with previous results. We also successfully analysed samples from coastal waters and a deep ocean hydrothermal plume. Samples from these environments could not be analysed with confidence using the forward titration, highlighting the effectiveness of the RT-CLE-ACSV technique in waters with high concentrations of inert Fe.

Tandem mass spectrometry of coprogen and deferoxamine hydroxamic siderophores.

Mechanisms of fragmentation of hydroxamic siderophores are proposed comparing deuterated and nondeuterated samples. Standard siderophores (e.g. deferoxamine and coprogen) were directly injected into both ion trap and linear quadrupole mass spectrometers with electrospray ionization (ESI). Four and two fragmentation steps were carried out for deferoxamine and coprogen (analyzed by positive and negative ESI, respectively). Deferoxamine cleavages occurred in both peptide and hydroxamic bonds while the coprogen fragmentation pattern is more elaborate, since it contains Fe(III) in its structure.

Validated HPLC assay for iron determination in biological matrices based on ferrioxamine formation.

A simple, robust and reproducible HPLC method has been developed and validated for iron determination in biological matrices. It is based on chelation with desferrioxamine (DFO) and the measurement of the chelate ferrioxamine (FO). The method was developed to permit monitoring of iron bio-kinetics and estimation of iron status in experimental animals. The chromatography was performed on a stainless steel XTerra MS C18 column (Waters; 250 mm x 4.6 mm i.d., 5 microm) using a gradient of Tris-HCl buffer (10mM, pH 5) and acetonitrile. The method was validated in terms of selectivity, linearity (0.3-80 nmol on-column), limit of detection (0.2 nmol on-column), low limit of quantification (0.3 nmol on-column), recovery (91-102%), intra- and inter-day reproducibility, stability, and robustness. The method's universal applicability was illustrated by monitoring plasma and heart iron kinetic profiles in rats after a single intraperitoneal (i.p.) injection of 200mg/kg iron dextran.

Electrospray ionisation-mass spectrometry of hydroxamate siderophores.

Electrospray ionisation-mass spectrometry (ESI-MS) was applied to the detection of the iron complexes of the hydroxamate type siderophores ferrioxamine (FO), ferrichrome (FC) and iron(III) rhodotoluate (FR). Mass spectra of the three siderophores produced by ESI-MS were dominated by the protonated (M + 1)+ parent ions, except for FR at pH 4.3, which was present as the positively charged 1:1 complex. On collision with He ions, fragmentation proceeded largely via cleavage of C-N bonds. Flow injection analysis of the siderophores with detection by ESI-MS produced detection limits of 1.9 fmol for FO, 31.1 fmol for FC and 524 fmol for FR.

Quantification of desferrioxamine, ferrioxamine and aluminoxamine by post-column derivatization high-performance liquid chromatography. Non-linear calibration resulting from second-order reaction kinetics.

Desferrioxamine B is widely used as therapeutic agent for removal of excess body iron and, more recently, for removal of aluminium. A HPLC-based method for direct sensitive and reliable determination of ferrioxamine, desferrioxamine, aluminoxamine and related metabolites has been developed for use in pharmacokinetic studies. The method consists of complete separation of the analytes by an optimized mobile phase avoiding conversion of desferrioxamine to ferrioxamine by the analytical system and overcoming problems due to peak tailing properties of desferrioxamine. A post-column derivatization reaction with colourless fluoro-complexed iron converts all iron free species to ferrioxamine and allows quantification at 430 nm avoiding interference of UV-absorbing coelutes. This reaction might be of interest for other analytical procedures concerning iron chelators. The influence of the post-column reaction system on the column plate number is characterized. As the reaction rate of desferrioxamine and aluminoxamine with iron(III) is of second-order kinetics, a quadratic calibration function is observed, resulting from a compromise between residence time and peak broadening. A solid-phase extraction procedure is presented for extraction of the analytes from plasma. Limits of detection (S/N ratio of 3) were determined as 1.95 ng for ferrioxamine, 3.9 ng for aluminoxamine and 15.7 ng for desferrioxamine, expressed as on-column load. A new iron-free metabolite was identified with fast atom bombardment-mass spectrometry as N-hydroxylated desferrioxamine.

Desferrioxamine suppresses experimental allergic encephalomyelitis induced by MBP in SJL mice.

