Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Kerner, A.
Determination of Superoxide anion by a HPLC Method in endothelial dysfunction induced by oxLDL.
[ Diplomarbeit/Master Thesis (UNI) ] University of Graz; 2017. pp.75.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Hallström Seth
- Altmetrics:
- Abstract:
- The superoxide anion (O2●─) is a biological radical which e.g.: can be formed by NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), a membrane-bound enzyme complex. Alternatively, O2●─ can also be formed in pathophysiological situations by uncoupling of nitric oxide synthase (NOS). Enhanced O2●─ formation plays a key role in the pathology of numerous cardiovascular and vascular dysfunctions including arteriosclerotic plaque formation. The primary aim of this thesis was to establish an optimized high performance liquid chromatography (HPLC) method capable of measuring intracellular O2●─ concentrations in endothelial cells. There are numerous methods for determination of O2●─ produced in biological samples. However, most of these methods contain pitfalls in the specificity for O2●─. We established a method described in literature based on the reduction of dihydroethidine (DHE) to 2-hydroxy-ethidium by O2●─ with simultaneous separation of the coformed ethidium by HPLC. This method enables a highly specific determination of O2●─. Other methods and their pitfalls used for O2●─ determination are also described in the introduction section. The second part and second aim of this thesis was to utilize the established sensitive and specific method for O2●─ determination in endothelial dysfunction induced by oxidized low density lipoprotein (oxLDL). Handling of DHE under cell culture conditions, extraction procedures for DHE and sample preparation for HPLC were optimized. Finally, measurements of O2●─ under the influence of various agonists of endothelial NOS (eNOS) were performed including oxLDL. It could be clearly demonstrated that oxLDL enhances O2●─ formation by inducing uncoupling of eNOS. In summary, a routine method for determination of O2●─ in cultured cells was established and its application under simulated pathophysiological conditions tested.