Browsing by Author "Gailer, Jürgen"
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- ItemOpen AccessA developed metallomics method reveals the biochemical fate of Cd2+ and Hg species in erythrocyte lysate(2017) Gibson, Matthew; Gailer, Jürgen; Prenner, Elmar; Thurbide, Kevin; Osthoff, HansBioanalytical techniques that can be employed to obtain health-relevant information from biological fluids (i.e. erythrocytes) are urgently needed to gain new insight into the bioinorganic chemistry of non-essential metal species in the human body. To this end, a metallomics method was developed to probe the interaction of Cd2+, Hg2+, CH3Hg+ and thimerosal in the erythrocyte based on the direct analysis of erythrocyte lysate by size exclusion chromatography coupled on-line to an inductively coupled plasma atomic emission spectrometer (SEC-ICP-AES). This method development entailed systematic investigations into the sample preparation of the erythrocyte lysate (i.e. filtration to remove cell debris) as well into the identification of the ideal stationary phase and mobile phase. Filtering (0.45 μm pore-size Millex syringe-driven filers) and diluting (5-fold with corresponding buffer) resulted in the most consistent and reproducible results for the analysis of the erythrocyte lysate. A Superdex 75TM Increase 10/300 GL (300 x 10 mm I.D., 8.6 μm particle size) column that offers a fractionation range of 3 – 70 kDa was best suited for the separation of the major Cu, Fe and Zn metallospecies that are present in the erythrocyte lysate. 100 mM Tris(hydroxymethyl)-aminomethane (Tris) buffer (pH 7.4) was identified as the optimal mobile phase as it allowed for the separation of all the metallospecies of interest and gave the highest metal recovery of all the mobile phases (Zn: 95.8 ± 2.8% , Fe: 88.0 ± 7.2%). This developed metallomics method was subsequently employed to probe the comparative interaction of Cd2+, Hg2+, CH3Hg+ and thimerosal in erythrocyte lysate over a 6 h time period. The results that were obtained at time points ≥2 h revealed that ~85% of Cd2+ weakly interacted with hemoglobin (Hb), while ~13% eluted as (GS)xCd and ~2% bound to a ≥70 kDa Cd-binding protein. In contrast, ~6% of Hg2+ co-eluted with Hb at all time points, while ~94% eluted as (GS)xHg. The results for CH3Hg+ showed that ~5% of Hg co-eluted with Hb (constant over the 6 h time period), while for thimerosal (THI), this percentage gradually increased to 12% over the 6 h time period. The remaining Hg eluted as GS–HgCH3 and GS–HgCH2CH3, respectively. The co-elution of Hg with Hb – regardless of whether Hg2+, CH3Hg+ or THI was added – indicates that these interactions may adversely affect the function of Hb. The results demonstrate that the developed metallomics tool provided is ideally suited to obtain new insight into the bioinorganic chemistry and the toxicology of non-essential metal species within erythrocytes.
