Exogenous N-linoleoyl tyrosine marker as a tool for the characterization of cellular oxidative stress in macrophages.
Oxidative stress and its resultant products continue to attract investigators. Numerous endogenous substances have been suggested as potential markers for the identification of oxidative stress in tissues and organisms. In this study, we present a novel concept whereby an exogenous marker is designed and synthesized for the characterization of oxidative stress. The designed marker is constructed from tyrosine (Tyr) and linoleic acid (LA), which are attached covalently to form N-linoleoyl tyrosine (N-LT). Each of the two components (Tyr and LA) is known to be easily oxidized upon exposure to different types of reactive species. Combining the two allows their distinction from the endogenous Tyr and LA in the tested biological samples. The ability of the N-LT marker to characterize oxidative stress in macrophage cell lines was first studied using different types of ROS/RNS. N-LT was found to interact with macrophages, binding to the cell membrane. Upon treatment of J-774 A.1 macrophages with N-LT (40 microM) and with various oxidants; HOCl (0.2, 0.4 mM), copper ions (20 microM), SIN-1 (0.1, 1.0 mM), specific oxidized N-LT (Ox-N-LT) products were formed, depending on the type of oxidant used. Exposing cells to HOCl (0.2 mM) resulted in exclusive attack of the LA residue of N-LT, preferentially forming an adduct of HOCl to the LA double bond (N-L(HOCl)T, 4.3%). In contrast, when SIN-1 (0.1 mM) was applied as the oxidant, the Tyr moiety of N-LT was most reactive, yielding a nitration product of the Tyr aromatic ring (N-LT(NO(2)), 1.8%). Similar N-LT oxidation in cell-free systems yielded a significantly higher content of Ox-N-LT (10.8% N-L(HOCl)T, 7% N-LT(NO(2)). The designed marker was then tested with peritoneal macrophages taken from atherosclerotic apolipoprotein-deficient (E(0)) mice showing specific and selective oxidation of N-LT to yield N-LT-hydroperoxide (1.9% N-L(OOH)T), at significantly higher levels than resulted from similar experiments using peritoneal macrophages harvested from control BalbC mice (0.0% N-L(OOH)T). In contrast, the differences in N-L(epoxy)T level between BalbC and E(0) mice were not significant using both types of peritoneal macrophages (E(0) and BalbC), suggesting that N-L(OOH)T is characteristic of the atherosclerotic state. Thus, we show that the designed marker is sufficiently sensitive to detect oxidative stress imposed on cells and cell-free systems and to react selectively with the various ROS/RNS induced. Such a marker may be useful for characterizing oxidative stress in general, and possibly also in oxidative-stress-associated diseases.
Szuchman
Andrea
A
Laboratory of Natural Medicinal Compounds, Migal-Galilee Technological Center, Kiryat Shmona and Tel-Hai Academica College, Israel.
Aviram
Michael
M
Soliman
Khatib
K
Tamir
Snait
S
Vaya
Jacob
J
D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology; D019787:Linoleic Acid / Q000737:chemistry* / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
D014443:Tyrosine; C002385:linsidomine; D006997:Hypochlorous Acid; D003300:Copper; D019787:Linoleic Acid; D008981:Molsidomine