Lipid Peroxidation in Isolated Hepatocytes

Abstract

Intracellular lipid peroxidation was initiated by the addition of ADP-complexed ferric iron to isolated rat hepatocytes and the reaction monitored by the thiobarbituric acid method or by measurement of the formation of conjugated dienes. Both the production of malondialdehyde (thiobarbituric-acid-reacting substances) and of conjugated dienes was dependent, on the ADP · Fe³⁺ concentration in a dose-related fashion. Malondialdehyde formation stopped spontaneously within 20 min after the initiation of the reaction and the plateau reached was also related to the ADP · Fe³⁺ concentration. Control experiments revealed that more than 90%, of the malondialdehyde accumulating during the incubation period could be ascribed to intracellular production. The cellular NADPH/NADP⁺ ratio was always high and only slightly decreased upon ADP · Fe³⁺-induced lipid peroxidation which, however, was associated with a marked decrease in the cellular glutathione concentration. The rate of accumulation of malondialdehyde as well as the final level reached during ADP · Fe³⁺-initiated lipid peroxidation was increased by the addition of chloral hydrate. This apparent stimulatory effect could, however, be ascribed to the inhibition of the mitochondrial oxidation of the malondialdehyde formed during cellular lipid peroxidation, thus allowing more malondialdehyde to accumulate during the process. ADP · Fe³⁺-induced cellular lipid peroxidation was associated with a decrease in the concentration of glutathione. Also, lowering of the intracellular glutathione level by the addition of diethyl maleate or by simply preincubating the hepatocytes (up to 50 min) promoted the ADP · Fe³⁺ malondialdehyde production and formation of conjugated dienes. Furthermore, when cellular glutathione concentration had been lowered by preincubation of the hepatocytes, significant malondialdehyde production could be observed even at ADP · Fe³⁺ concentrations which were too low to induce measurable lipid peroxidation in fresh hepatocytes. It is thus concluded that glutathione has an important role in the cell defence against lipid peroxidation and suggested that the isolated hepatocytes provide a suitable experimental model system for the characterization of this and other possible cellular defence mechanisms and how they are affected by the nutritional status of the donor animal.