Thesis-2017-Fagieh.pdf (5.74 MB)
ICP-MS determination of Zn, Cu, Fe and Mn in muscle cells as potential markers of oxidative stress
thesisposted on 2017-10-19, 16:19 authored by Taghreed M. Fagieh
Oxidative stress is imbalance between oxidant and antioxidant levels in living systems. Human cells are protected from reactive oxygen species by endogenous enzymatic antioxidants. Most of these compounds require particular redox metals in their structures as cofactors to allow them to scavenge the free radicals such as Cu, Zn-SOD, Mn-SOD and catalase (Fe). The aim of this study was to quantify these metals in human cells to evaluate their effectiveness as novel biomarkers for measuring oxidative stress. The metals (Zn, Cu, Fe, Mn) were measured in vitro in skeletal muscle cells (C2C12) which were incubated under hypoxia/hyperoxia conditions generated by varying oxygen level from 1%-60% for 24 and 48 hours. Two methods were used to perform the analysis. ICP-MS was applied to liquid samples to quantify Zn, Cu, Fe and Mn in cell populations. And LA-ICP-MS was employed to solid samples to measure their intensity in individual cells. The data acquired from both techniques are positively correlated confirming the reliability of the two approaches. All elements of interest were successfully measured except Mn which was not detected in single cells using LA-ICP-MS due to the limit of detection. Interestingly, the results showed that their concentration increased dramatically in cells grown at 25%-60% O2, the most significant increase was in Cu at 60%O2. None showed any increase at 5%-15% O2 indicating normoxia states. At 1%O2, all elements except Fe showed a significant increase and the most remarkable growth was in Mn. More interestingly, increasing incubation to 48 hours for liquid samples had differing effects on the elements. Zn and Cu concentrations were unaffected by increasing incubation time except at 60%O2 where they showed further growth. In contrast, Mn concentration grew sharply over oxygen levels of 30%-50% with no further effect at 1%, while Fe concentration decreased at 1%O2 and grew steadily over oxygen levels of 5%-60%. It can be concluded that all four elements were significantly affected by stress conditions applied to cells, but at different rates. Importantly, a novel analytical method was introduced in this current study since there have been no previous reported investigations measuring changes in concentration of redox-active elements in human cells subjected to different controlled oxidative stress conditions in vitro.
Publisher© Taghreed Muhammad Fagieh
Publisher statementThis work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/
NotesA Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.