Data from several studies indicate that free radicals have a pathogenic role in experimental allergic encephalomyelitis (EAE). Iron can contribute to free radical damage by catalyzing the formation of hydroxyl radical, inducing secondary initiation of lipid peroxidation and by promoting the oxidation of proteins. The iron chelator, desferrioxamine, can limit these oxidative reactions and it can scavenge peroxynitrite independent of iron chelation. Two previous studies have examined the therapeutic value of desferrioxamine in EAE. One study observed an effect when disease was induced by spinal cord homogenates (J. Exp. Med. 160, p. 1532, 1984), but a second study found no therapeutic value of desferrioxamine for myelin basic protein (MBP)-induced EAE (J. Neuroimmunol. 17, p. 127, 1988). In the second study, the drug was only administered during the preclinical stages of disease. Since desferrioxamine scavenges free radicals and prevents their formation, we hypothesized that the drug should be given during the active stage of disease to have therapeutic value. We first demonstrated that the drug enters the CNS around inflammatory cells in EAE animals. In animals treated during the active stage of MBP-induced EAE, the clinical signs were significantly reduced compared to vehicle-treated animals. The iron-bound form of this drug, ferrioxamine, was without therapeutic value. A derivative of desferrioxamine, hydroxylethyl starch (HES)-desferrioxamine, has a greater plasma half-life than desferrioxamine and it was also tested. Although there was a suggestion of improvement in these animals, the effects were less than that observed for desferrioxamine which may be related to the greater molecular size of HES-desferrioxamine. In summary, these data suggest that chelation of iron is an effective therapeutic target for EAE.

Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation.

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.

Deferoxamine posttreatment reduces ischemic brain injury in neonatal rats.

BACKGROUND AND PURPOSE: Iron catalyzes the formation of damaging reactive species during cerebral reperfusion. Brain iron concentration is highest at birth, so the brain of the asphyxiated newborn may be at increased risk of iron-dependent injury. We investigated whether the ferric iron chelator deferoxamine could reduce hypoxic-ischemic brain injury in neonatal rats. Because deferoxamine has concentration-dependent activities other than iron chelation, we measured brain deferoxamine levels and calculated deferoxamine pharmacokinetic parameters. METHODS: We produced hypoxic-ischemic injury to the right cerebral hemisphere of 7-day-old rats by right common carotid artery ligation followed by 2.25 hours of hypoxia in 8% oxygen. At 5 minutes of recovery from hypoxia the rats received 100 mg/kg deferoxamine mesylate or saline subcutaneously. Rats (saline, n = 33; deferoxamine, n = 38) were killed at 42 hours of recovery to assess early acute edema by measurement of hemispheric water content. Other rats (saline, n = 31; deferoxamine, n = 32) were killed at 30 days of age for morphometric determination of right hemisphere atrophy. In still other rats, we measured deferoxamine levels in blood and brain after hypoxia-ischemia. RESULTS: Deferoxamine significantly reduced right hemisphere injury as measured by early water content (P < .01) and later atrophy (P = .019). Deferoxamine brain levels peaked between 100 and 200 mumol/L at 40 to 60 minutes after injection and exceeded serum levels by +/- 70%. CONCLUSIONS: Deferoxamine administered after induction of cerebral hypoxia-ischemia reduces injury in 7-day-old rats. Deferoxamine concentrates in the brain at levels between 100 and 200 mumol/L. At the concentrations achieved, deferoxamine might protect the brain through mechanisms unrelated to its ability to chelate iron.

Urinary and biliary excretion of aluminoxamine and ferrioxamine in dogs with various renal function.

We assessed the pharmacokinetics of aluminoxamine and ferrioxamine in dogs with sustained intermittent bile duct ligation and either normal renal function or stable chronic renal failure. A first group of male beagle dogs were given aluminoxamine and ferrioxamine, while a second group received desferrioxamine after loading them with iron and aluminum. Only minute amounts of ferrioxamine and aluminoxamine were found in the bile after administration of these compounds. The distribution volume of aluminoxamine and ferrioxamine appeared to be confined to the extracellular space and their renal excretion correlated with renal function. Administration of desferrioxamine to iron and aluminum-loaded dogs resulted in an increased biliary ferrioxamine but negligible aluminoxamine excretion. Renal clearance of the in vivo formed ferrioxamine and aluminoxamine in this group strongly correlated with renal function. Our observations indicate that biliary excretion of intravenously administered ferrioxamine and aluminoxamine is negligible even in renal failure. The data presented in this study provide indirect evidence that desferrioxamine administration to iron- and aluminum-loaded dogs results in the intra-hepatic formation of ferrioxamine which is partly excreted in the bile. Biliary excretion of aluminoxamine after desferrioxamine administration remained negligible.

Quantification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and simultaneous ultraviolet-visible/radioactive detection.

An HPLC-based method for quantification of desferrioxamine (DFO) and its iron chelating metabolites in plasma has been developed. This assay overcomes stability problems associated with DFO by the addition of radioactive iron to convert unbound drug and metabolites to radio-iron-bound species. A dual detection system utilizing uv-vis absorption and radioactive (beta-particle) detector was used to quantify total and radio-iron-bound species. The use of octadecyl silanol solid phase extraction cartridges permits concentration of samples and allows accurate quantification of drug and metabolites down to 0.1 nmol/ml.