NADPH-dependent lipid peroxidation capacity in unfixed tissue sections: characterization of the pro-oxidizing conditions and optimization of the histochemical detection |
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Authors: | M Thomas W M Frederiks C J F Van Noorden K S Bosch and A Pompella |
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Institution: | (1) Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands;(2) Institute of General Pathology, University of Siena, Strada del Laterino 8, 53100 Siena, Italy;(3) Present address: Department of Pathology, Ontario Cancer Institute, Princess Margaret Hospital, 500 Sherbourne Street, M4X 1K9 Toronto, Ontario, Canada |
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Abstract: | Summary Factors which influence the iron-stimulated lipid peroxidation in rat liver have been studied by incubating unfixed cryostat
sections with a pro-oxidant system and using an optimized histochemical detection method for lipid peroxidation products with
3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B. We used a method that was slightly different from the one described
previously. The final reaction product was exclusively localized in the cytoplasm of liver parenchymal cells with a homogeneous
distribution within the liver lobule. The absorbance maximum, as measured cytophotometrically, was found to be 550 nm. Maximum
lipid peroxidation was observed when the pro-oxidant system contained 0.2 mm NADPH, 1 mm ADP and 15 μm FeCl2. Some reaction product was found when NADPH was omitted. Iron concentrations higher than 180 μm prevented the formation of lipid peroxidation products in certain areas of the sections, whereas ADP concentrations higher
than 1 mm inhibited the reaction in the whole section. A pH dependency was also observed, with the highest lipid peroxidation at pH
7.2. Optimum lipid peroxidation was induced by incubating for 30 min at 37°C with the pro-oxidant system. A linear relationship
was found between the thickness of the sections (up to 20 μm) and the amount of lipid peroxidation products. The addition
of scavengers of O2- (superoxide dismutase), hydrogen peroxide (catalase) and OH · (mannitol) to the first step medium did
not affect the amount of final reaction product. These findings appear to confirm the hypothesis proposed for events occurring
in isolated microsomes, leading to the formation of hydroperoxides and ultimately lipid peroxidation-derived carbonyls. The
present method is a useful tool for studying the capacity of lipid peroxidation in tissues under different (patho)physiological
conditions. |
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