Fluorescent products of lipid peroxidation of mitochondria and microsomes

Abstract

Liver microsomes and mitochondria and heart sarcosomes from rats fed diets with varying α-tocopherol concentrations and lipid contents were peroxidized over a 6 hr time period. Lipid peroxidation was measured by absorption of oxygen, production of thiobarbituric acid (TBA) reactants and by development of fluorescence. The spectral characteristics of the fluorescent compounds were the same for all peroxidizing systems; the excitation maximum was 360 nm and the emission maximum was 430 nm. As time of peroxidation increased, uptake of oxygen and production of fluorescent compounds increased. These two parameters as well as production of TBA reactants were dependent upon dietary antioxidant and all three had an inverse relationship with the amount of dietary α-tocopherol. The relationship between absorption of oxygen and development of fluorescent compounds was also dependent upon dietary polyunsaturated fats (PUFA). Subcellular particles from animals fed higher levels of PUFA produced more fluorescent products per mole of oxygen absorbed than did those from animals on a diet with lower PUFA content. TBA reacting products increased with time during the initial phase of peroxidation: in the microsomal systems their production stabilized or decreased by 4–6 hr of peroxidation. Using the synthetic 1-amino-3-iminopropene derivative of glycine as standard for quantitation of fluorescence, the molar ratios of oxygen absorbed per fluorescent compound produced were calculated. This ratio for subcellular particles isolated from rats fed diets with PUFA ratios similar to those in the average American human diet was 393∶1. The fluorescent compounds had the same spectral characteristics as the lipofuscin pigment that accumulates in animal tissues as a function of age, oxidative stress or antioxidant deficiency. The fluorescent molecular damage represented by that accumulated in human heart age pigment by 50 years of age was calculated to have been caused by approximately 0.6 μmole of free radicals per gram of heart tissue.