- ItemOpen AccessAmeliorating the toxic side-effects of cisplatin by systematically modulating its metabolism in human plasma with chemoprotective agents(2015-04-20) Sooriyaarachchi, Melani Devindya; Gailer, JürgenUsing size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES), the effect of three chemoprotective agents − sodium thiosulfate (STS), N-acetyl-L-cysteine (NAC) and D-methionine − were investigated on the metabolism of the Pt-based anti-cancer drug cis-platin (CP) in human blood plasma in vitro. All three sulfur compounds resulted in the formation of Pt-S complexes independent of their order of addition to plasma while concomitantly reducing the plasma protein binding of CP-derived Pt species. Analogous experiments that were carried out with PBS-buffer instead of plasma allowed to gain more insight into the bioinorganic chemistry that unfolds in the absence of plasma proteins under physiologically similar conditions. Analysis of a PBS-buffer sample that was spiked with CP and STS by X-ray absorption spectroscopy revealed the presence of [Pt(S2O3)4]6- while the analysis of those corresponding to D-methionine and NAC by liquid chromatography coupled to electrospray ionization mass spectrometry revealed the presence of [Pt(NH3)Cl(D-methionine)]+, [Pt(D-methionine)2]+ and [(NAC)2Pt(NH3)2]-, respectively. Unlike CP, STS or D-methionine, NAC adversely effected the plasma protein distribution of Fe and Zn in a dose and a time dependent manner. Combined, the results present - for the first time - a feasible biomolecular mechanism by which these chemoprotective agents may reduce the CP-induced toxic side-effects in vivo. The unique capability of SEC-ICP-AES to simultaneously detect all Pt species in human plasma (CP, CP hydrolysis products and Pt-S complexes) suggests that the metabolism of CP can be deliberately modulated to develop strategies to reduce the severe toxic side-effects of this otherwise very effective anti-cancer drug in patients. Since combination therapy is a ii promising strategy to overcome drug resistance during chemotherapy and since SEC-ICP-AES allows to visualize the simultaneous metabolism of two metal-based anti-cancer drugs in human blood plasma, the latter was spiked with CP (Pt) and NAMI-A (Ru) and analysed over time. The results revealed that both drugs bind to the same apparent plasma proteins, but importantly, did not adversely affect each other’s metabolism in plasma which is promising with regard to investigating this particular drug combination in clinical studies with patients in the near future.
- ItemOpen AccessApplying a Metallomics Tool to Probe Interactions between Cadmium, Mammalian Blood Plasma Constituents and Chelating Agents(2013-09-23) Zeini Jahromi, Elham; Gailer, JürgenThe analysis of biological fluids in a top-down manner can be employed to gain new insight into the bioinorganic chemistry of toxic metals. To this end, a metallomics method has been previously developed to determine all major blood plasma Cu, Fe and Zn metalloproteins based on the direct analysis of plasma by size exclusion chromatography coupled on-line to an inductively coupled plasma atomic emission spectrometer (SEC-ICP-AES). In order to assess the viability of this metallomics method, the effect of various mobile phase buffers on the distribution of Cu, Zn and Fe metalloproteins was studied using rabbit plasma. 100 mM 3-(N-morpholino)propanesulfonic (Mops), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and tris(hydroxymethyl)-aminomethane (Tris)-buffers perturbed the Fe and/or Zn metalloproteome, whereas phosphate-buffered saline (PBS)-buffer produced results that were most consistent with literature data. This metallomics method was subsequently employed to probe the dose-dependent interaction of Cd2+ (0.05-200 µg/mL) with rabbit and human blood plasma metalloproteins in vitro. At 200 µg Cd/mL, Cd2+ displaced Zn2+ from a 150 kDa protein which likely corresponds to extracellular Cu, Zn superoxide dismutase (Cu, Zn-SOD) based on previous studies by others. In addition, this methodology was used to assess the efficiency of the abstraction of Cd2+ from plasma proteins (2.0 µg Cd/mL plasma) by three different chelating agents, meso-2,3-dimercaptosuccinic acid (DMSA), diethylenetriamine-pentaacetic acid (DTPA) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) in vitro using pharmacologically relevant doses. The unique capability to simultaneously observe the size distribution of Cd2+ and the essential elements Zn2+ and Ca2+ in plasma revealed that Na5DTPA was the most efficient in terms of abstracting Cd2+ from plasma proteins (100% removal, intended), but it also mobilized Zn2+ from plasma proteins and resulted in the dose-dependent formation of a [Ca-DTPA]3- complex (unintended). In order to minimize these unintended effects, the effect of the Zn salt of DTPA (ZnNa3DTPA) on the mobilization of Cd2+ from plasma proteins was investigated. ZnNa3DTPA was as efficient as Na5DTPA in abstracting Cd2+ from plasma proteins, but minimal or no effect on the distribution of Zn and Ca were observed. Thus, in vitro studies combined with the analysis of blood plasma by SEC-ICP-AES revealed an improved selectivity of a chelating agent. This inherent advantage of this metallomics tool can be used to develop more effective and selective chelating agents to mobilize Cd2+ and other toxic metals from afflicted organisms. Finally, the complexes that are formed between Cd2+ and the chelating agents DMSA and DMPS in aqueous solution at pH 7.4 were structurally characterized by using a combination of X-ray absorption spectroscopy (XAS), SEC-ICP-AES, electrospray ionization-mass spectrometry (ESI-MS) and density functional theory (DFT). The results indicate a complex chemistry in which multi-metallic forms are important, but are consistent with both DMPS and DMSA acting as true chelators, using two thiolates for DMPS and one thiolate and one carboxylate for DMSA.
