Novel designed probes for the characterization of oxidative stress in biological fluids, cells, and tissues.

Oxidative stress (OS) is linked to the development of human diseases. Early identification of OS-associated diseases is essential in the control of their progression and treatment. Efforts have been undertaken to identify reliable endogenous markers, which correlate with the progression of a disease in an organ undergoing OS. An ideal biomarker must be validated, utilize noninvasive sampling, and have a simple, specific and highly sensitive detection method. Among the currently used markers assessing OS, are those that are nonspecific (peroxide value [PV], conjugated dienes [CD], thiobarbitoric acid reactive substances [TBARS]), and others that measure end-products of oxidized degradation biomolecules (isoprostanes, oxysterols, keto-proteins, 8-oxodeoxyguanosine), whose accumulation is not necessarily correlated with augmented OS. The search for a more reliable marker necessitates new approaches to fulfill such requirements and overcome many of the obstacles associated with the current markers. We suggest a new strategy of using designed exogenous novel reporters, constructed from endogenous subunits, that are sensitive to reactive oxygen and nitrogen species (ROS/RNS) and commonly known to react with them, forming specific oxidized products. These subunits are tyrosine (representing proteins), bonded covalently to linoleic acid (representing polyunsaturated fatty acids) forming an amide bond, which can be further connected through an ester bond to a third unit, either to cholesterol (representing sterols) or to 2'-deoxyguanosine (representing DNA). Oxidation of the designed probe can outline, in real time, the formation of oxidation products and distinguish them from intrinsic biomolecules, provide information about the relative subunit susceptibilities to a specific oxidant challenge, and allow for the assessment of the utility of intervention, such as antioxidant supplementation. By utilizing such markers, it may be possible to correlate between the damaged fingerprints of the marker and the specific pathological conditions. The above markers were tested to characterize OS in in vitro and in in vivo experiments, such as in those carried out in human fluids (blood, serum, saliva), tissues (brain or muscle homogenates), and cells (macrophages, astrocytes, neurons), pertaining to OS-associated diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.