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-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-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-iron 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 h.p.l.c. spectra obtained from zones I and III. However, 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-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 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.

Evidence that 4-Hydroxynonenal Mediates Oxidative Stress-Induced Neuronal Apoptosis

“…FeSO 4 and A␤ are known to induce lipid peroxidation and to kill cultured neurons (Poli et al, 1985;Zhang et al, 1993;Behl et al, 1994;Goodman and Mattson, 1994;Muller and Krieglstein, 1995;Goodman et al, 1996). Exposure of PC12-V or PC12-Bcl2 cells to high concentrations of FeSO 4 , A␤, and HNE resulted in rapid decreases in levels of MTT reduction to Ͻ20% of basal levels within 6 hr of exposure ( Fig.…”

Increased levels of 4‐hydroxynonenal in human monocytes fed with malarial pigment hemozoin A possible clue for hemozoin toxicity

“…HNE analysis was performed as described [17][18][19][20] after derivatization of HNE with dinitrophenylhydrazine and separation of dinitrophenylhydrazones by thinlayer chromatography and subsequently by HPLC. Peak identification was performed by comparison of retention times of peaks of biological extracts and of standard extracts (pure HNE was a kind gift from Professor H. Esterbauer, Graz, Austria), by co-elution of biological extracts with the reference compounds and by comparison of spectra [19,20].…”

Histological Detection of Lipid Peroxidation Following Infusion of Tert-Butyl Hydroperoxide and ADP-Iron Complex in Perfused Rat Livers.

“…CC14 and related halogenomethanes are well-known to induce lipid peroxidation (1,4). Furthermore, a large amount of aldehydic compounds are reported to be produced dur ing lipid peroxidation in various biological sys tems including a halogenomethane-induced peroxidation system (7)(8)(9)(10)(11)(12). Therefore, it appears that the histologic Schiff positive reac tion may be applied for the detection of peroxidized lobular zones.…”