- ItemOpen AccessCharacterization of Planar Titanium Platforms for Flame Ionization Detection in Microfluidic Gas Chromatography(2016) Raut, Rahul Pandurang; Thurbide, Kevin; Hinman, Allen Scott; Gailer, Jürgen; Marriott, RobertGas chromatography (GC) with a Flame Ionization Detector (FID) has been widely used for the analysis of volatile organic compounds in various fields. Instrument portability and miniaturization of the FID for micro GC (μGC) applications are of increasing interest. This thesis describes the design and characterization of a planar on-chip counter current micro FID (μ-FID). The μ-FID fabricated in both quartz and Titanium (Ti) substrates were characterized and the Ti μ-FID showed great improvements in detection limit and sensitivity over other prototypes. This Ti μ-FID design is further integrated with a Ti separation column to fabricate a planar μGC-FID Ti tile for the first time. This Ti μGC-FID tile is explored for its detector performance attributes and some routine GC applications are examined. This novel Ti μGC-FID tile, with an on-chip single monolithic design, is found to provide a very low detection limit (9 pgC/s) and a high sensitivity (60 mC/gC).
- ItemOpen AccessCytotoxic, Cellular Uptake, and Photophysical Properties of Various Re(I) Tricarbonyl Complexes(2019-09-20) Capper, Miles S.; Jalilehvand, Farideh; Heyne, Belinda; Roesler, Roland; Gailer, Jürgen; Derksen, Darren J.A series of Re(I) tricarbonyl complexes with the general formula, fac-[Re(CO)3(2,2’-bipyridine)(X)]-/0 (X= L-cysteine; N-acetyl-L-cysteine; thiosulfate) were characterized using spectroscopic techniques and single-crystal X-ray diffraction. Photophysical, as well as singlet oxygen (1O2) generation and CO releasing properties were assessed. Cell viability of the complexes against the MDA-MB-231 breast cancer cell line were determined. Cellular localization and accumulation were investigated using synchrotron-based X-ray fluorescence microscopy (XFM). The results of this study show the cytotoxicity, cellular uptake and photophysical properties of fac-[Re(CO)3(bpy)X]+/0/- complexes (X= H2O, HCys-, NAC2-, S2O32-; bpy=2,2’-bipyridine). The cytotoxicity of fac-[Re(CO)3(bpy)(H2O)]+ is diminished when the aqua ligand is replaced by cysteine or thiosulfate.
- ItemOpen AccessDevelopment of Purge-and-Trap Sample Preconcentrator for Enhanced Detection of Alkyl Nitrates by Thermal Dissociation Cavity Ring-Down Spectroscopy(2016) Ye, Connie; Osthoff, Hans; Thurbide, Kevin; Gailer, Jürgen; Marriott, RobertThis thesis describes the development of a sample preconcentrator for enhanced detection of alkyl nitrates (∑ANs) in ambient air by thermal dissociation cavity ring-down spectroscopy (TD-CRDS). The recovery of isopropyl nitrate on Tenax adsorbent was (96±1)% and unaffected by potential interfering gases such as ozone and nitrogen dioxide. Mixtures of five alkyl nitrates were also recovered in high yield in dry (99±5)% and in humidified air (92±11)% with relative humidity of 83%. Results from the first field deployment of the new instrument from the Ozone-depleting Reactions in a Coastal Atmosphere (ORCA) campaign are presented. The ORCA campaign took place at the Amphitrite Point Observatory on the West coast of Vancouver Island in Ucluelet, B,C. from July 6-31, 2015. A signal to noise ratio of 16.8 and a limit of detection of 7 pptv were demonstrated in the field. Future applications include measurements of isoprene and monoterpene nitrates.
