Metabolic biomarkers for oxidative and nitrosative stress

Oxidative stress and nitrosative stress are caused by the excessive formation of -or insufficient protection against- reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively. ROS and RNS are molecules containing unpaired electrons, which render them exceptionally reactive to DNA, proteins and lipids. The superoxide radical (•O- ) is formed as a byproduct of different enzymatic reactions, including cellular respiration, and is converted to the hydroxyl radical (•OH) in the Fenton reaction which is catalyzed by transition metals such as copper and iron. •OH is primarily responsible for DNA damage and lipid peroxidation. Reactive nitrogen species arise from the regulated production of •NO from L-arginine by NO synthase. In combination with •O- , •NO will form the peroxynitrite anion (ONOO-), which causes DNA fragmentation, lipid peroxidation and protein modification (2).
Some biomarkers of oxidative and nitrosative stress have already been developed (3), but they generally lack sufficient stability, specificity or sensitivity and do not provide direct information on the pathophysiology. A major challenge in the design of biomarkers for oxidative and nitrosative stress is the instability of many reactive species. This can be overcome by measurement of more indirect, downstream targets of oxidative damage. Alternatively, we will work towards increased compound stabilization during sampling.
The aim of this project is to improve the coverage of metabolic biomarkers for different forms of oxidative and nitrosative stress and to correlate these to disease progression using a combination of in vitro and in vivo models.
F2-isoprostanes are formed through non-enzymatic reactions between superoxide and hydroxyl radicals and arachidonic acid, a common membrane lipid that holds four double bonds. F2- isoprostane formation is considered to be the golden standard in determination of levels of oxidative stress given their stability. We have set up a sensitive, robust and quantitative platform to determine ROS levels in plasma and urine on a triple-quadrupole LC-MS/MS platform. This platform has been validated (see outlined in detail below) and was transferred to the Demonstration and Competence Laboratory of the NMC in summer 2010.
 

Main project title: 
Biology driven improved coverage of the metabolome
AIO/PD: 
AIO 1-1-09
Researcher: 
Co-applicant: 
Code 1: 
QP2
Code 2: 
C
Status Project Proposal: 
Approved