Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions

Abstract

Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-Fe. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine and the derivatives were extracted and separated by TLC into 3 zones of non-polar materials and 1 fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by HPLC [high performance liquid chromatography] demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-Fe contained mainly 4-hydroxyhex-3-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-Fe was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the HPLC spectra obtained from zones I and III. The stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-Fe are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were metabolized very rapidly. Nonetheless, both CCl4 and ADP-Fe produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-Fe it was found that .apprx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium. This was not leakage from dead cells, as 90-95% of the hepatocytes had retained their integrity at the end of the incubation. Release of carbonyl products from cells stimulated to undergo lipid peroxidation may be a mechanism for spreading an initial intracellular disturbance to affect critical targets outside the parent cell.