- ItemOpen AccessEffect of the human serum albumin concentration on the metabolism of cisplatin in vitro(2014-05-14) Morris, Thomas; Gailer, JürgenSize-exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) was employed to study the effect of the human serum albumin (HSA) concentration on the metabolism of cisplatin (CP) in human blood plasma in vitro. Plasma from 14 healthy adults and 11 pediatric cancer patients was spiked with CP and the Pt distribution was determined at the 5 min and 2 hr time point. The results revealed that as the HSA concentration decreased, the percent of Pt-bound to HSA decreased; whereas the percent of Pt-hydrolysis products increased, while other Pt-species were largely unaffected. These changes were corroborated by fortifying the cancer patient plasma with HSA to healthy concentrations followed by the addition of CP. In summary, these findings imply that the infusion of cancer patients with HSA to healthy plasma levels may decrease the fraction of free hydrolysis products, which may alleviate the severe toxic side effects of CP.
- ItemOpen AccessInteractions of Inorganic Mercury and Inorganic Cadmium with Biomimetic and Complex Biological Membranes and their Influence on Membrane Packing and Size(2016) Kerek, Evan; Prenner, Elmar; Gailer, Jürgen; Zaremberg, Vanina; Sutherland, ToddInorganic mercury (Hg2+) and Inorganic Cadmium (Cd2+) are toxic heavy metals linked to the development of cancer, diabetes and neurological dysfunctions. The effect of these metals on the fluidity and phase transition (Tm) of biomimetic and polar extract membranes was investigated using Laurdan Generalized Polarization (GP) and Dynamic Light Scattering (DLS). Hg2+ and Cd2+ electrostatically target and induce rigidity in membranes containing cationic and anionic lipids respectively. Hg2+ also imparts rigidity by acting as a catalyst in the vinyl ether hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) plasmalogens. Cd2+-induced rigidity of anionic membranes results in a stabilization of the gel phase and a suppression of the Tm of membranes composed of phosphatidic acid (PA), cardiolipin (CL), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). Cd2+ induces more rigidity in rigid anionic membranes compared to more fluid anionic membranes. These results further our understanding of metal-lipid interactions.
- ItemOpen AccessMetal Species in Biology: Bottom-Up and Top-Down LC Approaches in Applied Toxicological Research(2013-06-10) Gailer, JürgenSince the inception of liquid chromatography (LC) more than 100 years ago this separation technique has been developed into a powerful analytical tool that is frequently applied in life science research. To this end, unique insights into the interaction of metal species (throughout this manuscript “metal species” refers to “toxic metals, metalloid compounds, and metal-based drugs” and “toxic metals” to “toxic metals and metalloid compounds”) with endogenous ligands can be obtained by using LC approaches that involve their hyphenation with inductively coupled plasma-based element specific detectors. This review aims to provide a synopsis of the different LC approaches which may be employed to advance our understanding of these interactions either in a “bottom-up” or a “top-down” manner. In the “bottom-up” LC-configuration, endogenous ligands are introduced into a physiologically relevant mobile phase buffer, and the metal species of interest is injected. Subsequent “interrogation” of the on-column formed complex(es) by employing a suitable separation mechanism (e.g., size exclusion chromatography or reversed-phase LC) while changing the ligand concentration(s), the column temperature or the pH can provide valuable insight into the formation of complexes under near physiological conditions. This approach allows to establish the relative stability and hydrophobicity of metal-ligand complexes as well as the dynamic coordination of a metal species (injected) to two ligands (dissolved in the mobile phase). Conversely, the “top-down” analysis of a biological fluid (e.g., blood plasma) by LC (e.g., using size exclusion chromatography) can be used to determine the size distribution of endogenous metalloproteins which are collectively referred to as the “metalloproteome”. This approach can provide unique insight into the metabolism and the plasma protein binding of metal species, and can simultaneously visualize the dose-dependent perturbation of the metalloproteome by a particular metal species. The concerted application of these LC approaches is destined to provide new insight into biochemical processes which represent an important starting point to advance human health in the 21st century.
