engleski

Subcellular 4-hydroxy-2-nonenal determination with immunogold method

Oxidative stress, a balance shift of oxido-reductive reactions to oxidation, is resulting in excessive production of reactive oxygen species (ROS). Oxidative stress is considered an important part of different disorders (malignant tumors, traumatic injuries etc.), as well as in physiological processes (before all in inflammation). Overproduction of ROS is cytotoxic, damages macromolecules and can lead to occurrence of lipid peroxidation and its final toxic products, among which is 4-hydroxy-2-nonenal (HNE), denoted as a “ second toxic messenger of free radicals” . The aim of this study was to determine distribution of HNE-histidine conjugates on subcellular level. For this purpose spleen tissue, peripheral blood leukocytes and peritoneal macrophages obtained from rats challenged by the i.p. application of tribomechanically micronized zeolites (non-specific stimulator of macrophages) were analyzed. As positive controls we used 100  M HNE treated samples. We used genuine monoclonal antibodies against HNE-histidine conjugates for immunoelectronmicroscopical analysis of HNE-histidine conjugates. HNE-untreated specimens had better preserved cell structures and moderate HNE positivity marked with colloidal gold 10 nm particles. HNE-histidine conjugates were detected inside mitochondria, near phagosomes and membranes structures, beside macrophage granules or attached to the cellular membrane, but were rarely present in the cytosol or the nuclei. Ultrastructural analysis of leukocytes, macrophages and spleen tissue that were treated with 100 μ M HNE showed very evident HNE-positivity and slightly damaged cytoplasmic structures. HNE positivity was remarkably present beside macrophage granules, near and on membrane structures and diffusely spread in the cytoplasm. Therefore, immunodetection of HNE-histidine conjugates may be used as analytical immunochemical method to study HNE formation in pathological and physiological processes, as well as addition of pathomorphological diagnostic procedures. A further goal of our research is to analyze biological effects of lipid peroxidation products on gene expression depending on the presence of HNE-histidine conjugates in inflammatory cells.

subcellular HNE; immunogold method

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