Lipid peroxidation in isolated rat hepatocytes measured by ethane and n-pentane formation

Abstract

Isolated rat hepatocytes (1×10⁷ cells/ml) were aerobically incubated in Eagle's Minimum Essential Medium which contained 2.0% albumin. As potential parameters of lipid peroxidation ethane and n-pentane formed were measured in samples obtained from the gas phase above the incubation mixture. 15–30 nmol ethane or n-pentane were produced by 10⁷ hepatocytes within 90 min. Carbon tetrachloride (CCl₄) or ADP-complexed ferrous ions stimulated ethane and n-pentane formation considerably, depending on the concentrations of the compounds. With CCl₄ 10⁷ cells formed max 180 nmol ethane and 140 nmol n-pentane within 90 min incubation, whereas with Fe(II) max 130 nmol ethane and 220 nmol n-pentane could be detected. When n-pentane was added to the gas phase above the incubation mixture containing either medium or medium plus hepatocytes its amount decreased by 30% within the first 5 min of incubation. However, afterwards only minor amounts of n-pentane disappeared, even in the presence of hepatocytes. This indicates that n-pentane equilibrates with the cell suspension under the conditions used. Cell viability, as determined by the release of lactate dehydrogenase into the medium and by the uptake of trypan blue by the cells, and the recovery of the cells decreased only in presence of relatively high concentrations of CCl₄, or Fe(II) respectively. However, a maximal effect on ethane and n-pentane formation was reached already with lower concentration.