Probing the Chemistry of Methylmercury in Mammalian Blood Plasma
Date
2022-01-04
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Abstract
A 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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Keywords
size-exclusion chromatography, inductively coupled plasma, atomic emission spectroscopy, methylmercury, blood plasma, thiol, cysteine, homocysteine, glutathione, metalloprotein
Citation
Bridle, T. G. (2022). Probing the Chemistry of Methylmercury in Mammalian Blood Plasma (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.