Gailer, JürgenGibson, Matthew2017-12-152017-12-1520172017http://hdl.handle.net/11023/4274Bioanalytical 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.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Chemistry--AnalyticalBiochemistryChemistry--Inorganicmaster thesis10.11575/PRISM/28741