טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSzuchman Andrea
SubjectCharacterization of Oxidative Stress Processes and their
Products by Chemically Synthesized Markers
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Michael Aviram
Professor Jacob Vaya


Abstract

The association between oxidative stress (OS) and human diseases has brought about the necessity for a marker that can identify and characterize OS, based on an oxidative damage fingerprint. Novel exogenous markers were designed to measure and characterize the OS damage, to identify the type of reactive oxygen or nitrogen species (ROS/RNS) involved and to study the protective effectiveness of antioxidants.

First, the level and type of oxidized products formed were compared after different ROS were applied to cholesterol (chol), linoleic acid (LA) and tyrosine (Ty). The resulting oxidation pattern was found to differ, if the substrates were exposed alone, or in a mixture to the various ROS. Since biological systems are composed of such molecules, we concluded that simultaneous detection of the major oxidized products is necessary for better characterization of the OS outcome.  Therefore, an exogenous marker was designed and synthesized from Ty and LA, to form N-linoleoyl tyrosine (N-LT). Combining the two, allowed their distinction from the endogenous Ty and LA in the tested biological samples. Treatment of the N-LT marker with various oxidants in macrophage cell lines resulted in specific oxidized N-LT products, depending on the oxidant employed. Exposing cells to HOCl resulted in the exclusive attack of the LA residue of N-LT.  In contrast, when SIN-1 was applied as the oxidant, the Ty moiety of N-LT was the most reactive. In peritoneal macrophages from atherosclerotic apolipoprotein-deficient (E0) mice, specific and selective oxidation of N-LT yielded significantly higher levels of N-LT-hydroperoxide (N-L(OOH)T), than in those from control BalbC mice, implying that N-L(OOH)T is characteristic of the atherosclerotic state. Subsequently, another exogenous marker was employed to characterize OS in diabetic and hypercholesterolemic (Hc) blood, based on the oxidative damage fingerprint. It was constructed from Ty, LA and Chol, to form the N-linoleoyl tyrosine cholesteryl ester molecule. By this designed marker, it was demonstrated that the ratios of oxysterol to cholesterol, and that of oxidative N- LT to N-LT were significantly higher, and specific to blood samples of diabetic patients. In diabetic patients' serum, 7-ketocholesterol was the major oxidized product, and 7β-hydroxy cholesterol was the most characteristic. Diabetic patients' serum oxidized predominantly the linoleate moiety to hydroperoxide, while an epoxidation reaction dominated in blood cells. Pomegranate juice consumption by diabetics, significantly reduced serum oxysterol-to-cholesterol level, with a major reduction in 7b-hydroxy cholesterol.

Our studies demonstrate that an exogenous marker characterizes OS and yields important information on the extent and type of OS.