- ItemOpen AccessMethylated Trivalent Arsenic-Glutathione Complexes are More Stable than their Arsenite Analog(2008-05-15) Percy, Andrew J.; Gailer, JürgenThe trivalent arsenic glutathione complexes arsenic triglutathione, methylarsonous diglutathione, and dimethylarsinous glutathione are key intermediates in the mammalian metabolism of arsenite and possibly represent the arsenic species that are transported from the liver to the kidney for urinary excretion. Despite this, the comparative stability of the arsenic-sulfur bonds in these complexes has not been investigated under physiological conditions resembling hepatocyte cytosol. Using size-exclusion chromatography and a glutathione-containing phosphate buffered saline mobile phase (5 or 10 mM glutathione, pH 7.4) in conjunction with an arsenic-specific detector, we chromatographed arsenite, monomethylarsonous acid, and dimethylarsinous acid. The on-column formation of the corresponding arsenic-glutathione complexes between 4 and 37∘C revealed that methylated arsenic-glutathione complexes are more stable than arsenic triglutathione. The relevance of these results with regard to the metabolic fate of arsenite in mammals is discussed.
- ItemOpen AccessProbing interactions between exogenous metal/metalloid compounds and endogenous constituents by hplc/lc-icp-aes(2009) Pei, Katie L.; Gailer, Jürgen
- ItemOpen AccessProbing interactions between iron chelation therapeutics and platinum based anti-cancer drugs with plasma constituents by sec-icp-aes(2011) Sooriyaarachchi, Melani Devindya; Gailer, Jürgen
- ItemOpen AccessProbing the Chemistry of Methylmercury in Mammalian Blood Plasma(2022-01-04) Bridle, Tristen G.; Gailer, Jürgen; Jalilehvand, Farideh; Thurbide, KevinA size-exclusion chromatography-inductively coupled plasma-atomic emission spectroscopy (SEC-ICP-AES) technique was developed for the analysis of mammalian blood plasma for endogenous metalloproteins and exogenous metal species. Parameters related to sample preparation, separation, and detection were optimized. The blood plasma was filtered through a 0.45 ?m pore size syringe filter to preserve the separation performance of the column. The 1:1 dilution of filtered human and rabbit blood plasma in phosphate-buffered saline (PBS) prior to analysis was needed to prevent peak tailing and shoulder formation. An SRT-10C SEC column from Sepax Technologies Inc., when compared to a previously used Superdex 200 Increase SEC column, gave the same number of peaks with similar resolution, but in approximately half the time and with higher peak intensities. The mass recovery of proteins on the SRT-10C SEC column was 96 ± 2%. To explore the role that small molecular weight plasma thiols play in the delivery of methylmercury (CH3Hg+) to transporters located at the placental and blood-brain barriers, the described SEC-ICP-AES technique was applied to the analysis of CH3Hg+-spiked rabbit plasma using PBS mobile phases in the absence and presence of the small-molecular weight (SMW) sulfur compounds L-cysteine (Cys), L-homocysteine (hCys), L-glutathione (GSH), and D-methionine (Met). While Met did not affect the binding of CH3Hg+ to the main plasma protein, rabbit serum albumin (RSA), Cys, hCys, and GSH did. The presence of 50 ?M Cys, hCys, or GSH in the mobile phase resulted in the mobilization of CH3Hg+ from RSA in rabbit plasma and from pure RSA in solution. The SMW-Hg species that formed when hCys was present in the mobile phase was qualitatively identified by electrospray ionization mass spectrometry as CH3Hg-hCys. Using the developed metallomics technique I have found evidence of the formation of SMW-CH3Hg+ species under near-physiological conditions that may be involved in the translocation of CH3Hg+ from blood plasma to the brain.
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